Weird baseline dips from gradient - Acquity TUV

RobBurgess

<p><span>Has any user experieneced any similar problem to the .bmp file I have attached where they suddenly see odd dips in the baseline when running steep gradients on their Acquity TUV? if so how have you solved the problem or what could be causing it. Is it perhaps a detector / flow cell problem?</span></p><p></p><p><span>For everyone's info:</span></p><p></p><p><span>Black chrom: Acquity (MPA = Millipore Water + 0.1% TFA; MPB = Merck premixed AcN + 0.1%) dips @ about 9.9, 12.2 and 15.9 minutes</span></p><p><span>Green chrom: Agilent 1100 ( same mobile phase / column and conditions as black chrom) - no dips</span></p><p><span>Blue chrom: Acquity (MP A - millipore water + 0.1% TFA; MP B = Romil ACN + 0.1% TFA)</span></p><p></p><p><span>This could be probelematic trying to detect low level imps at 0.05% or less and the imp happens to elue at the point of one of these dips so a long term solution would be required.</span></p>

lizh

Can you send a new file, I have tried a couple of times now and my cpu hangs.

MAny TX

Liz

ACQUITYEPM

Greetings Rob,

I am having the same problem with the attachment. Baseline disturbances in TFA gradients are, usually, chromatographic artifacts. The TFA is constantly being adsorbed and desorbed onto and off the column. In essence, these disturbances are a reflection of the relative concentrations of the binary solvent pair. When the strong solvent (ACN+TFA) is slightly higher than requested, small amounts of TFA will be desorbed. When there is a deficit of the strong solvent, the TFA is adsorbed (to be eluted later). Since TFA possesses a UV signature, this process results in baseline fluctuations (small peaks and dips). There are several factors that influence this behavior. Without the chromatographic evidence, I am only making guesses. However, some actions I would suggest:

Is the column the same in all cases? My guess is probably not. For debugging purposes, try the column used on the 1100 on the ACQUITY. The specific selectivity of the column is one of the factors that impact TFA noise.

Incomplete mixing is another cause of baseline disturbances in TFA gradients. Steep gradients at lower flow rates are very challenging to mixing dynamics. If this is a low pressure gradient 1100, there will be more complete mixing due to the added dwell volume of the pump. A typical, low pressure gradient, 1100 will have a dwell volume just under 1 mL (configuration dependant). A standard ACQUITY will have a dwell volume of approximately one tenth of this, or 100 uL. Without the chromatograms, I cannot estimate the approximate dwell volumes of the two systems, but replacing the standard 50 uL filter/mixer with the optional 100 uL filter/mixer (P/N 205000272) on the ACQUITY system may blend out the disturbances. Even with the added volume, the ACQUITY should still have a much lower dwell volume.

I appreciate that 0.1% TFA has become the "standard" concentration for TFA gradients. However, we are discovering that the smaller volumes of the ACQUITY system allow for a reduction in TFA concentration while still offering all of the chromatographic benefits. Historically, our standard peptide separation called for 0.1% TFA. More recently, this has been reduced to around 0.02% on the ACQUITY system. I understand that such a change may not be an option in the case of your method, but lower TFA concentrations will result in fewer TFA induced disturbances.

It is possible that the observed disturbances are related to the UV flow cell, but cell issues usually manifest themselves differently. Most often, flow cells cause long sweeping deflections of the baseline. It is uncommon that the cell would cause short period, discrete, peaks and valleys. Never the less, you could try changing the cell just to be sure.

Hope this helps,

pcb

RobBurgess

Thanks for your replies.

If this new .jpeg file attachment doesn't work, please let me know.

Alternatively, if you could let me have your email adressses I could send to you direct.

Could this evn be due to the detector itself or flow cell mis-alignement somehow? Has any other users seen this?