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DAD_VWD
Posted: Sat Dec 25, 2010 3:42 pm
by mohammad
Dear all,
Does any one has a remedy for my problem?
01) I have 2 HPLC (make Agilent) one with DAD & other with VWD, the problem is when ever i am performing Ampicillin Trihydrate related substances (gradient)analysis as per EP 6.0 on DAD, i have observed a negative baselines between retention of 22 & 24 minutes. Where as, when the analysis of same sample performed on VWD, i have ohserved a peak between 22 to 24 minutes. The interesting part is the blank/Mobile Phase-A in both the system shows a straight line at above mention retention time.
Please help!!!!!!
Regards
Mohammad.
Re: DAD_VWD
Posted: Sat Dec 25, 2010 4:29 pm
by tom jupille
Check to see if the reference wavelength is set on the DAD. If the peak at 22-24 minutes absorbs more at the reference wavelength than at the measurement wavelength, that will cause a negative offset.
If the reference is set, turn it off or set it to a different wavelength.
Re: DAD_VWD
Posted: Sat Dec 25, 2010 9:05 pm
by Consumer Products Guy
Exactly what I was thinkin' ....
Re: DAD_VWD
Posted: Sun Dec 26, 2010 3:18 pm
by mohammad
Dear Tom Jupille ,
Than q for your valuable guidenace. i have set the refrence wavelength to "off". Now the problem is solved.
With best regards
Mohammad.
REFERENCE WAVELENGTH UV / VIS (HPLC)
Posted: Tue Dec 28, 2010 2:53 pm
by HPLCCONSULT
Just saw this post and it reminded me that this is one of the most common problems we see in the chromatography laboratory. Since twenty years of requesting that the various vendors make the default value of the Reference Wavelength 'OFF' has been ignored, we instead do our best to educate client's about this feature.
*For future reference (no pun intended), here is a link which briefly describes the feature.
http://www.lc-ms.com/Tip%20102%20Refere ... %20DAD.htm
Re: DAD_VWD
Posted: Tue Dec 28, 2010 3:24 pm
by MSCHemist
In Agilent Chemstation the option is off by default. I never use the reference. I run filtered citrus juices and find there are too many matrix interferants to be subtracting out things. On cleaner samples though, I could see the value in it. I have a gradient method at 210nm that the baseline is very slanted.
Re: DAD_VWD
Posted: Tue Dec 28, 2010 5:43 pm
by HPLCCONSULT
MSCHemist: Sorry, but all versions of ChemStation software that I am aware of set the reference wavelength default to 'ON' (since the 1990's). The only version that I have not checked is the latest version, B.04.03. All the rest have reference 'ON' for the DAD/MWD units (Def LC Method is ON). *This is one of the main reasons it is a problem.