Related links:
- HPLC troubleshooting
- Regenerate HPLC column
Backflushing the HPLC column
CautionIn general, it is worth trying to clean and back flush an HPLC column with appropriate solvents before trying to replace the frit. There is always a danger that small amounts of packing material may be lost when a frit is replaced and column efficiency will decrease. In addition, the use of an 0.5 µm in-line filter to capture particulate material and routine column washes with an appropriate strong solvent are highly recommended.
Symptoms of column pluggingWhen a column becomes plugged, chromatographic performance generally suffers. System back pressure increases and, in some instances, peaks become distorted or split, as well.
Back Flush FirstWhen plugging of the inlet frit occurs there are two ways to restore column performance. Bach flush or change frit. The easiest and fastest way is to back flush particulates off the inlet frit.
Back-flushing the inlet frit:1. Connect the column to the chromatograph so that the flow now is in the reverse direction. Do not connect the column to the detector because dislodged particulates from the inlet frit may flow into the detector flow cell.
2. Back-flush the column with first with 100% HPLC grade water at a flow rate of 0.5ml per minute for 2 to three hours. Then repeat at the same rate with 100% HPLC Acetonitrile. If this does not work, try flushing with mix of 50% methanol and 50% water.
3. Connect the column to the chromatograph so that the flow is in the proper direction.
4. Check to see if chromatographic performance is acceptable.
If back flushing the column or column regeneration does not solve the problem, follow this procedure to replace the inlet frit.
How to replace the Column Inlet Frit:
1. Place the column in a ring stand or a bench vice with the column inlet pointing up.
2. Use a marking pen to mark the position of the end fitting relative to the column nut. A vertical mark directly across the end fitting and column nut is recommended. When you tighten the end fitting after replacing the inlet frit, these marks can help you torque the nut and end fitting back to the same position.
3. Allow the column to come to room temperature before removing the end fitting. Do not hold the column while replacing the frit. Heat from your hand can cause expansion of solvent in the column and may extrude packing material from the open end of the column.
4. Hold the column end fitting steady with one wrench while loosening the column nut counterclockwise with another wrench until the nut drops away from the end fitting. Lift the end fitting off the column.
5. Remove the column inlet frit by carefully sliding it to the side. If the frit is lodged in the end fitting, remove it by tapping it on a lab bench until the frit drops out.
6. Clean packing material from the top of the column by gently scraping across the top with a razor blade or a flat spatula. Be careful not to disturb the packing bed inside the column.
7. Place a new frit on the newly cleaned column inlet.
8. Remove all residual packing material from the column end fitting, tube ferrule, and column nut. This is easily done by flushing with isopropanol. A squirt bottle works well for this purpose.
9. Replace the column end fitting and tighten the column nut l/s to 1/4 turns past finger tight. Align the vertical marks that were placed on the end fitting and column nut in Step 2 so that the nut is tightened back to the proper torque.
10. Reconnect the column to the chromatograph.
11. Start flow through the column and check for leaks. If the column leaks, turn the flow off, allow the pressure to bleed off, and then tighten the nut slightly more. If the column still leaks, some residual packing material probably remains on a sealing surface (column ferrule, end fitting, threads of the column nut). Remove the end fitting again and reclean the surfaces.
REFERENCE:
1. MAC-MOD Analytical Inc.
Email About Main index Copyright Disclaimer
Signature : Delloyd's Lab Tech resources, reagents and Instrumentation
