For quantitative analysis of sugars and reduced sugars we usually use the Lane-Eynon method. But there's a spectrophotometric method that uses 3,5-dinitrissacilic and it's necessary to do a calibration curve, so it should be a more accurate method. The original paper is MILLER, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, v. 31, n. 3, p. 426 - 428, 1959.

If you want qualitative to identify the presenting sugars in your sample, you should look for a HPLC method, but unfortunately I'm not usued with HPLC.

And the classical method for protein content it's the determination of nitrogen content (Kjedhal method), first you find the nitrogen content in your sample and then convert to protein using differents constant according to your food.

All the best,

Antonio

I had once analysed sugar components using HPLC with C18 column and if you used proper standard you could also measured the concentration, there are quite a number of journal articles informing about sugar analysis and protein content of bakery products, see also AOAC methods Hope it is usefull,with best wishes,Hari Purnomo

You should go for HPLC-ELSD for sugar analysis.

For Bradford method for protein analysis

Sugar estimation can be done by Lane-Eynon method which is a titeration method. For protein estimation you can go for kjeldhal method and bradford method Other advanced methods are also available such as HPLC.

A HPLC with amperometric detector is the one used by researchers in Faculdade de Ciências Farmacêuticas - USP/SP. Try contact with [email protected] (Profa. Beatriz).

Thanx for all ur guidance..,

I think that one possibility, depending what do you want to determine, is the use of a LC column for carbohydrate analysis based on ionic exchange and size exclusion (Ca and Pb options, go to web page of Biorad or other suppliers). The use of ELSD as Robin suggested is a good choice for detection and gradient elution. However, if you have a RI detector could be useful; better if a MS is coupled to LC.

For biscuits i also want to see but for protein estimation we use kjeldhal method

Macro kjeldahl for proteins & HPLC c18 for sugars...!!!

You could use High pH anion exchange chromatography, coupled with a Pulsed Amperometric Detector (PAD). You could quantitate simple sugars and polyols (isomalt, maltitol, mannitol, sorbitol, etc. in one run with very low noise-to-signal. Check out J. Agric. Food Chem., 1999, 47, 157-163.

Anyone used an ligand exchange column, e.g. Hi-Plex Na, coupled to ELSD HPLC for oligosaccharide detection? I would like to know the conditions used. I have the Hi-Plex Na column using water as mobile phase with the RI detector but I want to change to ELSD. What should be the conditions used? Any application notes?

For sugar analysis, ACN:Water (78:28) with NH2 column for ELSD.

What about the Na column with water as mobile phase, anybody used before?

No. I never use. But I think you should go with ELSD. I can provide all conditions with references.

:)

IC (Ion Chromatography) is the best PAD (Pulsed Amperometric Detection) see Dionex application notes (now Thermo).

Carbohydrate column and Carbohydrate wave form. But for years I used the RI (Refractive Index) detector with HPLC. It does not have as much sensitivity, but you should not need that for your application. Grind sample to a fine powder possibly put in freezer a couple of times and reblend.

weigh out sample into whirlpak or volumetric flask. Add water (pipet or to volume) Shake, mix, vortex, etc.. The sugars will come out into the water. Syringe filer w/ 0.45um filter. into HPLC or IC vial. Estimate concentration to fall within the standards.

Kjeldahl for protein.

Megazyme International has a kit for measuring glucose, fructose and sucrose enzymatically. The results are read in a spectrophotometer.

Thanx for ur guidance bt can anyone say e the formla for kjeldahl method for protein n also the co factor or co efficient., used for calculation.,

Calculations

Calculate results as follows:

1.4007 x (mL HCL, sample - mL HCL, blank ) x normality HCL

Nitrogen, % = --------------------------------------------------------------------

g sample

F factor tables for some foods attached

The first question is, which sugar exactly you want to analyse?

"sugar" as in sucrose? all simple sugars (glucose, fructose and sucrose?)? For this one of the enzymic-colorimetric tests (from megazyme but also most furnishers of chemicals) is the easiest (only s spectrophotometer and basic lab equipment). The enzymes should be treated carefully and have definitely limited shelf-lives.

Metabolizables carbohydrates? You need to add starch, for which colorimetric-enzymic methods are also available, but more time consumming as they demand enzymic hydrolysis (at least 2 temperature and phs for a reasonably complete hydrolysis); this produces glucose, so as to be corrected for the free glucose.

All carbohydrates including fibers? The adds another method (gravimetric) for thedietary fibers.

I would not recommand HPLC (many published methods) as a first option, because either you use a classical column and RI detector (low sensitivity, prone to interference by free acids, sorbitol... you name it) or very low wavlength UN (210nm, same propension to interference), And PAD after an ionic column can give complex signals (you'll see the oligomers). It also used to be that the PAD electrodes gave signals that were not stable and demanded frequent (= start and end of day) calibration. This is better with the newer machines.

The analysis is extremely simple. The sugars can be quantified by Anthrone reagent and the proteins by Lowry method or Brodford reagent. Only for qualitative analysis either HPLC or 2DE may be required for sugars and proteins respectively

Sugars can be quantatively determined using HPLC with RI detector on amide/Carbohydrate H+ column. It gives you a good separation between glucose, fructose, sucrose, maltose etc.

the sugars can be detected quantitively as total sugars by phenol - sulfuric acid method The Phenol - Sulfuric Acid method isa colorimetric method that is widely used to determine the total concentration of carbohydrates present in foods. A clear aqueous solution of the carbohydrates to be analyzed is placed in a test-tube, then phenol and sulfuric acid are added. The solution turns a yellow-orange color as a result of the interaction between the carbohydrates and the phenol. The absorbance at 420 nm is proportional to the carbohydrate concentration initially in the sample. The sulfuric acid causes all non-reducing sugars to be converted to reducing sugars, so that this method determines the total sugars present. This method is non-stoichemetric and so it is necessary to prepare a calibration curve using a series of standards of known carbohydrate concentration.

protein determined by kjeldahl method