LAB 2

ESTIMATION OF CARBOHYDRATES-TOTAL AND REDUCING

Spectrophotometric Carbohydrate Assays

Introduction:

In this investigatory exercise, two quantitative carbohydrate assays will be carried out, the dinitrosalicylic acid (DNSA) and the phenol-sulfuric acid assays. The DNSA reagent detects reducing ends of carbohydrates and it useful in many experiments. From the students results concludes what aspect of carbohydrate molecular structure each assay reveals. They are then led to speculate about applications for each.

Materials & Methods:

A) Preparation of carbohydrates

Unknown concentration of carbohydrates to be given to the students:

1 mg/ml mg/mL aqueous solutions of five carbohydrates are prepared by the lecturer (X-the concentration of carbohydrate that will be decided by the lecturer).

a) Glucose - reducingb) Fructose - reducing

c) Sucrose

d) Maltose

e) starch

Standard curve preparation:

1. The students are asked to prepare a known concentration of carbohydrates solutions and serial dilutions need to be made from this solution to a certain

concentration of carbohydrates (at least five (5) different concentrations required). This is for the purpose of making standard curve.

2. The DNSA assay uses the serial dilutions of carbohydrates.

B) Preparation of DNSA Reagent

Materials:1. 3,5-dinitrosalicylic acid (DNSA)2. potassium sodium tartrate tetrahydrate3. 0.4 M NaOH

Methods:

1. 1 g DNSA dissolved in 20 ml 0.4M NaOH and 50 ml distilled water. 2. 30g of Rochelle salt (potassium sodium tartrate tetrahydrate:

KO2CCH(OH)CH(OH)CO2Na·4H2O) was added3. Volume was brought up to 100 ml with distilled water.

** It appears to be stable for a year or so; there is some darkening on longer storage, though older reagent still seems to function adequately.

C) Analysis using DNSA Reagent

1. 0.8-mL sample is mixed with 0.8 mL of DNSA reagent and heated in a boiling water bath for 10 minutes.

2. The mixture is then diluted with 8 mL of water giving a reasonable volume for absorbance determination and the absorbance is determined at 540 nm.

3. When there are no reducing ends present, the final color is yellow (the absorbance ranges from 0.03 to 0.05).

4. A positive result is formation of a yellow-orange color with absorbance that may range upward to well over 1.0.

Data analysis:

Students are asked to tabulate the data for the standard curve (Absorbance vs. Concentration of carbohydrates) and identify the unknown concentration of carbohydrates given by the lecturer. Students need to identify which of the given carbohydrates is the reducing and non-reducing sugar. Students need to identify the quantity and types of sugar exist in the starch. They are then asked to answer this question in their report under discussion section: What chemical features of carbohydrates appear to be measured by the assay?