**3. Sample preparation**


**33**

**Figure 2.**

*Advancement in Analytical and Bioanalytical Techniques as a Boon to Medical Sciences*

biomolecules from biological samples are summarized in **Table 1**.

and metabolites containing huge amounts of proteins and large numbers of endogenous compounds within these samples is very complicated. Direct injection of drug containing biological sample into a chromatographic column results in the precipitation or absorption of proteins on the column packing material, resulting in an immediate loss of column performance. A number of advances have made to convert sample preparation techniques, used for the cleanup of drugs in biological samples into formats that are acceptable for high-volume processing with or without automation. The most widely used cleanup methods for separation of

Because of this, sample preparation became a prominent step in the analysis of biological samples. In recent years, the necessity of new developed method is largely required. Frequently, it was earlier considered as a separate procedure prior to the analysis, while it nowadays has become a more or less integrated part of the analytical procedure. It is necessary to lay the foundation of their development on a systematic and scientific approach. Thus, fundamental understanding of the different processes involved in a sample preparation method is served as a basis for its optimization [10]. We should select the appropriate sample preparation method on the basis of requirements of the assay and time allowed to run sample preparation method.

The spectrophotometric technique is used to study interactions between electromagnetic radiations and analyte (**Figure 2**). The concentration of an analyte is determined by using a graph which is called standard analytical curve. An example is determination of iron in blood serum. The iron content of blood serum is determined after deprotonation (by precipitating protein) with trichloroacetic acid and reduction with hydroxyl ammonium sulfate. Iron (II) ions are reacted in the medium buffered with ammonium acetate and with diphenyl-1,10-phenanthrolinedisulfonic acid disodium salt (bathophenanthroline disulfonate–Na), and the absorbance of the complex formed is measured at 535 nanometer. The concentration belonging to the absorbance data of the test solution is read from the standard

Optical spectroscopy for biomedical applications covers up the plethora of medical technological and fundamental research areas. This includes screening and early detection of diseases which remain clinically silent over long periods. It is a noninvasive, fast spectroscopic technique. The technique is also capable of observations from femtosecond time scale at nanometer spatial resolution, so it can be applied in all areas of life sciences. This technique is to make an early, noninvasive, and patient-specific diagnosis near the source of the disease and then to treat the

*Schematic representation of biological sample determination using spectrophotometric technique.*

analytical curve and multiplied by three for threefold dilution [11].

**4.1 Optical spectroscopic techniques of human models**

*DOI: http://dx.doi.org/10.5772/intechopen.80279*

**4. Spectrophotometry**

Biological samples involve plasma, serum, CSF, bile, urine, tissue homogenates, saliva, seminal fluid, and frequently whole blood. Quantitative analysis of drugs

#### **Table 1.**

*Sample preparation techniques used for biological samples [32].*

#### *Advancement in Analytical and Bioanalytical Techniques as a Boon to Medical Sciences DOI: http://dx.doi.org/10.5772/intechopen.80279*

and metabolites containing huge amounts of proteins and large numbers of endogenous compounds within these samples is very complicated. Direct injection of drug containing biological sample into a chromatographic column results in the precipitation or absorption of proteins on the column packing material, resulting in an immediate loss of column performance. A number of advances have made to convert sample preparation techniques, used for the cleanup of drugs in biological samples into formats that are acceptable for high-volume processing with or without automation. The most widely used cleanup methods for separation of biomolecules from biological samples are summarized in **Table 1**.

Because of this, sample preparation became a prominent step in the analysis of biological samples. In recent years, the necessity of new developed method is largely required. Frequently, it was earlier considered as a separate procedure prior to the analysis, while it nowadays has become a more or less integrated part of the analytical procedure. It is necessary to lay the foundation of their development on a systematic and scientific approach. Thus, fundamental understanding of the different processes involved in a sample preparation method is served as a basis for its optimization [10].

We should select the appropriate sample preparation method on the basis of requirements of the assay and time allowed to run sample preparation method.

### **4. Spectrophotometry**

*Biochemical Testing - Clinical correlation and Diagnosis*

proteome and metabonome [9].

**3. Sample preparation**

Affinity separation: molecularly imprinted polymers (MIPs)/

Solid phase micro extraction

Ultrafiltration and microdialysis

*Sample preparation techniques used for biological samples [32].*

antibodies

(SPME)

(MD)

**Sample preparation techniques Advantages**

The type of preservatives should be known to protect the samples from degradation prior to cryopreservation at a reasonable cost. Cryopreservation is a process to store biological samples at very low temperature for prevention of damage. The purpose is to find readily accessible and data-rich biological samples. The stability of a wide range of bioanalytes and cells as a component of whole blood should be estimated, taking into account different anticoagulant (inhibition of coagulation of blood) media, at different temperatures and under varying transport conditions. Bioanalytes can be known biochemicals, such as DNA, defined proteins, and specific metabolites, or unknown analytes, such as the constituent plasma/serum

Design and testing of the sample handling protocol considered as key factors that affect the stability of biological samples, including anticoagulants, stabilizing agents, and temperature, elapsed time from collection to initial processing and endogenous degrading properties (enzymes, cell death). We also aim for cost-efficiency by avoiding collecting multiple sources of material for the same analyte. The samples undergo minimal processing locally in the assessment centers before being shipped to the main laboratory for processing with the aim of cryopreservation within 24 h of collection. Samples are protected against degradation during shipping by being chilled at 4°C (only peripheral blood lymphocytes, at 18°C). Once the samples get processed in the laboratory, they are placed in cabinets maintained at −80°C for the working archive or in nitrogen vapor at −180°C or below for the backup archive.

Biological samples involve plasma, serum, CSF, bile, urine, tissue homogenates, saliva, seminal fluid, and frequently whole blood. Quantitative analysis of drugs

Liquid phase extraction (LLE) LLE is one of the first methods used for extraction. It depends on the

by subsequent LLE and evaporations. Solid phase extraction (SPE) SPE is a method for the isolation and concentration of selected analytes

recovery, uses less organic solvent than LLE.

cross-reactivity and leaking of template.

through a semi-permeable membrane.

partitioning of analytes between two immiscible liquids. The resulting extract may be directly analyzed or further purified and concentrated

from a fluid sample by their transfer on a solid phase. The analytes are recovered by elution or thermal desorption. This method has high

The affinity sorbent may consist of an immobilized antibody or a molecularly imprinted polymer. This technique is highly specific and very sensitive, but the sorbent is difficult to prepare; it suffers from

SPME, a solvent free extraction method, consists of a single extraction step, but the experimental variables must be well controlled. It reduces solvent and sample volume needs and sample preparation time. Improves detection limits i.e., parts per trillion level detection.

Ultrafiltration consists of filtering the sample through a special size-excluding filter, either by applying pressure (10–100 psi) or by centrifugation. The method is widely used, simple, efficient, but suffers from ligand binding to the filter and shift of equilibrium. Dialysis and MD can be used to separate an analyte by diffusion

**32**

**Table 1.**

The spectrophotometric technique is used to study interactions between electromagnetic radiations and analyte (**Figure 2**). The concentration of an analyte is determined by using a graph which is called standard analytical curve. An example is determination of iron in blood serum. The iron content of blood serum is determined after deprotonation (by precipitating protein) with trichloroacetic acid and reduction with hydroxyl ammonium sulfate. Iron (II) ions are reacted in the medium buffered with ammonium acetate and with diphenyl-1,10-phenanthrolinedisulfonic acid disodium salt (bathophenanthroline disulfonate–Na), and the absorbance of the complex formed is measured at 535 nanometer. The concentration belonging to the absorbance data of the test solution is read from the standard analytical curve and multiplied by three for threefold dilution [11].

#### **4.1 Optical spectroscopic techniques of human models**

Optical spectroscopy for biomedical applications covers up the plethora of medical technological and fundamental research areas. This includes screening and early detection of diseases which remain clinically silent over long periods. It is a noninvasive, fast spectroscopic technique. The technique is also capable of observations from femtosecond time scale at nanometer spatial resolution, so it can be applied in all areas of life sciences. This technique is to make an early, noninvasive, and patient-specific diagnosis near the source of the disease and then to treat the

**Figure 2.** *Schematic representation of biological sample determination using spectrophotometric technique.*

disease at primary stage, for example, Alzheimer's disease and coronary disease. Thus, optics offers a wide variety of diagnostic methods and products of biomedical spectroscopy [12].

#### **4.2 Absorptions and reflectance spectroscopy**

This method involves investigations of brain dysfunction and mental health problems like depression, epilepsy, and Alzheimer's disease [13]. The direct absorption like near-infrared (NIR) techniques and instrumentation are particularly suitable in routine neonatal care applications.

However, diffused reflectance spectroscopy, in the UV-Vis-NIR region, can be used for biomedical applications, like studies on skin condition (vitiligo, psoriasis, skin cancer) and glucose concentration measurement [14].

#### **4.3 Photoacoustic spectroscopy (PAS)**

This includes measurement of concentration of biomolecular species. Examples are glucose determination, characterization of tissue status (biopsy tissue), and imaging application. PAS can provide information on three-dimensional distribution of specific molecular species in a specimen, by appropriate choice of excitation wavelength [15].

#### **4.4 Raman and infrared spectroscopy**

The "fingerprint" molecular specific technique will be of great advantage in understanding the biochemical interactions involved in induction, progression, therapeutic invention, and regression. This technique is suitable for biomedical applications such as breath analysis, drug-cell interaction, microscopy, and imaging of biopsy sample (tissue, fine needle aspiration) [16–18].

#### **4.5 Fluorescence spectroscopy**

Depending on excitation wavelength, the fluorescence peak has been observed in blood and urine spectra. A few examples are spectra observed from epithelial tissues, proteins, NAH, FAD, and hemoglobin [19].

#### **4.6 Mass spectrometry**

Mass spectroscopy (MS) measures masses within the sample. In mass spectroscopy, chemical species get ionized and ions get sorted on the basis of their mass-to-charge ratio (**Figure 3**). Major application of MS includes confirmation of immunoassay-positive drug screens, identification of inborn errors of metabolism, and analysis of steroid hormones [20]. Conclusive identification of molecules that range in size from tens of daltons (small molecules) to hundreds of thousands of daltons (biomolecules) is based on different principles.

Discovery of highly sensitive polymerase chain reaction (PCR) was a major step forward in the biomedical research and diagnostics. This technique is used for analysis of small quantities of short sequences of DNA and RNA without cloning. PCR can detect the presence of pathogens earlier than the culture tests. The miniaturization of MS systems allows a transportable device that minimizes the need of highly skilled operators and allows for rapid and accurate MS analysis in a point-ofcare format (near the physician's clinic) [21].

**35**

*Advancement in Analytical and Bioanalytical Techniques as a Boon to Medical Sciences*

Advances in medical imaging present a great opportunity in drug development. A number of different imaging technologies are available. These include computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), positron-emission tomography (PET), and single photon emission computed tomography (SPECT). If adequately qualified, imaging biomarkers can

An electrochemical sensor consists of a diffusion barrier, a sensing electrode (working electrode, measuring electrode, or anode), a counter electrode (cathode), and an electrolyte. Their fabrication includes various types of systems such as conductometry, voltammetry, potentiometry, and capacitance, and it is an important tool to detect various analytes in environmental, clinical, and biological fields due to their high sensitivity, cheapness, and miniaturization. These sensors have the potential to achieve sensitive, specific, and low-cost detection of biomolecules which is relevant to

the diagnosis and monitored treatment of disease [23]. A few are listed below:

recognition of urea by urease enzyme. The following reaction takes place:

Techniques based on measurement of potential sensor are termed as potentiometry, for example, determination of potassium in blood serum by direct potentiometry with an ion-selective electrode. The determination of urea is a frequent task of clinical laboratories. The basis of enzyme electrode function is the selective

Urease→2NH4

This reaction can be followed using different potentiometric electrodes [24].

MIP could be one of the important tailor-made systems for targeted analyte recognition exclusively even their presence in complex real biological samples in parts per million to parts per billion levels. The general idea is to create the cavity in the presence of the guest. The guest organizes and promotes energy-minimized interactions with polymer forming around it. Thus, after washing the guest out, the polymers retain

<sup>+</sup> + HCO3

− <sup>+</sup> OH<sup>−</sup>

(1)

be very helpful in the early stages of clinical development [22].

*DOI: http://dx.doi.org/10.5772/intechopen.80279*

*Schematic representation of mass spectrometry detection of sample.*

**4.7 Imaging techniques**

**5.1 Potentiometric sensors**

Urea <sup>+</sup> 3H2O⎯

**5.2 Molecularly imprinted polymer (MIP) sensors**

**5. Sensors**

**Figure 3.**

*Advancement in Analytical and Bioanalytical Techniques as a Boon to Medical Sciences DOI: http://dx.doi.org/10.5772/intechopen.80279*

**Figure 3.**

*Biochemical Testing - Clinical correlation and Diagnosis*

**4.2 Absorptions and reflectance spectroscopy**

suitable in routine neonatal care applications.

**4.3 Photoacoustic spectroscopy (PAS)**

**4.4 Raman and infrared spectroscopy**

**4.5 Fluorescence spectroscopy**

**4.6 Mass spectrometry**

skin cancer) and glucose concentration measurement [14].

of biopsy sample (tissue, fine needle aspiration) [16–18].

tissues, proteins, NAH, FAD, and hemoglobin [19].

daltons (biomolecules) is based on different principles.

care format (near the physician's clinic) [21].

spectroscopy [12].

wavelength [15].

disease at primary stage, for example, Alzheimer's disease and coronary disease. Thus, optics offers a wide variety of diagnostic methods and products of biomedical

This method involves investigations of brain dysfunction and mental health problems like depression, epilepsy, and Alzheimer's disease [13]. The direct absorption like near-infrared (NIR) techniques and instrumentation are particularly

However, diffused reflectance spectroscopy, in the UV-Vis-NIR region, can be used for biomedical applications, like studies on skin condition (vitiligo, psoriasis,

This includes measurement of concentration of biomolecular species. Examples are glucose determination, characterization of tissue status (biopsy tissue), and imaging application. PAS can provide information on three-dimensional distribution of specific molecular species in a specimen, by appropriate choice of excitation

The "fingerprint" molecular specific technique will be of great advantage in understanding the biochemical interactions involved in induction, progression, therapeutic invention, and regression. This technique is suitable for biomedical applications such as breath analysis, drug-cell interaction, microscopy, and imaging

Depending on excitation wavelength, the fluorescence peak has been observed in blood and urine spectra. A few examples are spectra observed from epithelial

Mass spectroscopy (MS) measures masses within the sample. In mass spectroscopy, chemical species get ionized and ions get sorted on the basis of their mass-to-charge ratio (**Figure 3**). Major application of MS includes confirmation of immunoassay-positive drug screens, identification of inborn errors of metabolism, and analysis of steroid hormones [20]. Conclusive identification of molecules that range in size from tens of daltons (small molecules) to hundreds of thousands of

Discovery of highly sensitive polymerase chain reaction (PCR) was a major step forward in the biomedical research and diagnostics. This technique is used for analysis of small quantities of short sequences of DNA and RNA without cloning. PCR can detect the presence of pathogens earlier than the culture tests. The miniaturization of MS systems allows a transportable device that minimizes the need of highly skilled operators and allows for rapid and accurate MS analysis in a point-of-

**34**

*Schematic representation of mass spectrometry detection of sample.*

#### **4.7 Imaging techniques**

Advances in medical imaging present a great opportunity in drug development. A number of different imaging technologies are available. These include computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), positron-emission tomography (PET), and single photon emission computed tomography (SPECT). If adequately qualified, imaging biomarkers can be very helpful in the early stages of clinical development [22].

### **5. Sensors**

An electrochemical sensor consists of a diffusion barrier, a sensing electrode (working electrode, measuring electrode, or anode), a counter electrode (cathode), and an electrolyte. Their fabrication includes various types of systems such as conductometry, voltammetry, potentiometry, and capacitance, and it is an important tool to detect various analytes in environmental, clinical, and biological fields due to their high sensitivity, cheapness, and miniaturization. These sensors have the potential to achieve sensitive, specific, and low-cost detection of biomolecules which is relevant to the diagnosis and monitored treatment of disease [23]. A few are listed below:

#### **5.1 Potentiometric sensors**

Techniques based on measurement of potential sensor are termed as potentiometry, for example, determination of potassium in blood serum by direct potentiometry with an ion-selective electrode. The determination of urea is a frequent task of clinical laboratories. The basis of enzyme electrode function is the selective recognition of urea by urease enzyme. The following reaction takes place: Urea <sup>+</sup> 3H2O⎯

$$\text{Urea} \star \text{3H}\_2\text{O} \xrightarrow{\text{Urea}} 2\text{NH}\_4^+ \text{ + } \text{HCO}\_3^- \text{ + OH}^- \tag{1}$$

This reaction can be followed using different potentiometric electrodes [24].

#### **5.2 Molecularly imprinted polymer (MIP) sensors**

MIP could be one of the important tailor-made systems for targeted analyte recognition exclusively even their presence in complex real biological samples in parts per million to parts per billion levels. The general idea is to create the cavity in the presence of the guest. The guest organizes and promotes energy-minimized interactions with polymer forming around it. Thus, after washing the guest out, the polymers retain

**Figure 4.** *Synthesis of molecularly imprinted polymer.*

a template cavity of the guest's size and shape which subsequently display binding selectivity toward the guest just like induced-fit model of enzyme (**Figure 4**). The MIP-modified sensors can be used for biological and pharmaceutical analyte determination from biological samples. An example is the development of a polyscopoletinbased MIP nanofilm for the electrochemical determination of elevated human serum albumin (HSA) in urine. The results suggest that MIP-based sensors may be applicable for quantifying high-abundance proteins in a clinical setting [25].

#### **5.3 Biosensors**

The need for rapid, simple handheld testing devices in medicine paves the way for introduction of biosensor. Biological sensors are optical, electrical, and piezoelectrical devices that have the ability to detect biological compounds, such as nucleic acids and proteins [26, 27]. Early diagnosis of inherited disease is important for effective treatment and is sometimes lifesaving. Methods, like enzyme-linked immunosorbent assay and PCR, can require highly skilled professionals and expensive chemicals and can be time-consuming. In this area, many biosensor schemes had developed as an alternative to classical methods.

The latest advancements in nanotechnology result in its application for cancer biomarker recognition [28]. Several other biosensors include nanomaterial-based biosensors [29], peptide nucleic acid-based biosensors, biosensors for medical mycology, optical DNA biosensors, and last but not least, the biosensors for the diagnosis of heart disease [30].

#### **6. Chromatographic separation techniques**

In chromatographic separation technique, various constituents of the mixture in the given sample travel at different speeds, causing them to separate. This technique involves two phases: a mobile phase and a stationary phase. The separation mainly depends on the differential partitioning between these two phases. A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing. The stationary phase is the substance fixed in place for the chromatography procedure, and the mobile phase is moving in a definite direction [31]. Examples include the silica layer in thin

**37**

**Figure 5.**

*Advancement in Analytical and Bioanalytical Techniques as a Boon to Medical Sciences*

layer chromatography (TLC). Archer John Porter Martin and Richard Laurence Millington Synge won a Nobel Prize in chemistry for chromatography invention [32]. Their work encouraged the rapid development of several advanced chromatographic methods such as paper chromatography, gas chromatography, and HPLC. The differences in a compound's partition coefficient bring about differential retention on the stationary phase and thus affect the separation process. Chromatography may be preparative or analytical. The preparative chromatography separates the components of a mixture for later use and is thus a form of purification. Analytical chromatography is done normally with smaller amounts of material and is for establishing the presence or measuring the relative proportions of analytes in a mixture. **Figure 5** describes a chromatogram for a biological system where the signal is proportional to the concentration of the specific analyte

Depending upon the shape of stationary phase, chromatography may be (i) planar chromatography, having one-dimensional bed support such as paper or TLC, or (ii) column chromatography with three-dimensional bed support. TLC is useful for separating mixtures of organic compounds and is often used to monitor

On the basis of physical state of mobile phase, chromatographic technique may be GC or LC. GC can be used to separate mixtures of volatile organic compounds. A GC consists of a flowing mobile phase, an injection port, a separation column containing the stationary phase, a detector, and a data recording system. LC is useful for separating mixtures of ions or molecules that are dissolved in a solvent. If the matrix support, or stationary phase, is polar (e.g., paper, silica, etc.), it is normal-phase chromatography; and if it is nonpolar (C-18), it is reversed-phase

In short, chromatography is a method of separating the constituents of a solution, based on one or more of its chemical or physical properties. This could be charge, polarity, or a combination of these traits and pH balance. The solution is passed through a medium which will hinder the movement of some particles more than others. These principles are used to isolate and analyze enzymes, pigments, amino acids, constituents of DNA, and almost any other molecule you can imagine. A wide variety of chromatography techniques had developed to allow mixed

*Chromatogram response of a biological sample. The retention time is plotted on X-axis and signal on Y-axis* 

*obtained from detector corresponding to the response created by the analytes exiting the system.*

the progress of organic reactions and to check the purity of products.

*DOI: http://dx.doi.org/10.5772/intechopen.80279*

separated.

chromatography.

substances to be separated [33].

#### *Advancement in Analytical and Bioanalytical Techniques as a Boon to Medical Sciences DOI: http://dx.doi.org/10.5772/intechopen.80279*

layer chromatography (TLC). Archer John Porter Martin and Richard Laurence Millington Synge won a Nobel Prize in chemistry for chromatography invention [32]. Their work encouraged the rapid development of several advanced chromatographic methods such as paper chromatography, gas chromatography, and HPLC. The differences in a compound's partition coefficient bring about differential retention on the stationary phase and thus affect the separation process. Chromatography may be preparative or analytical. The preparative chromatography separates the components of a mixture for later use and is thus a form of purification. Analytical chromatography is done normally with smaller amounts of material and is for establishing the presence or measuring the relative proportions of analytes in a mixture. **Figure 5** describes a chromatogram for a biological system where the signal is proportional to the concentration of the specific analyte separated.

Depending upon the shape of stationary phase, chromatography may be (i) planar chromatography, having one-dimensional bed support such as paper or TLC, or (ii) column chromatography with three-dimensional bed support. TLC is useful for separating mixtures of organic compounds and is often used to monitor the progress of organic reactions and to check the purity of products.

On the basis of physical state of mobile phase, chromatographic technique may be GC or LC. GC can be used to separate mixtures of volatile organic compounds. A GC consists of a flowing mobile phase, an injection port, a separation column containing the stationary phase, a detector, and a data recording system. LC is useful for separating mixtures of ions or molecules that are dissolved in a solvent. If the matrix support, or stationary phase, is polar (e.g., paper, silica, etc.), it is normal-phase chromatography; and if it is nonpolar (C-18), it is reversed-phase chromatography.

In short, chromatography is a method of separating the constituents of a solution, based on one or more of its chemical or physical properties. This could be charge, polarity, or a combination of these traits and pH balance. The solution is passed through a medium which will hinder the movement of some particles more than others. These principles are used to isolate and analyze enzymes, pigments, amino acids, constituents of DNA, and almost any other molecule you can imagine. A wide variety of chromatography techniques had developed to allow mixed substances to be separated [33].

#### **Figure 5.**

*Chromatogram response of a biological sample. The retention time is plotted on X-axis and signal on Y-axis obtained from detector corresponding to the response created by the analytes exiting the system.*

*Biochemical Testing - Clinical correlation and Diagnosis*

a template cavity of the guest's size and shape which subsequently display binding selectivity toward the guest just like induced-fit model of enzyme (**Figure 4**). The MIP-modified sensors can be used for biological and pharmaceutical analyte determination from biological samples. An example is the development of a polyscopoletinbased MIP nanofilm for the electrochemical determination of elevated human serum albumin (HSA) in urine. The results suggest that MIP-based sensors may be applicable

The need for rapid, simple handheld testing devices in medicine paves the way for introduction of biosensor. Biological sensors are optical, electrical, and piezoelectrical devices that have the ability to detect biological compounds, such as nucleic acids and proteins [26, 27]. Early diagnosis of inherited disease is important for effective treatment and is sometimes lifesaving. Methods, like enzyme-linked immunosorbent assay and PCR, can require highly skilled professionals and expensive chemicals and can be time-consuming. In this area, many biosensor schemes

The latest advancements in nanotechnology result in its application for cancer biomarker recognition [28]. Several other biosensors include nanomaterial-based biosensors [29], peptide nucleic acid-based biosensors, biosensors for medical mycology, optical DNA biosensors, and last but not least, the biosensors for the

In chromatographic separation technique, various constituents of the mixture

in the given sample travel at different speeds, causing them to separate. This technique involves two phases: a mobile phase and a stationary phase. The separation mainly depends on the differential partitioning between these two phases. A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing. The stationary phase is the substance fixed in place for the chromatography procedure, and the mobile phase is moving in a definite direction [31]. Examples include the silica layer in thin

for quantifying high-abundance proteins in a clinical setting [25].

had developed as an alternative to classical methods.

**6. Chromatographic separation techniques**

diagnosis of heart disease [30].

**36**

**5.3 Biosensors**

**Figure 4.**

*Synthesis of molecularly imprinted polymer.*
