Bioseparation II

1. Bioseparation II Chromatography Techniques

2. Chromatography • Most widely used purification technique used for biomolecules. • Useful for analyzing samples with many different components eg. drug mixtures, urine samples. • Refers to a number of techniques which are based on the differential interaction of molecules between stationary and mobile phases. • Stationary phase: immobile matrix • Mobile phase: liquid or gas phase which moves past the stationary phase and elutes molecules.

3. Different chromatography methods. Partition chromatography: separation is based on differing polarities or relative water solubility (GC, HPLC, TLC). Ion exchange chromatography: separation is based on differing charges of the molecules (GC, HPLC) Gel permeation chromatography: separation is based on differing molecular weights (IEC, cf gel electrophoresis) Permeation chromatography: separation based on the hydrophobic properties of the molecules. Affinity chromatography: separation based on the specific binding properties of the molecules in the mixture.

4. Choosing a chromatography method Based on the type of separation - i.e. what property of the molecule will be exploited to achieve the best separation? Methods differ on the level of technology required, some require expensive equipment and some require a simple set-up and few materials.

5. Thin Layer Chromatography • a thin layer of stationary phase material (eg. silica or aluminum oxide) is spread on a glass or plastic plate. The mobile phase passes through the stationary phase by capillary action or gravity.

6. Components of a mixture are resolved as "spots" which can be visualized by staining with dyes or use of alternate light sources (UV).

7. Identification of mixture components • Use Rf values and comparison to purified “known” compounds run in an adjacent lane. • Rf = distance moved by component distance moved by solvent front

8. Column Chromatography • the stationary phase is packed into a cylindrical column. • The mobile phase (or eluent) passes through the column by gravity or is pumped mechanically with an electric pump. • Fractions of the mobile phase are collected and assayed as they leave the column.

9. Elution of the column

10. High performance chromatography

12. the stationary phase is a column of harder particles than those used for column chromatography • Components are identified on chromatograms by their retention times

13. Gas Chromatograph.

14. Gel permeation chromatography • technique used for separating molecules by relative size and molecular weight using a column filled with porous gel particles. • GPC is also known asmolecular sievingor size exclusion chromatography. • Equipment and column look similar to column chromatography

16. Purification problems • Most commonly, loss of product “somewhere”! • Sample at each stage and determine yield and purity. • Precautions 1. The fewer the number of purification steps the better (higher yield). 2. Stability is a primary concern. Constant temperature and pH. 3. NEVER discard any fractions during a separation procedure.

17. Mass Spectrometry Aim a beam of high energy electrons at the sample. • electrons are lost and they acquire a positive charge (“ions”) • decompose into small fragments • magnetic field will separate them according to their mass

18. Mass spectrum • No two substances have the same fragmentation pattern. • Only need 0.000001 grams • Widest application in identification of drugs.

19. Gas chromatography-mass spectrometry • Gas chromatograph can be linked directly to the mass spectrometer for definitive identification. • How a GC/MS works