Is there anybody out there who can help me. I am trying to analyse a compound by LC which has the same chromophore as Toluene and unfortunately the sample work-up has toluene in it. I cannot seem to be able to separate the two contituents by any system I have tried.
Am I overlooking something obvious?
I will await your replies.
By Anonymous on Wednesday, October 20, 1999 - 09:25 am:
Have you tried using a chromatographic media which separates compounds by structure ? I refer to Hypercarb. This is one of the very few medias I know which will do this. You should have no problems with co-elution of your compounds. Give Rachel Phillips at ThermoQuest a ring, or mail her on RachelP@Hypersil.com . She developed my HPLC method on Hypercarb for similar types of compounds.
By Anonymous on Thursday, October 21, 1999 - 07:32 pm:
At the risk of being a little pedantic here, the real question is "how does your analyte *differ* from toluene?". That will point the way to an appropriate separation technique.
By Steph on Friday, October 22, 1999 - 08:35 am:
The structure differs by a long chain fatty acid and a nitro group.
Hope this is of some use to you.
By Anonymous on Sunday, October 24, 1999 - 12:54 am:
Separation of toluene from an aromatic compound bearing a long-chain fatty acid and a nitro group should work on a wide range of reversed-phase columns. If they should co-elute, you should be able to get them apart by changing the organic solvent from acetonitrile to methanol (or vice versa).
How many other analytes are in the sample? and what have you tried so far?
By Steph on Monday, October 25, 1999 - 08:38 am:
There are no other analytes in the sample.
So far I have tried changing the eluent composition i.e. from MeCN to MeOH and the water phases to buffers etc.
I have also tried to separate them using normal phase LC and have tried chaning the solvent systems in that to no avail!!!
I've even tried GC.
Any more help is appreciated.
By Bruce Freeman on Monday, October 25, 1999 - 10:24 am:
You have thus far provided almost no information to go on. Perhaps you should describe exactly at least one fo the column/mobile phase combinations you've tried, along with all instrument conditions.
By Steph on Tuesday, October 26, 1999 - 01:25 am:
The two systems I started with are;
The normal phase conditions were:
Column: Lichrosorb Si 100
Eluent: Hexane: Ethyl acetate 80:20
Flow rate: 1.5ml/min
The reverse phase conditions wre:
Column: IB-SIL BDS
Eluent: MeCN: water 50:50
Flow rate: 1.0ml/min
I have tried variations among these i.e. changing the solvents to those of differing strengths, making the solvent system quaternary, changing columns - Spherisorb ODS, Hypersil Elite C18 etc to almost no avail.
Is this any more use??
By Anonymous on Tuesday, October 26, 1999 - 05:48 am:
How do you know that your compound and toluene are co-eluting? And, how do you know that your compound of interest (quite hydrophobic?) has eluted at all? Have you used enough organic to elute it? Have you run a gradient of H20 and ACN up to 100% ACN?
By Steph on Tuesday, October 26, 1999 - 08:37 am:
I know that both are eluting because I have run them both separately - with a wash inbetween. I have spiked each sample with each. I have run a gradient with each solvent system I have used i.e ACN, MeOH, etc. I know that the compound is pure by NMR, IR MS etc before I made up the sample and ran it. I have tried all these things by RP and NP.
By Anonymous on Tuesday, October 26, 1999 - 07:18 pm:
When you ran the reversedd-phase separation using 50/50 ACN/water:
1. what was the column size (length x diameter)?
2. What was the retention time for the toluene and your analyte?
The reason I ask is to find out if you had adequate retention to do the separation.
By Steph on Friday, October 29, 1999 - 03:53 am:
The column dimensions were 250x4.6mm.
The retention time was around 2.5 - 3.0 mins.
By Tom Jupille on Friday, October 29, 1999 - 12:28 pm:
OK, I think I see the problem: your peaks are unretained. As a reasonable approximation, the internal volume of a 4.6-mm reversed-phase HPLC column is 0.1 x L where L is the column length in cm. Your 25-cm column has about 2.5 mL of internal volume, so the dead time at a flow rate of 1 mL/min is about 2.5 min. In order to do decent chromatography, the retention time of your analytes should be at least twice the dead time. This means you need to use a *weaker* mobile phase (i.e., less ACN, more water).
For now you might try a series of experiments with your analyte, decreasing the concentration of ACN each time: 40%, 30%, 20%, 10%. Allow at least 10 minutes (and preferably 25 minutes) equilibration time after each change of mobile phase and do each run in duplicate (to ensure that the column has in fact equilibrated).
In the future, you can save yourself a lot of wasted effort by following a procedure like the one outlined on LC Resources' web site:
In essence, you could have gotten the requisite information from only two initial gradient runs (plus the use of appropriate software!)
Let us know how it works out!
-- Tom Jupille
By Steph on Tuesday, November 9, 1999 - 04:25 am:
Thank-you to all who helped me.
The problem is now resolved!! The analyte was dropping out of solution in the column as soon as it touched water and toluene was washing it off the column - hence co-elution!
I have developed a mobile phase to counteract this.
Once again thanks for the time and assistance provided.
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