best condition for sugars analyses

[wahyu]

Hi.
I’m a new user of LC. I want to ask about the best condition of LC in order to observe sugars profile in food sample..

[Noser222]

If you do a search on this forum for sugars or carbohydrates you'll come up with a lot of hits. If you look at any manufacturer's catalog you'll also find a whole section on that.

The two easiest methods that I've used:

Amino phase most commonly with a mobile phase of 80% acetonitrile and 20% water, plus or minus a few %.

Sulfonated styrene/divinyl benzene in the H+, calcium, or lead form with a mobile phase of water.

These are commonly used with RI detectors, and the exact conditions depend on whether you want to separate certain monosaccharides or di,tri, and polysaccharides.

[Rob Burgess]

It all depends on what sugars you are looking to analyse. I'd recommend a look at the shodex website... lots of useful information here.

[wahyu]

Noser222: thanks for your advice. I have done what you said
I run using zorbax carbohydrate analysis column 4,6 x 150 mm (5 micro m), MP 75/25 (ACN/water), Flow 0,5 ml/min, 25 C column temperature, 20 microlit sample injection (dissolve in water).
The signal was too weak, overlap, difficult for quantitative purpose. I wonder that this is because sample preparation. The sample is feed sample (for poultry); consist of protein, lipid, and sugars. Sample was dissolved with water, centrifuge, through sep pac C18 and Millipore membrane (0,45 m) before injection.
Another problem: to separate dextrose and mannose. Their signals are always overlap, pile each other.
Do you (anyone) know what should I do, due to better sample preparation to given an optimum signals; and to separate dextrose and mannose.
Rob: thanks for your information.

[Uwe Neue]

I recommend to look at the Waters catalogue, which contains a listing of retention times of sugars with different columns. For the specific issue here, the separation of dextrose and mannose, I recommend to reduce the amount of water in the mobile phase. 80% should be OK.

You should have lost all of the anticipated interferences that you have described in the solid-phase extraction step. Please describe the SPE step in detail, there could be some weakness in this step.

Please describe your detection as well.

[Noser222]

Reducing the water content will help, as Uwe said. Also, are you injecting your sample in 100% water? Water is the strong solvent, so try diluting a to 80% acetonitrile and see if your resolutin improves.

[wahyu]

Uwe: thanks. This is the sample preparation steps, in detail..
Sample wash by chloroform for eliminate the lipid, then dried (oven, 80 C). Add the extraction solvent (I used three different solvent; Water, 50/50 EtOH/water, 70/30 EtOH/water). Shake at 80 C for 45 min, then centrifuge.
Sep pac C18 then through 0,45 micro m Millipore membrane, and injected (20 microlit).
Since water gave a better signal, I used water as extraction solvent.

Noser222: thanks, I’ll try it..

[Uwe Neue]

Yes, water is the better solvent to redissolve the sugars and leave other stuff behind. The C18 is functional, if properly prepared (activated with methanol first). You will get better results, i.e. remove more undesirables, by using mixed-mode ion-exchangers, both cation and anion exchanger in combination.

Take the redissolved sample in water and pass it through an Oasis MCX and then through an Oasis MAX cartridge. The sugars will fly through, and hydrophobic materials as well as anionic and and cationic contaminants will all be retained and stay behind.