Bioseparation II Chromatography Techniques

Chromatography Most widely used purification technique used for biomolecules. Most widely used purification technique used for biomolecules. Useful for analyzing samples with many different components eg. drug mixtures, urine samples. 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. 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.

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.

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.

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.

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

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

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.

Elution of the column

High performance chromatography

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

Gas Chromatograph.

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 as molecular sieving or size exclusion chromatography. Equipment and column look similar to column chromatography

Purification problems Most commonly, loss of product “somewhere”! Most commonly, loss of product “somewhere”! Sample at each stage and determine yield and purity. Sample at each stage and determine yield and purity. Precautions 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.

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

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

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