Chromatography Forum

lmh:
Well said! We don't really care how you got your training. We only care about what you have learned, and it appears that you have learned quite a lot.

Hi Ben,

Since we’re dealing with the basics now, it might be a good idea to get some definitions right.

1. RPC stands for reversed phase chromatography, which in turn means that the stationary phase is of hydrophobic (apolar) nature, whilst the mobile phase is some sort of a polar liquid (most often water, modified with a moderately apolar liquid, that is miscible with water). The above disqualifies hexane as a mobile phase in RP context – without knowing too much about chromatography i.e. no need for huge experience to figure it out either.
2. If you’re forced to use 90 % organic solvent to elute the analyte in question, at that long retention time (45 min) you’re losing quite a bit of the selectivity you so desperately need in order to separate all those compounds you’re dealing with. Otherwise the explanation lmh offered in regards to why suddenly C4, or phenyl, should be adequate. I personally do not exceed the self imposed limit of 80 % organic solvent (at the most) but a discussion of that will be misplaced in a forum like this, so I’ll spare you for that.
3. Finally, this forum has witnessed quite a bit of funny questions and suggestions over the time. And really, it’s not a shame to ask questions and suggest things that are not that well founded. What is a shame is; people having made up their minds asking questions, expecting the answers to that questions to confirm what they’ve already decided was the truth/solution.

Best Regards

You could say that I have learned enough to be dangerous.

I intend to hire a chemist and technician, ideally with a university lab, and then later an accredited laboratory. I would simply like to understand the theory and practice (concepts & instrument functions) of this specific application in enough detail to communicate and specify what is to be done regarding the columns, solvents, samples, instruments, lab, analysts, and measurements. I tend to think analytically myself, and I am legally liable for the results, which are subject to GLP. This is why I ask questions as much as anything else.

Is it not rational to start with the analyte and the sample? Most of all, the analyte is lipophilic and apolar. This is shown by the long retention time, using RP18 & MeOH:H20 90:10. It has a terminal lactone, long-caarbon chain, and 3 hydroxl groups.

Any C18 will do?

This is what appears to be an independent review of columns by Mac-Mod:

http://www.mac-mod.com/Comparison_Guide ... 4-cat.html

If the retention, the chemical bonding and interaction between the analyte and the stationary phase, is greatest if the characteristics are the same, then does it not make sense to start with an Inertsil ODS-3, Kromasil C18, or Develosil ODS-MG column, since these are most like the analyte, lipophilic (hydrophobic) and apolar, according to this review. It is still not clear to me how to explain the difference between lipophilicity and apolarity in the abstract, but I still have practical questions, such as the solvent.

If the elution depends on the interaction between the analyte and the mobile phase, then what is the problem with hexane ? If there is a problem with miscibility, then why not use 100% hexane as a solvent at a low flow or volume ? Hexane is said to be used in the vegetable oil industry. What are the practical problems that you referred to ? Can you please explain ?

The sample, the matrix, other compounds, and the analyte depend on SFE, which is like sample preparation on steroids, with some selectivity possible.

So if, just if, we had this method, either one of these three columns and 100% hexane, then what could we vary further or specify to the chemist & technician to vary to try to optimize the resolution/selectivity and run-time? The flow? The injection volumne? The heat? The particle size, column internal diameter, and column length?

What if we substituted ethanol for hexane? Is there any reason to believe that ethanol might not be possible to try, if it failed with 1 of the 3 columns in the Mac-Mod review and 100% hexane?

Detection by MS would still be possible and practical, still keeping some robustness, if ethanol interfered with UV detection. MS complications can depend on the interface between the LC & MS devices, from what I hear. The instruments can be calibrated, but can only meaure MW, not the formula uniquely, without tandem MS or equivalent method. Even then we depend on NMR to identify completelty, since our analyte is chiral, ultra-chiral, with 5-8 chiral centers.

Sincere thanks for your time and attention in responding to the LC.

Ben

During the time you write one of your lengthy posts (we are all volunteers and tend not to cotton to stomping through novels to get at the kernel) you could have tried hexane and tell us about the results.
Retention is determined by an equilibrium constant between the stat. phase and the mobile phase. The interaction between mobile phase and analyte is only one aspect of this. Normally, the interaction between stat. phase and analyte is considered the more important, but in borderline situation this might be different. For instance if the solubility of the analyte is extremely low in mobile phase this aspect might dominate, the analyte will just get stuck somewhere (probably not even make it to the column).
Just a hunch: I suspect that your compound will do this if hexane is used. If your compound is soluble in hexane it will elute at the dead time with hexane as mobile phase.
Chromatography involves a lot of chemical and physical aspects which are still poorly understood. For instance, I know of nobody who had an inclination to see what hexane does to a C-18 phase and whether this sort of interaction could be exploited.

I can see that you are having issue with this:

Any C18 will do.

I can understand the confusion, so I will take a stab at this. All C18 columns are different (as you have pointed out). However, they are not wildly different. So, if you gave me 3 C18 columns and a probe test mix and I ran them under identical conditions I would get different retention times for the compounds in the mix. By adjusting the various parameters at my disposal, solvent composition, solvent strength, acid/base modifiers, temperature, flow rate, I would bet I could get the retentions time to line up pretty well. This is why any C18 will do.

To try and answer one of your other question "what is the problem with hexane". Well, from an reverse phase LC perspective, it is a VERY strong solvent, meaning that it will elute everything off the column very quickly (usually in the void). Since most of the solvents that could weaken its elution strength (and are miscible) are themselves quite strong, its usefulness in RP is limited.

Dear Ben,

in your first post you said:

I am discovering that two months in the lab can save three to four hours in the library...

maybe...but I'm afraid in this case it is:

one hour in the lab can save weeks of fruitless discussion

Let me summarize, what I got out of the discussion so far:
You don't have any experience with HPLC, you don't even own a HPLC-system. But you have read something about "very lipophilic and apolar" (we really got the point after you repeated it the fourth time...). And you are convinced that you need whatever column your lipophilic apolar website recommended, probably with very lipophilic and apolar hexane as solvent.

If you really don't want to listen to what all the posters in this thread told you, I would recommend you buy all the neccessary equipment and especially a very lipophilic and apolar column, a very lipophilic and apolar solvent and try it out for yourself.

The alternatative would be to give your extracts to some experienced GLP lab and save lots of time and money....

Cheers,
Hartmut

BTW

Greetings, and forgive me posting this also as a new topic by mistake.

Sincere thanks for your responses and constructive criticism. I will try to keep it brief, because I understand that your time is valuable.

Yeah, it's still the statement that "any C18 will do" that I question, and I have no lab. I do intend to hire a contract lab for this method transfer and another lab for method validation. For now, I have no lab, but am trying to compare columns objectively, for this very specific application & method.

Because I am legally liable for the results, which are subject to GLP, the details matter to me, and yeah, I have no formal training nor experience with LC, just book-learning and talking with various experts over the last year. Before hiring a chemist (contract lab) for the required analytical (& preparative) chemistry, I would like to understand the specifications to the labs, the columns, the solvents, and the conditions, why these specs would work, and what to vary, if it fails.

It's not that I am not listening to the advice of experts, but various experts can have various opinions. Consultants, like experts, sometimes contradict themselves. The history of science is full of raging debates and bitter disputes of what the objective reality is and how to see it optimally. I'm also just naturally sceptical of experts.

Nevertheless, even if nobody has used C18 with hexane, then why not try it, as you suggest? I agree. I accept this advice, pending selection of a competent laboratory. As previous stated, I have no lab for now, so I value the collective advice from you.

Before hiring a lab, I still have questions concerning the specifications to the lab:

If hexane is too strong a solvent, then what if we mixed hexane with ethanol (though not in Germany, where it is taxed more) or methanol 50/50 ?

For less solvation, what if we lowered the flow or lowered the injection volume, or even used hexane:ethanol/methanol 10:90 ?

By the way, can someone please give me an expert opinion of the review of columns by Mac-Mod (linked to my previous posting and also available by searching the www.mac-mod.com website, and you better believe that I have nothing to do with Mac-Mod, so this most certainly is not a sales pitch for them nor their products), and one of the sponsors of this website (not some lipophilic antipolar website according to a poster) ? This review seems believable to me, but perhaps this is because I am naive and gullible, wet behind the ears, naked in the woods, so I ask you, the experts, about the selection of the columns. So I will take one or all of the three columns that would most match the analyte, according to this C18 column review, because no expert can tell me why not, specifically an Inertsil ODS-3, Kromasil C18, or Develosil ODS-MG column. What these columns appear to have in column, by the way, at least according to the websites of their suppliers, is a much greater internal surface area, particularly with smaller particle sizes, compared with other C18 columns, though the details ("carbon load", ...) were not clear to me. If you think that the C18 column review by Mac-Mod is nonsense, completely biased, unreliable,or incomplete, then please tell me. You are the experts.

I accept the advice to go back to the library and to read books about development of HPLC methods, but most of these books are not specific to lipids found in natural products nor to these specific three columns. Mostly, they are oriented to Rx products & methods. I did read the earlier website and other websites about lipids, but found no answers. Also, this topic is more about the modification and optimization of a method, than it is about the development of a method.

If you are as smart as you think you are, then you should have answers to the simple questions in this posting.

Sincere thanks for your time and attention.

Regards,

Ben

You want to kill the patient, but not before you are convinced that you can resuscitate him.
But why kill the patient in the first place?

If the stationary phase is too hydrophobic for the analyte i.e. you have a hard time getting it out of the column, then the most obvious action is to decrease the hydrophobicity of the stationary phase.

I don’t know why you obviously have fallen in love with hexane, but if you meant you interest in green technology seriously, I don’t think hexane is the right solvent for you – solely on that ground.

Also, I think it’s quite OK that you challenge us to prove how smart we are, but how would you confirm our wisdom (or the lack of it) if you do not examine the ideas given to you seriously. You lack the knowledge and experience necessary for evaluating our suggestions – for one. And then you only listen to your own ideas/hunches.

One day when you’ve gathered the required knowledge and experience (several years from now) you’ll be more appreciative of the efforts made by several posters here to guide you to a successful implementation of the analysis in question.

For now – taking the fact that you don’t have access to an HPLC lab in to account – I’m afraid your only options are either to go to the library or listen to the experts’ advice.

Best Regards

*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.

I felt a little guilty when I saw so many nice people dealing with an unpractical mission with so much patience. I am probably considered one of people “turns around, walks away from discussion with a low whistle and a shake of the head".

However, my experience tells me when LC-MS (or MS/MS) can do a job easily, I won't spend days and months to develop a nice, super LC-UV method to separate and quantify scores of structurally similar compounds in a difficult matrix. It's simply not worth it, economically or technically. So next time, I will do the same: tell them the approach is unpractical (but not impossible), turns around, walks away...............

BTW, I am not in an academia envrionment, so pursuing perfection is not my daily goal, my goal is to just make thing work with available resources.

Because I am legally liable for the results, which are subject to GLP, the details matter to me, and yeah, I have no formal training nor experience with LC, just book-learning and talking with various experts over the last year. Before hiring a chemist (contract lab) for the required analytical (& preparative) chemistry, I would like to understand the specifications to the labs, the columns, the solvents, and the conditions, why these specs would work, and what to vary, if it fails.

Ben,

If you are going to hire a GLP lab to validate the the method then it will be the Study director at the GLP lab who is legally responsible for the validation work according to GLP. Once the validation study is complete and reported you should, if the validation is robust enough, be able to transfer the method and repeat the validation in your own lab quite easily. That is the whole point of the GLP system, to enable others to follow exactly what has been done and documented. If problens arise they shoud be able to help with follow-up especially if you mate that known in initial discussions.

Any decent study director should be able to discuss the aspects of the analysis quite easily with you, after all you're paying for his expertise. I spent over 20 years in contract research doing exactly that and the vast majority of clients were not chemists.

Wow, entertaining.

A simple Google search reveals plenty of similar methods to start from,
eg SFE extracts on reverse phase columns. http://www.doctors.org.cn/doc/ucmed2008 ... 080110.pdf

Ben, there are plenty of options to choose from, but until you actually have some of the SFE extract to put down the HPLC, I wouldn't even bother worrying about any details of suitable HPLC systems.

Obtain your extracts, hire a CRO for the HPLC, and you'll be able to come back and tell us what worked.

Incidently, if you really wanted to learn about HPLC columns, you would have diligently Googled the topic, and should have discovered that Uwe Neue has written the main reference book. I hope your research skills are better than they appear here..

Sad, but entertaining, thread. Thanks everyone.

To those of you who answered the questions I asked about modifying and optimizing a published method (lipids, RP18 & MeOH), evaluating the Mac-Mod review of RP18 columns, selecting a column based on this review, selecting a solvent, and the affinities between the analyte, the column, and the solvent for the separation, I thank you, sincerely.

Most helpful was the posting with the reference to an actual method that already used RP18 and hexane:methanol 40:60. This implies that such a method is practical, or at least feasible, even if not common. On the other hand, some of the experts said “no, hexane is not miscible; this will create two phasesâ€

....
To those of you experts, would-be experts, self-styled experts, pundits, gurus, or otherwise qualified vagabonds, who have not answered these relatively simple, yet theoretical, questions, then I have only one last question for you: if you’re so smart, why aren’t you rich? Ben

Should I feed the troll? Why not, I've a few minutes waiting for an HPLC

The reference I pointed you towards ( a full paper - freely available to download ) included the following section that was the relevant part:-
" 3. Experimental Content
3.1 The assay of Squamocin A by HPLC
3.1.1 Chromatographic condition
Agilent 1200 HPLC; chromatographic column: Kromasil reverse phase C18; mobile phase: methanol-purified water (83:17); flow rate: 1ml/min; sample size was 10ul, column temperature was 30℃, column efficiency was accounted with Squamocin A content, number of theoretical plates n =7000, resolution exceed 1.5.

The HPLC chromatograms of standard and sample were following Fig. 1. The retention time is about 44.847 minutes."

One issue is that SFE; depending on the extraction conditions, use of cosolvents, primary solvent, etc.; may generate a diverse range of closely eluting compounds from the feedstock, as in the example I provided. The choice of chromatographic system will depend on the nature of the extracted fraction, and the desired separation.

The immiscible solvents issue. Anhydrous solvents may be miscible, but introduction of water results in two phases - reverse phase HPLC separations typically involve a water component, and even normal phase silica systems usually have a layer of water involved. You want to check my assertion?, simple - buy some gasoline without oxygenates, add methanol, then add water - it will separate. Just because somebody has used a certain solvent mixture doesn't make it the best option available.

The principal issue is that you don't even have a characterised extract, but you want to freely mine other peoples' IP as though their time has no value, and also ignore their advice.

Your research appears to be minimal, your assumptions about participants abusive and woeful. You attempt to justify your behaviour by making a virtue of willful ignorance.

Until I appeared, this thread demonstrated any amazing amount of goodwill and free knowledge to you. My tolerance is somewhat less than others, because you have consistently failed to respect sensible advice.

A lot of skilled people participate in these forums to help others learn, but some don't appear willing to learn.. Perhaps participants should charge our normal market rates - amazing as it may seem, some successful companies pay for, and heed , our advice. Fortunately, I don't encounter many potential customers like you.

Bruce Hamilton