Chromatography Forum

Dear colleagues,

We have been crushing in a problem of weird hunches (I dont know which name it should be) and we truly hope all of you can help us to solve:

1. When we used mobile phase containing acetonitrile - water (70:30) or acetonitrile - water - acid phosphoric (700:300:0,2), C18 column, DAD 215 nm, with the sample was mobile phase or tacrolimus solution (prepared in acetonitrile-water 10:1), there was a hunch repeatly with retention time (80 mins) about 10 times of the main peak (8 mins).
When we sent this sample and the same method to an other laboratory, the hunch appeared, too, but retention times were not repeat among peaks.

2. The same problem happened with mobile phase containing acetonitril - tetrabutyl ammonium dihydrogenphosphate 0,01 M (66:34), C18 column, DAD 214 nm, in different days, the chromatograph of blank (acetonitrile - water 50:50) had a hunch at different retention times (the first day: 120 mins (25-27 degree celcius), the second day: 60 mins (27 degree celcius), the third day: 120 mins at 27 degree celcius, 90 mins at 40 degree celcius and 127 mins at 24,5 degree celcius) (using the same column for 3 days). The chromatograph of mobile phase was the same but the hunch is higher (~10 mAu) (the hunch of blank: ~5 mAu). We tried with different degas time but nothing different.

3. We have some other mobile phases having the same rate of acetonitrile (ex: Acetonitrile - phosphate buffer pH 2,2 67:33, DAD 214 nm) but there has been not any hunches.

4. We have a mobile phase containg acetonitrile - tetrabutylammonium hydroxide 0,01 M buffer with acid phosphoric (pH 6,5) (25:75), there was no problem.

The hunch area has the width about 8-10 mins. Spectrum of it had max absortion at about 190-200 nm, and min at about 200 and almost horizon after that.

We are very confused and we put all believe in your help!
Thank you so muchhhhhh!

Hi Greyfield,

1. When we used mobile phase containing acetonitrile - water (70:30) or acetonitrile - water - acid phosphoric (700:300:0,2), C18 column, DAD 215 nm, with the sample was mobile phase or tacrolimus solution (prepared in acetonitrile-water 10:1), there was a hunch repeatedly with retention time (80 mins) about 10 times of the main peak (8 mins).
When we sent this sample and the same method to another laboratory, the hunch appeared, too, but retention times were not repeat among peaks.

So here, there is both a big hump in the baseline of two isocratic methods as well as variation in retention times in two labs.

2. The same problem happened with mobile phase containing acetonitrile - tetrabutyl ammonium dihydrogenphosphate 0,01 M (66:34), C18 column, DAD 214 nm, in different days, the chromatograph of blank (acetonitrile - water 50:50) had a hunch at different retention times (the first day: 120 mins (25-27 degree Celsius), the second day: 60 mins (27 degree Celsius), the third day: 120 mins at 27 degree Celsius, 90 mins at 40 degree Celsius and 127 mins at 24,5 degree Celsius) (using the same column for 3 days). The chromatograph of mobile phase was the same but the hunch is higher (~10 mAu) (the hunch of blank: ~5 mAu). We tried with different degas time but nothing different.

Different isocratic methods, and a similar trouble with a baseline hump. These methods may be buffered a bit--more info would be appreciated. Much variation is noted as the temp is varied.

3. We have some other mobile phases having the same rate of acetonitrile (ex: C) but there has been not any hunches.

4. We have a mobile phase containing acetonitrile - tetrabutylammonium hydroxide 0,01 M buffer with acid phosphoric (pH 6,5) (25:75), there was no problem.

And sometimes all is well with a buffered system.

Have you tried equilibrating the LC without a column in place? How do you make the eluents and degas them? Is the work done in 2. with or without a true buffer?


Thank you, will wait for your reply.

Hi Matt,

Thanks much for your help!
The hump apppeared in some mobile phases we used to try (paragraph 1-2). And the same point here is the very high rate of acetonitrile in mobile phase (66-70%). We tried 2 times with mobile phase containing only MeCN-water (70:30 or 66:34), the same hump appeared with the same retention to buffered mobile phases. I wonder if there is some interactions between MeCN (a weak base) and particles in column.

In paragraph 3, the same rate of MeCN (67%) but the aqueous phase has ion potassium. May be some interaction between MeCN and K+ help to neutralise them and there is no hump baseline.

In paragraph 4, the aqueous phase has no ion alkaline but the rate of MeCN is just small (25%).

But I dont understand how they interact to each other and what the hump baseline is.
I am trying once again with test 2, with the adding of monobasic sodium phosphat in mobile phase (pH 5.4). The result will be got in some hours.

Noone of you have met the same problem? I am truly confused.
Ah, in test 2, in 2016 and 2017 we running alright, no problem. I saw the temperature of column is 25 °C. But now at any temp the hump is still there. @@

Hope to see your reply soon. I will give you the result right when it's done

Hi Greyfield,

My thanks...I wasn't sure all of the eluents in paragraphs 1 and 2 were buffered or what the pH values were. Low wavelengths were used, which is a condition where the drift of the baseline is more readily observed in general.

Some possibilities:

1. "Column temperature fluctuation. (Even small changes cause cyclic baseline
rise and fall. Most often affects refractive index and conductivity
detectors, UV detectors at high sensitivity or in indirect photometric
mode.)

2. Nonhomogeneous mobile phase. (Drift usually to higher absorbance, rather
than cyclic pattern from temperature fluctuation.)

3. Contaminant or air buildup in detector cell (or column).

4. Mobile phase mixing problem or change in flow rate.

5. Slow column equilibration, especially when changing mobile phase.

6. Mobile phase contaminated, deteriorated, or not prepared with high-quality chemicals.

7. Strongly retained materials in the sample (high k’) can elute as very broad
peaks and appear to be a rising baseline. (Gradient analyses can aggravate the problem.)

8. Detector (UV) not set at absorbance maximum but at the slope of the curve."

Credit properly goes to Supelco for suggestions.

The separation runs you're doing are Long (> 80 minutes). Removing the column and running those eluent compositions can remove "dirty column" as a problem source. Cleaning the UV cell (nitric acid?) could remove another source.

Let us see how your test goes. With gradient chromatography I have seen the same behavior, not with isocratic runs, causes 1 - 3, 5 - 8 were determined most often in my experience.

Thanks Matt,

The test with monobasic sodium phosphate added have shown the same hump (but longer retention time) ((( I wonder the effect of air bubbles born when injecting, cause if mobile phase running without injection, there was no hump.

Today I'm trying a test with different sample (containing different rate of MeCN-water) to check effect of air bubbles.

Hello again,

Seems to me to be a reasonable next step. If the new eluent affords a hump, next will be to decide how the UV cell will be cleaned, I think.

Thanks Matt,

I have found out where the hump was from. It's membrance!!! When we injected MeCN without filting and MeCN filted through nilon, PTFE, Regenerated Cellulose 0,45 mcm membrance, only chromatograph of MeCN without filting didnot show any hump.

When injecting MeCN filted through nilon membrance and discarding the first 2 ml or 10 ml of the filtrate, the sample with 2 ml discarded show smalller hump.

And the hump didnt appear when injecting water into the column.

Thanks,

Excellent troubleshooting grayfield!! Glad it was only dirty filters.

I dont mean that the filter is dirty, Matt. This is the effect of filter material when using MeCN as diluent

Understood, grayfield. No worries! I've looked at extractables from filters in my past--that is what I meant by "dirt".

Ah yes, but I have a question. I have read some information about choosing a filter material suit for diluent, they said PTFE and RC are compatible with acetonitrile. In fact this is not true with my experiment.

Good point--perhaps a different vendor per each source of filter? PALL has been good to me, also Millipore.

Ah yes, but I have a question. I have read some information about choosing a filter material suit for diluent, they said PTFE and RC are compatible with acetonitrile. In fact this is not true with my experiment.

Even if the filter material is compatible with your solvent, it is good practice to always discard the first 3 - 5mL of filtrate, sometimes filtrate particulate can come out initially and into your solution.

Also make sure when you filter you do not push so hard on the syringe, you can blow out the filter and push more filter contaminants into your sample.