[Dhruv]

Dear Friends,

 I am trying to seperate a compound having antifungal activity by Preperative HPLC on Agilent 1100 system using Thermo Hypersil Gold column. Solvent A: 2% Acetic acid in DIW and Solvent B: Acetonitrile.

 I got a peak with very good antifungal acivity. But when i try to analyze it further then i realized that its nothing but having 3 subpeaks. But the 3 subpeaks are so closely associated that i am not able to seperate them.
 I tried different Solvent A and B combinations including

   Solvent A             Solvent B

1   Acetic acid 2%             0.5% AA in DIW n Acn (50:50)
2   Acetic acid 2%              Acne 100%
3   Formic acid 0.5%   Acne 100%
4   Orthophosphoric acid 0.2%   Acne 100%
5   Trifluoroacetic acid 0.2%    Acne 100%
6   Acetic acid 2%                          Meth 100%
7   Formic acid 0.5%               Meth 100%
8   Orthophosphoric acid 0.2%    Meth 100%
9   Trifluoroacetic acid 0.2%      Meth 100%
But nothing is working.
Please suggest something
waiting for expert comments
with regards and thanks in advance
Dhruv

[enahs]

What is the flow rate, temperature you are using? What is the retention time for the various solvents you have tried?

And what method of detection are you using?

And what ratios of A:B have you used and/or played with?

[Dhruv]

For Enahs: Thanks for your comments and here are the further details you had asked for:

1. Flow rate: 1 mL/min
2. Temp: 25 degree C
3. Retention time: Approx 20 mins plus minus 2 mins for all the above solvents
4. Detections is Agilent 1100 inbuilt Diode array detector
5. A: B Ratio: its a gradient solution from 5-100 percent for 50 mins

with regards,
Dhruv

[enahs]

A few things you can do. You can slow down the flow rate, it will make it take longer but things will have more time to interact with the column and can improve separation.

I have not looked up the specs on you column, so do that fist before using the following ideas.
Increasing the temperature from 25 to ~35C can sometimes have significant impact on peak separation and shape, so I would try that.
You probably need a less polar organic solvent, I would try THF or Toluene (again, check if that is ok for column).

It is also very possible that despite not having a chiral column, you are getting some slight chiral separation. Does your compound you are looking at have chiral centers? You can look at adding some Chiral Mobile Phase Additives and see if that helps separating them more. You then have to ask your self if you want to separate them or not, if it is the case.

Perhaps you could attach and example chromatogram?

[Ligte]

Its also helpful to know whether this compound is an acid or base or even better whats the pKa and thereby adjusting the pH of your mobile phase, by also adding a base. Its also important to play around with the polarity of your injection solvent and the amount you inject can sometimes play a huge role. 

[Dhruv]

For  Enahs: Thanks for your suggestions

1.   Regarding slow flow rate: I will give it a try
2.   Specs of my column: My current column is a Thermo Hypersil Gold (C18) 5 mm ID column. But previously i used a YMC Pack-ODS-A column with ID of 20 mm as preparatory column. That i used to separate the ethanolic extract of my plant sample into different peaks. Sample loaded 50 mg/mL, 500 microliter. Then i checked all the peaks for activity. and the peak with the best activity was pooled from more than 20 runs. That peak/ fraction i concentated and finally  dissolved in 1 mL to a 5 mg/mL concentration.  This 1 mL fraction i cant risk to put on a YMC 20 mm ID column as if anything goes wrong then my all the sample will go to waste so i am using the small 5 mm ID column. so that though it will take time but still if something goes wrong i will lose not more than 50-100 microliter in a go.
3.   Increasing the temp: As per your suggestion I have increased the column from 25 to 35 degree C. It did had some effects. The peaks became distinct but separation was not achieved.
4.   THF and Toluene: I will give them a try
5.   Chiral Separation: I am not having any idea if my compound is a chiral one or not. So cant say anything about that.
6.   Attaching the chromatogram: Herewith I am attaching two chromatograms. With the Hypersil Gold column with 10 microlit (5 mg/mL) with beautiful single peak and second chromatograph with 60 microlit injected with 4 peaks (giving me headaches).

For Ligte: Thanks for your comments

1.   I cant say if the compound is acidic or basic. I am following the general protocol which is followed by most of the authors to purify a phenolic compound from ethanolic compounds of plant extracts.
2.   Amount I inject: Ya it is definitively showing effect, as with 10 microlit I am getting a single peak but with 60 microlit it is showing that its not a single compound but atleast 4 compounds.


with regards,
Dhruv

[enahs]

I see you are using a Diode array detector and not just pure absorbance. Good.

Those extra peaks might not be extra peaks, you might just be overloading your column. Look at the spectra  at 15.8, and 17 min. Do they look virtually identical?

If they are identical, then it is very likely (though not guaranteed) you are either overloading the column or you are resolving enantiomers slightly. If they are very different, then yes, they are different compounds.

[Dhruv]

Dear Ehnas!

I couldnt understand what you want to say by
"Look at the spectra  at 15.8, and 17 min. Do they look virtually identical?"

But by looking at the spectra do you think that it is a single compound (overloaded) or 4 different compounds? Please comment.
By loading 60 microlit to me they appear to be 4 different compounds due to the peaks they show at 254 and 280 nm.
Your comments please!

waiting for your reply,
with regards,
Rakesh

[enahs]

What you have provided is a chromatogram.

Diode aray detectors not only provide absorbance chromatograms at one wave length, but can also provide a full UV-VIS absorbance spectra at all points. This provides much more information on the compound then just "when it comes out". You need to either enable the spectra taking in your runs or figure out how to access it from the old data if it is already doing so, and compare the absorbance spectra at the different times.

[Dhruv]

Dear Enahs!

I am sorry, but still cant understand what you want to convey. In my system i can take spectra at different wavelength and i am taking two wavelengths 254 and 280.
Thanks for your comments and suggestions,
with regards,
Dhruv

[enahs]

Your data says the detector is a Diode Array Detector; that means it can take a full UV-Vis spectrum. So, if you read here at the following website and it will briefly tell you about what kind of information you can get from a spectrum http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UV-Vis/spectrum.htm
It would tell you with great confidence if is overloading the column or some other compounds.

You need to ask around from somebody who has more experience with the instrument on how to either turn that feature on or access that data from the previous runs if it is turned on in your method.


The chromatogram is not a spectra/spectrum. Yes, it is using UV-Vis at a specified wavelength(s) to plot the chromatogram.

[Dhruv]

Dear Enahs!
Thanks for your help, i will go through the article and hope that some solution will come out.
with regards,
Dhruv