With the development of modern biotechnology, various polypeptide drugs have emerged, and polypeptides have great application value in clinical medicine. Reverse-phase high-performance liquid chromatography (RPLC) has been used for the separation, purification, and preparation of two chemically synthesized polypeptides - 32-peptide and 21-peptide, which can improve the purity of polypeptides. The following is a sharing of knowledge related to the use of reverse-phase high-performance liquid chromatography. ### Reverse-Phase High-Performance Liquid Chromatography – Method for Adjusting Mobile Phase pH Value Due to the denaturing effect of low pH and high organic solvents, reverse-phase high-performance liquid chromatography can be used to separate and quantify light and heavy chains with different drug loads. Reverse-phase liquid chromatography (RP-LC) is a commonly used chromatographic separation technique. The adjustment of the mobile phase pH value has an important impact on the retention behavior and separation effect of analytes. The adjustment of the mobile phase pH value can be carried out through the following steps: 1. Select an appropriate buffer salt according to the chemical properties of the target analyte and the requirements of the chromatographic column. Commonly used buffer salts include phosphate, citrate, acetate, and triethylamine. 2. Use a pH meter to accurately determine the buffer solution to ensure it meets the experimental requirements. The pH measurement should be performed at a predetermined temperature, as temperature changes can affect the pH value. 3. If the pH value of the buffer solution does not meet the requirements, dilute hydrochloric acid or dilute sodium hydroxide solution can be used for adjustment. Usually, the titration method is adopted to gradually adjust the pH value, and continuous stirring is performed to ensure uniform mixing. 4. After adjusting the pH value, the mobile phase needs to be degassed to avoid interference from bubbles in the chromatographic separation. Degassing can be carried out by ultrasonic degassing, vacuum degassing, or an online degassing device. 5. To prevent blockage of the chromatographic column, the mobile phase needs to be filtered. A 0.22 μm or 0.45 μm filter membrane is used for filtration. 6. After the mobile phase pH value is adjusted, degassed, and filtered, the chromatographic system should be fully equilibrated with the newly prepared mobile phase to ensure the stability and reproducibility of the chromatographic separation. 7. The prepared mobile phase should be stored in a clean container and kept in a low-temperature environment to prevent changes in pH value. ### Application of Reverse-Phase High-Performance Liquid Chromatography in the Detection of Synthetic Pigments #### Instruments and Reagents - **Instruments**: Detector, funnel, balance, and chromatographic column. The detector used is LC-20 AD, a high-resolution liquid chromatograph equipped with a diode array detector. The G3 sintered glass funnel specifications are XS 205 and LC-20AD. The C18 chromatographic column specifications are 4.6 mm × 250 mm, 5 μm, as well as an electronic balance and 0.45 μm aqueous phase filter membrane. - **Reagents**: Ultrapure water (manufactured by Yuanmei); ammonium dihydrogen phosphate, formic acid, and ammonia water, all of analytical grade; methanol and ethanol, all of chromatographic grade. #### Standard Samples - Brilliant blue: purity > 99.7% - Tartrazine: purity > 91.0% - Sunset yellow: specification GBW(E)10003a, 0.5 mg/mL - Erythrosine: purity > 91.7% - Allura red: purity > 80.0% #### Preparation of Standard Solutions Prepare standard solutions according to standard specifications with a concentration of 1.0 mg/mL. First, take an appropriate amount of the standard sample to be prepared, add it to a 25 mL volumetric flask, and dilute to the mark with water to gradually adjust the solution to the standard concentration. It should be noted that the concentration of the standard stock solution of sunset yellow is different from that of the normal solution, which is 0.5 mg/mL. #### Preparation of Mixed Solutions Mixed solutions require the mixing of multiple standard solutions. The predetermined standard solutions to be mixed can be mixed by first diluting them with water to prepare mixed solutions with different contents, namely 2 μg/mL, 5 μg/mL, and 10 μg/mL. After preparation, they are stored for later use. #### Chromatographic Conditions The main condition for pigments is the chromatographic column, with specifications of 4.6 mm × 250 mm, 5 μm. The detection wavelength is 254 nm, the column temperature is 30°C, the injection volume is 10 μL, and the flow rate is 1 mL/min. For specific gradient conditions, see Table 1. *Table 1: Mobile Phase Gradient Conditions* #### Sample Processing Weigh a certain amount of tablets and grind them until the tablets in the mortar become uniform fine powder, then weigh the sample. In addition to tablets, there are capsules and capsule shells. For capsule shells, take out all the powder inside the capsule, wipe the capsule shell with a cotton swab until it is completely clean with no powder residue, and then weigh the sample [3]. Take a certain amount of these samples and pour them into a 100 mL beaker, then pour 30 mL of water and heat the water to warm (60°C) to dissolve the sample. Take 1 g of polyamide powder and slowly adjust it with water to a porridge-like consistency, then pour it into the solution where the sample is dissolved and stir. Further wash it: first, wash 3 times with 60°C water filtered through a G3 sintered glass funnel, then wash 3 times with a mixed solution of 60% methanol and 40% formic acid until the solution is neutral. Then, desorb the solution with a mixed solution of 70% ethanol, 20% ammonia water, and 10% water, divided into 4 times, 5 mL each time. After desorption, neutralize the solution with acetic acid, then evaporate and dilute to volume, finally adjusting the solution to 5 mL. Finally, filter the solution with a 0.45 μm filter membrane for liquid chromatography detection. #### Stability Test Sample and analyze the standard mixed solution every 2 hours under the chromatographic conditions of liquid chromatography detection, and timely record the linear relationship and correlation coefficients of the standard working curves of the 6 colorant components. The experimental results show that each component concentration has a good linear relationship with the peak area, and the minimum detection limit is determined by the 3-fold noise ratio. For the linear range, regression equation, correlation coefficient, and detection limit, see Table 2. The reverse-phase high-performance liquid chromatography detection method was discussed according to the RP-HPLC method. Synthetic colorants were used as samples during the test, and targeted detection was carried out according to the characteristics of health food and the different dosage forms of the samples. The method adopted in this study is easy to operate, with good experimental speed. The separation of mixed pigments is smooth, with good resolution results, good sample stability, and ideal recovery rates. Therefore, this method is suitable for the separation and detection of various dosage forms of different colorants in health food. ### What is the Packing Material of C18 Chromatographic Column? The Thermo Hypersil ODS (C18) liquid chromatographic column is packed with a stationary phase where octadecyl groups (C18) are bonded to a silica gel matrix. It is a typical packing material for high-performance liquid chromatographic columns, suitable for the analysis of non-polar and neutral samples, including acidic, neutral, and lipophilic compounds. C18 chromatographic columns are the most commonly used and essential general-purpose chromatographic columns in every laboratory. The packing material is octadecyl-bonded silica gel matrix, with high carbon content and good hydrophobicity, suitable for most compounds, including non-polar, small polar molecules, and some polypeptides and proteins. With the research and development of each chromatographic column manufacturer, the types of C18 columns are constantly increasing. The applicable pH range has expanded from the original 2–8 to 1–14 now, and the compatibility with pure aqueous phases is getting better. Some chromatographic columns will not experience hydrophobic collapse of the packing even when using 100% aqueous phase for a long time. For each experimenter, the range of options is increasingly wide. #### Column Models The specifications of C18 chromatographic column packing types: octadecyl-bonded spherical silica gel, including 250 × 4.6 mm, 5 μm; 200 × 4.6 mm, 5 μm; 150 × 4.6 mm, 5 μm; 250 × 4.6 mm, 7 μm; 200 × 4.6 mm, 7 μm; 150 × 4.6 mm, 7 μm. Source: Kaogong, Modern Food