Liquid Chromatography

Separation Science HPLC Solutions

Separation Science HPLC Solutions is produced in collaboration with John Dolan, best known as one of the world’s foremost HPLC troubleshooting authorities. He has been responsible for training of thousands of scientists over the last 30 years, and is also known for his ongoing research with Lloyd Snyder, resulting in more than 100 technical publications and three books.

Recent Posts

Injecting Organic Solvents in Reversed-Phase

A reader wrote me recently asking what would happen if he injected his sample, which came dissolved in hexane, into a reversed-phase mobile phase of methanol-buffer. The first answer is that this is not a good habit, but is it possible? We’ll see.

Column Abuse, I: Does Air Damage the Column?

I recently had an inquiry from a reader of 'HPLC Solutions' that comes up with some regularity in the HPLC troubleshooting classes that I teach: “Will I damage the column by getting air in it?” Let’s consider the possibilities for a moment. One...

Column Abuse 2: Column Storage

A reader asked me if she was in danger of shortening the lifetime of an HPLC column by removing it after each batch of samples and storing it. This also raised the related question about expectations of shorter column lifetimes if the same...

Column Abuse 3: Oops, I Dropped It!

What happens if your favourite HPLC column accidentally rolls off the bench and lands on the concrete floor of the lab? Is it ruined?

Readers Share In-Line Degasser Experiences

After I wrote an article on in-line Degasser (HPLC solutions #72), I immediately received some reader feedback on a specific failure, which was published in 'Failed Degasser' (HPLC solutions #73). Since that time I have been collecting occasional...

Column Blockage

A reader reported a problem blockage of a column packed with one of the newer shell-type packings. The mobile phase A comprises a mixture of 0.1 M KH2PO4, 0.05% triethylamine (TEA), and 150 mg/L EDTA adjusted to pH 7.5 with NaOH. The B-solvent is...

Method Limits

A reader recently emailed me a question that went something like this: “I’m using LC-MS/MS to analyse a biomolecule that I have isolated from tissue by LC-MS/MS. The lowest point on the calibration curve that I prepared is 0.1 µM. All the samples...

Peak Purity

A reader recently asked a question about the use of the peak-purity function of his diode-array UV detector (DAD). The question related to whether or not he could detect the presence of enantiomers, stereoisomers, diastereoisomers, or epimers...

Dwell Differences

The dwell volume comprises all the HPLC system volume between the point the solvents are mixed to the head of the column. For high-pressure-mixing systems, this includes the mixer, connecting tubing, and sample loop. For low-pressure-mixing...

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...

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...

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