effect of acetonitrile or methanol on analyte response

[olivia]

Hi, the analyte is just an aromatic hydrocarbon. I am just using organic-aqueous
Thank you
Liv

[danko]

Liv,

Looking at the information you’ve provided I’m pretty convinced that you’re dealing with absorption wavelength shift caused by solvent polarity differences. You may care to follow the link Tom provided earlier.

Maybe you’d also like to confirm this hypothesis by dissolving the compound in question in methanol/water and ACN/water mixtures respectively and then obtain a spectrophotometric scan for each of the solutions.

Please remember to get back with the result, if you chose to perform the test.

Best Regards

[Tomasz]

Using methanol one usually gets longer retention times than using acetonitrile in the same percentage. Longer retention times usually give broader peaks. UV detector is concentration dependent and broader peaks will give you higher area.

[danko]

Using methanol one usually gets longer retention times than using acetonitrile in the same percentage. Longer retention times usually give broader peaks. UV detector is concentration dependent and broader peaks will give you higher area.

It would be true if the response was measured in peak height.
In this case the response is measured in area units and the area is constant – unless the flow rate is changed!

Best Regards

[danko]

It would be true if the response was measured in peak height.

Sorry! I meant peak breadth/width and not height.
The rest of the post is still valid.

Best Regards

[Alfred88]

Dear olivia:
For your MP, you should select either MeCH or MeOH based on your desired selectivity/retention/solvent_cost, not on the detector's response. In fact, you might adjust the gain in the detector or the inj volume to achieve the desired output.

As for the solvent effect on the detector output, I just found this explanation handy in a latest book (Analytical Chemistry; R. Kellner et al. Wiley, 2004; ISBN 3-527-30590-4):

Solvents can interact strongly with certain solutes and thus change the observed UV-VIS spectra, either by removing vibrational fine structures, or by shifting absorption band maxima, or both. When the UV spectra of phenol in water (...) and in isooctane (...) are compared, both a blue shift and the disappearance of splitting can be observed in the water case. The blue shift is typical for the stabilization of the ground state by hydrogen bonding solvents such as water (why?) , the splitting can only be observed in water-free solvents without hydrogen bonding (why?). Red shifts of band maxima can be observed in polar solvents when the excited states are more polar than the ground states...

In short, MeCN and MeOH will shift your aromatic hydrocarbon (what is the func. group here?) differently with different max abs. wavelengths, and will give you different detector outputs.

Alfred.

[olivia]

Hi Alfred, and everyone else who posted, thank you very much for all your help. The solute was toluene
Thanks
Liv

[Tomasz]

danko!

I and some of my co-workers observed this phenomenon in my lab. Shift in retention time causes different detection signal measured as area. There is no need to change flow-rate. It is enough to change column from an old to a new one to observe this phenomenon.
Nevertheles, the change of maximum wavelengh suggested by Alfred88 is more probable reason in this case.

[danko]

Hi Tomasz,

I’m sure you’ve experienced some anomalies connected to column performance issues.
The first I can think of is a small, very flat peak that resembles the baseline, more than a real peak.
In this case the integration of the peak could be anything from zero area to twice or even more than the “realâ€

[Bruce Hamilton]

OK, a quick and dirty experiment.

Within the experimental error, no solvatochrome effect observed, and peak areas were similar. The higher absorbance of methanol observed may be real - could be the quality of the reagents I had, or some property of the mixture at those wavelengths, and would affect peak area.

Agilent 1100 Quat system with DAD
A = Water, B = Acetonitrile, C=Methanol
Sample 5ul of 1ul AR Toluene in Milli-Q water.
Column Phenomenex Luna C18(2) 250 x 4.6 5um
Guard Security Guard 4 x 3 C18
Flow 1ml/min
Detector 262, 254, 268 nm
Band Width = 4 nm, No reference, Data = 1 nm

Mobile Phase 25:75 Water : Methanol, Pressure = 165 Bar
Toluene Retention Time = 8.7 mins
Absorbance Minima 233, 266 nm
Absorbance Maxima 262, 268 nm
Peak Area at 262 nm = 427

Mobile Phase 35:65 water : Acetonitrile, Pressure = 90 Bar
Toluene Retention Time = 9.0 mins
Absorbance Minima 233, 266 nm
Absorbance Maxima 262, 268 nm
Peak Area at 262 nm = 433

Note that column was not allowed to condition more than a couple of minutes, and the 75:25 Methanol : Water had 4 - 5 mAu positive baseline displacement compared to 65:35 Acetonitrile : Water.
Single injections only.

Will update if I get time to perform 35: 65 Water : Methanol, which should generate a higher backpressure and longer retention

Please keep having fun,

Bruce Hamilton

[ym3142]

Bruce,

Thank you. Good Job!

what does it mean by

Note that column was not allowed to condition more than a couple of minutes

,

And so your experiment is contrary to what Olivia found?

Olivia, right?

[Bruce Hamilton]

I didn't have time to allow the column more than a few minutes after the new solvent eluted before starting the injection, and the same vial was used for all samples - over time, toluene should decrease ( evaporation ). Not ideal, but I had other work for the instrument.

My understanding is that Olivia used 35:65 ratio for both MeOH and Acetonitrile, whereas I selected a MeOH ratio ( 25:75 ) that should give similar elution times. I don't recall the detection wavelength she used.

If I had used 35:65, the elution would be abouit 50% longer and the pressure about 190 Bar, more than double the 65:35 acetonitrile, perhaps with some effects that may confuse single injection results.

Ideally, multiple injections and samples in replicate sequences, would produce accurate numbers, but the experiment demonstrates that the touene didn't shift maxima/minuma wavelengths in the tests I performed, and peak areas were similar.

The experiment also showed that, for some reason, the methanol:water absorbed more than the acetonitrile:water. That could explain the lower values reported by Olivia, depending on the solvent grades and wavelengths she used.

Update.

I used the same sample solutions to run a 35:65 MeOH : Water system several hours later and, as expected, the pressure climbed to 190 Bar, and Retention of the toluene went to 15.3 mins. The peak maxima and minima were as reported above, and area was 411, which was similar to the peak area ( 414 ) of an immediately prior 35:65 CH3CN : water run.

Bruce Hamilton

Bruce Hamilton

[Tomasz]

Hi danko

This effect was observed for fast eluting peaks - not for late eluting peaks where wrong integration can change area. It was also observed in experiments where we used the same column but mobile phase prepared in a different day or just with diffrent pH. Some change in retention was observed here although the column was the same.
Please note that it is not mobile phase flow rate that changes peak area. The area is changed because of time during which analyte is present in a UV detector. One of the options of changing this time is to change the flow rate of the mobile phase. In my lab wider peaks are not because of lower flow rate but because of some different reasons. Thus, wider peaks have bigger area than narrower peaks even when the flow rate is constant.

Regards
Tomasz

[danko]

Hi Tomasz,

The described peak broadening, due to longer retention time, will indeed cause a longer presence of the compound in the flow cell, but the concentration will be correspondingly lower. Consequently, the resulting peak’s height will be reduced and at the end the peak area will be the same.

But you mentioned another thing which could explain your observation. Namely, different pH! As you know, many chromfores’ absorption intensity and spectra are pH dependant, so maybe that is what caused peak area variation in your case, but because retention too can vary with pH – as you’ve observed in this situation – one can be tricked to attribute the area increase to the retention time.

Best Regards

[Tomasz]

danko

Its the same situation like with changing mobile phase flow rate. Concentration (and peak hight) will also be lower when you decrease mobile phase flow rate. However, peak area will be higher.

Change of pH in the range +/- 0.5 does not always change signal. Moreover, I observed the phenomenon even for mobile phases with no buffer.

I'm not saying, I'm right for sure but I got these results from real experiments. I don't think you are completely wrong but your argumentation is not good enough for me. And vice versa, I believe

Regards