What is the internal standard method?

The internal standard method is a relatively accurate quantitative technique in chromatographic analysis, and its advantages become even more evident when there is no standard substance for comparison. The internal standard method involves adding a certain amount of a pure substance as an internal standard (see the entry on internal standards) to a given quantity of the sample mixture to be analyzed. Then, chromatographic analysis is performed on the sample containing the internal standard, and the peak areas (or peak heights) of both the internal standard and the component of interest are determined, along with their relative correction factors

The internal standard method is an indirect or relative calibration method. When analyzing and determining the content of a certain component in a sample, an internal standard is added to calibrate and eliminate the effects of variations in operating conditions on the analysis results, thereby improving the accuracy of those results. The internal standard method is an important technique in the quantitative analysis of gas chromatography. When using the internal standard method, a certain amount of a standard substance is added to the sample; this substance can be separated by the chromatographic column and is not affected by the peaks of other components in the sample. By measuring the peak areas and relative response values of the internal standard and the component of interest, it is possible to determine the percentage concentration of that component in the sample. When quantification is performed using the internal standard method, the selection of the internal standard is a very important task. Ideally, the internal standard should be a known compound for which pure samples are available, so that it can be added to the sample in an accurate and known quantity. It should have essentially the same or as similar physicochemical properties (such as chemical structure, polarity, volatility, and solubility in solvents) as well as chromatographic behavior and response characteristics as the component being analyzed; preferably, it should be a homolog of the substance being analyzed. Of course, under chromatographic analysis conditions, the internal standard must be able to be fully separated from the various components in the sample. It should be noted that in a few cases, analysts may be more concerned with the recovery rate of a compound in a complex process; in such cases, they can use a compound that can be easily completely recovered in that process as an internal standard to determine the percentage recovery of the compound of interest, without having to follow the selection principles mentioned above. When quantification is performed using an internal standard, what factors can affect the ratio of the peak height or peak area of the internal standard to that of the component being analyzed? The factors that influence this ratio fall into three categories: chemical, chromatographic, and instrumental. Changes in the area ratio caused by chemical reasons often occur when analyzing repeated samples. Chemical factors include: 1. Poor mixing of the internal standard in the sample ； 2. A reaction occurs between the internal standard and the sample components, 3. The purity of the internal standard can vary, etc. For a relatively mature method, chromatographic problems are more likely to occur. Common chromatographic issues such as leaks have a significant impact on the absolute area, while their effect on the area ratio is smaller. However, if the change in the absolute area is large enough to cause a significant change in the area ratio, then there must be some serious chromatographic problem at hand, such as a too large change in the injection volume, a large difference between the concentrations of the sample components and those of the internal standard, or detector non-linearity. The injection volume should be small enough and remain constant, so as to avoid saturating the detector and the integration device. If the method is considered reliable and the chromatography appears normal, attention should be paid to checking the integration device and settings, as well as the slope and peak width determination. The strongest evidence suggesting a problem with the integration device is that the area ratio varies, while the peak height ratio remains relatively constant. What should be taken into account when creating a standard curve using an internal standard? In colorimetric quantitative analysis using the internal standard method, a mixture of the component to be analyzed and the internal standard in a certain weight ratio is first prepared for chromatographic analysis; the peak areas are measured, and a relationship curve between the weight ratio and the area ratio is created – this curve constitutes the standard curve. The chromatographic conditions used in the analysis of actual samples should be as consistent as possible with those used when preparing the standard curve. Therefore, when creating the standard curve, it is necessary to specify not only the chromatographic conditions (such as the stationary phase, column temperature, carrier gas flow rate, etc.), but also the injection volume and the concentration of the internal standard. When preparing the internal standard calibration curve, the points do not lie entirely on a straight line; in such cases, it is necessary to determine the ratio of area ratios to weight ratios as well as the standard deviation of their average values. During use, single-point corrections should be performed regularly. If the deviation of the obtained values from the average value is less than 2, the curve can still be used; if it is greater than 2, the curve needs to be redrawn. If the curve shows changes over a short period of time, it is not suitable for quantitative analysis using the internal standard method.

The internal standard must meet the following requirements: (1) It should not be present in the sample； (2) Relatively similar in properties to the component being tested ； (3) Does not undergo a chemical reaction with the sample ； (4) The peak emergence position should be near the component being measured, with no influence from other component peaks. Characteristics of the internal standard method: (1) The internal standard method offers high accuracy, and slight changes in operating conditions or sample volume have little impact on the quantitative results. (2) For the analysis of each sample, two weighings are required, which is not suitable for the rapid analysis of a large number of samples. (3) If the sample volume and the amount of internal standard added in the internal standard method are kept constant,

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