Methods and requirements for liquid chromatography sample pretreatment

In liquid chromatography analysis, sample pretreatment is the primary step to ensure the accuracy and reliability of analytical data. It covers a series of pretreatment steps from sample collection to final injection, aiming to remove impurities in the sample, adjust sample properties, enrich target analytes, and protect the liquid chromatography system from potential damage. Correct sample preparation methods not only increase analytical sensitivity, but can also significantly improve the clarity and resolution of chromatograms.

1. Liquid chromatography sample pretreatment method 1. Sample purification: (1) Solid phase extraction (SPE): Use solid adsorbent to selectively adsorb the target compound in the sample, and then elute with an appropriate solvent to achieve separation. and purification purposes. (2) Liquid-liquid extraction (LLE): Based on the difference in distribution coefficients of different components in the sample in two immiscible solvents, the target compound is transferred from one solvent to another. (3) Supercritical fluid extraction (SFE): Use supercritical fluid (such as supercritical carbon dioxide) as the extraction agent, and control its solubility by adjusting pressure and temperature to extract target compounds in the sample. 2. Sample concentration: (1) When the concentration of the target compound in the sample is low, concentration needs to be performed to improve detection sensitivity. Commonly used methods include rotary evaporation, freeze drying, nitrogen blowing and concentration, etc. 3. Sample derivatization: (1) For some compounds that are difficult to detect directly, easy-to-detect groups (such as fluorescent groups, chromophores, etc.) can be introduced through chemical reactions to improve detection sensitivity and selectivity. 4. Sample dilution: (1) If the sample concentration is too high, it may exceed the detection range of the liquid chromatograph or cause damage to the instrument. In this case, appropriate dilution is required. 5. Sample pH adjustment: (1) Some liquid chromatography columns have strict requirements on the pH value of the sample, so it is necessary to adjust the pH value of the sample by adding acid or alkali to protect the chromatographic column and optimize the separation effect. 6. Sample filtration: (1) Before injecting a sample, it is usually necessary to use an appropriate filter (such as a microporous membrane) to remove particulate matter and suspended solids in the sample to prevent clogging of the chromatographic column or affecting the analysis results. 7. Sample stability treatment: (1) For samples that are easily decomposed, oxidized or volatile, appropriate measures (such as refrigeration, light protection, adding stabilizers, etc.) need to be taken to maintain their stability. 8. Automated pre-processing system: (1) With the development of science and technology, more and more laboratories are beginning to use automated pre-processing systems to simplify operating procedures, improve processing efficiency and reduce human errors. These systems usually integrate a variety of pre-treatment steps, have automatic injection functions, and can be directly connected to the liquid chromatograph.

2. Requirements for liquid chromatography sample pre-processing 1. Sample preparation (1) Representativeness: Ensure that the sample taken is representative and can truly reflect the characteristics of the overall sample. (2) Stability: For samples that are easily decomposed, oxidized or volatile, appropriate measures (such as refrigeration, light protection, adding stabilizers, etc.) should be taken to maintain their stability. 2. Purification and separation (1) Method selection: According to the characteristics and analysis requirements of the sample, select the appropriate purification and separation method, such as solid phase extraction (SPE), liquid-liquid extraction (LLE), etc. SPE: Use solid adsorbent to selectively adsorb the target compound in the sample, and then elute it with an appropriate solvent. LLE: Extraction based on the difference in partition coefficients of different components in the sample between two immiscible solvents. (2) Condition control: Strictly control conditions such as temperature, pH value, solvent type and dosage during purification and separation to ensure effective extraction and separation of target compounds. 3. Concentration and dilution (1) Concentration: For samples with lower concentrations, methods such as rotary evaporation, freeze-drying, and nitrogen blowing concentration are used to improve detection sensitivity. (2) Dilution: If the sample concentration is too high, appropriate dilution is required to avoid exceeding the detection range of the liquid chromatograph or damaging the instrument. 4. Derivatization (1) Necessity: For some compounds that are difficult to detect directly (such as components with no UV absorption or fluorescence), derivatization is performed to improve the sensitivity and selectivity of detection. (2) Reaction conditions: Strictly control the conditions of the derivatization reaction (such as temperature, time, pH value, etc.) to ensure completeness and selectivity of the reaction. 5. Filtration and degassing (1) Filtration: Use appropriate filters (such as microporous membranes) to remove particulate matter and suspended matter in the sample to prevent clogging of the chromatographic column or affecting the analysis results. The filter membrane should be able to retain particles above 0.15μm. (2) Degassing: Degas the gases (such as oxygen, carbon dioxide, etc.) dissolved in the sample to prevent bubbles from forming in the chromatographic column and affecting the separation effect. 6. Instruments and reagents (1) Instrument cleaning: Keep the liquid chromatograph and its accessories clean and dry to prevent contamination and damage. (2) Reagent purity: Use chromatographically pure, superior grade pure or analytically pure reagents to ensure the accuracy of analysis results. Avoid using reagents containing impurities to avoid impurity peaks that may affect the measurement results. 7. Operating specifications (1) Follow the procedures: Strictly follow the operating procedures for sample preprocessing and analysis operations to reduce human errors. (2) Detailed records: Record every step of the sample pre-processing process and its conditions in detail for subsequent analysis and problem tracing. 8. Environmental control (1) Keep clean: The instrument room should be isolated from other laboratories and kept clean to prevent harmful gases, gas solutions, dust and other pollution from affecting the test results. (2) Temperature and humidity control: Air conditioning should be installed in the instrument room and attention should be paid to moisture-proof, anti-corrosion, shock-proof, and relative air humidity control (generally should be less than 70%) to ensure the normal operation of the instrument and extend its service life.

Liquid chromatography sample pretreatment is a complex and delicate process, which requires laboratory personnel to have a solid theoretical foundation and rich practical experience. By selecting appropriate pretreatment methods and strictly complying with operating requirements and precautions, interferences in the sample can be effectively removed, target analytes can be enriched, and the sensitivity and accuracy of analysis can be improved. At the same time, reasonable sample pretreatment can also protect the liquid chromatography system from contamination and damage and extend its service life.