Separation of 2 peaks elutaing at approimately the same RT

[seamoro]

I am developing a HPLC method Phenoxymethlypencillin (Antibiotic), Sodium Benzote (Perservative) and 5 related substances of the phenxymethlypencillin. I having problems separating the sodium benzoate and a related substance (Penilloic acid of Phenoxymethylpencillin) . I am using :
Sphereisorb 5u ODS 2
250 X 4.6MM
Mobile phase A:100ml KH2PO4 pH3.5,300ml MeOH, 600ml water
Moblie phase B:100ml KH2PO4 pH3.5, 550ml MeOH, 350ml water
Flow 0.700ml
Gradient:
Mobile A/Mobile B
0mins 60%/40%
25mins 60%/40%
45mins 100%
45.1mins 100%
60.0mins 60%/40%
I believe the mayor problem is the environ of so many peaks in the space of about 13minutes. That there is very little room for maneuver. I think the chances of separating so many peaks in such proximity is going to difficult! Any suggestions would be greatly appreicated. My boss wants to use this exsisting method due to it being in the EP. I have tryed lots of varitions allowed by the EP such as:
Column Length +/- 70%
Column Diameter +/- 25%
Particle size +/- 50%
pH +/- 0.2pH
Compostion of moblie phase +/- 30%
Flow rate +/- 50%
But with no sucess of separating the 2 peaks! HAVE YOU GUYS ANY OTHER SUGGESTIONS besides developing a whole new method! Is there any way i can stretch these peaks out to eluete over a longer time therefore hopefully allowing the problems peaks to separate!!

[grzesiek]

"My boss wants to use this exsisting method due to it being in the EP" - they all do, they even want to use pharmacopoeial methods for compunds not included in monographs moroever they even know which method is suitable ) at least before the experiment

I would say column first, if there is alternative column specified than try it, is this spherisorb recommended by pharmacopoeia or is it your idea?

I would say temperature second, if no temperature is specified, you could gain some selecivivty by varying column temp

"eluete over a longer time" - oh no, lets make them elute faster increase flow right now young man

[bisnettrj2]

I'd agree with grzesiek, typical flow rate for a column of the dimensions you're using is 1-2 mL/min, and if you're allowed a 50% increase in flow rate, I'd move that flow rate to 1 mL/min, and try a column temperature above ambient temp (30 degrees C is probably a good start) to at least get reliable retention times. As for the column, it would probably be more economical to tweak method parameters within guidelines than to buy a new column without trying to optimize conditions first.

[krickos]

"I would say column first, if there is alternative column specified than try it, is this spherisorb recommended by pharmacopoeia or is it your idea?

Hi

I agree that you should check the column equivalency, if I did not misread, Ph Eur indicates a Hypersil column (end capped), the one you use seems not to be end capped according to Waters website (http://www.waters.com/waters/partDetail ... =PSS833015).

Not sure it will help though.

[grzesiek]

"Ph Eur indicates a Hypersil column" - if the method was validated using hypersil and you're using spherisorb, then you can't say that you're using EP method, unless spherisorb is alternative column (used in the validation)

"Not sure it will help though." - I think it does help a great deal

How much theoretical plates do you get using spherisorb?

[seamoro]

its the related substances method (EP) for phenoxymethlypenicillin we are using......and why are we using a sphereisorb?...cause we already use this method for the raw material of phenoxymethylpenicillin...and now we want to use it for the finished product!

[grzesiek]

"its the related substances method (EP) for phenoxymethlypenicillin we are using" - does EP states that spherisorb was used in validation of this method?

[seamoro]

the EP states
column:size: l=0.25m, Diameter =4.6mm
Stationary phase: octadecylsilyl silica gel 5um

But we used sphereisorb when we vaildated for the raw material.

[grzesiek]

"the EP states
column:size: l=0.25m, Diameter =4.6mm
Stationary phase: octadecylsilyl silica gel 5um" - yes that is ok, but all methods are validated on particular column, it happens a lot that saying only L1 does not mean you are taking any L1 and achive required selectivity

I would advise you to email EP guys and ask for the column first

Going back to original question, if you interpret L1 as any L1 than i would advise experimenting with column type, cos there are many L1 to choose from

[HW Mueller]

One would think that playing with pH would be by far the most promising for influenzing the separation here, a distant second maybe the organic content of the mobile phase. Unless it is a bum column I would change that last.
(Incidentally, a pH of 3.5 for a phosphate buffer?)

[Uwe Neue]

Spherisorb ODS 2 is endcapped. However, you should be able to get quite significant selectivity differences by using different columns with the same mobile phase. I suggest Symmetry C18, SymmetryShield RP18, Atlantis T3. Reason: Spherisorb is based on an older silica, Symmetry C18 is based on a high-purity silica and the coating level is higher. SymmetryShield RP18 has an embedded polar group, and the selectivity will change because of that. Atlantis T3 is a fully endcapped packing with a low coating. Differences in coating levels of that type will move the peaks around.

Unfortunately, the detailed selectivity for your analytes is not predictable, but such an approach maximizes your chances of being successful.

[Bryan Evans]

Unison UK-C18, 250x4.6mm, 3um (USP L1)

You'll be within the EP limits of reducing particle size.
Efficiency will be much higher - and pressure will be ok on
your current HPLC

[lmh]

How sad. I'm sure the original thinking behind EP wasn't intended to force people to use methods that don't really work.

[seamoro]

well i am trying the water T3 column..and its doesnt look too good!
I have already tryed the waters sunfire Xterra 3.5u 4.6x100mm, Phenomenex Prodigy 3u 100x4.60mm, Varian Pursuit 5 C18 150X4.6MM and the phenmenex Luna 3u C18...all with no sucess

[grzesiek]

how is trying performed?

is XTerra L1? or prodigy? pursuit?