Chromatography Technique and its Applications
Chromatography is an important biophysical technique which is used in the separation. This technique is used for the separation, identification and purification of components from a mixture of compounds. This process yields qualitative as well quantitative results. Chromatographic technique is based on the principle in which molecule in the mixture when applied into solid stationary phase can be separated from each other while moving with the help of mobile phase. The factors which affects the separation process includes molecular characteristics related to adsorption, partition and difference between molecular weights. some components of the mixture stay longer in the stationary phase as they move slowly in the stationary phase while some components of mixture pass fast into the stationary phase and elutes faster than other molecules which pass slowly. Chromatographic techniques acquire unique positions among modern methods in the field of detection, and separation. These latest chromatographic techniques provides simple techniques for separation, purification and structure determination including most complicated natural and synthetic macromolecules in biological and geological systems and their metabolite and breakdown products.
Some common chromatographic techniques includes
(1) Thin Layer chromatography (TLC)
(2) Gas Chromatography
(3) High Performance liquid chromatograph
(4) Ion Exchange chromatograph
(5) Gel Permeation Chromatograph
Thin Layer Chromatography
In TLC stationary phase is a thin layer of adsorbent material coated on plate which is made up of glass or silica and mobile phase also called as developing solvent is a mixture of solvent for which analyte has strong affinity. This mobile phase ascends the thin layer of stationary phase by capillary action. Sample spots done on the stationary phase at the bottom then slowly comes in to contact with ascending mobile phase and then depending on the affinity to each phase analyte molecule are partitioned between mobile and stationary phase Based on the capacity to travel upward directions analyte then get separated and one single spot shows different spots in the capacity of separation. This technique is widely used in the monitoring progress of organic reactions. This is sensitive as well as rapid technique, it is economical but it has certain limitations as it is qualitative technique. Now a days an enhanced technique called high performance thin layer chromatography is used having same basic principle. This HPTLC technique can be used for quantitive purpose.
Gas Chromatograph
In Gas chromatography gas is used as mobile phase and samples are gasified and flow with gaseous mobile phase before entering in a column. This techniques has the advantage of higher sensitivity, smaller sample size, strong separation capacity, good selectivity and fast analysis. This techniques is further enhanced with mass detection and called as GCMS. In GCMS molecule is fragmented and fragments are detected.
High performance Liquid chromatography
HPLC technique is used to separate and identify the quality of components in a mixture. This technique is most commonly used in laboratory. This technique is used to perform structural and functional analysis. In HPLC mobile phase passes through stationery phase (Column) under high pressure and flow, this increases separation power.
HPLC can be coupled with MS-MS to identify unknown peaks. In HPLC separation takes place by 2 different processes as reverse phase and normal phase.
Ion exchange chromatography
This is also and important but selective technique and used to separate charged molecules based on affinity of individuals towards an ion exchanger. IC uses electrostatic interaction for separation of ionic chemicals. There are two types of exchangers used in this chromatography depending on the functional group as Cation exchange and anion exchange.
The detector used in the analysis is conductivity detector which measures the change in conductivity as ion passes through the detector.
Gel Permeation chromatograph
This is also a selective chromatographic technique and used to separate macro molecules based on their molecular weight. In this chromatography molecular weights of proteins can be detected. In ion chromatography stationery phase consists of inert molecules with small pores.
Pharmaceuticals applications of chromatographic techniques
In the Pharmaceutical and chemical industry chromatography techniques mainly used are High-performance liquid chromatography (HPLC), Thin Layer Chromatography (TLC), and Gas chromatography to give precise results which are required for the purity of drug substance, drug product, drug stability, separation of chiral compounds, analysing the purified compounds for traces of impurities, etc. The advantage of HPLC is in elucidating the structures and determining the quantities of impurities in pharmaceutical drug products and drug substances.
Environmental applications : Pollutants bio monitoring, detection of phenolic compounds in drinking water and monitoring air quality, etc.
Forensic applications: Chromatography has unique applications in Forensics like quantification of drugs in biological samples, analysis of textile dyes, identification of steroids in blood, urine, etc. Chromatography is very important for Forensic pathology work.
Food and flavour: Chromatography is used to analyse sugar in fruit juices, separating and analysing additives, proteins, amino acids, vitamins, quality of soft drinks and water, and preservative analysis. Food products contain a complex mixture of compounds (these compounds when present above the concentration level) that may be naturally occurring, at the manufacturing stage or the harvest stage. Chromatography techniques are used to determine nutritional values of food samples. In the food industry the technique of chromatography plays a vital role in the determination of shelf life of the food substances by helping in the analysis of the point at which food spoils.
Clinical Trials: Chromatography is used in clinical trials.
Molecular Biology: Chromatography is used in Molecular Biology.
Petroleum Industry: Chromatography is used in Petroleum industry.
Chemical Industry: The presence of toxic contaminant in oil and pesticides can be determined with the help of specialized chromatography techniques such as GC and HPLC.
FAQ’s
In the Pharmaceutical industry, chromatographic techniques used are High-performance liquid analysis (HPLC), the advantage of HPLC is in elucidating the structures, determining the quantities of impurities and efficient and accurate results, and ensuring high quality of drugs.
Impurities are present in pharmaceutical products or drug substances and need to be identified and characterized by using suitable analytical methods. HPLC is frequently used to determine the assay and impurities in drug substances and drug products.
Chromatography has wide applications and has a significant role in fields like the Pharmaceutical and Chemical Industry, Food and beverage Industry, Environmental analysis, Forensic science, and petroleum Industry, etc.
Chromatography is a laboratory technique used for separating and identifying the components of a mixture based on their physical and chemical properties. In chromatography, the mixture is first dissolved in a mobile phase, which is then passed through a stationary phase. The different components of the mixture interact with the stationary phase to varying degrees, leading to their separation.
References
- González-González, Mirna; Mayolo-Deloisa, Karla; Rito-Palomares, Marco (1 January 2020), Matte, Allan (ed.), “Chapter 5 – Recent advances in antibody-based monolith chromatography for therapeutic applications”, Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics, Elsevier, pp. 105–116.
- Alternative bioseparation operations: life beyond packed-bed chromatography T.M. Przybycien, N.S. Pujar and L.M. Steele Curr Opin Biotechnol, 15 (5) (2004), pp. 469-478.
- Center for Drug Evaluation and Research (CDER) Reviewer Guidance’ Validation of Chromatographic Methods. 1994, www.fda.gov/media/75643/download.
