Chromatography Forum

Hi all,

I am having a problem in my HPLC. I recently replaced the guard column (with new one), conditioned guard column (as per instructions) before attaching to the analytical column. After column equilibration (I allow good time 3-4 hrs) one of my drug (hydrophilic salt, RT 1.5 min) is showing extensive peak tailing and reduced area under the curve (sensitivity reduced). My second drug (hydrophobic, RT 4 min) is not at all affected. I used fresh mob phase (isocratic acn/water 30/70 each with 0.1% TFA). The two solvents are mixed using two different pumps. I am using zorbax XDB C18 guard column (5um, agilent) and same material analytical column (3.5 um, agilent). The process is same always and i always use fresh mob phase.This never happened with my first guard column but is happening with the second and third one. I only had to change guard column as previous one was blocked (after 200 injections). I do get some tailing when I started using guard column but my main issue is reduced sensitivity of my drug by around 60%. The second drug was unaffected.

There is not much change in ret. time as such but sensitivity of hydrophilic drug has reduced. Also when I remove guard column, the sensitivity issue is resolved. So looks like it is to do with guard column.

Any suggestions on which might have gone wrong.

Please help as I am stuck for a while now. I dont want to skip using the guard column.

Thanks a lot
Mamcy 10

Dear Mamcy:

From the troubleshooting you have already done, it would appear that you are correct in suspecting the guard column. Now you need to determine if the problem is associated with the HPLC packing in the guard, or possibly a distorted thread that is not allowing the tubing to seat properly in the guard inlet. If the nut or threads in the guard column holder inlet are distorted, the seal may be compromised, which does not let the tubing butt right up against the opening of the guard column, or if not using a guard, the column opening. This creates a mixing chamber in the inlet in front of the column opening where the sample swirls around then moves into the column or guard slowly. Movement into the column or guard slowly, rather than in a tight band, creates tailing, rounded peaks, sometimes split peaks, etc. Peak tailing can create less sensitivity; however, 60% drop is very high. Therefore, make sure the nuts and ferrules are new, and even if new, check to make sure that the threads in the guard and analytical inlet are not distorted. If all is well, then I would agree that the guard packing may be the issue.
Before contacting the manufacturer or place of purchase, using the same system and same batch of mobile phase, run a standard using the new guard, then remove it and put the old guard back on. Run the same standard for comparison of the system and column, no change other than the guard. If you still see peak tailing and loss of sensitivity, you will have the documentation needed to show the manufacturer of the guard. The only true test of differences in columns is switching one out for the other, no other changes. Also, when troubleshooting, change one thing at a time. Changing more than one thing at a time may result in extra work, trying to determine which change created the new result.

Regards,
Linda Koch

Thanks a lot Linda,

Let me try this. Will keep you posted.

It wouldn’t surprise me if the guard column needs to be saturated with your drug/s.
Just inject the standard several times and see if the area increases with each consecutive injection.
Best Regards

Unless the drug you are working with is extremely sensitive to variation in silica gel lots, strong chelator, I would not think priming the guard would be needed; however, it is a good suggestion, and if priming doesn't affect the results, it is one more parameter ruled out. With the high purity silica gel, bonding and endcapping procedures used by HPLC column manufacturers these days, peak tailing is becoming more and more less common. Of course one needs to make sure that the conditions are correct, such as the mobile phase pH, etc. If the compound is ionic and you are working with the compound hydrophobicity, the pH of the buffer needs to be such that the compound is neutralized. If not, it may partially ionize, which can result in tailing, what appears to be a split peak, but really is an ionized compound, etc. Make sure the column is made with type-B silica gel and endcapped.

Regards,
Linda Koch
Former, now retired, Supelco Technical Service