Use and maintenance of C18 liquid chromatography column

Before choosing a chromatographic column, you should know more about your samples and impurities, their type, structure, polarity, acidity and basicity, molecular weight, etc,
1. The sample is polar and weakly acidic; C18 can be selected for detection under the condition of 100% acidic aqueous solution, that is, the chromatographic column with 100% pure water and good retention of polar compounds should be selected
2. If the polarity of the sample is too strong or the acidity is too strong, CN, NH2, silica gel column, HILIC (hydrophilic chromatography) can be selected, and C18 + strong anion pair reagent or strong anion exchange chromatographic column can also be used (the disadvantage is that the balance time of ion pair reagent is long, and the pH requirement of the mobile phase is relatively precise, no It is very difficult to repeat the experiment. In addition, it is difficult to wash down the ion pair reagent. Basically, the chromatographic column with ion pair can not be used for other experiments.)
3. If the sample is alkaline, high-purity silica gel column (high-purity silica gel is lack of metal impurities, and the end group of silica gel is sealed) or some modified C18 column (such as polar embedding technology or alkali deactivation technology, etc.), they will reduce the tailing of alkaline compounds, and they will generally choose the medium or alkaline conditions, because this can increase the retention of alkaline samples.
4. If the polarity of alkaline compounds is too strong or the alkalinity is too strong, C18 column with wide pH can be selected for detection at high pH value (the advantage is that the method is simple to develop, but the disadvantage is the current practice) At present, there are few chromatographic columns in this technology, and the price is also high) or HILIC column (silica column is used in reversed phase condition, which is also a classic method for detecting alkaline samples) and strong ion exchange column (disadvantage) C18 + strong anion pair reagent or strong anion exchange chromatographic column is also used.
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C18 column is a commonly used reversed-phase chromatographic column, which uses octadecylsilane bonded silica gel as filler, so it has higher carbon content and better hydrophobicity. It has stronger adaptability to various types of biomacromolecules, and has a wide range of application. It is an optional column type in the analysis of liquid chromatography.
In the process of using C18 column, the following matters should be paid attention to:
1. The new C18 column should be washed with methanol or acetonitrile at a low flow rate of 20 times the volume of the column before use. The purpose is to make the stationary phase fully wetted and the carbon chain extended, so that the performance of the column can reach a good state. The specific operation is as follows: after washing the prepared 65% acetonitrile / water, connect the inlet end of the chromatographic column to the chromatograph, vent the outlet end (do not connect the detector to avoid polluting the detector), dry the clear solution in the chromatographic column at a low flow rate of 0.1ml/min ~ 0.5ml/min, connect the detector after 20 minutes, and continue to wash for 2 ~ 8 hours at the flow rate of 1.0ml/min;
2. The chromatographic column must be washed after the sample is detected and analyzed by liquid chromatograph. Especially when the mobile phase contains acid or salt, the acid or salt in the chromatographic column should be washed clean. It is generally recommended to wash 20 times the column volume with 10% methanol water;?
3. If the chromatographic column is idle for a long time, it is recommended to wash the column for more than 3-4 hours, and then keep it in pure organic reagent or high proportion of organic reagent solution (such as 90% methanol water);?
4. Due to the strong hydrophobicity of the stationary phase of C18 column, the high water phase condition should not be used in the use process. If the water phase ratio is too high, it is easy to cause the hydrophobic collapse of the stationary phase, resulting in the rapid decline of column efficiency and even irreversible negative effects. Due to the high carbon load, the performance is particularly obvious, so it is suggested that the proportion of water phase should not exceed 90% in the use process;
5. Column pressure increases and column performance decreases: the column is usually contaminated, and part of the column efficiency can be recovered by regenerating the column;??
6. Sudden pressure rise of chromatographic column: it is usually caused by salting out or blocking of sieve plate. In case of salting out, wash it with 10% methanol water at low flow rate until the pressure is restored; if it is judged that it is not salting out, flush the chromatographic column with pure methanol or 10% methanol water as the flow opposite (connect the chromatographic column reversely without connecting the detector). If the backwash is still ineffective for half an hour, it indicates that the backwash is invalid, and other reasons need to be found.
For more related technologies, please consult MS technologies technicians