CHROMATOGRAPHY

1. CHROMATOGRAPHY Magnet Analytical Chemistry Unit 4

2. Chromatography Physical separation method based on the differential migration of analytes in a mobile phase as they move along a stationary phase. Separation occurs because of differences in particle mass and attraction to the solvent. Mechanisms of Separation: •Partitioning •Adsorption •Size Exclusion •Ion Exchange •Affinity Chromatographic Separations: Based on the distribution (partitioning) of the solutes between the mobile and stationary phases, described by a partition coefficient, K: K = Cs/Cm where Cs is the solute concentration in the stationary phase and Cm is its concentration in the mobile phase.

3. Principle The level of Interaction (adsorption) packing material & sample A,B differs, resulting in different speeds of travel of A & B in a media (paper, column etc.). Usually sample to be analyzed is injected into a carrier (gas or liquid). Carrier is usually inert (does not react with packing materials). The components in sample, being separated after chromatography, are analyzed. Types of chromatography LC - Liquid (carrier & A,B) Chromatography GC - Gas (carrier & A,B) Chromatography HPLC - High Pressure Liquid Chromatography TLC – Thin Layer Chromatography Paper Chromatography

4. Chromatography Terms: The analyte is the substance to be separated during chromatography. Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample. A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing. A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture. Chromatography is a physical method of separation that distributes components to separate between two phases, one stationary (stationary phase), while the other (the mobile phase) moves in a definite direction.

5. The eluate is the mobile phase leaving the column. The eluent is the solvent that carries the analyte. An immobilized phase is a stationary phase that is immobilized on the support particles, or on the inner wall of the column tubing. The mobile phase is the phase that moves in a definite direction. Preparative chromatography is used to purify sufficient quantities of a substance for further use, rather than analysis.

6. The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. See also: Kovats' retention index The sample is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components. The solute refers to the sample components in partition chromatography. The solvent refers to any substance capable of solubilizing another substance, and especially the liquid mobile phase in liquid chromatography. The stationary phase is the substance fixed in place for the chromatography procedure.

7. Gel-filtration chromatography: (aka: size exclusion) particles are separated based on size. The separation is carried out on the basis of hyrdodynamic diameter of the molecules.[large molecules exit (elute) first] Ion-exchange chromatography: The mechanism of ion-exchange used allows the separation of analytes based on charge. Separation of charged compounds takes place through a charged stationary phase. Least charged particles will elute first. (two different modes: column and planar) Affinity chromatography: particles are passed through a column of beads containing a covalently bound high affinity group for the particle of interest. Bound particles are eluted by free high affinity group.

8. Adsorption chromatography: the analyte is passed over an adsorbent bed. Different compounds present in the mixture get adsorbed onto the bed at different rates. [large molecules exit (elute) first] Partition chromatography: Components of the analyte are separated through the use of partitions of a solute between two solvents. In this process one of the solvents is immobilized by the means of a substance present in the filter paper or column. High Performance Liquid chromatography: particles are separated on the basis of their idiosyncratic polarities. Interaction of these particles with the stationary phase of the column is also considered for analysis. A pump is used to push the mobile phase through the column. There is a detector following the stationary phase to analyze the retention time of the various particles.

9. Gas chromatography: the analyte is moved through the column via a high pressure gas (like helium). Flame ionization detectors (mass spec.) and thermal conductivity (pyrolysis) are used to evaluate the separated particles. Reverse-Phase chromatography: the stationary phase is made of hydrophobic compounds; they attract the hydrophobic compounds in the mobile phase allowing the elution of the hydrophobic particles. Thin Layer chromatography: particles are separated using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate.

10. Paper chromatography: the analyte is moved through the column via a paper and solvent. This paper is made of cellulose, a polar substance, and the compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far.

13. Size Exclusion chromatography: Biochemists refer to a protein's size in terms of its molecular weight, in kDa (a kilodalton, kD or kDa, is 1000 times the molecular mass of hydrogen) Each amino acid residue counts for about 110 daltons, that is, about 0.11 kDa.

18. Ion-exchange example with proteins:

26. Vertical Gel Electrophoresis: