By Anonymous on Monday, May 31, 2004 - 10:00 am:

i'm in a bind, yes i'm being strongly retained by this project even though i thought i could get through it fairly quickly *groan* and am looking for some directional advice..

i'm trying to prep a neutral species (MW 390) by reversed phase HPLC. i have ~2g of material to get through the column and am trying to figure out the best approach to minimise the number of runs needed. we have a simple 50:50 H2O/MeCN mobile phase which when run isocratically will bring out the cpd of interest (dissolved in MeCN) at k'~8 on the analytical and prep column at low load.

the sample is soluble to >400 mg/ml in MeCN. solubility is ~150 mg/ml in 25:75 H2O/MeCN and presumably <150 in the MP (for this discussion let's call it 100 mg/ml in MP.)

what steps should i take from here?

if i try to determine the loadability of the sample in pure MeCN on the analytical scale column i might find problems when scaling that up with the strong injection solvent..

determine the loadability, via increased injection volume, on analytical column of sample (close to saturated soln) dissolved in MP? scale up, with my fingers crossed, and hope for no peak distortions? :-)

The HPLC system only has a 1000 ul syringe and loop. Maybe i could have a low %MeCN hold at the start if i knew how to do repeated injections in the same run..

the column of choice is a Kromasil 5um C18, 100A 250 x 21.2 mm with an analytical equivalent of 250 x 4.6 mm.

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By Anonymous on Monday, May 31, 2004 - 10:05 am:

i just figured for increased injection volumes i could bodge it with a low %MeCN method of around 1 minute then another method in series with short low %MeCN hold to bring subsequent injections to the head of the column before stepping up to 50:50 for elution \o/

that's while i look into larger syringe and loop for the sample manager :-)

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By Uwe Neue on Monday, May 31, 2004 - 11:26 am:

for problems of this type, we have developed the at-column dilution technique. You may be able to implement it with the equipment that you have.

The sample is dissoved in an organic solvent, such as the acetonitrile used by you. The injector is put into the stream of acetonitrile used for the mobile phase. Just in front of the column, you have a T-connector that mixes this stream of acetonitrile with the water, 1:1 in your case. The sample is therefore carried to the column top in the intended mobile phase and is either retained or precipitates. Either is OK, since you want it to be retained on the column top.
Since I do not know your equipment, you need to figure out yourself what you need to do to plumb it. We use a very short connection between the point of mixing and the column top to prevent clogging of capillaries. Using this technique, you can slowly increase the load to see how far you can go from the standpoint of resolution in the chromatogram and solubility of the sample.

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By Uwe Neue on Monday, May 31, 2004 - 11:28 am:

PS: we had a publication in Chromatographia Vol 57 (2003), S121-127 on the subject.

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By Anonymous on Monday, May 31, 2004 - 01:26 pm:

thanks for the reply Uwe. I've read your publication and would like to implement the technique you describe there. i don't think i can sort it out for this particular job though (time).

because i'm limited to introducing the sample in the traditional manner, i am looking for advice on which approach others would take in terms of finding out the loadability limit of my method.

bearing in mind the aim is to use a little material as possible until the point at which the prep method is "ready to run" i feel i'm forced to study the analytical loadability in MP and scale up the injection volume only.

please feel free to add to the discussion

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By Uwe Neue on Tuesday, June 1, 2004 - 04:16 pm:

OK, I would take the sample at the solubility limit in acetonitrile, the sample at the solubility limit in the mobile phase, and also a sample at the solubility limit in about 30% MeCN 70 % water and inject increasing amounts on the analytical column. Let us know, which of the three solution wins the loadability contest!