Precautions for Use and Maintenance of HPLC Columns
The experimenters need to use and maintain chromatographic columns correctly. The improper use of chromatographic columns will cause the columns to decrease or lose effectiveness. They should focus on many things to protect the columns in the use process. Here we talk about several attentions.
a. Precautions for Use of HPLC Columns
Avoid changing pressure and temperature dramatically and any mechanical vibration.
Sudden temperature changes affect the filling conditions in the columns. Sudden changes in column pressure may loosen the fillers. Consequently, people should adjust the flow rate of the fillers slowly to protect the fillers.
Change the composition of the solvents gradually.
The experimenters should change the composition of the solvents gradually, especially in reversed-phase chromatography.
Do not backwash the columns.
Generally speaking, chromatographic columns cannot be backwashed. But, if the manufacturer specifies the columns can be backwashed, the experimenters can remove the impurities through backwashing.
Choose a suitable mobile phase.
A suitable mobile phase is chosen to avoid destruction by the stationary phase. Sometimes it is a better way to install a pre-column that has the same substrate and little impact on the reaction, where the mobile phase will be pre-reacted before entering the analysis column.
One of the main factors affecting the life of the liquid chromatographic column is the mobile phase. There are three aspects of the mobile phase factors.The pH value of the Mobile Phase
The fillers of different substrates apply to different pH value ranges. Under normal temperatures, the solubility of amorphous silica gel in pure water is about 100mg•L-1, and the dissolved amount of silica gel in the pH range of 1 to 9 is almost constant. In consideration of the influence of dissolution rate, the most suitable pH range of the mobile phase for the silica gel substrate is 1 to 8. However, the silica gel fillers of the bonded phase filler greatly increase the adaptability of the fillers to the mobile phase because of the shielding effects. The fillers with the improper pH value will quickly reduce the column efficiency and scrap it.
The Deterioration of Mobile Phase
The most widely used filler is the silicon-based bonded phase filler nowadays. When the mobile phase is an aqueous solution or contains a certain concentration of phosphate buffer, some bacteria growing in the solutions will block the gap between the stationary phase particles.
a. The solvent must be HPLC grade, and the reagent should be as high-purity as possible;
b. Does not react chemically with the stationary phase, has low viscosity, and has suitable solubility for the sample. It is required that k is in the range of 1 to 10 (available range) or 2 to 5 (optimum range), and the k value is too small, which is disadvantageous. Separation: The k value is too large, and may cause the sample to precipitate in the mobile phase.
c. Ensure that the solvents are mutually miscible;
d. The mobile phase must be matched to the detector. For example, when using a UV detector, a solvent with a cutoff wavelength greater than the detection wavelength cannot be used.The Purity of the Mobile Phase
A large number of mobile phases will pass through the column when the chromatographic columns are used. In that case, if the mobile phase contains a certain amount of insoluble impurity particles, the particles will be intercepted at the head of the column easily. The mechanical blockage of the columns caused by the particles results in an increase in column pressure, which means that the columns can not be used.
The stationary phase of HPLC Columns
a. The pH of the mobile phase should be maintained between 2.0-8.0 (unless otherwise specified).
b. Pre-saturated HPLC columns and guard columns can be used if necessary.
c. When using amino HPLC columns, avoid aldehydes and ketones in the mobile phase and sample.
b. Precautions for Maintenance of HPLC Columns
- Try to protect the HPLC column with a pre-column or guard column.
- The pH range of most HPLC columns is 2-8; try not to exceed the range.
- Avoid drastic changes in the composition or proportion of the mobile phase;
- The mobile phase must be filtered and degassed before use.
- If a polar or particulate buffer is used as the mobile phase, the column should be rinsed clean after the experiment.
C18 columns cleaning regeneration
Wash with the following 10 column volumes of solvent: (flow rate during forward flush: 0.5 ml/min)
95% water: 5% acetonitrile (or methanol)
(removal of buffer salts)
If the system does not have a gradient function, add 5 times column volume to a 50% water: 50% acetonitrile (or methanol) rinse step.
95% acetonitrile (or methanol): 5% water
Finally stored in 35% water and 65% acetonitrile (or methanol).
Regeneration of reversed-phase columns
Sequentially use 20-30 times the column volume of methanol: water = 10:90 (V / V), nitrile, and isopropanol as a mobile phase to wash the column, and then rinse the column in reverse order.
c. Precautions for Storage of HPLC Columns
- After the new HPLC columns are used for some time, the top packing may collapse, and the efficiency of the column may be reduced. At this time, the filler may be added to restore the efficiency of the column.
- After each work, rinse with a highly eluting eluent. When using a salt buffer solution as the mobile phase, rinse with a salt-free mobile phase after use. Compounds containing halogen elements (fluorine, chlorine, bromine) may corrode stainless steel pipes and should not be in contact with them for long periods.
- If the HPLC columns are installed on the HPLC instrument, they should be rinsed for 15 minutes every 4 to 5 days if it is not used frequently.
- When storing the HPLC columns, the column should be filled with acetonitrile or methanol, and the column joint should be tightened to prevent the solvent from evaporating and drying. Do not leave the buffer solution in the column for an overnight or longer period.
Finally, generally speaking, the columns always have problems at the end of them. If we can install a short column with the same stationary phase as the analysis column in front of the analysis column, which is about five to thirty millimeters, the column will have a longer lifetime. Although the effectiveness of a short protective column is less, the final result is worthwhile.
The chromatographic columns’ lifetime usually can reach two years or more when used correctly. One kind of column that is based on silica gel can only be used in pH values ranging from two to nine. Some adsorptive substances will be retained on the top of the column, especially some colored substances, after being used for a long time. For the new columns, the filler at the top of the columns may decrease and collapse, which reduces the column efficiency. And under these circumstances, the experimenters can restore the efficiency of the columns by adding fillers.
It is better to rinse the columns with eluent with strong eluting power after each work. When a hydrochloric acid buffer solution is used as the mobile phase, a salt-free mobile phase is used to rinse. The compounds containing the halogen family may corrode stainless pipes. It is inappropriate to be exposed to them for a long time.
