FAQ Analysis And Solutions About HPLC Columns
HPLC analysis, which is supposed to have more broader potential applications, is not limited by environment temperature and the boiling point of samples. After more than 30-year rapid development, the theory of HPLC has been more and more mature in the basic theory, the instruments and the chromatography columns, etc. And now HPLC has become one of the most advantageous separation and analysis methods in many fields, such as chemical industry, environment, pharmacy, food and so on.
According to the different separation mechanism, the liquid chromatography can be divided into five kinds: liquid-solid adsorption chromatography, liquid-liquid distribution chromatography, ion-exchange chromatography, ion-pair chromatography and molecular exclusion chromatography, which can be also called gel permeation chromatography.
For instance, in the process of the liquid-solid adsorption chromatography, the mobile phase is liquid and the stationary phase is solid adsorbent. The substances are separated according to the different adsorption effects. And when doing the liquid-liquid chromatography, the mobile phase and stationary phase are both liquid chromatography, which uses the distribution of sample components between two immiscible liquid phases for separation.
FAQ analysis and solutions about HPLC columns
1.The column pressure is too high, after a period of use.
Solution: First check whether the HPLC system is the cause. After eliminating the system cause, the reason is basically due to the accumulation of impurities in the column during the experiment. Try to improve the operating conditions. After the sample is finished, it should be rinsed off in time. For example, the mobile phase of the buffer salt must be washed with a high proportion of aqueous solution (such as 90%) and then stored with the organic phase.
2.Sieve plate blockage and column head collapse.
Solution: If it is determined that the filter screen of the column head is contaminated, the column can be flushed to normal pressure in the opposite direction with methanol, or the column head should be removed, the sieve plate should be carefully removed, and the solution should be sonicated for about 20 minutes with a 5% or so nitric acid solution. Then, it is sonicated with pure water for about 20 minutes and reloaded into the column.
3.The packing of the column head is contaminated by the sample.
Solution: If it is determined that the packing of the column head is contaminated, remove the stud screws, dig out the contaminated packing in the front part of the column, refill with the same column packing. And after careful repair, reinstall the stud screws.
4.The salt in the buffer in the column encounters a high concentration of methanol or other organic solvent to form crystals and precipitate.
Solution: If it is determined to be salt crystals, rinse the column with 10% methanol/water to dissolve all the salt in the column, and then change the high concentration of methanol.
1. Whenever possible, the column bed must be kept dry, especially during the injection test and column flushing process, the mobile phase should not be left empty for more than 30 minutes. Otherwise, the column will dry up and can be discharged into a large amount of non-ejectable bubbles which even cause partial collapse or central cracking of the bed.
2. For use and storage, it is strictly forbidden to use force to absolutely prevent accidental violent impact or falling at high places. Otherwise, it will easily cause mechanical damage to the column bed, then there will be no regeneration.
3. When the column and the chromatograph are coupled, the valve or pipe must be cleaned.