Selection, use, maintenance and troubleshooting of liquid chromatography columns
The columns of liquid chromatography are usually divided into normal phase columns and reverse phase columns. Most of the normal phase columns use silica gel as the column, or a bonded phase silica gel column bonded with functional groups such as -CN, -NH3 on the surface of the silica gel; The octaalkyl functional group (ODS) is called C18 column, and other commonly used reversed-phase columns include C8, C4, C2 and phenyl columns. There are also ion exchange columns, GPC columns, polymer packing columns, etc. This article focuses on the selection and use of reversed-phase chromatography columns:
1. Selection of reversed-phase chromatography column
1. The use range of the PH value of the column. The advantage of the reversed phase column is that the stationary phase is stable, and it is widely used. A variety of solvents can be used. However, silica gel-based fillers must pay attention to the PH range of the mobile phase when used. The pH range of a general C18 column is 2-8. When the PH value of the mobile phase is less than 2, it will cause hydrolysis of the bonded phase; when the pH value is greater than 7, silica gel is easy to dissolve; often use a buffer solution to degrade. Once this happens, the column population will collapse. Columns of different grades with the same packing are different. If the mobile phase PH is high or when buffers are often used, it is recommended to choose a column with a large pH range, such as the Diclaim company's Acclaim column PH2-9 or Zorbax's PH 2-11.5 column.
2. End-end sealing (or sealing) of packing
The residual silicon hydroxyl group of the filler is end-sealed by sealing technology, which can improve the adsorption or tailing of polar compounds; the carbon content is increased, which is conducive to the separation of the compound that is not easy to retain; the stability of the filler is good, and the component Reproducibility of retention time is good. If the sample to be analyzed is an acidic or basic compound, it is best to use a chromatographic column with end capped packing.
2. Use of Liquid Chromatography Columns Before using the chromatographic columns, it is best to perform column performance tests and save the results as a reference for future evaluation of column performance changes. The column performance test should be carried out according to the conditions in the column factory report (the conditions used in the factory test are the best conditions). Only in this way can the measured results be comparable. However, it should be noted that column performance may vary due to differences in the samples, mobile phase, and column temperature used.
1. Sample pretreatment a. It is best to use mobile phase to dissolve the sample.
b. Use the pretreatment column to remove the impurities with strong polarity or irreversible adsorption with the column packing.
c. Use a 0.45μm filter membrane to remove particulate impurities.
2. Preparation of mobile phase Liquid chromatography is the mass exchange of sample components between the column packing and the mobile phase to achieve the purpose of separation. Therefore, the mobile phase is required to have the following characteristics:
a. The mobile phase has a certain dissolving capacity to ensure that the sample components will not precipitate in the column (or remain in the column for a long time).
b. There is no chemical reaction between the mobile phase and the sample. c. The viscosity of the mobile phase should be as small as possible in order to obtain a good separation effect; reduce the pressure drop of the column and prolong the service life of the pump (you can use the method of increasing the temperature to reduce the viscosity of the mobile phase) .
d. The physical and chemical properties of the mobile phase should be compatible with the detector used. If a UV detector is used, it is best to use a solvent with low UV absorption.
e. The boiling point of the mobile phase should not be too low, otherwise bubbles will easily be generated, which will make the experiment impossible.
f. After the mobile phase is prepared, it must be degassed. Removal of trace gases dissolved in the mobile phase is not only conducive to detection, but also prevents trace oxygen in the mobile phase from interacting with the sample.
3. Selection of mobile phase flow rate Because the column efficiency is a function of the linear flow rate of the mobile phase in the column, different column efficiencies can be obtained by using different flow rates. For a particular column, to pursue the best efficiency, it is best to use the best flow rate. For a column with an inner diameter of 4.6mm, the flow rate is generally 1ml / min. For a column with an inner diameter of 4.0mm, a flow rate of 0.8ml / min is preferred.
When the optimal flow rate is selected, the analysis time may be extended. The method of changing the washing intensity of the mobile phase can be used to shorten the analysis time (for example, when using a reversed-phase column, the content of methanol or acetonitrile can be appropriately increased).
note:
a. Aqueous mobile phase is best prepared before the experiment, especially in summer, use buffer solution as mobile phase not overnight. It is best to add sodium azide to prevent bacterial growth.
b. The mobile phase requires 0.45 μm filter membrane to remove particulate impurities.
c. Use HPLC-grade solvent to prepare mobile phase. Using appropriate mobile phase can extend the life of chromatographic column and improve column performance.
3. Column maintenance
1. The equilibrated reversed phase column of the column is stored in acetonitrile / water after being tested by the factory. The new column should be flushed with 10-20 column volumes of methanol or acetonitrile. Please make sure that the mobile phase used in your sample analysis is compatible with acetonitrile / water. Use enough time every day to balance the column with the mobile phase, you will get the most "compensation" in dealing with the problem, and the life of your column will also become longer! Operation steps:
a. Slowly increase the flow rate at the beginning of equilibration, and equilibrate the column with the mobile phase until a stable baseline is obtained (if the flow rate of the buffer salt or ion pair reagent is low, it takes a long time to equilibrate)
b. If the mobile phase used contains buffer salts, you should pay attention to the "transition" with pure water, that is, you must rinse with pure water for more than 30 minutes before the analysis starts every day, and then balance with the buffer salt mobile phase; after the analysis, you must first use pure water Rinse for more than 30 minutes to remove the buffer salt and then rinse with methanol for 30 minutes to protect the column.
2. Regeneration of chromatographic columns. Chromatographic columns used for a long period of time tend to have lower column efficiency (the theoretical plate number of the column decreases). The column can be regenerated, and a cheap pump should be used to regenerate the column in a qualified laboratory.
Recommended solvent volume for rinsing the column Chromatography column size Column volume Volume of solvent used 125-4mm 1.6ml 30ml
250-4 mm 3.2ml 60ml
250-10mm 20ml 400ml
Choose regeneration method:
Regeneration of polar stationary phases (eg Si, NH2 *, DIOL-based chromatography packing):
N-heptane → chloroform → ethyl acetate → acetone → ethanol → water **
Regeneration of non-polar stationary phase (such as reversed-phase chromatography packing RP-18, RP-8, CN, etc.): water → acetonitrile → chloroform (or isopropanol) → acetonitrile → water Note:
a. When regenerating the NH2 modified column, because NH2 may exist in the form of ammonium ions, it should be washed with 0.1M ammonia water after washing, and then washed with water until the alkaline solution completely flows out.
b. 0.05M dilute sulfuric acid can be used to clean contaminated chromatographic column. If the simple treatment with organic solvent / water cannot completely remove the impurities adsorbed on the surface of silica gel, it is very effective to add 0.05M dilute sulfuric acid after water washing.
3. Maintenance of chromatographic column a. Use pre-column to protect analytical column (silica gel has certain solubility in polar mobile phase / ionic mobile phase)
b. The pH stability range of most reversed-phase chromatography columns is 2-7.5, try not to exceed the pH range of the column. c. Avoid drastic changes in the composition and polarity of the mobile phase d. The mobile phase must be degassed and filtered before use Treatment e. If a polar or ionic buffer solution is used as the mobile phase, the column should be rinsed out after the experiment and stored in methanol or acetonitrile. F. The solvent of the chloride is corrosive to it, so use it Note that such solvents should not be left in the column and connecting tube for a long time to avoid corrosion.
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