Chromatography Forum

hi everybody,
I'm an intern and for my graduation paper I have to determine a UV-stabilizer (HALS). They gave me the tip of detecting it with an ELSD, but nobody in the lab is familiar with the detector. Please understand that I'm kinda new in all of this, I'm graduating from university this year.
I found out which peak is the UV-stabilizer, it elutes at +- 35 minutes but it's very wide and the baseline is very noisy.

I'm using a Waters 2424 ELSD, the column of the HPLC is a C18 of 15cm.
the drift tube is on 60°C
the nebulizer is on cooling
gain 3
The Hostavin N30 is solved in tetrahydrofuran with concentrations of 10, 100 and 1000 ppm.

I use a mobile fase gradient:
A: milli q water + 0.5mg/l n-hexylamine
B: acetonitrile + 0.5mg/l n-hexylamine
C: isopropanol + 0.5mg/l n-hexylamine

According to the journal I've been following, the hexylamin is to block some C18 groups. Otherwise the stabilizer wouldn't elute.

time - A - B - C
0 - 30 - 70 - 0
2 - 30 - 70 - 0
34 - 0 - 100 - 0
36 - 0 - 0 - 100
41 - 30 - 70 - 0
45 - 30 - 70 - 0

Does somebody have any tips on getting a better baseline and why it is that the peak of the stabilizer is so wide?

thank is advance!

Hi Dana,

I think the tip you received is a bit odd. UV-stabilizers work by absorbing UV themselfs. So, what's more logical than using UV detection?
As for the peak width - well it's independant on the detection technique. If it's broad when detecting it by ELSD it'll be broad when UV is utilized and vice versa. So, you'll need to revise the chemistry in that context.

Best Regards

It's an UV-stabilizer from the HALS type (hindered amine light stabilizer). These work as free radical scavanger, they don't absorb UV-light. In fact, this UV-stabilizer doesn't absorb UV at all (I tested it), that's why we chose for ELSD.

Your peak may be wide because your retention time is pretty long for this column/mobile phase (I'm assuming it's a 4.6x150mm and you're running at 1 mL/min, so that assumption may be wrong). If you have no other competing peaks in your chromatography (if your matrix is clean, no other contaminant or target peaks), you could try a more aggressive gradient or an isocratic mobile phase with a higher proportion of organic, or a different column with a different selectivity to elute your peak faster. You could also try reducing the concentration of the n-hexylamine in your mobile phase to get less retention with your current gradient profile. You may also want to check out this paper (if you haven't already):

http://www.sciencedirect.com/science?_o ... archtype=a

I have no experience with the ELSD, but I suspect danko is right - the poor peak shape is probably chromatographically based, and not a function of the detector.

The first return in google also mentioned that HALS don´t absorb UV. I can´t see why they would not adsorb quite well at around 210 nm or below. Anybody have info on that?
Also, I have trouble to understand how hexylamine is blocking some C-18 groups, I have used amines only to block SiO-.

Dana,

OK I didn't look up this compound, so it was just an assumption - that it worked by absorbing UV light. So, the detection technique must be OK.
As for the chromatographic efficiency (or rather the lack of it) you still need to play around with the system chemistry.

Best Regards

P.S. Blocking the C18 groups is just some misconception

So actually the n-hexylamine is useless here?

@ HW Mueller: I tested it, the HALS doesn't give a signal in UV
@ bisnettrj2: I was using this paper already, thanks anyway

Nobody said the hexylamine is useless here, the explanation of its function is what bothered me. Now, especially with modern columns, one usually controls silanol/silanolate effects via pH and/or ionic strength rather than by the addition of an amine.
It would be interesting to get more info on your UV experiment: wavelength range and absorbance at what concentration. . . . .

I think the thread may have gotten a little off-course here. The original question was, essentialy "is my detector functioning properly, and are my settings correct?" Like I stated before, I have no experience with an ELSD, but I did a little poking around on the Waters website.

The operator's guide to the detector is here: https://www.waters.com/webassets/cms/su ... 1802rb.pdf

Page 3-31 of the manual details how to use the detector to auto-optimize the gain settings.

Page 3-45 gives a periodic maintenance guideline for the ELSD.

Page 5-2 begins the section on instrument diagnostics.

Page 5-21 begins the section on chromatography troubleshooting.

Chapter 6 gives a complete overview for optimizing detection and preparing solvents.

I would probably start at the periodic maintenance section, then instrument diagnostics, then chromatography diagnostics, and then to the rest. Hopefully this re-orients the thread, and gets someone who knows a lot about ELSD on-board with some specific tips. Also, Waters technical support should be able to give you some advice on what your particular situation might need, or some good first steps toward fixing your issue.


*Edit: After I posted, I remembered reading a post from a member recommending a company that specialized in ELSD. I searched the Forum and found the post, and the company is called Ticoscen (http://www.ticoscen.com/), and the Chromforum member is also Ticoscen. You may want to contact them as well.

Ticoscen posted this ELSD optimization guideline on a separate site:
http://reg.accelacomm.com/servlet/Frs.F ... d=50896125

hi!
here's a little update about how things go by now...

by changing the temperature of the drift tube from 70°C to 60°C the noise went down alot. The broad peak at 35 minutes seemed to be a pollution in the column, it had nothing to do with the stabilizer. By injecting a larger concentration of the stabilizer I (finaly) obtained a peak at 7.5 minutes, so it seems that all this time I was injecting not enough of the stabilizer. the peak at 7.5 minutes is narrow, now I'm going to try to get the background noice down even a bit more.

Thanks for the help everyone!