Liquid chromatography can handle mixtures with complex components, significant polarity differences, or low stability. By selecting appropriate stationary and mobile phase gradients, it can achieve effective separation of high boiling point, large molecules, and thermally unstable substances (such as proteins, polysaccharides, pesticide residues) without the need for high temperature heating, avoiding the problem of substance decomposition. Combining high-precision injection systems and high-sensitivity detectors (such as UV detectors, fluorescence detectors, mass spectrometry detectors, etc.), it can meet the detection needs of trace substances (concentrations as low as μ g/L or even ng/L). In terms of qualitative analysis, accurate identification of substance composition can be achieved through dual verification of retention time and characteristic spectra or mass spectra; In terms of quantitative analysis, mature methods such as external standard method and internal standard method can be used to achieve accurate calculation of substance concentration.

Liquid chromatography uses liquid as the mobile phase. Due to the high resistance experienced by the liquid flowing through the chromatographic column, high pressure (usually up to 150-35000KPa) needs to be applied to the carrier liquid to ensure that the mobile phase can quickly pass through the chromatographic column. At the same time, the flow rate of the mobile phase inside the column is much faster than that of classical liquid chromatography (generally up to 1-10 mL/min), greatly reducing the analysis time. High sensitivity detectors have been widely adopted, further improving the sensitivity of analysis. For example, the sensitivity of a fluorescence detector can reach the level of 10g, and the sample size is small, usually only a few microliters.

Compared with gas chromatography, liquid chromatography is not limited by sample volatility and can analyze organic compounds with high boiling points, poor thermal stability, and high relative molecular weight (greater than 400). These substances account for almost 75-80% of the total organic matter and can generally be separated and analyzed using high-performance liquid chromatography. Usually equipped with automated sampling systems and data processing software, the operation process is more convenient and efficient, reducing the risk of manual errors.

The determination steps of liquid chromatography:

1. Sample pretreatment

-Extraction: Weigh approximately 10g (accurate to 0.1g) of the sample into a 100ml conical flask, add 60ml of mixed extraction solution and 2g of sodium chloride, and shake for 30 minutes. Filter through a filter funnel with a pore size of 80-120 μ m, and wash the residue with 3 × 10ml of mixed extraction solution. Combine the filtrate and wash solution in a 250ml beaker. Heat in boiling water for 30 minutes, remove, centrifuge at 3000 r/min for 20 minutes, cool in a refrigerator at 4 ℃ for 20 minutes, and then filter through a filter funnel with filter holes of 40-80 μ m to collect the filtrate.

-Purification: If the sample composition is complex, solid-phase extraction and other methods can be used for further purification.

-Concentrate to volume: Concentrate the extract to near dryness using a nitrogen blower, dilute to an appropriate volume with methanol, and pass through a 0.22 μ m microporous membrane for testing.

2. Instrument preparation

-Mobile phase preparation: Select HPLC grade or higher solvents, prepare the mobile phase in proportion, and filter it through a 0.22 μ m filter membrane. When using, cover the bottle cap with an exhaust hole.

-Column installation: Install the chromatography column correctly in the column temperature chamber, connect the pipelines, and ensure no leakage.

-System equilibrium: Rinse the system with mobile phase for at least 30 minutes until the baseline is stable.

3. Injection analysis

-Sample loading: Inject the sample solution into the injection ring, taking care to avoid the formation of bubbles.

-Data collection: Start the chromatography workstation to collect data and record chromatograms.

4. Data processing

-Qualitative and quantitative: Qualitative analysis is conducted based on retention time, using either external or internal standard methods for quantification.

-Result report: Calculate the content of the target compound in the sample and issue a detection report.