Weak/Strong wash question

MikeT

<p>We have a UPLC/TQD and I need some help figuring out what to use for weak and strong needle wash. When they set up our instrument they put the weak wash and seal wash lines in the A, aqueous bottle and the strong needle wash line in the B, organic bottle. So right now the weak wash and seal wash is still in A which is 5 mM ammonium acetate and the strong needle wash is in B which is Acetonitrile (100%). One method I'm doing is an isocratic 30:70 and the other a gradient of 20:70 to 100%B.</p><p></p><p>But now I will be trying a method that will use methanol for B rather than Acetonitrile. So I'm assuming I need to change the wash solutions when I change over the mobile phase. I figure on getting separate small mobile phase bottles just for the wash lines.</p><p></p><p>Here's the question. What exactly should the strong wash be? I cannot find a clear answer. The Acquity UPLC System Quick Start Card is confusing. I get what to use for weak wash. But what for strong wash? </p><p></p><p>And if my weak wash is supposed to be as the initial gradient, which is 30:70, isn't that practically the same the strong wash I'm using now? I thought the weak wash is supposed to wash off the strong wash. Well 30:70, 70% acetonitrile seems rather strong to me.</p><p></p><p>TIA</p><p></p><p>Mike</p>

lizh

Mike:

The first question to ask is what injection mode are you using? And what is the method in detail is it just water and organic. The injection mode predicts how the sample will be introduced and hence how best to get rid of the sample left in the injection system.

Basically, the washes are designed to deal with carryover. This if you are not getting carryover I would not worry!

Full Loop - overfill the sample loop (usually 3 to 4X) to ensure that contents are uniform -Advantage: best precision, lowest dispersion -Disadvantage: sample consumption and multiple sample loops are required for multiple injection volumes.

Partial Loop Uses Needle Overfill - sampling needle draws extra sample to "rinse" the needle ensuring that the volume drawn into the sample loop is homogeneous -Advantage: good linearity, precision and accuracy. Low dispersion and sample sees only weak wash and initial mobile phase Disadvantage: sample consumption

Pressure Assisted Partial Loop - sample volume is drawn and placed between air gaps then transferred to the center of the sample loop. Advantage: lowest sample consumption, pressure assist reduces cycle time, good linearity and precision -Disadvantage: weak wash solvent, mobile phase and sample are co-injected, recoveries are method dependant

As a general rule weak wash is the weakest gradient condition and strong wash the strongest gradient condition. For isocratic the weak wash can be the same or weaker and the strong wash can be the same or stronger. However, if your sample, or in some cases one of the components is sticky you need to remove them and so strong wash should be as strong as is necessary. In the injector strong wash really serves to get any lingering sample out of the system and compatibility should be with the weak wash. As a MS method we often suggest putting Formic Acid, if used in the method, in the strong at reasonable high conditions 1 or 2%. I am going to assume that you are using PLNO and in this case the sample diluent does not really see strong wash and it can be pretty strong, so do not worry.

Hope this helps.

Liz

Edited by: Elizabeth Hodgdon on Dec 24, 2008

ACQUITYEPM

Greetings Mike,

All of the information supplied by Liz is accurate. I offer a few additional pieces of information. We do not, typically, recommend buffers in the wash solvents (seal or Sample Manager). However, this precaution, primarily, speaks to nonvolatile buffers (phosphates etc.). Your ammonium acetate should be fine, but if you do not feel you need it for sample solubility, you might consider excluding it from the wash solvents. All of the recommendations regarding the weak and strong wash solvents were developed prior to the introduction of the Partial Loop with Needle Overfill (PLNO) injection mode. The Partial Loop with pressure assist (PA) injection mode does co-inject weak wash solvent with the sample. For this reason, it should neither be too weak (samples falling out of solution) or too strong (early eluting peak distortion). The PLNO injection mode injects only sample and mobile phase. It (PLNO) allows a free hand when it comes to wash solvent selection. With PLNO you can go as strong as you need to prevent sample carry over. PLNO even allows the two wash solvents to be exactly the same and as strong as is necessary.

Hope this helps,

pcb