Gas Chromatography - A Complete Lab Guide
The sample injected into the instrument is vaporized and transported through the column by flow of carrier gas. The vaporized sample is separated and fractionated on account of the mobile gaseous phase and stationary phase held in the column.
Design:- Basic layout diagram as on Wikipedia
1. A tank of carrier gas:-
Mobile phase inside a gas chromatograph is achieved by the use of Helium, nitrogen, argon, carbon dioxide and hydrogen. Though the disadvantages of hydrogen is that it might explode, making Helium and nitrogen the preferred option.
Points to be considered while selecting carrier gas:-
- It should be chemically inert.
- It should be low in cost.
- Choice of carrier gas depends upon the detector used.
A cylinder containing carrier gas is provided with a gauge to control the pressure inside the tank and achieve control over flow of gas.
2. Sample injection port:-
The Injector port is connected to the carrier gas cylinder. The temperature of the sample port is maintained to ensure rapid vaporization of the sample without its thermal degradation. A microsyringe is used to inject the sample through the rubber septum into flash vaporizer port at the head of the column.
Manual injection of sample is now replaced by Autosamplers. Autosamplers allows automatic injection into the port inlet to obtain reproducible and accurate results.
Diagram 2: A cross-sectional view of a microflash vaporizer direct injector
Points to Remember:-
- Solids should be dissolved in appropriate solution before loading in the Injector port.
- Avoid air bubbles in the microsyringe
- Sample loading inside the port should be quickly done.
The most important part of the Gas chromatograph is "Column" maintained in temperature-controlled oven. The solutes from the vaporized sample are separated depending on their interaction with the packing inside the column. The rate of progression of the solutes in the sample is affected by the absorption of the analyte molecules on the column wall or in the column packing. Since each molecule has a different rate of progression, different molecules flow at a different rate through the column reaching the detector at different times.
Upon separation the solutes individually enter the detector.
Types of Columns:-
- Packed column
- Capillary column
As the name suggests, the column is packed with an finely divided, inert solid material (like diatomaceous earth) coated with liquid stationary phase.
a. Length - 1.5 to 10m
b. Diameter - 2 to 4mm
Capillary columns are thin with diameter of few millimeters and more efficient compared to the packed ones.
Types of Capillary column:-
1. Wall-coated open tubular (WCOT)
2. Support-coated open tubular (SCOT)
WCOT columns have a Capillary tube coated with liquid stationary phase. Whereas SCOT columns have a Capillary lined with a support material like diatomaceous earth on which the liquid stationary phase is absorbed.
A detector is used to monitor the stream of separated solutes from the column on basis of the time taken to reach the outlet. Substances can be analyzed by the retention time of the analyte in the column and the order in which the solutes emerge from the column.
Variety of detectors are available having different selectivity. Based on selectivity, detectors are characterized as selective, non-selective and specific. In addition to selectivity, detectors may also be classified as mass flow dependant and concentration dependant.
Selective detector:- Respond to compounds with same physical and chemical properties.
Non-selective detector:- Respond to all compounds except the carrier gas.
Specific detector:- Respond to single chemical compound.
Mass-flow dependant:- Destroy the sample and give a signal depending on the rate at which the solute molecules reach the detector.
Concentration dependant:- Gives signals based upon the concentration of the solutes in the detector.
Most commonly used detectors are Flame ionisation detectors (FID) and Thermal conductivity detectors (TCD).
Compiled By: - Kalyani Koli
Bio: - Kalyani Koli, a post-graduate in Biotechnology, from the University of Mumbai with extensive 8 years of experience in the field of Animal Cell Culture and Immunology. She works as a Senior Cell Culture Analyst at Department of Animal Cell Culture. She has also worked as Copy Editor for Elsevier, Williams & Wilkinson and America Online. Follow Kalyani on Facebook
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