When your chromatographic column is abnormal, do you know the reasons and preventive measures?

Today, I bring you a column maintenance report. Let us take a look at the common contamination of reversed-phase liquid chromatography columns, find out the reasons, and learn a variety of feasible prevention solutions.

1.Packing materials contamination

Abnormal performance:

The column pressure becomes higher, the column efficiency becomes lower, the peak shape is abnormal, etc.

Cause Analysis:

  • The composition of traditional Chinese medicine samples is complex. If the pretreatment method is not appropriate, it is easy to cause strong retained components to accumulate at the inlet end of the chromatographic column, resulting in increased column pressure, decreased column efficiency, abnormal peak shape, or “” Ghost Peak”.
  • Accumulation of protein samples: biological samples are easy to accumulate at the head of the column, resulting in an increase in the column pressure and a decrease in the column efficiency.
  • Others: samples such as gelatinous and flocculent samples cannot be removed by filtration, and will also accumulate on the sieve plate and the column head end, causing the column pressure to rise.
  • If the mobile phase is used for more than 12 hours at a time, it will deteriorate, and the generated flocs will lead to the contamination of the chromatographic column.

Prevention program:

  • Samples with complex components such as traditional Chinese medicine: develop appropriate pretreatment methods, use SPE cartridges, extraction and other methods to reduce easily contaminated components in the sample solution.
  • Accumulation of protein samples: Rinse and maintain the chromatographic column regularly. It is recommended to use acetonitrile-water-trifluoroacetic acid (50:50:0.1) as the elution reagent, and wash at low flow rate overnight.
  • Others: Samples such as gelatinous and flocculent samples cannot be removed by filtration, thereby contaminating the column frit and column head. It is recommended to optimize the solution in sample processing or method.
  • Use a guard column, and when the capacity of the guard column decreases, replace it with a new guard column in time. Column pressure rise > 10%; column efficiency drop > 10%; resolution drop > 10%, which are all signals that the guard cartridge needs to be replaced.
  • For the chromatographic column for detecting complex samples, it is recommended to regularly perform the regeneration and maintenance of the chromatographic column according to the abnormal regeneration method of the chromatographic column manual, in order to regularly remove the strongly retained contaminants at the column head.
  • The mobile phase and the liquid storage bottle should be replaced within 12 hours and no more than 24 hours.

2.Packing materials compaction

Abnormal performance:

  • The column pressure is increased, the column efficiency is low, and the peak shape is tailed or extended.
  • The chromatographic column packing is hardened. After opening the chromatographic column, it will be found that the color of the packing remains unchanged, but the packing is agglomerated in flakes, blocks, and crystals.

Cause Analysis:

  • The mobile phase configuration time is too long, the mobile phase grows bacteria, or the organic phase such as acetonitrile is placed for a long time to produce flocculent precipitation.
  • Among the reasons for the above-mentioned packing contamination, the sample composition is complex and is easily adsorbed by the packing at the head end of the column. Especially if the sample contains protein substances, enrichment in the packing at the head end of the column will easily lead to packing contamination and hardening.
  • Others: samples such as gelatinous and flocculent samples cannot be removed by filtration. After the samples enter the chromatographic column, they accumulate on the sieve plate and the column head end.

Prevention program:

  • The mobile phase should be configured as needed. The pure water mobile phase should be used for no more than 24 hours at a time, and other mobile phases containing organic phases should be used for no more than 3 days at a time.
  • For the chromatographic column contamination prevention plan for detecting complex samples, please refer to the packing contamination prevention in the previous section.

3.Packing materials collapse

Abnormal performance:

Generally, there will be high column pressure, low column efficiency, peak tailing or front extension, peak drift, etc. Such damaged columns generally cannot be improved by abnormal regeneration or repair.

Cause Analysis:

  • Except for special instructions, the long-term pH range of silica-based chromatographic columns is 2-8. Higher or lower pH of the mobile phase or sample solution will accelerate the shedding of the bonded phase and the fragmentation of the silica gel.
  • The higher column temperature accelerates the attack of the buffer salt on the silica matrix, causing the bond phase to fall off and the packing to collapse.
  • The bonding phase of small groups is easy to fall off by its own nature, which is a phenomenon of normal filler properties.
  • Long-term use of chromatographic columns under high pressure conditions, etc.

Prevention program:

There are two forms of packing anomalies in packing collapse: bond phase shedding and matrix fragmentation.

● Avoid the bond phase falling off

  1. The pH of the sample solution and mobile phase should be controlled within the appropriate range for long-term use. If the pH of the sample solution is abnormal, use a guard column, or replace the Yuexu LP (withstand pH≤2) series or Xtimate series (withstand pH≥8) .
  2. According to the nature of the bonded phase of the chromatographic column, select the appropriate reagent as the mobile phase. For non-polar bonded phases, avoid using non-polar solvents; for intermediate bonded phases, avoid using moderately polar solvents; for polar bonded phases, avoid using polar solvents.
  3. Refer to the storage conditions in the Welch LC column manual.

● Avoid matrix breakage

  1. Unless otherwise specified, the pH of the mobile phase and sample solution used in the silica-based chromatographic column should not exceed 8.
  2. According to the chromatographic column manual, select the appropriate column temperature and avoid high temperature.
  3. Avoid pressure pulse, high pressure, etc.

It should be noted that Welch’s reversed-phase liquid chromatography columns generally recommend forward use and reverse flushing. However, for the column with the collapsed packing, it is not recommended to flush the column in reverse, so as to avoid further damage to the column.

4.Frit blockage

Abnormal performance:

Generally, high column pressure, low column efficiency, and peak tailing will occur.

Cause Analysis:

  • The filter membrane used is of wrong material or of poor quality, and new substances are introduced into the mobile phase or sample, resulting in contamination of the sieve plate and packing.
  • The instrument system has not been thoroughly cleaned for a long time.
  • The buffer salts and ion pair reagents in the mobile phase have no transition and effective flushing, precipitation, and clogging of the sieve plate and packing before and after use.
  • In the process of gradient use, the proportion of organic phase is higher than 60%, the conversion rate is too fast, and there is buffer salt precipitation.
  • Particles falling off caused by improper transportation may block the sieve plate.

Prevention program:

  • Replace the filter equipment with more tolerance and better quality.
  • The cleaning solvent and needle washing solvent after the pump should be replaced regularly. It is recommended to replace it once a week. The instrument system should be thoroughly cleaned with 40-degree warm water and organic solvents on a regular basis.
  • Use the transition mobile phase to transition out the high organic solvent or high salt phase in the system before and after the column is used (refer to the method under the new column activation item in the Welch liquid chromatography column manual).
  • Slow down the rate of change of the mobile phase and reduce the proportion of the highest organic phase.
  • The new column should be used after activation in strict accordance with the instructions. If the pressure is found to be too high during activation, it means that the storage solution of the chromatographic column has been lost during transportation and storage. If the pressure is still high after activating the chromatographic column at a low flow rate overnight, it may be that the sieve plate is blocked due to improper transportation, and you need to contact the manufacturer to send it back for repair and investigation.
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