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By Anonymous on Wednesday, October 29, 2003 - 01:01 am:
I have both 0.1%v/v TFA in water and 0.1%v/v TFA in acetonitrile. When I run gradients the baseline at 210nm (and 220nm) still rises, although the concentration of TFA through the detector should be constant (??).
Is there a reason for this?
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By Anonymous on Wednesday, October 29, 2003 - 12:29 pm:
absorbance of the MeCN...
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By R.C. on Wednesday, October 29, 2003 - 12:53 pm:
That is most likely the change in refractive index that your detector reports as an absorbance. The magnitutde should be negligable. If it's a problem for your analysis, a different detector model may respond differently. This assumes you're sure that the acetonitrile doesn't have UV absorbing contaminant, isn't leaching something from your eluent bottle or system, etc.
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By Anonymous on Thursday, October 30, 2003 - 01:07 am:
When I run the gradient with 0.1%v/v TFA and acetonitrile (from the same supplier) the baseline drops.
So I guess its a detector - RI problem.
Thanks
Anonymous
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By Andreas Neumaier on Thursday, October 30, 2003 - 06:42 am:
Calculate molar, not v/v concentrations and the rise of baseline should be in a range you can live with.
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By dkm on Thursday, October 30, 2003 - 11:49 am:
I've run into the same situation. My gradient ran from 10 to 60% Acetonitrile. I tried using 0.1%TFA in Water and 0.1%TFA in Acetonitrile just as you did. I found that the baseline drifted. I then chose to premix 10% Acetonitrile and 60% Acetonitrile eluents (each containing 0.1%TFA). The baseline improved. I think it has to do with the mixing / outgassing. The premixed solvents were degassed prior to use. The neat eluents were not degassed prior to use.
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By Chris Pohl on Thursday, October 30, 2003 - 02:41 pm:
Another thing to keep in mind is that not only does TFA weakly absorb at 210, but the amount of TFA adsorbed on the stationary phase is solvent dependent. Even if the feed solution concentration is constant, a solvent gradient will add some additional TFA as it desorbs from the stationary phase. If you interrupt the gradient, does the baseline drift back down again? This may be an indication that desorption of TFA is partially responsible for the baseline drift. To correct for this component of the background you may need to either reduce the gradient ramp or introduce a corresponding decrease in TFA concentration in the gradient.
As mentioned above, another possibility is that this is related to the purity of your TFA. Are you using a fresh sample of TFA specified for peptide mapping? If not, you might want to obtain a better quality TFA sample for your work.
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By HW Mueller on Thursday, October 30, 2003 - 11:55 pm:
It is also very likely that the absorption of TFA differs between H2O and ACN (doesn´t anybdy have some exp. evidence on this?).
On dkm´s contribution: It was, partially, drifting baselines in isocratic HPLC (after mobile phase changes) that led to the experiments on gases in mobile phases mentioned in an earlier chain. According to this, outgassing would not be necessary (even unlikely! Outgassing should cause spikes or sawtooth peaks) for drifting, a changing DISSOLVED gas content will produce drifts.
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By MG on Friday, October 31, 2003 - 08:42 am:
HW: I don't have such data on TFA, but chloro- and bromo-acetic acids are retained in reverse phase on an ordinary C18 column. I have analyzed them by LC/MS.
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By HW Mueller on Monday, November 3, 2003 - 12:00 am:
Darn, was not clear, meant light absorption.
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By Anonymous on Tuesday, June 22, 2004 - 02:46 am:
This is proably a bit late but here goes anyway. See Winkler G, Wolschann P, Briza P, Heinz X, Kunz C (1985) J Chromatogr 347:83-88. The carbonyl function of TFA absorbs weakly at low wavelengths and the spectrum changes depending on its ionisation state. As the amount of acetonitrile increases during the gradient, the ionisation state of acid function changes and affects the UV signal.
I have both 0.1%v/v TFA in water and 0.1%v/v TFA in acetonitrile. When I run gradients the baseline at 210nm (and 220nm) still rises, although the concentration of TFA through the detector should be constant (??).
Is there a reason for this?
-------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, October 29, 2003 - 12:29 pm:
absorbance of the MeCN...
-------------------------------------------------------------------------------------------------------
By R.C. on Wednesday, October 29, 2003 - 12:53 pm:
That is most likely the change in refractive index that your detector reports as an absorbance. The magnitutde should be negligable. If it's a problem for your analysis, a different detector model may respond differently. This assumes you're sure that the acetonitrile doesn't have UV absorbing contaminant, isn't leaching something from your eluent bottle or system, etc.
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, October 30, 2003 - 01:07 am:
When I run the gradient with 0.1%v/v TFA and acetonitrile (from the same supplier) the baseline drops.
So I guess its a detector - RI problem.
Thanks
Anonymous
-------------------------------------------------------------------------------------------------------
By Andreas Neumaier on Thursday, October 30, 2003 - 06:42 am:
Calculate molar, not v/v concentrations and the rise of baseline should be in a range you can live with.
-------------------------------------------------------------------------------------------------------
By dkm on Thursday, October 30, 2003 - 11:49 am:
I've run into the same situation. My gradient ran from 10 to 60% Acetonitrile. I tried using 0.1%TFA in Water and 0.1%TFA in Acetonitrile just as you did. I found that the baseline drifted. I then chose to premix 10% Acetonitrile and 60% Acetonitrile eluents (each containing 0.1%TFA). The baseline improved. I think it has to do with the mixing / outgassing. The premixed solvents were degassed prior to use. The neat eluents were not degassed prior to use.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Thursday, October 30, 2003 - 02:41 pm:
Another thing to keep in mind is that not only does TFA weakly absorb at 210, but the amount of TFA adsorbed on the stationary phase is solvent dependent. Even if the feed solution concentration is constant, a solvent gradient will add some additional TFA as it desorbs from the stationary phase. If you interrupt the gradient, does the baseline drift back down again? This may be an indication that desorption of TFA is partially responsible for the baseline drift. To correct for this component of the background you may need to either reduce the gradient ramp or introduce a corresponding decrease in TFA concentration in the gradient.
As mentioned above, another possibility is that this is related to the purity of your TFA. Are you using a fresh sample of TFA specified for peptide mapping? If not, you might want to obtain a better quality TFA sample for your work.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Thursday, October 30, 2003 - 11:55 pm:
It is also very likely that the absorption of TFA differs between H2O and ACN (doesn´t anybdy have some exp. evidence on this?).
On dkm´s contribution: It was, partially, drifting baselines in isocratic HPLC (after mobile phase changes) that led to the experiments on gases in mobile phases mentioned in an earlier chain. According to this, outgassing would not be necessary (even unlikely! Outgassing should cause spikes or sawtooth peaks) for drifting, a changing DISSOLVED gas content will produce drifts.
-------------------------------------------------------------------------------------------------------
By MG on Friday, October 31, 2003 - 08:42 am:
HW: I don't have such data on TFA, but chloro- and bromo-acetic acids are retained in reverse phase on an ordinary C18 column. I have analyzed them by LC/MS.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Monday, November 3, 2003 - 12:00 am:
Darn, was not clear, meant light absorption.
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, June 22, 2004 - 02:46 am:
This is proably a bit late but here goes anyway. See Winkler G, Wolschann P, Briza P, Heinz X, Kunz C (1985) J Chromatogr 347:83-88. The carbonyl function of TFA absorbs weakly at low wavelengths and the spectrum changes depending on its ionisation state. As the amount of acetonitrile increases during the gradient, the ionisation state of acid function changes and affects the UV signal.
