- Written by Administrator
- Created: 26 January 2014
Chromatography is a physical method based on separation of the components by distribution between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. Quantitative chromatography is used to determine the concentration of analytes in a sample. The components is identified by its retention time and concentration calculated from intensity of detector signal. (In optimal system the signal is proportional to the concentration of the specific analyte separated).
Gas chromatography is a chromatographic technique used for separation of volatile organic compounds. The separation of compounds is based on in their different partitioning behavior between the mobile gas phase and the stationary phase in the column.
A gas chromatograph consists of:
• flowing mobile phase - inert gases ( helium, argon, or nitrogen )
• an injection port - the temperature of injection port is maintained higher than the boiling point of the least volatile component in the sample mixture
• a separation column with stationary phase. In most cases solid support is coated by liquid stationary phase (Gas-Liquid chromatography). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column).
• a thermostat-controlled oven for maintaining column temperature. The separation of compounds depends from temperature. In the case when necessary separate components with a wide range of boiling points temperature the gradient of column temperature is used. In this case separation starts at a low oven temperature followed by increasing the temperature over time to elute the high-boiling point components.
• a detector - flame ionization detector (FID), thermal conductivity detector (TCD), electron capture detector (ECD) , mass spectrometric detector (MSD), atomic-emission detector (AED), flame-photometric detector (FPD), chemiluminescence detector, photoionization detector (PID)
• data recording system.
Liquid chromatography (LC) is a chromatographic technique in which the mobile phase is a liquid. High performance liquid chromatography (HPLC) is liquid chromatography where column is packed with very small packing particles and a relatively high pressure is used.
There are different LC techniques divided by separation mechanism:
• normal phase liquid chromatography -stationary phase is more polar than the mobile phase. In normal phase chromatography, the most nonpolar compounds elute first and the most polar compounds elute last
• reversed phase liquid chromatography - mobile phase is more polar than the stationary phase. In reversed phase chromatography, the most polar compounds elute first with the most nonpolar compounds eluting last.
• Ion-exchange chromatography - the separation of ions and polar molecules based on their charge. The stationary phase surface contains ionic functional groups that interact with analyte ions of opposite charge.
• Size exclusion chromatography (gel permeation chromatography, gel filtration chromatography) - separates molecules according to their size. Smaller molecules are able to enter the pores of the media and, therefore, take longer to elute, whereas larger molecules are excluded from the pores and elute faster.
• Affinity chromatography – separating of biochemical mixtures, based on a highly specific biological interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.
Detectors used in LC - Diode array detector (DAD), Fluorescence detector, UV absorption detector, refractive index detector (RID), Chemiluminescence detector, The Evaporative Light Scattering Detector, The Electrical Conductivity Detector, The Electrochemical Detector.
Capillary electrophoresis (CE) is a analytical technique based on differences in electrophoretic mobilities of ions inside small capillaries filled with an electrolyte. CE is used mostly for the separation of larger biopolymers (polypeptides, proteins, oligonucleotides, DNA, RNA, and oligosaccharides) Capillary electrophoresis system consists from a sample vial, source and destination vials, a capillary, electrodes, a high-voltage power supply, a detector, and a data output and handling device. High voltage (10 - 30 kV) is used to decrease sample run times.
All ions, positive or negative, are pulled through the capillary in the same direction by electroosmotic flow. The analytes separate as they migrate due to their electrophoretic mobility are detected near the outlet end of the capillary. The detector signal is sent to data recording and handling device. Separated chemical compounds appear as peaks with different retention times in an electropherogram. Most common used detectors in CE are based on UV, UV-Vis absorbance or fluorescence detection.