Chromatography Forum

Hi,

I have developed a method for the separation of Eugenol from Clove Oil. This is for an undergraduate pharmacy practical. The problem is we have three systems (Perkin Elmer Series 200, autosampler, UV, pump) and are using a C18 15 cm x 4.6 mm colum with a TFA/MeOH gradient. We are getting a good separation on all three systems but with varying retention between systems. I have switched the columns between systems but the retention for that system stays the same. The three retentions are 5, 6.5 and 8 minutes. For the purpose of the practical this isn't an issue but sure as guns the undergrads will ask why there friends peak is at a different RT.

The only things I can think of are that either the tubing between systems is extremely varied (unlikely since they are all new systems within the year installed by PE engineers) or that the pumps proportioning valves are all operating slightly different.

The batch of mobile phase is also identical.

Cheers

James Mann
University of East Anglia

Is this this right: You have three identical systems; system1 gives an RT of 5, system2 gives 6.5, and system3 gives 8 minutes for your peak of intrest?

What about the RT's of the first peaks (unretained?) in the chromatogram of each system?

I would first look at the autosampler...Maybe one system has a bigger loop, etc?

OK - let's assume that the systems are identical and that you've run uracil injections on all 3 to confirm this (Uracil is basically unretained, so it will give you an idea of your total system volume).

We'll also assume that you've used volumetric glassware and a stopwatch to confirm that all pumps are running the same.

How about other environmental parameters? Are the systems in the same room? If not, what are you doing to ensure that they're all operating at the same temperature? If they have column compartments, how close are they in temperature?

Actually, the retention time for uracil will tell only part of the story: it will identify flow rate errors (remember, though, he's running a gradient, so a flow rate error will not translate directly into a retention time error the same way it does for an isocratic separation).

The other part(s) of the story are the gradient delay volume (also called "dwell volume") and, as suggested, the proportioning accuracy of the pumping system.

If this were my problem, the first thing I'd do would be to determine the dwell volume on each of the three systems. The general procedure is to remove the column, put something UV-transparent in A and something slightly UV-absorbing in B, the run a gradient and use the UV detector to monitor the composition. What you're interested in is the time it takes the gradient to get to the column. If you want an explicit procecedure, go the Rheodyne web site (they're at the top under "Sponsored by"), click on Products . . . Software, and download the evaluation version of the DryLab program. The help file includes instructions on measuring dwell volume.

Assuming that dwell volume differences don't explain all of the retention time shifts, the next thing to do is to program a series of 5% or 10% steps (same setup as above). If the proportioning system is working correctly, each step should show the same shift in absorbance.

Thanks for your help. I am measuring the dwell volumes of the three systems, if they are the same then I will try the step up procedure.

The columns aren't thermostatted but the three systems are located on the same bench so I don't think temprature can explain a retention difference of 3 minutes.

Cheers

James

Hi,

If you find that the dwell volumes are the same on all three of your systems I would suggest the you run a gradient formation check.

To do this set mobile phase A to 2% acetone/ water mixture and mobile phase B to acetonitrile. (Ensure that your system is properly purged of air)

Switch your column to a backpressure regulator (if you don't have one of these you can use a column instead)

Configure your UV detector to acquire at 254 nm.

Set up and run the following gradient (ensuring that the gradient steps are linear and not exponential or stepwise etc), acquire the UV trace. (If you cannot aquire the UV trace without injecting a sample inject a solvent blank to trigger the acquisition).

Flow rate = 1ml/min

Time mins %B

0 0
5 25
10 50
15 75
20 100
25 100

You should observe a UV trace where absorbace increases stepwise proportionally to %B increase, all three instruments should produce comparable results. If the steps are not linear this is indicative that your pump is not working properly.

I am not familiar with your pumps however outlet check valves would be a good place to start your troubleshooting...

James,
Is there any other information about Clove Oil that you are generating in this analysis, other than eugenol content?
(I routinely use GC to separate other volatiles in this oil and was just curious)
WK

Thanks for the advice Richard. If the dwell volumes match then we will try the run you suggested.
WK, no the only information we are after is the Eugenol concentration. We run an external calibration of pure Eugenol, and then inject clove oil from sigma and then the clove oil they have manufactured in the practical and compare concentrations. Its a 2nd year Pharmacy undergrad practical

Cheers

James