DAB by HPLC

[Perreman]

Hi everyone.

We've received a sample consisting of waste which may or may not contain 3,3'-DAB. Do anyone have any experience with analysis of DAB with LC-UV? And do anyone in that case have any method parameters to share?

We don't have a lot to go on from the customer, we dont know what else the waste do contain so were working a bit blindfolded. However, we've started analysing a reference and the samples using:
C8 column 150x2,0 mm x3µm. Column oven at 50 C
Mob. A: H20+0,1% TFA
Mob. B: 100% Acetonitrile
Gradient: 0-15 min 100% A -> 95% A/5% B, 15-20 min 95% A/5% B -> 100 % B. Keeping 100 % B to 24 min, 24-25 min 100% B - 100% A. Using PDA, detection at 254 nm.

Any suggestions would be very appreciated!

[Consumer Products Guy]

Any suggestions would be very appreciated!

My suggestion would be to explain what DAB is. And for others to also use names of compunds and not abbreviations in the titles of posts.

[sepscientologist]

If your analyte is diaminobenzidine then I think your method ought to work but it is far from perfect.
When does your reference sample of DAB come out? The first 15 minutes changing aqueous from 100 to 95 is
probably a waste of time. Start even higher in B. Going all in to 100% B in five minutes might be too fast.
Try stretching that out to at least 10 to 20 minutes during exploration to see if anything is coeluting.
Just my $0.02 worth

[Bintang]

Are you doing any kind of sample prep?
If it is diaminobenzidine then it is chromatographable in both HILIC and RP mode if the sample is dirty you could clean up your chromatogram by doing sample prep in one dimension and separation in the other.

[Perreman]

DAB, 3,3'-diaminobenzidine as sepscientologist figured it was.

I have changed the column to a C18, 250x4,6mm x5µm and the retention time is approximately 7 min. Unfortunately one peak from the samples would co-elute with DAB if I use higher %B in the gradient, but I do agree that it would have been more effective if I had used higher %B from the beginning.

The sample doesn't seem to be that dirty, and there doesn't seem to be any DAB in the samples (which falls in line with what customer thought it would). We didn't receive any information at all of what the sample consisted of, so I've refrained from doing any preparation either that filering it, which seems to have worked well.

It seems as we're doing ok with this method (slight changes of gradient times than mentioned before). Thanks for your advice anyhow, they might still come in handy. I just hope nothing screws up during the run on the weekend! (you never know...)