Use and maintenance of chromatographic column

Chromatography is a kind of separation and analysis method, and separation is the core. Therefore, the chromatographic column responsible for separation is the heart of the chromatographic system. Today, let's learn more about the chromatographic column!
1、 Column structure
The column consists of column tube, pressure cap, ferrule (sealing ring), sieve plate (filter), joint, screw (head) and column packing.
2、 Column classification
Chromatographic columns can be divided into analytical column and preparative column according to their use, and their sizes and specifications are also different
① Conventional analytical column (constant column) with inner diameter of 2-5 mm (commonly used 4.6 mm, domestic 4 mm and 5 mm), column length of 10-30 cm;
② Narrow bore column (also known as thin tube diameter column, semi micro column), inner diameter 1 ~ 2 mm, column length 10 ~ 20 cm;
③ Capillary column (also known as microcolumn), inner diameter 0.2 ~ 0.5 mm;
④ Semi preparative column, inner diameter > 5 mm;
⑤ The internal diameter of the column was 20-40 mm and the length was 10-30 cm;
⑥ The inner diameter of the column can reach tens of centimeters. The inner diameter of the column is generally determined according to the length of the column, the particle size of the filler and the reduced flow rate in order to avoid the wall effect.
The chromatographic column was separated by stationary phase matrix such as silica gel
① Reversed phase chromatographic column: the packing of reversed phase chromatography is usually based on silica gel, and its surface is bonded with relatively weak polar functional groups. The commonly used reversed phase fillers are C18 (ODS), C8 (MOS), C4 (b), C6H5 (phenyl), etc;
② Normal phase chromatography: silica gel is usually used as stationary phase for normal chromatography, and other bonded phase fillers with polar functional groups, such as amine group (NH2, APS) and cyanogen group (CN, CPS);
③ Ion exchange chromatographic column: sulfonated cross-linked strong anion / cation bonded silica gel column, common specifications: strong anion column (sax), strong cation exchange column (SCX).
3、 Good habit of chromatographic experiment
① When a new chromatographic column is obtained, the column efficiency shall be measured first;
② Keep the chromatogram measured on the new column and record the conditions;
③ The column efficiency was tested regularly.
4、 Cleaning of chromatographic column
During the use of chromatographic column, if the pressure increases, one may be that the sintered filter is blocked, so the filter should be replaced or taken out for cleaning; the other may be that macromolecules enter the column to pollute the column head; if the column efficiency is reduced or the chromatographic peak is deformed, the column head may collapse and increase the volume.
In case of the latter two cases, carefully screw off the column joint, take out the column head filler with clean small steel 1 ~ 2 mm height (pay attention to clean the contaminated filler), and then level the column packing. Then fill the column with a stationary phase (the same as in the column) wetted with appropriate solvent, flatten, and then tighten the column joint. However, it is difficult to recover to the level of the new column.
A short column (5 ~ 30 mm) with the same fixed phase as the analytical column is installed in front of the analysis column, which can protect and prolong the column life. It is worth while to use protective column.
Generally, the life of chromatographic column can be more than 2 years when it is used correctly. The filler based on silica gel can only be used in the range of pH 2 ~ 9. After the column has been used for a period of time, some substances with strong adsorption effect may be retained on the top of the column, especially some colored materials can be easily seen to be absorbed on the filler at the top of the column. When a new chromatographic column is used for a period of time, the packing at the top of the column may collapse and the column efficiency will decrease. At this time, the packing can also be added to restore the column efficiency.
After each work, it is better to wash with eluent with strong elution capacity, for example, ODS column should be flushed with methanol to baseline equilibrium. When salt buffer solution is used as mobile phase, salt free mobile phase shall be used for washing after use. Compounds containing halogenated elements (fluorine, chlorine, bromine) may corrode stainless steel pipes and should not be exposed to them for a long time. If the column installed on the HPLC instrument is not used frequently, it should be started and washed for 15 minutes every 4-5 days.
5、 Storage of chromatographic column
① Treatment before storage: remove impurities, salts, etc. to prevent bacterial growth;
② Suitable storage solvent;
③ The column should be filled with acetonitrile or methanol, and the column joint should be tightened to prevent solvent evaporation and drying. It is absolutely forbidden to leave the buffer solution in the column overnight or longer;
④ Avoid mechanical vibration;
⑤ Pay attention to the storage temperature.
6、 Notes on chromatographic column
The correct use and maintenance of chromatographic column is very important. A little carelessness will reduce the column efficiency, shorten the service life and even damage it. In the process of chromatographic operation, attention should be paid to the following problems in order to maintain the chromatographic column.
① Avoid any mechanical vibration and sharp temperature changes. The sudden change of temperature or the falling of chromatographic column will affect the filling condition of the column; the sudden increase or decrease of column pressure will also affect the packing in the column, so it should be carried out slowly when adjusting the flow rate, and the rotation of the valve should not be too slow when the valve is injected.
② The composition of solvents should be changed gradually, especially in reversed phase chromatography. It should not be changed from organic solvents to water, and vice versa.
③ Generally speaking, the chromatographic column can not be recoiled. Only when the producer indicates that the column can be recoiled, the impurities left in the column head can be removed by backflushing. Otherwise, recoil will reduce column efficiency rapidly.
④ Choose the appropriate mobile phase (especially pH) to avoid the destruction of stationary phase. Sometimes a pre column can be connected in front of the injector. When the analytical column is bonded silica gel, the pre column is silica gel, which can make the mobile phase "saturated" by silica gel before entering the analysis column, so as to avoid the dissolution of silica gel matrix in the analysis column.
⑤ To avoid injecting complex matrix samples, especially biological samples directly into the column, it is necessary to pretreat the samples or connect a protective column between the injector and the chromatographic column. The protective column is usually a short column filled with similar stationary phase. Protective posts can and should be replaced frequently. The pre column is for the mobile phase to protect the chromatographic column, the protective column is for the sample to protect the column.
⑥ Wash the chromatographic column with strong solvent to remove the impurities retained in the column. During the cleaning process, the replacement of mobile phase in the convection system should be gradually transited with miscible solvents. The volume of each mobile phase should be about 20 times of the column volume, that is, 50 ~ 75ml is needed for routine analysis.
The following is a list of some chromatographic column cleaning solvents and sequence, as a reference:
If the mobile phase for analysis is water (buffer salt) / methanol system, water and methanol should be used for cleaning. If water (buffer salt) / acetonitrile system is used for analysis, it is recommended to clean with water and acetonitrile, because the viscosity coefficient of methanol is higher and the elution strength is weaker than that of acetonitrile.
In general, do not use methanol: water (50:50) to wash the chromatographic column, because the viscosity coefficient of methanol: water (50:50) is the highest, which is easy to cause the pressure to exceed the upper limit value and easy to damage the chromatographic column.
Generally, the column of 15 cm should be washed with more than 50 ml pure water (20 times the volume of the column), and the column of 25 cm should be washed with more than 80 ml pure water to remove salt, and then the column can be washed with methanol for preservation.
If the analysis condition is heated, it is recommended to close the column temperature box after cleaning, because the decrease of temperature leads to the increase of the viscosity of the mobile phase, which easily causes the pressure to exceed the upper limit.
Finally, the pump, detector, column incubator and automatic injector are set to standby state.
Remember: when you get a column that you have never used, you must read the instructions first!
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