Chromatography Forum

i'm a new HPLC user and using Zorbax 300SB-C18 column running samples of pectin microbeads extract (filtered through 0.45um). The injected extract contained Phosphate Buffer Saline pH 5.6 (suspending medium), viscozyme (mixture of pectinolytic enzymes), soluble fiber and pectin (from the microbeads).

The injection of first (untreated) sample produced erratic peaks and increase in pressure. I flushed it 1hr water then 1 hr methanol - that's when the pressure started to steadily build up. When i later checked, the sample is not well dissolved in methanol - produces precipitate. I immediately stopped and now flushing with water.

Is there a way i can flush out the precipitate and recover the column? Also, I left the column attached to the detector, during flushing, should i continue to leave it to clean out entire flow by flushing?

Turn the column around and reverse-flush. Do not connect to the detector during the reverse-flush; the last thing you want to do is to plug your cell.

If the precipitate is well-embedded in the inlet frit, you may not be able to get it all out

Thanks!

turn up the temperature too to the maximum recommended for your column. The high temperature will aid in redissolving the precipitate.

thanks for the suggestion! but I'm not equipped with a column oven yet. Should the column be flushing when in high temperature?

I have since reverse-flushed with water, then flushed with 10% methanol, followed by 50 % methanol and 100 % methanol. Also changed the frit in the column guard and sonicated the filter several times, which brought the pressure down. Pressure when flushing 100 % methanol seems abit higher (91) than usual (80).

The column seems to be restored, sharp peak but the retention time drifted about 1 min (before contam 5.6 min, after contam 6.5min).

During flushing, i left it to flush for quite a longtime (5 - 12 hr water/10% methanol/50% methanol) Is there a time limit not too exceed when flushing? will excessive flushing time damage column?

Flush time should not adversely affect the column.

The shift in rt could simply be a result of the column not having been equilibrated with mobile phase long enough after washing.

Thanks for the tips =)

I've tested the column with standard solutions after 1hr flushing with mobile phase and the retention time returned to normal.

However, after injecting samples, i flushed with water for 3 hrs, then 10 % methanol for 4 hrs (0.5ml/min). Pressure seemed normal until i switched flushing to 50% methanol - pressure started to build up. When i swapped back to 10% methanol, the pressure became erratic, gradually increasing to a point, then decreasing, and then increasing again (gradual repetitive up-down trend).

I checked and found the problem to be in the pre-column in-line filter. The pressure fluctuated the same without the guard column or column attached. There were some clicking sounds when the pressure increased. I sonicated the filter in 10 % methanol, then 50% methanol, but still no improvement.

Anyone know what's wrong with the filter/how I can fix it?

Replace it. They are not intened to be cleaned.