Daily Use, Maintenance, and Repair of the C18 HPLC Column

Daily use and maintenance of the C18 HPLC column

In the daily separation and analysis work, whether the column is used properly or not directly affects the life of the column. The following are the matters that should be noted in the daily use of the C18 HPLC column.

(1) When loading and unloading, replacing the column, we should act lightly, and tighten the joint moderately. Stronger mechanical vibration must be prevented so as not to create voids in the column bed.
(2) If the instrument is used for routine analysis with a limited variety of samples but a large number of analyses, it is advisable to configure one column for each type of routine analysis.
(3) If a column temperature control device is used, care should be taken to ramp up the temperature only after the mobile phase is passed through.
(4) Before use, degas the mobile phase to avoid column efficiency reduction and affecting detection, and so on. The sample solution needs to be properly pretreated and filtered to reduce column contamination and blockage.
(5) When replacing the mobile phase type, attention should be paid to the mutual solubility of the solvent to prevent the occurrence of salt precipitation.
(6) The actual operating pressure of the column should be lower than the pressure at the time of filling, and it is recommended to be below half of the upper limit pressure. Generally not more than 20593.965 ~ 29419.95 kPa. If used in the range of low pressure (≤ 14 709.975 kPa), it can make the column maintain a long period of high column efficiency.
(7) C18 column is a non-polar bonded phase chromatography column, the pH of the mobile phase should be strictly controlled between 2~7 to avoid damage to the column.
(8) After finishing the separation and analysis work, it should not be stopped immediately, and the chromatographic analysis system needs to be flushed in time, generally for 0.5 h or more, to remove the impurities in the column.
(9) If there are salts in the mobile phase, first wash them with water fully. If amines are added to the mobile phase, rinse with a solvent mixture of 50% methanol and 0.05% phosphoric acid solution, not just with water.
(10) C18 HPLC column generally uses 100% methanol as the preservation solvent to prevent the column from drying out and damage.
(11) Choose a suitable C18 protective column to protect the analytical column from impurity particles and irreversibly adsorbed interferents. The granularity of the packing material inside the protective column should be as consistent as possible with the granularity of the analytical column packing.
(12) The storage period of the column should not be too long. If the C18 column is not used for a short period of time, it is flushed with methanol for 30-60 min, and then the column is sealed at both ends. If the column is not used for a long time, adopt the method of regular flushing and then sealing; after flushing, each end of the column is filled with a certain capacity of methanol (only one end is also acceptable) to supplement the evaporation of the solvent in the column during a longer storage period.

Repair of the C18 HPLC column

During the daily use of chromatographic columns, it is still difficult to avoid contamination of the column, loss of stationary phase, slabbing, the collapse of the column bed, and decrease of column efficiency after a long period of time, despite strict protection and sample and mobile phase pre-treatment. Some of these can be repaired to partially restore column efficiency.

1. Column contamination regeneration techniques
After the chromatographic column is contaminated, the column efficiency can be regenerated by washing with suitable solvents. the conventional regeneration washing method for the C18 HPLC column is to pass 60mL each of methanol, trichloromethane, and methanol/water through the column in turn, and then seal the column after equilibrating it with 60mL of 100% methanol, and the column efficiency will return to normal. If necessary, depending on the nature of column contamination (e.g. organic contamination, salt contamination, etc.), 0.05 mol/L H2SO4, 0.5 mol/L H3PO4, or 0.1 mol/L EDTA sodium salt is used to rinse the column, then rinse with water, and then equilibrate the column with 100% methanol and seal it. For the seriously contaminated C18 column, water, methanol, and ethane can be used to rinse in turn, then rinse again in reverse order, using 60 ml of solvent each time, without connecting the detector, and then seal the column with 100% methanol equilibration.

2. The repair of column contamination
If the column contamination regeneration is ineffective or known column contamination is serious, the column repair method can be used to solve it, but the depth of column contamination should not exceed 5 mm. The method is to dig out the contaminated part of the special small shovel, and then use the same stationary phase with the column packing and mobile phase mixture made of slurry, and then the slurry stationary phase carefully fill into the dug-out part (as far as possible after filling the stationary phase close to the original tightness), repair the end face can be. The end face can be flattened. If the aperture of the screen plate at both ends of the repaired column is the same, the column can be turned upside down and used for a general time, with the purpose of restoring the tightness of the column bed with the help of the mobile phase flushing effect.

3. Repair of column collapse
Column collapse can be caused by many reasons. For the collapse is not too serious, if the aperture of the sieve plate at both ends of the column head is the same, the column can be turned upside down and used for a general time to restore the performance. When the collapse is serious (about 5 mm), the repair method can be used to repair the column pollution.