Chromatography Forum

Dear All,

I am currently working with a Microsorb 100-5 NH2 250 x 4.6 mm column, trying to separate barley beta-glucan hydrolysates products (e.g. glucose, cellobiose, and other oligosaccharides DP<7). Unfortunately, after the first successful separation, when I run my standards I saw extra peaks in the trisaccahride and tetrasaccharide chromatographs and the backpressure was very high. What I did was to initially wash the column with multiple volumes of the mobile phase (70:30 ACN:water), both the right way and the reverseof the column, then with pure ACN, then with a gradient of 50:50 MeOH:water to 100% MeOH but still the pressure is very high. Namely, the backpressure is currently 200 bar at a flow rate of 0.5 mL/min. I am currently washing the column with 50:50 MeOH :Chorophorm as suggested in the booklet of the column. Would you have anything else to suggest? Thank you in advance.

It is likely that you have 'other' molecules in your hydrolysate that can 'gum up' a column (proteins?). Therefore, be sure to reverse the column when washing and 'acidify' your 50:50 Methanol:Water mobile phase (0.05M pH 3 Phosphate buffer). Use a 'guard' column with your samples.

Hi,

thanks for the useful response. Unfortunately, yesterday I noticed that when I took the column out and run just ACN or 70:30 ACN: water, the pump pressure increased to ~40 bar at a 0.2 mL/min flow rate. Apparently there is something wrong with the pump system...

Hi,

thanks for the useful response. Unfortunately, yesterday I noticed that when I took the column out and run just ACN or 70:30 ACN: water, the pump pressure increased to ~40 bar at a 0.2 mL/min flow rate. Apparently there is something wrong with the pump system...

Do you an in-line filter? My perkinelmer system has an in-line filter and I have seen that get worn out or clogged for some reason or another, and when I replace it with a new one it helps significantly with the pressure when I notice it gets very high with no column attached. You can try disconnecting this filter and connecting the lines without it to see how much it affects the pressure.