Understand it all in one article: normalization method, internal standard method, external standard method, and standard addition method

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Area normalization method, internal standard method, external standard method, standard addition method. With so many quantitative methods, which one should be used? What is the scope of application for each one? What are its advantages and disadvantages? What are the requirements for its standards? You’ll know everything after reading it. The normalization method is a quantification technique in which the sum of the contents of all components that show peaks is set at 100%. This method can be used when the correction factors for each component are consistent. It is simple and accurate; especially when it is difficult to control the sample volume accurately, the effects of changes in injection concentration and volume are minimal. Other operating conditions, such as flow rate and column temperature, also have little impact on the quantitative results. GC is more widely used than HPLC. The external standard method (standard curve method, direct comparison method) first involves preparing a standard working curve using standard samples of the component to be measured. The specific procedure is as follows: a standard series of different concentrations is prepared using standard samples; under the same chromatographic conditions as those used for the component to be analyzed, equal volumes of these samples are accurately injected. The peak areas or peak heights of each peak are measured, and a standard working curve is drawn by plotting these peak areas or peak heights against the sample concentration. This standard working curve should be a straight line passing through the origin. If the standard working curve does not pass through the origin, it indicates that there is a systematic error in the measurement method. The slope of the standard working curve is the absolute correction factor. When the concentration of the component to be measured changes little and its approximate content is known, it is also possible to avoid drawing a standard working curve and instead use the single-point correction method, that is, to perform quantification by direct comparison. The single-point correction method actually uses the origin as another point on the standard calibration curve. Therefore, when the method has systematic errors (i.e., the standard working curve does not pass through the origin), the error of the single-point correction method is large. Therefore, it is specified that y = ax + b. The absolute value of b should not be greater than 100%, and the response value is 2% of y. Advantages of the standard curve method: Once a standard working curve is prepared, the analysis becomes very simple; the concentration can be directly read from the standard working curve during calculations, which is highly suitable for analyzing large numbers of samples. In particular, once a standard working curve has been established, it can be used for a certain period of time; during this time, a standard sample can be used frequently to perform single-point corrections to the curve, in order to determine whether it is still suitable for use. Disadvantages of the standard curve method: The chromatographic conditions for each sample analysis (detector response, column temperature, flow rate and composition of the mobile phase, injection volume, column efficiency, etc.) are difficult to be exactly the same, which thus leads to significant errors. Furthermore, when drawing a standard working curve, standard samples of the component to be measured (or samples with a known exact content) are generally used; as a result, any changes in the concentration of the component to be measured during sample pretreatment cannot be compensated for. The internal standard method is a technique in which an appropriate substance is selected as a reference for the component to be measured and added quantitatively to the sample. Quantitative analysis is carried out based on the ratio of the response values (peak area or peak height) of the component to be measured to those of the reference at the detector, as well as the amount of the reference added. The key to the internal standard method is selecting an appropriate internal standard. The internal standard should be a pure substance that is not present in the original sample; its properties should be as similar as possible to those of the component being analyzed, it should not react chemically with the sample under examination, and it must be fully soluble in the sample. The peak of the internal standard should be as close as possible to the peak of the component being analyzed, or located between the peaks of several components being analyzed; however, it must not overlap with any of the peaks in the sample, that is, it must be completely separate from them. Isotopic variants of reference materials are generally chosen as internal standards. Advantages of the internal standard method: Changes in the injection volume or minor variations in chromatographic conditions have little impact on the quantitative results obtained using this method. In particular, by adding the internal standard before sample preprocessing (such as concentration, extraction, derivatization, etc.) and then proceeding with the preprocessing, it is possible to partially compensate for the loss of the component of interest during that process. To obtain highly accurate results, several internal standards can be added to improve the precision of quantitative analysis. Disadvantages of the internal standard method: It is difficult to select an appropriate internal standard, the weighing of the internal standard must be accurate, and the procedure is rather complicated. When using the internal standard method for quantification, it is necessary to measure the peak areas (or peak heights) of both the component to be analyzed and the internal standard. According to the principle of error superposition, among the errors associated with quantification using the internal standard method, the error resulting from peak area measurements is 2–2 times that of quantification using a standard curve. However, the errors caused by changes in the injection volume and chromatographic conditions are much smaller in the internal standard method compared to the standard curve method. Therefore, overall, the internal standard method provides better accuracy and precision for quantification. Standard addition method: The standard addition method is essentially a special form of internal standard technique. When no suitable internal standard can be found, a pure sample of the component to be analyzed is used as the internal standard and added to the sample to be tested. Under the same chromatographic conditions, the peak area (or peak height) of the component of interest is measured before and after adding this pure sample, thereby allowing for the calculation of the concentration of that component in the sample. Advantages of the standard addition method: no additional standard substance is required as an internal standard; only a pure sample of the component to be analyzed is needed, the injection volume does not have to be extremely precise, and the procedure is simple. If a known accurate amount of the component to be measured is added before the sample preprocessing, the loss of that component during preprocessing can be completely compensated for; this is a commonly used quantitative analysis method in chromatographic analysis. Disadvantages of the standard addition method: It requires that the chromatographic conditions for the two chromatographic analyses before and after adding the component to be measured be exactly the same, in order to ensure that the correction factors for both analyses are identical; otherwise, it will lead to errors in the analytical results.