How to extend the life of the column

Typically, a column will perform well after analyzing thousands of samples, but some columns will be almost scrapped after analyzing only a small number of samples. There are many factors that affect column life and other problems, and some factors are difficult for the operator to control. If the sample being analyzed (such as analyzing biological samples), how to purify the sample is also “dirty” and the effect on the column is very big. However, in the following cases, in most cases, it is always possible to artificially reduce the failure on the column to achieve the purpose of extending the life.

Column prevention and maintenance - avoid high pressure shock

Generally, the column can withstand high pressure, but cannot withstand sudden changes in high pressure shock. Sudden impact of high pressure, mainly due to the slow rotation of the sample valve, the pump start fast, the column switching operation. The flow from the pump to the column is momentarily cut off when the six-way injection valve is turned. The pressure on the pump side of the valve rises and the pressure on the column side of the valve decreases (more than 20% change). After the valve is turned to the bottom, the pressure suddenly hits and returns to normal. The manual injection valve does not change much. The automatic injection valve is slow, which may cause pressure shock. The helium gas can be used instead of air to drive the injection valve. Due to the small nitrogen compression coefficient. In addition, the pump should not be started too fast and can be operated step by step. If using a flow rate of 3 mL/min, first from 1 mL/min to 2 mL/min, then 3 mL/min, each interval should be greater than 20 s. Column switching technology is also widely used, and the pressure at the inlet of the column changes between zero and a large number during the switching process, which quickly causes the column to be scrapped.

Column Precautions and Maintenance - Separation Conditions

Most columns have a wide range of test conditions, but specific applications are limited, mainly in pH, column temperature, and mobile phase. The bonding phase of the silica gel matrix requires a pH between 2.5 and 7. The mobile phase of extreme pH can "dissolve" the silica gel and lose the bonded phase. As a result, the retention of the non-alkaline component is continuously reduced, and the retention of the alkaline component is increased, causing the peak of the alkaline component to broaden. If a high or low pH mobile phase must be used, a precolumn (saturated column) can be added. The pre-column is placed between the pump and the injection valve, filled with the same packing as the analytical column, or with ordinary silica gel. Silica gel saturates the mobile phase, reducing the loss of analytical column packing. The pre-column does not require high column efficiency, and is loosely packed with ordinary silica gel at a low price, and the dissolution of the silica gel is checked on schedule. The use of pre-columns also has an adverse effect, that is, the new mobile phase is difficult to balance, and the retention time is unstable or stable. Always use a flow filter to use the pre-column to prevent the trouble caused by silica gel particles. When the silica-based column and the anion exchange column exceed 60 ° C, the adsorption of chemicals in the mobile phase is increased. Using a small particle column at high temperatures causes the bed to collapse, reducing column efficiency and changing peak shape. A 3 μm column was used at 40 ° C or higher, and a 5 μm column was used at 70 ° C or higher, which lowered the value by 50%.

Column Precautions and Maintenance - Flow Filters and Guard Columns

The flow path filter is located immediately behind the injection valve and is located in front of the analytical column. The 0.5 μm sintered stainless steel sheet is clamped in a sleeve with a small dead volume, blocking the particles from the sample and the injection valve gasket into the column. Because filtering each sample is both laborious and error-prone, it is more difficult to filter less sample volume, so it is easier to install filters on the flow path. It is also possible to add a guard column to the flow path, place it between the flow path filter and the analytical column, or replace the flow path filter. The mission of the guard column is to collect chemical "garbage" from the sample that blocks the inlet of the column. This waste ultimately reduces column efficiency. The guard column is a consumable. After analyzing 50 to 100 dirty samples, it is necessary to replace it with a new one. The guard column should be small in size and filled with the same packing of the analytical column. The guard column is used properly and has no effect on the separation, as if the guard column was not installed. Some manufacturers' products connect the guard column and the flow path filter in one unit, which is very convenient to use. Filling the guard column by yourself is a short column, not more than 3cm long, and the inner diameter is 2~3mm. It is filled with a larger particle size (15~20μm), which will slightly reduce the analytical column efficiency.

Column Precautions and Maintenance—Rain the column regularly with a strong solvent

It is a good practice to rinse the column with strong solvent at the end of each work. The reversed-phase column can be washed with methanol and acetonitrile to wash away the strongly adsorbed components remaining on the column. Wash with methanol water as the mobile phase. The flushing procedure is described in the following sections. Sintered stainless steel filter on the stigma requires flatness, small dead volume and appropriate pore size (2~5μm). Poor selection of the filter will change the peak shape of the chromatogram, increasing the resistance or not blocking the contaminant (the filler will change color quickly).

Column Precautions and Maintenance - Purification of Samples

With the solvent-dissolved sample, most of the components can completely flow out of the column for a certain period of time without causing harm. Some samples may contain particulate matter. Some components (such as proteins) in the sample are deposited on the column head. The components remain strongly on the stationary phase. The solvent carries the column packing, etc., which will cause the column efficiency to drop or the column. For the impact of life, it is necessary to take measures to prevent the column from getting bad.

If the sample is turbid or milky white, it must be filtered before injection. Although the filter and guard column are installed on the flow path, it is not a substitute for the pretreatment of the sample. Excessive particles in the sample will overload the filter and the guard column, block quickly, or the particles enter the analytical column, so it must be injected before the injection. Filter the sample.

If you suspect that the sample is mixed with the mobile phase and there is a blockage on the column, you should first test it to see if the sample solution is added to the mobile phase with or without turbidity. If the pressure suddenly increases after injection and then slowly decreases, it indicates that there are particles or precipitation in the sample. If there is a precipitate, try to change the separation conditions, including changing the sample solvent and mobile phase or treating the sample to remove insoluble materials. A small volume of sample should be used as much as possible.

Some samples can be strongly adsorbed on the column packing, which will reduce the number of plates, change the retained material of the sample, and add a guard column to clean the column regularly. Sometimes, due to negligence, the sample is dissolved in a solvent harmful to the column, such as 6 mol/L sodium hydroxide. If the sample is 50 to 100 μL into the silica matrix column, the silica gel dissolves quickly and the column is scrapped. In this case, the sample should be neutralized immediately or the harmful components in the original solvent should be removed.

In summary, how to protect good column performance and column life:

1. Carefully read the instruction manual of the column;

2. Use a well-filled column;

3. Minimize pressure fluctuations and avoid mechanical and thermal shocks;

4. Use guard column and online filter;

5. Rinse the column often with a strong solvent;

6. Fully filter the sample and mobile phase to avoid impurity particles and strong retention components;

7. Use a stable stationary phase (C18 is the most stable);

8. At medium pH operation (6-8), use organic buffer solution;

9. The column temperature is preferably less than 40 ° C;

10. The silica gel matrix column should maintain the pH of the mobile phase in the range of 3.0 to 8.0;

11. Add 200 ppm of sodium azide to the water mobile phase and the buffer solution;

12. The mobile phase contains a buffer solution, which should be filtered with 95:5 water and organic solvent, the organic solvent should not be less than 5%;

13. At night or during storage, rinse off the salt and buffer and store in a pure organic solvent mobile phase.