Liquid Chromatography

Measuring Dwell Volume

In a previous article (Dwell Differences, HPLC Solutions #85) we saw an example of how the appearance of a chromatogram could change when a gradient method was run on two HPLC systems of differing dwell volume. In this article we’ll look at a simple...

Gradient Distortion

In a previous article (Measuring Dwell, HPLC SOlutions #86) we looked at a simple technique to measure the dwell volume of an HPLC system. In a prior discussion (Dwell Differences, HPLC Solutions #85), it was seen that differences in dwell volume...

Matrix Effects

Recently a reader sent me a question regarding a problem he was having with recovery of a vitamin he was measuring in pet foods. He found that the precision, measured as repeatability of peak area for a given sample, was adequate for multiple...

Standard Additions

In an earlier article (#88) we looked at the need to use a matrix-based calibration curve so as to correct for low recovery when extracting an analyte from a sample matrix. To do this, we used a blank matrix and spiked in known concentrations of...

Linearity Requirements

I recently had an email from a reader who wondered how it was possible to use an evaporative light scattering detector (ELSD) with an HPLC for quantitative analysis when instead of a linear response, the concentration vs response relationship...

Peaks Too Narrow

I recently had a reader send me an email complaining that the peaks in his chromatogram were too narrow. At first, this seems a bit odd – after all, most people complain because peaks are too wide, so narrow peaks usually are preferred over broader...

Calculated Peak Width

In a previous article (HPLC Solutions #91) we looked at a problem where we suspected that the data acquisition rate of the data system was too slow to gather a sufficient number of data points across a peak with a 2-µm particle column. The data of...

Poor Equilibration

A reader emailed in the following question: I am trying to analyze a peptide (2675 Da) on a 150 x 2.1 mm, 3 µm particle column by LC-MS using an ion trap MS in the positive electrospray ionization (ESI) mode. The first injection is OK, with the...

How to Calculate k*

In a previous article (Poor Equilibration) we looked at a problem gradient method where peaks in the second and subsequent chromatograms were eluted too early. Our conclusion was that the most likely cause was insufficient equilibration time between...

UV Conditions

I recently had an “Ask the Doctor” question that went something like this: “I need to report impurities for my product at levels of 0.1-0.5% of the main ingredient. Occasionally when I show my folks my chromatograms, I’m accused of making the sample...

Premix Mobile Phases

A reader asked me when it is appropriate or necessary to premix mobile phases when on-line mixing is available. For example, if a gradient is run from 95/5 buffer/organic (5% B) to 5/95 buffer/organic (95% B), should one bottle contain 95% buffer...

Late Elution

A reader asks, “I have a nice isocratic method for compound A, which elutes at about 8 min, so I can inject a new sample every 10 min. Occasionally, however, my samples contain another compound B that comes out at about 40 min. I have no way of...

Where’s My Pesticide?

A reader is trying to figure out the fate of a pesticide in water. For the test, he takes a soil sample (e.g., 5 g) and adds 100 mL of an aqueous solution containing 10 ppm of a pesticide. He takes 1 mL aliquots of the liquid phase each hour and...

HPLC Resurrection

What do you do if you receive the gift of an HPLC system of unknown breeding? In a recent enquiry, a reader indicated that when he moved into a new lab, the prior occupants left an HPLC system sitting on the bench. They thought it was working well...

Column Regeneration

How do you go about recovering a column that you suspect has some problems? Or perhaps you’ve pulled a used column off the shelf and want to make sure that it will work OK. Do you just dust it off before trying it, or is there a better approach?...

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