Chromatography Forum

Hello Members,

I've got a curious case here that I'm hoping someone can help me diagnose. I'm working with a client to develop a purity method for a small-molecule API. The method they've developed is quite conventional (C18 column running an ACN/Water gradient with phosphate buffer).

MP A: 20mM Phosphate buffer, pH 3.0 in 5% ACN
MP B: 20mM Phosphate buffer, pH 3.0 in 70% ACN
Column: Waters Acquity BEH Premier C18, 2.1x150, 1.7µm
Flow rate: 0.25 mL/min
Injection vol: 1 µL
Detection: VWD @ 230 nm
Gradient: shown as overlayed red trace in the chromatogram pasted below

Client is exclusively using HPLC-grade reagents, and I've instructed them to check pH of MP on a separate aliquot of MP to avoid contamination. We can't seem to figure out why the baseline drifts downward as % MP B increases. Theoretically, MP B should have a higher response at 230nm than MP A, right?

Note that they have been running this method for months, and it has always looked like this (ie, not a new issue). Any ideas what might be causing the downward drift as the % MP B increases?

Simply ,It seems the buffer has some contamination ( from water and chemicals ).

You may check the purity of each step, starting from the water ,during the preparation of buffer , using a spectrophotometer at 230nm ( if available ).

Simply ,It seems the buffer has some contamination ( from water and chemicals ).

Thank you for the response. We're preparing a stock buffer (at 100 mM) and adding equal amounts of that stock to each mobile phase (A and B). Could you explain how contaminated buffer would result in downward baseline drift if the buffer concentration remains constant throughout the gradient?

Contaminated buffer percentage is much more in solvent A

Contaminated buffer percentage is much more in solvent A

Contaminated buffer percentage is much more in solvent A

Sorry if this wasn't clear, but the buffer concentration is identical (20mM) across the two mobile phases. We take a 100mM stock buffer solution and add 200 mL of that per liter of mobile phase. For MP A, it's 200 mL stock buffer, 50 mL ACN, and 750 mL Water. For MP B, it's 200 mL stock buffer, 700 mL ACN, and 100 mL Water.

So, to me, that's the same amount of buffer across the two mobile phases. Am I missing something there?

so , the water is contaminated.

or it's just old and degasser is sub-par

or it's just old and degasser is sub-par

Thanks for the idea! I hadn't considered degassing as a contributing factor here. Could you expound a bit on your hypothesis? Specifically, how/why would insufficient degassing result in MP A (5% ACN) having a higher response than MP B (70% ACN)?

The site is almost exclusively using relatively new Waters Acquity H-Class UPLCs (with quaternary pumps). My assumption has been that those systems all have properly functioning in-line degasses (I believe they are integrated in to the 'solvent manager' component), but I'll definitely confirm this. I'll also float the idea of degassing each MP offline after prep.

Unrelated to degassing: I read one of your previous posts regarding pre-mixed vs. non-premixed mobile phases. I've recommend that the lab use premixed MPs for this procedure, as I didn't want them to have buffer salting issues. Since they're using quaternary pumps, I wonder if the performance may improve if they only pre-mix MP B. This way, they can avoid pumping from line B at <5%, which I know some quat pumps have trouble with. Any thoughts on that?

I'm not certain of the mechanism, but experience shows that gradient related baselines tend to dive instead of rise for RP setups only when the MP is getting old or if your degasser is not quite keeping up with things.
By "quarternary" (from Waters), I'll assume you are talking about 4 lines feeding a proportioning valve that then feeds a single 2 stage pump.
The only additional caveat here is that you have to go out of your way to make sure that ALL 4 lines get flushed with something before you run, or else sir will find a way to foul you up. The best way to start an H class is by using the Smart Start(or is it Instrument Start?) option.

High pressure mixing systems (where A and B meet while they're under pressure) tend to be better at preventing outgassing on mixing but tend to do not as good a job at mixing, low pressure mixing systems do better with the actual passive mixing but can generate bubbles if things are not adequately degassed.

Premixing is always good - anything you can do to make your gradient formation closer to the "100% A to 100% B in X minutes in a linear fashion" ideal is best for reproducibility across different types of HPLCs. Once you have this, all you need to worry about is any significant difference in dwell volume from system to system.

By "quarternary" (from Waters), I'll assume you are talking about 4 lines feeding a proportioning valve that then feeds a single 2 stage pump. The best way to start an H class is by using the Smart Start(or is it Instrument Start?) option.

Yes, their systems use a proportioning value w/ low-pressure mixing. I'll have to look into their standard practices regarding system startup / line flushing. I'll be surprised if they flush more than just the two lines they're using, but you're right, if they routinely use something with higher absorbance on the other lines, these should be flushed out as well. Good tip.

High pressure mixing systems (where A and B meet while they're under pressure) tend to be better at preventing outgassing on mixing but tend to do not as good a job at mixing, low pressure mixing systems do better with the actual passive mixing but can generate bubbles if things are not adequately degassed.

I've always preferred LCs w/ binary (high-pressure) pumps, myself, but they've got a very limited number of systems with binary pumps at their site. I'll see if I can get them to trial the method on one of those systems. I've also asked them to trial pre-mixing of MP B only to see if that makes a difference.

Thanks for all the input!

A few comments:
(1) That is NOT a degassing issue. Degasser issues result in poor baseline stability etc, not perfect downward drift. *The "drift" in your example follows the change in solvent composition. BTW:This does not mean that their integrated vacuum degasser is working properly. It most likely is broken if the system is more than four years old. When they break they introduce contamination into the mobile phase flow path and of course provide little to no actual degassing of mobile phase (By the time any degasser "errors" appear on the system, the degasser has been broken for some time. The system will not show an error until long after the system is destroyed, So never wait for an error, instead have the degasser profesionally serviced or replaced every 4-5 years of use).
(2) High grade Water and ACN have similar low UV abs. Ask them for details regarding the water quality used. Example: lab grade RO water that is filtered and polished..
(3) You did not say which VWD they have. Some Waters models have a "reference" wavelength software feature which can be INCORRECTLY enabled by the user. Depending on the Ref wavelength selected, this can have the effect of reversing the signal somewhat as it subtracts out the original signal from the reference signal creating a third signal which is the one that is shown on the chromatogram (If the user has the 'Reference' turned ON, this might be the cause).

One more thought... if the buffer solution was pH adjusted with the wrong acid (e.g. Acetic), then the addition of the impure acid with higher absorbance may also result in the same downward drift as the ACN composition is increased. In the case of acetic acid, it is often of low purity and goes bad quickly resulting in an observable brown color.

This might be a bit silly, but I only suggest it because I have done this before and achieved similar results. Check that your mobile phase lines are not backwards. i.e. A plugged into B at some point and vice versa...yeah, i did that.

(1) So never wait for an error, instead have the degasser profesionally serviced or replaced every 4-5 years of use).

These systems are in a high-throughput cGMP lab, so they are serviced routinely (annual PM schedule, I believe). I've never actually looked into what is typically done during routine PM to verify Degasser performance. I'll have to look into that one, but I agree with you that the specific trend we're investigating doesn't really fit a degasser issue.

(2) High grade Water and ACN have similar low UV abs. Ask them for details regarding the water quality used. Example: lab grade RO water that is filtered and polished..

Yeah, reagent quality was definitely high on my list. They were originally using water from their Milli-Q system, so I asked them to switch to HPLC-grade bottled water to rule out the water system. The baseline smoothed out a bit after that change, but the downward drift issue persisted. They've gone through several lots of of HPLC-grade bottle water at this point with no change in the profile, but I suppose we could always try water from a different vendor.

(3) You did not say which VWD they have. Some Waters models have a "reference" wavelength software feature which can be INCORRECTLY enabled by the user. Depending on the Ref wavelength selected, this can have the effect of reversing the signal somewhat as it subtracts out the original signal from the reference signal creating a third signal which is the one that is shown on the chromatogram (If the user has the 'Reference' turned ON, this might be the cause).

I'm going to have to get back to you on this one. I've asked the lab to confirm which specific detector they've been using, and whether or not a reference wavelength is being used. Inappropriate reference wavelength correction definitely fits the trend (ie, if ACN has high absorbance at the ref wavelength, would result 'overcorrection' as %ACN increases).

Thank you very much for the thoughtful response to my post. I really appreciate your time.