Rekha Singh M.

Phil Botany, UOM

Introduction Classification History Significance Principles Applications Conclusion

Chromatography is a physio-chemical method of separation. The word chromatography is derived from the Greek words khroma" meaning colour and "graphein" meaning to write An analytical technique employed for the purification and separation of organic and inorganic substances. Useful for the fractionation of complex mixture, separation of closely related compounds, such as isomers and in the isolation of unstable substances.

Separation of ionic species and molecular species ranging in size from the lightest and smallest, helium and hydrogen, to particulate matter such as single cells Separation of several hundreds of components of unknown identity and unknown concentrations, leaving the components unchanged Amounts in the picogram or parts per billion range can be detected with specific detectors.

MOBILE PHASE

GAS

MECHANISM

Gas-Liquid Chromatography (Partition)

Gas-Solid Chromatography (Adsorption)

STATIONARY PHASE

Liquid

Solid

Ion exchange Size exclusion

MOBILE PHASE

LIQUID

Affinity

MECHANISM

Liquid-Liquid Chromatography (Partition)

Liquid-Solid (TLC) Chromatography (Adsorption)

STATIONARY PHASE

Liquid

Solid

Normal Phase Mobile Phase - Nonpolar Stationary phase - Polar

Reverse Phase

Normal Phase

Reverse Phase

Stationary phase - Nonpolar

1855 German chemist Friedlieb Ferdinand Runge descried the use of paper to analyze dyes. 1861 German chemist Friedrich Goppelsroder described chromatography as Capillary Analysis 1906 the technique was first described in detail by Russian Biologists, Dr Mikhail Tswett by the separation of colored plant pigments on a column of alumina. 1938 Ion exchange chromatography - T.I Taylor & Harold C. Urey

1938 TLC - Soviet pharmacists Nicolay & Maria and realized after intensive research by German chemists Egan Stahl in 1956 1940s extensive work done on Paper chromatography by British chemist Archer J.P. Martin & his associates. 1941 Martin & Richard L.M. Synge - Partition chromatography 1944 Gas chromatography Erika Cremer and first practical exploitation- Martin & James 1952 1957 Open tubular columns by Marcel J. E. Golay

1959 Size exclusion chromatography Per Flodin & Jerker Porath from Sweden 1962 Supercritical fluid chromatography - Ernst Klesper German chemist 1964 HPLC theory Calvin Giddings American chemist 1966 Field- flow fractionation Giddings 1968 Affinity chromatography Pedro Cuatrecases

Chromatography is adaptable to wide range of compounds, because variety of separation principles (retention mechanisms) and types of experimental setup (planar or column -gas and liquid phase elution) Separated analyte is immediately available for identification or quantification Today various types of chromatography are used to separate almost any given mixture whether coloured or colourless into its component.

Two immiscible phases i.e. Stationary and Mobile, brought in contact with each other. The mobile phase either percolates through or moves over the stationary phase. The sample mixture carried by the mobile phase interacts with the mobile and stationary phase The element which interacts more with mobile phase & least with stationary phase travels fast and vice versa. The differential movement of the elements leads to their separation.

SEPARATION PHENOMENON MOBILE PHASE

STATIONARY PHASE

PRINCIPLE MECHANISM

Normal Phase Adsorption Chromatography Reverse Phase

Liquid or gas Hydrophilic

Hydrophilic Solid that attracts the solutes Solutes move at different rates according to the hydrophilic or hydrophobic forces of attraction to the stationary phase

Liquid or gas Hydrophobic

Hydrophobic Solid that attracts the solutes

Partition Chromatography

Liquid or gas

Capillary action & Solubility. Solutes Thin film of liquid formed on the equilibrate between the two phases surface of a solid inert support according to their partition or adsorbent on solid surface coefficients

Ion Exchange Chromatography

Liquid containing electrolytes

Solid resin that carries fixed ions & mobile counter-ions of opposite charge attached by covalent bonds

Solute ions of opposite charge to the fixed ions are attracted to the resin by electrostatic forces & replace the mobile counter-ions

SEPARATION MECHANISM

MOBILE PHASE

STATIONARY PHASE

PINCIPLE MECHANISM

Molecular (Size) Exclusion Chromatography

Liquid

Porous gel with no attractive action on solute molecules

Molecules separate according to their size. Smaller molecules enter the pores of the gel, and need a larger volume of eluent

Affinity Chromatography

Liquid

Ligands on solid support that have the ability to recognize one of the structural motif of a molecule or group of structurally similar molecules

Selective non-covalent interaction between an analyte and specific molecules

Displacement Chromatography

Liquid

Liquid, solid column or bonded liquid

A molecule with a high affinity matrix (the displacer) will compete more effectively for binding sites, leaving the mobile phase enriched in the lower-affinity solute

PC - Chromatogram

Used as a qualitative method for identifying the components in a mixture. The characterization and isolation of acids, alcohols, glycols, alkaloids, amines, proteins, peptides, carbohydrates, antibiotics, steroids etc. For quality control in the food industry, by separating and analyzing additives, vitamins, preservatives, histamines and amino acids Used to detect traces of pollutants in water food or in soil Analysis of metabolites of drugs in blood and body fluids In forensic studies in CSI and DNA & RNA fingerprinting To look for traces of flammable chemicals in burned material from possible arson sites Used as an analytical chemistry technique for identifying and separating colored mixtures like pigments

Is simple, inexpensive and rapid method that provides graphic and clear results The separated spots on the finished and dried chromatogram can be cut out and re-dissolved to obtain a pure sample of component of the sample mixtures. Sugars, amino acids, lipids and nucleic acids and other biomolecules can be easily identified by spraying with appropriate reagents to detect these specific compounds. Paper chromatography can be reproduced easily as long as the conditions are controlled and maintained.

Thin-Layer Chromatography Uses thin plastic or glass slabs to embed the stationary phase

Pharmaceuticals and Drugs Identification, purity testing and determination of the concentration of active ingredients, auxiliary substances and preservatives in drugs and drug preparations and process control in synthetic manufacturing processes. Clinical Chemistry, Forensic Chemistry and Biochemistry Determination of active substances and their metabolites in biological matrices, diagnosis of metabolic disorders such as PKU (phenylketonuria), cystinuria and maple syrup disease in babies. Cosmetology Dye raw materials and end products, preservatives, surfactants, fatty acids, constituents of perfumes. Food Analysis Determination of pesticides and fungicides in drinking water, residues in vegetables, salads and meat, vitamins in soft drinks and margarine, banned additives in Germany (e.g. sandalwood extract in fish and meat products), compliance with limit values (e.g. polycyclic compounds in drinking water, aflatoxins in milk and its products).

Environmental Analysis Groundwater analysis, determination of pollutants from abandoned armaments in soils and surface waters, decomposition products from azo dyes used in textiles. Analysis of Inorganic Substances Determination of inorganic ions (metals). In organic chemistry used for:
Checking the purity of samples, As purification process, For identification of organic compounds For studying various organic reactions In characterizing and isolating a number of compounds such as acids, alcohols, glycols, amides, alkaloids, vitamins, amino acids, antibiotics, aromatic amines, food stuffs etc. Examination of reaction

Used in checking separation processes and purification processes like distillation, molecular distillation etc.

The technique is non-destructive, i.e. the molecules in the mixtures are separated physically without being chemically altered In comparison with column chromatography, it only requires small quantities of the compound (~ng) and is much faster Often used to monitor the progress of organic reactions and to check the purity of products

In separation & study of : Carbohydrates Amino acids, proteins Vitamins, A, D, E, K Nucleosides (purines and pyrimidines) Fatty acids, fats Aflatoxins Antioxidants Contaminants of packaging materials Carotenoids, chlorophylls Saccharines

Biotechnological analysis of amino acids

Purity control, quality management and certification Environmental analysis Detection of Polycyclic aromatic hydrocarbons Pharmaceutical analysis Detection of basic drugs Biotechnological analysis of amino acids Analysis of main trace components (% to ppm) Reversed phase chromatography commonly used HPLC technique for compounds with diverse polarity & molecular mass RPC allows precise control of variables such as organic solvent type and concentration, pH, and temperature In pharmaceutical industry - for the separation of Chiral compounds

Simulated Moving Bed chromatography a variant of HPLC used in the pharmaceutical industry, where resolution of molecules having different chirality must be done on a very large scale. For the production of Fructose (in High fructose corn syrup or amino-acids, biological-acids) Displacement chromatography was applied to preparative separations of amino acids and rare earth elements and has also been investigated for isotope separation. Can be readily carried out using a variety of resin systems including, ion exchange, HIC and RPLC Two dimensional chromatography utilizing displacement rather than elution mode in the upstream chromatography step represents a potentially powerful tool for analysis of trace components, modifications, and identification of minor expressed components of the proteome.

In preparation of high purity water for power engineering, electronic and nuclear industries; i.e. polymeric or mineralic insoluble ion exchangers are widely used for water softening, purification and decontamination etc. Widely used in household (laundry detergents and water filters) to produce soft water Important industrial application extraction and purification of biologically produced substances such as proteins (amino acids) and DNA/RNA Ion-exchange processes are used to separate and purify metals, including separating uranium from plutonium and other actinides, including thorium, and lanthanum, neodymium, ytterbium, samarium, lutetium, from each other and the other lanthanides. PUREX process Used in nuclear reprocessing and the treatment of radioactive waste. Dealkalization - removal of alkali ions from a glass surface. Chemically strengthened glass, produced by exchanging Na+ for K+ in glass surfaces using KNO3 melts.

Used primarily for analytical assays and semipreparative purifications used as a measure of both the size and the polydispersity of a synthesized polymer Useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions Applied to the separation of biopolymers such as proteins and nucleic acids, i.e. water-soluble polymers For separation/clean-up of platelets from blood plasma before their chemical investigation

In the purification of antibody from blood stream Nucleic acid purification, protein purification from cell free extracts, and from blood Common use is for the purification of recombinant proteins The process has the ability to remove pathogens, which is necessary if the purified biomolecules are to be used in clinical applications. Used to study drug-protein binding interactions. For cleaning up and removing excess albumin and macroglobulin contamination from samples To separate glycoproteins from non- glycosylated proteins

Auto sampler for liquid samples

Injection vials auto sampler

Flame Ionizing Detector & Thermal Conductivity Detector

Mass Selective Detector

GC oven with column(s)

Flow rates & Temp. control Injector

Chlorinated pesticide analysis

Used in gas refinery for petrochemical analysis In environmental and petrochemical monitoring and consequently necessary remediation processes Is a novel method of detecting the nerve agent VX and its hydrolysis products

To determine the role of olfaction in the feeding behavior of Anopheles gambiae, one of the most active and efficient malaria vectors known to date. Agricultural Research Service (ARS) of the US Department of Agriculture are working on how to save Avocado trees from the threat of fungal infection caused by the Redbay Ambrosia beetle. Led by Paul Kendra, the team used gas chromatography mass spectroscopy to determine the volatile compounds (VOCs) released by the trees that attract the beetle, leading them to infect the trees. Yale Center for Environment Law and Policy GC for air pollution sound study

Research team from Israel and Colorado GC for non invasive breath analysis to diagnose lung cancer Used to determine the level of CP & CPO insecticide in wine University of Ontario Institute of Technology use GC to check the accuracy of the kits that law enforcement units use for cadaver dog training University of Regensburg- GC determined toxins in black tattoo ink, which can cause irritation and tissue abnormalities

Utilized for the separation of underivatized triterpene acids, estimation of caffeine from tea and conjugated bile acids Analysis of pesticide residues in canned foods, fruits and vegetables wherein pyrethroids, herbicides, fungicides and carbamates have been tested For analysis of high molecular weight lipids like triacylglycerols Due to the high efficiency, fast separation, low temperature analysis and applicability to wide variety of detectors, SFC has now become an attractive alternative for chiral drug separation The biggest advantage that SFC holds over GC is the ability to separate thermally labile compounds, which is a very significant application in the pharmaceutical field as 20% of all drug candidates fall in this category Applied to analysis of polyethoxylated alkylphenols, polyolefinic antioxidants /light stabilizers and polynuclear aromatic hydrocarbons in automobile exhaust

Breakthrough in the field of synthetic drugs has increased the need for new chromatographic process Synthetic oligonucleotides are an exciting new class of biomolecules capable of treating many disorders, which are currently not amenable to existing drugs, including viral infections, respiratory disorders, cancers and rare diseases. HPLC- MS used for characterization API. Currently complete characterization of API & impurities is a challenge Further advances in chromatographic purification of macromolecules and nanoparticulates demand close collaboration between inventors and manufacturers of new products and the suppliers of chromatographic hardware and consumables There are more than 50 acclaimed companies invested in research & production of chromatographic columns for laboratories and industries Identification of new detectors, cost effective techniques yielding compounds with no impurities comparatively faster to match with the growing demand

Chromatography represents one of the greatest methodical phenomenon of the Twentieth century, with an extremely fruitful output for the future Influenced discoveries in scientific disciplines as Chemistry, Biology, Medicine and applied scientific fields as Food, Drugs and Environmental studies Responsible for the future of microanalysis The technique is probably the most powerful and versatile technique available to the modern analyst