Jan 01, 2012

LCGC North America

One of the privileges of teaching liquid chromatography (LC) classes for a living is that I get to meet chromatographers from around the world during my travels. As I write this installment, I'm just finishing six weeks of classes, where I have had a chance to discuss problems that attendees were having with a variety of LC applications. Although the topics in this column originate in Poland, Turkey, and Argentina, they are no different than those that arise in Singapore, Galway, Charlottetown, or Huntsville. Let's look at a couple of problems that students had related to both conventional LC and ultrahigh-pressure LC (UHPLC). These problems apply equally to both techniques.
A good rule of thumb (1) is that you can inject ≈15% of the volume of the first peak of interest without causing a more than ≈5% increase in peak broadening, as long as you use mobile phase for the injection solvent. We need to determine the volume of the first peak of interest to see how this applies in the present case. We will assume a retention factor, First, we need to estimate a column plate number (efficiency),
where Next, it is necessary to estimate the column volume,
where With the knowledge of the column dead time, we can calculate the retention time,
This gives The next to last step is to estimate the peak volume of the first peak. We estimated
where
For Using our 15% rule of thumb from the beginning of our discussion, an injection of (0.15 × 11.8 µL) = 1.8 µL, or ≈ 2 µL would be allowed in the mobile phase. This is less than half the desired 5-µL injection, so it is expected that excess peak broadening would be observed with a 5-µL injection. Armed with this knowledge, I would perform a series of injections of 1, 2, 3, 4, and 5 µL and observe the effect on the peak width of the first peak of interest. I agree wholeheartedly with Izaak Kolthoff, the father of analytical chemistry, who is credited with the quote, "Theory guides, experiment decides." So, use the results of the injection-volume experiment for the final decision. |