Analytical Method..Help or suggestion needed.

[San2222]

This is my first Analytical method experiment.I have to develop a Method for the Analysis of Coumarin And its Major Metabolite 7-Hydroxycoumarin
and to icorporate an internal standard into the method.

Ok this is what I have:

1. Coumarin
2. 7-OHC (7-Hydroxycoumarin)
3. O-Coumaric acid ( possible Internal Standard)
4. 7-Amino-4-Methyl-coumarin (possible Internal Standard)

The HPLC system will use:
UV-Visible detector or Photodiode array detector.
Mobile phase (ACN,MeOH,Water)
C18 uBondapak coloumn
Pump(2.5ml/min)


Now,these are the things I know and dont know:

1.Methanol is more polar than water.I dont know how polar is ACN and how it relates to Water and MeOH.

So which is the best mobile phase I can use here?
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2.Coumarin,7-OHC,O-Coumaric acid,7-Amino-4-Methyl-coumarin are all polar (to me at least)

How do you know,which is the most polar among this ,followed by the less polar one?I know the structures of each of this componds,from this how can I guess/calculate which is more polar than the rest?
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3.Of the two Internal standards(O-Coumaric acid AND 7-Amino-4-Methyl-coumarin),the latter one is more structurally similar to Coumarin and 7-OHC than the latter.So

Which of these should I choose as the internal standard?What would happen(to the chromatogram) if I choose the wrong standard?
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Any help will be appreciated,and if you have any external links that would help me in this,please do post it.
Thank you

[Kostas Petritis]

1) You are wrong, water is more polar than methanol and ACN is the least polar of all. To begin with I would recommend to start from 5:95 ACN:water up to 80:20 ACN:water and maybe an hour gradient, then you'll be able to adjust it.

2) If you inject your compounds one by one the most polar will elute first, the second most polar will elute after the first one etc... Sometimes elution order will also depend on the pH of the mobile and if your compounds are charged at that particular pH etc...

3) Your internal standard must not co-elute of course with your compounds of interest. Nothing (bad) will happen to your chromatogram if you choose one over the other... I would choose the first one as I wouldn't have to worry about the amino group

[San2222]

1) You are wrong, water is more polar than methanol and ACN is the least polar of all. To begin with I would recommend to start from 5:95 ACN:water up to 80:20 ACN:water and maybe an hour gradient, then you'll be able to adjust it.
Sometimes elution order will also depend on the pH of the mobile and if your compounds are charged at that particular pH etc...

Thank you so much,and sorry for the mistake.You taught me something which i get confused.So you say to use both water and ACN in a ratio as the mobile phase. But why will I have to do this step? Why cant I use one of them alone?And you said something about an hour gradient and that I can adjust it?Well,may I ask,what is this "hour gradient" and to adjust what?

Now can you or some one here can provide me a link or explanation on how elution depends on pH.

Im sorry for asking all these.Im only a student,and so I may ask sily or stupid questions.So,please bear with me..
Thanks

[JA]

Somebody is really going to suggest you read up on the very basics before undertaking your work

From the solvents you suggested, and the column you are proposing to use, we can see you are about to embark into the realms of reversed-phase HPLC. Here the stationary phase is typically considered "non-polar" and the mobile phase is often a polar mixture of water with an organic "modifier".

For me, I hardly think about the polarity of the solvents with respect to their strength. The HPLC founders have kindly performed a lot of work and produced references with which we can figure out the 'elution-' or 'elutropic-' strength of a solvent. For typical reversed-phase applications, water is the weakest solvent. The strengths of other common solvents employed in RPLC were fitted to the empirical relationship;

log k' = log kw - SΦB

kw = retention factor with water as the mobile phase.
S = tabulated below.
ΦB = volume fraction of chosen solvent in water-solvent mixture.



To summarise, using only water as your mobile phase is in most cases not strong enough to elute the compounds of interest; you can sit there for days and nothing may happen. Using 100% organic solvent has the opposite effect: everything elutes too quickly, often without any separation at all.

Isocratic and gradient chromatography

Chromatographers don't usually hit on the "correct" or "best" mixture of water-organic solvent straight away. You could, for example, employ an iterative approach by adjusting the organic percentage downwards in 10% steps starting at 90% (it's best to begin with things eluting too quickly than not at all) or, if your equipment allows, use a scouting gradient run. The gradient begins from a low percentage of organic and, usually linearly, is increased over a defined period of time (Kostas suggests 60 minutes). The utility is obvious.

Elution, or more appropriately, the retention of your chosen compounds will vary with pH according to their state of ionisation. I have to admit I'm not certain on the keto-enol tautomerism of the coumarins and how they will respond to pH under reversed phase conditions. A generalisation for retention of acids and bases with changes in pH is depicted below.



edit: This isn't the nicest example I've seen of acid & base retention with pH... almost like the equivalence points are the wrong way around. I'll pretend it's something like a phenol and substituted pyridine/nitrogen heteroaromatic.