XBridge PH-3 vs Thermo Hypersil silica columns

[scherza]

Does anyone have any expertise with comparing the performance of an X-Bridge phenyl-3 column versus Hypersil silica, specifically? Or had any experience analyzing the same analyte with both of these columns? Especially with a steroid (cortisol) assay? I do know that these are very different column chemistries (and that this is not trivial): X-bridge is reverse-phased and Hypersil is normal-phased. X-Bridge has functional groups attached to the stationary phase (probably three phenyl groups?) and silica doesn't have anything attached to the silica stationary phase. X-Bridge probably has better selectivity and silica is probably an older techology.... I am a novice analytical biochemistry technician - and was reviewing published cortisol assay methods - and noticed that one method for a very similar assay used X-Bridge and the other Hypersil silica columns.... Can anyone shed some light on this question?

Thank you everyone/anyone for the kind indulgence of your time with this question....S

[Uwe Neue]

Unless the mobile phase conditions are weird, I would go with the reversed-phase assay. Generally, you get much better reproducibility from RP.

[scherza]

Actually the mobile phase conditions are wierd for the Hypersil silica column method - and the extraction procedures are wierd for the X-bridge method. My supervisor bought the X-bridge column with the expectation that we would be doing that method, but the extractions are difficult to reproduce. I have played with both of these methods - and with some ideas from other published methods - and I have gotten it down to a simpler and faster method - even though I am not a biochemist. What I have to do now is be able to defend why I am using one column over the other - and the other issues with the parts of the method I have changed. I can do most of this - now I am looking at column chemistry and pH...etc. The results are fantastic...and well within the standards set by the FDA. That should be enough - but it isn't. Hence, I am asking for expert input...:)

[Uwe Neue]

Well, what do you need? The column came with a Certificate of Analysis, hwere you can look up the ranges of the specs and where this particular column fell into the range. It also gives a lot of information on the properties of the packing.

If you describe your extraction method and the analyte, I can comment on it as well to tell you what is good (or bad) about it.

[scherza]

I really appreciate your very generous offer to critique my extraction method - but I have not yet published it - and my supervisor would be very unhappy with me if I disclosed those details in an online forum. This would be different if I had met you at a conference and we were talking method troubleshooting on bar napkins at 2am.... I am really sorry....

What I am asking for here is anything that anyone knows about the differences between these column stationary phases. If there is anything that goes beyond the obvious specifications. I have noticed that sometimes techs with decades of experience or scientists that just love the science have these unpublished tricks of the trade - or knowledge about what works better - and sometimes we don't know why - but they just do....

I honestly do appreciate your offer....S

[kerri]

Since you won't release any info to substantiate your method/separation, I'll give my experience:

XB Phenyl separation of polar (accessible) aromatic compounds is better than normal phase. Try acidic and basic conditions. You might be surprised.


Kerri

[bisnettrj2]

In addition, Uwe works for Waters and wrote the book on HPLC columns (literally). If there's anyone you should talk to about the vagaries of column chemistry, it would be him.

[HW Mueller]

Cortisol is not amenable to base/acid manipulation as far as HPLC mobile phase is concerned.
I used a C-18 column as main column in two different 3 step (3 dimensional) methods. If the cortisol of blood is to be analyzed I don´t see why one would use normal phase. The reason for the three steps was that the low levels of cortisol in most patients was responsible for the coextraction of a lot of interferences.
(This was published in J Chrom about 1996, I gave the ref. here before, don´t have it in my head).

[bisnettrj2]

I believe HW Mueller is referring to the paper below:

Mueller H W, Eitel J. Quality control in the determination of cortisol in plasma/serum by using, on every sample, two different three-step separation methods including ultrafiltration, restricted-access high-performance liquid chromatography and reverse-phase high-performance liquid chromatography and contrasting results to immunoassays. J Chromatogr B. 1996;678:137–150.

[HW Mueller]

That´s it, thanks.

[scherza]

Please believe me that I never ever intended to insult anyone...honestly. I do appreciate all of the feedback - I really do. Please do not be insulted by my inability to reveal my extraction method. My boss would blow a gasket....

Kerri, I actually use both basic and acidic conditions - in the sample prep protocol. This seems to clear the sample of many artifacts really well much of the time. You are right about the phenyl stationary phase being better than the normal phase silica. I am just gathering information as I wrap up my article for publication. The last piece I am working on is the detail about the column chemistry.

Uwe Neue & bisnettrj2: You are quite correct about Dr Neue being an authority about column chemistry.... I could not find a copy of his book in the local medical center library so I have just ordered a personal copy. It should be very helpful. Thanks!

HW Mueller: Thank you for your input! I have your paper. I am honored to hear from you, as well!! You are so right about there being many interferences in this assay. My blood and urine samples are from very critically ill patients - and this adds so much more to my challenge. Urine can be real bug-a-bear! The acid-base manipulation that does seem to help with my method is in the sample prep protocol - and with adding TFA to the mobile phase. TFA in the mobile phase has drammatically improved the assay for me - especially with reducing the S/N ratio among the lower concentrations. I learn about TFA from reading Michael Dong's book....

Thanks so much everyone!!!

BTW: When my method is published, Waters will be very pleased with it. They have been very helpful to me over the last 2 years that I have been working on this. :)

[Uwe Neue]

Here is the link to the XBridge brochure, where you will find everything that you ever wanted to know about XBridge columns:

http://www.waters.com/webassets/cms/lib ... 1255en.pdf

(you may need to register, if you have not already done so)

[scherza]

Thank you so much, Uwe Neue!