Column 1.0 x 100 mm

<p>Which would be the recommendations for the column 1 x 100?</p><p>I want to develop a method with the same one.</p><p></p><p>(inj.vol, loop and needle volume, mode injection, outlet tubing, inlet filter?)</p><p></p><p>Manuel</p>


  • Greetings Manuel,

    I hope you did not grow too weary waiting for an answer. I've been meaning to respond for several days, but work and life have prevented me from doing so. One mm columns are challenging because they are very sensitive to extra column volume. That is, all of the system volume that is not related to the column, itself. The contributors to extra column volume are all tubing and devices from the injection valve to the detector(s). In an ACQUITY UPLC system, this includes the injection valve, the sample loop, the solvent stabilizer, the column outlet tubing and the detector cell(s). The last item, the detector cells, is true unless your application employs MS detection only. In that case, there are no detector "cells", but there is a longer piece of connection tubing to the MS. The reason why extra column volume is so critical is that it determines system dispersion or, as it is called, extra column variance (? 2) (also known as band spreading). In addition, the manner in which ACQUITY transfers sample into the sample loop can, slightly, increase sample dispersion if not managed properly.

    The reality is that the ACQUITY is not scaled, in an optimum fashion, for use with 1 mm columns. The standard ACQUITY is, primarily, plumbed with 0.005"X1/16" stainless steel tubing. We made this choice for historical reasons and to make tubing connections easier to accomplish. There are limits to how precisely 1/16 inch stainless steel tubing can be manufactured. The practical limit is +/-0.001". This means that, nominal, 0.005' tubing can range from 0.004" to 0.006". This doesn't seem like a lot, but it can have profound impacts on system back pressure and system dispersion. We have investigated using smaller (nominal) ID tubing, but the +/-0.001" tolerance limit begins to become a very large percentage difference (+/-50% for 0.002" tubing) and begins to create performance inconsistencies from system to system. This is not what we (Waters) desire for the ACQUITY UPLC product line.

    I do not know what you have in mind for a chromatographic method, but I strongly suggest the use of a gradient with 1 mm columns. If the gradient is constructed correctly, it can help to, significantly, reduce the extra column variance prior to the column. If the initial solvent conditions are weak enough they will focus the sample on the head of the column. This will mean that you will, effectively, "buy back" most of the sample dispersion that occurred before the column. The initial solvent conditions should be just strong enough to keep your sample in solution, but not strong enough to initiate elution from the column. Even if your, original, concept was for isocratic chromatography, I would still recommend the use of a "step" gradient where the is sample loaded onto the column with a weak mobile phase and then the mobile phase conditions are immediately changed to the elution conditions.

    So, after this long winded preamble, these are my suggestions. Use some form of gradient chromatography per the above. Install a 15 uL needle. Your injection volumes will all be around 1 uL, or below. Because of this, you should install the 2 uL or 1 uL sample loop (I'd use the 2 uL loop). Install the 50 uL sample syringe. All of these changes will increase the time it will take to characterize your system, but it is something you only need to do once (or, maybe, twice for accuracy). Install 0.0025" PEEK tubing after the column to connect to the detector. Use the Partial Loop with Needle Overfill (PLNO) injection mode unless you are, truly, sample limited. If you have very little sample and cannot sacrifice the 15 uL per injection that PLNO requires, the overfill volume can be reduced. However, this may increase sample dispersion and decrease injection precision. If you cannot use PLNO at all, you really must use some type of gradient scheme to focu...

  • I should have been a little more precise with my language in the last paragraph. The injection mode that is most favorable to dispersion is full loop injections. If you have no need for variable injection volumes, I would employ full loop injections with the 1 uL sample loop. Even with the full loop overfill factors you will not use as much sample as you would use in PLNO. If you cannot use either Full Loop or PLNO and must use Partial Loop with pressure assist, then the use of the gradient focusing technique is, essentially, mandatory for maximum column efficiency on 1mm columns.



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