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Gradient DistortionIn the last installment o HPLC Solutions (#86) we looked at a simple technique to measure the dwell volume o an HPLC system. In a prior

discussion (#85), it was seen that dierences in dwell volume between two HPLC systems could result in oset retention times as well aspossible changes in resolution or early-eluted peaks. Another potential problem can be diagnosed using the same dwell-volume test, as isdiscussed below.

The SetupIn the current example, the experimental setup is the same as that o thestandard dwell-volume test discussed last week, with the exception that thecolumn was let in the system. Instead o water-based solvents, this test needsto be done with methanol-based solvents, because the column is installed. Souse A = methanol and B = methanol + 0.1% acetone. In this case, the columnwas 100 x 2.1 mm, operated at 0.2 mL/min on a system with 1 mL o dwellvolume (1). In the upper chromatogram o Figure 1, as can be seen by the redline, a 20-50% B gradient was programmed over 10 min, resulting in a gradiento 3% B/min. There was a short isocratic hold, then a step back to the initialconditions. In the lower chromatogram, the gradient was 20-40% B in 1.5 minor a 14% B/min gradient. A 3-min isocratic hold was ollowed by a step back to the initial conditions.

The ResultsIn Figure 1, the blue trace shows the gradient as it appears at the detector. Inthe ideal world, this would be expected to closely track the gradient program(red line). In the upper chromatogram, the blue line ollows the red one quite

well during the gradient portion. On the step back to the initial conditions, ittakes a while or the actual gradient to catch up with the program. But this isnot surprising – ater all, isn’t this why we wait between runs or the columnto re-equilibrate? However, note the contrast with the lower example. In thiscase the actual gradient cannot keep up with the program, and it takes theentire 3-min hold or the gradient to reach its maximum concentration. So,although a linear 14%/min gradient was programmed, this is not at all whatwas produced by the HPLC system. Imagine how dierent the chromatogrammight look i it were developed on a system that closely tracked the program,similar to the top case, and were transerred to a system that gave a distortedgradient, as is the lower case!

Furthermore… The scary part about this example is that it is common to program a stepgradient as part o a gradient method. This might link two isocratic segmentsor might be a very steep gradient beore or ater a shallow segment. HPLCsystem controllers allow you to program any gradient conditions you want –even i they are not realistic. So the take-home message here is that i you plan

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to use steps or steep segments in your gradient programs, it is a good idea touse the methanol-acetone gradient technique to see what the gradient reallylooks like. I your system does not produce gradient segments that closelytrack those you have programmed into the system, you could be in or a veryunpleasant surprise when you try to transer the gradient to another HPLCsystem that may have dierent mixing characteristics.

Reerence(1) G. Hendriks, J.P. Franke, and D.R.A. Uges,  J. Chromatogr. A,1089 (2005) 193.

John Dolan is best known as one o the world’s oremost HPLC troubleshooting

authorities. He is also known or his ongoing research with Lloyd Snyder,

resulting in more than 100 technical publications and three books.

Contact John at TechTips@sepscience.com

Figure 1

0 5 10 15 20 25time (min)

0 3 6 9 12 15

time (min)

     %     B

     %     B

20-50%B/10 min

3%/min

20-40%B/1.5 min

14%/min

A

B

D

C

A

B, C

D

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Figure 1: Gradients run on a 100 x 2.1 mm column at 0.2 mL/min with a 1 mL dwell volume system. Adapted from (1).

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Tech TipIn the next HPLC Solutions John Dolan looks at areader’s problem with low recovery of a vitamin frompet food.

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