Chromatography Forum

Hi folks

We have some problem. we are doing HPLC Asaay of glycerol dibehenate using

column 0.6m x 7mm with styrene-divinylbenzene copolymer R 54um with pore size 10nm

Mobile phase Tetrahydrofuran

Flow rate 1.0ml/min
Detector differential refractive index

Inj vol 40ul

St 0.1g glycerol to 25ml with THF

We have done everything including freshly opened bottles of THF but no response.

THF now we are using that is for HPLC without inhibitor

even higher concentration of glycerol in THF were tried

Would some one help us out

Hi -

As far as I can tell, you're using a size exclusion column for this analyte. It's possible that the GDB is eluting in the void, which on an RI would likely give you no result.

I might suggest analysis using either reversed phase, non-aqueous reversed phase, or normal phase LC. The MW is ca 740, which shouldn't be too large for a typical analytical column to deal with. RI should be fine, though method development will be slow - ELSD (if available) would be quicker.

I'd grab a C8 column and run your sample in THF down that using a THF mobile phase initially, then dilute the THF with neat methanol or methanol/0.1% acetic acid/10% water 'til the GDB is retained.

Hi thanks for quick reply
but
I want to make one correction

column is 0.6m x 7mm with styrene-divinylbenzene copolymer R 5um with pore size 10nm not 54um it was typing error

We are contract lab and unfortunately we have to follow this method blindly

This is Euporean Pharmacopoeial method for glycerol dibehenate

If your problem is " no peaks", then look at the following:

1. Is your detector functioning properly. Test it according to the manufacturer's standard test procedure (not just a "quick and dirty" check!). If it does not pass, then clean it, again, following the manufacturer's recommended procedure.

2. If the detector checks out OK confirm that you are using the exact same column (including the exact manufacturer and part number) specified by the client (not just an "equivalent" column, the exact same kind).

3. If the detector checks out OK and the column is the correct one, then I would contact the client and ask them for a chromatogram run in their lab (to confirm that the method actually worked in their hands). If it has not been run successfully by them, then you have a method development problem. If they have run it successfully, then you will have to go over the hardware, column, mobile phase, temperature, etc. one bit at a time, comparing your system to theirs to find out the difference.

Is this it? (see below, please)

Assuming it is, a series of stupid questions (please don't be insulted):

Is your run time long enough? (Do you see a clear void dip / spike?)

Have you equilibrated your RI sufficiently?

Is your RI properly set up (sensitivity, etc...) to detect the level you're after?

Are you sure your RI cell isn't in purge mode when you're running?

Are you sure your column's in good shape? Have you tested it?

Is the column correctly labeled? I once bought a GFC column that ended up being mislabeled from the factory (incorrect pore size) - what a pain that was!

As far as I can tell, it seems a straightforward enough method...I'd assume preserved THF should be OK to use as BHT wouldn't be retained and your detector is an RI.

Assay

Size-exclusion chromatography (2.2.30).

Test solution

Into a 15 ml flask, weigh about 0.2 g (m), to the nearest 0.1 mg. Add 5 ml of tetrahydrofuran R, warm the flask slightly (about 35¡ãC) and shake until dissolution is obtained. Reweigh the flask and calculate the total mass of solvent and substance (M).

Reference solutions

In four 15 ml flasks, weigh to the nearest 0.1 mg about 2 mg, 5 mg, 10 mg and 20 mg respectively of glycerol R. Add 5 ml of tetrahydrofuran R and shake until well mixed. Weigh the flasks again and calculate the concentration of glycerol in milligrams per gram for each reference solution.

Column:
size: l = 0.6 m, Ø = 7 mm,

stationary phase: styrene-divinylbenzene copolymer R (5 mm) with a pore size of 10 nm.

Mobile phase : Tetrahydrofuran R.

Flow rate : 1 ml/min.

Detection: Differential refractive index.

Injection

40 ml; when injecting the test solution, maintain the flask at about 35¡ãC in order to avoid precipitation.

Relative retentions

With reference to glycerol: monoacylglycerols = about 0.82; diacylglycerols = about 0.76; triacylglycerols = about 0.73.

From the calibration curve obtained with the reference solutions determine the concentration (C) in milligrams per gram of glycerol in the test solution.

Exactly what is the problem?

I don't think he's seeing a peak...

Also...check your injector. If you're not getting your sample into the HPLC, you'll see no peaks!

Hi folks thanks for ur interest

I checked everything regarding detector and injector.
It was a bran new column and after yesterdays attemp i kept 0.1 ml flow of THF for whole night

I got peak but

that is at 20 min instead of 15 min
and
there are two negative peaks right after the main peak

even for THF which is mobile phase when injected those two negative peaks were there almost of the same magnitude.

this is what i am unable to understand. how can i get rid of those negative peaks

The negative peak(s) are most likely your void and there's no way to get rid of them. Just quantify your analyte peak and don't sweat it as long as your analyte is sufficiently resolved from the void. If it isn't sufficiently resolved, get a smaller pore column, run at a lower flow rate, and/or increase your data acqisition rate.

I think you're probably OK...

Sorry to bother u again guys
everything was going well. I started getting response but negative peaks were with the peak of glycerine.

Iadded 4% water in THF and chromatogram became very good with a big difference in retention times with negative peaks

but just now its beahiour is changed base line sharply goes negatine (negative peak) before analyte.

also after sample blank chromatogram was ok but in standard chromatogram it was peak split.

what colu be the reason for this