This experiment is from the official website of Wuhan University School of Pharmacy
Operation method
Gas chromatography quantitative analysis experiment
Principle
Quantitative analysis by chromatography is based on the fact that the amount of a substance to be measured is proportional to its peak area (or peak height) on the chromatogram. Data processing software (workstations) can give a variety of chromatographic data including peak height and peak area. Because peak height is more susceptible to fluctuations in analytical conditions than peak area, and because the linear range of the standard curve for peak height is narrower than that for peak area, it is common to use peak area for quantitative analysis. 1. Correction Factor Quantification 2. Normalization 3. External Standard Method 4. Internal Standard Method 5. Standard Addition Calibration Factor Quantification: Absolute Correction Factor FI: Concentration (or mass) of the substance measured per unit of peak area, i.e., the ratio of peak area of a sample component to the amount of sample component. The absolute calibration factor fi: the concentration (or mass) of the substance to be measured per unit peak area, i.e., the concentration of the component to be measured is obtained by multiplying the peak area of the sample component with the calibration factor of the standard substance of the component under the same conditions. The absolute correction factor is affected by the experimental conditions, and the correction factor of the standard substance must be determined under the same conditions as the actual sample for quantitative analysis. Relative correction factor f': the ratio of the absolute correction factor of a substance i to that of a selected standard substance S. This means that the relative correction factor is only the same as the detection factor. That is, the relative correction factor is related only to the detector type and not to the chromatographic conditions. Normalization method Normalization method is to multiply the peak areas of all components by their relative correction factors and then sum up, that is, the so-called "normalization", the content of the measured component X can be obtained by the following formula: the prerequisite for quantitative analysis using the normalization method is that all the components in the sample should be able to elute from the chromatographic column, and be detected by the detector. The normalization method is mainly applied in gas chromatography. External Standard Direct Comparison Method: Quantification by direct comparison of the peak area of a substance in an unknown sample with the peak area of a standard for that substance. Usually, the concentration of the standard is close to the concentration of the measured component to minimize the quantification error. Standard curve method: The standard substance of the component to be measured is prepared into standard solutions of different concentrations, and a standard curve is made after chromatographic analysis, i.e. the relationship curve between the concentration of the substance and its peak area (or peak height). According to the chromatographic peak area (or peak height) of the component to be measured in the sample, the corresponding concentration is found from the standard curve. The slope of the standard curve is related to the nature of the substance and the characteristics of the detector, and is equivalent to the correction factor of the component to be measured. Internal standard method: A certain amount of pure substance (internal standard) is added to an accurately weighed specimen, and the content of a component is determined by the weight of the specimen and the internal standard and its peak area ratio. Internal standard calibration curve method: A series of control solutions of different concentrations are added to the same amount of internal standard, Ai and As are measured, and Ai/As is plotted against the concentration of the control solution. After finding the slope and intercept. Then, the same amount of internal standard is added to the sample solution as to the control solution, and Ai/As is measured, and finally the content of the sample is calculated. Internal Standard Comparison Method (Internal Standard One-Point Method): Standard Addition Method Standard Addition Method: It is a widely used test method for checking the accuracy of an instrument, and is particularly suitable for checking the presence of interfering substances in a sample. A certain amount of a standard solution of known concentration is added to the sample to be tested, and the concentration of the sample before and after the addition is determined. The concentration after the addition of the standard solution will be higher than before, and the increase should be equal to the amount of the substance to be measured contained in the standard solution added. If an interfering substance is present in the sample, the increase in concentration will be less or greater than the theoretical value. Move 1. Chromatographic conditions For more product details, please visit Aladdin Scientific website.
The analytical column was HP-1 capillary column 30m×0.25um×0.25um with FID detector. The inlet temperature was 140 0C, column temperature was 60 0C, detector temperature was 150 0C, and the injection volume was 0.2 ul. The carrier gas was nitrogen or hydrogen at a flow rate of 15-20 mL/min (post-column).
2. Determination of relative weight correction factor
On an analytical balance, a binary mixture of cyclohexane and benzene was prepared by weighing cyclohexane and benzene in a 5mL tube with a milled mouth in the ratio of about 2:1 by weight. After the baseline of the chromatograph was stabilized, the sample was injected to analyze the binary mixture, and the procedure was repeated 3~5 times. The peak areas of hexane and benzene were measured and the relative weight correction factor of cyclohexane to benzene was found according to the formula.
As an example, the relative weight correction factor of cyclohexane to toluene was determined and found.
3. Quantitative determination
Accurately measure cyclohexane, benzene and toluene mixed by volume 1:1:1, as the standard reserve solution.
Take 0.2 microliter of the sample to be measured and inject into the chromatographic analysis, repeat 3 times, take the average peak height (or peak area), and use the internal standard one-point method to determine the concentration of the substance to be measured in the sample.