Liquid Chromatography

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...

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...

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...

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...

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?...

Estimating Column Volume

A question that comes up now and then in our Master Classes is how to figure out the volume of an HPLC column. I’d like to share a couple of rules of thumb that I find very useful for estimating column volume, VM. Figuring out the volume inside...

Acid Wash

Did you ever feel like someone was out to get you? I remember the story one of my Master Class students told about leaving on a vacation. She left instructions with her lab mate to shut down her HPLC system the following morning after all the...

Blocked Detector Cell

A reader recently asked, “The flow cell on my UV detector is completely blocked with some kind of white substance. I tried using a syringe to flush with water and with isopropanol, but neither was successful (and the process was very slow). The...

Choosing the Injection Solvent

A reader recently submitted this question: “Most HPLC methods call for preparing the sample in mobile phase as the solvent. I like to avoid using some of the organic solvents for sample preparation because of toxicity issues, so most of the time...

Flow Splitters

A reader asks, I would like to split HPLC column effluent between an evaporative light scattering detector (ELSD) and a fraction collector. What is the best way of doing this, particularly as we may want a biased split, i.e., 10% to detector and...

Too Much Ion Pairing Reagents

A reader asks, “I am using a shared equipment where somebody used tetrabutyl ammonium (TBA) as an ion pair in their mobile phase for chromatography. It looks to me like the ion pairing reagent was not washed completely from the column, even...

Subscribe to FREE learning from the experts!