*WARNING - LONG-WINDED POST AHEAD*
benrock:
What is your intention with these questions? Do you intend to use this information to determine whether the lab you commission to perform method development has done a good job or not? Or do you intend to use this information in conjunction with the method development that you will contract out to troubleshoot any problems you may experience when you are using the developed method in your own lab?
I ask, because while I can understand having a rudimentary understanding of the principles behind the analysis, if you won't be doing it yourself, you probably should not be imposing your ideas upon those that have experience in this field. You can submit a request for bids from as many labs as you see fit to perform method development using "green" technology (probably in the form of the solvents used to constitute the mobile phase, total solvent consumption, etc.), and you can possibly specify the means of detection of your analyte to suit the future capabilities of the instrumentation you might acquire, or of that which is widely available and understood, and which has the lowest limit of detection (MS over UV in your analyte's case, I would suppose). However, from there on I believe you should leave the choices of columns and solvents in the hands of those submitting the bids, and leave your opinions out of it, as the professionals at those labs will be better suited to make those determinations. Part of the bid request/submission should relate to the requirements you are being legally required to meet - and the ability of a third-party validator/auditor to verify that the lab can meet those requirements, and whether they have met the requirements when they submit the developed method.
In regard to your mac-mod C18 study query - mac-mod probably has done a good job characterizing columns via the particular conditions they used in their characterization studies. However, they used well-characterized probe analytes in absolutely clean matrices, which you will
not be doing. The number of factors that go into developing quality HPLC methods could fill a book - and have filled many. Look up my post in the 'Water Cooler' section of this forum (
viewtopic.php?t=12365) to get a good list of reference books to help understand all the facets of chromatography and analytical chemistry. The fact that no one specifically addressed the three columns you mentioned is most likely due to the fact that the particular brand of column is quite secondary to the overall needs and purpose of this analysis. I think most will agree a C18 is a good start point - but it is not an end-all, be-all. And believe me, there are more C18s out there than what mac-mod tested - there may be a more lipophillic apolar column out there yet! Just try not to get hung up on one particular (or three) brand(s) of columns - books have been written about this subject as well (the poster who said you can use 'any' C18 is Uwe Neue - Google his name and HPLC, then see my list of books in the aforementioned forum post).
In regard to using hexane as a choice of mobile phase in reversed-phase chromatography, yes, it is possible (you've seen Bryan Evans' post in your other thread posted in this section of the forum). I think the hexane question is almost beside the underlying point, which, in my opinion, is in regard to the mechanisms of retention and separation in HPLC, and how the choices of temperature, solvent, pH, and stationary phase affect those mechanisms. You should think of HPLC as the interaction of the analyte with the mobile phase
and the stationary phase in tandem. When you have a lipophilic, apolar analyte, you can run a mobile phase that has a high polar:less polar ratio (e.g. high water content) to 'load' the analyte onto your nonpolar (C18, C8, C4, etc) column because the analyte will have a greater affinity for your stationary phase than your mobile phase. Then, in a gradient elution, as you increase the proportion of less-polar (but still miscible) solvent (e.g. not hexane), the affinity of the analyte changes from the stationary phase to the mobile phase, and at some proportion of organic solvent to water, the analyte is eluted from the column. The fact that many ignored your hexane suggestion is that hexane just isn't used (usually) in reversed-phase chromatography, which is the most common form of chromatography used today. Hexane is not miscible with many polar solvents, and as I stated before, the interaction between your mobile phase and stationary phase is influenced by many factors - if you have a nonpolar stationary phase and a nonpolar mobile phase, your nonpolar analyte will not interact with your column. You can add in a miscible alcohol, but you will sacrifice UV-detection limits due to the relatively high UV-cutoff for most alcohols compared to the low UV-absorbance maxima of your analyte. Hexane is a great normal-phase chromatography solvent, and your analyte may be amenable to that mode of chromatography (perhaps more so than reversed phase, but that's where method development comes into play). In terms of solvents, using different solvents will have varying effects on your chromatograpic separation - e.g. methanol versus acetonitrile at the same percentage in an isocratic mobile phase may lead to different elution patterns, longer retention of certain analytes, or less resolution between analytes. A lot depends on the needs of the analyst and laboratory. We don't know whether you'll be screening an herbal product for impurities or trying to isolate and purify this analyte from a matrix for use in animal testing, or some other purpose. But the mobile phases suggested - namely, acetonitrile:water gradients - are the most common in HPLC, and will serve you well in terms of reduced viscosity over methanol or ethanol (lower backpressure, higher flow rates), and very low UV-absorbance.
In short (hehehe), I personally feel you may have come into this discussion with too many pre-suppositions, and not an open-enough mind to the opinions and suggestions of the posters to this forum - namely Uwe Neue, danko, HW Mueller, and mbicking (note - I'm not trying to diminish the rest of us who've posted, I just think those mentioned tend to make some very good points, and have a lot of experience in this field, so I mention them specifically). Instead of continuing on about why we're not answering your specific questions, perhaps you should be inquiring as to how to best judge a developed method when you are a novice, or how to best choose a lab to perform method development, or perhaps how to determine which lab would be the right choice to award your method development work when you want to make sure your method is as 'green' as possible. Just my two cents.
Incidentally, I found a Phytochemistry journal article describing the analysis of Squamocin - they use a high MeOH (10:1 MeOH:H2O) mobile phase to separate a fraction of an extract, and then run a MeOH:ACN:H2O:IPA mobile phase on another column to separate the components of this extract.
Hiroshi Araya, Mahendra Sahai, Shubhra Singh, Ajit Kumar Singh, Mitsuzi Yoshida, Noriyuki Hara, Yoshinori Fujimoto, "Squamocin-O1 and squamocin-O2, new adjacent bis-tetrahydrofuran acetogenins from the seeds of Annona squamosa", Phytochemistry, Volume 61, Issue 8, December 2002, Pages 999-1004, ISSN 0031-9422, DOI: 10.1016/S0031-9422(02)00351-5.
(
http://www.sciencedirect.com/science/ar ... 42129c4d6d)
"This fraction showed a broad peak about 12.5 min (14.0 min for squamostatin-A) when analyzed by HPLC (column: Shimadzu Shim-Pack CLC-ODS (25 cm × 10 mm i.d.); solvent: MeOH–H2O 10:1; flow rate 1.0 ml/min). The separation of the peak afforded a mixture of compounds 1 and 2 (46 mg). This was further separated by HPLC (column: STR PREP-ODS (25 cm × 10 mm i.d.); solvent, MeOH–CH3CN–H2O–iPrOH (120:40:30:1); flow rate 6.0 ml/min; typical retention times, 39 min for 1, 43 min for 2) to furnish compounds 1 and 2."
The first column used is essentially a means to separate two analytes from the original matrix, with little on-column retention. The second is a complex mobile phase. Both are detecting relatively large amounts of material - which allows you to use a different wavelength than the absorbance maxima, which you could not do if you were trying to analyze trace amounts of the analyte in question. This article seems to vaguely agree with some of the claims you've made about your analyte in this thread (90:10 MeOH:H2O mobile phase, RT around 35-40 minutes, ODS columns), so I wonder if you aren't deriving some of your arguments from the analysis done within this paper?
P.S. - This discussion has reminded me of my favorite Donald Rumsfeld quote - "There are known knowns. These are things we know that we know. There are known unknowns. That is to say, there are things that we know we don't know. But there are also unknown unknowns. There are things we don't know we don't know."
Ok, for those brave enough to read all of this, reply away.
