I think I've seen it previously, but the current example is from this publication
https://www.mdpi.com/1420-3049/14/5/1825
where they used the following gradient on HPLC:
min ... % MeOH
0 ... 2
20 ... 55
30 ... 35
Based on the chromatograms in Fig 5, they eluted compounds up to 40th min, so that's probably not equilibration to starting condition (plus, those are obviously not starting conditions).
So what would be the reasoning to go with the elution buffer first up and then down again?
As presented, I would not use the paper as a reference. Too much missing information. It does not appear to follow some basic chromatography fundamentals. The authors do not provide any column dimensions (that is a rather important omission). The use of 210nm for all samples makes no sense for their samples, as they ALL absorb at 210nm so the wavelength used is not selective and subject to error. It implies little training in HPLC as does their odd change in gradient profile (*as you mentioned. Maybe it was a typo or they wanted to slow down the elution rates for the last few peaks to improve resolution? This is rare, but sometimes their is a good reason for the change. e.g. Adjusting the slope of a gradient midstream to increase or decrease retention after some of the compounds of interest have eluted. Probable reason in their case, but a linear slope should have worked fine for this. It looks more like a lack of experience more than anything). Unfortunately, many published papers and articles are found to be missing key information or are invalid. Publication in any form does not in fact add to their credibility. An understanding of the basic fundamentals is the tool by which we check the methods and findings with. Sometimes they just do not make scientific sense.
*Update: The copy of the article that I initially downloaded omitted the column dimensions (now I see them shown in the pdf copy, and that they were 2.1 x 250 mm, 5u which is appropriate for use at ~ 200 ul/min). Also, their injection volume of 20 ul is past the acceptable max volume for such a small column. The method analysis at 210nm (Fig 7) shows the expected poor quality baseline and does not appear to be 'improved'.
Dear Tomáš,
let us assume that they aimed simply to decrease the elution power in their reversed phase chromatographic mode after time 20 min to improve the resolution between the eluted peaks after that time.
Best regards,
Sami
Dear William Letter, thank you for your input. I was also wondering about the columns, but they do mention it in the Methods section (250×2.1 mm; 5 um columns). And the pick of 210 nm seem odd as well. Like for standards, that would be OK, but for real samples... anything will absorb there.
What's so strange about the mobile phase and ternary mixture? We use something similar and ternary mixture as well.
Sami El Deeb OK, but shouldn't such compounds be already eluted by higher %MeOH? In other words, would they get eluted with such low concentration, if they weren't already eluted?
Well, now that I'm thinking about it, I wouldn't care if they used isocratic elution to improve the resolution, so going even lower is possibly not *that* strange idea...
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