On the quantitative analysis of liquid chromatography - Database & Sql Blog Articles

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Quantitative analysis is based on qualitative analysis and requires pure material as a standard sample. The quantification of liquid chromatography is a relative quantitative method, that is, the amount of the sample to be tested is derived from a known standard sample.

Basis for quantitative determination by liquid chromatography

The amount (W) of the component to be tested is proportional to the response value (A) (peak height or peak area), W = f × A. Quantitative correction factor (f): is the proportional constant of the quantitative calculation formula, and the amount of the measured component represented by the unit response value (peak area) in the physical sense. A quantitative correction factor can be determined from the amount of known standard sample and its response value. The response value of the unknown component is determined, and the amount of the component can be determined by quantitative correction factor. Quantitative analysis Common term: sample: A solution containing a sample for chromatography. Divided into standard and unknown samples. Standard: A pure product with a known concentration. Unknown (unknow): The concentration of the mixture to be tested. Sample weight: The original sample weight of the sample to be tested. Dilution: An unknown dilution factor. Competence: A chromatographic peak to be quantified, that is, an analyte of unknown content. Amount: The content (or concentration) of the substance to be tested. Integerity: The calculation of the peak area measurement by a computer on a chromatographic peak. Calibration curve: A linear curve of component content versus response, established from a known amount of standard used to determine the unknown content of the analyte.

Commonly used quantitative methods 1

External standard method

The standard curve method is divided into an external standard method and an internal standard method. The external standard method is the most used in liquid chromatography. The internal standard method is accurate but cumbersome and is used most in standard methods.

Pure samples of the test compound were used as standard samples to prepare a series of standards of known concentrations. The response of the column to its response (peak area).

Within a certain range, there is a good linear relationship between the concentration of the standard sample and the response value, that is, W=f×A, and a standard curve is prepared.

Under the same experimental conditions, an unknown sample is injected to obtain a response value of the component to be tested. The concentration of the component to be tested can be determined from the known coefficient f.

Advantages of the external standard method:

Simple operation and calculation is a commonly used quantitative method; no need to detect and elute each component; standard samples are required; the measurement conditions of the standard sample and the unknown sample should be consistent; the injection volume should be accurate.

The disadvantages of the external standard method:

The experimental conditions are high, such as the sensitivity of the detector, the flow rate, and the composition of the mobile phase cannot be changed; each injection volume should have good repeatability.

2

Internal standard method

operating:

A known amount of internal standard is added to the standard sample to make a mixed standard and a series of working standards of known concentration are prepared. The molar ratio of the standard sample to the internal standard sample in the mixed standard is unchanged. Inject the column with the (standard peak area / internal standard peak area) as the response value. A standard curve is prepared based on the linear relationship between the response value and the working standard concentration, that is, W = f × A.

A known amount of the internal standard is added to the unknown sample and injected into the column to obtain the response value of the component to be tested. The concentration of the component to be tested can be determined from the known coefficient f.

Characteristics of the internal standard method: During the operation, the sample and the internal standard are mixed and injected into the column, so as long as the ratio of the measured component to the internal standard is constant, the change of the loading volume will not affect the quantitative result. . The internal standard method offsets the loading volume, and even the influence of the mobile phase and the detector, so it is more accurate than the external standard method.

case analysis

Case study one

What I did was phenol hydroxylation. The product contained phenol, benzenediol (o-, p-), benzoquinone, tar, analyzed by Agilent 1100 liquid chromatography, and the external standard method was used to calculate selectivity and conversion. But for a long time, the calculated results are always unsatisfactory. Occasionally, a good result is calculated, but it is not possible to repeat it. Sometimes it is different to repeat it several times. It is very depressed. Recently, I have calculated that the selectivity is over 100%. I started to suspect the dilution error (the sample was measured after dilution), but now I am very careful when I operate, I suspect the problem of the standard curve, but I have done a few more. The result was very strange, I thought about it: 1. Because the liquid chromatograph is common, I specially bought a dedicated column. However, when it is often measured, it is found that the column pressure is not the same. Is it because this affects the peak area, which in turn affects the result? Is the effect of column pressure so great? 2. Is the external standard method inaccurate? Should I use the single point calibration method? I felt that it was a little troublesome to have a standard before each sample, so it was useless. 3. What else is the reason? Supplement: The column used is ZORBAX SB-C18, using methanol: water = 3:7, each peak is very open. After sampling, the catalyst was removed by centrifugation, and then quantitatively diluted and then measured.

Netizens are screaming:

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Sometimes it is true that the difference in column pressure will have an effect on the results, but this will happen if the column pressure is very different. Is the column not washed, or you need to see if the bubble is in, as this may result in inaccurate results.

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If the difference in column pressure is very large, for example, 30 bar is already very large. When it is used normally, there is a column oven, and the temperature is generally controlled. The basic agreement condition does not change so much, up to 10 bar up and down, usually Less than 5bar.

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Is it through the mobile phase of the buffer salt? Rinse with low flow rate for a long time, then try acetonitrile rinse. Is there something left in the column that is not flushed in time and the column pressure is too high? Try using isopropyl alcohol.

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The change in column pressure mainly affects the retention time and has little effect on the peak area. What you said is not reproducible, it is the same sample in the liquid phase into the same sample. If different samples are the problem you are reacting to. As for selectivity over 100%? Is it a calculation method error?

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Simply put, calculate the selectivity of benzenediol:

Assume that the benzene peak area is substituted into the standard curve to obtain the concentration C1 (g/ml); the phenanthrene peak area corresponds to the concentration C2, the phenol peak area corresponds to the concentration C, the total solution volume V, the dilution factor of 20, and the initial phenol addition amount m (g). as follows

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If the calculation formula is correct, you can see if you have not done any blanking and component testing when reacting.

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As for the single point or external standard method?

Single point calibration is relatively simple, and if the requirements are not very strict, single point calibration can meet the requirements. If the single point standard concentration is near the measured sample concentration, the result is more accurate than the external standard method. When doing the assessment, we generally use the external standard to stipulate the approximate concentration range, and then quantify it by the single point method.

A single point is generally suitable for linear curve intercepts below 2% of the 100% concentration point. Above 20%, single point calibration and external standard cannot be used. The external standard method and the arch sample concentration should be consistent, and the calibration curve can be linearly matched.

Case study 2

I want to use liquid chromatography for quantitative analysis, using the external standard method. The question is: Is the standard curve done every time the sample is taken? I have 200 samples, but not once, I can measure 20 samples each time. Do I have to do a standard curve before each measurement? I am the same instrument, the same condition.

Netizens are screaming:

Strictly speaking, it should be a standard curve every day. If the requirements you are doing are not too strict (no need to approve), you can consider making a day's mark every few days. It is undeniable that making a standard curve every day can reduce the error of the measured data.

However, if it is a drug test, it should be done with a standard curve or a single point calibration standard solution analysis before starting the sample measurement. However, if the general chemical analysis requirements are not strict, there is no need to make a curve every time, because the difference is not big. However, if the test unit detects the sample, it must be performed according to the standard. To put it bluntly, it is actually a matter of normative regulation.