The poor service life of chromatographic columns has been bothering many partners in the laboratory. In view of the fact that the service life of chromatographic columns of various brands is lower than their common service life, many customers have said that their glass hearts are unable to withstand the shock of dying new chromatographic columns. In this regard, Welch Materials has carefully studied the “Immortality” technique of chromatographic column for you to solve the problem of unsatisfactory service life of chromatographic column. Let’s follow the footsteps and look down.
- Whether to use the same packing guard column (same brand);
- The mobile phase should be used within 12 hours from configuration to disposal;
- The mobile phase bottle is strictly cleaned, dried and sealed;
- Whether the material and quality of the filter head and filter membrane are wrong, pay attention to check whether there is dissolution after use;
- Flush the column using the flushing method recommended by the manufacturer.
If the above self-inspection items cannot be solved after improvement, the second step is to take the second step: communicate with the manufacturer, and suggest sending it back to the manufacturer for “dissection” of the chromatographic column. The possible reasons are summarized as follows:
Reason 1: Column contamination
- The sample components are adsorbed on the front end of the chromatographic column
a. It may be precipitated due to the low solubility of the sample in the mobile phase;
b. There are strong retained substances in the sample (substances with similar polarity to the bonded phase);
c. A weak and unpredictable chemical reaction occurs with the surface of the filler in the sample or mobile phase;
d. The pH value of the sample solvent itself is near the tolerance critical value of the chromatographic column, and the injection volume is large (inner diameter is 4.6 mm, and the injection volume is above 50 μL).
Usually, such a situation is accompanied by an increase in the column pressure; or when the column pressure is normal, there are some abnormal peak shapes in a chromatogram, and some peak shapes are normal.
a. Try to solve the problem from the root cause, select the appropriate sample pretreatment technology, remove the strong retained substances, and obtain a more “clean” and single-component sample;
b. Use a guard column. The choice of the guard column should be the same as the analytical column, and the guard column with the same bonding method. If you use a guard column with a different brand of packing material and a different packing process, it may not be the best in terms of separation capacity and protection. Do not use a larger particle size guard column, because a larger particle size or a poorly packed guard column will reduce the separation ability due to its peak broadening effect.
- The mobile phase is “dirty”
The amount of mobile phase used in the chromatographic column is much larger than that of the sample, and the chromatographic column is constantly flushed. If the mobile phase is deteriorated or there are other contaminants, the contamination of the chromatographic column will be greater than that of the sample, and the chromatographic column will affect the flow rate. Instead it’s a cleanup, and when running gradients, ghost peaks also appear.
a. Use the mobile phase as soon as possible after configuration, and no more than 12 hours at most;
b. The mobile phase mixed with the organic phase and the salt phase is more likely to lead to the precipitation of salt. Try to use online mixing, and the change rate should not be too fast when running the gradient to avoid salt precipitation;
c. Use professional passivated solvent bottles to pack the mobile phase, do not use plastic bottles, seal them well, and replace the reagent bottles when replacing the mobile phase;
d. The water will deteriorate after it comes into contact with the air or leaves the water making equipment. Use it now and use it as soon as possible after opening.
Reason 2: The stationary phase is damaged
The damage of the stationary phase caused by the various effects of chromatographic conditions is a problem that cannot be solved by the current chromatographic technology. Under normal circumstances, the bonding phase of small groups is more likely to be hydrolyzed and fall off, which accelerates the falling off of the bonded phase. The exposed silica gel is exposed to the mobile phase, which increases the attack of salts on the silica skeleton, resulting in the thinning of the skeleton and the decrease in mechanical strength, which is manifested as column head collapse.
a. Salts in the mobile phase, especially phosphate buffer salts, have a strong attack on silica gel;
b, high column temperature, accelerate the rate of salt movement;
c. High oxygen, the oxygen that is not excluded in the mobile phase will accelerate the breaking of the internal bonds of the silica gel;
d. At high pH (>8), OH radical ions attack the Si-O-Si bond inside the silica gel, resulting in the direct fragmentation of the silica gel;
e. At low pH (<2), H ions attack the Si-C bond on the surface of the silica gel, and after the bonded phase falls off, the silica gel is exposed;
f. High water is also an important cause for the exposure of bonded phase hydrolyzed silica gel.
a. When using salt, the column temperature is less than or equal to 30℃;
b. The concentration of salt is used below 50mmol;
c. Use an online degasser or ultrasonic degassing (10 minutes, too long will heat up and cause the composition of the mobile phase to change);
d. Except for special markings, the pH value of the chromatographic column is between 2-8. If the pH of the sample is harsh, please use a guard column to resist the damage caused by pH, and replace it in time;
e. Do not use chromatographic columns that are not marked as being able to withstand pure water under the condition that the water phase is greater than 95% of the mobile phase.