Everything you need to know about quantitative analysis in liquid chromatography is right here!

Quantitative analysis, built upon qualitative analysis, requires pure substances as standard samples. Quantitative determination in liquid chromatography is a relative method, meaning that the quantity of the analyte in the sample is inferred from known standard samples.

The basis for quantitative determination in liquid chromatography: The quantity (W) of the analyte and the response value (A) (peak height or peak area) are directly proportional: W = f × A. Quantitative correction factor (f): This is a constant proportionality constant in the quantitative calculation formula, representing the amount of the analyte per unit response value (peak area). The quantitative correction factor can be obtained from the known quantity of standard samples and their response values. To determine the response value of an unknown component, its quantity can be calculated using the quantitative correction factor.

Common terms in quantitative analysis:

Sample: A solution containing the analyte for chromatographic analysis, divided into standard samples and unknown samples.

Standard: A pure substance with a known concentration.

Unknown: A mixture with an unknown concentration to be measured.

Sample weight: The original weight of the sample to be analyzed.

Dilution: The multiplication factor by which an unknown sample is diluted.

Component: The chromatographic peak for quantitative analysis, representing the analyte of unknown concentration.

Amount of component: The quantity (or concentration) of the analyte.

Integration: The process of calculating the peak area measurement of a chromatographic peak by a computer.

Calibration curve: A linear curve relating the content of a component to its response value, established with known quantities of standard substances, used to determine the unknown content of the analyte.

Common Quantitative Methods

1. External Standard Method

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

   Using a pure sample of the analyte as a standard sample, a series of known concentration standards are prepared. These standards are injected into the chromatographic column, and their response values (peak areas) are recorded. Within a certain range, there is a good linear relationship between the concentration of the standard and its response value, i.e., W = f×A, resulting in the creation of a standard curve. Under identical experimental conditions, unknown samples are injected, and the response values for the desired component are obtained. Using the known coefficient f, the concentration of the desired component can be determined.

   Advantages of External Standard Method:

   • Simple operation and calculation, commonly used for quantitative analysis.
   • Does not require the detection of all components or elution.
   • Requires standard samples.
   • Determination conditions for standard and unknown samples must be consistent.
   • Accurate injection volumes are necessary.

   Disadvantages of External Standard Method:

   • High experimental requirements, such as detector sensitivity, flow rate, and unchanged composition of the mobile phase.
   • Good repeatability of injection volume is required.

2. Internal Standard Method

   Operation: Known amounts of an internal standard sample are added to standard samples, creating a mixed standard sample. A series of working standard samples with known concentrations is prepared. The molar ratio of the standard to the internal standard remains constant in the mixed standard sample. This mixed sample is injected into the chromatographic column with the response value calculated as the ratio of the peak area of the standard to the peak area of the internal standard. A standard curve is then created based on the linear relationship between the response value and the concentration of the working standard sample.

   Known amounts of the internal standard sample are added to unknown samples, which are then injected into the chromatographic column. The response values for the desired component are obtained. Using the known coefficient f, the concentration of the desired component can be determined.

   Characteristics of Internal Standard Method:

   • During the sample injection into the chromatographic column, the sample and the internal standard are mixed together. Therefore, changes in the injection volume do not affect the quantitative results as long as the ratio of the analyte to the internal standard in the mixed solution remains constant.
   • The internal standard method compensates for the effects of injection volume, mobile phase, and detector, making it more precise than the external standard method.

Case Analysis

Case Analysis One:

I am conducting the hydroxylation reaction of phenol, and the products include phenol, catechol (ortho and para), benzoquinone, and tar. I am using an Agilent 1100 liquid chromatograph for analysis, employing the external standard method to calculate selectivity and conversion rates. However, the results have consistently been unsatisfactory. Occasionally, a good result is obtained, but it cannot be consistently reproduced. Recently, the selectivity has been calculated to exceed 100%. I initially suspected dilution errors (samples were measured after dilution), but I am now very careful during the operation. I also suspected issues with the standard curve, but I have redone it several times. The results are still strange, so I have been considering:

1. Since the liquid chromatograph is shared, I specifically purchased a dedicated chromatographic column. However, I often find that the column pressure is different during measurements. Could this affect the peak area and, consequently, the results? Is the influence of column pressure significant?

2. Is the external standard method inaccurate? Should I use a single-point correction method? I initially found it a bit troublesome to prepare a standard sample before each measurement, so I didn't use it.

3. Or is it some other reason?

   Additional Information: I am using a ZORBAX SB-C18 column, with a methanol: water ratio of 3:7. Each peak is well separated. After sampling, the catalyst is removed by centrifugation, then quantitatively diluted before measurement.

Responses from Netizens:

1. Differences in column pressure can indeed affect results, but this usually happens when there is a significant disparity in column pressure. Check if the column has been adequately rinsed or if there are air bubbles, as these could lead to inaccurate results.

2. If the column pressure varies significantly (e.g., 30 bar is considered high), it might be due to improper column rinsing or the presence of residues not promptly washed away, causing an elevated column pressure. Try using isopropanol for rinsing.

3. Changes in column pressure mainly affect retention time, with minimal impact on peak area. Your mention of poor repeatability could be due to issues in the reaction itself rather than the chromatography.

4. For the calculation of catechol's selectivity, make sure your formula is correct. Have you performed a blank test and tested the components when introducing reactants?

5. Regarding single-point versus external standard method: Single-point correction is simpler and can meet requirements when precision is not stringent. If your standard concentration is close to the analyte concentration in the sample, the single-point method may be more accurate than the external standard method.

Case Analysis Two:

I want to use liquid chromatography for quantitative analysis, using the external standard method. The question is: Do I need to make a standard curve every time I analyze samples? I have 200 samples, but I can only analyze 20 at a time. Do I have to make a standard curve before each analysis, considering it's the same instrument and conditions?

Responses from Netizens:

1. Strictly speaking, you should create a standard curve every day. If your requirements are not too strict (no need for official approval), you can consider making a standard curve every few days. Making a standard curve daily can reduce measurement data errors.

2. For pharmaceutical testing, it is recommended to make a standard curve or single-point correction standard solution analysis every time before analyzing samples. However, for routine chemical analysis with less stringent requirements, it may not be necessary to make a standard curve every time, as the differences are usually not significant. The necessity depends on regulatory standards.