By Anonymous on Tuesday, June 8, 2004 - 07:59 am:

hi,
i would like to know bout Ascorbic acid.does it show tendency to degrade after 5hrs.and also its stability in solvents.pls do write tyo me bout it.
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By A.Mouse on Tuesday, June 8, 2004 - 07:06 pm:

Yes, ascorbic acid can degrade easily. The details can either be found in textbooks or you have to do the experiments.
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By Anonymous on Thursday, June 10, 2004 - 07:49 am:

please can u specify as my friend is stabilisg l-ascorbic acid and it seems that it degrades by 6%after 5 hrs
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By Anonymous on Wednesday, June 23, 2004 - 11:43 am:

Has anybody know Tacrolimus Assay HPLC method and dissolution method?
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By kobykat on Thursday, July 1, 2004 - 09:37 am:

Hello everyone. I was wondering what the significance in determining the signal to noise ratio might be at the start of a method and how to calculate this ratio.
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By Kaoula on Friday, July 2, 2004 - 07:30 am:

Hi all
Please I wont your opinions of this state it is correct or not :

The pH of mobile phase used in analytical method is about 3.5 and the pH of dissolution medium is 9.2

and what is the procedure when the pH of dissolution medium and mobile phase is very different

Thank you for your advise
Kaoula
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By Chris Pohl on Saturday, July 3, 2004 - 11:31 am:

Kaoula,

The answer to your question depends in part upon whether or not any of your analytes are weakly acidic. If all of your analytes are neutral or basic, then there there won't be any major problems with the wide disparity between your eluent pH and your sample pH (although it may be necessary to boost the buffer capacity of your mobile phase somewhat in order to prevent overloading your buffer system) since the higher pH of the sample is either irrelevant in the case of neutral compounds or suitable for "focusing" in the case of basic compounds. However, if one of your analytes is weakly acidic and you are using a low pH in order to achieve retention via ion suppression then injecting a high pH sample can significantly distort the peak shape of weakly acidic solutes because the ionized form of acidic analytes invariably are more weakly retained then the fully protonated form. In this case, it would be preferable to adjust the pH of the sample to more closely matched that of the mobile phase. If, however, the high pH for the sample is connected to difficulty of dissolving the sample, it might be worth trying a two-step protocol: preparing the sample at high pH and a somewhat higher concentration and then diluting the solution to the final injection concentration into a buffer system matching the eluent pH.
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By Kaoula on Sunday, July 4, 2004 - 12:14 am:

Hi ALL:

Please i need a clarification about the rule for choice between L7 coulmn and L1 coulmn ??
thank you ver match
Kaoula
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By Anonymous on Sunday, July 4, 2004 - 11:47 am:

Exactly what do you need clarified?
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By kaoula on Wednesday, July 7, 2004 - 06:12 am:

Hi Chris Pohl:
thank you for yor advice.
i wont to aske you if the Destroyed peak appear as tow peaks and the resolution between them is good 4.5 as example?

thank you
Kaoula
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By Kaoula on Wednesday, July 7, 2004 - 06:13 am:

Hi ALL:
What is the rule for the choice between L7 coulmn and L1 coulmn ??

thank you ver match
Kaoula
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By tom jupille on Wednesday, July 7, 2004 - 08:29 am:

L1 = C18
L7 = C8

If you are adapting a USP method, then you should use an "equivalent" column (i.e. one from the same "L" class as the column specified). The catch is that there are several hundred commercially available columns which fit the L1 description, and they are *not* all equivalent. It is up to you to demonstrate equivalence for the specific column you use.

If you are not dealing with a USP method, then the "L" designations are irrelevant.
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By kobykat on Wednesday, July 7, 2004 - 11:39 am:

O.k., trying to develop a good method for the seperation of pseudoephedrine in a matrix of other cold product ingredients (acetaminophen, etc). I've tried some different methods from various websites but I'm not obtaining any great results. Does anyone have some suggestions on a method, or how I can tweek the seperation via ion-pair, etc.? Thanks!
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By SIELC_Tech on Wednesday, July 7, 2004 - 01:59 pm:

Check this method it was developed for NyQuil/Advil type of formulations (pseudoephedrine, acetaminophen, doxylamine, dextromethorphan). Concentrations in the mixture match the real life sample (acetaminophen 500 mg other in 10-50 mg range. No ion-pairing reagent, base line separation short method run - 10 minutes. Check also a comparison with regular C18 method and you will see the difference. Problem is, that all compounds in the mixture are quite different in terms of hydrophobic properties, polarity and pKa (acetaminophen and pseudoephedrine vs. dextromethorphan) and on traditional columns achieving good separation with time efficient method is quite challenging:

http://allsep.com/makeCmp.php?cmp=Cmp_116
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By THP5 on Thursday, July 8, 2004 - 08:31 pm:

SILEC,

Do you have LC/MS compatible method with formic acid or ammonium formate? DO you make columns with 1 mm ID?
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By Chris Pohl on Saturday, July 10, 2004 - 12:56 pm:

Kaoula,

I'm not sure I understand your question regarding your question regarding a "destroyed peak which appears as two peaks and the resolution between them is good". Is this still in the context of sample pH being significantly different than mobile phase pH? Are you saying that you think the sample pH has caused peaks splitting? I guess I need to know that more about the analyte to say one way or the other.
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By Kaoula on Sunday, July 11, 2004 - 05:07 am:

Hi Chris Pohl:

My problem is as follow:

The analyte is weak acid material it exist tow method of analysis:
the first is based on ion suppression principle (pH about 3.5) and the second is based on ion pairing principle (pH about 7.5).

When we are used these tow methods to carried out the dissolution test which the pH of dissolution method is 9.2 the results is as follow:

Using ion pairing method: the chromatogram show one peak
Using ion suppression method: the chromatogram show tow peaks which the resolution between them is about 4.5.

Note: the tow peaks appear in the standard(prepared in dissolution medium) and the samples of dissolution but doesn’t appear in the standard which prepared in methanol as solvent

Thank you very match for your advice
Kaoula
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By Pantiano on Monday, July 12, 2004 - 08:07 am:

hello,i´m student university of granada, i need to konw how i can purify tetradecane. i have found information,that i can purify with column of Al2O4,alumine, but i don´t know how much Al2O3/tetradecane i need to do column.If someone can help me...
thank anyone
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By Chris Pohl on Monday, July 12, 2004 - 12:15 pm:

Kaoula,

Even if we consider all of the factors you have mentioned, it's still not possible to make a definitive assessment. Comparing two different retention modes like ion suppression and ion pairing, it's easy to imagine that an interference might be observed in one method and not in the other, so the discrepancy between the two methods could easily be due to either the sample pH or the fact that the two methods are fundamentally different in terms of retention mode. So this observation doesn't conclusively indicate a problem with your sample preparation on the chromatography. Furthermore, the fact you do not observe two peaks when injecting a sample which has been dissolved in methanol doesn't prove anything either since this discrepancy could be due to the fact that you are dissolving your sample in an "eluent" significantly more potent than your mobile phase. Generally, it's not advisable to dissolve your sample in pure solvent when using reversed phase (although you can sometimes get away with it by injecting a very small sample volume). My suggestion is that you try dissolving your sample in mobile phase (or better still, a solution containing a lower solvent content than your mobile phase) and see if you still see only one peak. If so, then this does suggest the problem is due to the high pH of your sample solution and I would suggest that you spike your sample with sufficient acid to bring the sample pH down to 3.
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By Kaoula on Tuesday, July 13, 2004 - 12:12 am:

Hi Chris Pohl:
Thank you for your advice.
I wont to ask you :

why it's not advisable to dissolve the sample in pure solvent when using reversed phase ?

Kaoula
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By DR on Tuesday, July 13, 2004 - 07:15 am:

Putting a sample on a (reverse phase) column with a solvent that is higher in organic content than your mobile phase can cause broader peaks, poor resolution and (sometimes) more tailing. These problems will be more pronounced with larger injection volumes. Using a weaker sample diluent results in narrower peaks, which allows for better resolution. This is because injecting sample in a slug of high organic solvent will allow some of the sample to move fairly far down the column before engaging in the normal mp/stationary phase partitioning that results in getting the separation in the first place.
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By kobykat on Wednesday, July 21, 2004 - 10:11 am:

Hello Everyone. I'm currently working with chlorpheniramine but after injecting the standard I'm getting two distinct peaks. Is one a derivative of the other? Does anyone have knowledge about this drug that might help? My purpose is to quantitate the active in a product. Thank you
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By M.Sai LAXMI on Monday, July 26, 2004 - 01:25 am:

hi, can i get a validated hplc method with florescence detector for analysing metformin and tolbutamide. please send it to my e. mail given below.
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By kobykat on Wednesday, July 28, 2004 - 06:29 am:

Anyone know how to derivate an amino acid to be detected using UV?
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By Uwe Neue on Wednesday, July 28, 2004 - 03:19 pm:

There are commercial kits around, including descriptions how to do this. Check at the Waters website for the AccQFluor regant or for the PicoTag reagent. The chemistry is described there... too long to outline here.
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By Anonymous on Friday, December 20, 2002 - 10:23 pm:

How do I separate Paracetamol, Phenylpropanolamine hydrochloride and Triprolidine hydrochloride on Reverse Phase coulmn?
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By Anonymous on Sunday, December 22, 2002 - 10:56 pm:

Check the following:

Simultaneous quantification of ephedrines in urine by high-performance liquid chromatography
Journal of Chromatography B: Biomedical Sciences and Applications Volume 661, Issue 2 18 November 1994 Pages 357-361
P. J. van der Merwe, L. W. Brown and S. E. Hendrikz
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By Anonymous on Tuesday, December 24, 2002 - 09:41 pm:

To Anonymous
Thank you for your advice. Definately I will go through the Journal of Chromatography.
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By rao, sree on Wednesday, December 25, 2002 - 08:59 am:

why the Cyano column is unstable than C18 column
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By Anonymous on Wednesday, December 25, 2002 - 09:02 am:

what is the main difference between Normal phase and Reverse phase
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By Anonymous on Thursday, December 26, 2002 - 07:34 am:

Simple but complicated question. 'main difference'?

reverse phase is based on solubility or dipole weakness, normal phase is based on retardation based on hydrogen bonding to silica or induced dipole strength(polarity)?

normal phase elutes peaks from non-polar to most polar by time.
reverse phase elutes peaks in reverse order to that of normal phase?

main difference? water should be avoided with normal phase Liquid chromatography but with planar chromatography it is OK.
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By Anonymous on Friday, December 27, 2002 - 10:21 pm:

Thanks to everyone who reply to my question.
I try with phenyl column to separate paracetamol,Phenylpropanolamine Hydrochloride and triprolidine hydrochloride with following chromatography condition.
Column : hypersil phenyl column 10micron particle size, 4.6 x 250 mm length.
Mobile phase :
Buffer : Methanol :Acetonitrile (60:20:20)
BUFFER (N2HPO4 500MG TO 500ML DILUTE WITH WATER AND ADJUST THE PH TO 6.5 WITH GLACIAL ACETIC ACID)
Detector : 220nm
Flow rate : 2.0ml/min
I am able to separate Paracetamol and Phenylpropanolamine hydrovchloride. Their Retention time are 2.5min and 3.5 min resp.
My problem is Triprolidine does not elute even after 30minutes. Any one can help me ?
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By Anonymous on Sunday, December 29, 2002 - 08:34 am:

Thanks to everyone who reply to my question.
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By Anonymous on Friday, January 3, 2003 - 05:24 am:

did you use the same analytical conditions as in the reference? what were the retention times for Paracetamol and Phenylpropanolamine hydrochloride in your reference?
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By Anonymous on Monday, January 6, 2003 - 05:07 am:

Yes, I used the same analytical conditions as in the reference. The Rt of Paracetamol and Phenylpropanolamine hydrochloride is 2.5min and 3.5min resp.
Just for your information every component used is reference substance and concentration of each component is 1mg/ml and 20 microlitre were injected so there is no question of response is very small which can not be detectable.
Thanks for your answer.
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By Anonymous on Monday, January 6, 2003 - 10:51 pm:

what about triprolidine retention time in the reference? Have you seen triprolidine eluting at all? At what time? What equipment are you using compared to the ref? Any change you are having difference in a delay volume?

Check and double check the mobile phase (pH meter!) and column. I have worked one month with a method and realized I was having wrong column id...frustrating.

Good luck
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By Anonymous on Saturday, January 11, 2003 - 12:57 pm:

Cyano column is unstable than C18 column ?
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By Chris Pohl on Tuesday, January 14, 2003 - 12:40 pm:

Cyano phases are definitely less stable than conventional C18 columns. This stems from 3 problems: first, the chemical bond to the silica surface is more accessible because of the small size of the ligand; second, the higher polarity of functional group improves the compatibility of reagents responsible for the hydrolysis process (i.e. acids or bases) which results in accelerated hydrolysis relative to more hydrophobic environments and third, the cyano group itself is not hydrolytically stable (although, this latter factor is probably not the predominant cause of capacity loss since hydrolysis of the cyano group under acid conditions is quite slow and proceeds rapidly only under alkaline conditions not generally utilized with silica columns). Agilent sells a product (Zorbax SB-CN) which reportedly has significantly improved hydrolytic stability do to the incorporation of two bulky isopropyl side groups into the cyano ligand. This helps restrict access to the cleavage site and renders the vicinity of the leakage to the surface more hydrophobic and thus less prone to hydrolytic attack.
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By Anonymous on Tuesday, January 14, 2003 - 07:03 pm:

hi
Chris

Thank you very much for replying to my question.
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By Anonymous on Tuesday, January 14, 2003 - 07:15 pm:

Does any one know about Sertraline.Hcl HPLC Separation conditions, there are three Impurities. one of the Imp coming along with drug in Reverse phase conditions. pl respond
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By Jan on Wednesday, January 15, 2003 - 04:37 am:

Anonymous, you might check this paper :

Journal: Journal of Pharmaceutical and Biomedical Analysis
ISSN : 0731-7085
Volume : 26
Issue : 3
Date : Oct-2001
pp 505 - 508
Assay of sertraline in tablets and drug substance by liquid chromatography
A.I.H. Adams, A.M. Bergold
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By Anonymous on Wednesday, January 15, 2003 - 07:21 pm:

THANKS A LOT FOR YOUR INFORMATION,
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By Anonymous on Thursday, January 16, 2003 - 09:43 pm:

ANONYMOUS, You will get HPLC method for Sertraline Hydrochloride in USP FORUM VOLUME NO 24
Try this method wheather all impurities get separated with this method.
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By Anonymous on Friday, January 17, 2003 - 07:34 pm:

Thank you very much,
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By Anonymous on Sunday, May 25, 2003 - 12:56 am:

Hi everyone,
Does someone, by any (small) chance, knows something, anything, about an HPLC method of IODANTIPYRINE?
Thanks in advance,
Any.
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By Anonymous on Friday, July 4, 2003 - 09:13 pm:

Not sure this is the correct palce to post this. I am using a Starion AS300 column to separate the Cr(III) and Cr (VI). The mobile phase is HNO3 at pH 3. I am trying to understand the dynamic of the chromatography on the column as AS300 is an anion exchange column and in low pH the species being investigated are presumably not anions. Thanks in advance.
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By somnath on Friday, August 22, 2003 - 06:59 am:

Hi everyone
Please suggest some method for the determination of related substances of Cyclosporin A.
Thanks
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By Anonymous on Saturday, August 30, 2003 - 07:20 pm:

does anyone know how to separate 4-aminophenol from paracetamol using RP HPLC?
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By Anna on Monday, October 6, 2003 - 08:24 am:

Could some one suggest a HPLC method for Ibuprofen? Initial conditions for method development?
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By A.Nonymous on Monday, October 6, 2003 - 08:54 am:

Anna, try the search engines, you can find a lot of starting material there.

Here is one application:

Link


Regards
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By Anna on Monday, October 6, 2003 - 10:13 am:

Thank You A.Nonymous. Your suggestion is much appreciated.
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By ad on Monday, October 6, 2003 - 11:10 am:

i you have a new question, start a new treat. at least use a name. in this line there are a lot of queswtions from anonymous. i can't figure out who is anwsering whos question.
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By venksin on Saturday, October 11, 2003 - 04:50 am:

Hi,

Could anybody suggest a method for analysis of an acid chloride (such as benzoyl chloride) by HPLC. Is derivatisation essential
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By Anonymous on Saturday, October 11, 2003 - 12:26 pm:

Venksin: It is a very labile compound. No chance in reversed-phase due to the use of aqueous mobile phases. In normal phase, I expect it to react with silica or with residual silanols on those packings that are derivatized.
The best and simplest way is a derivatization, with any amine, followed by reversed-phase.
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By venksin on Monday, October 13, 2003 - 02:49 am:

Thanks for the info. But how would you ensure complete derivatisation. Since the compound (the benzoyl chloride) is not stable on exposure, there are chances for the acid impurity to be present. Will it also get derivatised, or acid will remain free
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By Anonymous on Monday, October 13, 2003 - 12:25 pm:

Well, maybe you want to just analyse the acid
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By Chris Pohl on Tuesday, October 14, 2003 - 12:42 pm:

Venskin:

Relative to your question about the viability of directly performing an essay on an acid chloride: as mentioned above, you could well have problems with such an essay in the presence of an aqueous mobile phase although hydrolysis in the absence of a catalyst is not all that rapid so you might be able to get away with it in an unbuffered eluent system. It seems to me that you should also be able to make an ester with high enough yield to be able to perform the assay on the ester. For example, react the acid chloride with methanol under conditions suitable for quantitative conversion to the methyl ester.
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By Jeff on Tuesday, October 14, 2003 - 01:14 pm:

Hi,

Can any one help explain how the response factor and recovery test are used in the method validation of organic impurity quantification assays (HPLC or GC). I will appreciate if someone can provide a detailed procedure.

Thanks,
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By venksin on Monday, October 20, 2003 - 08:59 pm:

Hi

On the derivatisation of acid chloride, of several methods tried we were more comfortable with a morpholine derivative (basically an amine derivative). The derivative will be formed in the presence of a strong base such as KOH. The acid was remaining free and thus could be estimated. Thanks for all suggestions
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By kapil on Tuesday, October 21, 2003 - 01:02 am:

methanol,THf,acetone&acetonitrilwhich is more polor &why pl give incereasing orderof polarity
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By Chris Pohl on Tuesday, October 21, 2003 - 09:26 am:

The answer to your question depends somewhat upon your intended use of these solvents. For normal phase applications, the most polar of the solvents listed is acetonitrile. Burdick and Jackson have, or at least used to have a nice solvent handbook which lists the polarity index for these solvents as acetonitrile: 5.8, methanol: 5.1, acetone: 5.1 and THF: 4.0. But if you look at the elutropic value on alumina you get the following values: methanol is the most potent at 0.95, acetonitrile: 0.65, acetone: 0.56 and THF: at 0.45. A physical parameter which correlates roughly with the polarity index is the dipole moment of these solvents. The dipole moments for the solvents are: acetonitrile: 3.44, methanol: 2.87, acetone: 2.69 and THF: 1.75. Another physical parameter which correlates roughly with the polarity index is the dielectric constant. The dielectric constant for these solvents are: acetonitrile: 37.5, methanol: 32.7, acetone: 20.7 and THF 7.58. Of course, in reversed phase work methanol is invariably the weakest eluent followed by acetonitrile which is weaker than THF (I don't have any experience with acetone so I couldn't place it in this sequence for reversed phase elution power), so obviously more than just polarity is involved in this order reversed phase elution power.
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By syx_interbat on Friday, November 7, 2003 - 05:14 am:

Anonymous or anyone,
May you give me the reference for determination paracetamol and phenylpropanolamine HCl above?
Thanks
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By lucia on Tuesday, November 18, 2003 - 09:35 am:

I´m trying to implement a method for the determination of sulfonamides in honey, but the five standars have very close retention times which make them imposible to resolve, is there any possibility without changing the mobile phase?
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By Anonymous on Tuesday, November 18, 2003 - 10:39 am:

when you want to implement a method using fluorescence detector, is there any problem in using the maximum gain possible? (18)has this any effect on the detector, the lamp, the memory used, etc,anyway, is this more desirable than injecting more volume into the column, I guess, if I need a very low detection limit (0.01ppb)
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By Anonymous on Tuesday, November 18, 2003 - 01:19 pm:

Gain increases both signal and noise, injecting a larger volume increases signal. Sensitivity is signal/noise.
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By Anonymous on Tuesday, November 18, 2003 - 04:49 pm:

Lucia,

It is always best to play with the mobile phase to get better resolution. However, you may get an improvement, if you reduce the flow rate by a factor of two (and wait two times longer for the peaks to come out).
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By Anonymous on Wednesday, November 19, 2003 - 03:20 am:

Hi, we are developing a method for the determination of an ACE drug and its metabolite by LC electrospray ionization mass sspectrometry. We are currently working with an aqueous/methanol mobile phase containing 0.05% formic acid and we cannot explain the serious instability of the ms signal of the metabolite. Do you believe that a small quantity of sodium hydroxide less than 0.01% could affect the ms signal? Notice that a less than 5% adduct with sodium was observed in the mass spectrum of the metabolite.
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By lucia on Wednesday, November 19, 2003 - 06:00 am:

supossed the same response and the noise is not a problem, which one is more desirable?
gain 18 volume 20ul
gain 10 volume 100ul

In other words, is there any extra expense of the detector using higher gains?
thank you in advance
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By Anonymous on Wednesday, November 19, 2003 - 03:25 pm:

To anomymous above: why should there be any sodium adduct if your mobile phase does not contain any sodium?
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By Basil on Thursday, November 20, 2003 - 01:23 am:

To anonymous: the sodium adduct comes from the sodium extracted from glass bottle or any other glass component.
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By Anonymous on Thursday, November 20, 2003 - 02:14 pm:

to Anon ACE drug:

do you see a 2M+Na signal? You may only get 5% on the M+Na adduct, but the dimer (from pi - cation-pi interaction) may be a significant. Also, check the 2M+H response.

I usually avoid ESI for quantitation since it is infamous for nonlinear response. Often, I use uv detector and MSD in tandem. MSD for identification, and uv for quant.
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By Anonymous on Thursday, November 20, 2003 - 10:52 pm:

To Anon of November 20, 2003 - 02:14 pm.
If you make a survey of the literature, ESI is the most used technique in LC/MS.
IT IS MOSTLY USED FOR QUANTITATION, AND GIVES LINEAR RESPONSE (you have to use the right concentration range, however, because saturation occurs as with any type of detector including UV).
Using a combination of MSD for identification and UV for quantitation reminds me of the people who used a chart-strip recorder in combination with the integrator, when the first computer-based data systems were introduced (just in case...)
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By Anonymous on Friday, November 21, 2003 - 11:28 pm:

A quick survey of the literature shows me that ESI is mostly used for proteomics. It can be used for quantitiation but that typically requires purchasing or synthesizing stable isotope standards to normalize the signal response.

I have found the uv detector to be inheritently more robust, reproducible, and trouble free than the MSD response. ie. the above Na interference would have no effect in delaying my project's completion.
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By Anonymous on Thursday, December 4, 2003 - 10:34 pm:

Am master student and he/she wanted to know as the the procediment to identify the chemical compuestes of a watery extract of Ambrosia cumanensis, using HPLC
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By ulm on Wednesday, December 10, 2003 - 04:00 am:

Hi

Can anybody help me to solve my problem:
How to quantify using HPLC with UV detector following compounds:
m-phenylene diamine,diaminoanisole and
2,4-diaminophenoxyethanol dihydrochloride

Many thanks
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By zelechonok on Wednesday, December 10, 2003 - 08:40 pm:

Take one of our Primesp columns (Primesep 200, 150 mm x 4.6 mm, for example) and analyze your mixture using MeCN/Water/TFA mobile phase. Keep TFA concentration 0.1% and adjust water/MeCN ratio until all peaks are resolved. The easiest way to do that is to add 0.1% TFA in each water and MeCN bottle.
Primesep columns specifically design to retain polar basic compounds that are not retaining by RP systems. Call us or e-mail if you have more questions. All information available on our website www.primesep.com
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By mitali on Thursday, January 1, 2004 - 06:29 am:

i want to know the method for seperation of fexofenadine HCLtablet and its impurities namely 4 impurities metafexofenadine,ketofexofenadime,
methyl ester of fexo and methyl ester of keto fexo HCL
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By Uwe Neue on Thursday, January 1, 2004 - 07:43 pm:

Mitali:
I have not done this assay, but we have worked with fexofenadine. Its retention varies nicely with pH, which allows you to optimize the method by playing with the pH. We worked with XTerra RP18 (also with XTerra MS C18). In 30% acetonitrile, the retention factor changed from 4 at very acidic pH to 2 around neutral pH to values abov 4 at pH 10 again.

Hope this gives you a good start.
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By mitali on Monday, January 5, 2004 - 06:08 am:

dear uwe,
thank u for ur suggestion.i will get back to u
after i research a more on it.
hope to keep in touch
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By Anonymous on Tuesday, March 2, 2004 - 10:46 am:

please give me an extraction method for TACROLIMUS from erythrocytes by LC/MSMS
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By Maximoose on Saturday, March 13, 2004 - 07:03 am:

Hi Every body there
thanks for your co-operation in advance..my question is
what is the powerful,accurate method to extract and analyse Diquat(Contact herbicide) from Potato Tubers and using HPLC in analysis...
Wish u all the best
thanks
bye
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By Anonymous on Saturday, March 13, 2004 - 09:24 am:

Please check this method and related methods: www.waters.com/pdfs/Oasis117.pdf. These methods have been used to determine paraquat and diquat in a variety of matrices.
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By arvind on Sunday, March 14, 2004 - 04:12 am:

Hi every body
Any of you have an idea to calculate theoratically a polarity of methanol,water or any
solvent used in H.P.L.C.
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By Dimitris Hatzinikolaou on Tuesday, March 23, 2004 - 03:56 am:

Hello,
This is an urgent inquiry, please.
Is it possible to use a LichrossorbNH2 column
to separate and quantify a sample that
has glucose, gluconic acid and fructose.
If yes at what approximate conditions??
Thanks to everyone in advance
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By Zelechonok on Tuesday, March 23, 2004 - 06:57 pm:

Most likely this will not work. Usually when sugars are analyzed on NH2 columns the mobile phase is ACN/water. Acidic compounds like gluconic acid will not elute from NH2 column with this mobile phase. If you add a buffer to the MP then sugars separation can be compromised.
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By Zelechonok on Thursday, March 25, 2004 - 06:41 am:

What is your detection system? MS, IR, UV, ELSD?
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By Anonymous on Thursday, March 25, 2004 - 04:35 pm:

Hi,
I am trying to develop a method for quantitation of Hexadecanol using HPLC and ELSD for detection.
Any help pertaining to known mobile phase composition and/or column type would be great.
Thanks in advance!
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By Anonymous on Thursday, March 25, 2004 - 07:36 pm:

Any C18, Gradient from 50%Water to no Water.
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By HARSHA on Friday, April 2, 2004 - 06:58 am:

We are getting unknown zone(peak) in Residual solvents by GC method.It is not listed in the ICH Guidelines.Could you recommend that what level we have to fix the specification.We have no idea about the toxicology data.
Can we discard the peak which S/N is below 10.

Can we fix the limit of Quantification level as Limit for such compounds.

In manufacturing process we are using the some reagent which we are going check in Residual solvents methods.
For those reagents how we have to fix the specification limit(Not menioned in ICH )

Pl clarify our doughts.Waiting for your reply
Here I am giving my mail id

id:harsha_gsr@hotmail.com

THANKING YOU
Regards
Harsha
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By Anonymous on Friday, April 2, 2004 - 08:04 am:

Could some one suggest a HPLC method for sodium diclofenac? Initial conditions for method development?
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By Uwe Neue on Friday, April 2, 2004 - 03:03 pm:

Here is a link for diclofenac:

http://www.waters.com/pdfs/YMC111B.pdf
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By Anonymous on Tuesday, April 6, 2004 - 04:45 am:

I have been trying to analyze ACC (1-aminocyclopropane-1-carboxylic acid, ethylene plant hormone precursor) using HPLC coupled to fluorescence detector. ACC was derivatized using 9-carbazolyl acetic acid chloride in alkaline media (pH 8.8 with borate buffer) for 2 min. After that time a cleavage reagent (hidroxilammonium hydrochloride and methyl-thio-ethanol in 0.850 M NaOH) was added, and after 3.5 min a quenching reagent (acetonitrile:water:acetic acid) was added. After this procedure resulting solution was ready to inject in the HPLC system without further purification (Fan, X et al.1998. Anal. Chim. Acta. 367:81-91). Everything seemed to be very beautiful, standard mixture of ACC was easily derivatized and separation in a 100x2.0 mm C18 colum only was 7.0 min. The problem is that in plant extracts (extracted from 0.5 g fresh tissue in 5 mL 80% EtOH, after extraction supernatant was evaporated under vaccuum and then pellet resuspended in ultrapure water, water layer was washed three times with chloroform and kept at -20ºC until analysis) derivatization did not take place, or at least this is our first proposal. Even in spiked samples at 400 uM level was not possible to see any peak at the aforementioned retention time. I have tried with spe with c18 cartridges, solvent partition, but none of these techniques seems to improve the situation. What can I do? please help me...
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By sonal on Thursday, April 15, 2004 - 02:37 am:

It is mentioned that the pH of the mobile phase be +/-2 of the pKa of the drug. I have to develop a method for its analysis by HPLC. I wanted to know whether +2/-2 is preferred. My drug is an ionizable molecule with a pKa of 4.5.
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By sonal on Thursday, April 15, 2004 - 02:44 am:

If a drug is converted to its lactone form is there any way that we can prevent the conversion.Can we add an antioxidant to the stock solution of the drug?
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By Uwe Neue on Thursday, April 15, 2004 - 01:45 pm:

Sonal: the retention time of a compound will vary with the pH, if it is in the range of +2/-2 around the pK of the compound. You can either stay away from this range, or you can make sure that the pH is always adjusted accurately. If you do the latter, the retention can be as reproducible inside this range as outside this range. If an acetate buffer is fine for your application, there is nothing fundamentally wrong with using this buffer.
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By sonal on Thursday, April 15, 2004 - 09:35 pm:

Uwe Neue: We would definitely not be using the range of +2 to -2 rather only one pH ,either +2 or -2. I wanted to know which pH is preferred as the compound has to be in one state only either ionized or unionized. If we use -2 whole of the compound will be in the unionized state and if we use +2 it will be in the ionized state. Also I have no separations to be done as I have only one drug whose method I have to develop. My drug is an acidic drug.
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By Chris Pohl on Friday, April 16, 2004 - 08:11 am:

Sonal

As a general rule, it's usually preferred to operate with analytes that are in their neutral form when using a reversed phase column. This generally gives better retention, better peak shape and better reproducibility. The major exception to this would be basic solutes where the pH necessary to produce the neutral form of the analyte might result in significantly shorter column life, depending upon the basicity of the analyte. Of course, the other exception to this is in cases where you need to adjust selectivity in order to improve the resolution of two closely eluting analytes. Increasing the ionization of one of the poorly resolved components can be an effective tool in adjusting resolution.
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By Anonymous on Saturday, April 17, 2004 - 02:22 am:

how do i separate Phenoxy Ethanol and Methyl paraben on reverse phase column
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By sonal on Monday, April 19, 2004 - 01:06 am:

Thankyou Chris Pohl and Uwe Neue for your reply. regarding my second question, to prevent the lactone conversion of the compound is there any other method besides maintaining the ph to a specified value for eg by adding an antioxidant so that I can manipulate my ph to control the retention characteristics.
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By Chris Pohl on Monday, April 19, 2004 - 06:13 pm:

Sonal,

Depending upon the stability of the lactone in question, you may be able to convert your compound back to the free hydroxy acid by simply elevating the pH of your sample for a sufficient time prior to injection. Generally, the rate of formation of the lactone will be too slow to occur to a significant extent during the chromatographic separation. Of course, performing the separation at a pH significantly higher than the pKa the acid will help prevent formation of the lactone during chromatography.
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By sonal on Monday, April 19, 2004 - 10:32 pm:

Chris Pohl

In one of the references it is mentioned that the pH has to be maintained at around 4 and the pKa is around 4.5. According to what you have said that the lactone formation takes time then can I take the ph around 2.5 so that the compound remains in the unionized or shall I maintain the stated ph i.e around 4 only.
Also in the stock solution which I shall prepare everyday is there any chance of conversion to the lactone?
Can adding an antioxidant be of any use?
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By Anonymous on Monday, April 19, 2004 - 11:15 pm:

Does anyone know about the HPLC (RP or Normal) method for Tacrolimus
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By Chris Pohl on Tuesday, April 20, 2004 - 08:13 am:

Sonal,

Formation of the lactone is nothing more than formation of an internal ester. Depending upon the size of the ring formed during the lactone formation reaction, there can be considerable differences in the stability and rate of formation. Without knowing just how stable your specific lactone is, I can't give you a definitive answer. However, if you're chromatographic parameters are compatible with operating at higher pH (i.e. if you have sufficient retention and suitable selectivity at higher pH) then your best option would be to operate at a pH equal to or greater than the pKa since the lactone can only be formed when the hydroxy acid is in the nonionized form. Regarding your stock solution, your best option would be to adjust the pH such that there is essentially none of your compound present in the nonionized form (i.e. at least two pH units higher than your pKa). This should prevent formation of lactone in your stock solution. Finally, an antioxidant should have no effect on this reaction since lactone formation is esterification reaction not an oxidation reaction.
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By sonal on Wednesday, April 21, 2004 - 07:30 am:

Thankyou Chris Pohl for your reply. Actually I am new to this area and have not yet started with my work. So whenever I read something new I have queries in my mind.
The reference I mentioned in my earlier message says that the pH should be maintained at 4 to prevent the conversion of the compound to lactone and you are saying to maintain the ph above the pka of the compound.
Also is TLC necessary before proceeding for the method development by HPLC. What inference can we get from it? Can the solvents optimized with this method be extended to hplc. Can we use mixed solvents for tlc like mixture of buffer and the organic solvent or just the pure organic solvent needs to be tested?
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By Chris Pohl on Wednesday, April 21, 2004 - 08:03 am:

Sonal,

I can't imagine a good reason for recommending pH 4 in order to minimize lactone formation unless it's related to the optimal conditions for retention of your analyte. As I said previously, the higher the pH the better with regard to preventing lactone formation.

Regarding the use of TLC prior to HPLC method development, I would say that generally there is relatively little benefit to this approach. While I suppose it might be conceivable that one could screen a wide variety of mobile phases more efficiently using TLC, the fact is that conditions used for TLC are not exactly analogous to conditions used for HPLC since in TLC one does not normally move analyte bands all the way across the plate during development. Since in HPLC one must use mobile phases sufficient to carry the analyte bands through the column, the conditions which are optimal for TLC will not necessarily be directly relevant to optimal HPLC conditions. I would say that a better strategy would be to use a "generic" gradient from 5% acetonitrile to 95% acetonitrile with a suitable buffer to scan for appropriate eluent conditions for a given set of analytes. From there you can identify suitable conditions for isocratic operation, assuming isocratic conditions are feasible.
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By Anonymous on Friday, April 23, 2004 - 07:41 am:

for anonymous with separation problem of Phenoxy ethanol and methyl parabene

Try a Caltrex AIII column 250x4 with ACN/water 20:80 (v/v); 1ml/min; 40°C; 225nm!
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By sonal on Thursday, April 29, 2004 - 07:59 am:

Thankyou Chris Pohl for the reply
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By Bharat Kumar Agarwal on Thursday, May 6, 2004 - 04:55 am:

Repeatability: Repeatability should be assessed using:
a) a minimum of 9 determinations covering the specified range for the procedure (e.g.
3 concentrations/3 replicates each)
or
b) a minimum of 6 determinations at 100% of the test concentration.

Please explain me above statement of ICH
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By Bharat kumar agarwal on Thursday, May 6, 2004 - 05:21 am:

Hi everybody,

If two drug have not the same Maximum UV absorpation suppose one shows the 322 and another shows 254 nm, in that conditions can we develope a simultaneous HPLC method.What is the basic criteria for develope a simultaeous method developement?

Please suggest me ?

Thanks in advance
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By Bharat Kumar Agarwal on Thursday, May 6, 2004 - 05:34 am:

How we measure the column effecincy or number of theoritical plate ? If we made 1000 run during validation of a method then is it necessary to calculate the column effecincy or number of theoritical plate for every run ?
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By tom jupille on Thursday, May 6, 2004 - 08:50 am:

Re: ICH. the statement means just what it says. You have a choice:
a) do three runs at each of three concentrations, or
b) do 6 runs at the "target" concentration of your analyte.

Re: wavelength. No simple answer here, depends on what interferences are present and how well your two compounds are separated. For method development, use a photodiode array detector; you can then reprocess the data at different wavelengths to get an idea of how to proceed. Safest would be to switch wavelengths between the two peaks (but they must be reasonably well resolved for this to work). An intermediate wavelength (e.g., 280 nm) may also work, but may have robustness problems.

Re: plate number. That is a *very* basic question. We have a free on-line course called "Getting Started in HPLC" that will answer it (and many more!). You can access it here:
http://www.lcresources.com/resources/reslinksform.html

If you measure retention times and resolutions in each run, then plate number need not be specified separately. On the other hand, most data systems will do the calculation for you automatically, so why not?
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By Uwe Neue on Saturday, May 8, 2004 - 09:23 pm:

Here is a bit of basic thought:
In order to determine the quality of your separation, the general measure is the resolution between peaks. Resolution has several components: the retention time difference between the peaks, and the peak width(s). In order to get at retention time difference, you need to know the retention times. Retention times are one set of the important parameters from the standpoint of troubleshooting: did one peak move, did both move, etc.? The other set of important parameters are the peak widths. Did one peak become wider, or both? Is the peak width where it is expected to be?
Retention time differences are primarily caused by changes in the chemistry of the separation - differences in the mobile phase or aging of the stationary phase or similar things.
Peak width differences are often caused by changes in the physics of the separation - deterioration of plate count, differences in the sample make-up, differences in the plumbing of the system etc.
While these are not 100% solid rules, they are good starting points for troubleshooting.
Since you are getting all these other pieces of information for free when you calculate resolution, it is worth keeping an eye on the more basic parameters.
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By Anonymous on Wednesday, February 25, 2004 - 07:57 am:

Any suggestions do develop an HPLC method for, Benzyl alcohol, Benzyl Peroxide, Phenoxyethanol, Parsol MCX, Parsol 1789, Octyl Salicylate,and methyl Salicylate (7 in one peak). Any help would be appreciated.
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By belle on Thursday, August 12, 2004 - 09:45 am:

hi,
i need some help, it's for a research project and i have no idea how to begin.

should i use liquid chromatography or HPLC to test for methyl salicylate from menthol liquid and eucalyptus oil? and why?

thank you.
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By GC Dave on Friday, August 13, 2004 - 08:10 am:

Belle,
Have you no access to GC? Look at column manufacturers websites for GC chromatograms.
Varian,Restek,Agilent etc.
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By John on Friday, August 13, 2004 - 10:05 am:

Has anybody help me on Oxandrolone Tablet Assay/Rc, if you have any method or any ideas i would really apprecite....