Technical Tip: Split Peaks


Do your peaks need couples therapy?

Nothing a little personality assessment can’t handle!

There are a few possible causes for such troubled peaks and, luckily, a few ways to overcome them.

Problem #1: pH of the mobile phase is close to the pKa of a functional group on the compound, or inadequately buffered.

Solution #1Adjust the mobile phase pH +/- 2 units above or below the pKa of the compound’s ionizable functional group, and buffer at that pH with an appropriate buffer (of sufficient buffer capacity to ensure the pH is controlled to the desired pH).

Problem #2A physical blockage is obstructing the flow path.

Solution #2Preventative measures such as filtering the samples prior to injection and/or using a guard cartridge to protect the column are the best steps to take.

Problem #3There’s a void or channel in the column.

Solution #3Discard column and replace with a new one. A void or channel in the column packing bed cannot be repaired or regenerated. If this occurred prematurely (expected column lifetime is quite application-specific), consider the mobile phase components, backpressures, and sample to confirm if the running conditions are within the tolerances of the particular column.

With these simple troubleshooting steps, you’ll be sending your peak on a second honeymoon!

3 Replies to “Technical Tip: Split Peaks”

  1. Good morning.
    in our analyses of substance Prednisolone on column phenomenex luna c18 column we should have peak-to-valley ratio=3 as result. Instead we did not have any separation between peaks of prednisolone and impurity A. Please help to find out the reason of the problem.
    P.S: in another column recommended by EDQM we have peak to valley 2,7.

    thank you

    1. Hi Olga! Thanks so much for reaching out.

      Peak to valley ratio requirements are commonly used when a critical pair has a wide range in response (large peak next to a small peak), which can make traditional resolution calculations ineffective. The same contributing factors will influence this (efficiency/peak capacity, peak shape, and selectivity). If all peaks are sharp with good peak shape, then selectivity would be the primary influencing factor. More likely for this EP method, it is broader or tailing peaks that are contributing to a peak to valley failure, and this particular method can be challenging to run on a traditional HPLC instrument.

      If this is a first trial, I would encourage exploring Kinetex 5u C18 150 x 4.6mm, which will give much more efficiency, lower backpressure (shorter length than the 250mm), and is still within the allowable changes of the EP.

      If you are running this routinely without issues, please submit your serial number with some illustrative chromatograms here:

      Let us know if you have any other questions! We are happy to help.

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