Preface

This book discusses several aspects of and recent advances in analytical liquid chromatography (LC), referring mainly to high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC). It is not intended to be comprehensive and thus only some selected but important subjects are included. Chapter 1 addresses one such subject, which is the technology of the chromatographic columns used in HPLC and UPLC. The chromatographic column is at the core of HPLC/UPLC separation and significant effort is being made to make improvements to the column. This effort involves improving the type of columns that are widely used in everyday work for practical analyses and developing exploratory new materials for the stationary phase of the columns and miniaturization.

One of the newest developments in HPLC/UPLC is the use of mixed-mode stationary phases, which is discussed in Chapter 2. Mixed-mode phases have at least two different functionalities that can be involved in different types of interaction with analytes. Examples of such different interactions include reversed-phase/hydrophilic interaction, reversed-phase/ion-exchange, hydrophilic interaction/ion-exchange, and even trimodal-type interactions. Different procedures are used to obtain mixed-mode stationary phases, such as mixing particles with different types of active phases, phases containing different ligands, or phases having two (or more) embedded functionalities in the active stationary phase. The chapter discusses the subject of mixed-mode types of stationary phases and presents some of the newest applications of mixed-mode phases in the analysis of pharmaceuticals and biological samples.

Another development in analytical LC is related to multidimensional separations. Although the interest in multidimensional separation in LC is not new, the increasing need for the analysis of complex samples has made the technique more relevant. In practice, the most common type of multidimensional LC is bidimensional, which is examined in Chapter 3. The chapter presents different techniques used in bidimensional LC such as on-line, stop-and-go, and off-line. Also, the chapter describes various applications of multidimensional LC such as in proteomics, lipidomics, environmental analysis, polymer and oligomer separation, metabolomics, and chiral separations.

Chapter 4 is dedicated to the use of ionic liquids in HPLC/UPLC. The chapter discusses stationary phases with attached structures of ionic liquids, the use of ionic liquids as additives in the mobile phase of HPLC separations, and the retention behavior of ionic liquids studied as analytes. It also presents the advantages and shortcomings of using ionic liquids, and the reasons ionic liquids are more and more frequently used in HPLC.

Finally, Chapter 5 discusses the ion mobility (IM) method of separation. In modern instrumentation, IM typically follows an HPLC separation and is used in connection with a time of flight (TOF) mass spectrometric (MS) detection. While HPLC separation provides separations in the range of seconds, IM provides the separation of sample components in the range of 10-1 to 10-3 seconds, and TOF–MS produces the separation of ions by their mass on a microsecond scale. The IM separation significantly cleans up mass spectral data of co-eluting peaks not separated by the liquid chromatography and adds descriptive information for each ion. The combination LC–IM–MS allows separation based on the LC retention time, cross-sectional area differences in IM, as well as m/z and mass spectral fragmentation in the MS. The chapter describes several aspects regarding the types of IM and presents in detail the results of using LC–IM–MS in three case studies.

Each chapter is written by scientists with considerable experience in the field and recognized academic experience. This allows for the material to be clearly presented as well as very informative from a scientific point of view.

> **Dr. Serban C. Moldoveanu** R.J. Reynolds Tobacco Co., Winston-Salem, NC, United States

> > **Victor David** Professor, University of Bucharest, Bucharest, Romania

**Chapter 1**

HPLC

**Abstract**

Progress in Technology of the

Chromatographic Columns in

Chromatographic column is an essential part of a any HPLC separation, and significant progress has been made in developing columns with better performance to provide better separation, a shorter separation time, resilience to a wider pH range of the mobile phase, longer lifetime, use of lower volumes of mobile phase, etc. All these characteristics were achieved by the introduction of novel technologies and improvements of the older ones. These include smaller particle used to fill the column, more homogeneous spherical particles, core-shell particles, monolithic columns, more pure silica as a stationary phase support, use of ethylene bridge silica, a wider variety of active phases, use of mixed mode stationary phases, use of polymers as stationary phase, use of various endcapping techniques, etc. Miniaturization and progress in the instrumentation played an important role for the chromatographic column develop-

**Keywords:** chromatographic column, silica, support derivatization, reversed phase,

The chromatographic separation is based on the differences in the retention of the components of a sample dissolved in a mobile phase when passing through a stationary phase typically contained in a chromatographic column. In HPLC, the mobile phase is a liquid and the characteristics of high performance (of the separation) and high pressure (used for the mobile phase) lead to the acronym HPLC. Although cartridges and micro-fluidic chips can be used to contain the stationary phase, a column is much more frequently utilized for this purpose [1, 2]. The external body of the column is a tube made from stainless steel or a strong polymer (e.g., polyether ether ketone or PEEK). This tube is filled with the stationary phase. Stationary phase can be in the form of particles or as monoliths. Both particles and the monoliths usually have a rigid porous support that may also act as the active phase, but more frequently the support has on the surface a chemically bonded or physically coated active phase used for the separation. The progress in the making of chromatographic columns is very important for the development of HPLC. A large body of information

*Serban C. Moldoveanu and Victor David*

ment. All these aspects are summarized in the present chapter.

HILIC, ion exchange, chiral columns

**1. Introduction**

#### **Chapter 1**

## Progress in Technology of the Chromatographic Columns in HPLC

*Serban C. Moldoveanu and Victor David*

#### **Abstract**

Chromatographic column is an essential part of a any HPLC separation, and significant progress has been made in developing columns with better performance to provide better separation, a shorter separation time, resilience to a wider pH range of the mobile phase, longer lifetime, use of lower volumes of mobile phase, etc. All these characteristics were achieved by the introduction of novel technologies and improvements of the older ones. These include smaller particle used to fill the column, more homogeneous spherical particles, core-shell particles, monolithic columns, more pure silica as a stationary phase support, use of ethylene bridge silica, a wider variety of active phases, use of mixed mode stationary phases, use of polymers as stationary phase, use of various endcapping techniques, etc. Miniaturization and progress in the instrumentation played an important role for the chromatographic column development. All these aspects are summarized in the present chapter.

**Keywords:** chromatographic column, silica, support derivatization, reversed phase, HILIC, ion exchange, chiral columns

#### **1. Introduction**

The chromatographic separation is based on the differences in the retention of the components of a sample dissolved in a mobile phase when passing through a stationary phase typically contained in a chromatographic column. In HPLC, the mobile phase is a liquid and the characteristics of high performance (of the separation) and high pressure (used for the mobile phase) lead to the acronym HPLC. Although cartridges and micro-fluidic chips can be used to contain the stationary phase, a column is much more frequently utilized for this purpose [1, 2]. The external body of the column is a tube made from stainless steel or a strong polymer (e.g., polyether ether ketone or PEEK). This tube is filled with the stationary phase. Stationary phase can be in the form of particles or as monoliths. Both particles and the monoliths usually have a rigid porous support that may also act as the active phase, but more frequently the support has on the surface a chemically bonded or physically coated active phase used for the separation. The progress in the making of chromatographic columns is very important for the development of HPLC. A large body of information describes the progress in column construction including peer reviewed papers, books, and information on the Internet [3–11]. Present chapter describes some of the more recent progress in column construction and indicates potential for new developments. This progress takes place into two main directions: 1) the improvement of mainstream-type columns that are widely used in everyday work for practical analyses and 2) the development of exploratory new materials for the stationary phase and miniaturization.

#### **2. Short theoretical background**

In a chromatographic separation, the components of a mixture are eluting from the column, then are detected, and the detection electric signal is converted into a graphic output as *peaks* in a chromatogram. The peaks have ideally a Gaussian shape. Each peak has a specific *retention time tR*. For a compound *X*, *tR(X)* is the time (usually measured in min) from the injection of the sample into the chromatographic system to the time of elution of the compound. A time slightly longer than the retention time of the last peak in a chromatogram is indicated as *run time.* The retention time for an unretained compound is known as *dead time t*0. The dead time *t*<sup>0</sup> is defined by the ratio *L*/*u* where *u* is the *linear flow rate* of the mobile phase and *L* is the *column length*. In HPLC instrumentation, the controlled parameter by the user is the *volumetric flow rate U* and not *u*. The two parameters, *U* and *u*, are related by the expression:

$$U = \frac{\varepsilon^\* \,\pi d^2}{4} u \tag{1}$$

In formula (1), *d* is the inner diameter of the column, and ε\* is a constant depending on column packing porosity (an average value for ε\* is 0.7 although this may vary considerably depending on the stationary phase particle size and structure).

The separation in a chromatographic process between two compounds *X* and *Y* (*X* eluting first in the separation) is overall characterized by a parameter termed *resolution R*. The expression for *R* is given by the formula:

$$R = \frac{1}{4}(a - 1)\frac{k'(Y)}{1 + k'(Y)}N^{1/2} \tag{2}$$

In formula (2) parameter *k'* termed *retention factor* is defined by the formula:

$$k'(X) = \frac{t\_R(X) - t\_0}{t\_0} \tag{3}$$

Parameter *k'* depends on chemical nature of the separated compound *X*, on the nature of the mobile phase, on the chemical composition and physical characteristics of the stationary phase as well as on a parameter termed *phase ratio Ψ*. The value of *Ψ* is given by the ratio *Vst*/*V*<sup>0</sup> where *V*st is the volume of the active part of the stationary phase involved in separation process, and *V*<sup>0</sup> is the dead volume of the column (*V*<sup>0</sup> = *t*<sup>0</sup> *U*). The retention factor *k'* is proportional with *Ψ*. Parameter *α* is the *selectivity*, which is defined as the ratio *k'*(*Y*)/*k'*(*X*), and *N* is a parameter termed *theoretical plate number*, which describes the peak broadening in a separation and estimate the column efficiency. The value of *N* depends on column length and a

*Progress in Technology of the Chromatographic Columns in HPLC DOI: http://dx.doi.org/10.5772/intechopen.104123*

related parameter to *N* independent on *L* is the *height equivalent to a theoretical plate H* (HETP) defined as *L*/*N*.

For achieving a good separation, the value of *R* should be higher than 1.0 and *R* is larger when *α*, *N* and *k'* are larger. These parameters can be increased by improving the columns properties. Since a larger *k*' indicates a longer retention time *tR*, which is not usually desired, the increasing of *R* is achieved mainly by increasing *α* and *N*. The increase of *N* for a given *L* is achieved by decreasing *H*. The value of *H* depends on the linear flow rate *u* by the following expression known as van Deemter equation:

$$H = A'd\_p + B'\frac{D}{u} + C'\frac{d\_p^2}{D}u \tag{4}$$

In formula (4), *D* is the diffusion coefficient of the mobile phase, *dp* is the diameter of the particles in the column, and *A'*, *B*<sup>0</sup> , and *C*<sup>0</sup> are coefficients that depend on the nature of stationary and mobile phase.

In addition to *R*, many other parameters are used for the characterization of an HPLC separation such as peak asymmetry *As*, which shows the deviation from the ideal Gaussian shape of the chromatographic peak, column backpressure *Δp*, which is the difference between the pressure at the column inlet and that at the outlet of the column, etc. Column backpressure is described by the following formula (known as Darcy equation):

$$
\Delta p = \frac{\eta u \phi\_r L}{d\_p^2} = \frac{\eta \phi\_r L^2}{d\_p^2 t\_0} \tag{5}
$$

In formula (5), *η* is the mobile phase viscosity, and *ϕ<sup>r</sup>* is a column flow resistance factor. Also, the columns are characterized by several other parameters and properties such as the construction of particles (porous, core-shell, etc.), uniformity of particles dimensions, porosity of the stationary phase, percent coverage of the solid support with the active phase, resilience of the stationary phase to a specific pH range of the mobile phase, resilience to dewetting, etc. A more detailed description of many parameters and properties describing a separation and characterizing the chromatographic column can be found in various books about HPLC (e.g., [12]). Significant progress in HPLC is being made such that to obtain better separation (higher *R*), shorter run times, lower values for *As* and *Δp*, reproducible separations, etc. A main source of progress is the improvement in the making of the chromatographic column by modifying parameters such as *L*, *d*, *dp*, ε\*, *k'*, *H*, *As*, *ϕr*, etc., in a manner that will lead to better chromatography. Other properties of the modern columns that are not captured with these parameters are also being improved and will be further discussed.

#### **3. Trends in the column physical dimensions**

The column physical dimensions are its length and internal diameter (i.d.). The common column lengths are between 30 mm and 250 mm with typical lengths of 50, 100, 150, 250 mm. The i.d. of the column is used to classify the columns as standard (3.0–4.6 mm i.d.), minibore (2.0–3.0 mm i.d.), microbore (0.5–2.0 mm i.d.), capillary (0.2–0.5 mm i.d.), and nanoscale (0.05–0.2 mm i.d.). The tendency of modern

columns is to have them shorter and narrower leading to shorter run times and the use of less solvent. However, for a given *H*, the decrease in column length *L* leads to a lower value for *N*. The improvements in the stationary phase such that the columns have lower *H* allow the use of shorter columns maintaining a desirable *R*.

The use of narrower columns leads to higher linear flow rate *u* for a given volumetric flow rate *U* resulting in shorter retention times. This can be seen based on Eq. (3) and from the dependence of *<sup>t</sup>*<sup>0</sup> on *<sup>u</sup>* that give *tR* <sup>¼</sup> *<sup>k</sup>*<sup>0</sup> <sup>þ</sup> <sup>1</sup> *<sup>L</sup>=<sup>u</sup>* . Although sorter retention times are desirable, the increase in *u* is limited by the decrease in the value of *H* (as indicated by van Deemter Eq. (4)) and by the increase in column backpressure (as indicated by formula (5)). For this reason, the most commonly utilized columns in current practice are those with standard and minibore i.d. The use of microbore, capillary, and nanoscale column encounters problems with a decrease efficiency (decreased *α*, increased *H*) [13, 14]. In addition to that, the microbore and narrower columns have a low loading capacity (maximum amount of sample that can be loaded on the column) leading to requirements for the increased sensitivity of the detector. For narrower columns, a compromise in setting *U* must be made such that a faster chromatography is obtained but the associated increase in the value of *H* does not preclude a good separation. A study for the evaluation of the possibilities to use narrower columns indicated that an optimum i.d. is around *d* = 1.5 mm, which achieves short retention times and low solvent use with good column performance [5].

The tendency to use shorter and narrower column in order to achieve shorter run times and use of less volume of mobile phase will continue in the future [15]. The production of columns with smaller *H*, higher *α*, and the progress in the instrumentation allowing the use of higher backpressure for the chromatographic columns as achieved currently with ultrahigh-pressure chromatographs (or ultra-performance LC, UPLC) that can generate up to 1300 bar, will continue to allow the decrease in column length and diameter. In parallel with the developments of commonly used columns for routine analytical laboratories, significant effort is made in developing novel experimental columns using miniaturization, very high column backpressure, as well as special active phases, etc. The progress in the sensitivity of the detectors used in HPLC/UPLC will allow the use of smaller and more diluted samples to overcome the lower loading capacity of smaller columns.

#### **4. Trends in the structure and composition of solid support of stationary phase**

The most common type of stationary phase in HPLC and UPLC is made from small particles (typically 1.7–10.0 μm in diameter), which are packed in the body of the column. Monolithic columns are also utilized and are made from a single rod of a solid porous material. Because hydrated porous silica can have a very large surface and can be derivatized to bind an active phase, it is the most common material used as solid support to make the particles and also some monoliths for HPLC. This silica usually has a bonded, grafted, or coated layer of organic material. This organic layer is the active part of stationary phase involved in the separation process, but the silanol groups from the uncovered surface of silica also participate in the separation. In case of hydrophilic interaction liquid chromatography (HILIC) and in normal phase chromatography (NPC), bare silica can be used as active phase without additional coverage due to its polar character.

Not only silica can be used as support for the active stationary phase. Materials such as hybrid organic–inorganic still based on a hydrated silica but containing organic groups such as -CH2-CH2- in its structure can be used as support. Also hydrated zirconia, titania, ceramic hydroxyapatite, or organic polymers can be used as solid support. The progress regarding the solid support is made in two directions, one being the physical characteristics of the support and the other its chemical properties. These characteristics are further discussed separately.

#### **4.1 Physical characteristics of stationary phase support**

For particles used as solid support, one first characteristic is the physical type, which can be fully porous, core-shell, or pellicular. Porous particles (1.7–10 μm in diameter) have a porous structure for the entire particle, core-shell have a solid nonporous core 1.5–3 μm in diameter surrounded by a porous outer shell 0.3–0.5 μm in depth. Pellicular particles are solid nonporous spheres covered with a thin layer of stationary phase. Fully porous and core-shell particles are widely utilized in common HPLC practice, while pellicular particles are less common because of their reduced loading capacity. Core-shell particles offer better peak shape (lower *H* values) compared with fully porous particles and are likely to continue to be used even more frequently in the future. Generally, they are characterized by higher values for phase volume ratio *Ψ* than monolithic columns, but lower *Ψ* than fully porous particles [16].

The particles are also characterized by (average) diameter *dp*, the shape of the particles, which can be irregular or spherical, the uniformity of the particles dimension, the surface area, the pore size and volume, the tortuosity and the uniformity of the channels in the particle, the structural rigidity. The dimension of particles *dp* with diameters of 5 μm, 3 μm, 2.1 μm, 1.8 μm, 1.7 μm is commonly used, and smaller particles lead to lower *H* values as indicated by formula (4). An empirical formula shows how *N* depends on *dp* as follows:

$$N \approx \frac{1000 \text{ L}}{\text{Ct} \cdot d\_p} \tag{6}$$

In formula (6), *Ct* can be 2, 2.5, or even 3, depending on other particle characteristics. The use of core-shell particles and dimensions of 1.7–1.8 μm leads to columns having the values for *N* per m (*L* = 1 m) as high as 200,000–300,000 [17].

At the same time with increasing *N*, lower *dp* leads to higher *Δp* as indicated by formula (5). This increase imposes limitations on how small the particles can be made. However, the tendency to use smaller particles to obtain columns with higher *N* is likely to continue in the future in spite of the increase in the backpressure, as the capability of the HPLC/UPLC pumps to deliver higher pressures increases.

Regarding particle shape, spherical particles show lower *H* values compared with particles of irregular form, and more uniform values of particle size show lower *H* values compared with particles of various sizes (particle size distribution is described by a parameter *d*90/*d*<sup>10</sup> and values lower than 1.2–1.3 indicate good homogeneity). Particles in modern columns have spherical shape and low *d*90/*d*<sup>10</sup> values, and these characteristics will continue to be maintained. Similar to the external aspect of particles, the internal structure regarding the channels in the porous material can be more or less homogeneous. The uniformity of particles interior is also contributing to a lower *H* value for the column.

Another physical characteristic of the stationary phase is its surface area [18]. For silica, common values for surface area are between 100 m<sup>2</sup> /g and 300 m<sup>2</sup> /g. The trend for modern stationary phases is to have particles with larger surface area since they can be coated with more active phase (increased *Ψ* value). However, the strength of stationary phase tends to decrease when stationary phase surface area increases. High silica strength (HSS) support is available now, allowing its use without restrictions in UPLC-type conditions where the back pressure of the column can be up to 900–1000 bar.

The pore size of the porous solid support is commonly characterized as small pores (with diameter below 60 Å), medium (in the range 60–150 Å), and large (of about 300 Å or larger). Common silica pore size is around 100 Å. However, the selection of the pose size depends on the type of molecules to be separated on the stationary phase. For small molecules (with *Mw* lower than about 3000 Da), the pore size of 100 Å is adequate, but for larger molecules, special phases with large pore size (about 250 Å) should be used. The adequacy of the pore size for the type of molecules to be separated, and in particular for the separation of proteins, is a field where significant development takes place [19].

Monoliths have a porous structure characterized by mesopores (pores between 2 and 50 nm in diameter) and macropores (about 4000–20,000 nm in diameter). For silica monoliths, the silica skeleton is 1–2 μm thick and has a void volume of almost 80% of the entire column volume. Polymeric monoliths have similar void volume. Since monolithic columns produce a lower pressure drop as compared with columns containing particles with similar characteristics, the monoliths are a promising material to be used as support for chromatographic columns. Monoliths are also successfully utilized in the construction of capillary and nano-LC columns [20].

#### **4.2 Chemical characteristics of stationary phase support**

The two main aspects of the chemical characteristics of solid support, which are of interest, include: 1) its internal chemical composition and 2) the chemical functionalities allowing the binding of the active phase (in cases when the solid support does not act itself as the active phase). Regarding the internal chemical composition, the solid support can be made from silica, ethylene or propylene bridged silica, hydrated zirconia, hydrated alumina, aluminosilicates, porous graphitic carbon, zeolites, or various organic polymers such as polystyrene cross-linked with divinylbenzene (PS-DVB) [21], methacrylates, etc. More recently, metal–organic frameworks (MOFs) were experimentally evaluated as support for HPLC stationary phases [22].

The most common support material is hydrated silica, which is obtained in principle from a chemical reaction that generates silicic acids followed by condensation reaction of the type:

The resulting material contains numerous silanol groups that are further used for bonding the active phase. The purity of resulting hydrated silica is very important

#### *Progress in Technology of the Chromatographic Columns in HPLC DOI: http://dx.doi.org/10.5772/intechopen.104123*

since the presence of metal ions in its structure leads to undesired effects such as peak tailing in chromatography. Very high purity silica (indicated as *Type B*) is now common as support in chromatographic columns. The ethylene bridge silica (indicated as BEH technology by Waters or TWIN technology or EVO by Phenomenex) [23] is also a common support offering excellent resilience to the strong acidic or basic character of the mobile phase (pH range of stability 1 to 12). This is a significant advantage compared with the range of pH stability of common silica, which is between 2.5 and 7.5. Ethylene bridge silica can be prepared from hydrolytic condensation of bis (triethoxysilyl)ethane and tetraethoxysilane using a small amount of water in a reaction schematically written as follows:

Ethylene bridge silica is an excellent material to be used as solid support for the stationary phase in HPLC, and its use will continue probably becoming even more common.

Regarding the other materials, columns based on hydrated zirconia are commercially available, but in general, they have lower chromatographic performance compared with those based on silica mainly due to numerous Lewis acid sites present on the stationary phase. Commercially available are also porous graphitic carbon columns. In order to achieve a large surface area, graphitic stationary phases are made using silica as template on which a layer of an organic material is applied followed by pyrolysis in an inert atmosphere to generate graphite. This is followed by the dissolution of silica template [24]. This type of column has a strong hydrophobic character, but some problems with surface homogeneity remain to be solved.

For the organic polymers, various procedures are used to obtain porous materials [21]. In some cases, these porous polymers may also act as the active phase, and in other cases they contain reactive groups on which an active phase is further bound. Among the problems with organic polymers as support material are the limitation of their structural rigidity and propensity to swelling in certain mobile phase compositions. Although for some types of HPLC (e.g., reversed phase or HILIC), the use of silica-based support is by far more common, organic polymers are frequently used in ion exchange chromatography and in size exclusion chromatography. Also, organic polymers are frequently used for making monolith-type columns [25].

The second important property of the stationary phase support is its capacity to react with a derivatization reagent with the goal of attaching a desired type of functionality such as aliphatic chains of 8 or 18 carbon atoms (C8 or C18) typically used in reversed phase (RP) type of HPLC. For silica support, the reacting capacity is assured by the presence of numerous silanol groups on the silica surface. The number of OH groups per unit mass of silica is characterized by silanol density *αOH* expressed by the formula:

$$a\_{OH} = 602.214 \frac{\delta\_{OH}}{\mathcal{S}\_{surf}} \tag{9}$$

In formula (9), *Ssurf* is the surface in m2 /g, and *δOH* is the amount of silanol groups (in mmol/g). The value of *αOH* typically varies between 4.1 and 5.6 OH groups per nm2 .

A different type of phase support still based on silica but with different active groups is hydride-based silica (known as type C silica [26]). This material is obtained using a reaction of the type indicated below:

This type of silica can be used as normal phase without further derivatization or can react to attaching further organic groups that will operate as active phase.

Reactive groups used for further derivatization can also be present in various organic polymers. For example, many acrylate-type polymers are synthesized to contain glycidyl groups. These act as reactive sites on the porous polymer surface on which the desired functionalities can be bound. The use of organic polymers as solid support is an attractive alternative in particular related to the efforts toward miniaturization of HPLC columns, where 3D printing technology can be applied to make capillary and nano columns [27].

As described in this section, the most common stationary phase support is based on silica. Although numerous other types of support are continuously evaluated, significant progress is also being made in generating silica with better properties. One of the most promising directions is the preparation of ethylene-bridge silica, which offers an excellent stationary phase support, with good reactivity for binding the desired functional groups and with high resilience to the mobile-phase extreme pH values or composition. The use of ethylene-bridge silica in combination with core-shell type phase will continue to expand, and further progress is likely to continue for this type of phases.

#### **5. Trends in the making of the active part of stationary phase**

In many types of chromatographic columns, the active phase intended to be involved in the separation is bound or coated on a porous solid support with a large surface area. In some types of HPLC/UPLC, the solid support acts as the active phase without being further chemically modified, and some details about this type of phases will also be further presented, but this section is dedicated to bonded active phases. The bonded phase on a solid support is a key part of the type of chromatography for which the phase is made. For example, for RP-HPLC, which is the most common type of chromatography, the bonded phase is made to have a hydrophobic character. For this purpose, hydrocarbon moieties with different number of carbon atoms are attached to the porous support. The most common such groups contain 18 aliphatic linear carbon chains (C18) or eight carbons chains (C8), but other hydrophobic groups can be bound. For HILIC-type columns, organic fragments containing diol groups, amide, amino, sulfonylethyl, etc., can be bound. For ion-exchange-type chromatography, the bonded groups can be -COO, -SO3 , or -NH3 + ,- N(CH3)3 + , etc.

#### **5.1 Progress in chemical reactions used for generating the active phase**

Various chemical reactions are utilized for derivatizing the solid porous support of a stationary phase. The active phase can be directly attached to the silica surface, but

variants of this procedure including the use of a pre-derivatization followed by a second one are also used. A typical derivatization reaction can be written as follows:

$$\begin{array}{c} \begin{array}{c} \text{(11)} \\ \text{(12)} \\ \text{(13)} \\ \text{(14)} \\ \end{array} \end{array} \begin{array}{c} \begin{array}{c} \text{(12)} \\ \text{(14)} \\ \text{(15)} \\ \text{(16)} \\ \end{array} \end{array} \begin{array}{c} \begin{array}{c} \text{(16)} \\ \text{(17)} \\ \text{(18)} \\ \text{(19)} \\ \end{array} \end{array} \begin{array}{c} \begin{array}{c} \text{(16)} \\ \text{(19)} \\ \text{(19)} \\ \text{(19)} \\ \end{array} \end{array} \begin{array}{c} \begin{array}{c} \text{(17)} \\ \text{(18)} \\ \text{(19)} \\ \text{(19)} \\ \text{(19)} \\ \end{array} \end{array} \begin{array}{c} \begin{array}{c} \text{(18)} \\ \text{(19)} \\ \text{(19)} \\ \text{(19)} \\ \text{(19)} \\ \end{array} \end{array} \begin{array}{c} \begin{array}{c} \text{(19)} \\ \text{(10)} \\ \text{(10)} \\ \text{(10)} \\ \text{(10)} \\ \end{array} \end{array} \begin{array}{c} \begin{array}{c} \text{(10)} \\ \text{(10)} \\ \text{(10)} \\ \text{(10)} \\ \text{(11)} \\ \text{(11)} \\ \end{array} \end{array}$$

The reactive substituent X can be Cl, but also OCH3, OC2H5, etc. The substituent *R* will determine the active phase (C8, C18, amino, cyano, and many others). Numerous variants of reaction (11) were applied for the derivatization of the silica solid support. In some of these variants, the CH3 groups are replaced with other reactive substituents such as OC2H5, and the resulting material has the capability to further react. The procedure of using di- or tri-functional reagents (containing two or three reacting groups) leads to surfaces with different degree of coverage [28]. One important type of variant in derivatization is the formation of a single layer of attached active groups (indicated as *horizontal polymerization*) [29], or the formation of a multiple layer polymer on the silica surface (*vertical polymerization*). In vertical polymerization, a small amount of water is usually added during the derivatization process such that some of the bounded groups containing reactive fragments such as OC2H5 will be hydrolyzed generation active -OH functionalities that can be further reacting with the derivatization reagent. In this manner, the derivatization can be repeated a number of times [30]. For the preparation of hydrophobic phases with the use of vertical polymerization, various levels of carbon load (C%) can be placed on silica surface, C% varying depending on the procedure between 5% and 30%.

As the sensitivity of detection in HPLC/UPLC is becoming higher and higher in particular with the development of mass spectrometric (MS) and MS/MS detectors, one important quality of the chromatographic columns is to have a very low background that may be caused by small molecule "leaking" into the mobile phase left from the manufacturing process of the stationary phase. The use of trifunctional reagents and new procedures to achieve the derivatization of the solid support (usually of silica) led to chromatographic columns with very low bleed, higher resilience to a wide range of composition for the mobile phase and good reproducibility of the separation.

After derivatization, silica surface (and also the surface of hydrated zirconia or alumina) still remains with a considerable number of underivatized OH groups (silanols in case of silica). These silanol groups interact with the analytes from the mobile phase such that not only the *R* groups forming the active phase influence the separation but also the silanols. This effect is undesirable in some cases, and the process of endcapping is used for diminishing (or removing) the silanol interference. The endcapping consists of additional derivatization that places on silanols small organic groups such as Si(CH3)3. Steric hindrance that precludes the dense covering of the silica surface with larger groups such as C8 or C18 is avoided by using derivatization with small groups such as trimethylsilyl (TMS). Repeated derivatization with the endcapping reagent such as chlorotrimethylsilane is usually performed when most silanol groups are intended to be covered.

The process of silica surface derivatization offers numerous possibilities to generate stationary phases with different properties [31]. A large variety of columns is

commercially available, and they are tailored for specific utilization. Derivatization and endcapping are used to obtain stationary phases with higher resistance to extreme pH of the mobile phase (e.g., controlled surface charge or CSH type columns), with extra dense bonding (XDB) of the active phase, with different degrees of hydrophobicity, with polar endcapping groups (e.g., CH2OH), or with embedded polar groups in the hydrophobic chain of the active phase [32, 33]. Besides the procedures summarily indicated above to derivatize the solid support, various other derivatization procedures are reported in the literature [34]. Also, alternative procedures to obtain the active phase such as direct synthesis of silica materials with an active bonded phase surface [35] can be used. Progress in the synthesis of monoliths, as well as of stationary phases based on organic polymers, is also being made [36]. One such example is the production of latex-agglomerated ion exchangers.

A variety of other procedures are available for producing the active phase for HPLC and UPLC columns (e.g., [37]). Some of these procedures are kept undisclosed by the column manufacturers and some are reported in the literature. Also, a variety of novel procedures for attaching the active phase on the solid support are developed, such as grafting of pre-synthesized polymers [38, 39], or direct synthesis of the stationary phase containing the desired functionalities [40, 41].

Stationary phases with better performance including better resolution *R*, lower asymmetry *As*, resilience to a wider pH range of the mobile phase, capability to work in 100% aqueous mobile phase (resilience to de-wetting), and production of phases with more complex structure than a single functionality are achieved using a diversity of procedures to derivatize the porous solid support. The use of trifunctional derivatization reagents and special endcapping techniques was among the important procedures to achieve this goal, and the use of these procedures is likely to continue to be improved in the future.

#### **5.2 Improved properties of active stationary phase**

The modern columns have various benefits from the improvements in the synthesis of the active phase. For example, from the derivatization with trifunctional reagents, the active phase is more homogeneous and stable, with reduced access of the analytes to the free silanols and more reproducible chromatography. The horizontal polymerization (derivatization) has the advantage of higher homogeneity and reduced presence of free silanols, while vertical polymerization leads to phases with a higher mass of active phase (larger *Ψ*). The new active phases allow the separation to be based on wider types of interaction, and preparation of phases with mixed mode functionalities is more and more common. The progress made in the endcapping process, the capability to use polar endcapping, and the introduction of controlled surface charge (CSH) contribute to the extension of pH range of column stability. Also, the stability of columns in time (to be used for a larger number of injections), the low bleed allowing the use of the columns with very sensitive detectors without generating a high background signal are important factors in the increase of column quality.

Besides the making of columns with improved characteristics, the increased variety of available columns is another direction in which considerable progress is being made. This variety of columns allows a better selection for a specific task, and also, as bidimensional HPLC is sometimes needed for the separation of complex samples, the column variety offers choices for orthogonal separations [42].

#### **6. Diversity of HPLC columns**

Under the acronyms HPLC or UPLC are included a number of similar techniques that have significant differences regarding the mechanism involved in the separation. According to the separation mechanism, a specific type of chromatographic column is used. Some of HPLC techniques are common, and some are more special having lower utilization. One main type of common HPLC/UPLC is RP-HPLC, which is used for the separation of molecules having in their structure hydrophobic moieties but frequently additional polar groups. Other common HPLC types are HILIC used for the separation of strongly polar molecules, ion exchange HPLC used for the separation of molecules capable to ionize, chiral HPLC used for the separation of enantiomeric molecules, size exclusion HPLC used for the separation of molecules based on their molecular size (more precisely hydrodynamic volume), and affinity/immunoaffinity HPLC. Various other techniques less frequently utilized are derived from the main types, and examples of such techniques are ion pair chromatography, hydrophobic interaction, normal phase, ion moderated, etc. The active stationary phase for each of those techniques has specific structures. Regardless of the column type, all modern columns benefit from the progress in the solid support in particular by using high-purity silica and ethylene bridge silica, from the use of core-shell particle construction and the advances in the making of monoliths. Some specific aspects for different types of HPLC/UPLC are further discussed.

#### **6.1 Columns for RP-HPLC**

Frequently used for the analysis of a large range of compounds, from small molecules to proteins and from highly hydrophobic to rather polar ones, RP-HPLC is the most commonly applied HPLC technique. To this extensive use is associated a significant number of RP type columns many of them commercially available. For RP-HPLC the active stationary phase contains hydrophobic groups, the most common being C18 and C8 phases. The hydrophobic character of the stationary phase in RP-HPLC can be modified by using the active phase with specific groups. Besides C18 and C8 that are very common, aliphatic C2, C4, C12, C14, C20, C22, C27, C30, cyclohexyl, phenyl, diphenyl, C6-linked phenyl, pentafluorophenyl, cyanopropyl, etc., can be used to create a hydrophobic surface. The hydrophobic character of these phases represents one criterion to differentiate them. However, even for columns containing the same type of phase, such as C18, many variations in the active phase structure are possible. The variations may include the type of bonding (mono, di, or trifunctional), the type of polymerization (horizontal or vertical), the carbon load, the density and uniformity of the coverage of solid support (e.g. of silica), and the variations in endcapping. Some hydrophobic stationary phase may contain polar imbedded groups [43]. Various imbedded groups in aliphatic chains were reported in the literature [32], and some are present in commercially available columns. Some of these groups include ether, amide, urea, carbamate, sulfone, thiocarbamate, etc. These groups are used to modulate separation of many types of organic compounds that have in their molecule polar groups. In addition, the imbedded polar groups "shield" the silica residual silanols for interacting with the analytes (in particular with highly basic ones) leading to a reduced silanol activity of the stationary phase (as in Symmetry Shield type columns) and also to better resilience to extreme pH values of the mobile phase.

Evolution of stationary phases in RP-HPLC (and RP-UPLC) took place in two directions: 1) perfecting common columns such as C18 or C8 columns and 2) exploring the binding of various less common groups on the solid support. Perfection of common columns is being done by working with either fully porous or core-shell particles, using special substrates usually high-purity silica or ethylene bridged silica, controlling the derivatization to be very homogeneous, and using special endcapping. By endcapping with TMS groups, the polarity of the silanols is reduced, but the extent of this process can vary from column type to column type, and some C18 columns are intentionally left with some silanol activity for interacting with polar molecules. The use of endcapping with small polar groups also brings distinctive properties to the RPtype columns. Adding special procedures such as CSH or XDB technologies, the variety of RP columns becomes even larger. CSH technology takes advantage that the silica surface is usually slightly negatively charged due to the dissociation of silanols. This charge can be neutralized by adding specific reagents such that the surface reactivity is decreased. The technology is applied to ethylene bridge particles by incorporating a low level of surface charges on stationary phase particles. Also, the construction of phases with C18 or C8 active phase but based on silica with specifically larger pores (e.g., 250 Å) is a promising path for the separation of large molecules such as proteins.

Regarding the binding of various less common groups on the solid support, special phases with bonded cholesterol or fullerene moieties were made, as well as columns with aliphatic chains having an unusual number of carbons (e.g., C3 or C4 for lower hydrophobicity or C30 for intended higher hydrophobicity) [44]. However, these types of experimental bonded phases did not generate columns with much different hydrophobic properties. The intimate mechanism of hydrophobic interactions caused by the "rejection" of the molecules containing hydrophobic moieties from a polar solvent and their "acceptance" in a hydrophobic stationary phase leads to a nonunique process of separation, as long as the accepting phase is less polar than the mobile phase (e.g., [12]). As a result, the choice of mobile phase composition in RP-HPLC plays an important role in the separation, and the differences in the properties of columns used in RP-HPLC are basically obtained by modulating the ratio of hydrophobicity and residual polar interactions and less by changing the phase hydrophobicity.

The use of hydrated zirconia as solid support, the use of coating of a silica base and not binding it, the use of organic polymers to make phases for RP-HPLC, or the use of porous graphitic carbon as stationary phase, although leading to a variety of columns to be used in RP-HPLC remained with a relatively limited utilization. Both trends of improving columns with common stationary phase such as C18, C8, phenyl, cyanopropyl, and experimenting with new active phases are likely to continue in the future. However, a considerably more impact for the progress is still expected from the improvements of common stationary phases.

#### **6.2 Columns for HILIC and NPC**

Important progress has been made in the construction of columns dedicated to HILIC separation. The active phase for these columns must be polar, and it is used with a mobile phase less polar than the stationary phase and containing water plus an organic solvent. Similar phases are used for NPC, but in this case the mobile phase is non-aqueous. Bare silica can be used as stationary phase in HILIC, and the improvements in the silica purity and homogeneity of silanol coverage made these columns

#### *Progress in Technology of the Chromatographic Columns in HPLC DOI: http://dx.doi.org/10.5772/intechopen.104123*

rather common. Bonded phases with groups such as diol, ether embedded+diol, amide terminal, polyamide, cyano (also used in RP HPLC) are common. Propylamine, diethylamine, or triazole groups are used to generate weak anionic active phases, sulfonylethyl groups are used to generate weak cationic active phase, and amino +sulfonic, amino+carboxylic groups are used to generate zwitterionic phases. Various other types of phases for HILIC applications were synthesized [45]. These phases have various polarities, but the spacer (handle) molecular fragment connecting the polar group with the silica base plays an important role in the separation. The same features as for RP-HPLC columns, including the coverage of support with the bonded phase, the pH resilience, the preparation procedure using mono-, bi-, or tri-functional reagents, the phase ratio are important for the column quality. Since in the HILIC separations not only the polar interactions are important in the separation, but also the hydrophobic interactions play a role, the carbon load (caused by the spacer) also influences the separation characteristics. Some HILIC columns are also endcapped, and this process changes the stationary phase characteristics in a similar manner as for the RP-HPLC. Besides common phases used in HILIC separations, special stationary phases were also known. Such phases were made with bonded cyclodextrin, bonded perhydroxyl-cucurbit[6]uril, polyhydroxyethyl-aspatamide, polysuccinimide [46], etc. One example of a structure of a zwitterionic stationary phase containing sulfonylalkylbetaine groups used in HILIC separations is indicated below:

Because of the proximity of the positive and negative charged groups in the structure, the phase is not used as a zwitterionic ion exchanger.

Stationary phases based on organic polymers are also used for HILIC separations [47]. However, more common are still the silica-based columns.

#### **6.3 Columns for ion exchange HPLC and related techniques**

Ion exchange (IC) stationary phases are classified as cation exchange phases (weak, medium, and strong), anion exchange phases (also weak, medium, and strong), zwitterionic, and amphoteric. The phases contain groups attached through a handle on silica or on an organic polymeric support. Specific groups such as -COO, -PO3H, -SO3 , etc., generate cationic phases, groups such as -NH3 + , -NH2(CH3)]+ , -N(CH3)3] + , [N(CH3)2(CH2CH2OH)]+ , [N(C2H5)(CH3)2] <sup>+</sup> generate anionic phases, and groups such as -N(CH3)2 + -(CH2)n-SO3 or -CH(SO3 )-(CH2)n-N(CH3)3 + generate zwitterionic phases. While for RP-HPLC and HILIC phases, the use of organic polymeric support is less common, for ion exchange phases the use of polymeric support is more common. A specific type of polymeric support is the latex agglomerated type. The latex agglomerated ion exchange particles contain an internal core that has ionic groups on its surface. On this surface is attached a monolayer of small diameter particles that carry functional groups having bonded ions with an opposite charge with those of the support. The groups of the outer particles have the double role of attaching the small particles to the support and also to act as an ion exchanger for the ions in the mobile phase. The advantages of this type of phase

include its stability to a wide range of pH of mobile phase and resilience to higher column backpressure compared with common polymeric columns. This is possible because the cross-linking of the polymer from the core particles can be very high.

Because the loading capacity for the same amount of stationary phase is typically larger for IC columns compared with RP or HILIC columns, and because the separation mechanism is based on ionic interactions, which is different from that in RP-HPLC and HILIC, the capillary columns in IC are more successfully utilized. Such columns are used with a low flow rate (e.g., 0.01–0.02 mL/min) that increases the sensitivity of the conductivity detector used frequently in IC separations [48, 49].

Ion chromatography is extensively used in the separations of proteins and nucleic acids [50], and continuous progress is being made with new phases of IC type. Many such new phases are commercially available [10].

Special ion chromatographic columns are also applied in ion-moderated and ligand exchange chromatography. These types of columns are used for the separation of carbohydrates, sugar acids, as well as lipids. For example, difficult separation such as those between cis and trans lipids and fatty acids can be achieved using an ionmoderated columns containing Ag<sup>+</sup> ions [51]. In spite of the need for ion-moderated chromatography for the separation of important types of analytes, some of the existent columns dedicated for ion-moderated chromatography require relatively long run times for the separation. For this reason, development of new ion-moderated type columns would be highly desirable.

The ion exchange stationary phases and columns are in continuous development, and in particular mixed mode phases containing ion exchange type moieties are demonstrated to be very useful in separations. A discussion dedicated to mixed mode phases is also included in this chapter.

#### **6.4 Columns for chiral separations**

The increased demand of analysis of a variety of pharmaceutical drugs, many of them with chiral character, required constant development of chiral columns. Other fields of chemical analysis also required chiral separation. For example, the increased use of vaping and the proliferation of companies producing synthetic nicotine required the development of sensitive methods for the analysis of nicotine enantiomers [52]. Active stationary phase for chiral separation can be of different types, which include: brush or "Pirkle" type, cellulose based, cyclodextrin or cyclofructanbased, amylose-based, crown-ether-based, macrocyclic antibiotic type, protein based, ligand exchange type, chiral synthetic polymer type [53, 54], etc. All these phases contain various types of chiral centers. In spite of the existence of such a variety of columns, the need for stationary phases offering better enantioresolution is still actual. Many chiral columns must be used in non-aqueous mobile phase (NPC type chromatography), and fewer phases allow the use of water in the mobile phase for RP, HILIC, or IC-type utilization. However, many chiral compounds are highly polar and some are even insoluble in non-aqueous media. In addition, the widespread electrospray type of MS detection (ESI-MS) generates weak or no response when a mobile phase with no water is used for the separation. For these reasons, continuous effort is made to develop chiral columns that work in RP, HILIC, or IC mode.

The improvements of stationary phases for chiral separations follow the same lines as the one utilized for other types of columns. The use of core-shell type particles (e.g., [55]), smaller particle size, monoliths, various types of phases containing chiral centers such as glicopeptides, and macrocyclic antibiotics, as well as more common ones

such as derivatized polysaccharides [56, 57] is providing important tools for obtaining better, more efficient types of chiral chromatographic columns [58].

#### **6.5 Columns for size exclusion HPLC**

Size exclusion HPLC (SEC) is a technique used for the separation of analytes according to their molecular size (hydrodynamic volume), and it is applied for the separation of macromolecules of different sizes and of macromolecules from small molecules. Ideally, only the size of the molecule should contribute to the separation, but it is common that some energetic interactions (e.g., of polar type) also take place between the stationary phase and the analytes. These energetic effects can modify the intended purpose in which only the size affects the separation. As the molecular size is usually proportional with the molecular weight *Mw* of a molecule, size exclusion is also used for the evaluation of *Mw* for macromolecules. Depending on the solubility of the polymers in an aqueous solvent or in an organic solvent, SEC is indicated as gel filtration chromatography (GFC) or as gel permeation chromatography (GPC), respectively. The stationary phases in SEC can be based on porous silica or on other inorganic materials, but very commonly on organic polymeric materials. Polymeric materials for making the stationary phase are more frequently used in SEC than in other HPLC procedures [59]. A common material for SEC stationary phase is polystyrene-divinylbenzene (PS-DVB) with different cross-linking degrees, but also gels based on dextran or agarose, hydroxylated poly(methyl methacrylate) (HPMMA), and polyvinylalcohol (PVA) copolymers are used. The separation phase should be made with large and controlled pore dimensions.

Among the requirements for a good stationary phase in SEC is to have homogeneous pores, to be as inert as possible and have minimal energetic interactions with the analytes, and to be resilient to high HPLC-type backpressure. These requirements are not very simple to achieve. The control of pose size such that they are as uniform as possible can pose difficulties during manufacturing. Silica-based SEC columns can be made using bare silica, but also bonded phases containing, for example, diol groups on silica are produced. The use of silica with large pores leads to lower resilience to the backpressure. In addition, the reduction of energetic interactions with the silanol groups on silica is not simple. For the polymeric phases, the problem of resilience to higher backpressure is even more stringent than it is for the silica-based phases. The use of special cross-linked polymers alleviates this problem. Also, SEC columns usually require long run times for separations, but new developments such as making core-shell type stationary phases shorten the separation time. Also, as the pressure resistance of the used materials is better, the reduction in the particle size of the phase contributes to improvements in SEC chromatography [60]. New stationary phases use all those procedures to improve the chromatographic columns for SEC.

#### **6.6 Columns in affinity, immunoaffinity, and aptamer-type HPLC**

In affinity/immunoaffinity chromatography (IAC), the stationary phase contains on its surface an immobilized biological complement of the analytes from the mobile phase [61]. Examples of pairs of biological complement and the analytes are antigens and their antibody, lectins and glycoproteins, metal ions and proteins containing amino acid residues that have affinity for the ion (e.g. histidine), biotin and avidin, etc. The solid support for the stationary phase can be silica, synthetic organic polymers, agarose (the neutral gelling fraction of the complex natural polysaccharide

agar), cross-linked agarose, cross-linked dextrans (sepharose, sephacryl), cellulose, etc. It is typical for the solid support in affinity chromatography to have large pores, between 300 Å and 500 Å because the technique is used for the separation of large molecules (e.g., proteins and nucleic acids). The stationary phase particles can be porous] or nonporous [62] and also can be monolithic [63]. A variety of techniques are used to make stationary phases for IAC, using different procedures for the immobilization of biological complement ranging from covalent attachment to adsorptionbased methods. For example, the immobilization of antibodies can be done through their amine groups by using a support that has been activated with reagents such as N, N0 -carbonyldiimidazole, cyanogen bromide, N-hydroxysuccinimide, or tresyl chloride/tosyl chloride [64]. New phases are continuously reported for this technique, with a variety of active phases including different types of proteins, aptamers [65], and dye ligands [66]. Continuous progress is also made regarding stationary phases for biomimetic LC that mimic the interactions in natural biological systems [67, 68].

#### **6.7 Mixed-mode HPLC columns**

Preparation of stationary phases with mixed-mode active groups in which the separation is based on two or more types of main interactions is currently an important direction of development in HPLC [11]. Mixed-mode phases offer special separation capabilities and could be a simpler alternative to bidimensional separations that use orthogonal columns [69]. These phases may have reversed-phase and HILIC capabilities, reversed-phase and ion exchange capability, HILIC and ion exchange, or even more than two types of capability allowing for example reversed-phase/hydrophilic interaction/ion-exchange-type separations [70]. Some of the mixed mode phases also have chiral centers such that can be used for special chiral separations [71]. Porous or core-shell silica can be used for the preparation of mixed mode phases, and common functionalities such as C18, NH2, diol, SO3 �, etc., that are specific for one type of phase are used to obtain the mixed-mode phases. The main difference from single type of phase is that multiple functionalities are simultaneously present on the solid support. Synthesis of such phases frequently requires a sequence of derivatizations and strict control of the quality of the final product [72, 73]. The preparation of mixed-mode phases with organic polymers support, in the form of monoliths or using covalent organic frameworks, has also been described in the literature [74, 75]. Mixedmode stationary phases can also be made as having the active functionality based on ionic liquids moieties [71, 76].

#### **7. Conclusions**

The chromatographic column is a key component of HPLC instrumentation, and the extensive use of HPLC promoted the effort for obtaining better columns. These columns provide better separations in a shorter time, generating reproducible chromatography, have minimal bleed avoiding background for the detectors, are resilient to a wide pH range of the mobile phase, can be used with the mobile phase having 100% water, and have a longer utilization life. Progress in making the chromatographic columns has been achieved by various procedures such as the optimizing the chromatographic column dimensions, the use of smaller particles for the stationary phase, the use of monoliths, the use of core-shell type particles. Significant progress was also made in chemistry of stationary phase, both regarding the solid support and

#### *Progress in Technology of the Chromatographic Columns in HPLC DOI: http://dx.doi.org/10.5772/intechopen.104123*

the active phase bonded on it. Future progress is expected on the same lines of development for columns used in routine analyses. At the same time, experimental columns for HPLC miniaturization and enhanced efficiency are experimented and reported in the literature (e.g., [20, 77]). The parallel progress regarding the pumping system of HPLC instrumentation that can provide higher backpressure and wellcontrolled low flow rates, the precision of injecting systems (autosamplers), as well as the unprecedent increased sensitivity of detection in particular of MS and MS/MS type, were key for making possible some of the improvements in chromatographic column construction.

#### **Author details**

Serban C. Moldoveanu<sup>1</sup> \* and Victor David2

1 R.J. Reynolds Tobacco Co., Winston-Salem, NC, USA

2 Faculty of Chemistry, Department of Analytical Chemistry, University of Bucharest, Bucharest, Romania

\*Address all correspondence to: smoldov@aol.com

© 2022 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.

#### **References**

[1] https://www.waters.com/waters/en\_ US/ionKey-MS—microflow-UPLC-Sepe ration-with-iKey/nav.htm?cid= 134782630&locale=en\_US

[2] Bao B, Wang Z, Thushara D, Liyanage A, Gunawardena S, Yang Z, et al. Recent advances in microfluidicsbased chromatography - a mini review. Separations. 2021;**8**:3

[3] Vissers JPC. Recent developments in microcolumn liquid chromatography. Journal of Chromatography A. 1999;**856**: 117-143

[4] Recent developments in LC column technology, Supplement to LC/GC North America 36. 2018. No. s6

[5] Fekete S, Murisier A, Losacco GL, Lawhorn J, Godinho JM, Ritchie H, et al. Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems. Journal of Chromatography A. 2021;**1650**: 462258

[6] Bell DS. New chromatography columns and accessories for 2018. LC/ GC North America. 2018;**36**:234-247

[7] Bell DS. New liquid chromatography columns and accessories. LC/GC North America. 2019;**37**:232-243

[8] Recent developments in LC column technology, Supplement to LC/GC North America 38. 2020. No. s6

[9] Lopez DA, Green A, Bell DS. What is on your HPLC particle? A look at stationary phase chemistry synthesis. LC/GC North America. 2020;**38**:488-493

[10] Bell DS. New chromatography columns and accessories for 2020. LC/ GC North America. 2020;**38**:211-219

[11] Bell DS. Modern trends in mixed mode liquid chromatography columns. LC/GC North America. 2021;**39**:56-60

[12] Moldoveanu SC, David V. Essentials in Modern HPLC Separations. Amsterdam: Elsevier; 2013

[13] Lestremau F, Wu D, Szücs R. Evaluation of 1.0 mm i.d. column performances on ultra-high pressure liquid chromatography instrumentation. Journal of Chromatography A. 2010; **1217**:4925-4933

[14] Eggleston-Rangel R. Why use miniaturized columns in liquid chromatography? Benefits and challenges. The Column. 2021;**17**:21-25

[15] Blumberg LM. Column length is a structure-independent measure of solvent consumption in liquid chromatography. Journal of Chromatography A. 2022;**1662**: 462727

[16] Caiali E, David V, Aboul-Enein HY, Moldoveanu SC. Evaluation of the phase ratio for three C18 high performance liquid chromatographic column. Journal of Chromatography A. 2016;**1435**:85-91

[17] Henry RA. Impact of particle size distribution on HPLC column performance. LCGC. 2014;**32**:12-19

[18] Giaquinto A, Liu Z, Bach A, Kazakevich Y. Surface area of reversedphase HPLC columns. Analytical Chemistry. 2008;**80**:6358-6364

[19] Schuster SA, Wagner BM, Boyes BE, Kirkland JJ. Optimized superficially porous particles for protein separations. Journal of Chromatography A. 2013; **1315**:118-126

*Progress in Technology of the Chromatographic Columns in HPLC DOI: http://dx.doi.org/10.5772/intechopen.104123*

[20] Ghanem A, Marzouk AA, El-Adl SM, Fouad A. A polymer-based monolithic capillary column with polymyxin-B chiral selector for the enantioselective nano-high performance liquid chromatographic pharmaceutical analysis. Journal of Chromatography A. 2022;**1662**:462714

[21] Lloyd LL. Rigid macroporous copolymers as stationary phases in highperformance liquid chromatography. Journal of Chromatography. 1990;**544**: 201-217

[22] Xuan QQH, Zhang K, Chen X, Ding Y, Feng S, Xu Q. Core-shell silica particles with dendritic pore channels impregnated with zeolite imidazolate framework-8 for high performance liquid chromatography separation. Journal of Chromatography A. 2017; **1505**:63-68

[23] Wyndham KD, O'Gara JE, Walter TH, Glose KH, Lawrence NL, Alden BA, et al. Characterization and evaluation of C18 HPLC stationary phases based on ethyl-bridged hybrid organic/inorganic particles. Analytical Chemistry. 2003;**75**:6781-6788

[24] Pereira L. Porous graphitic carbon as a stationary phase in HPLC: Theory and applications. Journal of Liquid Chromatography Releted Technologies. 2008;**31**:1687-1731

[25] Nischang I, Teasdale I, Brüggemann O. Porous polymer monoliths for small molecules separations: Advancements and limitations. Analytical and Bioanalytical Chemistry. 2010;**400**:2289-2304

[26] Borges EM. Silica, hybrid silica, hydride silica and non-silica stationary phases for liquid chromatography. Journal of Chromatographic Science. 2015;**52**:580597

[27] Matheuse F, Vanmol K, Van Erps J, De Malsche W, Ottevaere H, Desmet G. On the potential use of two-photon polymerization to 3D print chromatographic packed bed supports. Journal of Chromatography A. 2022; **1663**:46273

[28] Hetem M, Van de Ven L, de Haan J, Cramers C. Study of the changes in mono-, di- and trifunctional octadecylmodified packings for reversed-phase high-performance liquid chromatography with different eluent compositions. Journal of Chromatography. 1989;**479**:269-295

[29] Wirth MJ, Fatumbi HO. Horizontal polymerization of mixed trifunctional silane on silica. 2. Application to chromatographic silica gel. Analytical Chemistry. 1993;**65**:822-826

[30] Kirkland JJ. Development of some stationary phases for reversed-phase high-performance liquid chromatography. Journal of Chromatography A. 2004;**1060**:9-21

[31] Qui H, Liang X, Sun M, Jiang S. Development of silica-based stationary phases for high-performance liquid chromatography. Analytical and Bioanalytical Chemistry. 2011;**399**: 3307-3322

[32] O'Sullivan GP, Scully NM, Glennon JD. Polar-embedded and polarendcapped stationary phases for LC. Analytical Letters. 2010;**43**:1609-1629

[33] Zhang Y, Chen M, Zhou S, Han H, Zhang M, Qiu H. A carbonylative coupling approach to alkyl stationary phases with variable embedded carbamate groups for high-performance liquid chromatography. Journal of Chromatography. A. 2022;**1661**: 462718

[34] Ashu-Arrah BA, Glennon JD, Albert K. Synthesis and characterisation of bonded mercaptopropyl silica intermediate stationary phases prepared using multifunctional alkoxysilanes in supercritical carbon dioxide as a reaction solvent. Journal of Chromatography. A. 2012;**1222**:38-45

[35] Hasegawa I. Co-hydrolysis products of tetraethoxysilane (TEOS) and methyltriethoxy-silane in the presence of tetramethylammonium ions. Journal of Sol-Gel Science and Technology. 1993;**1**: 57-63

[36] Oláh E, Fekete S, Fekete J, Ganzler K. Comparative study of new shell-type, sub-2μ fully porous and monolith stationary phases, focusing on mass-transfer resistance. Journal of Chromatography. A. 2010;**1217**: 3642-3653

[37] Moldoveanu SC, David V. Selection of the HPLC Method in Chemical Analysis. Amsterdam: Elsevier; 2017

[38] Yu S, Sha X, Zhou X, Guo D, Han B, Huang S, et al. Cyclodextrin-dendrimers nanocomposites functionalized high performance liquid chromatography stationary phase for efficient separation of aromatic compounds. Journal of Chromatography A. 2022;**1662**:462730

[39] Fan C, Quan K, Chen J, Qiu H. Comparison of chromatographic performance of co-grafted silica using octadecene respectively with vinylpyrrolidone, vinylimidazole and vinylpyridine. Journal of Chromatography A. 2022;**1661**:462690

[40] Wang X, Cheng S, Chan JCC. Propylsulfonic acid functionalized mesoporous silica, synthesized by in situ oxidation of thiol groups under template free conditions. Journal of Physical Chemistry C. 2007;**111**:2156-2164

[41] Zheng Y, Wan M, Zhou J, Dai X, Yang H, Xia Z, et al. One-pot method for the synthesis of β-cyclodextrin and covalent organic framework functionalized chiral stationary phase with mixed-mode retention mechanism. Journal of Chromatography A. 2022; **1662**:462731

[42] Pellett J, Lukulay P, Mao Y, Bowen W, Reed R, Ma M, et al. Orthogonal separations for reversedphase liquid chromatography. Journal of Chromatography A. 2006;**1101**: 122-135

[43] Silva CR, Bachmann S, Schefer RR, Albert K, Jardim ICSF, Airoldi C. Preparation of a new C18 stationary phase containing embedded urea groups for use in high-performance liquid chromatography. Journal of Chromatography A. 2002;**948**:85-95

[44] Rimmer CA, Sander LC, Wise SA. Selectivity of long chain stationary phases in reversed phase liquid chromatography. Analytical and Bioanalytical Chemistry. 2005;**382**: 698-707

[45] Bo C, Li Y, Liu B, Jia Z, Dai X, Gong B. Grafting copolymer brushes on polyhedral oligomeric silsesquioxanes silsesquioxane-decorated silica stationary phase for hydrophilic interaction liquid chromatography. Journal of Chromatography A. 2021; **1659**:462627

[46] Alpert AJ. Cation-exchange high performance liquid chromatography of proteins on poly(aspartic acid) –silica. Journal of Chromatography A. 1983;**266**: 23-37

[47] Xu M, Peterson DS, Rohr T, Svec F, Fréchet JM. Polar polymeric stationary phases for normal phase HPLC based on monodisperse macroporous

*Progress in Technology of the Chromatographic Columns in HPLC DOI: http://dx.doi.org/10.5772/intechopen.104123*

poly(2,3-dihydroxypropyl methacrylateco-ethylene dimethacrylate) beads. Analytical Chemistry. 2003;**75**:1011-1021

[48] Kuban P, Dasgupta PK. Capillary ion chromatography. Journal of Separation Science. 2004;**27**:1441-1457

[49] Wouters B, Bruggink C, Desmet G, Agroskin Y, Pohl CA, Eeltink S. Capillary ion chromatography at high pressure and temperature. Analytical Chemistry. 2012;**84**:7212-7217

[50] Mant CT, Hodges RS, editors. High-Performance Liquid Chromatography of Peptides and Proteins, Separation, Analysis, and Conformation. Boca Raton: CRC Press; 1991

[51] Momchilova S, Nikolova-Damyanova B. Chapter: Silver Ion Chromatography of Fatty Acids. Springer, Dordrecht: Encyclopedia of Lipidomics; 2016

[52] Moldoveanu SC. Interconversion of nicotine enantiomers during heating and implications for smoke from burn-down cigarettes, heat not burn devices, and vaping, Chirality. 2022;**34**:667-677

[53] Pei Y, Li X, Zeng G, Gao Y, Wen T. Chiral stationary phases based on lactide derivatives for high-performance liquid chromatography. Journal of Chromatography A. 2022;**1661**:462705

[54] Xie S-M, Yuan L-M. Recent development trends for chiral stationary phases based on chitosan derivatives, cyclofructan derivatives and chiral porous materials in high performance liquid chromatography. Journal of Separation Science. 2019;**42**:6-20

[55] http://www.azypusa.com/columnstationary-phases

[56] Younes AA, Mangelings D, Vander Heyden Y. Chiral separations in normal phase liquid chromatography: Enantioselectivity of recently commercialized polysaccharide-based selectors. Part I: Enantioselectivity under generic screening conditions. Journal of Pharmaceutical and Biomedical Analysis. 2011;**55**:414-423

[57] Chankvetadze B. Recent trends in preparation, investigation and application of polysaccharide-based chiral stationary phases for separation of enantiomers in high-performance liquid chromatography. TrAC - Trends in Analytical Chemistry. 2020;**122**:115709

[58] Wang X, Li H, Quan K, Zhao L, Li Z, Qiu H. Anhydride-linked β-cyclodextrinbonded silica stationary phases with enhanced chiral separation ability in liquid chromatography. Journal of Chromatography. A. 2021;**1651**:462338

[59] Wu C-S, editor. Handbook of Size Exclusion Chromatography. New York: M. Dekker; 1995

[60] Bouvier ESP, Koza SM. Advances in size-exclusion separations of proteins and polymers by UHPLC. TrAC - Trends in Analytical Chemistry. 2014;**63**:85-94

[61] Urh M, Simpson D, Zhao K. Affinity chromatography: General methods, in methods in enzymology, volume 463. Elsevier. 2009;**2009**:417-438

[62] Schiel JE, Mallik R, Soman S, Joseph KS, Hage DS. Applications of silica supports in affinity chromatography. Journal of Separation Science. 2006;**29**:719-737

[63] Sproβ J, Sinz A. Monolithic media for applications in affinity chromatography. Journal of Separation Science. 2011;**34**:1-16

[64] Hermanson GT, Mallia AK, Smith PK. Immobilized Affinity Ligand Techniques. Ney York: Academic Press; 1992

[65] Chi J, Zhu D, Chen Y, Huang G, Lin X. Online specific recognition of mycotoxins using aptamer-grafted ionic affinity monolith with mixed-mode mechanism. Journal of Chromatography A. 2021;**1639**:461930

[66] McGettrick A, Worrall M. Dyeligand affinity chromatography. In: Cutler P, editor. Methods in Molecular Biology. Vol. 244. Totowa: Humana Press; 2004. pp. 151-157. DOI: 10.1385/ 1-59259-655-x:151

[67] Verzele D, Lynen F, De Vrieze M, Wright AG, Hanna-Brown M, Sandra P. Development of the first sphingomyelin biomimetic stationary phase for immobilized artificial membrane (IAM) chromatography. Chemical Communications. 2012;**48**:1162-1164

[68] Carrasco-Correa EJ, Ruiz-Allica J, Rodríguez-Fernández JF, Miró M. Human artificial membranes in (bio) analytical science: Potential for in vitro prediction of intestinal absorption - a review. TrAC - Trends in Analytical Chemistry. 2021;**145**:116446

[69] Kazarian AA, Barnhart W, Long J, Sham K, Wu B, Murray JK. Purification of N-acetylgalactosamine-modifiedoligonucleotides using orthogonal anionexchange and mixed-mode chromatography approaches. Journal of Chromatography A. 2022;**1661**:462679

[70] Wang L, Wei W, Xia Z, Jie X, Xia ZZ. Recent advances in materials for stationary phases of mixed-mode highperformance liquid chromatograph. TrAC - Trends in Analytical Chemistry. 2016;**80**:495-506

[71] Zhou J, Ren X, Luo Q, Gao D, Fu Q, Zhou D, et al. Ionic liquid functionalized β-cyclodextrin and C18 mixed-mode stationary phase with achiral and chiral

separation functions. Journal of Chromatography A. 2020;**1634**:461674

[72] Sykora D, Rezanka P, Zaruba K, Kral V. Recent advances in mixed-mode chromatographic stationary phases. Journal of Separation Science. 2019;**42**: 89-129

[73] Fan F, Lu X, Liang X, Wang L, Guo Y. Preparation of hydrogel nanocomposite functionalized silica microspheres and its application in mixed-mode liquid chromatography. Journal of Chromatography A. 2022; **1662**:462745

[74] Yang F, Bai Q, Zhao K, Gao D, Tian L. Preparation of a novel weak cation exchange/hydrophobic interaction chromatography dual-function polymerbased stationary phase for protein separation using "thiol-ene click chemistry". Analytical and Bioanalytical Chemistry. 2015;**407**:1721-1734

[75] Zheng Y, Wan M, Zhou J, Luo Q, Gao D, Fu Q, et al. Striped covalent organic frameworks modified stationary phase for mixed mode chromatography. Journal of Chromatography A. 2021; **1649**:462186

[76] Liu D, Wang H, Liang M, Nie Y, Liu Y, Yin M, et al. Polymerized phosphonium ionic liquid functionalized silica microspheres as mixed-mode stationary phase for liquid chromatographic separation of phospholipids. Journal of Chromatography A. 2021;**1660**:462676

[77] Li H, Liu C, Zhao L, Xu D, Zhang T, Wang Q, et al. A systematic investigation of the effect of sample solvent on peak shape in nano- and microflow hydrophilic interaction liquid chromatography columns. Journal of Chromatography A. 2021;**1655**:462298

#### **Chapter 2**

## Perspective Chapter: Mixed-Mode Chromatography

*Ngoc-Van Thi Nguyen*

#### **Abstract**

In this chapter, we present mixed-mode stationary phases and their applications in the determination of nonpolar, polar, and charged compounds, as well as larger molecules such as peptides or proteins using a single column. Mixed-mode chromatography (MMC) has been growing rapidly in recent years, owing to the new generation of mixed-mode stationary phases and a better understanding of multimode interactions. Mixed-mode chromatography provides a wide range of selectivities and adequate retention of a variety of compounds, especially polar and charged molecules. In summary, this technique is particularly useful in the pharmaceutical analysis of drugs, impurities, biopharmaceuticals, and polar compounds in natural products.

**Keywords:** mechanisms, intermolecular interactions, mixed-mode chromatography, mixed-mode stationary phases

#### **1. Introduction**

The development of liquid chromatography is one of the most active areas of research in separation science, with applications in various fields, such as drug analysis, medicinal chemistry, agriculture, food chemistry, and bioanalysis. This study aims to determine the optimal working conditions for the effective and selective separation of chemical compounds. In the research process, the choice of optimal chromatography conditions is of prime importance, including the determination of a suitable liquid chromatography mode and the investigation of mobile phase characteristics (pH, type of organic modifier, mobile phase additive, etc.) [1].

Chromatographic retention processes can be divided into many types, such as normal-phase, reversed-phase, ion-exchange, hydrophobic interaction, hydrophilic interaction, and metal coordination chromatography. These chromatographic methods are known as single-mode chromatography because the retention of solutes in these chromatograms is dependent on a single-retention mechanism. For instance, in reversed-phase chromatography, problems may be encountered during the analysis of highly polar (or charged) compounds. Hydrophilic interaction chromatography (HILIC) is designed for the analysis of polar compounds; however, it is still affected by a range of challenges, such as low solubility in highly organic media, the amount of organic solvents used, the sample matrix that affects retention, and the retention extent of hydrophobic analytes that can be controlled. Ion-exchange chromatography can be used for charged molecules, but not for neutral analytes. Therefore,

mixed-mode chromatography (MMC) can be utilized to resolve some of the problems associated with each of the other mechanisms [2].

Mixed-mode chromatography (MMC) separates solutes by using a stationary phase that involves in the separation two or more types of interactions. Compared to single-mode chromatography, mixed-mode chromatography can simultaneously act on different functional groups of the solute, such as hydrophobic and ionic groups [3]. Mixed-mode chromatography is not a new technique. Many chromatographic matrices are based on rigid supports, such as cellulose, agarose, polyacrylamide, or silica gel, which are modified to produce specific functionalities on their surfaces. If the solute is a substance with numerous functional groups, such as amino acids, nucleic acids, peptides, and proteins, which are commonly found in biological samples, mixed-mode chromatography will exhibit a distinct behavior as opposed to that of single-mode chromatography [4].

Recently, MMC has been receiving increasing attention as an alternative or complementary tool to traditional chromatography (reversed phase, ion exchange, and normal phase) in pharmaceutical and biopharmaceutical applications because of its efficient selectivity and adequate retention of a variety of compounds—particularly polar and charged molecules. To achieve better solute retention characteristics, selectivity, and separation capabilities, mixed-mode stationary phases must be designed and synthesized based on the specific structural characteristics of different compounds. Additionally, the diversity of the mixed-mode stationary phase depends on the diversity of the analyte structure and its properties. It is expected that the applications of mixed-mode chromatography will increase in the future and serve as a power resolution for the separation and purification of biological substances.

#### **2. Mechanisms of mixed-mode chromatography**

#### **2.1 Mechanisms related to stationary phases**

#### *2.1.1 Classification of stationary phases by chemistry design*

In MMCs, stationary phases have been prepared using several types of stationary phases involving different mechanisms. According to the study design, mixed-mode stationary phases can be divided into four categories [2, 3].

*Type 1*: A mixed-mode stationary phase is created by combining two types of stationary phase particles (each with a single chemistry) and packing them into a single column (**Figure 1**). However, the major drawbacks of this approach are the non-homogeneity of the stationary phases and low batch-to-batch reproducibility.

*Type 2:* The surface of the stationary phase is modified with a mixture of ligands of different chemistries. This is a second-generation approach, but its disadvantages are similar to those of Type 1. Thus, Types 1 and 2 are not commonly used because of their performance limitations.

*Types 3 and 4:* Embedded (Type 3) and tipped ligands (Type 4) are the thirdgeneration mixed-mode phases that improve the reproducibility and homogeneity of the stationary phase. The functional groups (polar or ionic groups) of the embedded ligands are close to the pore surface, and the hydrophobic parts of the ligands extend to the mobile phase. In contrast, the tipped ligands have functional groups at the ends of the hydrophobic chains.

*Perspective Chapter: Mixed-Mode Chromatography DOI: http://dx.doi.org/10.5772/intechopen.104545*

**Figure 1.** *Types of mixed-mode stationary phases classified by chemistry designs [5].*

#### *2.1.2 Combinations of separation modes in MMC*

#### *2.1.2.1 Reversed-phase ion-exchange stationary phases (RP-IEX)*

Polar compounds, such as biologically active molecules, natural products, and drug metabolites containing several functional groups, tend to be weakly retained in the reversed phase, resulting in poor separation. With the combination of hydrophobic and ion-exchange mechanisms in the mixed-mode stationary phases, the selectivity and retention of both the hydrophobic and polar compounds are improved [4]. In addition, it is the most popular ligand in MMC and is mainly used for the separation of peptides, nucleotides, basic drugs, and their metabolites. The ligands consist of a hydrophobic part (alkyl chains or aromatic hydrocarbons) and an ionic part embedded in the end, middle, or vicinity of the hydrophobic part. Depending on the structure of the ionic part, four ion-exchange modes can be classified: quaternary amines are used as strong cation-exchange groups (SCX); primary, secondary, or tertiary amines are used as weak cation-exchange groups (WCX); sulfonic acids are used as strong anion-exchange groups (SAX); and carboxyl groups are used as weak anion-exchange groups (WAX) (**Figure 2**) [6]. The retention mechanism of this mixed-mode phase was based on the formation of a divalent complex involving hydrophobic and oppositely charged analytes. Moreover, repulsive ionic interactions with identically charged functional groups also affect analyte retention in mixed-mode stationary phases. Thus, separation can be optimized by adjusting the mobile phase parameters, such as pH, ionic strength (including the concentration of buffers and modifiers), and solvent strength [4]. For example, the C18/SAX column shows strong retention of acidic compounds due to electrostatic attraction under basic conditions. In addition, the retention values increase with increasing alkyl chain length of the analytes. Elution can be achieved using acidic conditions with high percentages of organic solvents, and/or high ionic strength to make neutral the acidic compounds and weaken the hydrophobic interactions. In contrast, the RP/ SCX and RP/WCX columns can effectively retain base compounds, such as peptides

**Figure 2.**

*Structures of some RP-IEX mixed-mode stationary phases: (a) RP-WCX, (b) RP-SCX, (c) RP-SAX, and (d) RP-WAX [6].*

and alkaloids, under acidic conditions. If a mobile phase with a neutral pH and a low ionic strength is used, the retention of these compounds is strongly influenced by hydrophobic interactions [7].

In the field of mixed-mode reversed-phase/ion-exchange stationary phase, mixed-mode RP/AX based on ethylene-bridged hybrid (BEH) organic/inorganic particles was recently developed, named Atlantis BEH C18 AX. The intermediate C18 surface concentration (1.6 μmol/m<sup>2</sup> ) together with tertiary alkylamine groups) makes BEH C18 AX compatible with highly aqueous mobile phases. The BEH particles used for the BEH C18 AX stationary phase have an average pore diameter of 95 Å that increases retention, stemming from the 46% higher surface area. Furthermore, hydrophilic anion-exchange group of them create positive surface charge, which show stronger retention of negatively charged compounds in a wider pH range while using

#### *Perspective Chapter: Mixed-Mode Chromatography DOI: http://dx.doi.org/10.5772/intechopen.104545*

with buffers of pH 3.0–6.9 in the survey. The extended upper pH limit of BEH C18 AX allows it to be used with a wider range of mobile phase pH values. For samples containing ionizable analytes, mobile phase pH has been demonstrated to be a key variable to use in optimizing RP separations [8, 9].

#### *2.1.2.2 Reversed-phase hydrophilic stationary phases (RP-HILIC)*

RP-HILIC mixed-mode stationary phases have shown advantages in the separation of both hydrophobic and hydrophilic compounds, especially proteins. This combination is equivalent to combining the HILIC properties with the reversed-phase properties to analyze complicated compounds and matrices with a wide range of polarities in a single run. The ligands are composed of hydrophobic and polar groups. The hydrophobic parts can be alkyl or aromatic groups, and the hydrophilic parts can be charged or neutral functional groups, such as diol, amide, cyano, and ionic groups (**Figure 3**) [10]. For compounds with hydrophilic and polar parts, ligands containing nonpolar and polar groups can interact separately with their corresponding nonpolar and polar groups. Therefore, it is possible to improve analyte retention and separation selectivity through multivalent effects, including hydrophobic and hydrophilic interactions. In recent years, many mixed-mode stationary phases have been synthesized and applied to the analysis of surfactants, peptides, nucleotides, and proteins (**Table 1**).

#### *2.1.2.3 Hydrophilic ion-exchange stationary phases (HILIC-IEX)*

The combination of hydrophilic and ion-exchange groups presented strong advantages for analyzing charged polar compounds. The multivalent effects of these mixedmode phases provide unique selectivity, higher retention efficiency, and a wider range of application than any single-mode phase for peptide analysis [16]. The main application of this combination is the separation of proteins and peptides. In this mode, the retention mechanism of polar compounds depends on the percentage of an organic solvent (such as acetonitrile (ACN)) in the mobile phase. If a mobile phase has a low percentage of the organic solvent, the analyte retention is dominated by ion-exchange mechanisms. An increase in the percentage of acetonitrile promoted more hydrophilic interactions than ionic ones. At a high concentration of acetonitrile, the electrostatic interactions decreased significantly, whereas the hydrophilic interactions dominated the analyte retention. Bo et al. prepared a HILIC-IEX phase with adjustable selectivity to separate nucleosides and β-agonists, which were synthesized by controlling the mixture ratio of the two functional monomers [17]. In addition, the use of ionic liquids to develop HILIC-IEX stationary phases can provide an environment for multiple interactions, such as electrostatic, dipole-dipole, and π-π interactions, and hydrogen bonding. Quiao et al. developed a new HILIC-SAX phase by using glucaminium-based ionic liquids to separate nucleosides [18]. According to studies reported by Mant et al. [16], the hydrophilic cation-exchange column (HILIC-CEX) has a higher separation efficiency than the RP-LC for peptide analysis, and the highly charged peptides are best resolved by this column [16]. Hartmann et al. [19] separated amphipathic α-helical peptides using a HILIC-CEX column and an RP-LC column. Both columns presented an adequate efficiency but displayed different selectivities. With the HILIC-CEX column, the temperature had a stronger influence on the separation of peptide columns than that with the RP-LC column. The results showed that both the resolution and retention of peptides in the HILIC-CEX phase significantly improved with increasing temperature [19].

**Figure 3.**

*Structures of some RP-HILIC mixed-mode stationary phases with (a) diol, (b) amide, and (c) amine polar groups [6].*

#### *2.1.3 Other combinations*

*Inclusion hydrophobic mixed-mode:* The ligands are composed of hydrophobic parts and cavities, cages, or cryptates, which form an inclusion complex with the analytes. Thus, the multivalent effects of this mode include both the inclusion complexation and the hydrophobic interactions. A representative example of this combination is crown ether immobilized on a solid matrix. A tripartite hydrogen bond can form between the six oxygen atoms of the crown ether and three hydrogen atoms of the protonated primary amine. Therefore, these ligands can be used to retain and separate the primary amines or other protonated molecules. Additionally, hydrophobic


#### **Table 1.**

*Applications of reversed-phase hydrophilic stationary phases.*

interactions can form between the methylene on the crown ether and the alkyl chain of the analytes, resulting in improvement of analyte retention [4].

*Inclusion hydrophilic mixed-mode:* In this mode, the ligands are composed of a cavity, cage, or cryptate, and a polar group. An example of this chromatography process is the binding of crown ethers with primary amines. The hydrogen bonding between the primary amines and crown ether can be enhanced with a polar organic solvent mobile phase, leading to enhanced inclusion effects.

*π-π hydrophilic mixed-mode:* The ligands are designed by combining two groups: a π-electron donor or π-electron acceptor group, and a polar group. In π-π interactions, the electron-rich π system (π-electron donor) can interact with the electron-deficient π system or other π-electron acceptor groups, through electrostatic interactions. In this chromatography process, the π-interacting groups of ligands can interact with the π-interacting groups of analytes through π-π interactions, and the polar parts of the ligands can interact with the polar parts of the analytes through hydrogen bonding and/or dipole-dipole interactions. The main application of this combination is the separation of chiral compounds. An example of this mode is Pirkle-type chromatography ligands [20].

*Π-π ion-exchange mixed-mode:* In this mode, the stationary phase can interact with analytes through π-π interactions, dipole-dipole interactions, van der Waals forces, and electrostatic interactions. A representative example of this combination is the cinchona alkaloid derivative phase developed by Lämmerhofer M and Lindner W. Depending on the structure of the derivatives, this phase can exhibit many types of separation, such as anion exchange, cation exchange, and amphoteric ion exchange for chiral chromatography. Therefore, the main application of this phase is the separation of chiral acids, ionic chiral compounds with a wide range of polarities, and amphoteric compounds such as amino acids and small peptides. In particular, the amphoteric ion exchange can also be considered as an example of a multifunctional stationary phase because this ligand can present three ion-exchange modes for chiral separation under the conditions of a polar organic mobile phase. Thus, the anion-exchange mode is utilized for chiral acid separation, the cation-exchange mode for chiral amine separation, and the zwitterion mode for amphoteric compound separation [21].

*Polymeric mixed-mode:* Several novel polymeric MMC sorbents have been designed specifically for the separation of proteins, mainly serum albumins and immunoglobulins (IgGs). Heterocyclic compounds are unique as MMC ligands with specific aromaticity/hydrophobicity and dissociation properties compared with common aliphatic and aromatic compounds with capability to relatively selectively interact with some proteins, albumins, also antibodies and monoclonal antibodies [22].

*Capillary-channeled polymer (C-CP) fiber stationary phases:* The unique shape of C-CP gives them high surface area and when packed into columns, the fibers selfalign, providing a monolith-like structure with parallel channels of 1–5 μm size. In relation to the size of proteins, the C-CP fibers surface is nonporous, which significantly reduce mass transfer resistance. Thus, separation can be run at high linear velocities and at low pressures without detrimental effect on the separation efficiency. All the studied C-CP stationary phases were able to separate a BSA/hemoglobin/ lysozyme mixture at high mobile phase velocity and with acceptable elution characteristics [22].

#### **2.2 Composition of mobile phases and their effects on mixed-mode chromatography**

#### *2.2.1 Polar organic solvents*

The mobile phase used in mixed-mode chromatography usually involves a polar organic solvent, water, or a buffer. The following four properties of the solvent have significant effects on the retention and separation of analytes: solvent viscosity, dielectric constant, dipole moment, and surface tension. Solvent viscosity affects the chromatography process in various ways, especially when gradient conditions are used. Firstly, an increase in the viscosity of the mobile phase is the prime reason for an increase in the backpressure. Moreover, the column efficiency is influenced by the viscosity of the mobile phase. For example, a mixed solution of methanol and water has a higher viscosity than pure methanol. As a result, it reduces the diffusion coefficient of the solutes and exhibits a slow mass transfer, leading to a reduction in the column efficiency [23]. The dielectric constant (ε) and dipole moment (μ) characterize the polar nature of the solvent. A solvent with a higher ε value is usually considered a weaker eluent in reversed-phase chromatography, whereas the dipole moment is related to solvent polarity and has important effects on the interactions between the analytes and the ligands in hydrophilic chromatography. Finally, the surface tension of a solvent can affect analyte separation. A mobile phase with higher surface tension can lead to stronger analyte retention. In addition, the UV wavelength cutoff of the solvent must also be considered when a UV-Vis detector is used to measure the concentration of the analytes [24].

In RP-IEX mixed-mode chromatography, polar organic solvents (such as methanol, acetonitrile, ethanol, and tetrahydrofuran) were used as strong eluotropic components. Furthermore, organic solvents can control the retention and elution of analytes in the chromatography process, thereby providing scope to increase the solubility of analytes in the mobile phase. An increase in the organic solvent concentration causes the polarity of the mobile phase to decrease, and the hydrophobic interactions between the analytes and the ligands decrease, resulting in a decrease in retention. According to the eluotropic strength, the order of solvents is water < methanol < acetonitrile < propanol < isopropanol < tetrahydrofuran [23]. The most commonly used polar organic solvents are methanol and acetonitrile (ACN). To analyze peptides

#### *Perspective Chapter: Mixed-Mode Chromatography DOI: http://dx.doi.org/10.5772/intechopen.104545*

and proteins, acetonitrile is preferred over methanol because the mixed solution of acetonitrile and water has a low viscosity, leading to excellent mass transfer.

A binary mixture of organic solvents and buffers is commonly used in HILIC-IEX chromatography. An increase in the organic solvent concentration can reduce the polarity of the mobile phase, leading to a strengthening of the hydrophilic interaction between the analytes and the ligands. In contrast, decreasing the organic solvent concentration can weaken the hydrophilic interactions, facilitate ionic interactions, and lead to compound elution. Thus, the organic solvent acts as a polarity modifier. One of the most commonly used solvents is acetonitrile [4]. As acetonitrile is an aprotic solvent that does not possess a hydrogen bond donor capacity, it cannot compete with the analytes for the ligands. If the mobile phase has a high level of acetonitrile, analytes can be adsorbed to the stationary phases through polar interactions, and they can be resorbed by reducing the acetonitrile content. Therefore, in the HILIC-IEX mixed-mode, acetonitrile has an important effect on the retention and separation of analytes. At high acetonitrile levels (up to 90%, v/v), the hydrophilic interactions may dominate the electrostatic interactions, and this may become the main factor affecting analyte retention.

Furthermore, to elute proteins that are strongly bound to the mixed-mode stationary phase and are significantly affected by hydrophobic interactions, reducing the polarity of the mobile phase by increasing the organic solvent content can be used as a severe elution method instead of reducing the salt concentration.

#### *2.2.2 Buffers and pH*

In mixed-mode chromatography, buffers are usually added to the mobile phase to either maintain the pH at an almost constant value or to adjust the pH value. Buffer systems can be selected depending on the required pH range. Buffer systems are classified into two categories according to their components.

*Type 1:* A buffer system is composed of a weak acid and its conjugate base, or a weak base and its conjugate acid, such as acid acetic/sodium acetate or ammonium chloride. For example, when acetate buffers are used in anion-exchange mixed-mode chromatography, CH3COO− can participate in the ion-exchange process by binding to the positively charged ligands. Therefore, a buffer system with buffer ions having the same charge as the ligands is ideal for mixed-mode chromatography involving ion-exchange mechanisms. Positively charged buffer ions are preferred when using an anion-exchange mechanism (having positively charged ligands), and negatively charged buffer ions are recommended for the cation-exchange mechanism.

*Type 2:* A buffer system contains an organic amine or an amphoteric compound that can be used in both the anion-exchange and the cation-exchange chromatography. The examples of such a buffer system are N-2-hydroxyethylpiperazine-N-2′-2 ethanesulfonic acid (HEPES) and N, N-dihydroxyethylglycine (BICINE).

The mobile phase pH can influence the charged properties of the analytes and the nature of the ligands; therefore, it can be used to promote the adsorption and elution of the target compounds [24]. Certain rules are applicable for selecting a suitable pH value for the mobile phase. Firstly, the ideal pH should be selected according to the pKa of the analytes and the ionic groups of the ligands. For example, a target compound with amine groups will be positively charged when the pH value is lower than its pKa, thus resulting in adsorption by cation-exchange ligands. Generally, for the adsorption process, the pH should be selected to charge the analytes and facilitate the electrostatic interactions between the analytes and the oppositely charged ligands. Therefore, the pH should be lower than the pKa of the analytes by approximately 1–2 pH units when the adsorption is carried out on a cation-exchange ligand, while the pH should be higher than the pKa of analytes by approximately 1–2 pH units on anionexchange ligands. In contrast, for the elution process, the pH should be adjusted to weaken or disrupt the interaction between the target compounds and ligands by charge repulsion. Secondly, the pH of the mobile phase should be within the stability range of the stationary phase. Finally, for protein analysis, it is necessary to select a pH value at which the proteins are stable and retain their biological activity [4].

In mixed-mode chromatography involving ion-exchange mechanisms, the charged properties of weakly acidic and basic ligands can be significantly affected by the pH value. For example, if the mixed-mode stationary phase contains weakly basic groups when the pH of the mobile phase is higher than the pKa of the ligand, then the ligand is neutrally charged and its hydrophobicity increases. Contrastingly, the ligand is positively charged and has a high hydrophilicity when the pH is lower than the pKa of the ligand. In the elution stage, pH gradient changes can be utilized to obtain a higher selectivity of the separation when the change in solvent polarity and the change in ionic strength produce no improvement in the separation efficiency. By changing the pH, the analytes and the ligands can have the same charge; therefore, the analytes can be eluted by charge repulsion. For example, in the experiment of Hostein et al. [25], α-Lactalbumin, β-lactoglobulin A, and trypsin inhibitor with pIs (protein's isoelectric point) of 4.5, 5.1, and 4.5, respectively, were separated by using a linear pH gradient from pH 3.8 to 8.0 (0.05 pH units/min) on the multimodal cation exchanger Capto MMC. When the pH value of the mobile phase was higher than that of the pIs, these proteins were negatively charged, as with the ligands, and eluted by electrostatic repulsion. The farther the pH value is from the pIs, the more negatively charged these proteins are, leading to their stronger hydrophilicity. It was observed that a shallower gradient (0.05 pH units/min to 0.01 pH units/min) reduces the sharpness of the peaks but improves the protein resolution.

#### *2.2.3 Salts*

In MMCs, salts are usually added to the mobile phase to adjust their ionic strength. Sodium chloride is usually used in the ion-exchange mode, whereas salts with higher solubility in organic solvents (such as sodium perchlorate and ammonium perchlorate) are preferred in the hydrophilic mode. In the hydrophobic mode, salts are classified into two categories: salting-out and salting-in. Salting-out salts, such as sodium sulfate, ammonium sulfate, and potassium sulfate, can be used to stabilize proteins and promote hydrophobic interactions between the proteins and the ligands. In contrast, salting-in salts, such as calcium chloride, magnesium chloride, and zinc nitrate, can increase the solubility of the proteins in water and promote protein denaturation and unfolding [26].

The ionic strength of the mobile phase has a significant effect on the retention and the elution of analytes in both the ion-exchange and the hydrophobic modes. In the mixed-mode chromatography involving ion-exchange mechanisms, because an increase in the ionic strength can suppress the electrostatic interaction between the analytes and the charged groups of the ligand, it may result in the weakening of analyte binding on the ligands, thereby leading to a decrease in the analyte retention or elution [24]. Moreover, in the hydrophobic mode, the increase in the ionic strength of the mobile phase can cause the analytes to strengthen their binding to the hydrophobic parts of the ligands, leading to an increase in the analyte retention. Hydrophobic

*Perspective Chapter: Mixed-Mode Chromatography DOI: http://dx.doi.org/10.5772/intechopen.104545*


**Table 2.**

*Commonly used additives in mixed-mode chromatography process.*

mixed-mode stationary phases are typically used for protein separation. In this mode, the proteins are adsorbed at high salting-out salt concentrations and eluted at low salt concentrations. Therefore, reducing the salt concentration in the mobile phase can be used in the elution mode [4].

#### *2.2.4 Other additives*

For protein separation, proteins can be eluted with ease by changing the pH or by reducing the polarity or the ionic strength of the mobile phase. However, when the proteins are firmly bound to the ligand, the recovery and the biological activity of the protein can decrease during the elution step. Therefore, additives are usually added to the mobile phase to reduce its polarity, resulting in the weakening of protein binding to the hydrophobic parts of ligands, and leading to an enhanced protein recovery. Some of the commonly used additives and their functions are listed in **Table 2**.

#### **3. Pharmaceutical analysis application of mixed-mode stationary phases**

#### **3.1 Drugs and impurities in drugs**

A mixed-mode column with a stationary phase of 50% hydrophobic C18 phase and 50% strong cation exchanger allows for a simultaneous detection of the ionic and hydrophobic analytes [33]. Acetaminophen and its related impurities, which ionize based on the mobile phase pH, are often separated for drug examination using ion-pair chromatography, which is a technique for organic charged compounds. Despite its numerous advantages, the corrosive effect of a large number of counterions on the stationary phase of the column is a practical drawback of the ion-pair

chromatography. As a result, the mixed-mode stationary phases can overcome the limitations of ion-pair chromatography, allowing for the simultaneous separation of ionic and neutral organic molecules without practical constraints [34]. Furthermore, because of the lack of UV chromophores in most drugs, refractive index (RI) and evaporative light-scattering detection (ELSD) detectors have been utilized. However, these approaches are insensitive or have compatibility issues with gradient elution. Recently, charged aerosol detection (CAD) has been developed as a new type of detector for high-performance liquid chromatography (HPLC) applications. CAD is a universal detection technique for nonvolatile and semi-volatile substances with higher sensitivity and reproducibility than other types of detectors. It is highly convenient in usage as it eliminates the necessity for parameter optimization [35]. **Table 3** shows the combinations of MMC and CAD detectors, as well as the applications of various types


chromatography (mmSEC)

#### **Table 3.**

*Application of mixed-mode stationary phases in drugs and impurity.*

of MMCs for drugs and impurities. Therefore, it is also a viable analytical tool for concurrently determining a wide range of drugs, pharmaceuticals, and their related compounds in a particular procedure [33].

#### **3.2 Metabolomics applications**

A common target of pharmacokinetic studies is the development of a biological analysis method for simultaneous observation of a wide range of drugs in a biological matrix. The tandem usage of reversed-phase and ion-exchange chromatography in MMCs has shown favorable results on the retention of polar and nonpolar small molecules in a single run [42]. In addition, efficient retention and separation of the above compounds were obtained under common and MS-friendly RP conditions, reaching a high point of selectivity and sensitivity. Therefore, MMC tandem mass spectrometry has been commonly applied in metabolic analysis. For instance, the study by Roverso et al. [43] demonstrated the effective retention of selected highly polar metabolites, which was performed by using a mixed cationic-RP column, and simultaneously obtained an efficient separation in the analysis without ion pair and derivatization of 2,4-diaminobutyric acid (DAB) and isobaric beta-methylamino-L-alanine (BMAA) [43]. The metabolomics applications are summarized in **Table 4**.

In addition, combination of MMC with molecular imprinting technology is also improving recognition selectivity for protein BSA, which proved a potential combination of other chromatography modes and molecular imprinting technology [22].

#### **3.3 Biopharmaceuticals and polar compounds in natural products**

For biopharmaceutical analysis, Capto and HEA HyperCel MMC ligands with multimodal functionality have been commercialized. Capto includes a carboxyl group that exhibits the characteristics of a phenyl group involved in hydrophobic interactions, and a weak cation exchanger. HEA HyperCel contains a hexyl group that is involved in hydrophobic interactions, and a protonable amine localized in the spacer arm. The application of this type of MMC has been demonstrated in the research by Sophie Maria et al. for mAb determination [46]. Meanwhile, tri-mixed-mode


#### **Table 4.**

*Metabolomics application of mixed-mode stationary phases.*


#### **Table 5.**

*Application of mixed-mode stationary phases in biopharmaceuticals and polar compounds in natural products.*

chromatography and another dual combination of MMC are also useful tools for biopharmaceutical analysis. The applications are listed in **Table 5**.

**Table 5** also illustrates the determination of polar compounds in natural products. Strong anion-exchange and reversed-phase mechanisms were analyzed in both the polar and more apolar ionic and nonionic compounds and have been used to determine Chinese herbal medicines that provide good retention for separation [49]. Thus, the combination of reversed phase and hydrophilic interactions is a common mechanism in this field because of its suitable characteristics for the detection of polar compounds, especially those of natural origin.

#### **4. Conclusion**

In this chapter, advanced applications of mixed-mode stationary phases are reviewed. By adjusting the ratio of organic matter and the mobile phase *Perspective Chapter: Mixed-Mode Chromatography DOI: http://dx.doi.org/10.5772/intechopen.104545*

concentration, the reversed-phase, HILIC, and IEX modes can be successively used. In conclusion, RP-IEX, RP-HILIC, and HILIC-IEX are the most commonly preferred mixed-mode stationary phases.

#### **Acknowledgements**

We would like to express their hearty gratitude to Can Tho University of Medicine and Pharmacy. We also thank all of our colleagues for their excellent assistance. We would like to thank Editage (www.editage.com) for English language editing.

#### **Author details**

Ngoc-Van Thi Nguyen Can Tho University of Medicine and Pharmacy, Can Tho City, Vietnam

\*Address all correspondence to: nguyenthingocvanct@gmail.com; ntnvan@ctump.edu.vn

© 2022 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.

## **References**

[1] Taraji M, Haddad PR, Amos RI, Talebi M, Szucs R, Dolan JW, et al. Chemometric-assisted method development in hydrophilic interaction liquid chromatography: A review. Analytica Chimica Acta. 2018;**1000**:20-40

[2] Taylor T. Mixed-mode HPLC separations: What, why, and how. LCGC North America. 2014;**32**:226

[3] Zhang K, Liu X. Mixed-mode chromatography in pharmaceutical and biopharmaceutical applications. Journal of Pharmaceutical and Biomedical Analysis. 2016;**128**:73-88. DOI: 10.1016/j. jpba.2016.05.007

[4] Wan QH. Mixed-mode Chromatography Principles, Methods, and Applications. Chapter 3. Berlin, Germany: Springer; 2021. pp. 71-117

[5] Liu X, Pohl CA. HILIC behavior of a reversed-phase/cationexchange/anionexchange trimode column. Journal of Separation Science. 2010;**33**:779-786. DOI: 10.1002/jssc.200900645

[6] Eric L, Caroline W, Elise L, Philippe H, Sophie B. Mixed-mode chromatography—A review. LCGC Supplements. 2017;**30**(6):22-33

[7] Lemasson E, Richer Y, Bertin S, et al. Characterization of retention mechanisms in mixed-mode HPLC with a bimodal reversed-phase/cation-exchange stationary phase. Chromatographia. 2018;**81**:87-399. DOI: 10.1007/ s10337-018-3477-5

[8] Walter TH, Alden BA, Field JA, Lawrence NL, Osterman DL, Patel AV, et al. Characterization of a highly stable mixed-mode reversed-phase/weak

anion-exchange stationary phase based on hybrid organic/inorganic particles. Journal of Separation Science. 2021;**44**:1005-1014. DOI: 10.1002/ jssc.202001136

[9] Kadlecová Z, Kozlík P, Tesařová E, Gilar M, Kalíková K. Characterization and comparison of mixed-mode and reversed-phase columns; interaction abilities and applicability for peptide separation. Journal of Chromatography A. 2021;**1648**:462182. DOI: 10.1016/j.chroma.2021.462182

[10] Yang Y, Geng X. Mixed-mode chromatography and its applications to biopolymers. Journal of Chromatography A. 2011;**1218**:8813- 8825. DOI: 10.1016/j.chroma.2011.10.009

[11] Liu X, Pohl C. New hydrophilic interaction/reversed-phase mixedmode stationary phase and its application for analysis of nonionic ethoxylated surfactants. Journal of Chromatography A. 2008;**1191**:83-89. DOI: 10.1016/j.chroma.2007.12.012

[12] Aral T, Aral H, Ziyadanoğulları B, Ziyadanoğulları R. Synthesis of a mixed-model stationary phase derived from glutamine for HPLC separation of structurally different biologically active compounds: HILIC and reversed-phase applications. Talanta. 2015;**131**:64-73. DOI: 10.1016/j.talanta.2014.07.060

[13] Li Y, Xu Z, Feng Y, Liu X, Chen T, Zhang H. Preparation and evaluation of poly-L-lysine stationary phase for hydrophilic interaction/reversedphase mixed-mode chromatography. Chromatographia. 2011;**74**:523-530. DOI: 10.1007/s10337-011-2120-5

[14] Ray S, Takafuji M, Ihara H. Chromatographic evaluation of a newly *Perspective Chapter: Mixed-Mode Chromatography DOI: http://dx.doi.org/10.5772/intechopen.104545*

designed peptide-silica stationary phase in reverse phase liquid chromatography and hydrophilic interaction liquid chromatography: Mixed mode behavior. Journal of Chromatography A. 2012;**1266**:43-52. DOI: 10.1016/j. chroma.2012.10.004

[15] Ohyama K, Inoue Y, Kishikawa N, Kuroda N. Preparation and characterization of surfactin-modified silica stationary phase for reversedphase and hydrophilic interaction liquid chromatography. Journal of Chromatography A. 2014;**1371**:257-260. DOI: 10.1016/j.chroma.2014.10.073

[16] Mant CT, Hodges RS. Mixed-mode hydrophilic interaction/cationexchange chromatography: Separation of complex mixtures of peptides of varying charge and hydrophobicity. Journal of Separation Science. 2008;**31**:1573-1584. DOI: 10.1002/jssc.200700619

[17] Bo C, Wang X, Wang C, Wei Y. Preparation of hydrophilic interaction/ ion-exchange mixed-mode chromatographic stationary phase with adjustable selectivity by controlling different ratios of the co-monomers. Journal of Chromatography A. 2017;**1487**:201-210. DOI: 10.1016/j. chroma.2017.01.061

[18] Qiao L, Wang S, Li H, Shan Y, Dou A, Shi X, et al. A novel surfaceconfined glucaminium-based ionic liquid stationary phase for hydrophilic interaction/anion-exchange mixedmode chromatography. Journal of Chromatography A. 2014;**1360**:240-247. DOI: 10.1016/j.chroma.2014.07.096

[19] Hartmann E, Chen Y, Mant CT, Jungbauer A, Hodges RS. Comparison of reversed-phase liquid chromatography and hydrophilic interaction/cationexchange chromatography for the separation of amphipathic a-helical

peptides with L- and D-amino acid substitutions in the hydrophilic face. Journal of Chromatography A. 2003;**1009**:61-71. DOI: 10.1016/ S0021-9673(03)00620-4

[20] Pirkle WH, Pochapsky TC. Considerations of chiral recognition relevant to the liquid chromatographic separation of enantiomers. Chemical Reviews. 1989;**89**:347-362. DOI: 10.1021/ cr00092a006

[21] Lämmerhofer M, Lindner W. Quinine and quinidine derivatives as chiral selectors I. Brush type chiral stationary phases for high-performance liquid chromatography based on cinchonan carbamates and their application as chiral anion exchangers. Journal of Chromatography A. 1996;**741**:33-48. DOI: 10.1016/0021-9673(96)00137-9

[22] Sýkora D, Řezanka P, Záruba K, Král V. Recent advances in mixed-mode chromatographic stationary phases. Journal of Separation Science. 2019;**42**(1):89-129. DOI: 10.1002/ jssc.201801048

[23] Snyder LR, Kirkland JJ, Glajch JL. Practical HPLC Method Development. 2nd ed. Hoboken: John Wiley & Sons, Inc; 1997

[24] Lee TD. Introduction to modern liquid chromatography. Journal of the American Society for Mass Spectrom. 2011;**22**:196. DOI: 10.1002/9780470508183

[25] Holstein MA, Nikfetrat AA, Gage M, Hirsh AG, Cramer SM. Improving selectivity in multimodal chromatography using controlled pH gradient elution. Journal of Chromatography A. 2012;**1233**:152-155. DOI: 10.1016/j.chroma.2012.01.074

[26] Kang B, Tang H, Zhao Z, Song S. Hofmeister series: Insights of ion specificity from amphiphilic assembly and Interface property. ACS Omega. 2020;**5**(12):6229-6239. DOI: 10.1021/ acsomega.0c00237

[27] Durkee KH, Roh BH, Doellgast GJ. Immunoaffinity chromatographic purification of Russell's viper venom factor X activator using elution in high concentrations of magnesium chloride. Protein Expression and Purification. 1993;**4**(5):405-411. DOI: 10.1006/ prep.1993.1053

[28] Hou Y, Cramer SM. Evaluation of selectivity in multimodal anion exchange systems: A priori prediction of protein retention and examination of mobile phase modifier effects. Journal of Chromatography A. 2011;**1218**(43):7813- 7820. DOI: 10.1016/j.chroma.2011.08.080

[29] Vagenende V, Yap MG, Trout BL. Mechanisms of protein stabilization and prevention of protein aggregation by glycerol. Biochemistry. 2009;**48**(46): 11084-11096. DOI: 10.1021/bi900649t

[30] Zheng W, Borgia A, Buholzer K, Grishaev A, Schuler B, Best RB. Probing the action of chemical denaturant on an intrinsically disordered protein by simulation and experiment. Journal of the American Chemical Society. 2016;**138**(36):11702-11713. DOI: 10.1021/ jacs.6b05443

[31] Hirano A, Shiraki K, Kameda T. Effects of arginine on multimodal chromatography: Experiments and simulations. Current Protein and Peptide Science. 2019;**20**(1):40-48

[32] Holstein MA, Parimal S, McCallum SA, Cramer SM. Mobile phase modifier effects in multimodal cation exchange chromatography. Biotechnology and Bioengineering. 2012;**109**(1):176-186. DOI: 10.1002/ bit.23318

[33] Bergqvist Y, Hopstadius C. Simultaneous separation of atovaquone, proguanil and its metabolites on a mixed mode high-performance liquid chromatographic column. Journal of Chromatography B: Biomedical Sciences and Applications. 2000;**741**(2):189-193. DOI: 10.1016/s0378-4347(00)00082-7

[34] Calinescu O, Badea IA, Vladescu L, Meltzer V, Pincu E. HPLC separation of acetaminophen and its impurities using a mixed-mode reversed-phase/ cation exchange stationary phase. Journal of Chromatographic Science. 2012;**50**(4):335-342. DOI: 10.1093/ chromsci/bmr043

[35] Zhang K, Dai L, Chetwyn NP. Simultaneous determination of positive and negative pharmaceutical counterions using mixed-mode chromatography coupled with charged aerosol detector. Journal of Chromatography A. 2010; **1217**(37):5776-5784. DOI: 10.1016/j. chroma.2010.07.035

[36] Abdighahroudi MS, Lutze HV, Schmidt TC. Development of an LC-MS method for determination of nitrogencontaining heterocycles using mixed-mode liquid chromatography. Analytical and Bioanalytical Chemistry. 2020;**412**:4921-4930

[37] Liu XK, Fang JB, Cauchon N, Zhou P. Direct stability-indicating method development and validation for analysis of etidronate disodium using a mixed-mode column and charged aerosol detector. Journal of Pharmaceutical and Biomedical Analysis. 2008;**46**(4):639- 644. DOI: 10.1007/s00216-020-02665-x

[38] Wang Q, Long Y, Yao L, Xu L, Shi ZG, Xu L. Preparation, characterization and application of a reversed phase liquid chromatography/ hydrophilic interaction chromatography mixed-mode C18-DTT stationary

*Perspective Chapter: Mixed-Mode Chromatography DOI: http://dx.doi.org/10.5772/intechopen.104545*

phase. Talanta. 2016;**146**:442-451. DOI: 10.1016/j.talanta.2015.09.009

[39] Nováková L, Vlčková H, Petr S. Evaluation of new mixed-mode UHPLC stationary phases and the importance of stationary phase choice when using low ionic-strength mobile phase additives. Talanta. 2012;**93**:0-105. DOI: 10.1016/j. talanta.2012.01.054

[40] Kühnreich R, Holzgrabe U. Impurity profiling of L-methionine by HPLC on a mixed mode column. Journal of Pharmaceutical and Biomedical Analysis. 2016;**122**:118-125. DOI: 10.1016/j. jpba.2016.01.057

[41] Yan Y, Xing T, Wang S, Daly TJ, Li N. Coupling mixed-mode size exclusion chromatography with native mass spectrometry for sensitive detection and quantitation of homodimer impurities in bispecific IgG. Analytical Chemistry. 2019;**91**(17):11417-11424. DOI: 10.1021/ acs.analchem.9b02793

[42] Hsieh Y, Duncan CJG, Liu M. A mixed-mode liquid chromatographytandem mass spectrometric method for the determination of cytarabine in mouse plasma. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences. 2007;**854**(1-2):8-12. DOI: 10.1016/j. jchromb.2007.03.034

[43] Roverso M, Di Gangi IM, Favaro G, Pastore P, Bogialli S. Use of a mixed cationic-reverse phase column for analyzing small highly polar metabolic markers in biological fluids for multiclass LC-HRMS method. Applied Sciences. 2020;**10**(20):7137. DOI: 10.3390/ app10207137

[44] Zheng Y, Liu H, Ma G, Yang P, Zhang L, Gu Y, et al. Determination of S-propargyl-cysteine in rat plasma by mixed-mode reversed-phase and

cation-exchange HPLC–MS/MS method and its application to pharmacokinetic studies. Journal of Pharmaceutical and Biomedical Analysis. 2011;**54**(5):1187- 1191. DOI: 10.1016/j.jpba.2010.11.027

[45] Hinterwirth H, Lämmerhofer M, Preinerstorfer B, Gargano A, Reischl R, Bicker W, et al. Selectivity issues in targeted metabolomics: Separation of phosphorylated carbohydrate isomers by mixed-mode hydrophilic interaction/ weak anion exchange chromatography. Journal of Separation Science. 2010; **33**(21):3273-3282. DOI: 10.1002/ jssc.201000412

[46] Maria S, Joucla G, Garbay B, Dieryck W, Lomenech A-M, Santarelli X, et al. Purification process of recombinant monoclonal antibodies with mixed mode chromatography. Journal of Chromatography A. 2015;**1393**:57-64. DOI: 10.1016/j.chroma.2015.03.018

[47] He Y, Friese OV, Schlittler MR, Wang Q, Yang X, Bass LA, et al. On-line coupling of size exclusion chromatography with mixed-mode liquid chromatography for comprehensive profiling of biopharmaceutical drug product. Journal of Chromatography A. 2012;**1262**:122-129. DOI: 10.1016/j. chroma.2012.09.012

[48] Yang X, Zhang Y, Wang F, Wang LJ, Richardson D, Shameem M, et al. Analysis and purification of IgG4 bispecific antibodies by a mixedmode chromatography. Analytical Biochemistry. 2015;**484**:173-179. DOI: 10.1016/j.ab.2015.06.014

[49] Li D, Dück R, Schmitz OJ. The advantage of mixed-mode separation in the first dimension of comprehensive two-dimensional liquid-chromatography. Journal of Chromatography A. 2014;**1358**:128-135. DOI: 10.1016/j. chroma.2014.06.086

*Analytical Liquid Chromatography - New Perspectives*

[50] Sun WY, Lu QW, Gao H, et al. Simultaneous determination of hydrophilic and lipophilic constituents in herbal medicines using directlycoupled reversed-phase and hydrophilic interaction liquid chromatographytandem mass spectrometry. Scientific Reports. 2017;**7**:7061. DOI: 10.1038/ s41598-017-07087-x

#### **Chapter 3**
