Biomedical Chromatography Includes Research On Chromatographic Application Biology Essay

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Biomedical chromatography utilizes the analytical separation principles for isolation and analysis of biological specimen. It uses biological interactions for separation technique. As chromatographic techniques are reliable its application in biomedicine has become increasingly important in clinical and forensic pharmacy. Though biomedicine is related to patients health analysis of complex mixture is essential for diagnosis and safety of patients. This review includes the various chromatographic methods such as Affinity chromatography, HPLC, GC, Multidimensional chromatography, Ion exchange chromatography, Planer chromatography along with their clinical applications. It also includes characteristics and quantitative methods of chromatography. This various chromatographic methods used for the diagnosis of disease from the biological specimen such as blood, urine, saliva and hair. Currently different analytical methods used in the laboratory for obtaining concentration of drug in the blood and urine. Clinical application of biomedical chromatography includes determination of biomarkers in cancer cells, purification of human immunoglobulins, metabolomics in the cancer, isolation of proteins and amino acids from plasma and urine. As food is mainly related to health this technique is also used in the food analysis.

Key words

Biomedical chromatography, Affinity chromatograpy, Multidimentional chromatography, Biomarkers, cancer,


Biomedical chromatography includes research on chromatographic application and related techniques in biological and medical sciences. It makes use of biological- like interactions for the specific analysis and separation of sample components [1].

Although chromatographic methods in biomedicine are in the area of basic research still they are used widely in clinical applications [2-3][4].

Recognition of the diseases mainly depend upon the abnormal molecules or components in body fluids and abnormal conditions in the body that mainly responsible for cause of disease. The diagnosis and therapeutic treatment of such conditions in healthy as well as diseased state require its study. To perform such investigation there is need of identification, quantification and at the end purity of components. Chromatography is one of the technique which is highly successful in obtaining all these three objectives and biomedical chromatography includes [5].

Chromatographic methods represents the most useful and powerful technique for the separation of components of a complex mixture. Because of the rapidity and effectiveness, chromatography has been used in all fields particularly in chemistry, biology, medicines, forensic departments and clinical studies with much advantage over other methods. A notable advantage of Chromatographic methods is that they are relatively 'gentle' methods and decomposition of substances does not occur. This is important especially for substances from biological origin [6].

For the development of new drugs, therapeutic drug monitoring, forensic toxicology, diagnosis of patient, and biomonitoring of human exposure to the hazardous chemicals, the Biomedical analysis of drugs, metabolites, poisons, environmental and occupational pollutants, disease biomarkers and endogenous substances in body fluids and tissues are important. [7].

Biomedical analysis of lower-molecular-mass organic molecules mainly includes the analysis of drugs, poisons, metabolites, chemicals of environmental exposure and endogenous substances in body fluids and tissues. As biomedicine includes bio-physical and bio-chemical views of medicines and it creates great interest in analyst [4].

Biomedical chromatography has become a very important technique in medical analysis as chromatography is a group of methods for separating very small quantities of complex mixtures, with very high resolution. Recently there are too much advanced techniques involved in chromatography it detects specific compounds at lower levels in complex matrices of animal tissues.[character.pdf].

In this review there is focus on the clinical application of biomedical chromatography. It includes application of various recent chromatographic techniques in biomedical analysis.


Chromatographic techniques are simple, rapid and require simple apparatus. The basis of all types of chromatography is the partition of the sample compounds between a stationary phase and a mobile phase which flows over or through the stationary phase. Separation may occur by adsorption Chromatography has increased the utility of several types of spectroscopy, by delivering separate components of a complex sample, one at a time, to the spectrometer. This combination of the separating power of chromatography with the identification and quantitation of spectroscopy has been most important in biomedical analysis. It has enabled analysts to cope with tremendously complex and extremely dilute samples.

Biomedical chromatography includes the use of this principle for identification, separation and quqntitation of drug samples from biological specimen. A biological specimen involves blood plasma, urine, sweat and saliva. There is need of drug analysis in biological specimen, as their presence or absence is necessary in forensic pharmacy.



As the quality of the pharmaceutical product is directly related to patient's health, so analysis of these complex matrices is essential. In the drug development its quality control its analysis plays vital role for the high efficacy and safety. The pharmaceutical quality control should ensure use of appropriate analytical methods to develop faster and more efficient techniques for economic and decline in the solvent consumption. [8].

UV/VIS spectrophotometric methods

Extensively For the quantification of pharmaceutical active ingredients Several UV/VIS spectrophotometric tests have been developed. In most of the pharmaceuticals have chromophore groups, that can be obtained by UV without derivatization reaction. The advantage of this method is simple, economic with fine precision and accuracy so this technique is mostly preferred [9]. This technique has been frequently used to extract information from overlapping bands of the analytes and interferences. It consists of calculating and plotting one of the mathematical derivatives of a spectral curve [10].

Thin-layer chromatography-

for the analysis of pharmaceutical products Thin-layer chromatography (TLC) method is used as it is simple, most sensitive, rapid, and inexpensive technique. TLC has great separation and qualitative and quantitative analysis of a wide range of organic and metal-organic compounds [11]. In pharmaceutical analysis, TLC is predominantly used in its semiquantitative mode, where spots of reference tests solutions are visually against the impurity spots in a chromatogram of the test sample.

Eg- A chromatographic and densitometric method development for identification and quantitative analysis of hydrochlorothiazide, triamterene, furosemide, and spironolactone in drugs used to treat hypertension [12].

Gas chromatography

Gas chromatography (GC) and especially GC-MS well adapted for the analysis and sepration of complex mixtures and this technique plays significant role in the analysis of drugs and pharmaceutical products. Gas chromatography (GC) and particularly GCMS is a sensitive, accurate, reproducible, versatile and quantitative technique used for analysis of product [13].

Eg- Developed of method for the extraction of valproic acid by hollow-fiber coated wire as a lab-made solid phase microextraction fiber and its determination by capillary gas chromatography in human serum and pharmaceutical formulations [14]

High and ultra performance liquid chromatography

High performance liquid chromatography (HPLC) is the most widely used instrumental technique for the analysis of pharmaceuticals [15] and reversed-phase HPLC (RP-HPLC) is by far the most accepted LC technique for pharmaceutical analysis [16].

eg- Method validation for the quantitative evaluation of quercetin in topical emulsions by HPLC [17]


Affinity chromatography

High performance Liquid Chromatography

Multidimentional chromatography

Gas chromatography

Micellar Electro kinetic chromatography

Size exclusion chromatography

Ion chromatography

Planer chromatography


Affinity chromatography

According to the International Union of Pure and Applied Chemistry [18], affinity chromatography is defined as a liquid chromatographic technique that makes use of a "biological interaction" for the separation and analysis of specific analytes within a sample

Affinity chromatography is a liquid chromatography the separation and specific analysis of biological samples is performed by using biological interactions [1].

Affinity chromatography is a technique that utilizes highly specific interactions between one kind of solute molecule and a second molecule covalently attached means immobilized to the stationary phase. The covalently attached molecule can be an antibody to a specific protein.

As affinity chromatography is highly specialized form of adsorption chromatography, it is preferred in conditions where very specific separations are desired [19].

Some typical biological interactions used in affinity chromatography are-

Antigen - Antibody, virus, cell.

Enzyme - substrate analogue, inhibitor, cofactor.

Lectin - Polysaccharide, glycoprotein, cell surface receptor, cell.

Hormone, vitamin - receptor, carrier protein.

Glutathione - glutathione-S-transferase or GST fusion proteins.

Applications of affinity chromatography

Isolation and purification of human immunoglobulins

There is need of preparation of highly purified immunoglobulins to overcome the various immunological problems. The purification of immunoglobulin is the first step of obtaining the standard antibodies [20].

The antibodies are isolated from serum by affinity chromatography by use of specific antigen. The antibodies obtained by this way are highly specific, pure and highly suitable for conjugation [21].

Purification of fusion protein

DNA binding proteins and coagulation factor [19].

High performance Liquid chromatography

Liquid chromatography has become one of the most versatile techniques available to the analyst as it is simple and having high resolution capacity. It can be performed in which mobile phase allowed to flow through the packed column under the influence of gravitational force with high pressure [19].In both clinical research and routine clinical analysis, Liquid chromatography has great application [5].


Detection of Bladder Cancer in Human Urine

Bladder cancer is the second most common genitourinary malignancy. There is reappearing of bladder cancer in many patients but patient do not die by this disease. This bladder cancer may be detected by metablonomics with HPLC. In this metabolomics study of HPLC coupled on line with MS there is identification of variations in metabolite that may be used to differentiate between urine specimens from healthy individuals and patients with bladder cancer [22].

Detection of HIV Protease inhibitors in Biological matrices-

In HIV infection the mortality and morbidity has significantly decreased by the introduction of PIs [23]. These PIs inhibits the HIV protease enzyme and interferes with viral infection [24, 25].As there is increasd interest in the bioanalysis of these drugs number of HPLC methods has been published for each PIs [26-27]. The most suitable HPLC method for the bioanalysis of PI in biological matrix appeared as Reversed-phase or ion-pair chromatography [28].

C. Sepration of antidiabetic drug in human plasma

Diabetes mellitus is a heterogeneous group of disorders characterized by abnormalities in carbohydrate, protein, and lipid metabolism [29,30].Measuring glycated haemoglobin, especially type lc, is a means of monitoring longer term plasma glucose control in diabetic patients. As all the la, b, and c forms of glycated haemoglobin are less positively charged than normal haemoglobin at neutral pH, a method that separates proteins by charge should separate glycated from normal haemoglobin. Methods based on chromaography from

small cation exchange columns as well as various electrophoresis methods have been used successfully to resolve glycated from non-glycated haemoglobins [5, 30].

D. Investigation of amino acid from plasma.

HPLC is the method of choice for the diagnosis of many metabolic disorders as HPLC is having capacity for resolution of closely related compounds. This method has been found suitable for the identification, separation and quantification of amino acid in plasma and urine. In RP-HPLC method chemical pretreatment to amino acid increases the solubility of amino acid and helps in resolution of amino acid. The investigation of maple syrup disease and phenylketonuria the relative values of such method are considered or applied [5].

E. In separation of protein hormone-

Proteins can be isolated from biological specimen such as plasma, urine, saliva or sweat. Then these are fractionized or extracted with acid followed by centrifugation. Now the resolution is carried out by HPLC method by selection of suitable mobile phase. RP-HPLC method is most appropriate method for resolution of protein hormones [31].

F. IN therapeutic drug monitoring-

In hospitalized chronically ill patients and patients with failure organ, the pharmacokinetic properties of the many drugs may differ from normal condition. During treatment of anti-infective and anti-neoplastic treatment as well as neonatal care there may be toxic drug deposition or low concentration of drug in blood may appear. HPLC-MS/MS is having the potential that enables the routine monitoring of all essential drugs which are used in hospitals.

Prevalent utilization of HPLC-MS/MS might improve the influence of laboratory medicines with new markers. Eg - Methylmaloic acid [MMA] as a marker for deficiency of cobalmine [32,33], For nucleotide denaturation and oxidative stress 8-hydroxy-deoxy guanosine (8-OHDG) as a marker [34], advanced glycation end-products (AGEs) as novel markers of glycaemic control [35-36, 27]


Multi-dimensional chromatography separation methods have been widely applied in all kinds of biological sample investigations [38].Multi-dimensional liquid chromatography (MDLC) coupled with bio-mass spectrometry (MS) is playing important roles in proteome research due to its high speed, high resolution and high sensitivity [39].

Multidimensional chromatography allows separation of complex mixtures by using multiple columns with different stationary phases. These columns are coupled orthogonally, which means that fractions from the first column can be selectively transferred to other columns for additional separation. This enables separation of complex mixtures that cannot be separated using a single column. [pdf-Applications Multi]

Common applications for multidimensional LC are:

Proteins and Identification of Signature Peptides [40,41]

Analysis of human plasma proteome [38].

Drug isolation from urine and plasma


Homopolymers, oligomers, copolymers


Polycyclic aromatic hydrocarbons

DNA fragments [pdf-Applications Multi]

Analysis of the yeast proteome by multidimensional protein identification technology on large scale [42]

GAS Chromatography

It is widely used technique for the separation of gaseous and volatile substances which difficult to separate and analyze. Though it is a technique of separation it also provides identification of compounds and its quantitative estimation also. This technique is applied for analysis of oils, fats, food and flavors, drugs and vitamins, blood and serum, steroids and alkaloids [43]. Gas chromatography is analytical technique that can be used to investigate various clinical problems [44].

Detection of Metabolomics of gastric cancer metastasis

One of the most common malignancy is gastric cancer and this may lead to death of the patients[45,46,47]. In this Metastasis of gastric cancer is one of the main causes for death.

Recently it has been shown that metbolomic method is having potential in identification of

new diagnostic markers and therapeutic targets of various cancer such as gastric cancer,breast cancer, liver, prostate and pancreatic cancer.[48-49] In the detection of gastric cancer GC-MS method help identification of metabolomic differences between metastasis and non- metastasis models [50,51].

Identification of biomarkers in cancer

Cancer is a DNA deregulation disease and for its development various exogenous and endogenous factors are responsible [52]. The normal cells get transferred into malignant cell by disturbance in important metabolic path leading to changes in cellular functions resulting into various signals [53] such as biomarkers. The proteins, genes or metabolites present in biological specimen if identified and correlated with presence of specific caner then they are termed as biomarkers [54].

For Identification of biomarkers in different forms of cancer the analysis of biological specimen such as plasma, urine, saliva and tissue is increased because in clinical analysis. Gas chromatographic method is mainly used for identification, diagnosis and treatment of cancer [55].

Determining Volatile Compounds in Biological Fluids

Since the first application of gas chromatography to determine ethanol concentrations ,this technique has undergone significant modification and enhancement [56] Headspace sampling, coupled with gas chromatography, is the preferred technique for the analysis of volatile compounds, such as ethanol, in biological fluids[57].

In food analysis

Now a days as GC is very commonly used it is also used for food analysis. It includes qualitative and quantitative analysis of composition of food, taste, odor and natural products. It also determines the presence of pesticides, pollutants, natural toxins and drugs.

Application of Gas chromatography indicates that the care must be taken while using the agricultural and instrumental chemicals that will harm the human health as well as ecosystem. Recently, GC is most commonly used in food analysis of carbohydrates, lipids, Pesticides and drugs [58].

Ion exchange chromatography-

Ion exchange chromatography uses an ion exchange resin as the stationary phase. The mechanism is based on ion exchange equilibrium. Mobile phase pass through this surface, ionic solutes are retained by forming electrostatic bonds with functional group [19].

In purification of an anti-histone H1 monoclonal immunoglobulin M antibody

During organ transplantation there is need of development of novel immunosuppressant for permanent administration and to determine serious side effect. Determination of mechanism of immunosuppressant is analytically determined by the use of anti-histone H1 monoclonal immunoglobulin M antibody. The monoclonal antibody used for organ transplantation should of high purity and should not contain any allogenic protein. The monoclonal antibody can be purified by the use of Anion exchange chromatography from the serum-free culture supernatant [59].

In Pharmaceutical and Drug Analysis

On the basis of nature of analyte IC can be applied in all aspects to the pharmaceutical product manufacturing. It includes characterization of drug substances, additives, active ingredients including impurities and degradation products. By IC the raw materials, formulated products, additives and diluents can be analyzed. The method is especially valuable in the pharmaceutical industry for ionic analytes [60].

Size exclusion chromatography-

Size exclusion chromatography is also called as gel filtration or molecular-sieve

Chromatography. In this technique the solutes are separated on the basis of their size in

solution. Extensively this chromatographic method is used for preparative separation of

macromolecules from biological origin such as proteins, nucleic acid and polysaccharides[19].


Isolation and purification of platelets from blood plasma

In haemostasis platelates plays a vital function and their identification is necessary. For the determination of their concentration in blood and volume are measured by automated

laboratory analyzer. When their chemical investigation needed, a procedure for their separation and clean up is required, because their porous structure and active surface retains components from their environment being obstructive in obtaining data about their chemical composition. Size exclusion chromatography used for the separation of platelets from blood plasma [61].

Planer chromatography

Thin -layer chromatography (TLC) is one of the planer chromatography that is most popularly and widely used technique as it performs rapid screening [62]. In TLC a finely divided solid spreaded as a thin on a rigid plate act as solid phase over which mobile phase of liquid allowed to pass across the surface of plate [19].


Diagnosis of metabolic disease and cancer by separation and detection of cellular phosphoinositides from specefic phosphoinositide binding domains [63].

TLC with matrix solid phase dispersion used for screening of enrofloxacin

and ciprofloxacin residues in milk with isolation and concentration [64].

In complex matrixes detection of xanthine oxidase inhibitors and superoxide scavengers [65] and glucosidase inhibitors [66].

From feed sample spiramycin, virginiamycin, and tylosin can be determined by TLC method. [67, 68].

Micellar Electro kinetic chromatography

The use of surfactants as pseudo stationary phases in CE is called "micellar electrokinetic chromatography (MEKC)." Micelles are employed typically to enable separation of nonionic compounds [69].


The Rapid Analysis of Heroin Drug Seizures

Micellar electrokinetic chromatography (MEKC) is one the technique of CE that has been applied for the analysis of opiates in heroin seizure samples. This analysis is important in forensic pharmacy. The analysis of heroin seizures by MEKC with the use of the short-end injection is a rapid and accurate method [70].

Study on urinary metabolic profile of phenylketonuria

Phenylketonuria (PKU) is an autosomal recessive disorder and it is the most common metabolic cause of mental retardation. In this disease there is deficiency in phenylalanine hydroxylase activity, which results in the increasing of phenylalanine concentration in the blood and tissues [71]. In phenylketonuric the urinary metabolic marker compounds such as phenylpyruvic acid (PPA), 2-hydroxyphenylacetic acid (oOPAA), 4-hydroxyphenylacetic acid (pOPAA), phenyllactic acid (PLA) and phenylacetic acid (PAA) can be detected by micellar electrokinetic capillary Chromatography [72].

Determination of cortisol in urine samples

The human adrenal adrenal glands secrets the major glucocorticoid is Cortisol. It has anti-inflammatory and immunosuppressive effects regulate a myriad of biological function. it is also a biological biomarker of stress, anxiety and depression.In clinical laboratories, steroid hormones in urine are usually analyzed by immunological techniques [73,], HPLC [74,75-76], GC [77], LC [78-79], TLC [80] and capillary electrophoresis (CE) [81-82]. The immunoassay methods are rapid and simple, but it is having limitation that for urinary free cortisol along with cortisol metabolites or cortisol obtained for adrenal [83].


From above all information it is concluded that biomedical chromatography includes all chromatographic techniques such as affinity chromatograohy, HPLC, GC, Ion, that can be used for clinical application. It utilizes advanced analytical techniques for the diagnosis of disease. As biomedicine is related to human health we can say that the biomedical chromatography helps in control of human health. Although these technologies are more popular