Question about UV response

[Chicama]

A 0.2 mg/mL solution of an API in MeOH gives me 100000 area response, in buffer pH 5.7 gives me 80000 and the API is soluble in both at concentrations far greater than what is being prepared, what is this? I know there is a technical name for this. On the other hand, a dilution of the API in MeOH into buffer pH 5.7 (giving 1% MeOh content) gives me 100000 area response, same as in methanol, what is going on? It can not be a solubility problem as the compound is at least 10 times more soluble in buffer pH 5.7 than the concentration that is being prepared, please any help would be really appreciated?

[zheyin]

A 0.2 mg/mL solution of an API in MeOH gives me 100000 area response, in buffer pH 5.7 gives me 80000 and the API is soluble in both at concentrations far greater than what is being prepared, what is this? I know there is a technical name for this. On the other hand, a dilution of the API in MeOH into buffer pH 5.7 (giving 1% MeOh content) gives me 100000 area response, same as in methanol, what is going on? It can not be a solubility problem as the compound is at least 10 times more soluble in buffer pH 5.7 than the concentration that is being prepared, please any help would be really appreciated?

I think it IS a solubility issue.
Although visually clear in buffer,
it is not trully dissolved as in methanol.
confirmed by the dilution w/ buffer from the stock in methanol.

[Chicama]

Thank you Zheyin, that is what I originally thought. The problem is that solubility results indicate that this API is freely soluble in MeOH and 400 ug/mL in buffer and when a 20 ug/mL solution is prepared in both MeOH and buffer the areas do not match, I am lost and I am new to this. Thanks for the reply, any other help would be greatly appreciated.

[tom jupille]

http://en.wikipedia.org/wiki/Solvatochromism

[Kwet80]

Hello Tom,

Solvatochromism can appear with only 1% MeoH compared to 0% MeOH (aqeous buffer only)? And being the same for 100% MeOH or 1% MeOH? I'm not sure I have really understand!

[Chicama]

Thank you Tom for the information. I read about it and it mentions that this effect is observed in changes on the absorption spectra and the peak absorption spectra looks the same in either case, could this effect manifest in different way? Thank you

[carls]

Are the area counts you refer from an HPLC separation?

If so, which HPLC system are you using?

With the Acquity UPLC, for example, the volume of sample injected is known to vary with the viscosity of the sample solvent. There was a thread discussing this here previously. Not sure if this has been fixed in later versions of this instrument since it seemed to be related to the complex injection scheme used for partial loop injection.

[tom jupille]

what is this? I know there is a technical name for this.

I was responding to the question about what the effect is called.

In fact, the solvatochromic effect is unlikely to be responsible for the difference in response if only the diluent is different, because the analyte molecules and the diluent get separated during the chromatography. It's only the mobile phase that matters.

Upon reflection, carls's suggestion about viscosity effects makes sense. One easy way to check would be to add an internal standard (actually, any other compound which is separable from your API) to the standards and re-inject. If it's an injection volume issue, both peaks should respond the same way. If the internal standard stays constant but the API peak area changes, then you are looking at some sort of chemical effect.

[M. Farooq]

A 0.2 mg/mL solution of an API in MeOH gives me 100000 area response, in buffer pH 5.7 gives me 80000 and the API is soluble in both at concentrations far greater than what is being prepared, what is this? I know there is a technical name for this. On the other hand, a dilution of the API in MeOH into buffer pH 5.7 (giving 1% MeOh content) gives me 100000 area response, same as in methanol, what is going on? It can not be a solubility problem as the compound is at least 10 times more soluble in buffer pH 5.7 than the concentration that is being prepared, please any help would be really appreciated?

I think it could be due to the fact refractive index is different for an aqueous vs. methanolic solutions. In order to detect solvatochromic effects, can you use multiple wavelenghts?

[Chicama]

Thanks Tom, Carls and Farooq, I will take your advice and try a few things. I am using a waters system alliance HPLC. Thank you guys very much.

[Klaus I.]

Upon reflection, carls's suggestion about viscosity effects makes sense.

Maybe this can occur durring UHPLC measurments (I think the compressibility is not the most contributing factor, in many cases), but I have observed this phenomenon many times when I was doing usual RP-HPLC:

In most of the cases, surely the componend was eluting early in the chromatogram. In most of the cases I have thought that the pH will cause the different areas, since many chromophores are pH-dependent.

But I have also this phenomenon observed several times, when I have done RP-HPLC with uncritical (on pH terms) molecules but with strange solvents. These comparisons were usually done during 'Final Rinse' Studies, and I have then that the Area von the API solved in acetone is higher than if mobile phase was used as solvent.
Since this wer special methods für 'Final Rinses' the retention times wher not long but surely greater than 5 minutes.

I'm wondering about these phenomenona since 12 years. Can it be really possible that some structures will bind a highly UV-absorbant solvent (like aceton) during RP-HPLC. But if this is really possible, the retention time should not be constant in most of the cases?

Its confusing.

[M. Farooq]

Upon reflection, carls's suggestion about viscosity effects makes sense.

Maybe this can occur durring UHPLC measurments (I think the compressibility is not the most contributing factor, in many cases), but I have observed this phenomenon many times when I was doing usual RP-HPLC:

In most of the cases, surely the componend was eluting early in the chromatogram. In most of the cases I have thought that the pH will cause the different areas, since many chromophores are pH-dependent.

But I have also this phenomenon observed several times, when I have done RP-HPLC with uncritical (on pH terms) molecules but with strange solvents. These comparisons were usually done during 'Final Rinse' Studies, and I have then that the Area von the API solved in acetone is higher than if mobile phase was used as solvent.
Since this wer special methods für 'Final Rinses' the retention times wher not long but surely greater than 5 minutes.

I'm wondering about these phenomenona since 12 years. Can it be really possible that some structures will bind a highly UV-absorbant solvent (like aceton) during RP-HPLC. But if this is really possible, the retention time should not be constant in most of the cases?

Its confusing.

I see two issues with peak area comparison of a sample with UV under different conditions and using different mobile phases. The UV detector in the HPLC system is not a true double beam UV absorbance measurement, in other words there is not true, in time, background correction.

More sneaky reason the hidden system peaks (which arise due to adsorbed additives in the mobile phase). System peaks can be positive or negative. What we might see is the "sum" of the analyte peak+ the species desorbed from the stationary phase as result of sample injection in a different solvent composition. Until and unless one does a thorough study by employing multiwavelength analysis one cannot be sure of the reasons leading peak area changes. A system peak can "elute" right under the analyte peak as well as a negative peak.

We recently made a new stationary phase and were testing DL-malic acid. We saw two peaks, equal area, which made me happy thinking that this stationary phase has some capability of enantiomer separation (note that it was a carbon based phase). When I did multiwavelength analysis, turned out that the other peak was a "system" peak. Note our eluent was very simple- acetonitrile + ammonium formate, yet we would see system peaks.

Regards,

Farooq Wahab

[HPLCaddict]

Erm, "multiwavelength analysis"? I always use blank runs to identify system peaks and injections of pure solvent to identify solvent peaks Am I missing something?

Concerning the original problem: I don't think that this is a solvatochromism issue, since as already pointed out, the "solvent" is the mobile phase here and that's the same in all cases.
A solvent viscosity effect is also unlikely - we're talking about buffer vs. 1% MeOH in buffer as solvents here, correct? For pure buffer vs. pure MeOH it's surely possible.

For me, it really sound like a solubility issue. Question to the OP: How were the solubilities in buffer and MeOH determined? Just visually?
Oh yes and one further question before we get lost in theories: Is this effect reproducible?

[HPLCaddict]

One further question just popped up : Just to be sure (no offending): You're doing "decent" chromatography meaning your peak of interest shows enough retention (far enough away from the t0 noise/solvent-peak)?

[M. Farooq]

Erm, "multiwavelength analysis"? I always use blank runs to identify system peaks and injections of pure solvent to identify solvent peaks Am I missing something?

I am afraid yes . System peaks arise due to the presence of analyte in the sample, competing with the species present in the eluent. The insidious system peaks can only be properly assigned when we are either using several detection modes or more easily, couple of wavelengths. But this is more of an academic curiousity. In principle, there should be no system peak, when the composition of the blank is identical as the eluent.

Regards,

Farooq