Liquid Chromatography

If you use gradient elution for your HPLC separations, sooner or later you will run into problems with peaks that appear in the blank. Variously described as ghost peaks, background peaks or gradient interferences, these peaks can drive you crazy,...

This article discusses pipette calibration that shouldn't be ignored as it may be the key for consistent results.

Q: I have a problem with the linearity of my HPLC method. The method involves derivatization of the analyte, followed by reversed-phase separation with UV detection. To my surprise, the calibration curve fits a quadratic form very nicely, and not a...

Q: I’m hoping you might be able to shed some light on an issue we’ve had with unknown peaks in nearly every method we run in our lab. I’ve been working at µg/mL levels for a cleaning method for one product, while a colleague has been working at...

A reader recently sent me an email with this question: I run an LC-MS system and was recently tempted to try a recent published method using ion-pairing chromatography with 10 mM tributylamine (TBA).After the trial, I converted back to the regular...

Q: I have developed a method based on an article from literature for testing an analyte along with its impurities and degradants. This is a gradient method that utilizes a C18 column with 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in...

I recently had a reader ask me a question about what size the internal standard (IS) peak should be for a bioanalytical method (drugs in plasma). He was torn between advice that the IS should be about the same size as the smallest peak of interest,...

The following question was submitted by one of the readers of HPLC Solutions.

In previous instalments of this Back-to-Basics series, we have looked at the retention factor, k, the selectivity, α, and the column plate number, N. Nice as these measurements are, they don’t tell us much about the quality of the separation. For...

In an earlier article we examined how to calculate peak tailing. This issue, let’s take a closer look at some examples of peak tailing and see what they have to say in practical terms. For purposes of discussion, let’s consider the peaks in Figure...

This technical article shows how excessive peak tailing is an indication that something is wrong and how it is a good idea to include a measure of peak tailing as part of the system suitability measurements.

This technical article discusses what is meant when a column is referred to as end-capped, and what the function of the end-capping is. To understand end-capping, we need to step back and look at the bonded phase on the HPLC column. Reversed-phase...

Previously we looked at the consequences of metal contamination of the silica used for packing HPLC columns. In this article we’ll consider another aspect of the silica – the nature of the silanol groups.

Today we often take for granted the quality of the silica in the HPLC column. This may be a reasonable assumption for the high-quality columns produced today, but it is not a guarantee, especially for columns that have been on the market for more...

Earlier this year, I was in London, teaching an HPLC troubleshooting class in conjunction with Separation Science. I love to teach, so the presentation of short courses is one of the highlights of my job. In addition, these classes often serve as...