To analyze the composition of a mixed sample of amino acids using liquid chromatography, you can follow these steps:1. Choose the appropriate chromatographic technique: High-performance liquid chromatography HPLC is a widely used technique for the separation and analysis of amino acids. For even better resolution and sensitivity, you can use ultra-high-performance liquid chromatography UHPLC .2. Select the appropriate column: For amino acid analysis, a reversed-phase column e.g., C18 is commonly used. The column should be packed with small particles 3-5 m to ensure high resolution.3. Prepare the sample: Dissolve the mixed amino acid sample in a suitable solvent, such as water or a buffer solution e.g., phosphate buffer . If necessary, you can perform a pre-column derivatization to improve the detection and separation of amino acids. Common derivatization agents include o-phthalaldehyde OPA , 9-fluorenylmethyl chloroformate FMOC-Cl , and phenylisothiocyanate PITC .4. Choose the mobile phase: The mobile phase should be a gradient of water and an organic solvent e.g., acetonitrile or methanol containing an ion-pairing agent, such as trifluoroacetic acid TFA or formic acid. The gradient should start with a high percentage of water and gradually increase the percentage of organic solvent to elute the amino acids from the column.5. Set the flow rate and temperature: The flow rate should be optimized to ensure proper separation of amino acids. A typical flow rate for HPLC is 0.5-1.5 mL/min, while UHPLC may use a flow rate of 0.2-0.5 mL/min. The column temperature should be maintained at a constant temperature e.g., 30-40C to ensure reproducibility.6. Choose the detection method: The most common detection method for amino acid analysis is UV-Vis absorbance, typically at 214 nm or 280 nm. Fluorescence detection can also be used, especially when a pre-column derivatization agent is used.7. Run the chromatographic analysis: Inject the prepared sample into the HPLC or UHPLC system and monitor the elution of amino acids using the selected detection method. The retention times and peak areas of the amino acids can be used for identification and quantification.8. Data analysis: Compare the retention times of the amino acids in the sample with those of known amino acid standards to identify the amino acids present in the sample. To quantify the amino acids, create a calibration curve using known concentrations of amino acid standards and determine the concentration of each amino acid in the sample based on its peak area.By following these steps, you can successfully analyze the composition of a mixed sample of amino acids using liquid chromatography.