Hi, i've injected 5 microliters of pure CHCl3 in a 250 mm C18 column, with ACN:H2O 80:20 as solvent, 1,0 ml/min. Something appears at 3,72 min, absorbing strongly at 210 nm. Is the CHCl3 absorbing itself or could it be something else in the CHCl3? I want to use CHCl3 to extract the components of some creams with vaseline as vehicle, wich only dissolves in CHCl3, diethylether, hexane and carbon disulfide. I choosed CHCl3 as first option of this 4 alternatives. The CHCl3 injected is solvent of HPLC/Spectroscopy grade.
It seems there will be problems with compounds that could elute near this time, what do you think?
Thanks a lot for your help,
It's definitely not the chloroform. CHCl3 absorbs at considerably higher wavelength.
Could be Ethanol which is sometimes used as a preservative (to prevent peroxide formation).
Possibly. The UV cutoff for 'pure' Methanol (HPLC grade) is 205 nm (Ethanol would not be far away, look it up online). It also depends on the 'purity' of the Ethanol and the concentration (usually ppm). A 'technical' grade of Ethanol would have a much more increased UV spectrum.
amylenes or ethanol as they are used as stabilizers, please check the solvent bottl.
thank you
Thank yo very much! I suspected of the ethanol but wasn't sure if it could absorb that much.
Now i've to try to resolve this peak from one of the analytes extracted
Bye!
Hey Salvador Valero,
I do not aggree with Julius Pavlov, your spectrum of the HPLC-peak belongs to clearly to chloroform. CHCl3 absobs until 245nm but with an extinction coefficient of 0.1 L/(M*cm) in heptane. Your solvent is acetonitrile/water which is much more polar (chloroform is also slightly polar, possible slight hypsochromic shift to shorter wavelength). As I see from your results you are using a Agilent HPLC with an DAD and Chemstation software. All DAD detectors have problems with stray light, so extinction above 2.0/cm is about 1% straylight. Your absorption below 220nm is total-absorption > 3/cm. At 220 nm you have about extinction 2.0/cm and at 230nm about 0.2/cm. The absorption of CHCl3 in heptane is about 10/cm at 220nm and 1/cm at 230nm. Your peak in the detector is of course diluted by the HPLC-eluent about a factor of 5 (halfwidth of your peak is bigger than 6 sec at a flow of 1mL/min).
So the absorption cutoff fits well to CHCl3. Make a logarithic plot of your spectrum and you will get a nealy straight line from 220nm down to 245nm.
Instead of guessing on this, why not do two injections one with chloroform and one with ethanol. You may need to vary the concentration of these solvent to mimic the real concentration e.g. don't inject 100% ethanol.
Peak enhancement is also highly recommended, I do support Narong suggestion
Thanks, Manfred and Narong. I'll give it a try and inject ethanol diluted with water, to check If the peak appears at the same time more or less. I'll post the results. Thanks a lot to all for your support.
Hello, back in the lab i've injected again the undiluted chloroform and a solution of 0,8 % w/v Ethanol in mobile phase (80:20 ACN:H2O). These are the chromatograms at 210 nm, wavelenght of maximum absorbance of the unknown peak. I think this results means that the peak is chloroform, don't you think? There's not a trace of the small peak at 2.0 min of the ethanol in the chloroform. The rest of the conditions are the same as my initial post. Thank you!!
I would inject much less of chloroform till you get nice sharp peak (to confirm retention time) and confirm with the UV absorption spectra.
Hey Narong Chamkasem,
the chromatogram at the top is a 240nm trace and the chromatogram 6 days ago is a 210nm. To compare both chromatograms only the 240nm trace of second chromatogram should be plotted. But it looks already pretty good 3.70min and 3.72min (only about 1 sec difference).
Ever consider that you might be cleaning the column? Try swapping for a new column if this one is not new.
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