How to Activate and Equilibrate a New Chromatographic Column?
“I have never used this type of chromatographic column before. Are there any special aspects to its activation process? There is no instruction manual in the column box, so how should I use it correctly?”
Operational Guide:
Chromatographic columns come with a wide range of packing types, mainly including reversed-phase, normal-phase, HILIC (Hydrophilic Interaction Liquid Chromatography), size exclusion, and ion exchange columns. The activation procedures for different types of chromatographic columns vary. For unfamiliar chromatographic columns, you must refer to their corresponding Usage and Maintenance Guide.
How to Effectively Flush a Chromatographic Column When Its Pressure Rises?
“Yesterday, my chromatographic column could run the sequence normally, but today when I turned on the instrument, it showed an overpressure phenomenon. The column pressure increases by 10 MPa every day. What should I do to avoid this situation?”
Operational Guide:
When the pressure of a chromatographic column rises, troubleshooting should generally be carried out from the following aspects: the instrument system, mobile phase, sample status, and chromatographic column faults. In most cases, the pressure increase during use is caused by sample accumulation. Therefore, the primary solution is to flush the chromatographic column.
Flushing Protocol for Reversed-Phase Chromatographic Columns:
Clean the chromatographic column with 20 column volumes of solvent. The recommended solvent options are as follows:
Flushing Protocol for HILIC Chromatographic Columns:
Flush the column with 20 column volumes of 50:50 acetonitrile-water solution. If the effect is not ideal, gradually transition from 100% acetonitrile to 0% acetonitrile and perform flushing in several gradients.
Note: For other types of chromatographic columns, please refer to the Usage and Maintenance Guide.
Daily Maintenance Tips
- Improve the cleanliness of samples. If necessary, use the SPE (Solid Phase Extraction) method to remove excess matrix as much as possible.
- After each experiment, first perform isocratic elution with a high aqueous phase for 10-20 column volumes to ensure that buffer salts and components with high polarity (such as sugars, alcohols, etc.) are thoroughly flushed out. Then, use a gradient elution method to elute components with medium polarity, gradually increasing the proportion of organic phase until pure organic phase is used. Finally, perform isocratic elution with pure organic phase for 10-20 column volumes to ensure that non-polar and strongly retained components are eluted.
- Within the temperature tolerance range of the chromatographic column, the column temperature can be increased by 2-5 degrees. Turn off the column temperature promptly after the method runs.
- When the sample volume is large, it is recommended to clean the chromatographic column every 30-40 injections during the analysis process.
Unable to Reproduce the Chromatogram of an Old Column with a New Chromatographic Column? You May Have Fallen into These Pitfalls!
“I used this type of chromatographic column for my previous project, but this new column cannot achieve the same separation results. My client uses this type of column, so why can’t our laboratory get the same results?”
Troubleshooting Directions:
- Recheck the column efficiency of the new chromatographic column.
- On the same instrument, use the same batch of mobile phase to run the target sample on both the new and old chromatographic columns respectively.
- Consider the impact of pH value on the target analyte, and fine-tune the concentration of additives or salts.
Method Development Tips
- In the early stage of method development, try to test different batches of chromatographic columns as much as possible to verify the robustness of the method.
- Increase the concentration of additives or buffer salts, or age the new chromatographic column with a high-concentration buffer salt before use.
- For pH-sensitive projects (such as peptide drugs), precise pH control is required. The mobile phase can be prepared using the weighing method.