Peaks separation in HPLC

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Dhruv
Dhruv's picture
Peaks separation in HPLC

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

 

 

 

 

Dr. Analytical
Dr. Analytical's picture
We need more information

We need more information about your system.  Please provide the name of the column (C18, C8, Phenyl, etc.), the size (diameter, width, particle size), and other parameters (flow rate, injection volume, detector settings), and mobile phase composition.  You listed the combinations that you tried, but did not tell us how much of each solvent was used.

In general, if you need more resolution, you must increase the retention of the compounds.  With the information requested we can give you a better idea.

Sami Tuomivaara
Sami Tuomivaara's picture
Dhruv,

Dhruv,

It looks like you only considered acid additive and the organic solvents as parameters in your optimization.

I'd first try to lengthen the gradient time, then try to optimize flow rate. I think these suggestions can be quite robustly applied to various hplc resins.

Another thing to try is to load less material, maybe one tenth or fifth of what you loaded previously, if your detector can handle those small amounts.

Cheers,

 

Dhruv
Dhruv's picture
For Dr. Analytical: Thanks

For Dr. Analytical: Thanks for your comments and here are the further details you had asked for:

1.The Column: The Column is Thermo Hypersil Gold : One of my senior gave me this column for use and told me that its a C18 column and i started using it. But when i checked litrature from Thermo guide i got the following information: 

Hypersil GOLD columns are based on improved, highly pure silica and a novel proprietary derivatization and endcapping procedure using alkyl chain chemistry. This gives Significant reduction in peak tailing while retaining C18 (USP L1) selectivity

So from the definitation its a bit confusing, either its a silica column or C18 column or C18 like column. 

2. Each solvent used: It was a gradient of these solvent, with Solvent B from 0-100% in 50 mins.

3. I tried to increase the retention of compunds but of no use. The peaks become more flat instead of separating.

For Mr. Suola: Thanks for your comments and here are the further details you had asked for:
 

1. The acid additive: Actually i went ahead with phenolic extraction and found that most of the authors have used these combinations so i also followed that. It worked to some extent but now i am stuck. 

2. Lengthening the gradient time and optimizing flow rate: I i increase the gradient time it normally flatens the peaks but no improvement in resolutions. 

3. Loading less material: In fact i am getting the opposite results, as with loading less material (2.5 to 20 microliter of that isolated peak(fraction) on a 5 mm diam column i am again getting a sharp single peak for the fraction but when i load from 50 to 100 microliter of that fraction then i am getting that peak fractionated into three subpeaks though the resolution i want is not there and that is my problem.

Dr. Analytical
Dr. Analytical's picture
Dhruv:

Dhruv:
Your column has a silica "support" with a C18 bonded to the surface.  Therefore, it is a C18 column.

These columns usually do not operate correctly with 100% aqeuous mobile phase.  You should use at least 5% organic solvent as your starting conditions, and then program to 100%.  I do not think that will change your results here, but it is good information to have for future experiments.

If your column has 5 mm internal diameter, then it is an analytical scale column, not preparative.  Loading 50 - 100 uL will overload the column.  "Flat" peaks results from the large injection volume.  You said peak shape was normal for smaller injection volume.  The peaks are probably flat becuase you have saturated your detector - it has reached the upper response limit.  If this is an absorbance detector, the scale will probably be about 2 - 5 AU (or 2000 - 3000 mAU).  Under these conditions, the individual peaks that you are seeing may not be real.  They are noise in the detector caused by the high absorbance.  You must develop your separation under conditions that produce good peak shape.  Then you can make multiple injections and collect fractions.  Making large injections on small columns will probably not be successful.

Dhruv
Dhruv's picture
Dear Dr. Analytical:

Dear Dr. Analytical:
Thanks for your suggestions.
My current column is indeed a 5 mm ID column. But previously i used a YMC Pack-ODS-A column with ID of 20 mm. 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.
Further the peaks are not totally flat but are not sharp. The peak height is not more than 200 mAU for around 5-10 microliter of sample injected. And the upper limit is definitely above 2000 mAU as i have seen the maximum readings till 1500 mAU.
As far as noise peak is concrened i dont have much knowledge about that. 
Someone suggested me to change the column to c4 or c8 or even change the brand to get some better results. Also someone suggested me to go for Normal phase HPLC, though i have never done that before. Your comments please.
Still i am working on the issues. 
Thanks once again.
regards,
Dhruv.

Dr. Analytical
Dr. Analytical's picture
We need to see some

We need to see some chromatograms.

Also, you must realize that not all C18 columns are the same, so the YMC and Hypersil columns will not always give the same separations, so it is difficult to determine what the differences are in the two separations.

I do not think that normal phase will be a good option.  You would have to dissolve your sample in a solvent like hexane or dichloromethane.  And a C4 or C8 column would give less retention but probably the same selectivity.  You must find a column with much different selectivity.  Perhaps a phenyl, cyano, or PFP column might produce a different separation.

Dhruv
Dhruv's picture
Dear Dr. Analytical: Thanks

Dear Dr. Analytical: Thanks for your comments and
suggestions:
1.      
1. Chromatograms: 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)
2.      
2. Different columns: Somehow I am getting similar,
though not identical, elution profiles for both hypersil gold as well as
YMC  column.
3.      
3. Somhow I don’t have any of the phenyl, cyano or
PFP column with me. I have the following columns with me currently: let me know
if either of these can be used: YMC carotenoid (This one I am going to try next
time), Phenomenex Pherex 10 Silica (Normal phase, in my opinion), Thermo
Hypersil 100 amino (for amino acids, normal phase, I think), Hypersil HS silica
(normal phase, in my opinion), Phenomenex PolySep-SEC 1000 (Size exclusion) and
Phenomenex Rezex Ram Carbohydrate (Ion exclusion).

with regards,
Dhruv
 
 

Dr. Analytical
Dr. Analytical's picture
Dhruv:

Dhruv:
What solvent is your sample dissolved in, and which mobile phase are you using?  If your sample is in a "strong" solvent such as methanol or acetonitrile, then most of the peak shape changes in the large volume injection are caused by the sample solvent?  The column can tolerate a small volume of a strong solvent, but not a large volume like 60 uL.

I am not familiar with the YMC column, but that does look like the best option.  The amino column could also be used, but you must check what solvent it contains.  If it contains hexane or another water immiscible solvent, then you can not use it with your existing mobile phase.

Dhruv
Dhruv's picture
Dear Dr. Analytical:

Dear Dr. Analytical:

I am using DMSO as the solvent for my sample. Since i wanted to use the heighest concentration of my sample for prep HPLC (I could do it till 50 mg/mL).  So even now i am using the same DMSO for 5 mg/mL also. Can it effect the elution profile at higher concentration/ injection volume?

From the peak shapes using both 254 and 280 nm. Do you think that its a single compound or 4 different compounds? 

Your comments please!
waiting for your reply,
with regards,
Dhruv