Mixed product liquid chromatography comprehensive analysis method

Abstract

The invention relates to the technical field of liquid chromatography, and discloses a mixed product liquid chromatography comprehensive analysis method which comprises the following steps: S1, mixing products: respectively mixing aldehyde compounds and ketone compounds with different mobile phases, and then injecting into an injection chromatographic system; s2, liquid chromatographic analysis: separating and analyzing a mixed product by using a liquid chromatograph; s3, performing qualitative analysis; on the basis of liquid chromatographic analysis, each peak in a chromatogram is identified. The method aims at qualitative and quantitative analysis of main substances (groups) in an organic reaction mixed product system; the method is suitable for analyzing the relative percentage content of products related to organic chemical reactions such as raw materials (nonylphenol) related to a core production process, nonylphenol formylation reaction, nonylphenol esterification reaction, phenolic ester Fris rearrangement reaction and the like, and samples in a refining process; the influence of absorption peaks and chromatographic line oscillation of other non-target substances on the content analysis result can be effectively avoided.

Description

Technical Field

[0001] The invention relates to the technical field of liquid chromatography analysis, in particular to a liquid chromatography comprehensive analysis method for a mixed product. Background Art

[0002] At present, the analysis of samples in the process is an important means of real-time monitoring, quality control and process evaluation. Among them, the analysis of process samples by gas chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, liquid chromatography and other instruments is more in line with the actual needs of production enterprises.

[0003] Taking into account the properties of nonylphenol and its synthetic products, combined with the previous GC-MS sample analysis, it was determined that instruments such as gas chromatography and GC-MS could not meet the detection needs of intermediate process samples (crude ketone, refined nonylphenol acetone, etc.).

[0004] Mass spectrometry analysis has high requirements for sample purity and requires ultra-high performance liquid chromatography to achieve more complete separation. It also places certain requirements on the mass-to-charge ratio of the sample (samples that do not meet the requirements need to be bombarded and crushed to decompose). Therefore, a comprehensive analysis method of mixed product liquid chromatography is proposed. Summary of the invention

[0005] The object of the present invention is to provide a comprehensive analysis method of mixed product by liquid chromatography to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: comprising the following steps:

[0007] S1. Mixed product: aldehyde compounds and ketone compounds are mixed with different mobile phases respectively, and then injected into the chromatography system;

[0008] S2. Liquid chromatography analysis: Use liquid chromatography to separate and analyze the mixed product;

[0009] S3. Qualitative analysis: Based on the liquid chromatography analysis, the peaks in the chromatogram are identified to determine the components in the mixture;

[0010] S4. Establishment of standard curve of target substance: After the components are determined by qualitative analysis, a standard curve of the target substance is established (this is usually done by analyzing a series of standard samples with known concentrations);

[0011] S5. Quantitative analysis: Using the established standard curve, the content of the target substance in the sample to be tested is determined by measuring the peak area of ​​the sample to be tested. This is done by substituting the peak area of ​​the sample to be tested into the functional relationship of the standard curve to calculate the corresponding concentration.

[0012] Preferably, the mobile phase for aldehyde compounds is: 85% methanol, 15% water, the detector wavelength is 254nm, the analysis time is 17min, and the mobile phase flow rate is controlled to be 1.0ml / min;

[0013] The mobile phase for ketone compounds is: 48% acetonitrile, 37% methanol, 15% water, the detector wavelength is 230nm, the analysis time is 20min, and the mobile phase flow rate is controlled at 0.6ml / min.

[0014] Preferably, the method for quantitative analysis of two different compounds of aldehydes and ketones (such as aldehyde method-hydrolysis aldehyde, aldehyde method-distillation aldehyde, ketone method-phenol ester, ketone method-hydrolysis ketone, ketone method-rectification ketone) is manual integration method:

[0015] (1) Determine the integration level baseline and perform calibration;

[0016] (2) Removal or adjustment of interfering peaks (e.g., polymer multiple peaks, solvent peaks);

[0017] (3) Use front and tail processing to modify the target peak to ensure the accuracy of integration;

[0018] (4) Use the "Split Peak" tool to split complex peaks for more accurate integration. The integration results can reflect the actual content of the target substance.

[0019] Preferably, the quantitative analysis adopts the target substance standard curve method, and the specific steps are as follows:

[0020] (1) Perform chromatographic analysis on the sample under the same chromatographic conditions and record the absolute value of the peak area of ​​the target substance;

[0021] (2) Substituting the absolute value of the peak area of ​​the sample to be tested into the standard curve function to calculate the theoretical equivalent standard sample amount;

[0022] (3) Calculate the target substance content using the following formula:

[0023] The percentage calculation formula is: the theoretical equivalent standard sample injection volume ÷ the actual injection volume of the sample to be tested × 100%.

[0024] Preferably, the method for establishing the target substance standard curve is as follows:

[0025] (1) Weigh standard substances such as nonylphenol, tetrachloroethylene, and toluene, dilute them to a certain volume, and prepare samples with different injection volumes;

[0026] (2) Perform chromatographic analysis on the standard sample and record the absolute value of the peak area at different injection volumes;

[0027] (3) Use statistical software to fit the absolute values ​​of peak areas at different injection volumes and establish a standard curve;

[0028] (4) The standard curve can be linear, logarithmic or other functional relationship as needed to ensure R 2 ≥0.99.

[0029] Preferably, the concentration of the target substance is obtained according to the area or peak height of the chromatographic peak and the standard curve. If the sample has been diluted or treated, the final result needs to be corrected accordingly according to these steps, and the absolute value of the peak area of ​​the sample to be tested is substituted into the curve function to calculate the corresponding theoretical equivalent standard sample amount. The quotient of the actual injection amount of the sample to be tested is the target substance content.

[0030] In summary, this application includes the following beneficial technical effects:

[0031] This method is aimed at the qualitative and quantitative analysis of the main substances (groups) in the mixed product system of organic reactions. It is suitable for the relative percentage content analysis of products and refining process samples involved in organic chemical reactions such as raw materials (nonylphenol) related to the core production process, nonylphenol formylation reaction, nonylphenol esterification reaction, phenol ester Fries rearrangement reaction, etc. It can effectively avoid the influence of absorption peaks of other non-target substances and chromatographic line oscillations on the content analysis results, but requires that the accuracy and precision of the sample preparation process must always be maintained at a high level and the data must be stable on average. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The present invention provides a technical solution comprising the following steps:

[0034] Firstly, a mixed product is prepared: aldehyde compounds and ketone compounds are mixed with different mobile phases respectively, and then injected into the chromatography system;

[0035] For aldehyde compounds, the mobile phase was: 85% methanol, 15% water, the detector wavelength was 254nm, the analysis time was 17min, and the mobile phase flow rate was controlled at 1.0ml / min;

[0036] The aldehyde method uses gradient elution from 0 to 3 minutes, with the initial mobile phase ratio at 0 minutes, 100% methanol at 3 minutes, and isocratic elution of the methanol from 3 to 17 minutes. When the analysis of a single sample is completed, the mobile phase immediately returns to the initial ratio, and the column pressure increases at this time. The next automatic injection can only be started after it stabilizes;

[0037] For ketone compounds, the mobile phase was: 48% acetonitrile, 37% methanol, 15% water, the detector wavelength was 230nm, the analysis time was 20min, and the mobile phase flow rate was controlled at 0.6ml / min;

[0038] When switching the ketone method, attention should be paid to factors such as the unstable column pressure and temperature caused by the heat absorption and cooling after the mixture of acetonitrile and methanol. The mobile phase should be allowed to run at no load for 15 to 20 minutes before the first automatic injection can be started.

[0039] Liquid chromatography analysis is performed using a liquid chromatograph: the mixture is injected into the chromatographic system. When passing through the chromatographic column, different components will be separated due to different interactions with the stationary phase and the mobile phase. The separated components pass through the detector in turn to produce a chromatogram.

[0040] The quantitative analysis method for two different compounds, aldehydes and ketones (such as aldehyde method - hydrolysis aldehyde, aldehyde method - distillation aldehyde, ketone method - phenol ester, ketone method - hydrolysis ketone, ketone method - distillation ketone) is manual integration (based on the manual integration of the percentage of the chromatographic line peak area defined by the data processing software to characterize the relative proportion relationship). The following are adjustments made to the original spectrum using the manual integration tool:

[0041] (1) Aldehyde method - hydrolysis of aldehydes:

[0042] ① Take the peak valley at the end of the absorption peak of nonylphenol salicylaldehyde as the manual integration horizontal baseline, and remove the triangular area below the multiple peaks of the polymer (if it is inconvenient to remove, the time range of the area can be adjusted horizontally);

[0043] ② Prohibit integration of the region after the polymer multiple peaks and the region before the nonylphenol absorption peak; ③ Modify the nonylphenol salicylaldehyde absorption peak to highlight the main content of nonylphenol aldehyde (front treatment / tailing treatment);

[0044] ④ Manually calibrate the integration horizontal baseline of the remaining integration range.

[0045] (2) Aldehyde method - distillation of aldehydes:

[0046] ① Take the horizontal line at the rear end of the absorption peak of nonylphenol salicylaldehyde as the manual integration baseline;

[0047] ② Prohibit integration of the area before the nonylphenol absorption peak;

[0048] ③ Manually calibrate the integration horizontal baseline of the remaining integration range.

[0049] (3) Ketone method - phenol ester:

[0050] Determine the height of the integral horizontal baseline and calibrate it, and mark the relative content of nonylphenol and phenol ester

[0051] (4) Ketone method - hydrolysis of ketone

[0052] ① Determine and calibrate the height of the integration horizontal baseline (based on the lowest point of the initial chromatographic line signal value or the average horizontal baseline height of the chromatographic line), and add an integration area to supplement the missing chromatographic peaks;

[0053] ② Use the "Split Peak" tool to split the substance corresponding to the nonylphenol acetone peak time into four sections, use the front processing on the left side and the tail processing on the right side (pay attention to determine the end point of the nonylphenol acetone peak time!) Use tail processing for the overlap on the right side of the tetrachloroethylene peak;

[0054] ③ Remove the solvent tetrachloroethylene peak to obtain the relative content of the target substance in the hydrolyzed ketone sample.

[0055] (5) Ketone method - distillation of ketone

[0056] ① Determine the height of the integration horizontal reference line and calibrate it

[0057] ② Use the "Split Peak" tool to split the substance corresponding to the peak time of nonylphenol acetophenone into at least four sections according to the monotonicity or concavity of the chromatographic line, perform front processing on the left side of the main peak and tailing processing on the right side, and the relative content of the target substance in the distilled ketone sample can be obtained.

[0058] So as to achieve the effect of qualitative analysis.

[0059] Prepare a series of standard solutions with known concentrations. These solutions should contain the target substances you want to quantify. Use the same chromatographic conditions as for sample analysis to analyze the standard solutions. Record the chromatographic peak area or peak height at each concentration. Use the concentration of the standard solution as the abscissa and the corresponding peak area as the ordinate to draw a standard curve. This is usually a straight line passing through the origin. This has achieved the purpose of the target substance standard curve (established but sometimes it may also be a curve, especially when there is a nonlinear response);

[0060] The target substance standard curve method (based on the absolute value of the peak area corresponding to the peak time of the target substance, combined with the injection volume absolute content algorithm of the standard substance curve) is:

[0061] Nonylphenol, tetrachloroethylene, toluene and other substances that are easy to obtain standard samples are first fitted, and the corresponding function relationship is fitted by counting the absolute values ​​of the peak areas in the corresponding methods under different injection volumes. When accurate calculation is required (R 2≥0.99), the functional relationship is not necessarily linear, and is not necessarily a single functional relationship in the entire domain. For example, the fitting curve of the solvent tetrachloroethylene at different injection amounts may show two sections of linear (low injection amount) and logarithmic (high injection amount) functional relationships.

[0062] ①Basic calculation formula: Sample weight × Injection volume = Injection volume

[0063] ②General identity calculation formula: Sample weight × Sample injection volume ÷ The absolute peak area of ​​the sample to be tested value = Standard sample weight × Standard sample injection volume ÷ Absolute value of standard sample peak area

[0064] ③Calculation formula for percentage content: Theoretical equivalent standard sample injection volume ÷ Actual injection volume of the sample to be tested ×100%.

[0065] In actual calculation, first fit the standard curve function relationship of the target substance. (Weigh 0.01g-0.02g standard sample, dilute to 10ml, measure 6 1.5ml samples respectively, and set the injection volume gradient during analysis, such as 1μL, 2.5μL, 5μL, 7.5μL, 10μL, 12.5μL, 15μL). Count the absolute value of the peak area under each injection volume and fit it into the standard curve function relationship. Substitute the absolute value of the peak area of ​​the sample to be tested into the curve function and calculate the corresponding theoretical equivalent standard sample volume. The quotient of the actual injection volume of the sample to be tested is the target substance content.

[0066] Embodiment 2

[0067] Based on the first embodiment, the following method may also be adopted:

[0068] S1. Use the manual integration tool to pre-process the chromatogram of the sample to be tested to ensure the accuracy of the integration.

[0069] S2. Substitute the absolute value of the peak area after pretreatment into the standard curve established by the standard sample.

[0070] S3. Check whether the calculation results are reasonable and readjust the manual integration parameters or standard curve if necessary.

[0071] This combined approach ensures accurate quantification of target substances in complex mixtures while reducing human errors and improving analytical efficiency.

[0072] That is, it is difficult to obtain standard samples of substances such as nonylphenol ethyl ester, nonylphenol ethyl ketone, and nonylphenol salicylaldehyde. Intermediates with higher purity can be used as benchmarks, and then corrected according to the preliminary analysis results of the HNMR spectrum (the HNMR spectrum method mainly identifies the different chemical shifts of H atoms directly connected to the benzene ring and infers the percentage content of different substances); thin layer plate chromatography and column chromatography can also be used to further separate and purify the above substances to obtain high-purity products as benchmark substances. Under the same dilution concentration multiple, the absolute values ​​of the peak areas of the target peak substances in the corresponding methods of different injection volumes of the system are statistically analyzed, and the functional relationship is fitted;

[0073] After correcting the integral baseline of the original spectrum of the mixed system of the product to be tested, determine the injection volume of the mixed system and the absolute value of the peak area corresponding to the peak time of each target substance, substitute the absolute value of the peak area into the fitting function that conforms to the injection volume range, and calculate the absolute content of each target substance in the injection volume of the mixed system, and then calculate the percentage content. (The specific method is basically the same as that of raw materials and solvents).

[0074] The calculation of the mixed system must first calculate the absolute content of the solvent and deduct it; the percentage content of other target substances related to the reaction conversion rate evaluation is based on the absolute content after deducting the solvent. A small amount of simple organic matter, water, inorganic salts and other substances that cannot be detected may make the result lower than the actual content.

[0075] Taking the most complex phenol ester Fris rearrangement hydrolyzed ketone as an example, the standard curve method of the sample to be tested is demonstrated:

[0076] 1. After completing the preprocessing of the spectrum on the workstation, first substitute the absolute value of the absorption peak area of ​​tetrachloroethylene into the tetrachloroethylene fitting curve to calculate the mass (g) of tetrachloroethylene in the hydrolyzed ketone sample. After deduction, the mass of the residue (g) is obtained, and the mass of the residue is defined as the actual injection amount of the sample to be tested.

[0077] 2. Substitute the absolute values ​​of the peak areas of nonylphenol, nonylphenol ethyl ester, nonylphenol ethyl ketone, etc. into the corresponding standard fitting curves to obtain the theoretical injection volume (g) of the three target substances, and find the quotient with the total actual injection volume (g) to get the percentage content.

[0078] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0079] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for comprehensive analysis of a mixed product by liquid chromatography, comprising the following steps: S1. Mixed product: aldehyde compounds and ketone compounds are mixed with different mobile phases respectively, and then injected into the chromatography system; S2. Liquid chromatography analysis: Use liquid chromatography to separate and analyze the mixed product; S3. Qualitative analysis: Based on the liquid chromatography analysis, the peaks in the chromatogram are identified to determine the components in the mixture; S4. Establishment of standard curve of target substance: After the components are determined by qualitative analysis, a standard curve of the target substance is established (this is usually done by analyzing a series of standard samples with known concentrations); S5. Quantitative analysis: Using the established standard curve, the content of the target substance in the sample to be tested is determined by measuring the peak area of ​​the sample to be tested. This is done by substituting the peak area of ​​the sample to be tested into the functional relationship of the standard curve to calculate the corresponding concentration.

2. The method for comprehensive analysis of a mixed product by liquid chromatography according to claim 1, characterized in that: For aldehyde compounds, the mobile phase was: 85% methanol, 15% water, the detector wavelength was 254nm, the analysis time was 17min, and the mobile phase flow rate was controlled at 1.0ml / min; The mobile phase for ketone compounds is: 48% acetonitrile, 37% methanol, 15% water, the detector wavelength is 230nm, the analysis time is 20min, and the mobile phase flow rate is controlled at 0.6ml / min.

3. The method for comprehensive analysis of a mixed product by liquid chromatography according to claim 1, characterized in that: The method for quantitative analysis of two different compounds, aldehydes and ketones (such as aldehyde method - hydrolysis aldehyde, aldehyde method - distillation aldehyde, ketone method - phenol ester, ketone method - hydrolysis ketone, ketone method - distillation ketone) is manual integration method: (1) Determine the integration level baseline and perform calibration; (2) Removal or adjustment of interfering peaks (e.g., polymer multiple peaks, solvent peaks); (3) Use front and tail processing to modify the target peak to ensure the accuracy of integration; (4) Use the "Split Peak" tool to split complex peaks for more accurate integration. The integration results can reflect the actual content of the target substance.

4. The method for comprehensive analysis of a mixed product by liquid chromatography according to claim 1, characterized in that: The specific steps for quantitative analysis are as follows: (1) Perform chromatographic analysis on the sample to be tested under the same chromatographic conditions and record the absolute value of the peak area of ​​the target substance; (2) Substituting the absolute value of the peak area of ​​the sample to be tested into the standard curve function to calculate the theoretical equivalent standard sample amount; (3) Calculate the target substance content using the following formula: The percentage calculation formula is: the theoretical equivalent standard sample injection volume ÷ the actual injection volume of the sample to be tested × 100%.

5. The method for comprehensive analysis of a mixed product by liquid chromatography according to claim 1, characterized in that: The method for establishing the standard curve of the target substance is as follows: (1) Weigh standard substances such as nonylphenol, tetrachloroethylene, and toluene, dilute them to a certain volume, and prepare samples with different injection volumes; (2) Perform chromatographic analysis on the standard sample and record the absolute value of the peak area at different injection volumes; (3) Use statistical software to fit the absolute values ​​of peak areas at different injection volumes and establish a standard curve; (4) The standard curve can be linear, logarithmic or other functional relationship as needed to ensure R 2 ≥0.

99.

6. The method for comprehensive analysis of a mixed product by liquid chromatography according to claim 4, characterized in that: According to the area or peak height of the chromatographic peak and the standard curve, the concentration of the target substance is obtained. If the sample has been diluted or treated, the final result needs to be corrected accordingly according to these steps. Substitute the absolute value of the peak area of ​​the sample to be tested into the curve function to calculate the corresponding theoretical equivalent standard sample volume. The quotient of the actual injection volume of the sample to be tested is the target substance content.