Greetings
We are trying to determine what sugars are present in squirting cucumber (Ecballium elaterium) 'fruit'. We have access to an HPLC-DAD (unfortunately no RI detector) and to GC/MS. We can also do TLC if anyone have a good protocol. Do keep in mind that the samples are small, ~100 ul. All ideas are welcome! Many thanks. Yoram
Hi,
Do you have UV detector for the HPLC? I did separation of fermentation product (including sugar) using Sugar-pack Column and use RI and UV in tandem as detectors. The UV detector also give similar signal as the RI did. So you can try to use UV detector if you have one. I am not sure about the GC-MS, because sugars are not volatile compound and therefore I guess you need to react the sugar with other compound that can make it more volatile.
You can also try TLC method as describe on this paper: http://assbt-jsbr.org/JSBR/Vol20/JSBRVol20No3P251to254SeperationandAnalysisofSomeSugarsbyUsingThinLayerChromatography.pdf
Hope this help..
For sugar analysis in fruit juice you have to go for HPLC-ELSD detector using NH2 column, flow rate=0.8ml/min, ACN/Water (80/20), drift tube temp.=90C, gass pressure=2SLM.
Otherwise you have to go for RI detector but for isocratic elution for ACN/Water (80/20)
Gudluck
:)
Many thanks Safri. Maybe I am missing something here but I am not sure how UV detection (which is embedded in the DAD (diode array detector) suppose to work, since to the best of my knowledge sugar, in general, do not absorb light, even in the UV range.
Thanks you too Robin, we are have bought an ELSD that should arrive soon. When it arrives the conditions you supplied should be very useful.
You could derivatise the sugars to form ABEE (aminobenzylethylester) derivatives which will be visible using a UV detector. This works very well for all reducing sugars. It won't work for sucrose though. The sucrose is converted to glucose and fructose during the derivatisation if you derivatise for long (30minutes+). I suspect if you derivatise for only 10 minutes the sucrose won't be broken down. So, you could do a short derivatisation to get the concentrations of all the reducing sugars and then do a long derivatisation to get the fructose and glucose contribution from sucrose (test this with standards first to see if it works). These ABEE sugar monomers separate the best on a C8 column. On a C18 the arabinose and xylose co-elute. For sucrose you could also try with the DAD and see if you can see the underivatised sugar standard at all. Sugars do have minimal absorbance and your sensitivity won't be great. You'll have to make sure mobile phase is as low as possible in UV absorbance. For underivatised sucrose you can use and amine or HILIC column for separation and the DAD for detection. Unfortunately I think the detection limit will be too high for sucrose with the UV.
Perhaps it would be better to determine sucrose using the GC-MS. I'm not sure which derivatisation agent is used for this. You could determine all the sugars by GC-MS, but I know most reports I've read about this method seems to indicate that it is a cumbersome method and you do get several derivatisation products per sugar. However, In your case I think you'll have to use GC-MS for sucrose at least.
Here is a good review paper of techniques to use with GC-MS. If you are not sure about the identity of sugars in your sample GC-MS would be the better option.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 May 15;879(17-18):1226-40. doi: 10.1016/j.jchromb.2010.11.013. Epub 2010 Dec 8.
Derivatization of carbohydrates for GC and GC-MS analyses.
Ruiz-Matute AI, Hernández-Hernández O, Rodríguez-Sánchez S, Sanz ML, Martínez-Castro I.
Source
Instituto de Fermentaciones Industriales-CIAL (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain.
Hi Yoram,
Yes literature indicate that sugars do not absorb in UV wavelength. However, since the instrument I used was having tandem detectors (RID and PDA), I tried to set the PDA at 4 wavelength 195, 200, 210, and 220 nm. Of course RID gave better results, but I observed signal at the PDA detector also. So, in case of emergency UV detector might be used, even though the results is not as good as using RID.
Cheers..
In case you are limited with sample size (100ul) I would suggest capillary electrophoresis method. Beckman Coulter offers Carbohydrate separation Kit and protocol - you can separate a panel of mono and di-saccharides without derivatization with DAD detection.
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