Installation and Preparation Before Use HPLC Column
Installation of HPLC column
(1) First open the package to check the type, size, and storage solvent of the HPLC column. Also, please check the manufacture date.
(2) Unscrew the sealing plugs of the joints at both ends of the HPLC column and put them back into the packing box for spare.
(3) According to the mobile phase flow direction marked on the HPLC column tube, connect the inlet end of the chromatographic column to the outlet of the injection valve through a connecting tube (for example, if conditions permit, it is recommended to use an HPLC guard column in front of the column); the outlet of the HPLC column and the detector connection.
The connecting pipe is a stainless steel pipe with an outer diameter of 1.57 mm and an inner diameter of 0.1 to 0.3 mm. The connecting pipe’s both ends are equipped with hollow screws and pressure rings for sealing. Insert the connecting pipe as hard as possible after it passes through the hollow screw and the pressure ring.
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.
① First use a suitable homogenate (mostly organic solvents) to fully disperse the filler (It can be stirred, ultrasonic, etc.);
② In the process of packing homogenization, complete the preparation work, that is, connect the HPLC chromatographic column ( CN HPLC Column, NH2 HPLC 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;
③ After the homogenization tank is completed, connect the upper end of the homogenization tank and start pressurizing;
④ Choose the right pressure, you can compact the packing;
⑤ After 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.
Preparation before use
In addition to the structure and installation, we all should know the preparation before use well. Because of the complexity of biology, the higher the degree of sample purification, the longer the service life of the HPLC chromatographic column, so sample pretreatment is very important, for example, solvent selection, sample filtration, etc.
We can choose pure water for chromatography as the solvent, and use the solvent inlet filter to filter impurities, particles, etc., which can be more “pure” and will greatly help extend the service life of the HPLC chromatographic column. Before using a chromatographic column, other influencing factors should be considered.
For example, certain properties of the analyte can also affect the service life of the HPLC chromatographic column. Strong acids, strong alkaline substances, and protein-based biological macromolecules can interact with stationary phase fillers or form an irreversible adsorption layer, change the surface characteristics of the fillers, change the performance of the HPLC chromatographic column, and ultimately lead to separation failure.
Operating procedures and standards of HPLC Column
When the HPLC column is preserved, the plug should be tightened to avoid volatilization of the solvent in the HPLC column. Avoid violent shocks. Before the HPLC column is used, understand the preservation solvent after the previous use, and replace the transition with the appropriate solvent if necessary.
HPLC column tolerance pH range of silica gel matrix is generally 2-8. If the mobile phase needs to add acid or alkali, the pH value must be measured before the experiment, so as not to damage the HPLC column. When the sugar column is used, the flow rate is lower and the HPLC column temperature is higher.
In the experiment, the flow rate was corresponding to passing through the HPLC column at a small flow rate and gradually increased to the specified speed with the full expansion of the filler. Furthermore, the HPLC column should be stored in a solvent with a high organic phase ratio.
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.
