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Topic: mobile phase in HPLC  (Read 5299 times)

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veronika

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mobile phase in HPLC
« on: October 14, 2005, 12:19:13 PM »
I want to analyse HMF and according to literature there are two ways:
1) Mobile phase is 5% acetonitrile : 95% water. UV detection at 284 nm
2) Mobile phase is 0.08M sodium acetate buffer. UV detection at 280 nm
All analysis are made on ODS column.
As I don't have DAD, just UV detector, I can't see UV spectra of HMF. Is it possible that because of mobile phase I have shift in maximum of HMF in UV spectra (stationary phase is the same) ?
If I want to make analysis on the same column using 10% methanol : 90% water, should I measure HMF at 280 or 284 nm ?
Thanks for the answer in advance.
Veronika
« Last Edit: October 14, 2005, 12:20:54 PM by veronika »

Offline Mitch

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Re:mobile phase in HPLC
« Reply #1 on: October 14, 2005, 12:22:47 PM »
Why do you assume we know what HMF is?
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veronika

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Re:mobile phase in HPLC
« Reply #2 on: October 14, 2005, 02:20:52 PM »
Sorry,
HMF = 5-hydroxymethyl furfural (CAS No. 67-47-0)

Veronika

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Re:mobile phase in HPLC
« Reply #3 on: October 14, 2005, 04:40:28 PM »
It really depends on what you want to do;

1.  If you are looking for trace levels of HMF, then you need optimum sensitivity.  If you don't have a diode array detector, run the sample in a solvent resembling your HPLC solvent (assuming it is not a gradient) on an external UV spectrometer and use the peak maximum.  If you don't have an external UV spectrometer, run iterative runs using the same sample injection size and different nm settings on the HPLC detector and use the nm with the highest response.

2.  If you are looking for a crude purity determination (for example after a lab or process reaction), then you are likely assuming that the impurities have an identical response to your expected compound (which is a bad assumption if some impurities are aliphatic, for example).  The difference between measuring at 280 and 284 nm is inconsequential.

3.  For optimum analytical rigor (for example in the pharaceutical business), all of the unknowns in a process are identified with mass spec, characterized for their molar absorptivity coefficient at a particular wavelength and then quantified using accurate response factors for each of the known impurities.

veronika

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Re:mobile phase in HPLC
« Reply #4 on: October 15, 2005, 06:16:04 AM »
Thank you Dude !
I am looking for a trace levels of HMF. I will run iterative runs using the same sample injection size and different nm settings.
Thank you very much once more !
Best regards,
Veronika

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