With the advantages of accurate quantitative analysis results, short analysis cycle, wide analysis range, and low analytical detection limit, the high performance liquid chromatography, HPLC for the shot has developed very rapidly in recent years and it can be seen in almost all the macromolecular organics analysis.
It can be predicted that the application of HPLC instruments will be ubiquitous and omnipresent in various fields such as agriculture, light, heavy, sea, land, air, food, clothing, and use. However, how to make good use of the HPLC column is a very critical issue. Based on HAWACH’s long-term practice, the following 7 key issues will be discussed from multiple angles such as instrumental theory, analytical error theory, and analytical chemistry. Welcome to contact HAWACH for more information.
2. Related problems of the HPLC chromatographic column and outside the column 1) Chromatographic peak tailing: It is related to the column, the flow rate of the mobile phase, the sample, etc., and the cause of the tailing must be found from these aspects. 2) How to extend the life of the chromatographic column: Maintenance is very important. Soak it in methanol when not in use for a long time. Strictly control the column washing time or the volume of the column washing solvent. Generally, you should use methanol (or water: methanol is 20: 80) wash for 45 minutes or 20 times the bed volume. 3) Attention must be paid to the control of “extra-column effects”. The so-called “extra-column effect” refers to the chromatographic peak broadening effect caused by the dead volume of the tubing, connections, injector, and flow cell in addition to the column system in the HPLC system. 4) Sampling: During automatic sampling, the autosampler should be tested frequently, and the instrument injector is required to be stable and reliable; when manual sampling, special attention should be paid to the details of sampling and the feeling.
3. HPLC column quality 1) Column efficiency of the chromatographic column: the higher the number of plates is better, and special attention should be paid to the factors that affect the efficiency of the column. The column is scrapped when the number of plates drops to a certain extent. 2) Repeatability: When a column is used repeatedly, the maximum RSD can be kept at less than 0.1%. 3) Durability (lifetime): Because column efficiency is easily reduced, it is necessary to pay attention to column protection. 4) The chromatographic column must be cleaned after use to avoid corrosion, blockage, and reduction of the number of plates. Generally, it should be flushed with 20 times the bed volume. For HPLC that is used every few days, it should be washed with 20% methanol: 80% water for about 30 minutes, and then washed with pure methanol for 20 minutes before storage.
4. Mobile phase issues 1) The pH value is particularly important: Generally, when the pH of a C18 column is less than 3, it is easy to damage the chromatographic column, but a small pH value can be used for acid-resistant columns. 2) Please pay attention to the choice of the cut-off wavelength of the reagent: such as acetonitrile cut-off wavelength 215nm, acetone cut-off wavelength 330nm, n-butane 210nm, etc. 3) Flow rate: The flow rate should be appropriate, otherwise the peak shape will be affected and solvent will be wasted. Generally, 1ml/min is usually selected for routine analysis.
5. Solvent pretreatment It is best to use HPLC-grade high-quality solvents, which must be filtered and degassed before use. Note the following:
1) Purpose of filtering: The solvent should be filtered to remove the tiny particles and microorganisms in the solvent before entering the pump and before the sample injection, to ensure that the pump and the chromatographic column will not be blocked or damaged, and the analytical data should be reliable.
2) The requirements for the filter and the selection method of the maximum pore size: The general requirements for the filter: fast speed, small dissolution rate, small dead volume, precise pore size, proper volume, good chemical compatibility, etc.
For the pore size selection method: if the column packing diameter is less than 3μm or sterile filtration, generally choose 0.2μm pore size; if the column packing diameter is greater than 5μm, choose 0.45μm; if it is pre-filtration or filtering difficult to filter samples, generally choose 1µm aperture. Especially for buffers prepared with inorganic salts, you must pay more attention to choosing the right filter. Otherwise, it is impossible to get reliable analysis and test data.
3) Degassing: The main purpose is to remove bubbles dissolved in the mobile phase or generated by mixing. The most commonly used mobile phase degassing in liquid chromatography are offline ultrasonic vibration degassing, online inert gas bubbling purge degassing, and online vacuum degassing. Air bubbles in the mobile phase entering the liquid phase pump will cause pressure fluctuations, which are very harmful and must be eliminated. The drain valve can be opened to flush at a high flow rate.
6. Buffer solution 1) The buffer must be filtered before use. 2) Solvents and samples are susceptible to bacteria and mold, so clean them. 3) Generally, organic solvents cannot be used directly for cleaning and washing. 4) For gradient elution, choose a buffer with a low absorption value, for example: in gradient elution, the buffer is premixed with the organic corresponding to prevent salt precipitation. Under the condition that the peak is not tailed, use a low concentration of buffer salt as much as possible to prevent salt precipitation; the buffer salt concentration is generally 20 ~ 30 mm basically.
If the organic phase content in the mobile phase is high, the salt buffer concentration in the mobile phase should not be too high, and the salt concentration should at least ensure that it will not precipitate under the mobile phase conditions. The concentration of ion-pair buffer should be higher, generally 50-100mm.
7. Cleaning of the injector For the injector or injection needle, please pay attention to three points: A. Requirements for the sampler: automatic sample injection and fast sampling speed, some instruments only take 17 seconds to sample. B. The sampling chamber requires controllable: generally, Peltier cooling is used, and the temperature control range is 4℃-100℃. C. Special attention should be paid to the cleaning of the injection residue (mainly cleaning the needle).