Methods of Molecules Chemical Analysis

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

isolation of plant enzymes and organelles. Methods in Enzymology.

Genetics Laboratory; 1994

PNAS. 1969;**62**:1151-1158

2011;**124**(2):311-314

2003;**315**(1):85-89

2009:1-10

2015;**12**(3A):543-548

Biology. 1993;**63**(2):161-165

[20] Gordon CKR, Edward AD, Donella HMJS, Cohen OJM. The mechanism of action of ribonuclease.

[21] Shepherd LD, McLay TG. Two micro-scale protocols for the isolation of DNA from polysaccharide-rich plant tissue. Journal of Plant Research.

[22] Michiels A, Van den Ende W, Tucker M, Van Riet L, Van Laere A. Extraction of high-quality genomic

[23] Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1982

[24] Tan SC, Yiap BC. DNA, RNA, and protein extraction: The past and the present. BioMed Research International.

[25] Jadhav KP, Ranjani RV, Senthil N. Chemistry of plant genomic DNA extraction protocol. Bioinfolet.

[26] Cullis P, Elsy D, Fan S, Symons M. Marked effect of buffers on yield of single-and double-strand breaks in DNA irradiated at room temperature and at 77 K. International Journal of Radiation

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DNA from latex-containing plants. Analytical Biochemistry.

[19] de León DG. Laboratory Protocols. CIMMYT: CIMMYT Applied Molecular

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**142**

**145**

**Chapter 9**

**Abstract**

Principles of Chromatography

This chapter aims to explain the key parameters of analytical method development using the chromatography techniques which are used for the identification, separation, purification, and quantitative estimation of complex mixtures of organic compounds. Mainly, the versatile techniques of ultra−/high-performance liquid chromatography (UPLC/HPLC) are in use for the analysis of assay and organic impurities/related substances/degradation products of a drug substance or drug product or intermediate or raw material of pharmaceuticals. A suitable analytical method is developed only after evaluating the major and critical separation parameters of chromatography (examples for UPLC/HPLC are selection of diluent, wavelength, detector, stationary phase, column temperature, flow rate, solvent system, elution mode, and injection volume, etc.). The analytical method development is a process of proving the developed analytical method is suitable for its intended use for the quantitative estimation of the targeted analyte present in pharmaceutical drugs. And it mostly plays a vital role in the development and

*Narasimha S. Lakka and Chandrasekar Kuppan*

**Keywords:** analytical method development, ultra performance liquid

pharmaceuticals for separating the drug (API) mixture in particular.

impurities, impurity profiling study, forced degradation study

chromatography (UPLC), high-performance liquid chromatography (HPLC), assay,

It is well known that chromatography is a laboratory technique used for separation and quantification of complex organic mixtures which cannot be separated effectively by other purification techniques. The constituents of a mixture dissolved in solvent get separated gradiently according to their affinities to the stationary phase with the help of mobile phase one after another. Chromatography is invented by *Mikhail Semenovich Tswett* in 1903 during his research on plant pigments such as chlorophylls, xanthophylls, and carotenoids [1] which got extended for analyzing organic molecules of different kinds especially pharmaceutical from the year 1920 [2]. Invention of chromatography made the jobs of organic chemist and the whole industry relying on them especially pharma industry easier. Keeping in mind the various fields where this technique has been used, this chapter focuses on the use of chromatography in

Nowadays, many different kinds of chromatography techniques, such as thinlayer chromatography (TLC), paper chromatography, and liquid chromatography (e.g., HPLC, UPLC, and preparative HPLC), supercritical fluid chromatography, and gas chromatography (GC)) have been designed and utilized for the separation

Method Development

manufacture of pharmaceuticals drugs.

**1. Introduction**

#### **Chapter 9**

## Principles of Chromatography Method Development

*Narasimha S. Lakka and Chandrasekar Kuppan*

#### **Abstract**

This chapter aims to explain the key parameters of analytical method development using the chromatography techniques which are used for the identification, separation, purification, and quantitative estimation of complex mixtures of organic compounds. Mainly, the versatile techniques of ultra−/high-performance liquid chromatography (UPLC/HPLC) are in use for the analysis of assay and organic impurities/related substances/degradation products of a drug substance or drug product or intermediate or raw material of pharmaceuticals. A suitable analytical method is developed only after evaluating the major and critical separation parameters of chromatography (examples for UPLC/HPLC are selection of diluent, wavelength, detector, stationary phase, column temperature, flow rate, solvent system, elution mode, and injection volume, etc.). The analytical method development is a process of proving the developed analytical method is suitable for its intended use for the quantitative estimation of the targeted analyte present in pharmaceutical drugs. And it mostly plays a vital role in the development and manufacture of pharmaceuticals drugs.

**Keywords:** analytical method development, ultra performance liquid chromatography (UPLC), high-performance liquid chromatography (HPLC), assay, impurities, impurity profiling study, forced degradation study

#### **1. Introduction**

It is well known that chromatography is a laboratory technique used for separation and quantification of complex organic mixtures which cannot be separated effectively by other purification techniques. The constituents of a mixture dissolved in solvent get separated gradiently according to their affinities to the stationary phase with the help of mobile phase one after another. Chromatography is invented by *Mikhail Semenovich Tswett* in 1903 during his research on plant pigments such as chlorophylls, xanthophylls, and carotenoids [1] which got extended for analyzing organic molecules of different kinds especially pharmaceutical from the year 1920 [2]. Invention of chromatography made the jobs of organic chemist and the whole industry relying on them especially pharma industry easier. Keeping in mind the various fields where this technique has been used, this chapter focuses on the use of chromatography in pharmaceuticals for separating the drug (API) mixture in particular.

Nowadays, many different kinds of chromatography techniques, such as thinlayer chromatography (TLC), paper chromatography, and liquid chromatography (e.g., HPLC, UPLC, and preparative HPLC), supercritical fluid chromatography, and gas chromatography (GC)) have been designed and utilized for the separation and purification of pharmaceutical drugs [3]. In this chapter, the authors discuss the principles for chromatography method development using ultra/high-performance liquid chromatography (UPLC/HPLC) techniques for the analysis of assay and organic impurities/related substances/degradation products of pharmaceuticals (any drug product/drug substance/intermediate/raw material of pharmaceuticals). These techniques are developed substantially as a result of the work of *Archer John Porter Martin* and *Richard Laurence Millington Synge* during the 1940s and 1950s, for which they won the 1952 Nobel Prize in Chemistry [4]. Commonly used characterizing technique in pharma industry is liquid chromatography (e.g., HPLC, UPLC, and LC–MS). Each one varies in the stationary phase and operational conditions. HPLC and UPLC can be used as a quantitative technique if coupled with a mass detector (MS) to elucidate the structure of the molecule and quantification.

In pharma industry specific, stability-indicating HPLC/UPLC methods have to be developed to estimate the assay and to quantitatively determine the impurities of new drug substances and drug products [5]. Assay is a quantitative test of a substance to determine the amount of an individual components present in it. Impurity is an unknown component of drug substance that is not the chemical entity. Assay and impurity tests are major and critical quality attributes of the pharmaceutical dosage forms which help to check and ensure the quality, safety, and efficacy of drug substances and drug products. This chapter will discuss the various parameters that have to be chosen to run the chromatography in order to have a better separation and maximum purity. The process of changing the conditions in order to design a best method run for a particular drug mixture or compound is called the analytical method development.

#### **2. Analytical method development**

Analytical method development is a process of proving that the developed chromatography method is suitable for its intended use in the development and manufacturing of the pharmaceutical drug substance and drug product. The basic separation techniques and principles involved in the analytical method development using the HPLC and UPLC are listed as follows:

**147**

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

• Experimentation to finalize the method

• Forced degradation studies (stress resting)

• Selection of diluent

• Samples to be used

• Methods of extraction

• Evaluation of stress testing

• Finalization of wavelengths

• Robustness of the method

• Relative response factor

• Quantification methods

formulated products.

**2.1 Literature search**

• Mass balance study

• Stability of solution

• System suitability

• Selection of solvent delivery system (elution mode)

• Selection of test concentration and injection volume

i.Before starting an analytical method development, literature on some of the column characteristics as mentioned below has to be referred for the target molecules or similar molecules or precursors from open resources like articles, books, pharmacopeia reports, etc. This will give a tentative choice in designing a method for initial or test experiments, which will be further modified or updated to develop a method which fits the separation process for better results in terms of reproducibility, quantification, etc. *Solubility profile* of drug substance in different solvents at different pH conditions is useful while selecting the diluents for standard solutions and extraction solvents for test solutions.

ii.*Analytical profile* is useful in understanding the physicochemical properties (e.g., *pKa*, melting point, degradation pathways, etc.) and absorption characteristics of drug in selecting the detector wavelength for analysis.

iii. *Stability profile* of the drug substance with respect to storage conditions

iv.*Impurity profile* collects the information of impurities and degradation profile of the drug substance during their formation pathways. This helps

(sensitivity of the drug towards light, heat, moisture etc.) is useful as it helps in adopting the suitable/adequate precautions while handling drug and its

	- Buffer and its strength
	- pH of buffer
	- Mobile-phase composition

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*


*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

analytical method development.

**2. Analytical method development**

• Selection of chromatography mode

• Selection of column (stationary phase)

○ Buffer and its strength

• Selection of organic modifiers

• Selection of ion-pair reagents

○ Mobile-phase composition

• Selection and optimization of mobile phase

• Selection of detector

○ pH of buffer

• Selection of flow rate

ment using the HPLC and UPLC are listed as follows:

and purification of pharmaceutical drugs [3]. In this chapter, the authors discuss the principles for chromatography method development using ultra/high-performance liquid chromatography (UPLC/HPLC) techniques for the analysis of assay and organic impurities/related substances/degradation products of pharmaceuticals (any drug product/drug substance/intermediate/raw material of pharmaceuticals). These techniques are developed substantially as a result of the work of *Archer John Porter Martin* and *Richard Laurence Millington Synge* during the 1940s and 1950s, for which they won the 1952 Nobel Prize in Chemistry [4]. Commonly used characterizing technique in pharma industry is liquid chromatography (e.g., HPLC, UPLC, and LC–MS). Each one varies in the stationary phase and operational conditions. HPLC and UPLC can be used as a quantitative technique if coupled with a mass detector (MS) to elucidate the structure of the molecule and quantification.

In pharma industry specific, stability-indicating HPLC/UPLC methods have to be developed to estimate the assay and to quantitatively determine the impurities of new drug substances and drug products [5]. Assay is a quantitative test of a substance to determine the amount of an individual components present in it. Impurity is an unknown component of drug substance that is not the chemical entity. Assay and impurity tests are major and critical quality attributes of the pharmaceutical dosage forms which help to check and ensure the quality, safety, and efficacy of drug substances and drug products. This chapter will discuss the various parameters that have to be chosen to run the chromatography in order to have a better separation and maximum purity. The process of changing the conditions in order to design a best method run for a particular drug mixture or compound is called the

Analytical method development is a process of proving that the developed chromatography method is suitable for its intended use in the development and manufacturing of the pharmaceutical drug substance and drug product. The basic separation techniques and principles involved in the analytical method develop-

**146**


#### **2.1 Literature search**


a lot in developing the method for separation of all possible impurities and degradation products of targeted analyte. It should be borne in mind that impurity profile may vary depending on the manufacturing process (which uses different methods, precursors, and conditions), which makes it clear that not all manufacturing processes yield the same impurity profile.


#### **2.2 Selection of chromatography mode**

Chromatography can be operated by two ways, normal mode and reverse phase modes. The choice of the mode is very important, which is dependent on the type of sample which has to be separated. In general, the usage of reversed-phase chromatography (in which the mobile phase is polar and stationary phase is nonpolar in nature) is the preferred mode for most of the molecules, except in the case of isomer (enantiomers) separation where the normal-phase chromatography (in which the mobile phase is nonpolar and stationary phase is polar in nature) is used. Revered-phase chromatography separates the components with a good resolution based on their hydrophobicity. A compound with a greater polarity elutes earlier, and those with the least polarity elute later.

#### **2.3 Selection of detector**

Detector plays an important role in the finalization of any analytical method. Generally most of the organic/drug molecules are aromatic or unsaturated in nature, which has an absorption in the UV–vis region. This comes as an advantage in quantifying and analyzing the molecules and its associated impurities. The absorbance maxima of the compound shall be collected by analyzing the UV–vis spectrophotometer or diode array detector (DAD) of HPLC/UPLC. From the area intensity of the test compound using calibration curves, the quantification of the test compound can be done [9–10].

**149**

at the polar end.

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

be coupled in order not to miss any impurity.

**2.4 Selection of column stationary phase**

degradation products.

character.

If the compounds do not absorb and if they do not have chromophores, other detectors like refractive index detector (RID) and evaporative light scattering detector (ELSD)/corona-charged aerosol detector (CAD) can be used for the quantitative determination of assay and impurities [11]. If the compounds of interest contain a part, which is non-chromophoric, which may likely be cleaved and produce a nonchromophoric impurity, then both UV and other detectors like RI/ELSD/CAD can

Alternatively, non-chromophoric compounds can also be analyzed by UV after converting it into a derivative which will be active. But the usage of derivatives has to be carefully assessed keeping in view the functional group involved in the derivatization reaction [12, 13]. In case the molecule of interest is having fluorescence properties, a fluorescence detector (FLD) can be used for compounds for which structural information is available [14]. But when FLD is to be used for estimation of unknowns, it needs to be carefully assessed whether

The choice of the right column (stationary phase) is the basis of the whole technology. Most chromatographic separations are achieved due to a wide variety of columns available in the market and due to their flexibility in changing and controlling the parameters. A widely used choice of column material is silica either as neat or modified depending on the nature of the solute mixture in normal-phase chromatography, wherein the eluent (mobile phase) is nonpolar an organic solvent. The silanol groups on the surface of the silica give it a polar

Though silica remains the most common support for liquid chromatography (LC) columns, other commonly used materials are cross-linked organic polymers, zirconia, etc. The silica support for columns was gradually modified for the betterment through the years by three different manufacturing technologies commonly described as "evolution through three generations." The initial process started with type A silica where the raw material used is from inorganic sols. A slightly modified type A silica by performing a chemical treatment to remove the metal impurities is termed as a second-generation material which is called as base-deactivated silica. Third generation silica (type B) is an altogether new process which uses organic sols instead of inorganic sols. These materials are similar in properties to the second-generation silica because both have a minimum level of metal impurities. Silica-based liquid chromatography columns with a different percent of cross-linking and functionalization of silanol groups with substituted aliphatic and aromatic moieties were designed for varying polarities of the separating medium. An increasing order of functionalized silica is represented below with alkyl groups at the nonpolar end, phenyl and amino functionalized in the moderate polar region, and cyano and silica groups

fluorescence properties are available in all possible impurities and

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

a lot in developing the method for separation of all possible impurities and degradation products of targeted analyte. It should be borne in mind that impurity profile may vary depending on the manufacturing process (which uses different methods, precursors, and conditions), which makes it clear that not all manufacturing processes yield the same impurity profile.

v.*Metabolic pathway* is a chemical reaction which occurs within a cell when the drug molecule reacts with an enzyme and forms a metabolite [6]. Metabolic pathway gives the information on oxidation, reduction, and hydrolysis products which gives critical inputs on the possible degradation products.

vi.Stability-indicating method is to identify the closely related structures by collecting the structures of the molecule and its impurities and degradation products. This helps to develop a specific and stability-indication method

vii.*Checking the polarity* of the drug molecule using the functional groups as elucidated from structural analysis techniques. By comparing the structures of impurities and degradation products with the structure of drug molecule, it will help in understanding the polarity based on the nature of functional groups. This makes the scientists' job easy in choosing the right solvents with

either lesser or higher in polarity than the compound of interest.

**2.2 Selection of chromatography mode**

and those with the least polarity elute later.

**2.3 Selection of detector**

test compound can be done [9–10].

viii.*Estimation of maximum daily dose (MDD).* Calculate the reporting, identification, and qualification thresholds of drug substance and drug product based on the maximum daily dose as per ICH Q3A guideline [7, 8]. MDD info can also be obtained from physical desk reference (PDR), innovator product information leaflet (PIL), and the website of RX-list (www.rxlist.com).

Chromatography can be operated by two ways, normal mode and reverse phase modes. The choice of the mode is very important, which is dependent on the type of sample which has to be separated. In general, the usage of reversed-phase chromatography (in which the mobile phase is polar and stationary phase is nonpolar in nature) is the preferred mode for most of the molecules, except in the case of isomer (enantiomers) separation where the normal-phase chromatography (in which the mobile phase is nonpolar and stationary phase is polar in nature) is used. Revered-phase chromatography separates the components with a good resolution based on their hydrophobicity. A compound with a greater polarity elutes earlier,

Detector plays an important role in the finalization of any analytical method. Generally most of the organic/drug molecules are aromatic or unsaturated in nature, which has an absorption in the UV–vis region. This comes as an advantage in quantifying and analyzing the molecules and its associated impurities. The absorbance maxima of the compound shall be collected by analyzing the UV–vis spectrophotometer or diode array detector (DAD) of HPLC/UPLC. From the area intensity of the test compound using calibration curves, the quantification of the

with a good resolution between the closely related structures.

**148**

If the compounds do not absorb and if they do not have chromophores, other detectors like refractive index detector (RID) and evaporative light scattering detector (ELSD)/corona-charged aerosol detector (CAD) can be used for the quantitative determination of assay and impurities [11]. If the compounds of interest contain a part, which is non-chromophoric, which may likely be cleaved and produce a nonchromophoric impurity, then both UV and other detectors like RI/ELSD/CAD can be coupled in order not to miss any impurity.

Alternatively, non-chromophoric compounds can also be analyzed by UV after converting it into a derivative which will be active. But the usage of derivatives has to be carefully assessed keeping in view the functional group involved in the derivatization reaction [12, 13]. In case the molecule of interest is having fluorescence properties, a fluorescence detector (FLD) can be used for compounds for which structural information is available [14]. But when FLD is to be used for estimation of unknowns, it needs to be carefully assessed whether fluorescence properties are available in all possible impurities and degradation products.

#### **2.4 Selection of column stationary phase**

The choice of the right column (stationary phase) is the basis of the whole technology. Most chromatographic separations are achieved due to a wide variety of columns available in the market and due to their flexibility in changing and controlling the parameters. A widely used choice of column material is silica either as neat or modified depending on the nature of the solute mixture in normal-phase chromatography, wherein the eluent (mobile phase) is nonpolar an organic solvent. The silanol groups on the surface of the silica give it a polar character.

Though silica remains the most common support for liquid chromatography (LC) columns, other commonly used materials are cross-linked organic polymers, zirconia, etc. The silica support for columns was gradually modified for the betterment through the years by three different manufacturing technologies commonly described as "evolution through three generations." The initial process started with type A silica where the raw material used is from inorganic sols. A slightly modified type A silica by performing a chemical treatment to remove the metal impurities is termed as a second-generation material which is called as base-deactivated silica. Third generation silica (type B) is an altogether new process which uses organic sols instead of inorganic sols. These materials are similar in properties to the second-generation silica because both have a minimum level of metal impurities. Silica-based liquid chromatography columns with a different percent of cross-linking and functionalization of silanol groups with substituted aliphatic and aromatic moieties were designed for varying polarities of the separating medium. An increasing order of functionalized silica is represented below with alkyl groups at the nonpolar end, phenyl and amino functionalized in the moderate polar region, and cyano and silica groups at the polar end.

The following are the parameters of a chromatographic column which need to be considered while choosing a column (stationary phase) for separation of assay, impurities, and degradation products:

i.Length and diameter of column


v.Percent (%) of carbon loading

**Column dimension**: Length and internal diameter of packing bed.


A column with a diameter of 2.1 mm leads to a high resolution.

**Particle size**: Decrease in particle size leads to increase in resolution but with a corresponding increase in back pressure. In general smaller particles offer higher efficiency, but there is a chance to get high back pressure limiting the separation efficiency. Less (3 μm) particles are usually used for resolving complex and multicomponent samples, where the lesser surface area induces better resolution and separation characteristics.

**Pore size and surface area**: Larger pores allow larger solute molecules to be retained for a longer time through maximum surface area exposure. High surface area generally provides greater retention, capacity, and resolution for multicomponent samples. Low surface area materials generally equilibrate quickly and provide lesser separation efficiency but can be highly preferred and important in gradient analyses.

**Carbon loading**: Higher carbon loads generally offer greater resolution and longer run times. Low carbon loads shorten run times, and many show a different selectivity. A pictorial representation of difference in carbon loading is as shown below.

**End capping**: End capping reduces peak tailing of polar compounds that interact excessively with the otherwise exposed, mostly acidic silanols. Non-end capped packing provides a different selectivity than do end-capped packing, especially for polar compounds. A pictorial representation of difference in end capping is shown below.

**151**

mobile phase.

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

**2.5 Selection and optimization of mobile phase**

• The right choice of buffer and its eluting efficiency

• pH of the buffer or pH of the mobile phase

• Triethylamine/diethylamine buffers

mobile phase.

*2.5.1 Buffer and its strength*

Though adsorption is the principle behind chromatography, real separation happens only when the adsorbed compound is eluted using a mobile phase of the required polarity. The selection of mobile phase is done always in combination with the selection of column (stationary phase). The following are the parameters which shall be taken into consideration while selecting and optimizing the

• Mobile-phase composition inclusive of binary and tertiary solvent mixture

Buffer and its efficiency play an important role in deciding the peak symmetries (shapes) and peak separation. Various types of organic/inorganic buffers are employed for achieving the required separation. The most commonly used buffers are:

• Phosphate buffers—KH2PO4, K2HPO4, NaH2PO4, Na2HPO4, H3PO4, etc.

• Buffers with various ion-pair reagents like tetrabutyl ammonium hydrogen sulfate, butane sulfonic acid, hexane sulfonic acid, heptane sulfonic acids, etc.

The choice of buffer is to reduce the tailing factor for each peak separated which occurs due to varying ionic strength. The retention time of analyte(s) is delayed and got separated well when more concentrated buffer is used [15]. Better separation happens when the molarity of buffer used is in the range of 0.05 to 0.20 M. The concentration of buffer is chosen by carefully choosing the composition of organic

Depending on the need of the chosen mixture of separation, the strength of the buffer can be increased or decreased if necessary to achieve the required separation, and it can be varied between 10 and 20%, and the effect of variation has to be studied in detail before using. But it should be ensured that increased or decreased buffer strength should not result in precipitation or turbidity either in mobile phase during operation or during storage in refrigerator. Before using the chosen buffer of specific strength to run a column, test experiments have to be done in optimizing

the separation to avoid peak tailing, better separation, and reproducibility.

• Acetate buffers—Ammonium acetate, sodium acetate, etc.

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

impurities, and degradation products:

ii.Packing material

iii.Shape of particles

iv.Size of particles

separation characteristics.

analyses.

below.

i.Length and diameter of column

v.Percent (%) of carbon loading

The following are the parameters of a chromatographic column which need to be considered while choosing a column (stationary phase) for separation of assay,

**Column dimension**: Length and internal diameter of packing bed.

A column with a diameter of 2.1 mm leads to a high resolution.

• Short (30–50 mm)—can result in short run times and low back pressure

• Long (250–300 mm)—can result in higher-resolution and long run times

**Particle size**: Decrease in particle size leads to increase in resolution but with a corresponding increase in back pressure. In general smaller particles offer higher efficiency, but there is a chance to get high back pressure limiting the separation efficiency. Less (3 μm) particles are usually used for resolving complex and multicomponent samples, where the lesser surface area induces better resolution and

**Pore size and surface area**: Larger pores allow larger solute molecules to be retained for a longer time through maximum surface area exposure. High surface area generally provides greater retention, capacity, and resolution for multicomponent samples. Low surface area materials generally equilibrate quickly and provide lesser separation efficiency but can be highly preferred and important in gradient

**Carbon loading**: Higher carbon loads generally offer greater resolution and longer run times. Low carbon loads shorten run times, and many show a different selectivity. A pictorial representation of difference in carbon loading is as shown

**End capping**: End capping reduces peak tailing of polar compounds that interact excessively with the otherwise exposed, mostly acidic silanols. Non-end capped packing provides a different selectivity than do end-capped packing, especially for polar compounds. A pictorial representation of difference in end

**150**

capping is shown below.

#### **2.5 Selection and optimization of mobile phase**

Though adsorption is the principle behind chromatography, real separation happens only when the adsorbed compound is eluted using a mobile phase of the required polarity. The selection of mobile phase is done always in combination with the selection of column (stationary phase). The following are the parameters which shall be taken into consideration while selecting and optimizing the mobile phase.


#### *2.5.1 Buffer and its strength*

Buffer and its efficiency play an important role in deciding the peak symmetries (shapes) and peak separation. Various types of organic/inorganic buffers are employed for achieving the required separation. The most commonly used buffers are:


The choice of buffer is to reduce the tailing factor for each peak separated which occurs due to varying ionic strength. The retention time of analyte(s) is delayed and got separated well when more concentrated buffer is used [15]. Better separation happens when the molarity of buffer used is in the range of 0.05 to 0.20 M. The concentration of buffer is chosen by carefully choosing the composition of organic mobile phase.

Depending on the need of the chosen mixture of separation, the strength of the buffer can be increased or decreased if necessary to achieve the required separation, and it can be varied between 10 and 20%, and the effect of variation has to be studied in detail before using. But it should be ensured that increased or decreased buffer strength should not result in precipitation or turbidity either in mobile phase during operation or during storage in refrigerator. Before using the chosen buffer of specific strength to run a column, test experiments have to be done in optimizing the separation to avoid peak tailing, better separation, and reproducibility.

#### *2.5.2 pH of buffer*

pH plays an important role in achieving the chromatographic separations as it controls the elution properties by controlling the ionization characteristics. The pH of buffer or mobile phase should be selected based on the *pKa* of analyte or test mixture, which is based on the structure of the molecule. Depending on the *pKa*, drug molecules change retentions, e.g., acids show an increase in retention as the pH is reduced, while the base shows a decrease. If the *pKa* of the compound is high, lower pH or acidic mobile phase has to be chosen as it will stop unwanted association with the stationary phase. For basic compounds, the use of high pH or basic mobile phase and, for neutral compound, neutral mobile phase is highly preferable for better separation.

It is important to maintain the pH of the mobile phase in the range of 2.0 ~ 8.0 as most columns do not withstand the pH which is outside this range. This is due to the fact that the mostly used silica column gets deactivated at high pH (<2) and at low pH (>8) due to cleavage of siloxane linkages. If a pH outside the range of 2.0 ~ 8.0 is found to be necessary, stationary phase which can withstand the range shall be chosen [16–18].

#### *2.5.3 Mobile-phase composition*

It is well reported in literature that to achieve better efficiency, binary and tertiary solvent mixtures are used along with other components like buffer and acids or bases. The ratio of the organic versus (vs.) aqueous or polar vs. nonpolar solvents is varied accordingly to get better separation. This is due to the fact that a fairly large amount of selectivity can be achieved by choosing the qualitative and quantitative composition of aqueous and organic portions. Experiments shall be conducted with mobile phases having buffers of different pH and different organic phases to check for the best separations between the impurities. Most chromatographic separations can be achieved by choosing the optimum mobile phase composition [18].

#### **2.6 Selection of organic modifiers**

Most widely used solvents in reverse-phase chromatography are methanol and acetonitrile. Tetrahydrofuran (THF) is also used but to a lesser extent [19, 20]. In most of the systems, acetonitrile is used as the default organic modifier because of favorable UV transmittance and low viscosity. It is recommended to mix acetonitrile with 5–10% of the aqueous solution(s) to avoid the pumping problems associated with a higher percent (%) of acetonitrile usage. Methanol is also the second most widely used solvent in liquid chromatography, but it gives the back pressure to LC column. Though THF has some disadvantages like higher UV absorbance, reactivity with oxygen, and slower column equilibration, sometimes it gives very unique selectivity for closely eluting peaks. Intermediate selectivity (if needed for a particular sample) can be obtained by blending appropriate amounts of each of these solvents.

Order of polarity: methanol > acetonitrile > ethanol > THF > propanol. Order of solvent strength: propanol > THF > ethanol > acetonitrile > methanol.

#### **2.7 Selection of ion-pair reagents**

Ion pair reagents are necessary as a mobile-phase additive when structurally or chemically or polarity wise inseparable closely related compounds are to be separated [21, 22]. For example, if a mixture of ionic and nonionic analyte(s) having the same polarity and same retention time is required to be separated, start by optimizing for one of the analytes by adding an ion pair reagent in a mobile phase which

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*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

**2.8 Selection of flow rate**

**2.9 Selection of column temperature**

ultimately the elution and resolution [25–28].

prior to achieving better separation.

reduces or increases the polarity of component and helps in increasing the elution time difference. Careful choice of an appropriate ion-pair reagent is required in such cases to get the necessary selectivity. A dedicated LC column is used when an ion pair reagent (0.0005 M to 0.02 M) is intended to employ for specific analysis, but an appropriate cleaning procedure has to be established to enhance the lifetime of the column material. Alkyl ammonium salts (tertiary or quaternary) and alkyl sulfonate salts are the most useful in the separation of acidic and basic compounds,

Separation of mixtures is highly influenced by the flow of mobile phase inside the column [23, 24]. The flow rate is highly crucial in having well-separated peaks with no tailing. The flow rate of the mobile phase can be optimized based on the retention time, column back pressure, and separation of closely eluting adjacent peaks or impurities and peak symmetries from the test run. Preferably the flow rate is fixed not more than 2.0 mL/minute. The flow which gives the least retention times, good peak symmetries, least back pressures, and better separation of adjacent peaks/impurities could be the chosen as an optimized flow rate for the analysis.

Temperature is another criterion which has to be optimized for any sample, as the flow rate and the rate of adsorption vary with temperature. It is generally believed that with increasing temperature, it can help to improve the resolution between the adjacent/closely eluting peaks and peak merging. So a careful choice of the temperature is a must which might change the pressure of the column and

Choosing ambient temperature for the analysis is always preferred as it will minimize the degradation of the test sample; however, higher temperatures are also advisable under unavoidable conditions after confirming the stability of the compound. The temperature range which is usually allowed in liquid chromatography is 25 and 60°C. Higher temperatures above 60°C are preferred if the peak symmetry is

Chromatographic separations with a single eluent (isocratic elution: all the constituents of the mobile phase are mixed and pumped together as a single eluent) are always preferable. However, the gradient elution is a powerful tool in achieving separation between closely eluting compounds or compounds having narrow polarity difference [29–31]. An important feature of the gradient elution mode which makes it a powerful tool is that the polarity and ionic strength of the mobile phase are changed (increased or decreased) during the run. Experiments using different mobile-phase combinations and different gradient programs have to be performed

In a gradient run, two mobile phases which have different compositions of polar and nonpolar solvents are premixed using a single pump before introducing to the column which is called as *low pressure gradient (LPG),* and when the mobile phases are pumped at different flow rate and mixed in a chamber, then introduced into the column is known as *high pressure gradient (HPG)*. It is better to select the gradient run, whether *LPG* or *HPG*, while optimizing the chromatography method. HPG can be only preferred for use when more than 80% organic

not good and to increase the retention time for closely occurring peaks.

**2.10 Selection of solvent delivery system (elution mode)**

respectively. Sodium perchlorate can also be used for acidic components.

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

reduces or increases the polarity of component and helps in increasing the elution time difference. Careful choice of an appropriate ion-pair reagent is required in such cases to get the necessary selectivity. A dedicated LC column is used when an ion pair reagent (0.0005 M to 0.02 M) is intended to employ for specific analysis, but an appropriate cleaning procedure has to be established to enhance the lifetime of the column material. Alkyl ammonium salts (tertiary or quaternary) and alkyl sulfonate salts are the most useful in the separation of acidic and basic compounds, respectively. Sodium perchlorate can also be used for acidic components.

#### **2.8 Selection of flow rate**

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

pH plays an important role in achieving the chromatographic separations as it controls the elution properties by controlling the ionization characteristics. The pH of buffer or mobile phase should be selected based on the *pKa* of analyte or test mixture, which is based on the structure of the molecule. Depending on the *pKa*, drug molecules change retentions, e.g., acids show an increase in retention as the pH is reduced, while the base shows a decrease. If the *pKa* of the compound is high, lower pH or acidic mobile phase has to be chosen as it will stop unwanted association with the stationary phase. For basic compounds, the use of high pH or basic mobile phase and, for neutral compound, neutral mobile phase is highly preferable for better separation. It is important to maintain the pH of the mobile phase in the range of 2.0 ~ 8.0 as most columns do not withstand the pH which is outside this range. This is due to the fact that the mostly used silica column gets deactivated at high pH (<2) and at low pH (>8) due to cleavage of siloxane linkages. If a pH outside the range of 2.0 ~ 8.0 is found to be necessary, stationary phase which can withstand the range shall be

It is well reported in literature that to achieve better efficiency, binary and tertiary solvent mixtures are used along with other components like buffer and acids or bases. The ratio of the organic versus (vs.) aqueous or polar vs. nonpolar solvents is varied accordingly to get better separation. This is due to the fact that a fairly large amount of selectivity can be achieved by choosing the qualitative and quantitative composition of aqueous and organic portions. Experiments shall be conducted with mobile phases having buffers of different pH and different organic phases to check for the best separations between the impurities. Most chromatographic separations

Most widely used solvents in reverse-phase chromatography are methanol and acetonitrile. Tetrahydrofuran (THF) is also used but to a lesser extent [19, 20]. In most of the systems, acetonitrile is used as the default organic modifier because of favorable UV transmittance and low viscosity. It is recommended to mix acetonitrile with 5–10% of the aqueous solution(s) to avoid the pumping problems associated with a higher percent (%) of acetonitrile usage. Methanol is also the second most widely used solvent in liquid chromatography, but it gives the back pressure to LC column. Though THF has some disadvantages like higher UV absorbance, reactivity with oxygen, and slower column equilibration, sometimes it gives very unique selectivity for closely eluting peaks. Intermediate selectivity (if needed for a particular sample) can be obtained by blending appropriate amounts of each of these solvents. Order of polarity: methanol > acetonitrile > ethanol > THF > propanol.

Order of solvent strength: propanol > THF > ethanol > acetonitrile > methanol.

Ion pair reagents are necessary as a mobile-phase additive when structurally or chemically or polarity wise inseparable closely related compounds are to be separated [21, 22]. For example, if a mixture of ionic and nonionic analyte(s) having the same polarity and same retention time is required to be separated, start by optimizing for one of the analytes by adding an ion pair reagent in a mobile phase which

can be achieved by choosing the optimum mobile phase composition [18].

*2.5.2 pH of buffer*

chosen [16–18].

*2.5.3 Mobile-phase composition*

**2.6 Selection of organic modifiers**

**2.7 Selection of ion-pair reagents**

**152**

Separation of mixtures is highly influenced by the flow of mobile phase inside the column [23, 24]. The flow rate is highly crucial in having well-separated peaks with no tailing. The flow rate of the mobile phase can be optimized based on the retention time, column back pressure, and separation of closely eluting adjacent peaks or impurities and peak symmetries from the test run. Preferably the flow rate is fixed not more than 2.0 mL/minute. The flow which gives the least retention times, good peak symmetries, least back pressures, and better separation of adjacent peaks/impurities could be the chosen as an optimized flow rate for the analysis.

#### **2.9 Selection of column temperature**

Temperature is another criterion which has to be optimized for any sample, as the flow rate and the rate of adsorption vary with temperature. It is generally believed that with increasing temperature, it can help to improve the resolution between the adjacent/closely eluting peaks and peak merging. So a careful choice of the temperature is a must which might change the pressure of the column and ultimately the elution and resolution [25–28].

Choosing ambient temperature for the analysis is always preferred as it will minimize the degradation of the test sample; however, higher temperatures are also advisable under unavoidable conditions after confirming the stability of the compound. The temperature range which is usually allowed in liquid chromatography is 25 and 60°C. Higher temperatures above 60°C are preferred if the peak symmetry is not good and to increase the retention time for closely occurring peaks.

#### **2.10 Selection of solvent delivery system (elution mode)**

Chromatographic separations with a single eluent (isocratic elution: all the constituents of the mobile phase are mixed and pumped together as a single eluent) are always preferable. However, the gradient elution is a powerful tool in achieving separation between closely eluting compounds or compounds having narrow polarity difference [29–31]. An important feature of the gradient elution mode which makes it a powerful tool is that the polarity and ionic strength of the mobile phase are changed (increased or decreased) during the run. Experiments using different mobile-phase combinations and different gradient programs have to be performed prior to achieving better separation.

In a gradient run, two mobile phases which have different compositions of polar and nonpolar solvents are premixed using a single pump before introducing to the column which is called as *low pressure gradient (LPG),* and when the mobile phases are pumped at different flow rate and mixed in a chamber, then introduced into the column is known as *high pressure gradient (HPG)*. It is better to select the gradient run, whether *LPG* or *HPG*, while optimizing the chromatography method. HPG can be only preferred for use when more than 80% organic

phase is pumped. To avoid the pumping problems due to the low viscous solvents like acetonitrile in mobile phase, at least 10% aqueous portion could be added to the organic phase.

While optimizing the gradient program, it is important to monitor the following. Pressure graph is needed to be monitored so as to ensure that the overall system pressure will not cross 400 bar or 6000 psi at any point during the run. Flow rate has to be physically cross-checked by collecting the output from the detector during the run at different time intervals, especially when the gradient is running with higher organic-phase composition so as to ensure that there were no pumping problems during the run when mobile phases of different compositions are pumped. It is also important to optimize the program for initialization after each run and before going for the next injection. The program for initialization shall be optimized such that there shall be no carry-over to the next run and the system stabilizes with initial composition before the next injection.

One standard program which can be used for optimizing is discussed below. For starting a method development, a solvent gradient system is always preferred. Initially, start with a gradient of 50:50 buffer and mobile phase, and change the program linearly up to 5:95, and retain the ratio for at least 30 minutes. Then try with a gradient of 95:5 and the program linearly changed up to 5: 95, and retain for at least 30 minutes. The typical gradient program is as follows:

#### **2.11 Selection of diluent**

Diluent is an aqueous solution or a solvent used to dissolve and extract the drug moiety for analysis. Select a diluent in which impurities, starting material, by-product, intermediates, degradation products, and the analyte are soluble. It is advisable to check first in the mobile phase. All the analytes should be completely soluble and the solution should be clear [32]. Diluent should be compatible with the mobile phase to obtain the good peak shape.


Diluent is selected initially based on solubility of the substance. However, the finalization of diluent is based on its extraction efficiency, peak symmetries, resolution of impurities, and diluent blank injection interference. Inject the diluent blank and test solution spiked with known impurities into the chromatographic system, and establish the noninterference of blank in estimation of the drug and the effect of diluent on resolution of impurities from drug peak and peak symmetry.

#### **2.12 Methods of extraction**

General methods followed for extraction are sonication, rotary shaking, or seldom both. In some cases where the analyte cannot be extracted by the above

**155**

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

**2.13 Samples to be used for analysis**

development.

the case of drug API.

about 0.5 or 1.0 mg/mL.

show any turbidity or precipitation.

**2.16 Forced degradation studies (stress testing)**

**2.14 Experimentation to finalize the method**

placebo components in the case of the formulation.

nonpolar impurities are still un-eluted from the column.

**2.15 Selection of test concentration and injection volume**

cipitate upon cooling to room temperature [33–34].

degradation products to establish the separations.

for the dose in which higher placebo content is expected.

procedures, heating can be adapted if the substance is stable and should not pre-

• Use mixture of impurities, starting material, by-product, intermediates, and

• Use the reaction mass/mother liquor/what if study samples for the above study if all the impurities samples are not available in the beginning for method

• Use forced degradation samples, if degradation products are not available in

• Prepare a mixture of known impurities spiked on API at a test concentration of

• Prepare a placebo (mixture of excipients to be used in formulation) solution at a concentration equivalent to test concentration (of about 0.5 or 1.0 mg/mL)

Inject individual solution of standard and impurities to confirm the retention times. Check for the interference from blank. Check for the interference from

Gradient program will provide an assessment of the elution pattern of polar and nonpolar impurities. Also, run an isocratic run with a mobile phase of a buffer with a suitable pH and acetonitrile in the ratio of acetonitrile: buffer (90:10) using a 250 × 4.6 mm, 5 μm silica column. This will help to know whether any highly

The test concentration and injection volume are generally chosen based upon the response of API peak at the selected detector wavelength [35]. However, the test concentration shall be finalized after it is proven that drug (API) is completely extractable at the selected test concentration. After finalizing the test concentration and diluent, prepare a test solution, and keep the filtered solution in closed condition on a bench top, and check whether the solution has any precipitation or turbidity after 24 hours. Generally, the test solution must be clear and should not

It's a method of subjecting the drug substance or drug product to stress with varied strengths of stressing agents to obtain the degradation. The stressed samples were analyzed using an LC system equipped with a PDA detector and monitored for the separation of degradation products formed under the stressed conditions and the peak purity of the analyte peak. The method is considered as stability-indicating for the estimation of the drug if it meets the peak purity requirement [36, 37].

procedures, heating can be adapted if the substance is stable and should not precipitate upon cooling to room temperature [33–34].

#### **2.13 Samples to be used for analysis**

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

the organic phase.

composition before the next injection.

mobile phase to obtain the good peak shape.

temperature as ambient (25–30°C).

ably <1 AU) by diluting the test preparation.

**2.11 Selection of diluent**

phase is pumped. To avoid the pumping problems due to the low viscous solvents like acetonitrile in mobile phase, at least 10% aqueous portion could be added to

While optimizing the gradient program, it is important to monitor the following. Pressure graph is needed to be monitored so as to ensure that the overall system pressure will not cross 400 bar or 6000 psi at any point during the run. Flow rate has to be physically cross-checked by collecting the output from the detector during the run at different time intervals, especially when the gradient is running with higher organic-phase composition so as to ensure that there were no pumping problems during the run when mobile phases of different compositions are pumped. It is also important to optimize the program for initialization after each run and before going for the next injection. The program for initialization shall be optimized such that there shall be no carry-over to the next run and the system stabilizes with initial

One standard program which can be used for optimizing is discussed below. For starting a method development, a solvent gradient system is always preferred. Initially, start with a gradient of 50:50 buffer and mobile phase, and change the program linearly up to 5:95, and retain the ratio for at least 30 minutes. Then try with a gradient of 95:5 and the program linearly changed up to 5: 95, and retain for

Diluent is an aqueous solution or a solvent used to dissolve and extract the drug moiety for analysis. Select a diluent in which impurities, starting material, by-product, intermediates, degradation products, and the analyte are soluble. It is advisable to check first in the mobile phase. All the analytes should be completely soluble and the solution should be clear [32]. Diluent should be compatible with the

• Selection of diluent based on extraction efficiency and peak shapes: Select the diluent for finished dosage forms, in which the analyte should be extracted at least 95% for assay and 90% for organic impurities. Calculate the % extraction against pure standard compound in the concentration of linear range, (prefer-

• The peak shapes of all compounds should be good in the selected diluent: Select an initial flow rate of 1.0 mL/min or 1.5 mL/min and select column

Diluent is selected initially based on solubility of the substance. However, the finalization of diluent is based on its extraction efficiency, peak symmetries, resolution of impurities, and diluent blank injection interference. Inject the diluent blank and test solution spiked with known impurities into the chromatographic system, and establish the noninterference of blank in estimation of the drug and the effect of diluent on resolution of impurities from drug peak and peak

General methods followed for extraction are sonication, rotary shaking, or seldom both. In some cases where the analyte cannot be extracted by the above

at least 30 minutes. The typical gradient program is as follows:

**154**

symmetry.

**2.12 Methods of extraction**


#### **2.14 Experimentation to finalize the method**

Inject individual solution of standard and impurities to confirm the retention times. Check for the interference from blank. Check for the interference from placebo components in the case of the formulation.

Gradient program will provide an assessment of the elution pattern of polar and nonpolar impurities. Also, run an isocratic run with a mobile phase of a buffer with a suitable pH and acetonitrile in the ratio of acetonitrile: buffer (90:10) using a 250 × 4.6 mm, 5 μm silica column. This will help to know whether any highly nonpolar impurities are still un-eluted from the column.

#### **2.15 Selection of test concentration and injection volume**

The test concentration and injection volume are generally chosen based upon the response of API peak at the selected detector wavelength [35]. However, the test concentration shall be finalized after it is proven that drug (API) is completely extractable at the selected test concentration. After finalizing the test concentration and diluent, prepare a test solution, and keep the filtered solution in closed condition on a bench top, and check whether the solution has any precipitation or turbidity after 24 hours. Generally, the test solution must be clear and should not show any turbidity or precipitation.

#### **2.16 Forced degradation studies (stress testing)**

It's a method of subjecting the drug substance or drug product to stress with varied strengths of stressing agents to obtain the degradation. The stressed samples were analyzed using an LC system equipped with a PDA detector and monitored for the separation of degradation products formed under the stressed conditions and the peak purity of the analyte peak. The method is considered as stability-indicating for the estimation of the drug if it meets the peak purity requirement [36, 37].

Forced degradation studies are conducted basically to meet the following objectives:


The major forced degradation studies which are to be carried out are as follows:

#### a.Thermolytic degradation

This stress testing method studies the degradation that is caused by exposure to temperature high enough to induce bond breakage. Solid-state reactions often proceed in an autocatalytic pathway involving an induction period (lag), followed by a period of rapidly increasing degradation and then slowing down of the degradation rate as the compound is consumed. Thus, solid-state reaction kinetics will often follow an S-shaped curve when degradation vs. time is plotted. Thus, before conducting thermolytic degradation, determine the melting point of the compounds of interest. Then, choose a temperature of 70°C for all the drugs for which melting point is <100°C, or choose a temperature which is 40°C below the melting point.

For the compounds for which melting point is >150°C, stress the samples at 105°C. Keep the samples directly exposed in the oven for 1 week or until about 2–20% degradation is achieved, whichever is earlier. Stress the drug substance, placebo, and drug product separately. In the case of the multicomponent drug products, stress testing of placebo with other actives excluding the one at a time shall be performed additionally.

#### b.Hydrolytic degradation

Drug degradation that involves hydrolysis reaction is called hydrolytic degradation. Hydrolysis reactions are typically acid or base catalyzed. Acidic, neutral, and basic conditions should therefore be employed in order to induce potential hydrolytic reactions. As these hydrolytic stress studies are to be conducted in aqueous solutions, solubility of the drug molecule of interest in water has to be estimated first. Many small molecule drugs are not soluble in water at the concentrations typically used for analytical evaluations (0.1 to 1 mg/mL); in those cases either a slurry or suspension must be used to examine the hydrolytic stability of a compound, or a cosolvent must be added to facilitate the dissolution under the conditions of low solubility. Two most commonly used cosolvents are acetonitrile and methanol. Methanol has the potential of participating in the degradation chemistry which has to be used with caution especially under acidic conditions when the compound being tested contains a carboxylic acid, ester, or amide.

Acetonitrile is generally regarded as inert solvent and is typically preferable to methanol in hydrolytic stress testing studies. However, acetonitrile is not completely inert and can participate in the degradation reactions, leading to art factual degradation results.

The other cosolvents that are recommended for the hydrolytic stress testing studies are shown below.

**157**

additionally.

c.Humidity stress

shall be performed additionally.

d.Oxidative degradation

Arrhenius kinetics.

product separately.

and 200 W-hr/m<sup>2</sup>

additionally.

e.Photolytic degradation

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

The hydrolytic degradations (using water/0.1 M HCl/0.1 M NaOH with or without cosolvent) are recommended to be performed at a temperature of about 70°C

Stress the samples to 90% humidity for 1 week. Stress the drug substance, placebo, and drug product separately. In the case of the multicomponent drug products, stress testing of placebo with other actives excluding the one at a time

Oxidative degradation is one of the most common mechanisms of drug degradation. Oxidative drug degradation reactions are typically autoxidative, that is, the reaction is radical initiated. Radical initiated reactions start with an initiation phase involving the formation of radicals followed by propagation phase and eventually a termination phase. Thus, the reaction kinetics will often follow S-shaped curve when the degradation vs. time is plotted and will not follow

In oxidative stress study, the use of temperature > 30°C is not recommended because the reaction rate in solution may reduce at higher temp due to the decrease in oxygen content of the solvent. Thus, it is always suggested to perform the degradation with 3% hydrogen peroxide at room temperature (25–30°C) with constant stirring in the dark. Stress for 24 hours or until about 1–20% degradation is achieved or whichever is earlier. Stress the drug substance, placebo, and drug

In the case of the multicomponent drug products, stress testing of placebo with

UVA. Stress the drug substance, placebo, and drug product

Photolytic degradation is the degradation that results from exposure to UV or visible light. Expose the samples to 3 times to 1.2 million lux-hr visible

separately. In the case of the multicomponent drug products, stress testing of placebo with other actives excluding the one at a time shall be performed

other actives excluding the one at a time shall be performed additionally.

with a reflux condenser installed to avoid the loss of evaporation. Reflux until about 2–20% degradation is achieved. Stress the drug substance, placebo, and drug product separately. Neutralize the stressed solutions before injection.

**Acidic pH Neutral pH Basic pH** Acetonitrile Acetonitrile Acetonitrile DMSO N-methyl pyrrolidine DMSO Acetic acid Diglyme Propionic acid p-Dioxane

Prepare a stressed solution at a higher concentration than that of test concentration. In the case of the multicomponent drug products, stress testing of placebo with other actives excluding the one at a time shall be performed *Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

• Ensure the force degradation limit of 2–20%.

a.Thermolytic degradation

shall be performed additionally.

b.Hydrolytic degradation

being tested contains a carboxylic acid, ester, or amide.

objectives:

melting point.

Forced degradation studies are conducted basically to meet the following

• To investigate the likely degradation products; this, in turn, helps to establish the degradation pathways and the intrinsic stability of the drug molecule.

• To provide a foundation for developing a suitable stability-indicating method.

The major forced degradation studies which are to be carried out are as follows:

This stress testing method studies the degradation that is caused by exposure to temperature high enough to induce bond breakage. Solid-state reactions often proceed in an autocatalytic pathway involving an induction period (lag), followed by a period of rapidly increasing degradation and then slowing down of the degradation rate as the compound is consumed. Thus, solid-state reaction kinetics will often follow an S-shaped curve when degradation vs. time is plotted. Thus, before conducting thermolytic degradation, determine the melting point of the compounds of interest. Then, choose a temperature of 70°C for all the drugs for which melting point is <100°C, or choose a temperature which is 40°C below the

For the compounds for which melting point is >150°C, stress the samples at 105°C. Keep the samples directly exposed in the oven for 1 week or until about 2–20% degradation is achieved, whichever is earlier. Stress the drug substance, placebo, and drug product separately. In the case of the multicomponent drug products, stress testing of placebo with other actives excluding the one at a time

Drug degradation that involves hydrolysis reaction is called hydrolytic degradation. Hydrolysis reactions are typically acid or base catalyzed. Acidic, neutral, and basic conditions should therefore be employed in order to induce potential hydrolytic reactions. As these hydrolytic stress studies are to be conducted in aqueous solutions, solubility of the drug molecule of interest in water has to be estimated first. Many small molecule drugs are not soluble in water at the concentrations typically used for analytical evaluations (0.1 to 1 mg/mL); in those cases either a slurry or suspension must be used to examine the hydrolytic stability of a compound, or a cosolvent must be added to facilitate the dissolution under the conditions of low solubility. Two most commonly used cosolvents are acetonitrile and methanol. Methanol has the potential of participating in the degradation chemistry which has to be used with caution especially under acidic conditions when the compound

Acetonitrile is generally regarded as inert solvent and is typically preferable to methanol in hydrolytic stress testing studies. However, acetonitrile is not completely inert and can participate in the degradation reactions, leading to art factual

The other cosolvents that are recommended for the hydrolytic stress testing

**156**

degradation results.

studies are shown below.


The hydrolytic degradations (using water/0.1 M HCl/0.1 M NaOH with or without cosolvent) are recommended to be performed at a temperature of about 70°C with a reflux condenser installed to avoid the loss of evaporation. Reflux until about 2–20% degradation is achieved. Stress the drug substance, placebo, and drug product separately. Neutralize the stressed solutions before injection. Prepare a stressed solution at a higher concentration than that of test concentration. In the case of the multicomponent drug products, stress testing of placebo with other actives excluding the one at a time shall be performed additionally.

#### c.Humidity stress

Stress the samples to 90% humidity for 1 week. Stress the drug substance, placebo, and drug product separately. In the case of the multicomponent drug products, stress testing of placebo with other actives excluding the one at a time shall be performed additionally.

#### d.Oxidative degradation

Oxidative degradation is one of the most common mechanisms of drug degradation. Oxidative drug degradation reactions are typically autoxidative, that is, the reaction is radical initiated. Radical initiated reactions start with an initiation phase involving the formation of radicals followed by propagation phase and eventually a termination phase. Thus, the reaction kinetics will often follow S-shaped curve when the degradation vs. time is plotted and will not follow Arrhenius kinetics.

In oxidative stress study, the use of temperature > 30°C is not recommended because the reaction rate in solution may reduce at higher temp due to the decrease in oxygen content of the solvent. Thus, it is always suggested to perform the degradation with 3% hydrogen peroxide at room temperature (25–30°C) with constant stirring in the dark. Stress for 24 hours or until about 1–20% degradation is achieved or whichever is earlier. Stress the drug substance, placebo, and drug product separately.

In the case of the multicomponent drug products, stress testing of placebo with other actives excluding the one at a time shall be performed additionally.

#### e.Photolytic degradation

Photolytic degradation is the degradation that results from exposure to UV or visible light. Expose the samples to 3 times to 1.2 million lux-hr visible and 200 W-hr/m<sup>2</sup> UVA. Stress the drug substance, placebo, and drug product separately. In the case of the multicomponent drug products, stress testing of placebo with other actives excluding the one at a time shall be performed additionally.

#### **2.17 Evaluation of stress testing**

Peak purity can be evaluated for the main peak and the major degradants which have the peak heights less than 1 AU. Identify the degradation products by coinjection, in case of known impurities and have comparable spectra.

If any known impurity is observed to be increased in stress, it can be examined properly. If process impurity is found to be increased in stress study, it needs to be assessed whether there is any secondary pathway of formation of this impurity via some other degradant route.

After conducting these studies, verify the chromatograms, and observe any peaks merging with respect to main peak and any critical pairs. If any situations were arrived, adjust the mobile-phase compositions, column parameters, etc. and conclude the method parameters.

After method finalization, check the method using different detectors (RI/ ELSD/CE/LC–MS), and compare the data with other detectors like UV, fluorescence, etc. The UV inactive components can be found with these experiments. Identify the mass of major degradant which may be formed greater than 1.0% in stress studies, and try to establish the structures.

#### **2.18 Mass balance study**

Mass balance is a process of adding together the assay value and levels of degradation products to see how closely these add up to 100% of the initial value. It is important to have methods that detect all major degradation products. This is generally done by performing the assay of forced degraded samples and assesses the mass balance. Mass balance has to be achieved at least up to 95% level. If it is less than the required criteria, investigation has to be done and justified. The following are some of the reasons for not achieving the mass balance.:

	- Not eluted from the LC column
	- Not detected by the detector used
	- Lost from the ample matrix, due to insolubility, volatility, or adsorption losses
	- Co-eluted with the parent compound
	- Not integrated due to poor chromatography

#### **2.19 Detector wavelengths**

After separation of all impurities and degradation products, absorption spectra of all the compounds are recorded and compared by taking overlay spectra of all known impurities along with the main analyte in each stress condition and finalizing a wavelength where all impurities are detected and quantified and have the

**159**

separations.

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

have poor UV character.

**2.21 System suitability**

**2.20 Stability of analytical solutions**

the various deliberate changes in method.

**2.22 Robustness of the method**

**2.23 Relative response factor**

maximum absorbance. In case this is not feasible, select different wavelengths to estimate all impurities. It is also recommended to extract the chromatograms at lower wavelengths like 210 nm–220 nm to see if there is any additional impurities found, which are found to be missing at higher wavelengths; this is likely the case when parent compound breaks into two parts during forced degradation study with one part highly UV active and second part an alkyl chain where alkyl chain will

The stability of analytical solutions (sample or standard) can be established on auto-injector for at least 12 hours continuously in a sequence mode to know the stability of all components and ruggedness of the method (peak shapes, column back pressure over the period of time). To get better results, choose a diluent in which a test solution is stable for at least 12 hours. If the solution is found to be unstable by

System suitability tests verify and ensure whether the system's performance is acceptable at the time of analysis in accordance with the criteria set forth in the procedure or not. System suitability parameters are chosen based on the criticality of separation. In general, resolution factor for the two adjacent peaks or closely eluting peaks is selected as a system suitability requirement. If the separation of impurities from each other and from API peak is found to be satisfactory, there is no need to keep a resolution factor as a system suitability parameter. In such a case, only a diluted standard reproducibility can be adopted as a system suitability requirement. Before finalizing the system suitability parameters, the separation needs to be studied during the robustness study to understand its behavior during

System suitability checking must be performed on two different make of HPLC systems whenever the separation of any impurities is critical. For in-process-related impurity issues, the quantification limit (QL) concentration is to be injected, and

Robustness by definition means the reliability of an analysis with respect to deliberate variations in method parameters. After finalizing all chromatographic conditions, robustness study with regard to mobile phase composition (±10%), pH (±0.2), gradient (±0.2%/min), flow rate (±0.2 mL/min), and temperature (±5°C) can be carried out to ensure that the developed method is stability-indicating. If the method of analysis is in a gradient mode, it needs to be checked on two different brands of HPLC or different HPLC to check the effect of the system volumes on

The relative response factor is used to correct the difference in the detector response of impurities with respect to the main analyte peak. It is mainly used to control the impurities or degradation products in a drug substance or drug product. RRF is established for all the known impurities using any of the slope methods. The standard solutions of API and all impurity can be prepared in at least five different

signal to noise ratio (S/N) must be kept as a system suitability parameter.

its nature, then incorporate the stability of solution in test method.

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

Peak purity can be evaluated for the main peak and the major degradants which

If any known impurity is observed to be increased in stress, it can be examined properly. If process impurity is found to be increased in stress study, it needs to be assessed whether there is any secondary pathway of formation of this impurity via

After conducting these studies, verify the chromatograms, and observe any peaks merging with respect to main peak and any critical pairs. If any situations were arrived, adjust the mobile-phase compositions, column parameters, etc. and

After method finalization, check the method using different detectors (RI/ ELSD/CE/LC–MS), and compare the data with other detectors like UV, fluorescence, etc. The UV inactive components can be found with these experiments. Identify the mass of major degradant which may be formed greater than 1.0% in

Mass balance is a process of adding together the assay value and levels of degradation products to see how closely these add up to 100% of the initial value. It is important to have methods that detect all major degradation products. This is generally done by performing the assay of forced degraded samples and assesses the mass balance. Mass balance has to be achieved at least up to 95% level. If it is less than the required criteria, investigation has to be done and justified. The following

○ Lost from the ample matrix, due to insolubility, volatility, or

• Parent compound may be lost from the sample matrix, due to insolubility, vola-

After separation of all impurities and degradation products, absorption spectra of all the compounds are recorded and compared by taking overlay spectra of all known impurities along with the main analyte in each stress condition and finalizing a wavelength where all impurities are detected and quantified and have the

have the peak heights less than 1 AU. Identify the degradation products by co-

injection, in case of known impurities and have comparable spectra.

**2.17 Evaluation of stress testing**

some other degradant route.

**2.18 Mass balance study**

conclude the method parameters.

• Degradation products are:

adsorption losses

tility, or adsorption losses

**2.19 Detector wavelengths**

stress studies, and try to establish the structures.

are some of the reasons for not achieving the mass balance.:

○ Not eluted from the LC column

○ Not detected by the detector used

○ Co-eluted with the parent compound

○ Not integrated due to poor chromatography

• Inaccurate quantification due to differences in response factors

**158**

maximum absorbance. In case this is not feasible, select different wavelengths to estimate all impurities. It is also recommended to extract the chromatograms at lower wavelengths like 210 nm–220 nm to see if there is any additional impurities found, which are found to be missing at higher wavelengths; this is likely the case when parent compound breaks into two parts during forced degradation study with one part highly UV active and second part an alkyl chain where alkyl chain will have poor UV character.

#### **2.20 Stability of analytical solutions**

The stability of analytical solutions (sample or standard) can be established on auto-injector for at least 12 hours continuously in a sequence mode to know the stability of all components and ruggedness of the method (peak shapes, column back pressure over the period of time). To get better results, choose a diluent in which a test solution is stable for at least 12 hours. If the solution is found to be unstable by its nature, then incorporate the stability of solution in test method.

#### **2.21 System suitability**

System suitability tests verify and ensure whether the system's performance is acceptable at the time of analysis in accordance with the criteria set forth in the procedure or not. System suitability parameters are chosen based on the criticality of separation. In general, resolution factor for the two adjacent peaks or closely eluting peaks is selected as a system suitability requirement. If the separation of impurities from each other and from API peak is found to be satisfactory, there is no need to keep a resolution factor as a system suitability parameter. In such a case, only a diluted standard reproducibility can be adopted as a system suitability requirement. Before finalizing the system suitability parameters, the separation needs to be studied during the robustness study to understand its behavior during the various deliberate changes in method.

System suitability checking must be performed on two different make of HPLC systems whenever the separation of any impurities is critical. For in-process-related impurity issues, the quantification limit (QL) concentration is to be injected, and signal to noise ratio (S/N) must be kept as a system suitability parameter.

#### **2.22 Robustness of the method**

Robustness by definition means the reliability of an analysis with respect to deliberate variations in method parameters. After finalizing all chromatographic conditions, robustness study with regard to mobile phase composition (±10%), pH (±0.2), gradient (±0.2%/min), flow rate (±0.2 mL/min), and temperature (±5°C) can be carried out to ensure that the developed method is stability-indicating. If the method of analysis is in a gradient mode, it needs to be checked on two different brands of HPLC or different HPLC to check the effect of the system volumes on separations.

#### **2.23 Relative response factor**

The relative response factor is used to correct the difference in the detector response of impurities with respect to the main analyte peak. It is mainly used to control the impurities or degradation products in a drug substance or drug product. RRF is established for all the known impurities using any of the slope methods. The standard solutions of API and all impurity can be prepared in at least five different

concentrations in the range of 0.1–1.0% (e.g., 0.1, 0.3, 0.5, 0.7, and 1.0%) and analyzed using the liquid chromatography. RRF is calculated by using the slope of the respective impurity and slope of the main drug (API) [38, 39].

#### **2.24 Quantification methods**

The following methods can be used for the quantitative determination of assay and organic impurities [40, 41]:


#### **3. Conclusion**

Principles involved in chromatography method development, especially for the analytical method development for the separation, identification, purification, and quantitative estimation of organic compounds using the liquid chromatography techniques (HPLC, UPLC, LC–MS, preparative HPLC, etc.), were emphasized in this chapter. Though many different types of chromatography techniques are currently in use, the liquid chromatographic methods HPLC, UPLC, and LC–MS are most widely utilized for the separation and quantitative determination of organic compounds. This chapter mainly focused on and explained the major and critical parameters of the liquid chromatography for the method development and optimization of a suitable stability-indicating LC method and impurity profiling studies. Each and every parameter which controls the purification of most of the organic compounds inclusive of drug, its precursors, and degraded products has been explained in detail in this chapter. The information given in this chapter will help the reader in choosing the right conditions for a particular compound to quantitatively separate from the reaction mixture or drug composition.

**161**

**Author details**

Limited, Hyderabad, India

Narasimha S. Lakka1,2 and Chandrasekar Kuppan1

\*Address all correspondence to: ramachan16@gmail.com

provided the original work is properly cited.

\*

1 Department of Science and Humanities, VIGNAN'S Foundation for Science,

2 Department of Analytical Research and Development, Jodas Expoim Private

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Technology and Research (VFSTR), Guntur, Andhra Pradesh, India

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

**2.24 Quantification methods**

and organic impurities [40, 41]:

and bioanalytical methods).

factors.

**3. Conclusion**

concentrations in the range of 0.1–1.0% (e.g., 0.1, 0.3, 0.5, 0.7, and 1.0%) and analyzed using the liquid chromatography. RRF is calculated by using the slope of

The following methods can be used for the quantitative determination of assay

a.*External standard method:* This method is used for the assay and impurity estimation in a given sample, where the impurities are estimated using the respective impurity standard and without the API standard peak. It's possible

b.*Area normalization:* If the RRF value of known impurity is close to the API (analyte), i.e., 0.9–1.1, the area normalization method is chosen for quantification. The recovery needs to be established without using the response

c.*Diluted standard method:* If the RRF values of impurities are different from the

Principles involved in chromatography method development, especially for the analytical method development for the separation, identification, purification, and quantitative estimation of organic compounds using the liquid chromatography techniques (HPLC, UPLC, LC–MS, preparative HPLC, etc.), were emphasized in this chapter. Though many different types of chromatography techniques are currently in use, the liquid chromatographic methods HPLC, UPLC, and LC–MS are most widely utilized for the separation and quantitative determination of organic compounds. This chapter mainly focused on and explained the major and critical parameters of the liquid chromatography for the method development and optimization of a suitable stability-indicating LC method and impurity profiling studies. Each and every parameter which controls the purification of most of the organic compounds inclusive of drug, its precursors, and degraded products has been explained in detail in this chapter. The information given in this chapter will help the reader in choosing the right conditions for a particular compound to quantita-

d.*Internal standard method:* If the sample preparation procedure involves different extraction steps to avoid the error in the extraction procedure, internal standard procedure shall be chosen (normally for derivatization techniques

the respective impurity and slope of the main drug (API) [38, 39].

to estimate the concentration from calibration curve.

analyte, the diluted standard method can be chosen.

tively separate from the reaction mixture or drug composition.

**160**

### **Author details**

Narasimha S. Lakka1,2 and Chandrasekar Kuppan1 \*

1 Department of Science and Humanities, VIGNAN'S Foundation for Science, Technology and Research (VFSTR), Guntur, Andhra Pradesh, India

2 Department of Analytical Research and Development, Jodas Expoim Private Limited, Hyderabad, India

\*Address all correspondence to: ramachan16@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[3] Zlatkis A, editors. 75 Years of chromatography: A historical dialogue. Journal of Chromatography Library. Elsevier; 1979:7. ISBN 0-444-41754-0 (Vol. 17); ISBN 0-444-41616-1 (Series)

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[12] Rigas PG. Review: Liquid chromatography—post-column derivatization for amino acid analysis: Strategies, instrumentation, and applications. Instrumentation Science & Technology. 2012;**40**(2-3): 161-193. DOI: 10.1080/10739149. 2011.651669

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[14] Scott RPW. liquid chromatography detectors. Chapter 3. The fluorometric detector. Journal of Chromatography Library. 1977;**11**:121-130. DOI: 10.1016/ S0301-4770(08)61018-0

[15] McCalley DV. Choice of Buffer for the Analysis of Basic Peptides in Reversed-Phase HPLC. LCGC

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[24] Du Q, CaijuanWu GQ, Wu P, Ito Y.

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Relationship between the flowrate of the mobile phase and retention of the stationary phase in counter-current chromatography. Journal of Chromatography A.

and Column Thermostatting in Liquid Chromatography. White Paper. WP71499-EN 09/16S. Thermo Scientific. Available from: http:// tools.thermofisher.com/content/sfs/ brochures/WP-71499-LC-Temperature-Column-Thermostatting-WP71499-EN.

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[27] Thermo Scientific Poster Note PN71314. Thermostatting in UHPLC: Forced Air Mode, Still Air Mode, and Method Transfer. Germering, Germany. 2014. Available from: http:// www.thermoscientific.com/content/ dam/tfs/ATG/CMD/cmd-documents/

sci-res/posters/chrom/lc/sys/

1999;**835**(1-2):231-235

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composition on retention

[17] Zapala W. Influence of mobile phase

factors in different HPLC systems with chemically bonded stationary phases. Journal of Chromatographic Science. 2003;**41**. Available from: https://pdfs. semanticscholar.org/2310/bbddc ddcf462b99666f1d7cc351fcab 48952.pdf [Accessed: 10 July 2019]

[18] Janeček M, Šlais K. Design of mobile phase composition for liquid chromatography with an internal pH gradient. Chromatographia. 1993;**36**(1):246-250. DOI: 10.1007/

[19] Joshi VS, Kumar V, Rathore AS. Role of organic modifier and gradient shape in RP-HPLC separation: Analysis of GCSF variants. Journal of Chromatographic Science. 2015;**53**(3):417-423. DOI:

[20] Shimada K, Yoshida H, Komine Y. The effect of organic modifier in the mobile phase on the separation of bile acids and its fluorescent derivatives in inclusion chromatography. Journal of Liquid Chromatography.

[21] Ion-pair Reagents for HPLC. TCI/ A1084E 20170303. Available from: https://www.tcichemicals.com/a-cmn/ en/common/support-download/ brochure/ion-pair\_reagents\_for\_hplc.

10.1093/chromsci/bmu222

1991;**14**(4):605-617. DOI: 10.1080/01483919108049274

pdf [Accessed: 10 July 2019]

10 July 2019]

10 July 2019]

BF02263872

*Principles of Chromatography Method Development DOI: http://dx.doi.org/10.5772/intechopen.89501*

ASIA PACIFIC VOLUME 8. 2005. Available from: http://alfresco. ubm-us.net/alfresco\_images/ pharma/2014/08/22/806d27ce-71f0-4239-b9a6-f0541666d136/ article-254604.pdf [Accessed: 10 July 2019]

[16] Chemical Analysis. ZORBAX HPLC Columns. Available from: http://quimica.udea.edu.co/~carlopez/ cromatogc/ph/95121tb.html [Accessed: 10 July 2019]

[17] Zapala W. Influence of mobile phase composition on retention factors in different HPLC systems with chemically bonded stationary phases. Journal of Chromatographic Science. 2003;**41**. Available from: https://pdfs. semanticscholar.org/2310/bbddc ddcf462b99666f1d7cc351fcab 48952.pdf [Accessed: 10 July 2019]

[18] Janeček M, Šlais K. Design of mobile phase composition for liquid chromatography with an internal pH gradient. Chromatographia. 1993;**36**(1):246-250. DOI: 10.1007/ BF02263872

[19] Joshi VS, Kumar V, Rathore AS. Role of organic modifier and gradient shape in RP-HPLC separation: Analysis of GCSF variants. Journal of Chromatographic Science. 2015;**53**(3):417-423. DOI: 10.1093/chromsci/bmu222

[20] Shimada K, Yoshida H, Komine Y. The effect of organic modifier in the mobile phase on the separation of bile acids and its fluorescent derivatives in inclusion chromatography. Journal of Liquid Chromatography. 1991;**14**(4):605-617. DOI: 10.1080/01483919108049274

[21] Ion-pair Reagents for HPLC. TCI/ A1084E 20170303. Available from: https://www.tcichemicals.com/a-cmn/ en/common/support-download/ brochure/ion-pair\_reagents\_for\_hplc. pdf [Accessed: 10 July 2019]

[22] Shibue M, Mant CT, Hodges RS. Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography. Journal of Chromatography A. 2005;**1080**(1):68- 75. DOI: 10.1016/j.chroma.2005.03.035

[23] 1.14.4 High-Performance Liquid Chromatography: The International Pharmacopoeia. 8th ed2018. Available from: http://apps.who.int/phint/ pdf/b/7.1.14.4.1.14.4-High-performanceliquid-chromatography.pdf [Accessed: 10 July 2019]

[24] Du Q, CaijuanWu GQ, Wu P, Ito Y. Relationship between the flowrate of the mobile phase and retention of the stationary phase in counter-current chromatography. Journal of Chromatography A. 1999;**835**(1-2):231-235

[25] Heidorn M. The Role of Temperature and Column Thermostatting in Liquid Chromatography. White Paper. WP71499-EN 09/16S. Thermo Scientific. Available from: http:// tools.thermofisher.com/content/sfs/ brochures/WP-71499-LC-Temperature-Column-Thermostatting-WP71499-EN. pdf [Accessed: 10 July 2019]

[26] Thermo Scientific Product Spotlight SP71195: Best in UHPLC Column Thermostatting to Fit All Needs. 2014. Available from: http:// www.thermoscientific.com/content/ dam/tfs/ATG/CMD/cmd-documents/ bro/bro/chrom/lc/sys/SP-71195- Vanquish-Column-Thermostatting-SP71195-EN.pdf [Accessed: 10 July 2019]

[27] Thermo Scientific Poster Note PN71314. Thermostatting in UHPLC: Forced Air Mode, Still Air Mode, and Method Transfer. Germering, Germany. 2014. Available from: http:// www.thermoscientific.com/content/ dam/tfs/ATG/CMD/cmd-documents/ sci-res/posters/chrom/lc/sys/

**162**

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

https://database.ich.org/sites/default/ files/Q3B\_R2\_\_Guideline.pdf; https:// www.ich.org/fileadmin/Public\_Web\_ Site/ICH\_Products/Guidelines/Quality/ Q3B\_R2/Step4/Q3B\_R2\_\_Guideline.pdf

[10] Silverstein B. Morrill: Spectroscopic Determination of Organic Compounds.

Technologies. 2010;**33**:1130-1150. DOI:

[11] Swartz M. HPLC detectors: A brief review. Journal of Liquid Chromatography & Related

10.1080/10826076.2010.484356

[12] Rigas PG. Review: Liquid chromatography—post-column derivatization for amino acid analysis: Strategies, instrumentation, and applications. Instrumentation Science & Technology. 2012;**40**(2-3): 161-193. DOI: 10.1080/10739149.

[13] Santa T, Al-Dirbashi OY,

in liquid chromatography/

2007;**1**(2):108-118

S0301-4770(08)61018-0

Fukushima T. Derivatization reagents

electrospray ionization tandem mass spectrometry for biomedical analysis. Drug Discoveries & Therapeutics.

[14] Scott RPW. liquid chromatography detectors. Chapter 3. The fluorometric detector. Journal of Chromatography Library. 1977;**11**:121-130. DOI: 10.1016/

[15] McCalley DV. Choice of Buffer for the Analysis of Basic Peptides in Reversed-Phase HPLC. LCGC

[Accessed: 10 July 2019]

ja00472a008

5th ed1991

2011.651669

[9] Liljefors T, Allinger NL. Conformational analysis. 128. The Woodward-Fieser rules and α,βunsaturated ketones. Journal of the American Chemical Society. 1978;**100**(4):1073. DOI: 10.1021/

[1] Ettre LS. Milestones in Chromatography. LCGC North America. 2003. Available from: http:// files.pharmtech.com/alfresco\_images/ pharma/2014/08/22/e5b0c0c2-2e1c-463d-8d4f-4993f7245f47/article-56954.

**References**

pdf [Accessed: 10 July 2019]

[2] Blomberg L. Stationary phases for capillary gas chromatography. Trends in Analytical Chemistry. 1987;**6**(2):41-45

[3] Zlatkis A, editors. 75 Years of chromatography: A historical dialogue. Journal of Chromatography Library. Elsevier; 1979:7. ISBN 0-444-41754-0 (Vol. 17); ISBN 0-444-41616-1 (Series)

[4] The Nobel Prize in Chemistry. 1952. Available from: nobelprize.org

[5] FDA, United States-Published in the Federal Register. Q6A Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances. 2000. Vol. 65; 83041-83063. Available from: https://database.ich.org/sites/default/

[6] Nicholson DE. An Introduction to Metabolic Pathways by S. Dagley. 59, No. 2 ed. Sigma Xi. The Scientific Research Society; 1971. 266p. Available from: https://onlinelibrary.wiley.com/doi/ pdf/10.1002/food.19710150432

[7] Q3A(R2) Impurities in New Drug Substances: FDA, United States-Published in the Federal Register on June 2008. Available from: https://www. ich.org/fileadmin/Public\_Web\_Site/ ICH\_Products/Guidelines/Quality/ Q3A\_R2/Step4/Q3A\_R2\_\_Guideline.pdf

[8] Q3B(R2) Impurities in New Drug Products. FDA, United States-Published in the Federal Register. 14 November 2003; 68; 220; 64628-9. Available from:

[Retrieved: 25 August 2016]

files/Q6A\_Guideline.pdf

[Accessed: 10 July 2019]

PN-71314-ISC2014-UHPLC-Thermostatting-PN71314-EN.pdf [Accessed: 10 July 2019]

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[32] Wrezel PW, Chion I, Pakula R. Key factors in sample diluent selection for HPLC assays of active pharmaceutical ingredients. LCGC North America. 2005;**23**(7):682-686

[33] Slack GC, Snow NH. 8 HPLC sample preparation. Separation Science and Technology. 2007;**8**:237-268. DOI: 10.1016/S0149-6395(07)80014-6

[34] LCGC Editors. Overview of sample preparation. 2015;**33**(11):46- 51. Available from: http://www. chromatographyonline.com/overviewsample-preparation [Accessed: 10 July 2019]

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[36] Singh S, Junwal M, Modhe G, Tiwari H, Kurmi M, Parashar N, et al. Forced degradation studies to assess the stability of drugs and products. Trends in Analytical Chemistry. 2013;**49**:71-88

[37] Jain D, Basniwal PK. Forced degradation and impurity profiling: Recent trends in analytical perspectives. Journal of Pharmaceutical and Biomedical Analysis. 2013;**86**:11-35

[38] Kalyana Chakravarthy VV, Kishore Babu G, Lakshmana Dasu R, Prathyusha P, Aparna Kiran G. The role of relative response factor in related substances method development by high performance liquid chromatography (HPLC). Rasayan Journal of Chemistry. 2011;**4**(4):919-943

[39] Bhattacharyya L, Pappa H, Russo KA, Sheinin E, Roger L. Williams: U.S. pharmacopeia. The use of relative response factors to determine impurities. Pharmacopeial Forum. 2005;**31**(3). Available from: https://www.researchgate.net/ publication/295423011\_The\_use\_ of\_Relative\_Response\_Factors\_to\_ determine\_impurities

[40] Altria KD. Essential peak area normalisation for quantitative impurity content determination by capillary electrophoresis. Chromatographia. 1993;**35**(3-4):177-182

[41] de Oliveira EC, Muller EI, Abad F, Dallarosa J, Adriano C. Internal standard versus external standard calibration: an uncertainty case study of a liquid chromatography analysis. Química Nova. 2010;**33**:4. DOI: 10.1590/ S0100-40422010000400041

**165**

**Chapter 10**

**Abstract**

mated in-tube SPME.

**1. Introduction**

Online Automated Micro Sample

Liquid Chromatography

*Hiroyuki Kataoka, Atsushi Ishizaki and Keita Saito*

Preparation for High-Performance

Sample preparation is one of the most labor-intensive and time-consuming operations in sample analysis. Sample preparation strategies include the exhaustive or non-exhaustive extraction of analytes from matrices. Online coupling of sample preparation with the separation system is regarded as an important goal. In-tube solid-phase microextraction (SPME) is an effective sample preparation technique that uses an open tubular fused-silica capillary column as an extraction device. In-tube SPME is useful for trace enrichment, automated sample cleanup, and rapid online analysis. Moreover, this method can be used to determine the analytes in complex matrices by direct sample injection or merely by simple sample treatment such as filtration. In-tube SPME is frequently combined with high-performance liquid chromatography (HPLC) using online column-switching techniques. Various operating systems and new sorbent materials have been reported to improve extraction efficiency, such as sorption capacity and selectivity. This chapter discusses efficient micro sample preparation techniques for HPLC, especially online auto-

**Keywords:** sample preparation, online automated analysis, column switching, in-tube solid-phase microextraction, high-performance liquid chromatography

Sample analysis consists of various analytical steps, including sampling, sample preparation, separation, detection and data analysis. One of the most important steps is sample preparation, which involves the extraction, isolation and concentration of target analytes from complex matrices. Sample preparation [1–18] is the most labor-intensive and error-prone process in analytical methodology and markedly influences the reliability and accuracy of analyte determination. In addition, sample preparation requires large amounts of sample and organic solvents, and is therefore difficult to automate. An ideal sample preparation technique should be simple and fast; be specific for analytes through the efficient removal of coexisting components; provide high sample throughput; utilize fewer operation steps to minimize analyte losses; and be solvent-free, inexpensive, and compatible with chromatography systems. Online automated sample preparation [19–29], in which sample preparation is directly connected to chromatographic separation systems, eliminates further sample handling between the trace-enrichment and separation

#### **Chapter 10**

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

780-785. Available from: http://www. chromatographyonline.com/how-muchcan-i-inject-part-i-injecting-mobilephase [Accessed: 10 July 2019]

[36] Singh S, Junwal M, Modhe G, Tiwari H, Kurmi M, Parashar N, et al. Forced degradation studies to assess the stability of drugs and products. Trends in Analytical Chemistry. 2013;**49**:71-88

[37] Jain D, Basniwal PK. Forced degradation and impurity profiling: Recent trends in analytical perspectives.

Journal of Pharmaceutical and Biomedical Analysis. 2013;**86**:11-35

[38] Kalyana Chakravarthy VV, Kishore Babu G, Lakshmana Dasu R, Prathyusha P, Aparna Kiran G. The role of relative response factor in related substances method development by high performance liquid chromatography (HPLC). Rasayan Journal of Chemistry.

[39] Bhattacharyya L, Pappa H,

U.S. pharmacopeia. The use of relative response factors to determine impurities. Pharmacopeial Forum. 2005;**31**(3). Available from: https://www.researchgate.net/ publication/295423011\_The\_use\_ of\_Relative\_Response\_Factors\_to\_

[40] Altria KD. Essential peak area normalisation for quantitative impurity content determination by capillary electrophoresis. Chromatographia.

[41] de Oliveira EC, Muller EI,

S0100-40422010000400041

Abad F, Dallarosa J, Adriano C. Internal standard versus external standard calibration: an uncertainty case study of a liquid chromatography analysis. Química Nova. 2010;**33**:4. DOI: 10.1590/

Russo KA, Sheinin E, Roger L. Williams:

2011;**4**(4):919-943

determine\_impurities

1993;**35**(3-4):177-182

PN-71314-ISC2014-UHPLC-Thermostatting-PN71314-EN.pdf [Accessed: 10 July

[28] Dolan J. Separation Science. HPLC Solutions # 53. Temperature and Retention. Available from: https://owl.english.purdue.edu/owl/ resource/747/08/ [Accessed: 10 July

[29] Gradient HPLC Solvent Delivery System: WPP10. Waters. Available from: https://gimitec.com/file/wpp10.pdf

[30] Gradient Design and Development, Breaking the Bad Gradient Cycle: Agilent Technologies. Available from: https://www.agilent.com/cs/library/ slidepresentation/public/Gradient%20 Design%20and%20Development.pdf

[31] Good Habits for Successful Gradient Separations: Getting the Most From Your Method. Agilent Technologies. Available from: https://www.agilent.com/cs/ library/slidepresentation/Public/

Successful%20Gradient%20Separations.

[32] Wrezel PW, Chion I, Pakula R. Key factors in sample diluent selection for HPLC assays of active pharmaceutical ingredients. LCGC North America.

[33] Slack GC, Snow NH. 8 HPLC sample preparation. Separation Science and Technology. 2007;**8**:237-268. DOI: 10.1016/S0149-6395(07)80014-6

[34] LCGC Editors. Overview of sample preparation. 2015;**33**(11):46- 51. Available from: http://www. chromatographyonline.com/overviewsample-preparation [Accessed: 10 July

[35] John W. Dolan: How much can i inject? Part I: Injecting in mobile phase. LCGC North America. 2014;**32**(10):

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2019]

2019]

**164**

2019]

## Online Automated Micro Sample Preparation for High-Performance Liquid Chromatography

*Hiroyuki Kataoka, Atsushi Ishizaki and Keita Saito*

#### **Abstract**

Sample preparation is one of the most labor-intensive and time-consuming operations in sample analysis. Sample preparation strategies include the exhaustive or non-exhaustive extraction of analytes from matrices. Online coupling of sample preparation with the separation system is regarded as an important goal. In-tube solid-phase microextraction (SPME) is an effective sample preparation technique that uses an open tubular fused-silica capillary column as an extraction device. In-tube SPME is useful for trace enrichment, automated sample cleanup, and rapid online analysis. Moreover, this method can be used to determine the analytes in complex matrices by direct sample injection or merely by simple sample treatment such as filtration. In-tube SPME is frequently combined with high-performance liquid chromatography (HPLC) using online column-switching techniques. Various operating systems and new sorbent materials have been reported to improve extraction efficiency, such as sorption capacity and selectivity. This chapter discusses efficient micro sample preparation techniques for HPLC, especially online automated in-tube SPME.

**Keywords:** sample preparation, online automated analysis, column switching, in-tube solid-phase microextraction, high-performance liquid chromatography

#### **1. Introduction**

Sample analysis consists of various analytical steps, including sampling, sample preparation, separation, detection and data analysis. One of the most important steps is sample preparation, which involves the extraction, isolation and concentration of target analytes from complex matrices. Sample preparation [1–18] is the most labor-intensive and error-prone process in analytical methodology and markedly influences the reliability and accuracy of analyte determination. In addition, sample preparation requires large amounts of sample and organic solvents, and is therefore difficult to automate. An ideal sample preparation technique should be simple and fast; be specific for analytes through the efficient removal of coexisting components; provide high sample throughput; utilize fewer operation steps to minimize analyte losses; and be solvent-free, inexpensive, and compatible with chromatography systems. Online automated sample preparation [19–29], in which sample preparation is directly connected to chromatographic separation systems, eliminates further sample handling between the trace-enrichment and separation

steps. Online automated sample preparation methods usually improve data quality, increase sample throughput, reduce costs, and improve the productivity of personnel and instruments.

In-tube solid-phase microextraction (SPME), using a capillary tube as an extraction device, was introduced by Eisert and Pawliszyn [30] to overcome the problems inherent to conventional fiber SPME. These drawbacks included fragility, low sorption capacity, bleeding from thick-film coatings on fibers, limited effectiveness for extraction of weakly volatile or thermally labile compounds not amenable to gas chromatography (GC) or GC-mass spectrometry (MS), and reduced stability in solvents used in high performance liquid chromatography (HPLC). In-tube SPME was also developed to completely automate the sample preparation process and to enable direct online coupling of in-tube SPME with HPLC using capillary column switching systems [31].

This chapter reviews the configurations and characteristics of in-tube SPME technology and discusses current and future directions, including the strategies involved in extraction efficiency and method development. The details of in-tube SPME have been described in well documented reviews [27, 32–50].

#### **2. Configurations of in-tube SPME**

In-tube SPME is an efficient sample preparation technique for extraction in capillary columns using stationary phases coated on the inner wall of the capillary or on the surface of the packing material (**Figure 1**). Various in-tube SPME capillary devices have been developed, such as inner wall-coated fused-silica open tubular (**Figure 1A**), fiber-packed (**Figure 1B**), sorbent-packed (**Figure 1C**), and rod-type porous monolith (**Figure 1D**) capillaries [16, 31]. The capillaries are easily fixed with the autosampler injection system, and are generally reusable without plugging or breaking the column and without exfoliation of coating materials.

**Figure 1.**

*Capillary devices for in-tube SPME: (A) polymer coated, (B) sorbent-packed, (C) fiber-packed, and (D) monolith capillary tubes.*

**167**

**Figure 2.**

*(extraction), and (B) injection position (desorption).*

*Online Automated Micro Sample Preparation for High-Performance Liquid Chromatography*

Flow-through systems (**Figure 2**), in which sample solutions are continuously passed in one direction through a capillary column; or as repeated draw/ejection systems (**Figure 3**), in which sample solutions are repeatedly aspirated and dispensed from a capillary column, are used as an operating system of in-tube SPME [18]. These systems are operated by column switching techniques under computer control. In flow-through systems, the complete analytical system consists of an automatic six-port valve, two pumps (a sample pump and a wash pump) and a liquid chromatography (LC) system. A capillary column is installed in the six-port valve or sometimes placed in the loop. Although one or two six-port valves are available, one valve mode is used more frequently than others. The procedure consists of four steps, conditioning, extracting, washing and desorbing. After conditioning of capillary column with water, the aqueous sample is pumped through the column under the load position (**Figure 2A**). Remaining matrix and residues in capillary are removed by washing with water. After switching the six-port valve to the injection position, the LC mobile phase is passed through the column (dynamic desorption), with the flow-rate of the LC pump (**Figure 2B**). The desorbed analytes are subsequently transferred to the analytical column for separation and detection. The flow-through extraction system, however, may include systematic troubles, such as

contamination of the switching valve by sample matrix [18, 31, 37, 41].

Repeated draw/ejection systems include the placement of a capillary column for extraction between the injection loop and the injection needle of the autosampler. Since the sample solution moves only in the capillary, the metering pump and switching valve are not contaminated by sample matrix [18, 31, 37, 41]. A built-in UV diode array detector (DAD) or fluorescence detector (FLD) between the HPLC and the MS can enhance the multidimensional and simultaneous multi-detections, improving analyte identification. During the extraction and concentration step (**Figure 3A**), the injection syringe is programmed to repeatedly draw and eject sample solution from the vial until the concentration of the analyte reaches distribution equilibrium between the sample solution and the stationary phase. After switching the six-port valve to the injection position, the extracted analytes can be directly desorbed from the capillary coating by LC mobile phase flow (dynamic

*Schematic diagrams of a flow-through extraction system used for online in-tube SPME. (A) Load position* 

*DOI: http://dx.doi.org/10.5772/intechopen.89079*

**2.1 Operating systems of in-tube SPME**

*Online Automated Micro Sample Preparation for High-Performance Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.89079*

#### **2.1 Operating systems of in-tube SPME**

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

nel and instruments.

switching systems [31].

**2. Configurations of in-tube SPME**

steps. Online automated sample preparation methods usually improve data quality, increase sample throughput, reduce costs, and improve the productivity of person-

This chapter reviews the configurations and characteristics of in-tube SPME technology and discusses current and future directions, including the strategies involved in extraction efficiency and method development. The details of in-tube

In-tube SPME is an efficient sample preparation technique for extraction in capillary columns using stationary phases coated on the inner wall of the capillary or on the surface of the packing material (**Figure 1**). Various in-tube SPME capillary devices have been developed, such as inner wall-coated fused-silica open tubular (**Figure 1A**), fiber-packed (**Figure 1B**), sorbent-packed (**Figure 1C**), and rod-type porous monolith (**Figure 1D**) capillaries [16, 31]. The capillaries are easily fixed with the autosampler injection system, and are generally reusable without plugging

SPME have been described in well documented reviews [27, 32–50].

or breaking the column and without exfoliation of coating materials.

*Capillary devices for in-tube SPME: (A) polymer coated, (B) sorbent-packed, (C) fiber-packed,* 

In-tube solid-phase microextraction (SPME), using a capillary tube as an extraction device, was introduced by Eisert and Pawliszyn [30] to overcome the problems inherent to conventional fiber SPME. These drawbacks included fragility, low sorption capacity, bleeding from thick-film coatings on fibers, limited effectiveness for extraction of weakly volatile or thermally labile compounds not amenable to gas chromatography (GC) or GC-mass spectrometry (MS), and reduced stability in solvents used in high performance liquid chromatography (HPLC). In-tube SPME was also developed to completely automate the sample preparation process and to enable direct online coupling of in-tube SPME with HPLC using capillary column

**166**

**Figure 1.**

*and (D) monolith capillary tubes.*

Flow-through systems (**Figure 2**), in which sample solutions are continuously passed in one direction through a capillary column; or as repeated draw/ejection systems (**Figure 3**), in which sample solutions are repeatedly aspirated and dispensed from a capillary column, are used as an operating system of in-tube SPME [18]. These systems are operated by column switching techniques under computer control.

In flow-through systems, the complete analytical system consists of an automatic six-port valve, two pumps (a sample pump and a wash pump) and a liquid chromatography (LC) system. A capillary column is installed in the six-port valve or sometimes placed in the loop. Although one or two six-port valves are available, one valve mode is used more frequently than others. The procedure consists of four steps, conditioning, extracting, washing and desorbing. After conditioning of capillary column with water, the aqueous sample is pumped through the column under the load position (**Figure 2A**). Remaining matrix and residues in capillary are removed by washing with water. After switching the six-port valve to the injection position, the LC mobile phase is passed through the column (dynamic desorption), with the flow-rate of the LC pump (**Figure 2B**). The desorbed analytes are subsequently transferred to the analytical column for separation and detection. The flow-through extraction system, however, may include systematic troubles, such as contamination of the switching valve by sample matrix [18, 31, 37, 41].

Repeated draw/ejection systems include the placement of a capillary column for extraction between the injection loop and the injection needle of the autosampler. Since the sample solution moves only in the capillary, the metering pump and switching valve are not contaminated by sample matrix [18, 31, 37, 41]. A built-in UV diode array detector (DAD) or fluorescence detector (FLD) between the HPLC and the MS can enhance the multidimensional and simultaneous multi-detections, improving analyte identification. During the extraction and concentration step (**Figure 3A**), the injection syringe is programmed to repeatedly draw and eject sample solution from the vial until the concentration of the analyte reaches distribution equilibrium between the sample solution and the stationary phase. After switching the six-port valve to the injection position, the extracted analytes can be directly desorbed from the capillary coating by LC mobile phase flow (dynamic

#### **Figure 2.**

*Schematic diagrams of a flow-through extraction system used for online in-tube SPME. (A) Load position (extraction), and (B) injection position (desorption).*

**Figure 3.**

*Schematic diagrams of a draw/eject extraction system used for online in-tube SPME (reproduced from Ref. [37]). (A) Extraction and concentration step, and (B) desorption and injection step.*

desorption) or by an aspirated desorption solvent (static desorption) (**Figure 3B**) [31]. The desorbed analytes are subsequently transferred to an LC column. The computer controls the drawing and ejection of sample solution; switching of the valves; control of peripheral equipment, such as the HPLC and MS; and analytical data processing, thus reducing labor and enhancing precision. In addition, the autosampler can automatically process a large number of samples without carryover, because the injection needle and capillary column are washed in methanol and the mobile phase before the sample is extracted.

#### **2.2 Extraction sorbent materials**

The amount of analyte extracted into the stationary phase of the capillary during in-tube SPME is dependent on the characteristics of the capillary coating and the target analyte. Among the commercially available GC capillary columns, silica modified columns have been found more suitable for the analysis of nonpolar compounds. Porous polymer type capillary columns such as Supel-Q PLOT (divinylbenzene polymer, film thickness 17 μm) have shown better extraction efficiencies due to their large surface area for most organic compounds than other liquid-phase type capillary columns, such as CP-Sil 5CB (100% polydimethylsiloxane, film thickness 5 μm), Quadrex 007–5 (5% phenyl polydimethylsiloxane, film thickness 12 μm), CP-Sil 19CB (14% cyanopropyl phenyl methylsilicone, film thickness 1.0 μm), and CP-Wax 52CB (polyethylene glycol, film thickness 1.2 μm). CP-Sil 19CB was superior for extraction of polyaromatic hydrocarbons, although the film layer was thin. In contrast, some compounds were effectively extracted with other PLOT type coatings, including Carboxen-1006 PLOT (carboxen molecularsives, film thickness 17 μm) and CP-Pora PLOT amine (basic modified styrene divinylbenzene polymer, film thickness 10 μm).

Several unique phases and technical solutions have been developed to improve extraction efficiency and selectivity when extended to microscale applications

**169**

**Figure 4.**

*Online Automated Micro Sample Preparation for High-Performance Liquid Chromatography*

[44, 51–53]. These include polypyrrole (PPY) coated capillaries; PEEK tube capillaries packed with molecularly imprinted polymer (MIP) particles [54–61]; and highly biocompatible SPME capillaries packed with alkyl-diol-silica (ADS) particles as restricted access media (RAM) [62, 63], immunosorbents [64], ionic liquids [65–67], monolithic materials [68–73], carbon nanomaterials [74–82], silica-coated magnetite (SiO2-Fe3O4) [83–86], and temperature responsive polymers [87, 88]. Novel extraction sorbent materials for in-tube SPME are shown in

For example, chemically or electrochemically deposited PPY coatings have higher extraction efficiencies than commercial GC coatings due to the various types of interactions (e.g., π–π, polar, hydrogen bonding, and ionic interactions) between these multifunctional PPY coatings and the analytes. Capillary tubes have been coated with MIP, consisting of cross-linked synthetic polymers produced by copolymerizing a monomer with a cross-linker in the presence of a template molecule (**Figure 4A**), and PEEK tubes have been packed with MIP particles. By removing the template after polymerization, it is possible to leave open sites of a specific size and shape suitable for binding the same or similar chemicals in a sample.

*Novel extraction sorbent materials for in-tube SPME (eproduced from Ref. [37, 42, 84]). (A) Molecularly imprinted polymers, (B) restricted access media, (C) immunosorbents, (D) monolithic polymers, (E) carbon* 

*nanotubes, (F) silica-coated magnetite, and (G) temperature responsive polymers.*

*DOI: http://dx.doi.org/10.5772/intechopen.89079*

**Figure 4**.

*Online Automated Micro Sample Preparation for High-Performance Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.89079*

[44, 51–53]. These include polypyrrole (PPY) coated capillaries; PEEK tube capillaries packed with molecularly imprinted polymer (MIP) particles [54–61]; and highly biocompatible SPME capillaries packed with alkyl-diol-silica (ADS) particles as restricted access media (RAM) [62, 63], immunosorbents [64], ionic liquids [65–67], monolithic materials [68–73], carbon nanomaterials [74–82], silica-coated magnetite (SiO2-Fe3O4) [83–86], and temperature responsive polymers [87, 88]. Novel extraction sorbent materials for in-tube SPME are shown in **Figure 4**.

For example, chemically or electrochemically deposited PPY coatings have higher extraction efficiencies than commercial GC coatings due to the various types of interactions (e.g., π–π, polar, hydrogen bonding, and ionic interactions) between these multifunctional PPY coatings and the analytes. Capillary tubes have been coated with MIP, consisting of cross-linked synthetic polymers produced by copolymerizing a monomer with a cross-linker in the presence of a template molecule (**Figure 4A**), and PEEK tubes have been packed with MIP particles. By removing the template after polymerization, it is possible to leave open sites of a specific size and shape suitable for binding the same or similar chemicals in a sample.

#### **Figure 4.**

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

desorption) or by an aspirated desorption solvent (static desorption) (**Figure 3B**) [31]. The desorbed analytes are subsequently transferred to an LC column. The computer controls the drawing and ejection of sample solution; switching of the valves; control of peripheral equipment, such as the HPLC and MS; and analytical data processing, thus reducing labor and enhancing precision. In addition, the autosampler can automatically process a large number of samples without carryover, because the injection needle and capillary column are washed in methanol and

*Schematic diagrams of a draw/eject extraction system used for online in-tube SPME (reproduced from Ref. [37]).* 

The amount of analyte extracted into the stationary phase of the capillary during in-tube SPME is dependent on the characteristics of the capillary coating and the target analyte. Among the commercially available GC capillary columns, silica modified columns have been found more suitable for the analysis of nonpolar compounds. Porous polymer type capillary columns such as Supel-Q PLOT (divinylbenzene polymer, film thickness 17 μm) have shown better extraction efficiencies due to their large surface area for most organic compounds than other liquid-phase type capillary columns, such as CP-Sil 5CB (100% polydimethylsiloxane, film thickness 5 μm), Quadrex 007–5 (5% phenyl polydimethylsiloxane, film thickness 12 μm), CP-Sil 19CB (14% cyanopropyl phenyl methylsilicone, film thickness 1.0 μm), and CP-Wax 52CB (polyethylene glycol, film thickness 1.2 μm). CP-Sil 19CB was superior for extraction of polyaromatic hydrocarbons, although the film layer was thin. In contrast, some compounds were effectively extracted with other PLOT type coatings, including Carboxen-1006 PLOT (carboxen molecularsives, film thickness 17 μm) and CP-Pora PLOT amine (basic modified styrene divinylbenzene polymer, film thickness 10 μm). Several unique phases and technical solutions have been developed to improve extraction efficiency and selectivity when extended to microscale applications

the mobile phase before the sample is extracted.

*(A) Extraction and concentration step, and (B) desorption and injection step.*

**2.2 Extraction sorbent materials**

**Figure 3.**

**168**

*Novel extraction sorbent materials for in-tube SPME (eproduced from Ref. [37, 42, 84]). (A) Molecularly imprinted polymers, (B) restricted access media, (C) immunosorbents, (D) monolithic polymers, (E) carbon nanotubes, (F) silica-coated magnetite, and (G) temperature responsive polymers.*

MIPs recognize chemicals through combination of shape, hydrogen bonding, and hydrophobic and electrostatic interactions [16, 18, 31]. RAM materials possess defined diffusion barriers with small sized pores and biocompatible outer particle surfaces (**Figure 4B**). The bifunctionality of ADS particles used as a RAM SPME device can prevent fouling of the capillary by protein adsorption while simultaneously trapping the analytes in the hydrophobic porous interior. Furthermore, a simple SPME device has been fabricated for use in online immunoaffinity capillaries packed with immunosorbent materials, consisting of covalently immobilized antibodies (**Figure 4C**).

An alternative approach consists of in-tube SPME using monolithic capillary columns comprised of one piece of organic polymer or silica rods with a unique flow-through double-pore structure (**Figure 4D**). Monoliths are also highly permeable to liquids and biological samples, enabling reduced solvent use, varied support formats, and/or automation. Monolithic capillaries are especially suitable for intube SPME media due to the low pressure drop, allowing a high flow-rate to achieve high throughput and a total porosity greater than that of particle-packed capillaries. Hydrophobic main chains and acidic pendant groups of poly (methacrylic acidethylene glycol dimethacrylate) enhance the ability to extract basic analytes from aqueous matrices. The physicochemical properties of graphene-based sorbents and carbon nanotubes (**Figure 4E**) enable their use in extraction, with these combinations showing excellent results when used for in-tube SPME. In addition, various cationic, anionic and zwitterionic liquid-mediated sol–gel coatings have been developed for effective in-tube SPME.

Other innovative extractive phases that enhance the affinity of the analytes include silica magnetite (SiO2-Fe3O4; **Figure 4F**) and poly (N-isopropylacrylamide; **Figure 4G**), which have been used in new microextraction processes involving magnetism and thermal energy, respectively. Magnetic and temperature controlled in-tube SPME are performed using flow-through systems, due to the need for additional equipment providing a magnetic or thermal field, which is easier to implement using flow-through devices. Other techniques include wire-in-tube SPME, using modified capillary columns with inserted stainless steel wires, and fiber-in-tube SPME, using PEEK tubes packed with fibrous rigid-rod heterocyclic polymers. These methods increase extraction efficiency by reducing capillary volume or increasing the extracting surface and have shown improved extraction efficiency when extended to microscale applications.

#### **3. Method development and characteristics of in-tube SPME**

#### **3.1 Optimization of in-tube SPME**

In-tube SPME depends on the distribution coefficient of each analyte. Extraction conditions may be optimized by increasing the distribution factor in the stationary phase. The selectivity and efficiency of extraction depend on the type of stationary phase and on the internal diameter, length, and film thickness of the capillary column. Sorption equilibrium is attained by optimizing various extraction parameters for each type of analyte. These parameters include extraction rate, sample volume, sample pH, flow-rate, number of draw/eject cycles (only draw/ eject system), and desorption conditions. As described in the preceding section, the choice of capillary coating is important for optimizing extraction selectivity and efficiency. Generally, low and high polarity columns selectively retain hydrophobic and hydrophilic compounds, respectively. Stationary phase consisting of a thicker film and longer column can extract larger amounts of compound, but quantitative desorption of compounds from capillary columns may be difficult. PLOT-type

**171**

**Table 1.**

*Online Automated Micro Sample Preparation for High-Performance Liquid Chromatography*

columns have a larger adsorption surface area and thicker film layer than liquid-

Generally, the optimal length and internal diameter of a capillary column used in combination with HPLC is 20–80 cm and 0.25 or 0.32 mm, respectively. Although thick-film capillaries often show higher sample capacity and extraction sensitivity, it is extremely difficult to reliably bind thicker chemical coatings to the inner surfaces of fused-silica capillary tubes using conventional approaches. In contrast, thin-film capillaries can minimize the time to reach extraction equilibrium due to their low sample capacity. Capillary columns with chemically bonded or cross-linked liquid phases are very stable in water and organic solvents and can

The volume of sample passed through a capillary is usually 0.2–2 mL in flowthrough extraction systems, and their optimum extraction flow rates are 0.25–4 mL/ min depending on the volume of the column. Although increases in the number and volume of draw/eject cycles can enhance extraction efficiency in draw/ejection systems, peak broadening is often observed [16]. Optimal conditions for a capillary column of inner diameter 0.25 mm and length 60 cm include a draw/ejection volume of 30–40 μL, a draw/ejection flow rate of 50–100 μL/min and 10–15 draw/ejection cycles. Below this rate, extractions require an inconveniently long time, and above this rate, bubbles form on the inside of the capillary, reducing extraction efficiency. Furthermore, the extraction efficiency of the analyte to the stationary phase varies with the pH of the sample solution. The presence of hydrophilic solvents such as methanol in the sample reduces the extraction efficiency. The analyte extracted on capillary coatings can be easily desorbed statically or dynamically without carryover [18].

**Table 1** summarizes the characteristics of in-tube SPME. The main advantage is that the series of processes can be automated, which enables continuous extraction,

> • Tendency of the capillary to clog • Limited to particulate-free samples • Stripping of non-bonding thick-film

• Switching of valves, extraction columns,

• Possible peak broadening

and pumps required • Complicated switching system • Relatively low enrichment factor • Relatively long extraction time

coatings

phase-type columns, enabling more analytes to be extracted [16, 18].

*DOI: http://dx.doi.org/10.5772/intechopen.89079*

prevent loss of phase by LC mobile phase [18].

**3.2 Characteristics of the in-tube SPME technique**

• Minimal sample adjustment

• Low solvent consumption

• Lower likelihood of carryover

and efficient extraction

• Large injection volume (flow-through system) • Applicable to polar and thermolabile liquid samples

• Decreased handling of biohazardous samples • Less sample loss due to online closed system

• Higher mechanical stability of capillaries

• Commercially available autosamplers • Improvements in selectivity and sensitivity

*Advantages and disadvantages of in-tube SPME.*

• Better precision and accuracy

• Reusability of capillaries without plugging or breaking • Commercially available GC capillary columns • Applicability of various unique adsorbents to specific

• Easy on-line coupling with liquid chromatography • Enabling of full automation by column switching

**Advantage Disadvantage**

#### *Online Automated Micro Sample Preparation for High-Performance Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.89079*

columns have a larger adsorption surface area and thicker film layer than liquidphase-type columns, enabling more analytes to be extracted [16, 18].

Generally, the optimal length and internal diameter of a capillary column used in combination with HPLC is 20–80 cm and 0.25 or 0.32 mm, respectively. Although thick-film capillaries often show higher sample capacity and extraction sensitivity, it is extremely difficult to reliably bind thicker chemical coatings to the inner surfaces of fused-silica capillary tubes using conventional approaches. In contrast, thin-film capillaries can minimize the time to reach extraction equilibrium due to their low sample capacity. Capillary columns with chemically bonded or cross-linked liquid phases are very stable in water and organic solvents and can prevent loss of phase by LC mobile phase [18].

The volume of sample passed through a capillary is usually 0.2–2 mL in flowthrough extraction systems, and their optimum extraction flow rates are 0.25–4 mL/ min depending on the volume of the column. Although increases in the number and volume of draw/eject cycles can enhance extraction efficiency in draw/ejection systems, peak broadening is often observed [16]. Optimal conditions for a capillary column of inner diameter 0.25 mm and length 60 cm include a draw/ejection volume of 30–40 μL, a draw/ejection flow rate of 50–100 μL/min and 10–15 draw/ejection cycles. Below this rate, extractions require an inconveniently long time, and above this rate, bubbles form on the inside of the capillary, reducing extraction efficiency. Furthermore, the extraction efficiency of the analyte to the stationary phase varies with the pH of the sample solution. The presence of hydrophilic solvents such as methanol in the sample reduces the extraction efficiency. The analyte extracted on capillary coatings can be easily desorbed statically or dynamically without carryover [18].

#### **3.2 Characteristics of the in-tube SPME technique**

**Advantage Disadvantage** • Minimal sample adjustment • Large injection volume (flow-through system) • Applicable to polar and thermolabile liquid samples • Low solvent consumption • Decreased handling of biohazardous samples • Less sample loss due to online closed system • Lower likelihood of carryover • Higher mechanical stability of capillaries • Reusability of capillaries without plugging or breaking • Commercially available GC capillary columns • Applicability of various unique adsorbents to specific and efficient extraction • Easy on-line coupling with liquid chromatography • Enabling of full automation by column switching • Commercially available autosamplers • Improvements in selectivity and sensitivity • Tendency of the capillary to clog • Limited to particulate-free samples • Stripping of non-bonding thick-film coatings • Possible peak broadening • Switching of valves, extraction columns, and pumps required • Complicated switching system • Relatively low enrichment factor • Relatively long extraction time

**Table 1** summarizes the characteristics of in-tube SPME. The main advantage is that the series of processes can be automated, which enables continuous extraction,

#### **Table 1.**

*Advantages and disadvantages of in-tube SPME.*

• Better precision and accuracy

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

developed for effective in-tube SPME.

**3.1 Optimization of in-tube SPME**

efficiency when extended to microscale applications.

**3. Method development and characteristics of in-tube SPME**

In-tube SPME depends on the distribution coefficient of each analyte. Extraction conditions may be optimized by increasing the distribution factor in the stationary phase. The selectivity and efficiency of extraction depend on the type of stationary phase and on the internal diameter, length, and film thickness of the capillary column. Sorption equilibrium is attained by optimizing various extraction parameters for each type of analyte. These parameters include extraction rate, sample volume, sample pH, flow-rate, number of draw/eject cycles (only draw/ eject system), and desorption conditions. As described in the preceding section, the choice of capillary coating is important for optimizing extraction selectivity and efficiency. Generally, low and high polarity columns selectively retain hydrophobic and hydrophilic compounds, respectively. Stationary phase consisting of a thicker film and longer column can extract larger amounts of compound, but quantitative desorption of compounds from capillary columns may be difficult. PLOT-type

MIPs recognize chemicals through combination of shape, hydrogen bonding, and hydrophobic and electrostatic interactions [16, 18, 31]. RAM materials possess defined diffusion barriers with small sized pores and biocompatible outer particle surfaces (**Figure 4B**). The bifunctionality of ADS particles used as a RAM SPME device can prevent fouling of the capillary by protein adsorption while simultaneously trapping the analytes in the hydrophobic porous interior. Furthermore, a simple SPME device has been fabricated for use in online immunoaffinity capillaries packed with immunosorbent materials, consisting of covalently immobilized antibodies (**Figure 4C**). An alternative approach consists of in-tube SPME using monolithic capillary columns comprised of one piece of organic polymer or silica rods with a unique flow-through double-pore structure (**Figure 4D**). Monoliths are also highly permeable to liquids and biological samples, enabling reduced solvent use, varied support formats, and/or automation. Monolithic capillaries are especially suitable for intube SPME media due to the low pressure drop, allowing a high flow-rate to achieve high throughput and a total porosity greater than that of particle-packed capillaries. Hydrophobic main chains and acidic pendant groups of poly (methacrylic acidethylene glycol dimethacrylate) enhance the ability to extract basic analytes from aqueous matrices. The physicochemical properties of graphene-based sorbents and carbon nanotubes (**Figure 4E**) enable their use in extraction, with these combinations showing excellent results when used for in-tube SPME. In addition, various cationic, anionic and zwitterionic liquid-mediated sol–gel coatings have been

Other innovative extractive phases that enhance the affinity of the analytes include silica magnetite (SiO2-Fe3O4; **Figure 4F**) and poly (N-isopropylacrylamide; **Figure 4G**), which have been used in new microextraction processes involving magnetism and thermal energy, respectively. Magnetic and temperature controlled in-tube SPME are performed using flow-through systems, due to the need for additional equipment providing a magnetic or thermal field, which is easier to implement using flow-through devices. Other techniques include wire-in-tube SPME, using modified capillary columns with inserted stainless steel wires, and fiber-in-tube SPME, using PEEK tubes packed with fibrous rigid-rod heterocyclic polymers. These methods increase extraction efficiency by reducing capillary volume or increasing the extracting surface and have shown improved extraction

**170**

desorption and injection with column switching using a standard autosampler, and online coupling with the LC system [16, 18, 31]. In-tube SPME may be suitable for the determination of polar and thermolabile compounds. Compared with manual techniques, automated sample-handling procedures not only shorten the total analysis time but are more accurate and precise. Automated techniques are also suitable for miniaturization, high-throughput performance, and online coupling with analytical instruments, and reduce the consumption of solvent. Online procedures can limit contact with dirty and hazardous samples, reducing sample contamination and loss. Online column-switching systems are highly sensitive due to pre-concentration resulting from the injection of large sample volumes into the extraction support without loss of chromatographic performance. The main disadvantage is that the capillaries tend to clog, which may be avoided by removing interfering phases such as particles or macromolecules by filtration or centrifugation before extraction. Although the absolute recovery rate of the in-tube SPME method is generally low, it can be extracted and concentrated reproducibly using an autosampler, and all extracts can be introduced into the LC column [16, 18, 31].

The online in-tube SPME method can be applied to polar and nonpolar compounds in liquid samples, and can be coupled with various analytical methods, such as HPLC and LC–MS. Early applications of online in-tube SPME have involved draw/eject extraction systems and commercially available open-tubular GC capillaries such as Supel Q PLOT and Carboxen 1006 PLOT capillaries. The subsequent development of various operating systems and new sorbent materials improved extraction efficiency, such as sorption capacity and selectivity, and extended the range of applications. Last decade, numerous applications of online in-tube SPME methods have been reported to many types of pharmaceutical and biomedical [86, 89–124], food [125–137], and environmental [138–178] analyses.

#### **4. Conclusions and future directions**

The online in-tube SPME techniques described in this chapter have many desirable features for automated separation of analytes, using column-switching techniques. These methods are especially well suited to the analysis of samples requiring significant cleanup and concentration to improve their selectivity and sensitivity, as well as being useful for high-throughput sampling. Since the in-tube SPME method using capillaries as an extraction device is useful for online sample preparation to extract and concentrate polar and non-polar compounds from aqueous solution, it has become an effective technique for convenient analysis of a wide variety of compounds in complex matrices such as biological, pharmaceutical, food and environmental samples [31]. Furthermore, various operating systems and new sorbent materials have been developed to improve extraction efficiency and sorption capacity and selectivity, and to extend the range of applications. These include MIPs, RAM, immunosorbents, monolithic materials, carbon nanoparticles, ionic liquids, temperature responsive polymers and magnetic hybrid adsorbents.

The main future direction in sample preparation is the development of more sensitive and selective extraction sorbents [31]. Chiral active phases, ionic liquids, dendrimers, aptamer modified sorbents, magnetic materials, temperature responsive materials may be available as new polymer devices for effective sample preparation. Furthermore, biomimetic coating materials including ultrasound and light responsive polymers may be available as a selective extraction device in the future. These customized coating materials, differing in type, shape, and size, are expected to result in highly efficient extraction of various samples. Biocompatible RAM and monolithic sorbents are useful for direct analysis, without pre-treatment other than

**173**

**Author details**

Hiroyuki Kataoka\*, Atsushi Ishizaki and Keita Saito

provided the original work is properly cited.

The authors declare no conflict of interest.

\*Address all correspondence to: hkataoka@shujitsu.ac.jp

School of Pharmacy, Shujitsu University, Nishigawara, Okayama, Japan

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Online Automated Micro Sample Preparation for High-Performance Liquid Chromatography*

dilution and centrifugation of biological samples. As another future direction, better integration of sampling/sample preparation and instrumental analysis will allow wider use of automated online analysis. Especially, the use of column-switching systems involving microextraction techniques and/or microdevices will offer convenient integration of sample preparation with various analytical instruments such as HPLC as well as other chromatographic systems, electrophoresis, direct MS, etc. Finally, this chapter provides an overview of the configurations and characteristics of in-tube SPME technology for online automated micro sample preparation for HPLC. We hope that this chapter will serve as a guide to choosing the most effective

sample preparation techniques for the analysis of various complex samples.

This work was supported by a Grant-in-Aid for Basic Scientific Research (C, No.

*DOI: http://dx.doi.org/10.5772/intechopen.89079*

**Acknowledgements**

**Conflict of interest**

17 K08259).

*Online Automated Micro Sample Preparation for High-Performance Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.89079*

dilution and centrifugation of biological samples. As another future direction, better integration of sampling/sample preparation and instrumental analysis will allow wider use of automated online analysis. Especially, the use of column-switching systems involving microextraction techniques and/or microdevices will offer convenient integration of sample preparation with various analytical instruments such as HPLC as well as other chromatographic systems, electrophoresis, direct MS, etc.

Finally, this chapter provides an overview of the configurations and characteristics of in-tube SPME technology for online automated micro sample preparation for HPLC. We hope that this chapter will serve as a guide to choosing the most effective sample preparation techniques for the analysis of various complex samples.

#### **Acknowledgements**

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

desorption and injection with column switching using a standard autosampler, and online coupling with the LC system [16, 18, 31]. In-tube SPME may be suitable for the determination of polar and thermolabile compounds. Compared with manual techniques, automated sample-handling procedures not only shorten the total analysis time but are more accurate and precise. Automated techniques are also suitable for miniaturization, high-throughput performance, and online coupling with analytical instruments, and reduce the consumption of solvent. Online procedures can limit contact with dirty and hazardous samples, reducing sample contamination and loss. Online column-switching systems are highly sensitive due to pre-concentration resulting from the injection of large sample volumes into the extraction support without loss of chromatographic performance. The main disadvantage is that the capillaries tend to clog, which may be avoided by removing interfering phases such as particles or macromolecules by filtration or centrifugation before extraction. Although the absolute recovery rate of the in-tube SPME method is generally low, it can be extracted and concentrated reproducibly using an autosampler, and all extracts can be introduced into the LC column [16, 18, 31]. The online in-tube SPME method can be applied to polar and nonpolar compounds in liquid samples, and can be coupled with various analytical methods, such as HPLC and LC–MS. Early applications of online in-tube SPME have involved draw/eject extraction systems and commercially available open-tubular GC capillaries such as Supel Q PLOT and Carboxen 1006 PLOT capillaries. The subsequent development of various operating systems and new sorbent materials improved extraction efficiency, such as sorption capacity and selectivity, and extended the range of applications. Last decade, numerous applications of online in-tube SPME methods have been reported to many types of pharmaceutical and biomedical [86,

89–124], food [125–137], and environmental [138–178] analyses.

The online in-tube SPME techniques described in this chapter have many desirable features for automated separation of analytes, using column-switching techniques. These methods are especially well suited to the analysis of samples requiring significant cleanup and concentration to improve their selectivity and sensitivity, as well as being useful for high-throughput sampling. Since the in-tube SPME method using capillaries as an extraction device is useful for online sample preparation to extract and concentrate polar and non-polar compounds from aqueous solution, it has become an effective technique for convenient analysis of a wide variety of compounds in complex matrices such as biological, pharmaceutical, food and environmental samples [31]. Furthermore, various operating systems and new sorbent materials have been developed to improve extraction efficiency and sorption capacity and selectivity, and to extend the range of applications. These include MIPs, RAM, immunosorbents, monolithic materials, carbon nanoparticles, ionic liquids, temperature responsive polymers and magnetic hybrid adsorbents.

The main future direction in sample preparation is the development of more sensitive and selective extraction sorbents [31]. Chiral active phases, ionic liquids, dendrimers, aptamer modified sorbents, magnetic materials, temperature responsive materials may be available as new polymer devices for effective sample preparation. Furthermore, biomimetic coating materials including ultrasound and light responsive polymers may be available as a selective extraction device in the future. These customized coating materials, differing in type, shape, and size, are expected to result in highly efficient extraction of various samples. Biocompatible RAM and monolithic sorbents are useful for direct analysis, without pre-treatment other than

**4. Conclusions and future directions**

**172**

This work was supported by a Grant-in-Aid for Basic Scientific Research (C, No. 17 K08259).

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Hiroyuki Kataoka\*, Atsushi Ishizaki and Keita Saito School of Pharmacy, Shujitsu University, Nishigawara, Okayama, Japan

\*Address all correspondence to: hkataoka@shujitsu.ac.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[111] Saito A, Hamano M, Kataoka H. Simultaneous analysis of multiple urinary biomarkers for the evaluation of oxidative stress by automated online in-tube solidphase microextraction coupled with negative/positive ion-switching mode liquid chromatography-tandem mass spectrometry. Journal of Separation Science. 2018;**41**:2743-2749

[112] Yasuhara R, Ehara K, Saito K, Kataoka H. Automated analysis of salivary stress-related steroid hormones by online in-tube solidphase microextraction coupled with liquid chromatography−tandem mass spectrometry. Analytical Methods. 2012;**4**:3625-3630

[113] Kataoka H, Ehara K, Yasuhara R, Saito K. Simultaneous determination of testosterone, cortisol, and dehydroepiandrosterone in saliva by stable isotope dilution on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry. Analytical and Bioanalytical Chemistry. 2013;**405**:331-340

[114] Moriyama E, Kataoka H. Automated analysis of oxytocin by on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry. Chromatography. 2015;**2**:382-391

[115] Ishizaki A, Uemura A, Kataoka H. A sensitive method to determine melatonin in saliva by automated online in-tube solid-phase microextraction coupled with stable isotope-dilution liquid chromatography-tandem mass spectrometry. Analytical Methods. 2017;**9**:3134-3140

[116] Kataoka H, Inoue T, Saito K, Kato H, Masuda K. Analysis of heterocyclic amines in hair by on-line in-tube solidphase microextraction coupled with liquid chromatography-tandem mass spectrometry. Analytica Chimica Acta. 2013;**786**:54-60

[117] Yamamoto Y, Ishizaki A, Kataoka H. Biomonitoring method for the determination of polycyclic aromatic hydrocarbons in hair by online in-tube solid-phase microextraction coupled with high performance liquid chromatography and fluorescence detection. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 2015;**1000**:187-191

[118] Inukai T, Kaji S, Kataoka H. Analysis of nicotine and cotinine in hair by on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry as biomarkers of exposure to tobacco smoke. Journal of Pharmaceutical and Biomedical Analysis. 2018;**156**:272-277

[119] Chen D, Ding J, Wu MK, Zhang TY, Qi CB, Feng YQ. A liquid chromatography-mass spectrometry method based on post column derivatization for automated analysis of urinary hexanal and heptanal. Journal of Chromatography. A. 2017;**1493**:57-63

[120] Ying LL, Ma YC, Xu B, Wang XH, Dong LY, Wang DM, et al. Poly(glycidyl methacrylate) nanoparticle-coated capillary with orientedantibody immobilization for immunoaffinity in-tube solid phase micro-extraction: Preparation and characterization. Journal of Chromatography. A. 2017;**1509**:1-8

[121] Hakobyan L, Tolos JP, Moliner-Martinez Y, Molins-Legua C, Ramos JR, Gordon M, et al. Determination of meropenem in endotracheal tubes by in-tube solid phase microextraction coupled to capillary liquid chromatography with diode array detection. Journal of Pharmaceutical and Biomedical Analysis. 2018;**151**:170-177

[122] Wang S, Hu S, Xu H. Analysis of aldehydes in human exhaled breath condensates by in-tube SPME-HPLC. Analytica Chimica Acta. 2015;**900**:67-75

[123] Li Y, Xu H. Development of a novel graphene/polyaniline electrodeposited coating for on-line in-tube solid phase microextraction of aldehydes in human exhaled breath condensate. Journal of Chromatography. A. 2015;**1395**:23-31

[124] Hu Y, Song C, Li G. Fiber-in-tube solid-phase microextraction with molecularly imprinted coating for sensitive analysis of antibiotic drugs by high performance liquid chromatography. Journal of Chromatography. A. 2012;**1263**:21-27

[125] Wang J, Zhao Q, Jiang N, Li W, Chen L, Lin X, et al. Urea-formaldehyde monolithic column for hydrophilic in-tube solid-phase microextraction of aminoglycosides. The Journal of Chromatography A. 2017;**1485**:24-31

[126] Pang J, Yuan D, Huang X. On-line combining monolith-based in-tube solid phase microextraction and highperformance liquid chromatographyfluorescence detection for the sensitive monitoring of polycyclic aromatic hydrocarbons in complex samples. Journal of Chromatography. A. 2018;**1571**:29-37

[127] Wang J, Jiang N, Cai Z, Li W, Li J, Lin X, et al. Sodium hyaluronatefunctionalized urea-formaldehyde monolithic column for hydrophilic in-tube solid-phase microextraction of melamine. Journal of Chromatography. A. 2017;**1515**:54-61

[128] Wu F, Wang J, Zhao Q, Jiang N, Lin X, Xie Z, et al. Detection of transfatty acids by high performance liquid chromatography coupled with in-tube solid-phase microextraction using hydrophobic polymeric monolith. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 2017;**1040**:214-221

[129] Wang TT, Chen YH, Ma JF, Hu MJ, Li Y, Fang JH, et al. A novel ionic liquidmodified organic-polymer monolith as the sorbent for in-tube solid-phase microextraction of acidic food additives. Analytical and Bioanalytical Chemistry. 2014;**406**:4955-4963

[130] Asiabi H, Yamini Y, Seidi S, Esrafili A, Rezaei F. Electroplating of nanostructured polyaniline-polypyrrole composite coating in a stainlesssteel tube for on-line in-tube solid phase microextraction. Journal of Chromatography. A. 2015;**1397**:19-26

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[138] Poorahong S, Thammakhet C, Thavarungkul P, Kanatharana P. Online

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[140] González-Fuenzalida RA, López-García E, Moliner-Martínez Y, Campíns-Falcó P. Adsorbent phases with nanomaterials for in-tube solidphase microextraction coupled on-line to liquid nanochromatography. Journal of Chromatography. A. 2016;**1432**:17-25

[141] Saito K, Uemura E, Ishizaki A, Kataoka H. Determination of perfluorooctanoic acid and

[142] Vitta Y, Moliner-Martínez Y, Campíns-Falcó P, Cuervo AF. An in-tube SPME device for the selective

[143] Bagheri H, Piri-Moghadam H, Es'haghi A. An unbreakable on-line approach towards solgel capillary microextraction. Journal of Chromatography. A.

determination of chlorophyll a in aquatic systems. Talanta.

Acta. 2010;**658**:141-146

2010;**82**:952-956

2011;**1218**:3952-3957

perfluorooctane sulfonate by automated in-tube solid-phase microextraction coupled with liquid chromatographymass spectrometry. Analytica Chimica

in-tube microextractor coupled with UV-vis spectrophotometer for bisphenol A detection. Journal of Environmental Science and Health. Part A, Toxic/Hazardous Substances and Environmental Engineering.

2013;**1317**:121-128

2013;**48**:242-250

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of polycyclic aromatic hydrocarbons in food samples by automated on-line in-tube solid-phase microextraction coupled with high-performance liquid chromatography-fluorescence detection. Journal of Chromatography.

[132] Ishizaki A, Saito K, Kataoka H. Analysis of polycyclic aromatic hydrocarbons contamination in tea products and crude drugs. Analytical

[133] Ying LL, Wang DY, Yang HP, Deng XY, Peng C, Zheng C, et al. Synthesis of boronate-decorated polyethyleneimine-grafted porous layer open tubular capillaries for enrichment of polyphenols in fruit juices. Journal of Chromatography. A.

[134] Andrade MA, Lanças FM. Determination of ochratoxin A in wine by packed in-tube solid phase microextraction followed by high performance liquid chromatography coupled to tandem mass spectrometry.

Journal of Chromatography. A.

[135] Saito K, Ikeuchi R, Kataoka H. Determination of ochratoxins in nuts and grain samples by in-tube solidphase microextraction coupled with liquid chromatographymass spectrometry. Journal of Chromatography. A. 2012;**1220**:1-6

[136] Wu F, Xu C, Jiang N, Wang J, Ding CF. Poly (methacrylic acidco-diethenyl-benzene) monolithic microextraction column and its

application to simultaneous enrichment and analysis of mycotoxins. Talanta.

[137] Wang X, Ma Q, Li M, Chang C, Bai Y, Feng Y, et al. Automated and sensitive analysis of

28-epihomobrassinolide in Arabidopsis

A. 2010;**1217**:5555-5563

Methods. 2011;**3**:299-305

2018;**1544**:23-32

2017;**1493**:41-48

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of polycyclic aromatic hydrocarbons in food samples by automated on-line in-tube solid-phase microextraction coupled with high-performance liquid chromatography-fluorescence detection. Journal of Chromatography. A. 2010;**1217**:5555-5563

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

monolithic column for hydrophilic in-tube solid-phase microextraction of aminoglycosides. The Journal of Chromatography A. 2017;**1485**:24-31

[126] Pang J, Yuan D, Huang X. On-line combining monolith-based in-tube solid phase microextraction and highperformance liquid chromatographyfluorescence detection for the sensitive monitoring of polycyclic aromatic hydrocarbons in complex samples. Journal of Chromatography. A.

[127] Wang J, Jiang N, Cai Z, Li W, Li J, Lin X, et al. Sodium hyaluronatefunctionalized urea-formaldehyde monolithic column for hydrophilic in-tube solid-phase microextraction of melamine. Journal of Chromatography.

[128] Wu F, Wang J, Zhao Q, Jiang N, Lin X, Xie Z, et al. Detection of transfatty acids by high performance liquid chromatography coupled with in-tube solid-phase microextraction using hydrophobic polymeric monolith. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences.

[129] Wang TT, Chen YH, Ma JF, Hu MJ, Li Y, Fang JH, et al. A novel ionic liquidmodified organic-polymer monolith as the sorbent for in-tube solid-phase microextraction of acidic food additives. Analytical and Bioanalytical Chemistry.

2018;**1571**:29-37

A. 2017;**1515**:54-61

2017;**1040**:214-221

2014;**406**:4955-4963

[130] Asiabi H, Yamini Y, Seidi S, Esrafili A, Rezaei F. Electroplating of nanostructured polyaniline-polypyrrole

composite coating in a stainlesssteel tube for on-line in-tube solid phase microextraction. Journal of Chromatography. A. 2015;**1397**:19-26

[131] Ishizaki A, Saito K, Hanioka N, Narimatsu S, Kataoka H. Determination

[119] Chen D, Ding J, Wu MK, Zhang TY, Qi CB, Feng YQ. A liquid chromatography-mass spectrometry method based on post column derivatization for automated analysis of urinary hexanal and heptanal. Journal of Chromatography. A. 2017;**1493**:57-63

2017;**1509**:1-8

[121] Hakobyan L, Tolos JP,

Ramos JR, Gordon M, et al. Determination of meropenem in endotracheal tubes by in-tube solid phase microextraction coupled to capillary liquid chromatography with diode array detection. Journal of Pharmaceutical and Biomedical

Analysis. 2018;**151**:170-177

2015;**900**:67-75

[122] Wang S, Hu S, Xu H. Analysis of aldehydes in human exhaled breath condensates by in-tube SPME-HPLC. Analytica Chimica Acta.

[123] Li Y, Xu H. Development of a novel graphene/polyaniline electrodeposited coating for on-line in-tube solid phase microextraction of aldehydes in human exhaled breath condensate. Journal of Chromatography. A. 2015;**1395**:23-31

[124] Hu Y, Song C, Li G. Fiber-in-tube solid-phase microextraction with molecularly imprinted coating for sensitive analysis of antibiotic drugs by high performance liquid chromatography.

Journal of Chromatography. A.

[125] Wang J, Zhao Q, Jiang N, Li W, Chen L, Lin X, et al. Urea-formaldehyde

2012;**1263**:21-27

Moliner-Martinez Y, Molins-Legua C,

[120] Ying LL, Ma YC, Xu B, Wang XH, Dong LY, Wang DM, et al. Poly(glycidyl methacrylate) nanoparticle-coated capillary with orientedantibody immobilization for immunoaffinity in-tube solid phase micro-extraction: Preparation and characterization. Journal of Chromatography. A.

**182**

[132] Ishizaki A, Saito K, Kataoka H. Analysis of polycyclic aromatic hydrocarbons contamination in tea products and crude drugs. Analytical Methods. 2011;**3**:299-305

[133] Ying LL, Wang DY, Yang HP, Deng XY, Peng C, Zheng C, et al. Synthesis of boronate-decorated polyethyleneimine-grafted porous layer open tubular capillaries for enrichment of polyphenols in fruit juices. Journal of Chromatography. A. 2018;**1544**:23-32

[134] Andrade MA, Lanças FM. Determination of ochratoxin A in wine by packed in-tube solid phase microextraction followed by high performance liquid chromatography coupled to tandem mass spectrometry. Journal of Chromatography. A. 2017;**1493**:41-48

[135] Saito K, Ikeuchi R, Kataoka H. Determination of ochratoxins in nuts and grain samples by in-tube solidphase microextraction coupled with liquid chromatographymass spectrometry. Journal of Chromatography. A. 2012;**1220**:1-6

[136] Wu F, Xu C, Jiang N, Wang J, Ding CF. Poly (methacrylic acidco-diethenyl-benzene) monolithic microextraction column and its application to simultaneous enrichment and analysis of mycotoxins. Talanta. 2018;**178**:1-8

[137] Wang X, Ma Q, Li M, Chang C, Bai Y, Feng Y, et al. Automated and sensitive analysis of 28-epihomobrassinolide in Arabidopsis thaliana by on-line polymer monolith microextraction coupled to liquid chromatography-mass spectrometry. Journal of Chromatography. A. 2013;**1317**:121-128

[138] Poorahong S, Thammakhet C, Thavarungkul P, Kanatharana P. Online in-tube microextractor coupled with UV-vis spectrophotometer for bisphenol A detection. Journal of Environmental Science and Health. Part A, Toxic/Hazardous Substances and Environmental Engineering. 2013;**48**:242-250

[139] Feng J, Wang X, Tian Y, Luo C, Sun M. Basalt fibers grafted with a poly(ionic liquids) coating for in-tube solid-phase microextraction. Journal of Separation Science. 2018;**41**:3267-3274

[140] González-Fuenzalida RA, López-García E, Moliner-Martínez Y, Campíns-Falcó P. Adsorbent phases with nanomaterials for in-tube solidphase microextraction coupled on-line to liquid nanochromatography. Journal of Chromatography. A. 2016;**1432**:17-25

[141] Saito K, Uemura E, Ishizaki A, Kataoka H. Determination of perfluorooctanoic acid and perfluorooctane sulfonate by automated in-tube solid-phase microextraction coupled with liquid chromatographymass spectrometry. Analytica Chimica Acta. 2010;**658**:141-146

[142] Vitta Y, Moliner-Martínez Y, Campíns-Falcó P, Cuervo AF. An in-tube SPME device for the selective determination of chlorophyll a in aquatic systems. Talanta. 2010;**82**:952-956

[143] Bagheri H, Piri-Moghadam H, Es'haghi A. An unbreakable on-line approach towards solgel capillary microextraction. Journal of Chromatography. A. 2011;**1218**:3952-3957

[144] Moliner-Martínez Y, Molins-Legua C, Verdú-Andrés J, Herráez-Hernández R, Campíns-Falcó P. Advantages of monolithic over particulate columns for multiresidue analysis of organic pollutants by in-tube solid-phase microextraction coupled to capillary liquid chromatography. Journal of Chromatography. A. 2011;**1218**:6256-6262

[145] Prieto-Blanco MC, Moliner-Martínez Y, López-Mahía P, Campíns-Falcó P. Ion-pair in-tube solid-phase microextraction and capillary liquid chromatography using a titania-based column: Application to the specific lauralkonium chloride determination in water. Journal of Chromatography. A. 2012;**1248**:55-59

[146] Kataoka H, Shiba H, Saito K. Automated analysis of oseltamivir and oseltamivir carboxylate in environmental water samples by online in-tube solid-phase microextraction coupled with liquid chromatography− tandem mass spectrometry. Analytical Methods. 2012;**4**:1513-1518

[147] Prieto-Blanco MC, Moliner-Martinez Y, Campíns-Falcó P. Combining poly(dimethyldiphenylsiloxane) and nitrile phases for improving the separation and quantitation of benzalkonium chloride homologues: In-tube solid phase microextraction-capillary liquid chromatography-diode array detection-mass spectrometry for analyzing industrial samples. Journal of Chromatography. A. 2013;**1297**:226-230

[148] Masiá A, Moliner-Martinez Y, Muñoz-Ortuño M, Pico Y, Campíns-Falcó P. Multiresidue analysis of organic pollutants by in-tube solid phase microextraction coupled to ultra-high performance liquid chromatography-electrospray-tandem mass spectrometry. Journal of Chromatography. A. 2013;**1306**:1-11

[149] Prieto-Blanco MC, Moliner-Martínez Y, López-Mahía P, Campíns-Falcó P. Determination of carbonyl compounds in particulate matter PM2.5 by in-tube solidphase microextraction coupled to capillary liquid chromatography/mass spectrometry. Talanta. 2013;**115**:876-880

[150] Moliner-Martinez Y, Vitta Y, Prima-Garcia H, González-Fuenzalida RA, Ribera A, Campíns-Falcó P, et al. Silica supported Fe3O4 magnetic nanoparticles for magnetic solid-phase extraction and magnetic in-tube solid-phase microextraction: Application to organophosphorous compounds. Analytical and Bioanalytical Chemistry. 2014;**406**:2211-2215

[151] González-Fuenzalida RA, Moliner-Martínez Y, Prima-Garcia H, Ribera A, Campins-Falcó P, Zaragozá RJ. Evaluation of superparamagnetic silica nanoparticles for extraction of triazines in magnetic in-tube solid phase microextraction coupled to capillary liquid chromatography. Nanomaterials. 2014;**4**:242-255

[152] Ahmadi SH, Manbohi A, Heydar KT. Electrochemically controlled in-tube solid phase microextraction. Analytica Chimica Acta. 2015;**853**:335-341

[153] Moliner-Martínez Y, Serra-Mora P, Verdú-Andrés J, Herráez-Hernández R, Campíns-Falcó P. Analysis of polar triazines and degradation products in waters by in-tube solid-phase microextraction and capillary chromatography: An environmentally friendly method. Analytical and Bioanalytical Chemistry. 2015;**407**:1485-1497

[154] Zhang J, Zhang W, Bao T, Chen Z. Polydopamine-based immobilization of zeolitic imidazolate framework-8 for in-tube solid-phase microextraction.

**185**

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[161] Bu Y, Feng J, Sun M, Zhou C, Luo C. Gold-functionalized stainlesssteel wire and tube for fiber-intube solid-phase microextraction coupled to high-performance liquid chromatography for the determination of polycyclic aromatic hydrocarbons. Journal of Separation Science.

[162] Sun M, Feng J, Bu Y, Luo C. Ionic liquid coated copper wires and tubes for fiber-in-tube solidphase microextraction. Journal of Chromatography. A. 2016;**1458**:1-8

[163] Bu Y, Feng J, Tian Y, Wang X, Sun M, Luo C. An organically modified silica aerogel for online in-tube solidphase microextraction. Journal of Chromatography. A. 2017;**1517**:203-208

[164] Bu Y, Feng J, Wang X, Tian Y, Sun M, Luo C. In situ hydrothermal growth of polyaniline coating for in-tube solid-phase microextraction

environmental water samples. Journal of Chromatography. A. 2017;**1483**:48-55

[165] Mei M, Huang X. Online analysis of five organic ultraviolet filters in environmental water samples using magnetism-enhanced monolith-based in-tube solid phase microextraction coupled with high-performance liquid chromatography. Journal of Chromatography. A. 2017;**1525**:1-9

[166] Serra-Mora P, Jornet-Martinez N, Moliner-Martinez Y, Campíns-Falcó P. In tube-solid phase microextractionnano liquid chromatography: Application to the determination of intact and degraded polar triazines in waters and recovered struvite. Journal of Chromatography. A. 2017;**1513**:51-58

[167] Wang X, Pan L, Feng J, Tian Y, Luo C, Sun M. Silk fiber for in-tube solid-phase microextraction to detect aldehydes by chemical derivatization.

towards ultraviolet filters in

2016;**39**:932-938

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Journal of Chromatography. A.

[155] Pla-Tolós J, Moliner-Martínez Y, Molins-Legua C, Herráez-Hernández R, Verdú-Andrés J, Campíns-Falcó P. Selective and sentivive method based on capillary liquid chromatography with in-tube solid phase microextraction for determination of monochloramine in water. Journal of Chromatography. A.

[156] Tan F, Zhao C, Li L, Liu M, He X, Gao J. Graphene oxide based in-tube solid-phase microextraction combined with liquid chromatography tandem mass spectrometry for the determination of triazine herbicides in water. Journal of Separation Science. 2015;**38**:2312-2319

[157] Sun M, Feng J, Bu Y, Luo C. Nanostructured-silver-coated polyetheretherketone tube for online in-tube solid-phase microextraction coupled with high-performance liquid chromatography. Journal of Separation

Science. 2015;**38**:3239-3246

[159] Feng J, Sun M, Bu Y, Luo C. Development of a cheap and accessible carbon fibers-in-poly(ether ether ketone) tube with high stability for online in-tube solid-phase microextraction. Talanta.

[160] Bu Y, Feng J, Sun M, Zhou C, Luo C. Facile and efficient poly(ethylene terephthalate) fibers-in-tube for online solid-phase microextraction towards polycyclic aromatic hydrocarbons. Analytical and Bioanalytical Chemistry.

2015;**1408**:41-48

2016;**148**:313-320

2016;**408**:4871-4882

[158] Sun M, Feng J, Bu Y, Luo C. Highly sensitive copper fiber-in-tube solidphase microextraction for online selective analysis of polycyclic aromatic hydrocarbons coupled with high performance liquid chromatography. Journal of Chromatography. A.

2015;**1388**:9-16

2015;**1388**:17-23

*Online Automated Micro Sample Preparation for High-Performance Liquid Chromatography DOI: http://dx.doi.org/10.5772/intechopen.89079*

Journal of Chromatography. A. 2015;**1388**:9-16

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

[149] Prieto-Blanco MC,

Moliner-Martínez Y, López-Mahía P, Campíns-Falcó P. Determination of carbonyl compounds in particulate matter PM2.5 by in-tube solidphase microextraction coupled to capillary liquid chromatography/mass spectrometry. Talanta. 2013;**115**:876-880

[150] Moliner-Martinez Y, Vitta Y, Prima-Garcia H, González-Fuenzalida RA, Ribera A, Campíns-Falcó P, et al. Silica supported Fe3O4 magnetic nanoparticles for magnetic solid-phase extraction and magnetic in-tube solid-phase microextraction: Application to organophosphorous compounds. Analytical and Bioanalytical Chemistry.

2014;**406**:2211-2215

[151] González-Fuenzalida RA, Moliner-Martínez Y, Prima-Garcia H,

superparamagnetic silica nanoparticles for extraction of triazines in magnetic in-tube solid phase microextraction

Heydar KT. Electrochemically controlled in-tube solid phase microextraction.

[153] Moliner-Martínez Y, Serra-Mora P, Verdú-Andrés J, Herráez-Hernández R, Campíns-Falcó P. Analysis of polar triazines and degradation products in waters by in-tube solid-phase microextraction and capillary

chromatography: An environmentally

[154] Zhang J, Zhang W, Bao T, Chen Z. Polydopamine-based immobilization of zeolitic imidazolate framework-8 for in-tube solid-phase microextraction.

friendly method. Analytical and Bioanalytical Chemistry.

2015;**407**:1485-1497

Ribera A, Campins-Falcó P, Zaragozá RJ. Evaluation of

coupled to capillary liquid chromatography. Nanomaterials.

[152] Ahmadi SH, Manbohi A,

Analytica Chimica Acta.

2014;**4**:242-255

2015;**853**:335-341

[144] Moliner-Martínez Y, Molins-Legua C, Verdú-Andrés J, Herráez-Hernández R, Campíns-Falcó P. Advantages of monolithic over particulate columns for multiresidue analysis of organic pollutants by in-tube solid-phase microextraction coupled to capillary liquid chromatography. Journal of Chromatography. A.

2011;**1218**:6256-6262

[145] Prieto-Blanco MC,

Moliner-Martínez Y, López-Mahía P, Campíns-Falcó P. Ion-pair in-tube solid-phase microextraction and capillary liquid chromatography using a titania-based column: Application to the specific lauralkonium chloride determination in water. Journal of Chromatography. A. 2012;**1248**:55-59

[146] Kataoka H, Shiba H, Saito K. Automated analysis of oseltamivir and oseltamivir carboxylate in

Methods. 2012;**4**:1513-1518

[147] Prieto-Blanco MC, Moliner-Martinez Y, Campíns-Falcó P. Combining poly(dimethyl-

diphenylsiloxane) and nitrile phases for improving the separation and quantitation of benzalkonium chloride homologues: In-tube solid phase microextraction-capillary liquid chromatography-diode array detection-mass spectrometry for analyzing industrial samples. Journal of Chromatography. A. 2013;**1297**:226-230

[148] Masiá A, Moliner-Martinez Y,

Campíns-Falcó P. Multiresidue analysis of organic pollutants by in-tube solid phase microextraction coupled to ultra-high performance liquid chromatography-electrospray-tandem

Muñoz-Ortuño M, Pico Y,

mass spectrometry. Journal of Chromatography. A. 2013;**1306**:1-11

environmental water samples by online in-tube solid-phase microextraction coupled with liquid chromatography− tandem mass spectrometry. Analytical

**184**

[155] Pla-Tolós J, Moliner-Martínez Y, Molins-Legua C, Herráez-Hernández R, Verdú-Andrés J, Campíns-Falcó P. Selective and sentivive method based on capillary liquid chromatography with in-tube solid phase microextraction for determination of monochloramine in water. Journal of Chromatography. A. 2015;**1388**:17-23

[156] Tan F, Zhao C, Li L, Liu M, He X, Gao J. Graphene oxide based in-tube solid-phase microextraction combined with liquid chromatography tandem mass spectrometry for the determination of triazine herbicides in water. Journal of Separation Science. 2015;**38**:2312-2319

[157] Sun M, Feng J, Bu Y, Luo C. Nanostructured-silver-coated polyetheretherketone tube for online in-tube solid-phase microextraction coupled with high-performance liquid chromatography. Journal of Separation Science. 2015;**38**:3239-3246

[158] Sun M, Feng J, Bu Y, Luo C. Highly sensitive copper fiber-in-tube solidphase microextraction for online selective analysis of polycyclic aromatic hydrocarbons coupled with high performance liquid chromatography. Journal of Chromatography. A. 2015;**1408**:41-48

[159] Feng J, Sun M, Bu Y, Luo C. Development of a cheap and accessible carbon fibers-in-poly(ether ether ketone) tube with high stability for online in-tube solid-phase microextraction. Talanta. 2016;**148**:313-320

[160] Bu Y, Feng J, Sun M, Zhou C, Luo C. Facile and efficient poly(ethylene terephthalate) fibers-in-tube for online solid-phase microextraction towards polycyclic aromatic hydrocarbons. Analytical and Bioanalytical Chemistry. 2016;**408**:4871-4882

[161] Bu Y, Feng J, Sun M, Zhou C, Luo C. Gold-functionalized stainlesssteel wire and tube for fiber-intube solid-phase microextraction coupled to high-performance liquid chromatography for the determination of polycyclic aromatic hydrocarbons. Journal of Separation Science. 2016;**39**:932-938

[162] Sun M, Feng J, Bu Y, Luo C. Ionic liquid coated copper wires and tubes for fiber-in-tube solidphase microextraction. Journal of Chromatography. A. 2016;**1458**:1-8

[163] Bu Y, Feng J, Tian Y, Wang X, Sun M, Luo C. An organically modified silica aerogel for online in-tube solidphase microextraction. Journal of Chromatography. A. 2017;**1517**:203-208

[164] Bu Y, Feng J, Wang X, Tian Y, Sun M, Luo C. In situ hydrothermal growth of polyaniline coating for in-tube solid-phase microextraction towards ultraviolet filters in environmental water samples. Journal of Chromatography. A. 2017;**1483**:48-55

[165] Mei M, Huang X. Online analysis of five organic ultraviolet filters in environmental water samples using magnetism-enhanced monolith-based in-tube solid phase microextraction coupled with high-performance liquid chromatography. Journal of Chromatography. A. 2017;**1525**:1-9

[166] Serra-Mora P, Jornet-Martinez N, Moliner-Martinez Y, Campíns-Falcó P. In tube-solid phase microextractionnano liquid chromatography: Application to the determination of intact and degraded polar triazines in waters and recovered struvite. Journal of Chromatography. A. 2017;**1513**:51-58

[167] Wang X, Pan L, Feng J, Tian Y, Luo C, Sun M. Silk fiber for in-tube solid-phase microextraction to detect aldehydes by chemical derivatization. Journal of Chromatography. A. 2017;**1522**:16-22

[168] Wang X, Feng J, Bu Y, Tian Y, Luo C, Sun M. Mesoporous titanium oxide with high-specific surface area as a coating for in-tube solid-phase microextraction combined with highperformance liquid chromatography for the analysis of polycyclic aromatic hydrocarbons. Journal of Separation Science. 2017;**40**:2474-2481

[169] Feng J, Wang X, Tian Y, Luo C, Sun M. Poly(ionic liquids)-coated stainless-steel wires packed into a polyether ether ketone tube for in-tube solid-phase microextraction. Journal of Separation Science. 2017;**40**:4773-4779

[170] Feng J, Tian Y, Wang X, Luo C, Sun M. Basalt fibers functionalized with gold nanoparticles for in-tube solid-phase microextraction. Journal of Separation Science. 2018;**41**:1149-1155

[171] Feng J, Mao H, Wang X, Tian Y, Luo C, Sun M. Ionic liquid chemically bonded basalt fibers for in-tube solidphase microextraction. Journal of Separation Science. 2018;**41**:1839-1846

[172] Wang X, Feng J, Tian Y, Luo C, Sun M. Co-Al bimetallic hydroxide nanocomposites coating for online in-tube solid-phase microextraction. Journal of Chromatography. A. 2018;**1550**:1-7

[173] Feng J, Wang X, Tian Y, Bu Y, Luo C, Sun M. Electrophoretic deposition ofgraphene oxide onto carbonfibers for in-tube solidphase microextraction. Journal of Chromatography. A. 2017;**1517**:209-214

[174] Jillani SMS, Alhooshani K. Urea functionalized surface-bonded sol-gel coating for on-line hyphenation of capillary microextraction with highperformance liquid chromatography. Journal of Chromatography. A. 2018;**1543**:14-22

[175] Pang J, Mei M, Yuan D, Huang X. Development of on-line monolith-based in-tube solid phase microextraction for the sensitive determination of triazoles in environmental waters. Talanta. 2018;**184**:411-417

[176] Prieto-Blanco MC, López-Mahía P, Campíns-Falcó P. On-line analysis of carbonyl compounds with derivatization in aqueous extracts of atmospheric particulate PM10 by in-tube solidphase microextraction coupled to capillary liquid chromatography. Journal of Chromatography. A. 2011;**1218**:4834-4839

[177] Fernández-Amado M, Prieto-Blanco MC, López-Mahía P, Muniategui-Lorenzo S, Prada-Rodríguez D. Ion-pair in-tube solid phase microextraction for the simultaneous determination of phthalates and their degradation products in atmospheric particulate matter. Journal of Chromatography. A. 2017;**1520**:35-47

[178] Ishizaki A, Kataoka H. A sensitive method for the determination of tobacco-specific nitrosamines in main- and side-stream smoke samples by online in-tube solidphase microextraction coupled with liquid chromatography-tandem mass spectrometry. Analytica Chimica Acta. 2019;**1075**:98-105

**187**

**Chapter 11**

Exchanger

exchange capacity for Na+

**1. Introduction**

**Abstract**

Preparation, Characterization

and Ion-Exchange Properties

Composite Cation Exchanger:

Iodovanadate Inorganic Ion

*Nainar Kohila, Kasi Sathiyaseelan and* 

*Mariyathanislas Sagaya Lourdhu Sumithra*

Polyaniline-Bi(III) Iodovanadate

Polyaniline based composite cation exchange materials have been used in industrial application for more than 100 years. The organic-inorganic composite cation exchanger was prepared by using sol-gel process. The organic polymer part furnishes the good chemical and mechanical properties, whereas the inorganic part improves the ion-exchange behavior and thermal stability. A new and novel polyaniline-Bi(III) iodovanadate composite cation exchanger prepared by using sol-gel process and was characterized by FT-IR, XRD, SEM-EDS studies. The ion exchange capacity, effect of size and charge of metal ion, eluent concentration, effect of time on IEC elution behavior and oxidizing properties of Bi(III) iodovanadate was also studied by column method. Polyaniline-Bi(III) iodovanadate composite exhibits ion

ion is 1.48 meq/g.

**Keywords:** elution behavior, ion exchange capacity, thermal stability, sol-gel process

Polyaniline has become one of the most interesting organic conducting polymer due to its high chemical and environmental stability, easy to synthesis in laboratory, feasibility of electrical conductivity and low cost of aniline monomer [1–3]. Literature survey reported that many ion exchange composite cation exchanger used for the separation of some toxic metal ions and dyes from the environment. New organic-inorganic conducting polymer composite cation exchanger developed by incorporation of organic polymer into the inorganic heteropolyacid moiety [4]. It is extensively used in rechargeable batteries, charge storage devices, protector shield

and Catalytic Properties of Bi(III)

of an Organic-Inorganic

### **Chapter 11**

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

[175] Pang J, Mei M, Yuan D, Huang X. Development of on-line monolith-based in-tube solid phase microextraction for the sensitive determination of triazoles in environmental waters. Talanta.

[176] Prieto-Blanco MC, López-Mahía P, Campíns-Falcó P. On-line analysis of carbonyl compounds with derivatization in aqueous extracts of atmospheric particulate PM10 by in-tube solidphase microextraction coupled to capillary liquid chromatography. Journal of Chromatography. A.

2018;**184**:411-417

2011;**1218**:4834-4839

2017;**1520**:35-47

2019;**1075**:98-105

[177] Fernández-Amado M, Prieto-Blanco MC, López-Mahía P, Muniategui-Lorenzo S, Prada-Rodríguez D. Ion-pair in-tube solid phase microextraction for the simultaneous determination of phthalates and their degradation products in atmospheric particulate matter. Journal of Chromatography. A.

[178] Ishizaki A, Kataoka H. A sensitive

method for the determination of tobacco-specific nitrosamines in main- and side-stream smoke samples by online in-tube solidphase microextraction coupled with liquid chromatography-tandem mass spectrometry. Analytica Chimica Acta.

Journal of Chromatography. A.

[168] Wang X, Feng J, Bu Y, Tian Y, Luo C, Sun M. Mesoporous titanium oxide with high-specific surface area as a coating for in-tube solid-phase microextraction combined with highperformance liquid chromatography for the analysis of polycyclic aromatic hydrocarbons. Journal of Separation

Science. 2017;**40**:2474-2481

[169] Feng J, Wang X, Tian Y, Luo C, Sun M. Poly(ionic liquids)-coated stainless-steel wires packed into a polyether ether ketone tube for in-tube solid-phase microextraction. Journal of Separation Science. 2017;**40**:4773-4779

[170] Feng J, Tian Y, Wang X, Luo C, Sun M. Basalt fibers functionalized with gold nanoparticles for in-tube solid-phase microextraction. Journal of Separation Science. 2018;**41**:1149-1155

[171] Feng J, Mao H, Wang X, Tian Y, Luo C, Sun M. Ionic liquid chemically bonded basalt fibers for in-tube solidphase microextraction. Journal of Separation Science. 2018;**41**:1839-1846

[172] Wang X, Feng J, Tian Y, Luo C, Sun M. Co-Al bimetallic hydroxide nanocomposites coating for online in-tube solid-phase microextraction. Journal of Chromatography. A.

[173] Feng J, Wang X, Tian Y,

Bu Y, Luo C, Sun M. Electrophoretic deposition ofgraphene oxide onto carbonfibers for in-tube solidphase microextraction. Journal of Chromatography. A. 2017;**1517**:209-214

[174] Jillani SMS, Alhooshani K. Urea functionalized surface-bonded sol-gel coating for on-line hyphenation of capillary microextraction with highperformance liquid chromatography. Journal of Chromatography. A.

2018;**1550**:1-7

2017;**1522**:16-22

**186**

2018;**1543**:14-22

Preparation, Characterization and Ion-Exchange Properties of an Organic-Inorganic Composite Cation Exchanger: Polyaniline-Bi(III) Iodovanadate and Catalytic Properties of Bi(III) Iodovanadate Inorganic Ion Exchanger

*Nainar Kohila, Kasi Sathiyaseelan and Mariyathanislas Sagaya Lourdhu Sumithra*

### **Abstract**

Polyaniline based composite cation exchange materials have been used in industrial application for more than 100 years. The organic-inorganic composite cation exchanger was prepared by using sol-gel process. The organic polymer part furnishes the good chemical and mechanical properties, whereas the inorganic part improves the ion-exchange behavior and thermal stability. A new and novel polyaniline-Bi(III) iodovanadate composite cation exchanger prepared by using sol-gel process and was characterized by FT-IR, XRD, SEM-EDS studies. The ion exchange capacity, effect of size and charge of metal ion, eluent concentration, effect of time on IEC elution behavior and oxidizing properties of Bi(III) iodovanadate was also studied by column method. Polyaniline-Bi(III) iodovanadate composite exhibits ion exchange capacity for Na+ ion is 1.48 meq/g.

**Keywords:** elution behavior, ion exchange capacity, thermal stability, sol-gel process

### **1. Introduction**

Polyaniline has become one of the most interesting organic conducting polymer due to its high chemical and environmental stability, easy to synthesis in laboratory, feasibility of electrical conductivity and low cost of aniline monomer [1–3]. Literature survey reported that many ion exchange composite cation exchanger used for the separation of some toxic metal ions and dyes from the environment. New organic-inorganic conducting polymer composite cation exchanger developed by incorporation of organic polymer into the inorganic heteropolyacid moiety [4]. It is extensively used in rechargeable batteries, charge storage devices, protector shield

in magnetic fields, sensors, biosensors, catalytic processes, microwave absorption, image processing and infrared optic applications [5–9]. Polymer based composite cation exchanger have extended enormous applications such as ion selective electrode, photocatalyst, antimicrobial, sensors, environmental remediation, etc. Several hetropolyacids are used as a catalyst in organic synthesis and they are extensively used for reagents in qualitative and quantitative analysis [10–12]. This paper deals with preparation, characterization, ion exchange studies, chemical stability and oxidizing ability of Bi(III) iodovanadate and newly fabricated polyaniline-Bi(III) iodovanadate composite cation exchanger. The structural analysis of polyaniline-Bi(III) iodovanadate composite was done by FT-IR, XRD and SEM-EDS studies.

#### **2. Experimental section**

#### **2.1 Reagents and instrument**

The reagents used for the preparation were analytical grade and used without any further purification. FT-IR spectra were recorded on a JACSO-4100 FT-IR Spectrometer. X-ray diffraction pattern was also recorded by using analytical system Shimadzu XRD-6000 model and the spectrum was recorded 10–90° using Cu-Kα radiation. The surface morphology and elemental composition was determined by using scanning electron microscope JSM-6390Lv energy dispersive X-ray detector.

#### **2.2 Preparation of polyaniline-Bi(III) iodovanadate cation exchanger**

Polyaniline gel was prepared by 0.2 M solution of aniline and potassium persulfate in 1 M hydrochloric acid [13] with constant stirring. Bi(III) iodovanadate inorganic ion exchanger was prepared by mixing 1:2:3 volume ratio of 0.2 M solution of bismuth nitrate, potassium iodate and sodium meta vanadate. The mixture of solution was adjusted to pH = 1 by using 1 M HNO3, and the precipitate was stirred for 1 hour [14]. The gel of polyaniline was mixed with inorganic precipitate of Bi(III) iodovanadate and the mixture was stirred thoroughly using magnetic stirrer. The green colored gel was kept for 1 day. The gel was filtered and dried it in oven at 50°C. The dried product of composite cation exchanger is crushed and the product is converted into H+ form by using 1 M HNO3 with occasional shaking for 1 day. The product is filtered and dried at 50°C. The H+ form of polyaniline-Bi(III) iodovanadate is used for ion exchange and chemical stability studies.

#### **2.3 Ion exchange capacity**

The ion exchange capacity of dry H<sup>+</sup> form of polyaniline-Bi(III) iodovanadate was determined by column process using 0.1 M NaCl as eluent. The liberated H+ ion was determined titrimetrically against NaOH solution by using phenolphthalein indicator and IEC was calculated by using formula,

$$\text{IEC} = \frac{\text{(N} \ast \text{V)}}{\text{W}} \,\text{meq/g} \tag{1}$$

**189**

**Figure 1.**

*Preparation, Characterization and Ion-Exchange Properties of an Organic-Inorganic Composite…*

different solvents and kept for 24 hours. After 24 hours the composite material was filtered and dried. The stability of composite cation exchanger was determined by

The catalytic function of heteropoly compounds has attracted much attention, particularly in the last two decades, because their acidic and redox properties can be controlled at atomic/molecular levels. As for the phase of these catalytic systems, various systems are possible: homogenous liquid, liquid/liquid (phase transfer), liquid/solid, gas/solid systems, and so on. There are actually several large-scale

Polyaniline gel was prepared by 0.2 M solution of aniline and potassium persulfate in 1 M hydrochloric acid with constant stirring. About 0.5 g of Bi(III) iodovanadate inorganic ion exchanger were added to the solution of acidic aniline with constant stirring for 1 hour. Aniline was polymerized into the green color polyaniline. The product of polyaniline was filtered and washed with DMW, ethanol,

The incorporation of organic polymer polyaniline into the inorganic matrix of Bi(III) iodovanadate was confirmed by carrying out FT-IR spectral studies. The characteristic peaks of polyaniline and Bi(III) iodovanadate were observed in the FT-IR spectrum of polyaniline-Bi(III) iodovanadate composite shown in **Figure 1b**.

benzenoid and quinoid stretching frequency obtained at 1477 and 1560 cm<sup>−</sup><sup>1</sup>

*(a) FT-IR spectrum of polyaniline; (b) FT-IR spectrum of polyaniline-Bi(III) iodovanadate.*

in neighboring quinoid ring [16]. The characteristic bands at 878, 784 and 671 cm<sup>−</sup><sup>1</sup>

The XRD pattern of polyaniline shows broad peak at two theta value of 25.43° which indicate low crystallinity of the conducting polymer XRD pattern of polyaniline-Bi(III) iodovandate composite (**Figure 2b**) exhibit high intensity peaks at two theta values 26.09, 33.6 and 32.8°. The observation in the XRD pattern of polyaniline composite shows that the composite is crystalline nature and calculated particle size is 14.96 nm.

is due to the -OH stretching vibrations. The

as singed to -CN stretching and -NH bending vibration

[15].

form of polyaniline-Bi(III) iodovanadate mixed with 25 ml of

*DOI: http://dx.doi.org/10.5772/intechopen.87064*

change in color and weight of composite cation exchanger.

**2.5 Inorganic heteropolyacid as oxidizing agent**

industrial processes that use heteropolyacid catalysts.

milligram of H+

acetone and dried at 50°C.

**3. Results and discussion**

Peaks at 1270 and 1654 cm<sup>−</sup><sup>1</sup>

The FT-IR peak observed at 3400 cm<sup>−</sup><sup>1</sup>

may be assigned to M-O stretching [17].

where N and V are the normality and volume of NaOH respectively and W is the weight in gram.

#### **2.4 Chemical stability**

To find out extent of dissolution of composite cation exchange material, chemical stability was studied in different organic and inorganic solvents. Two fifty

milligram of H+ form of polyaniline-Bi(III) iodovanadate mixed with 25 ml of different solvents and kept for 24 hours. After 24 hours the composite material was filtered and dried. The stability of composite cation exchanger was determined by change in color and weight of composite cation exchanger.

#### **2.5 Inorganic heteropolyacid as oxidizing agent**

The catalytic function of heteropoly compounds has attracted much attention, particularly in the last two decades, because their acidic and redox properties can be controlled at atomic/molecular levels. As for the phase of these catalytic systems, various systems are possible: homogenous liquid, liquid/liquid (phase transfer), liquid/solid, gas/solid systems, and so on. There are actually several large-scale industrial processes that use heteropolyacid catalysts.

Polyaniline gel was prepared by 0.2 M solution of aniline and potassium persulfate in 1 M hydrochloric acid with constant stirring. About 0.5 g of Bi(III) iodovanadate inorganic ion exchanger were added to the solution of acidic aniline with constant stirring for 1 hour. Aniline was polymerized into the green color polyaniline. The product of polyaniline was filtered and washed with DMW, ethanol, acetone and dried at 50°C.

#### **3. Results and discussion**

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

**2. Experimental section**

**2.1 Reagents and instrument**

product is converted into H+

**2.3 Ion exchange capacity**

1 day. The product is filtered and dried at 50°C. The H+

The ion exchange capacity of dry H<sup>+</sup>

indicator and IEC was calculated by using formula,

iodovanadate is used for ion exchange and chemical stability studies.

was determined by column process using 0.1 M NaCl as eluent. The liberated H+

was determined titrimetrically against NaOH solution by using phenolphthalein

IEC <sup>=</sup> (N <sup>∗</sup> V) \_\_\_\_\_\_\_ <sup>W</sup> meq/g (1)

where N and V are the normality and volume of NaOH respectively and W is the

To find out extent of dissolution of composite cation exchange material, chemi-

cal stability was studied in different organic and inorganic solvents. Two fifty

in magnetic fields, sensors, biosensors, catalytic processes, microwave absorption, image processing and infrared optic applications [5–9]. Polymer based composite cation exchanger have extended enormous applications such as ion selective electrode, photocatalyst, antimicrobial, sensors, environmental remediation, etc. Several hetropolyacids are used as a catalyst in organic synthesis and they are extensively used for reagents in qualitative and quantitative analysis [10–12]. This paper deals with preparation, characterization, ion exchange studies, chemical stability and oxidizing ability of Bi(III) iodovanadate and newly fabricated polyaniline-Bi(III) iodovanadate composite cation exchanger. The structural analysis of polyaniline-Bi(III) iodovanadate composite was done by FT-IR, XRD and SEM-EDS studies.

The reagents used for the preparation were analytical grade and used without any further purification. FT-IR spectra were recorded on a JACSO-4100 FT-IR Spectrometer. X-ray diffraction pattern was also recorded by using analytical system Shimadzu XRD-6000 model and the spectrum was recorded 10–90° using Cu-Kα radiation. The surface morphology and elemental composition was determined by using scanning electron microscope JSM-6390Lv energy dispersive X-ray detector.

Polyaniline gel was prepared by 0.2 M solution of aniline and potassium persulfate in 1 M hydrochloric acid [13] with constant stirring. Bi(III) iodovanadate inorganic ion exchanger was prepared by mixing 1:2:3 volume ratio of 0.2 M solution of bismuth nitrate, potassium iodate and sodium meta vanadate. The mixture of solution was adjusted to pH = 1 by using 1 M HNO3, and the precipitate was stirred for 1 hour [14]. The gel of polyaniline was mixed with inorganic precipitate of Bi(III) iodovanadate and the mixture was stirred thoroughly using magnetic stirrer. The green colored gel was kept for 1 day. The gel was filtered and dried it in oven at 50°C. The dried product of composite cation exchanger is crushed and the

form by using 1 M HNO3 with occasional shaking for

form of polyaniline-Bi(III) iodovanadate

form of polyaniline-Bi(III)

ion

**2.2 Preparation of polyaniline-Bi(III) iodovanadate cation exchanger**

**188**

weight in gram.

**2.4 Chemical stability**

The incorporation of organic polymer polyaniline into the inorganic matrix of Bi(III) iodovanadate was confirmed by carrying out FT-IR spectral studies. The characteristic peaks of polyaniline and Bi(III) iodovanadate were observed in the FT-IR spectrum of polyaniline-Bi(III) iodovanadate composite shown in **Figure 1b**. The FT-IR peak observed at 3400 cm<sup>−</sup><sup>1</sup> is due to the -OH stretching vibrations. The benzenoid and quinoid stretching frequency obtained at 1477 and 1560 cm<sup>−</sup><sup>1</sup> [15]. Peaks at 1270 and 1654 cm<sup>−</sup><sup>1</sup> as singed to -CN stretching and -NH bending vibration in neighboring quinoid ring [16]. The characteristic bands at 878, 784 and 671 cm<sup>−</sup><sup>1</sup> may be assigned to M-O stretching [17].

The XRD pattern of polyaniline shows broad peak at two theta value of 25.43° which indicate low crystallinity of the conducting polymer XRD pattern of polyaniline-Bi(III) iodovandate composite (**Figure 2b**) exhibit high intensity peaks at two theta values 26.09, 33.6 and 32.8°. The observation in the XRD pattern of polyaniline composite shows that the composite is crystalline nature and calculated particle size is 14.96 nm.

**Figure 1.** *(a) FT-IR spectrum of polyaniline; (b) FT-IR spectrum of polyaniline-Bi(III) iodovanadate.*

#### **Figure 3.**

*(a) SEM photograph for polyaniline; (b) SEM photograph for polyaniline-Bi(III) iodovandate; (c) EDS analysis for polyaniline-Bi(III) iodovanadate.*

**Figures 3a**, **b** represents SEM photographs of polyaniline and polyaniline-Bi(III) iodovanadate composites, respectively. SEM photograph of polyaniline salt suggests that agglomerates are randomly distributed on its surface, whereas polyaniline-Bi(III)

**191**

**Figure 5.**

**Figure 4.**

*Preparation, Characterization and Ion-Exchange Properties of an Organic-Inorganic Composite…*

iodovanadate composite have porous morphology with granular structure [18]. It is clearly evident from SEM study, inorganic ion exchanger homogeneously distributed on the surface of polyaniline. **Figure 3c** shows that presence of all the elements pres-

The ion exchange capacity is affected by the size and charge of metal ions. Ion exchange capacity of polyaniline-Bi(III) iodovanate composite for vari-

Ba2+ > Sr2+ > Mg2+ respectively. The ions with smaller hydrated ionic radii easily enter the pores of cation exchanger resulting in higher adsorption. Similar observation was observed by Mesalem et al. and Nachood et al. [19, 20]. Elution behavior was carried out to find out the volume of eluent (NaCl) required for complete elu-

**Figure 4b**. It is clear from **Figure 4b** 120 ml of NaCl is enough for complete elution

ity. The minimum molar concentration of eluent was found to be 0.1 M. The effect of time on ion exchange capacity shows constant: ion exchange capacity after 60 minutes. The observed datas of eluent concentration and effect of time show in

Chemical stability study was carried out to find the stability of prepared composite cation exchanger in different solvents of interest such as DMW, 2 M HCl, H2SO4, NaOH, ether, 1,2-dichloroethane, cyclohexane and benzene. The polyaniline-Bi(III) iodovanadate composite was more stable in DMW, Partially stable in mineral acid and organic solvents and unstable in 2 M NaOH because of dedoped

*(a) IEC of polyaniline-Bi(III) iodovanadate composite for various metal ions; (b) elution behavior of* 

*Na + IEC for polyaniline-Bi(III) iodovanadate as a function of (a) eluent concentration; (b) contact time.*

> Na+

> Li+

form and represented in

ion exchange capac-

and

*DOI: http://dx.doi.org/10.5772/intechopen.87064*

tion in H+

(**Figure 5**).

form of ion exchanger.

*polyaniline-Bi(III) iodovanadate composite.*

ions.

of H+

ent in the material which shows purity of composite.

ous alkali and alkaline earth metal cations follows the order K<sup>+</sup>

ion from 1 g composite cation exchanger on H+

Eluent concentration is main factor which affects the Na<sup>+</sup>

*Preparation, Characterization and Ion-Exchange Properties of an Organic-Inorganic Composite… DOI: http://dx.doi.org/10.5772/intechopen.87064*

iodovanadate composite have porous morphology with granular structure [18]. It is clearly evident from SEM study, inorganic ion exchanger homogeneously distributed on the surface of polyaniline. **Figure 3c** shows that presence of all the elements present in the material which shows purity of composite.

The ion exchange capacity is affected by the size and charge of metal ions. Ion exchange capacity of polyaniline-Bi(III) iodovanate composite for various alkali and alkaline earth metal cations follows the order K<sup>+</sup> > Na+ > Li+ and Ba2+ > Sr2+ > Mg2+ respectively. The ions with smaller hydrated ionic radii easily enter the pores of cation exchanger resulting in higher adsorption. Similar observation was observed by Mesalem et al. and Nachood et al. [19, 20]. Elution behavior was carried out to find out the volume of eluent (NaCl) required for complete elution in H+ ion from 1 g composite cation exchanger on H+ form and represented in **Figure 4b**. It is clear from **Figure 4b** 120 ml of NaCl is enough for complete elution of H+ ions.

Eluent concentration is main factor which affects the Na<sup>+</sup> ion exchange capacity. The minimum molar concentration of eluent was found to be 0.1 M. The effect of time on ion exchange capacity shows constant: ion exchange capacity after 60 minutes. The observed datas of eluent concentration and effect of time show in (**Figure 5**).

Chemical stability study was carried out to find the stability of prepared composite cation exchanger in different solvents of interest such as DMW, 2 M HCl, H2SO4, NaOH, ether, 1,2-dichloroethane, cyclohexane and benzene. The polyaniline-Bi(III) iodovanadate composite was more stable in DMW, Partially stable in mineral acid and organic solvents and unstable in 2 M NaOH because of dedoped form of ion exchanger.

#### **Figure 4.**

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

*(a) XRD pattern for polyaniline; (b) XRD pattern for polyaniline-Bi(III) iodovanadate.*

**190**

**Figure 3.**

**Figure 2.**

*analysis for polyaniline-Bi(III) iodovanadate.*

**Figures 3a**, **b** represents SEM photographs of polyaniline and polyaniline-Bi(III) iodovanadate composites, respectively. SEM photograph of polyaniline salt suggests that agglomerates are randomly distributed on its surface, whereas polyaniline-Bi(III)

*(a) SEM photograph for polyaniline; (b) SEM photograph for polyaniline-Bi(III) iodovandate; (c) EDS* 

*(a) IEC of polyaniline-Bi(III) iodovanadate composite for various metal ions; (b) elution behavior of polyaniline-Bi(III) iodovanadate composite.*

**Figure 5.** *Na + IEC for polyaniline-Bi(III) iodovanadate as a function of (a) eluent concentration; (b) contact time.*

#### **3.1 Bi(III) iodovanadate as oxidizing agent**

The hetropolyacid (iodovanadate) unit of inorganic ion exchanger like Bi(III) iodovanadate is made up of oxygen and hydrogen with some metals and nonmetal (iodine and vanadium). Bi(III) iodovanadate can act as a oxidizing agent, to the polymerization of aniline monomer into green colored polyaniline gels without adding oxidizing agent [10, 21–24].

### **4. Conclusion**

In this present paper, polyaniline-Bi(III) iodovanadate composite cation exchanger have enhanced Na+ ion exchange capacity compared to polyaniline and Bi(III) iodovanadate. The composite cation exchanger was prepared successfully by using sol-gel method. FT-IR and SEM-EDS studies confirmed that the inorganic ion exchanger incorporated into polyaniline matrix. XRD spectral studies proved that the composite cation exchanger is in nano size range and crystalline nature. Ion exchange capacity and chemical stability studies proved that the composite material act as good ion exchanger and stable in mineral acid and organic solvents. It was concluded that polyaniline-Bi(III) iodovanadate composite act as good potential for environmental remediation.

### **Author details**

Nainar Kohila, Kasi Sathiyaseelan\* and Mariyathanislas Sagaya Lourdhu Sumithra Department of Chemistry, Aditanar College of Arts and Science, M.S. University, Tirunelveli, Tamil Nadu, India

\*Address all correspondence to: sseelan800@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**193**

*Preparation, Characterization and Ion-Exchange Properties of an Organic-Inorganic Composite…*

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[12] Chalmers RA, Parker CA, Stephen WI, Mills AA, Hall RJ, Kirkbright GF. Determination of non-metals. Proceedings of the Society for Analytical Chemistry. 1966;**3**:157-164

[13] Zeng XR, Man Ko J. Structure-Conductivity relationships of iodine-doped polyaniline. Polymer.

[14] Buhra R, Naushad M, Adnan R, Alothman ad ZA, Rafathullah M. Polyaniline supported nanocomposite

[15] Furukawa Y, Ueda F, Hyodo Y, Nakajima T, Kawagoe T. Vibrational Spectra and structure of polyaniline. Macromolecules. 1988;**21**:1297

[16] King ET, Neoh KG, Tan KL. Polyanilne A polymer with many

in Polymer Science. 1998;**23**:277

interesting intrinsic redox state. Progress

[17] Socrstes G. Infrared Characteristics Group Frequencies. NJ: Wiley; 1980.

cation exchanger: Synthesis, characterization and application for efficient removal of Pb2+ ion from aqueous medium. Journal of Industrial and Engineering Chemistry.

& Sons Inc. 1991. p. 472

1998;**39**:1187-1195

2015;**21**:1112-1118

p. 145

2010;**70**:707-714

*DOI: http://dx.doi.org/10.5772/intechopen.87064*

[1] Ding X, Han D, Wang Z, Xu X, Niu L, Zhang Q. Micelle assisted synthesis of polyaniline magnetic nanorods by in-situ self-assembly process. Journal of Colloid and Interface Science.

[2] Kim BJ, Oh SG, Han MG, Im SS. Preparation of polyaniline nanoparticle in miceller solution as polymerization medium. Langmuir. 2000;**16**:5841-5845

[3] Gao Y, Zhou Y. Polyaniline nofibers

[4] Khan AA, Alam MM, Mohammed IF.

[5] Rivera-Utrilla J, Sanchez-Poloa M, Gomez-Serranob V, Alvarezc PM, Alvim MCM, Ferrazd MC, et al. Activated carbon modifications to enhance its water treatment applications. An overview. Journal of Hazardous

[6] Jing G, Zhou Z, Song L, Dong M. Ultrasound enhanced adsorption and desorption of chromium (VI) on activated carbon and polymeric resin. Desalination. 2011;**279**:423-427

[7] Gupta RK, Singh RA, Dubey SS. Removel of mercury ions from aqueous solution by composite of poly aniline with red mud. Separation

[8] Aia L, Jianga J, Zhang R. Synthetic

and Purification Technology.

2004;**38**:225-232

Metals. 2010;**160**:762

fabricated by electrochemical polymerization: A mechanistic study. European Polymer Journal.

Electrical conductivity and ionexchange kinetitic studies of a new crystalline type 'organic-inorganic' cation-exchange material: polyprrolr/ polyantimonic acid composite system, (Sb2O5)(-C4H4NH-).nH2O. Journal of Electroanalytical Chemistry.

**References**

2008;**320**:341-345

2007;**43**:2292-2297

2004;**572**:67-78

Materials. 2011;**187**:1-23

*Preparation, Characterization and Ion-Exchange Properties of an Organic-Inorganic Composite… DOI: http://dx.doi.org/10.5772/intechopen.87064*

#### **References**

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

The hetropolyacid (iodovanadate) unit of inorganic ion exchanger like Bi(III) iodovanadate is made up of oxygen and hydrogen with some metals and nonmetal (iodine and vanadium). Bi(III) iodovanadate can act as a oxidizing agent, to the polymerization of aniline monomer into green colored polyaniline gels without

In this present paper, polyaniline-Bi(III) iodovanadate composite cation

Bi(III) iodovanadate. The composite cation exchanger was prepared successfully by using sol-gel method. FT-IR and SEM-EDS studies confirmed that the inorganic ion exchanger incorporated into polyaniline matrix. XRD spectral studies proved that the composite cation exchanger is in nano size range and crystalline nature. Ion exchange capacity and chemical stability studies proved that the composite material act as good ion exchanger and stable in mineral acid and organic solvents. It was concluded that polyaniline-Bi(III) iodovanadate composite act as good potential for

ion exchange capacity compared to polyaniline and

**3.1 Bi(III) iodovanadate as oxidizing agent**

adding oxidizing agent [10, 21–24].

exchanger have enhanced Na+

environmental remediation.

**4. Conclusion**

**192**

**Author details**

Tirunelveli, Tamil Nadu, India

provided the original work is properly cited.

\*Address all correspondence to: sseelan800@gmail.com

Nainar Kohila, Kasi Sathiyaseelan\* and Mariyathanislas Sagaya Lourdhu Sumithra Department of Chemistry, Aditanar College of Arts and Science, M.S. University,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

[1] Ding X, Han D, Wang Z, Xu X, Niu L, Zhang Q. Micelle assisted synthesis of polyaniline magnetic nanorods by in-situ self-assembly process. Journal of Colloid and Interface Science. 2008;**320**:341-345

[2] Kim BJ, Oh SG, Han MG, Im SS. Preparation of polyaniline nanoparticle in miceller solution as polymerization medium. Langmuir. 2000;**16**:5841-5845

[3] Gao Y, Zhou Y. Polyaniline nofibers fabricated by electrochemical polymerization: A mechanistic study. European Polymer Journal. 2007;**43**:2292-2297

[4] Khan AA, Alam MM, Mohammed IF. Electrical conductivity and ionexchange kinetitic studies of a new crystalline type 'organic-inorganic' cation-exchange material: polyprrolr/ polyantimonic acid composite system, (Sb2O5)(-C4H4NH-).nH2O. Journal of Electroanalytical Chemistry. 2004;**572**:67-78

[5] Rivera-Utrilla J, Sanchez-Poloa M, Gomez-Serranob V, Alvarezc PM, Alvim MCM, Ferrazd MC, et al. Activated carbon modifications to enhance its water treatment applications. An overview. Journal of Hazardous Materials. 2011;**187**:1-23

[6] Jing G, Zhou Z, Song L, Dong M. Ultrasound enhanced adsorption and desorption of chromium (VI) on activated carbon and polymeric resin. Desalination. 2011;**279**:423-427

[7] Gupta RK, Singh RA, Dubey SS. Removel of mercury ions from aqueous solution by composite of poly aniline with red mud. Separation and Purification Technology. 2004;**38**:225-232

[8] Aia L, Jianga J, Zhang R. Synthetic Metals. 2010;**160**:762

[9] Salem MA. The role of polyaniline salts in the removal of direct blue 78 from aqueous solution: A kinetic study. Reactive and Functional Polymers. 2010;**70**:707-714

[10] Lu T, Niu M, Hou Y, Wu W, Ren S, Yang F. Catalytic oxidation of cellulose to formic acid in H5PV2Mo10O40 + H2SO4 aqueous solution with molecular oxygen. Green Chemistry. 2016;**18**:4725-4732

[11] Jeffery GH, Bassett J, Mendham J, Denny RC. Vogels Textbook of Quantitative Analysis. V ed. John Wiley & Sons Inc. 1991. p. 472

[12] Chalmers RA, Parker CA, Stephen WI, Mills AA, Hall RJ, Kirkbright GF. Determination of non-metals. Proceedings of the Society for Analytical Chemistry. 1966;**3**:157-164

[13] Zeng XR, Man Ko J. Structure-Conductivity relationships of iodine-doped polyaniline. Polymer. 1998;**39**:1187-1195

[14] Buhra R, Naushad M, Adnan R, Alothman ad ZA, Rafathullah M. Polyaniline supported nanocomposite cation exchanger: Synthesis, characterization and application for efficient removal of Pb2+ ion from aqueous medium. Journal of Industrial and Engineering Chemistry. 2015;**21**:1112-1118

[15] Furukawa Y, Ueda F, Hyodo Y, Nakajima T, Kawagoe T. Vibrational Spectra and structure of polyaniline. Macromolecules. 1988;**21**:1297

[16] King ET, Neoh KG, Tan KL. Polyanilne A polymer with many interesting intrinsic redox state. Progress in Polymer Science. 1998;**23**:277

[17] Socrstes G. Infrared Characteristics Group Frequencies. NJ: Wiley; 1980. p. 145

[18] Deepti B, Patle Wasudeo B, Gurnule W, Zade AB. Synthesis, characterization and ion exchange properties of a terpolymer derived from 4-hydroxybenzophenone, biuret and formaldehyde. Der Pharmacia Lettre. 2011;**3**:341-353

[19] Mesalem MA. Sorption kinetics of cooper, zinc, cadmium and nickel ions in synthesized silico-antimonate ion exchanger. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2003;**225**:1380-1384

[20] Nachood FC, Wood W. The reaction velocity of ion exchanger. Journal of the American Chemical Society. 1944;**66**:1380-1384

[21] Mizuno N, Makoto M. Heterogenous catalysis. Chemical Reviews. 1998;**98**:199-217

[22] kozhevnikov IV. Catalysis by heteropolyacid and multicomponent polyoxometalates in liquid-phase reactions. Chemical Reviews. 1998;**98**:171-198

[23] Illies S, Kraushaar-Czarnetzki B. Processing study on the stability of heteropolyacid catalyst in the oxidation of methacrolein to methylacrylic acid. Industrial and Engineering Chemistry Research. 2016;**55**:8509-8518

[24] Silva D, Marcio J, Oliverira D, Macedo C. Catalysis by Keggin heteropolyacid salts. Current Catalysis. 2018;**7**:26-34

*Biochemical Analysis Tools - Methods for Bio-Molecules Studies*

[18] Deepti B, Patle Wasudeo B, Gurnule W, Zade AB. Synthesis, characterization and ion exchange properties of a terpolymer derived from 4-hydroxybenzophenone, biuret and formaldehyde. Der Pharmacia Lettre.

[19] Mesalem MA. Sorption kinetics of cooper, zinc, cadmium and nickel ions in synthesized silico-antimonate ion exchanger. Colloids and Surfaces A: Physicochemical and Engineering

[20] Nachood FC, Wood W. The reaction velocity of ion exchanger. Journal of the American Chemical Society.

[21] Mizuno N, Makoto M. Heterogenous

Aspects. 2003;**225**:1380-1384

catalysis. Chemical Reviews.

[22] kozhevnikov IV. Catalysis by heteropolyacid and multicomponent polyoxometalates in liquid-phase reactions. Chemical Reviews.

[23] Illies S, Kraushaar-Czarnetzki B. Processing study on the stability of heteropolyacid catalyst in the oxidation of methacrolein to methylacrylic acid. Industrial and Engineering Chemistry

Research. 2016;**55**:8509-8518

[24] Silva D, Marcio J, Oliverira D, Macedo C. Catalysis by Keggin

heteropolyacid salts. Current Catalysis.

1944;**66**:1380-1384

1998;**98**:199-217

1998;**98**:171-198

2018;**7**:26-34

2011;**3**:341-353

**194**

## *Edited by Oana-Maria Boldura, Cornel Baltă and Nasser Sayed Awwad*

This book explores the role of nucleic acid analysis and the advances it has led to in the field of life sciences. The first section is a collection of chapters covering experimental methods used in molecular biology, the techniques adjacent to these methods, and the steps of analysis before and after obtaining raw DNA data. The second section deals with the principles of chromatography, method development, sample preparation, and industrial applications.

Published in London, UK © 2020 IntechOpen © Gio\_tto / iStock

Biochemical Analysis Tools - Methods for Bio-Molecules Studies

Biochemical Analysis Tools

Methods for Bio-Molecules Studies

*Edited by Oana-Maria Boldura,* 

*Cornel Baltă and Nasser Sayed Awwad*