What Is an Internal Standard?

The internal standard method is to add a certain weight of pure material as an internal standard (see the internal standard strip) to a certain amount of sample mixture to be analyzed, and then perform chromatographic analysis on the sample containing the internal standard The peak area (or height) and relative correction factor of the internal standard and the component to be measured are determined, and the percentage content of the component in the sample can be determined according to the formula. The internal standard method is a more accurate quantitative method in chromatographic analysis. Especially when there is no standard control, this method is more advantageous.

The internal standard method is to add a certain amount of pure substance as an internal standard to a certain amount of sample mixture to be analyzed. Calculate the content of the measured component ^[1]

There are mainly three types of factors that affect the internal standard and the peak height or peak area ratio of the measured component: chemical, chromatographic, and instrumental.

Changes in area ratios due to chemical reasons often occur when analyzing duplicate samples.

Chemical factors include:

1. The internal standard is not mixed well in the sample;

2. Reaction between internal standard and sample components,

3. The purity of the internal standard is variable.

For a more mature method, chromatographic problems are more likely to occur. Some common problems on chromatography have a greater impact on the absolute area and a smaller effect on the area ratio. The change is large enough to cause a significant change in the area ratio, then there must be an important chromatographic problem, such as a large change in the injection volume, a large difference between the sample component concentration and the internal standard concentration. Device non-linear and so on. The injection volume should be small and constant so that the detector and integration device are not saturated. If the method is considered reliable and the column appears to be normal, check the integration device and settings, slope, and peak width positioning. The strongest evidence for skepticism of the integrating device is that the area ratio is variable and the peak height ratio remains relatively constant.

When the same reference standard solution is used to prepare the reference solution for the correction factor determination and the test component solution containing the internal standard substance, the internal standard substance solution need not be accurately weighed (measured).

When using internal standard method for quantification, the selection of internal standard is a very important task. Ideally, the internal standard should be a known compound that can be obtained in pure form, so that it can be added to the sample in an accurate and known amount. It should be substantially the same as or as far as possible Consistent physical and chemical properties (such as chemical structure, polarity, volatility, and solubility in solvents, etc.), chromatographic behavior, and response characteristics, preferably a homologue of the substance being analyzed. Of course, under the conditions of chromatographic analysis, the internal standard must be sufficiently separated from each component in the sample. It should be pointed out that in a few cases, the analyst may be more concerned about the recovery of the compound in a complex process. At this time, he can use a compound that can be easily recovered in this process as an internal standard To determine the percent recovery of a compound of interest without having to follow the selection principles described above ^[3] .