Regenerate HPLC column
Backflushing the HPLC column
In 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 Ám in-line filter to capture particulate material and
routine column washes with an appropriate strong solvent are highly recommended.
Symptoms of column plugging
When 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 First
When 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
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
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
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
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.
1. MAC-MOD Analytical Inc.