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LC column maintenance and maintenance

Time:2018/10/24   Pageviews:0    Share:
 This is an article about LC column maintenance and maintenance. If you are interested, please contact us!

With enough time to maintain the column every day, you'll save more time to handle column failures, and your column life will be longer! The service life of the column is not only related to the sample and mobile phase and frequency of use analyzed, but Zui is mainly related to daily maintenance. The lifetime of a column is primarily measured by both column efficiency and column pressure. If a column is too low or the column pressure is too high, it is generally considered that the column has reached the end of its useful life. Therefore, the key to extending the life of the column is to eliminate the factors that cause the column to fall and the column pressure to rise.11mm snap chromatography vials

First, the pH of the mobile phase

Due to the presence of Si-C and Si-O bonds in the filler of the silica matrix, the mobile phase exceeds its pH range, which will cause the silica matrix to dissolve and the carbon phase of the bonded phase to be broken, resulting in a decrease in column efficiency and a shortened life. Due to damage to the column caused by improper pH control of the mobile phase, it is often difficult to recover the column, so care must be taken to strictly control the pH of the mobile phase. .

Second, remove the solid particles in the sample and mobile phase

The solid particulate matter contained in the sample and mobile phase will block the chromatographic sieve plate. Blocking the sieve plate will not only cause the column pressure to rise, but also cause the column efficiency to drop, because the blockage of the sieve plate will cause uneven flow, resulting in uneven flow. The peak shape of the chromatogram is tailed, widened, and even double peaks, resulting in a decrease in column efficiency. Therefore, it is recommended to use ultrapure water and chromatographic pure reagents, ultrasonic and syringe filtration of the sample before analysis, mobile phase over 0.45μm filter, especially the mobile phase added buffer salt (such as NaH2PO4, etc.), must be filtered.

Third, use the guard column or online filter
After filtration of the sample and mobile phase, the solid particulate matter cannot be completely eliminated, because the wear of the pump, the aging of the seal ring and the pipeline will also produce solid particles, which are carried by the mobile phase into the column, blocking the sieve plate and causing the column. The pressure rises and the efficiency of the column decreases. There are sieve plates on the guard column and the in-line filter, and the pore size is the same as that of the column sieve plate, so that solid particles can be prevented from reaching the column and the blockage of the column plate can be prevented. Since the increase in column pressure is a large percentage of analytical failures, it is recommended that you add a guard column or inline filter to the column injection end, in addition to filtering the sample and mobile phase.

Fourth, the correct use of buffer salts

The buffer provides ion-balanced counterions and maintains the mobile phase with a certain ionic strength and p H value, reducing tailing. The buffer salt is usually soluble in water and is insoluble in organic solvents. Therefore, improper use of the buffer salt causes precipitation, blocking pores between the pores and particles in the filler matrix, causing the filler to be kneaded, and the column pressure is increased; The bonded carbon chain is free to stretch, which reduces the retention capacity of the column and reduces the efficiency of the column. After the buffer salt is precipitated, removal is very difficult, so proper use of buffer salts is very important to extend the life of the column.

The correct use of buffer salts is to prevent the precipitation of buffer salts, so the correct use of buffer salts can be attributed to one sentence: to transition before use, rinse after use.

The specific method is as follows:11mm snap chromatography vials

1. Isocratic conditions: Before using buffered salt, flush the 20-30 column volume with a transient mobile phase at a flow rate of 1.0 mL/min, and then use a mobile phase containing buffer salts; the method of removing the buffer salt after use is to use an excessive mobile phase. The column volume was washed 30 times at a flow rate of 1.0 mL/min, and then stored in a pure organic solution for 30 column volumes.

2. Gradient conditions: Before the gradient elution with the mobile phase containing the buffer salt, rinse the 30-fold column volume with the same transient mobile phase with the initial mobile phase composition at a flow rate of 1.0 mL/min, and then start the gradient operation; Then, the column was washed with a transition mobile phase at 1.0 mL/min for 30 column volumes, and then washed with a pure organic solution for 30 column volumes.

5. In addition, the following points should be noted in the process of using buffer:

1. Avoid the use of hydrochloride, which has a corrosive effect on steel;

2. Buffer zui should be used now, often the buffer is a good fungus culture solution, many strange phenomena will occur when the experiment is placed every other day or for a long time;

3. Use buffer to master the pH range in time, so that there are counts in the chest;

4. When cleaning the liquid path and the column, the temperature control can be heated to 30 degrees Celsius and easy to rinse;

5. Use buffer solution for a long time to observe whether there is any precipitation at the joint. If white salt is precipitated, periodically rinse the liquid with 10% nitric acid (remove the column, rinse 30mL 10% nitric acid solution, then rinse with 5 times water) Avoid blockage of the liquid path;

6. Select a buffer to use reliable reagents to avoid unnecessary trouble caused by impure salt;

The transition mobile phase refers to a solution in which the organic phase and the aqueous phase have the same composition as the analytical mobile phase, but do not contain a buffer salt.

6. Prevent strong retained substances from remaining in the column

Strongly retained substances and macromolecular compounds accumulate in the column, causing additional retention behavior of the compounds in the sample, which not only causes the peak shape to widen and smear, but also causes the column efficiency to decrease, and also causes the retention time to change. The degree will also cause the column pressure to rise. Since the influence of strongly retained substances and macromolecular compounds on chromatographic separation is a cumulative effect, it takes a certain amount of time to be reflected, but for many samples, especially complex samples, it is difficult to judge whether they contain strong retained substances, 11mm snap chromatography vials so To prevent the accumulation of strongly retained materials, it is necessary to clean the column with 30 volumes of pure methanol or acetonitrile in daily maintenance.

Seven, the regeneration of the column

Reversed phase column

Rinse 30 column volumes in the following solvent sequence

Acetonitrile-water (5:95) → tetrahydrofuran → acetonitrile-water (5:95)

2. Normal phase column

Rinse 30 column volumes in the following solvent sequence

N-hexane → dichloromethane → isopropanol → dichloromethane → mobile phase

3. Silica column (Silica) water removal

Rinse the column with 30 mL of 2.5% 2,2 dimethoxypropane and 2.5% glacial acetic acid in n-hexane 11mm snap chromatography vials


This is the end of the introduction of LC column maintenance and maintenance. I hope it can help you.

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