High performance liquid chromatography has the characteristics of high separation efficiency, fast analysis speed, and wide application range, especially suitable for the separation and analysis of compounds with high boiling points, large molecules, strong polarity, and poor thermal stability.
At present, high-performance liquid chromatography has become an important separation technology in disciplines such as chemistry, biochemistry, medicine, industry, agriculture, environmental protection, commodity inspection, and legal inspection. It is an essential tool for analytical chemists and biochemists to solve various practical analysis and separation problems they face.
Although expensive and high-performance high-end precision instruments are used in detection and analysis, the lack of attention to sample pretreatment, preparation of standard solutions, determination of sample solutions, contamination during analysis, and common instrument malfunctions can cause significant systematic errors, resulting in the failure of the entire measurement and analysis.
A brief analysis will be conducted on several aspects of sample pretreatment in the application of liquid chromatography analysis, in order to achieve better detection results.
Sample pretreatment method: Sample pretreatment should include all operations before injection, except for weighing, dissolution, dilution and other steps. The sample needs to be: ① filtered; ② Extraction; ③ Derivative (pre column derivative); ④ Liquid chromatography (low-pressure column chromatography); These operations can be manual or automated, and the purpose of sample pretreatment is to remove interferences, increase detector sensitivity (enrichment), protect the chromatographic column, etc. Sample pretreatment is also to avoid chromatographic separation failures, among which sample extraction is a crucial step, and it is extremely difficult to extract trace components from a large amount of interfering substances.
Some samples cannot be analyzed by injection after pretreatment and need to be derivatized, so that some components without UV absorption or fluorescence can be detected by UV and fluorescence detectors after derivatization. This not only improves sensitivity but also improves separation (mass change). Sample pretreatment can also bring some problems, such as sample loss, sample contamination, incomplete derivatization reactions, or the generation of multiple reactants. Derivative reactions often affect the accuracy of experiments or introduce errors throughout the entire sample pretreatment process.
The sample solution used for liquid chromatography analysis must be uniform and free of particles, as particles can damage the injector and block the column head. The processed sample should be aligned with the light and shaken before being prepared for column placement to check for particles in the sample solution. As long as particles, turbidity, or emulsification are seen, they should be filtered. The filter membrane should be able to retain particles larger than 0.15 μ m. During the sample filtration process, it may cause contamination of the sample due to reduced content of sample components caused by filtration adsorption and errors caused by sample solvent evaporation.
The purpose of extraction is to separate the analyzed components from the co dissolved sample medium, or to reduce substances that damage the column (such as proteins) and interferences. Organic solvent extraction is generally used, which requires the solvent used for extraction to have low toxicity, good volatility, few impurities, good solubility of the test sample, and be immiscible with water.
Commonly used solvents include ether, ethyl acetate, dichloromethane, chloroform, benzene, or a mixture of two or more. After extraction, the sample can generally be directly injected, sometimes requiring concentration or drying concentration, and then dissolved in a fixed volume of liquid or mobile phase for injection. This increases the sample concentration, improves sensitivity, and avoids interference from solvent peaks on the sample peaks. In extraction, the solubility of sample molecules should be considered. In addition to fat soluble and water soluble components, fat soluble components are also used to make water soluble salts. The extraction method is as follows: water soluble samples, (1) acid components and generated salts extraction method: organic solvents extract impurities and then adjust them to acid, and then add organic solvents for extraction or sample injection, or blow dry under N2 flow, and then use appropriate solvents to dissolve before sample injection. (2) Extraction method of alkaline component and generated salt. (3) Neutral component extraction method: ① After extracting impurities with organic solvents, analyze them directly by reverse phase chromatography; ② Extraction method of lipophilic components: organic solvent extraction or injection, or drying under N2 flow, dissolving in appropriate solvent before sampling;
Pollution in analysis: The environment, containers, and reagents used in general testing are all factors that affect the measurement results environmental pollution. Harmful gases, gas solutions, dust, and other substances in the instrument room can cause pollution and affect the detection results, making it difficult to correct such pollution. Therefore, the instrument room should be isolated from other laboratories, kept clean, and equipped with air conditioning. Attention should be paid to moisture resistance, corrosion prevention, shock resistance, and the relative humidity of the air should be less than 70% Container. The commonly used containers in the laboratory include glass, porcelain, quartz, plastic, etc. When conducting analysis, the containers should be selected according to the requirements of the sample to be tested. Regardless of which type of container is used, cleaning the cleaning strip of the container is very important and is also the basic guarantee for obtaining good test results Reagents. In liquid chromatography analysis, the selected reagents must be of chromatographic purity, superior purity, or analytical purity. If reagents with impurities are used, impurity peaks may appear and affect the measurement results.
Preparation of standard solution. When preparing a standard solution, it is necessary to first prepare an internal standard solution of the current concentration. It is best to use an internal standard solution prepared from the same batch in both the standard solution and the sample solution to reduce errors. (1) The substance used to prepare the standard solution should be chromatographically pure and have stable properties. (2) Commonly used standard solutions should be stored in brown solution bottles for low-temperature preservation. (3) When preparing vitamin standards, they should be placed in brown volumetric flasks or in the dark to prevent decomposition.
