How to Install and Use HPLC Column?

How to pack an HPLC column

1. The HPLC chromatographic column should be directly connected to the homogenization tank, if not, an adapter is required. The size of the adapter is very important. It should not only prevent liquid leakage but also avoid damage to the thread of the column interface due to excessive rigidity or improper size;

2. The specific column loading process is generally divided into the following 5 steps.
①First use a suitable homogenate (mostly organic solvents) to fully disperse the filler (It can be stirred, ultrasonic, etc.);
②In the process of packing homogenization, complete the preparation work, that is, connect the HPLC chromatographic column to the lower end of the homogenization tank (the section of the chromatographic column that is not connected to the homogenization tank should be connected to the sieve plate and column head to ensure tightness but not screw down to avoid damaging the chromatographic column), open the joint between the upper end of the homogenization tank and the air pump for filling in the homogenate;
③After the homogenization tank is completed, connect the upper end of the homogenization tank and start pressurizing;
④Choose the right pressure, you can compact the packing;
⑤After the pressure is stabilized at the appropriate pressure for a certain period of time, release the pressure, remove the chromatographic column, level the packing in the section where the homogenization tank is connected, and install the sieve plate and column head for ready use.

3. Packing a column is a difficult process. It requires patience to explore parameters such as pressure and homogenate volume to ensure that the column bed is uniform and compact and achieves good column efficiency and peak shape. If the installation is not good, there may be problems such as insufficient column efficiency or tailing.

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HPLC Column use

1. First, confirm whether the pH range of the mobile phase of the sample you want to analyze is within the pH range of your column, so as not to damage the column silica gel.
2. The next step is to equilibrate the column with the mobile phase you used as the sample. If the mobile phase contains a buffer salt solution, be sure to use 5% methanol (acetonitrile)/water to transition 10 times the column volume before equilibrating the column. And then use the mobile phase with salt to equilibrate the column for enough time (until the baseline is very stable), then the sample can be injected for analysis.
3. No matter what kind of sample you analyze, there will always be some impurities in the sample due to various reasons. Therefore, it is recommended that you add a guard column to the front of the column to protect your expensive analytical column. Or use a 0.2um or 0.45um syringe filter to filter the sample before injecting analysis.