Gas chromatography technology has been widely used in various fields due to the advantages of strong separation efficacy, high sensitivity, fast analysis speed, small sample children, and automation, etc. In microbiology experiments, it is mainly used to analyze the composition of microorganisms, metabolites, substrate degradation products, and identification of microorganisms. Source: Experiments in Microbiology (Third Edition).

Operation method

basic program

Principle

Gas chromatography (gas chromatography, i.e. GC) is a kind of chromatographic technology, and its test instrument gas chromatograph mainly consists of carrier gas system, injection system, column, detector and recorder or data processing device, etc., such as Fig. 1. Fig. 1 Schematic diagram of gas chromatograph The basic principle of this technology is that the components in the test sample. With the mobile phase (gas) installed in the chromatographic column of the stationary phase and flow, adsorption or solubility of weak components is difficult to be adsorbed or dissolved in the stationary phase, the flow of fast, the first out of the column, and adsorption or solubility of the components of the strong after the outflow of the column, so that the components of the sample will be able to separate the components are separated from the components in turn so that the detector to detect the recorder in the paper is recorded in order to record the components separated from the components, each component is a chromatogram. The separated components can be detected by the detector in turn, and the recorder records the separated components in turn on the recording paper, and each component shows a chromatographic peak, or the test results can be calculated and printed out directly by the data processing device at the same time. According to the time from the beginning of sample injection to the highest point of the chromatographic peak (called retention time tR), the height of the chromatographic peak h and the width of the chromatographic peak W and other test data, or other complementary test data, can be qualitatively and quantitatively the components of the sample. The principle of gas chromatography for separation of sample components is shown in Figure 2.

Materials and Instruments

Two gram-negative bacteria Two gram-positive cocci Two bacilli
Sterile water Physiological saline
Gas Chromatograph Accessories for Sampling Systems Lysis Furnace Hydrogen Ion Flame Detector Stainless Steel Columns Centrifuge

Move

1. Inoculate the bacterial strains on agar nutrient slant, incubate at 37℃ for 24 h, take off the bacteria with inoculation ring, do not take the medium, put the bacteria into a small plastic centrifugal tube containing saline, centrifugation at 1000 r/min for 5 min, and then wash the bacteria with sterile water for three times, the centrifugation speed and time of washing are the same as the previous one, but the last one is centrifuged for 10 min, and then the washing liquid is discarded, then make a sample of the bacteria to be identified. Then the sample was dehydrated under the airflow of hairdryer for 15 min to make the sample to be identified. 2.

2. Select the lysis chromatography conditions are: lysis temperature 800 ℃, lysis time 10 s, inlet temperature 260 ℃, detector temperature 280 ℃, the beginning of the crutch constant temperature of 80 ℃ constant temperature for 4 min, 6 ℃ / min, 45 ml of hydrogen / min, 450 ml of air / min, the recorder paper speed 8 mm / min. 3.

3. After the debugging chromatograph is running normally, take 1.5 mg of the bacterial sample to be identified, place it in the platinum boat of the cracking head, and then insert it into the cracking furnace, stay cracking for 10 s. Test it according to the above-selected cracking conditions, and the separation time of each sample in the chromatograph is 25 min.

Chromatographic identification of each sample, must strictly maintain the same conditions of the cleavage chromatography.

4. Remove the recording paper with each chromatographic peak of the sample from the recorder, or remove the recording paper with each chromatographic peak of the sample directly calculated from the data processor.

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