Chromatography Forum

can somebody help me with columns and detectors best for sugar..i have aqueous extract showing activity but i do not see any peak in elsd or UV/PDA/SQMS dectors..is the activity froms sugar part of it which is not retaining in column?

"is the activity froms sugar part of it which is not retaining in column?" - if i understand you wonder if your compound of interest stays in the column?

if so then make an injection without the column and see if you got the response

Sugar molecules do not contain strong chromophores so unless they are derivatised with a molecule containing a strong chromophore will not be easily detected by UV/PDA detectors.

The best options are Evaporative Light Scattering (ELS or ELSD) or Refractive Index (RI) detectors. Both have drawbacks RI can only be used Isocratically and ELSD has narrow linear range (calibrations will usually be quadratic regressions).

can somebody help me with columns and detectors best for sugar..i have aqueous extract showing activity but i do not see any peak in elsd or UV/PDA/SQMS dectors..is the activity froms sugar part of it which is not retaining in column?

The most used columns for sugars are Propyl-amino with 75:25 ACN:Water mobile phase (MP) or those with ligand-exchange principle like Lead, Calcium, etc with just water as MP....Maybe your sugars are coming in the void volume, check it.

Your detectors are ELSD and MS. So normal phase is your best option.

What sugars will you be analyzing? I can post data on here if you can
offer up more specifics.

We routinely do sugars with an RI detector. We have tried ELSD in the past, but it was not nearly as sensitive as the RI, and had far greater noise; overall S/N was greatly reduced. We use an NH2 column, with Acetonitrile/H2O eluant. We're looking at changing from Acetonitrile now, though, as it is getting so hard to get, and very expensive.

RI detector is the most common for corn based Fuel ethanol sugars and acids. Maybe check with your vendor depending on your industry you may not need to reinvent the wheel.

hi all,

i'm going to hijack this thread a little;

has any of you done sugars on a MS before?
how do they behave? what would you say is the best ionization technique?

we need to do some sugars on the HPLC but I want to try it on the MS.. seeing as I have the standards available.

haven't done anything myself yet, as this would be a 'personal' exercise to do in my own time. ("playtime")

someone should put in a word for other techniques: GC is good for many sugars, and CE is absolutely stunning for sugars of all sorts of lengths. The resolution makes uplc look like paper chromatography done on an old coffee-filter.

MassSpecM, I've done some sugary things using an amino column with ammonium acetate buffer (I think!) versus acetonitrile (i.e. hilic) plus ESI-MS. It worked OK, though the separation and peak-shape were nothing particularly impressive. Separation seemed largely by chain-length, and I certainly wouldn't think it able to separate all the multitude of interesting isomers that sugars can achieve.
From an MS point of view sugars aren't too hard, but they are prone to cosuppression if you get something more strongly ionising eluting at the same time. They also (in my hands, and regardless of buffers) tend to form more ammonium adducts than other analytes, which can be a minor inconvenience. There are, of course, things that can be done to alter the balance of adducts.

Below are LC-MS saccharide applications separted on Unison UK-Amino:

Fructose & glucose: http://www.imtaktusa.com/site_media/fil ... TI515E.pdf
Fructose and sucrose: http://www.imtaktusa.com/site_media/fil ... TI516E.pdf

RI requires isocratic elution. Newer detectors (ex: MS, ELSD, ect.) can be used with gradient elution, but
conventional NH2 phases are not stable and can create a lot of noise on such detectors.
That was the reason behind Unison UK-Amino.

This data proves the stability of Unison UK-Amino:
http://www.imtaktusa.com/site_media/fil ... TI304E.pdf

We routinely do sugars with an RI detector. We have tried ELSD in the past, but it was not nearly as sensitive as the RI, and had far greater noise; overall S/N was greatly reduced. We use an NH2 column, with Acetonitrile/H2O eluant. We're looking at changing from Acetonitrile now, though, as it is getting so hard to get, and very expensive.

Below are saccharide separations done on Unison UK-Amino, using both acetonitrile / water and acetone / water:
http://www.imtaktusa.com/site_media/fil ... TI478E.pdf

I do not have RI detector but i have ELSD...if sugar is comming in void volume i can not see it for sure will try to buy suggested column..but i am not sure the activity is due to sugars or any very hydrophillic compound which is not retaining in column ...

Bryan ,,its a aqueous part of methnolic extract of a leaves ( i used partition chormatography to fractionate methanolic extract)..i do not know what kind of sugars are there..there are sugars i can tell buy looking at the water eluted fraction of aqueous part which i got using HP-20 separation..

Can somebody will help how to extract highly water soluble compounds

The best HPLC column for sugar is Sugar-D COSMOSIL. This one is so stable and especially designed for sugar. I know very well this one. If you need more info, send me a email.

mk12, for leaf sugars, if you're interested in highly water soluble things there are loads of methods in primary literature. The usual aim is to extract the material but also to kill enzymes as quickly as possible to avoid changes. This isn't so critical if you're interested in bulk material such as starch, where the amount is large compared to enzymatic capacity to degrade, but for minor things it becomes very important.
Since sugars in leaves aren't so minor, probably boiling in several changes of 50% MeOH or similar, and combining the soluble fractions, is as good as anything, but look at what people have done in literature.

If you have doubts about your chromatography, the simple sugars (glucose, sucrose, fructose) can be easily, quickly, and reliably measured by enzyme coupled assays with a UV spec (or plate-reader) capable of measurements at 340nm. If you need recipes, I can probably dig some out. There are other approaches too. For both extractions and measurement of leaf metabolites, you could look out for authors such as Geigenberger and Stitt, but many others have done good work too.

Hey, isn't it funny how everyone who works for a column manufacturer knows THE best column for sugars. Funny, too, how THE best column is always made by their employer.