As we all know, chromatography can solve the problem of separation and analysis of complex multi-component mixtures such as homologues and isomers with similar physical constants and similar chemical properties, and can not only identify compounds but also do quantitative determination. Chromatography is based on quantitative analysis based on the area or height of chromatographic peaks. There are many chromatographic quantitative calculation methods. At present, there are three widely used methods: normalization method, internal standard method and external standard method. Today, I will briefly share with you several quantitative methods commonly used in chromatography.

#### Normalization method

Since the amount of a component is proportional to its peak area, if all components in the sample can generate signals and obtain corresponding chromatographic peaks, the following normalization formula can be used to calculate the content of each component.

In the formula:

C represents the content

F stands for correction factor

A is the peak area

If the correction factor of each component in the sample is similar, the correction factor can be eliminated, and the peak area normalization can be directly used for calculation. The Chinese Pharmacopoeia uses the area normalization method without correction factor to determine the limit of each impurity and the total amount of impurities in the drug.

**Advantage:**

- Simple and fast quantitative process
- The injection volume is not strictly required

**Shortcoming:**

- All component peaks should elute
- All components need to be measured
- All peaks must be corrected

**Internal standard method**

The pure substance not contained in the sample is selected as the reference substance and added to the solution of the sample to be tested, and the method of determining the content of the tested component by comparing the response signals of the tested component and the reference substance is called the internal standard method. The origin of the “internal standard” is because the standard (control) substance is added to the sample, which is different from the external standard method. This control substance is called the internal standard.

Not all components can flow out of the chromatographic column in one analysis cycle (such as difficult gasification components), or the detector cannot generate a signal for every component, or only the content of a few components in the mixture needs to be determined , the internal standard method can be used.

Accurately weigh W grams of the sample, and then accurately weigh Ws grams of the internal standard, add it to the sample, mix well, and inject the sample. Measure the peak area Ai of the component i to be measured and the peak area As of the internal standard, then the weight Wi of the i component contained in the W gram sample has the following relationship with the weight Ws of the internal standard:

The percentage C% of the component i to be tested in the sample is:

**Requirements for internal standards:**

- The internal standard is a component not contained in the original sample, otherwise the peaks will overlap and the peak area of the internal standard cannot be accurately measured
- The retention time of the internal standard should be similar to that of the components to be tested, but they can be completely separated from each other (R≥1.5)
- The internal standard must be a pure substance with the required purity

**Advantage:**

- Within the range that the injection volume does not exceed the limit (the column is not overloaded), the quantitative results have nothing to do with the repeatability of the injection volume
- As long as the measured components and the internal standard have peaks and the resolution meets the requirements, they can be quantified, regardless of whether other components have peaks
- It is very suitable for measuring the content of trace active ingredients or impurities in medicines. Since the content of impurities (or trace components) is very different from the main components, the content cannot be determined by the normalization method, and it is very convenient to use the internal standard method.

**Shortcoming**: Add an internal standard equivalent to the amount of impurities. Increase the injection volume to highlight the impurity peak, and measure the ratio of the impurity peak to the internal standard peak area to obtain the impurity content. However, sample preparation is cumbersome and internal standards are difficult to find.

#### External standard method (calibration curve method, correction factor calculation algorithm)

External standard method refers to adding a certain amount of standard substance (reference substance) in a gradient to make a control sample in a blank solvent, and performing sample processing and detection in parallel with the unknown sample. Different concentrations of standard samples are injected, and the peak area is used as the value to draw a standard curve, so as to calculate the quantitative method of the concentration of the measured component in the unknown sample. External standard method can be divided into calibration curve method and correction factor calculation algorithm in operation. .

**Calibration curve method**: take the pure substance to prepare standard solutions of different concentrations, and use a chromatograph to make a standard curve of the relationship between peak area and concentration. The concentration of the component to be tested can be directly found out through the standard curve.

**Correction factor calculation algorithm:** It is to obtain the response signal and its content after analyzing the standard sample for many times to obtain its absolute correction factor, and then calculate the content in the sample to be tested according to the formula.

In the formula:

C means content

F stands for correction factor

A is the peak area

**Advantages:** Simple operation and calculation, no need for all peaks to be detected.

**Disadvantages**: The injection volume must be accurate, and the instrument requires good stability.

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