1. CHROMATOGRAPHY Dr. Gobinath.P

2. What is Chromatography? Chromatography is the science which is studies the separation of molecules based on differences in their structure and/or composition. In general, chromatography involves moving a preparation of the materials to be separated - the "test preparation" - over a stationary support. The molecules in the test preparation will have different interactions with the stationary support leading to separation of similar molecules. Test molecules which display tighter interactions with the support will tend to move more slowly through the support than those molecules with weaker interactions.

3. In this way, different types of molecules can be separated from each other as they move over the support material. Chromatographic separations can be carried out using a variety of supports, including immobilized silica on glass plates (thin layer chromatography), volatile gases (gas chromatography), liquids which may incorporate hydrophilic, insoluble molecules (liquid chromatography).

4. Chromatography and Biotechnology This discussion of chromatography will focus on the separation of proteins into relatively homogeneous groups because proteins are often the target molecules which must be purified for use as "biopharmaceuticals" or medicines. It is important to remember, that chromatography can also be applied to the separation of other important molecules including nucleic acids, carbohydrates, fats, vitamins, and more.

5. identification of a "target protein" which may have therapeutic value identification of the "target gene" -- the gene responsible for encoding the target protein isolation of the target gene insertion of the target gene into a host cell (such as E. coli) which will both grow well, and continue to produce the protein product encoded for by the target gene separation of the target protein from the many other host cell proteins

6. large scale production of the target protein under controlled manufacturing conditions large scale testing for efficacy as a medicine marketing of a new medicine Many different disciplines, including microbiology, molecular biology, chemistry, and others, are required to complete the steps listed above to bring a protein from the "scientifically interesting" state to that of a full-fledged drug to be used in treating a specific disease. This discussion will focus on the work and tools of the chromatographer.

7. The most commonly used of these techniques is liquid chromatography, which is used to separate the target molecule from undesired contaminants (usually host-related), as well as to analyze the final product for the requisite purity established with governmental regulatory groups (FDA, WHO…..etc).

8. Main Type and Advanced Chromatography Techniques 1.Thin Layer Chromatography (TLC: Used for Basic study but it is good) 2.Ion-Exchange Chromatography (ICE- Good for Amino acids separation) 3.Gel-Filtration Chromatography (GFC- Protein separation) 4.Affinity Chromatography ( AC- Protein ) 5.Gas chromatography/Mass Spectroscopy (GC/MS- Advanced and high standard- Volatile compounds) 6.Liquid chromatography /Mass Spectroscopy (LC/MS- Very best for non volatile compounds)

9. Thin Layer Chromatography Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures.chromatography Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose (blotter paper). adsorbentsilica gelaluminium oxide celluloseblotter paper This layer of adsorbent is known as the stationary phase.stationary phase After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.solventmobile phasecapillary actionanalytes

11. Thin layer chromatography can be used to: Monitor the progress of a reaction Identify compounds present in a given substance Determine the purity of a substance Specific examples of these applications include: determination of the components a plant contains analyzing ceramides and fatty acidsceramidesfatty acids detection of pesticides or insecticides in food and waterpesticidesinsecticides analyzing the dye composition of fibers in forensics, orforensics assaying the radiochemical purity of radiopharmaceuticalsradiochemical purity radiopharmaceuticals

12. Ion exchange chromatography Proteins are made up of twenty common amino acids. Some of these amino acids possess side groups ("R" groups) which are either positively or negatively charged. A comparison of the overall number of positive and negative charges will give a clue as to the nature of the protein. If the protein has more positive charges than negative charges, it is said to be a basic protein. If the negative charges are greater than the positive charges, the protein is acidic.

13. When the protein contains a predominance of ionic charges, it can be bound to a support that carries the opposite charge. A basic protein, which is positively charged, will bind to a support which is negatively charged. An acidic protein, which is negatively charged, will bind to a positive support. The use of ion-exchange chromatography, then, allows molecules to be separated based upon their charge. Families of molecules (acidics, basics and neutrals) can be easily separated by this technique. This is perhaps the most frequently used chromatographic technique used for protein purification.

14. Gel-Filtration Chromatography This technique separates proteins based on size and shape. The support for gel-filtration chromatography are beads which contain holes, called "pores," of given sizes. Larger molecules, which can't penetrate the pores, move around the beads and migrate through the spaces which separate the beads faster than the smaller molecules, which may penetrate the pores. This is the only chromatographic technique which does not involve binding of the protein to a support.

15. Affinity Chromatography This is the most powerful technique available to the chromatographer. It is the only technique which can potentially allow a one- step purification of the target molecule. In order to work, a specific ligand (a molecule which recognizes the target protein) must be immobilized on a support in such a way that allows it to bind to the target molecule. A classic example of this would be the use of an immobilized protein to capture it's receptor (the reverse would also work). This technique has the potential to be used for the purification of any protein, provided that a specific ligand is available. Ligand availability and the cost of the specialized media are usually prohibitive at large-scale.

16. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY – HPLC High performance liquid chromatography is a powerful tool in analysis. This page looks at how it is carried out and shows how it uses the same principles as in thin layer chromatography and column chromatography.

17. High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster. The other major improvement over column chromatography concerns the detection methods which can be used. These methods are highly automated and extremely sensitive.

18. Normal phase HPLC This is essentially just the same as you will already have read about in thin layer chromatography or column chromatography. Although it is described as "normal", it isn't the most commonly used form of HPLC. The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for example. A typical column has an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to 250 mm.

19. Polar compounds in the mixture being passed through the column will stick longer to the polar silica than non-polar compounds will. The non-polar ones will therefore pass more quickly through the column.

20. HPLC http://www.youtube.com/watch?v=kz_egMtdnL4&f eature=related

21. Reversed phase HPLC In this case, the column size is the same, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in them. A polar solvent is used - for example, a mixture of water and an alcohol such as methanol.

22. In this case, there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. There won't be as much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent.

23. Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of vander Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules, for example.

24. That means that now it is the polar molecules that will travel through the column more quickly. Reversed phase HPLC is the most commonly used form of HPLC. Injection of the sample Injection of the sample is entirely automated, and you wouldn't be expected to know how this is done at this introductory level. Because of the pressures involved, it is not the same as in gas chromatography

25. Retention time The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound.

26. Different compounds have different retention times. For a particular compound, the retention time will vary depending on: the pressure used (because that affects the flow rate of the solvent) the nature of the stationary phase (not only what material it is made of, but also particle size) the exact composition of the solvent the temperature of the column That means that conditions have to be carefully controlled if you are using retention times as a way of identifying compounds.

27. The detector Many organic compounds absorb UV light of various wavelengths. If you have a beam of UV light shining through the stream of liquid coming out of the column, and a UV detector on the opposite side of the stream, you can get a direct reading of how much of the light is absorbed.

28. LC/MS/MS MassSpectrometry

29. HPLC/MS/MS is able to provide such excellent sensitivity both because of its great raw sensitivity and the improved Signal to Noise (S/N) ratio inherent in tandem mass spectrometry. MS/MS also can be coupled to capillary electrophoresis, providing greater sensitivity along with structure confirmation and degradant detection for peptides and proteins. GC/MS/MS for the analysis of drugs, synthetic contaminants, metabolites, and degradants. HPLC/MS/MS and HPLC/MS can identify oxidation and reduction, phosphorylation, as well as deamidation and isoaspartate formation, in peptides, proteins, and small molecules.degradants

30. Using HPLC/MS/MS and HPLC/MS instruments with electrospray (ESI), NanoSpray, and atmospheric pressure chemical ionization (APCI) techniques, RML can solve many of your most difficult analytical problems, even ones which once seemed to be quite intractable. The detection of Tetracyclines in animal tissues, the analysis of cyclic peptides in whole blood, and the identification and quantitation of nucleotide and nucleoside analogues at sub-nanogram per milliliter levels are routine.

31. GAS CHROMATOGRAPHY Gas chromatography - specifically gas-liquid chromatography - involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

32. Gas Chromatography Mass Spectrometry (GC/MS) identifies volatile and semi-volatile compounds and separates them into individual components using a temperature-controlled gas chromatograph. During the process, a sample is injected into the chromatograph (or it may come from another sampling device) and passes through the chromatography column, which separates mixtures into individual components as they pass through at different rates. The result is a quantitative analysis of the components, along with a mass spectrum of each component.

33. Carrier gas The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependant upon the type of detector which is used. The carrier gas system also contains a molecular sieve to remove water and other impurities.

34. Sample injection port For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a "plug" of vapour - slow injection of large samples causes band broadening and loss of resolution. The most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column. The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample. For packed columns, sample size ranges from tenths of a microliter up to 20 microliters. Capillary columns, on the other hand, need much less sample, typically around 10 -3 mL. For capillary GC, split/splitless injection is used. Have a look at this diagram of a split/splitless injector;

36. GC/MS http://www.youtube.com/watch?v=08YWhLTjlfo&f eature=related

37. Application of GC and GC/MS: Volatile compound and properties study and analysis (Flavour and Fragrance) Bioactive compound analysis Drug discovery and composition analysis Unknown compound/drug analysis