I am using ion pair chromatography to separate some oligonucleotides. I found that my column pressure was increased. What is the reason?
after all runs in a day, I washed the column by water:methanol (95:5) for 20 min, then store it in 30:70 respectively. I do not khnow this procedure is right? Indeed, what is storage condition for this column after ion pair using?
Leila
Maybe also wash with water or 5%methanol in water for 20 min?
People also use CH3CN(less toxic) instead of MeOH.
I typically flush analytical columns with 1:1 acetonitrile:water for 20-30 column volumes after running a chromatographic system with mobile phases that contain ion-pairing modifiers. After flushing, I will change the mobile phase modifier back to the solvent system recommended by the manufacturer for long-term storage.
If operational back pressure is going up, you can try to reverse the direction of flow during the flush (if the column is designed for reverse flow - check the column guide). If this doesn't solve the issue, then ask the manufacturer for a column regeneration procedure for that column.
When you are ready to run the ion-pairing mobile phase conditions again on that column, it is a good idea to condition the column for 20-30 column volumes before starting your batch.
Guobing Xiang
Thomas D Horvath
thanks for your suggestion.
I also found a manual for this condition.
Excellent. Feel free to reach out to me directly should you have addition issues, I might have a troubleshooting suggestion that can help.
Have a great day!
A few comments as it appears you are new to HPLC and may not have anyone experienced in HPLC to help you at your place of work.
(1) Any column used with an ion-pairing agent, including TEAA, will have its surface chemistry changed forever. This means the column should never be used for any other methods and it should be labeled as being used with "TEAA", in this example. Dedicated column for this application. It will save you a great deal of money and time, doing things in this way and is what we teach to our HPLC students.
(2) The Column manufacturer for your specific column should always be consulted to determine which solvents (and pressure, pH etc) it is and is not compatible with (check the column user guide provided by the vendor).
(3) Washing the HPLC system and optionally the C-18 column in your example, should include some acidic modifier in an aqueous solution (for your example with TEAA). Multiple washes with an acidic solution will help to remove some, but not all of the ion-pairing reagent quickly to prevent further contamination. After the washes are complete, then use a solution which starts at a high percentage of water (i.e. 90%) and changes via gradient to a high percentage of organic solvent that is fully compatible with your method (i.e. to 10% Water / 90% Methanol). This will help remove any bound material from the column. This gradient flush wash method could then be followed by substituting ACN for the methanol which may remove additional material.
William Letter
thanks.
I am working in nucleic acid analysis field. I am not new in HPLC, but a few new in HPLC of nucleic acid. so I faced with some problems in this area when I work with C18 in ion pair (e.g. TEAA) mode.
I dedicated the column for this work. but I lost the resolution and quantitative analysis of oligonucleotides. thanks for your above suggestions and anymore if you have.
Leila Rastegar: If resolution is slowly lost over time, then this is often the result of column fouling. A few things that may help to reduce column fouling: (1) The column needs to be fully washed down after each analysis with a solvent system that is STRONGER than the mobile phase used. This will reduce the accumulation of any retained material on the column. (2) Samples should be dissolved in the mobile phase, then filtered (e.g. syringe filter) before being placed in a vial or injected into the HPLC. (3) When you receive a new column, run an in-house QC on the column to establish a "baseline" chromatogram for future reference (*For a dedicated application method, you could use some of the standards that apply to your sample). When resolution or performance changes, refer back to this baseline chromatogram. (4) Columns are disposable (consumable) items and need to be disposed of when they no longer perform. Depending on the method, a column could last from 100 to 3000 injections (lots of variation possible). Using a worn out or contaminated column is a waste of your time plus results in additional costs for the solvent, instrument wear and tear and waste disposal costs. New, spare HPLC columns should always be on hand.
- Leila; Your made-up column wash solution; "I washed the column by water:methanol (95:5)" is not appropriate for a C18 column as the wash solution should be a ramp starting with a large percentage of aqueous phase, then end in a large percentage of organic phase to ELUTE off bound material. Your current wash method will not actually wash most compounds off the column, it will retain them leading to column fouling. Please find someone with chromatography training to help you. You need to learn the basics.
Check out the autosampler-vial based sample vial filters produced by Thomson. They work well for sample applications that require filtering.
William Letter , Thomas D Horvath , Guobing Xiang
thanks for your guidance.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Column