Hello,
The extract of blood samples were injected into a Alltima C-18, 150 x 2.1 mm, 5 micron column maintained at 50 degree celcius. The mobile phase (60%acetonitrile in water) was pumped at 0.5 ml/min. The column was ran with guard Alltima C-18, 5 micron, 7.5 x 4.6 mm. We injected the extract 150 samples, resulting in high back pressure 3000 psi. Then we followed the protocol, regeneration procedure from Alltech with inlet to outlet. The pressure was still high. After that, we ran 100% ACN, 100% methylene chloride, 100% ACN and mobile phase. The pressure was 3500 psi.
We concluded that our column was bad now. Since we injected the same sample in a row, 5 times and got different area (more than 20%).
We have no experience with this kind of column before. My question was how to take good care if we plan to run routinely. Someone told us that we should filter all mobile phase with filter 0.2 micron. We plan to do for the next column.
Thank you very much in advance.

Did you ever run your column backwards? Most modern columns allow this. The neat solvents you mentioned all denature and precpitate proteins, you probably consolidate plugging by using them. We have had considerable succeess washing with aqueous Li-dodecylsulfate + dithiothreitol as a last resort. Any chaotropic material in water should be tried first. It is indeed prudent to filter out particles and to get rid of as many proteins as is possible (precipitation, ultrafiltration, size exclusion) prior to injection, and especially particles with microfilters. A guard-frit or column is practicaly a must.

I concur that you should try backflushing, assuming that changing the guard column didn't lower your pressure. Filtering is also a good idea if you suspect your samples contain particulates.

What was your pressure on the new column? 0.5 mL/min is a pretty high flow rate for that column dimension. If I'm doing the calculation right, it corresponds to ~ 2.4 mL/min on a 4.6 mm id column for the same linear velocity. A high backpressure alone is not surprising.

I would work on the sample preparation method to get a cleaner sample. How did you prepare your sample?

I would concur that 0.5 mL/min flow is rather high for a 2.1mm ID column. What was the backpressure at the beginning of the sample set?

Did you change the guard column and see if the backpressure dropped?

a possibility is the plug is before/after the column. Start disconnecting tubing from the end of the apparatus and note any significant decrease in pressure, then work your way all the way to the pump. This will help isolate the source of the plug.

It could be, stationary phase knocking loose into the detector/tubing, or your matrix ppt in the injector when exposed to high conc. ACN.

60% MeCN to water may cause precipitation of proteins. Add some blood extract to a vial of your mobile phase and see if solids/turbidity occurs. That might be gunking up your system.

Highly recommend one of the extraction methods above. Anyone can give you details/recipes if you need.