In case that we do not have HPLC at the moment, so while waiting for the solution, may I know if there is a way to measure the concentrations of xylos, xylan in a mixture of sugar using spectrometer? I need to estimate the conversion rate of native sugar to simple sugar.
thank you
In case you donot have HPLC, xylose can be estimated by Bial's assay or Orcinol Assay. Following is the protocol.
Orcinol Assay
1. Take 1ml of sample and to it add 2.5 ml of Orcinol reagent** (0.4 g orcinol to 200 ml of concentrated HCl + 500uL of 10% Ferric Chloride).
2. Prepare xylose standards (10- 40 µg/ml) and add 1 mL of sample and to it add 2.5 ml of Orcinol reagent.
3. Prepare reagent blank by addition of 1 ml deionised water and 2.5 ml Orcinol reagent.
Boil the samples for 10 minutes. Cool the samples to room temperature.
Take the readings at 620 nm using UV-Visible spectrophotometer.
Note**: The Orcinol reagent should be prepared preferably fresh every time. In case of storage, please ensure that the reagent is stored in dark colored bottles in a cool and dry place.
Since xylan contains sugar other than xylose, you can also go for reducing sugar estimation by DNS method. This method would help you to know the total reducing sugars released and Orcinol method would tell you that contribution of pentose sugars in the total reducing sugars...
Wishing you good luck
Best regards: Deepti
Thank you for the explanation. That will be helpful. I performed the DNS but I was struggling with the HPLC problem. I will go on with your suggestion.
Thank you sir, unfortunately, not. I will proceed with chemical analysis for the time being while waiting for other suggestions outside our campus.
I look into this. I did not know TLC can be used as well.
thank you
Most sugars have no absorption bands UV-Visible, you must adopt a technique by refractometer, and for the mixture of two sugars, using the standard addition method with a prior étalonage curve
Xylose estimation kits that are based on the principle of conversion of b-xylose by b-XDH into b-xylonic acid and H + NADH, where NADH (stoichiometrically: 1 NADH molecule formed comes from 1 xylose molecule) is measured at 340 nm can be purchased at Megazyme (catolog no: K-XYLOSE). I find them quite sensitive and specific as well as they dont act on L-arabinose.
I did not determine Xylan, instead, I have determined cellulose, hemicellulose and glucose and xylose (HPLC). After that, I calculated for xylan using:
"concentration of polymeric sugars (glucan, xylan, arabinan, galactan and mannan) was calculated from the concentration of the corresponding monomeric sugars, using anhydrous correction factors of 0.88 (or 132/150) for xylose and arabinose, and a correction of 0.90 (or 162/180) for glucose, galactose and mannose.
(Nieves et al., 2016)
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