How to take good care of HPLC column? Protection and regener

The strong retention substances in the sample gradually accumulated in the chromatographic column, which caused the pollution of the high performance liquid chromatography column, and the serious situation directly led to the scrapping of the chromatographic column. In order to prolong the service life of chromatographic column, the commonly used methods are column protection and regeneration.
High performance liquid chromatography column in the chromatographic analysis system mainly plays the role of separation and detection of substances, like the heart of the chromatographic system, but also vulnerable consumer goods.
When the chromatographic column adsorbs the sample, the salt, lipid, hydrophobic protein and other biological substances in the sample may interact with the chromatographic column. Some substances with smaller retention value, such as salts, are more likely to be washed out of the column, while the mobile phase solution of some components with strong retention is not enough to elute these substances. After several times of sample loading, these substances are adsorbed on the column Material on the surface of the column usually accumulates in the stigma.
When the components of the adsorbed samples accumulate to a certain extent to form a new stationary phase and change the separation mechanism, the retention time of the samples will fluctuate and the phenomenon of tailing will appear.
Chromatographic column structure: 1-plastic protection plug; 2-column head screw; 3-edge ring (ferrule); 4-ptfe O-ring; 5-porous stainless steel sintered sheet; 6-chromatographic column tube; 7-liquid chromatography stationary phase
Column protection
In order to prolong the service life of chromatographic column, it has been pointed out that a small volume protective column should be connected before the analytical column. The protection column is a short filled column with inner diameter of 1.0 mm, 2.1 mm, 3.2 mm, 4.6 mm or 10 mm, 20 mm, 40 mm, and 7.5 mm, 10 mm or 20-60 mm long. It is usually filled with the same filler (stationary phase) as the analysis column, which can be regarded as the shortened form of the analysis column and installed in front of the analysis column.
Its function is to collect and block the mechanical and chemical impurities from the injector to protect and extend the service life of the analytical column. A 1 cm long protective column can provide sufficient protection. If a longer protective column is selected, the chance of pollutants entering the analytical column can be reduced, but the spectrum band will be expanded. Therefore, the principle of selecting the protective column is to select the short protective column with low retention for the separated samples on the premise of meeting the separation requirements.
The protective column can also be filled with different fillers, such as silica gel (10-15um) or polymer filler with coarse particles, but the volume of the column should not be too large to reduce the influence of the external effect.
The packing of the protective column is less and the price is low. It is a consumable. It can analyze 50-100 samples. The pressure drop of the column tends to increase, which is the signal that the protective column needs to be replaced.
The protective column with novel structure and replaceable core design is now available in the market, which is composed of protective column sleeve and replaceable protective column core.
Connection diagram of protection column and analysis column
1-protection column sleeve; 2-protection column core; 3-peek standard universal joint; 4-analysis column joint; 5-connection six way injection valve joint
The pH value of the mobile phase should be kept between 2.5 and 7.0 for the bonded stationary phase based on silica gel. The mobile phase with an extreme value will dissolve the silica gel and cause the loss of the bonded phase.
When using water-soluble mobile phase, 0.01% sodium azide should be added to prevent the column blocking caused by microbial reproduction to inhibit microbial reproduction. For the unclean samples, 0.45um filter membrane should be used or purified by solid-phase extractor before injection.
Before the formal analysis, the chromatographic column should be flushed with a mobile phase of 10 times the volume of the column to keep the column in equilibrium. When ethanol, isopropanol, acetic acid and other high viscosity mobile phases are used, the equilibrium time of chromatographic column will be prolonged or even doubled.
For the reversed phase C18 bonded phase column, pure water should not be used to remove the buffer solution salts or water-soluble substances contained in the column. Because the C18 bond phase is like a long chain that binds the silica gel, when there is an organic solvent, its surface is wetted by the mobile phase, and the C18 long chain is completely expanded, like seaweed in seawater. When pure water or buffer solution passes through, the surface of C18 bonded phase will collapse and the solvent, sample molecules or inorganic salt molecules will be entrapped. At this time, the inclusion compound can not be washed out with pure water, and the inorganic salt or water-soluble substance can be removed only by washing with aqueous solution containing 5% organic solvent.
Silica gel, alumina or normal phase bonded phase columns should be kept in the mobile phase. The cyano column can not be stored in pure organic solvents (such as methanol or acetonitrile), but should be kept in the mobile phase to be used; the amino column should be kept in acetonitrile rather than the mobile phase, and it should be noted that acetone should not be used as the mobile phase. The C18 reversed phase column should be kept in pure methanol solvent, and the nonpolar stationary phase filled with styrene divinylbenzene copolymer microspheres with high crosslinking degree can also be preserved by this method.
Regeneration of chromatographic column
For the chromatographic column which has been used for a long time and the column efficiency has gradually decreased, the following methods can be used for rapid regeneration to remove the accumulation of trace impurities in the column head.
Normal phase column: wash the chromatographic column with the following organic solvents with strong polarity and mutual solubility. Each solvent is cleaned with 30ml each time. Wash the column in the order of heptane, chloroform, ethyl acetate, acetone (amino column is not used), methanol and 5% methanol aqueous solution (the polarity of eluent increases in turn). Finally, the water is carried out by pure methanol through the column, and the column is removed and placed in the gas chromatograph column box to raise the temperature to 75 To remove moisture. In addition, it should be noted that ethanol should not be used because it will make the column lose its column efficiency.
Reverse phase column: use 30 ml of polar solvent, and clean in the following order: 5% methanol aqueous solution, 0.5 mol / L H3PO4 (or 0.1 mol / L sodium EDTA), 5% methanol aqueous solution, methanol, methanol chloroform (1:1) mixture, methanol, 5% methanol aqueous solution. Finally, it was stored in methanol solvent.
When the performance of chromatographic column becomes very poor, the following two methods can be used as the final remedy.
One method is to repair the column head and replace the stainless steel sintering plate at the same time. When the column head collapses or the filler is contaminated by impurities, the fixed phase of about 0.5mm can be dug out, and then the same dry stationary phase can be refilled, wetted with a small amount of mobile phase, and then the dry stationary phase shall be filled flush with the column mouth (if necessary, it shall be pressed with smooth stainless steel rod), so as to repeat for 3-5 times.
Then replace with a new stainless steel sintered filter, tighten the junction head, connect with the high-pressure pump, wash with mobile phase for 20min, if the column pressure drop returns to normal, connect the detector to measure the column efficiency. The column can be used again. If the column pressure drop returns to normal, but the column efficiency is still low, it indicates that the column head is not filled tightly and there is still a gap. At this time, the above operation should be repeated until the column pressure drop returns to normal and the column efficiency returns to normal.
Another method is to back flush the column. This method is suitable for the chromatographic column which has been repaired and the column life has reached the middle and late stage. When the column is used in reverse direction, the column efficiency will be greatly lost (about 30%). The back flush column can be used to check whether the stainless steel sintering plate at the head of the column is blocked. If it is blocked in the back flush column, the column pressure drop can be observed to decrease. After the column pressure drop returns to normal, the chromatographic column can be installed in the original direction.
Precautions for use and maintenance of columns
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 sharp changes in pressure and temperature and any mechanical vibration. 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. The mobile phase must be degassed and filtered before use. 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 precolumn can be connected in front of the injector. When the analytical column is bonded silica gel, the precolumn 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, and the pressure rise is the signal that the precolumn needs to be replaced. 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. 6. Wash the chromatographic column with strong solvent to remove the impurities retained in the column. The volume of each mobile phase should be about 20 times of the column volume, that is, 50-75ml for routine analysis.
7. Avoid using high viscosity solvent as mobile phase; if polar or ionic buffer solution is used as mobile phase, column should be washed clean after experiment. 8. The sample should be purified; 9. The injection volume should be strictly controlled without overloading. 10. After the analysis and determination every day, the column should be cleaned with appropriate solvent, preferably 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. 11. The pH stability range of most RP-HPLC columns is 2-7.5, which should not exceed the pH range of the column. 12. Keep the chromatographic column according to the instructions. It is absolutely forbidden to leave the buffer solution in the column overnight. Pay attention to the reversed-phase column and do not wash it with pure water for a long time. 13. It is not recommended to dismantle the column and replace the filler by yourself
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