UNIT 4.2.

CARBOHYDRATES QUANTIFICATION

QUANTITATIVE ANALYSIS

After we have identified the carbohydrates samples in the

first part, we will quantify one of them using the DNS
method and a standard curve. DNS (3, 5-dinitrosalicylic
acid) is used to measure the total amount of reducing
sugars in a given sample. Oxidized DNS is yellow, and
when it gets reduced by the action of glucose (or other
reducing sugar) it turns orange-red and absorbs light at
λ=540 nm. This is a quantitative reaction that can be
calibrated to convert the absorbance values into
concentration values.

EXPERIMENTAL PROCEDURE
1º) Standards and sample dilutions calculations.
To quantify glucose concentration of your sample, you
need to performe a Standard Curve. The principle of the
standard curve is the same as the one you performed in
Unit 3: we need to prepare several tubes with a provided
known concentration of glucose, which are called
standards. You will prepare 5 standard tubes from a 4
mg/ml initial glucose solution.

Tub Glucose H2
Vf Concentratio
e (ml) O (ml n
4mg/ml (ml ) (mg/ml)
)
Std 0 0.5 0.5
0
Std 0.1 0.5
1
Std 0.2 0.5
2
Std 0.3 0.5
3
Std 0.4 0.5
4

At the same time you prepair your standards, you will

prepair some dilutions of your unknown sample, because
you don´t know the concentration range, and it could be
very concentrated to be used in your curve. Complete the
following tables:

SAMPLE Glucose (ml) unkown H2

Vf Concentration
DILUTION sample O (ml (mg/ml)
(ml )
)
1/2 0.5 (x2) ? POSSIBLE
RESULT
1/5 0.5 (x5) ? POSSIBLE
RESULT
1/10 0.5 (x10) ? POSSIBLE
RESULT

2º) Experimental protocol.

As you have 5 standards (including negative control) and
3 sample dilutions, you will prepair 8 reaction tubes:
1. Set up 8 glass tubes, and name them: Std 0-4, sample
1/2, sample 1/5, sample 1/10. Pipet the corresponding
amount of glucose and water you have calculated in
your tables.
2. Follow the protocol described above (*DNS method).
3. Read absorbance at 540 nm of all the samples.
Remember that you must record each value three times,
ant take the average.
4. Plot in Excel Abs vs Conc of the standard tubes and
obtain a standard curve with its equation.
5. Substitute the values obtained for the sample tubes (use
only the dilution that are in the apropiate range) and
calculate the concentration of the original sample by
interpolating in the curve. Don´t forget to undow the
dilution.

*DNS METHOD
The DNS method is performed as follows:
• Mix the samples with DNS reagent in equal
proportion: 0,5-0,5ml (glass or falcon tubes)
• Incubate at 100ºC for 5 min

• Cool in ice for 2 min

• Add 5 ml of H O, let stand for 5 min

2

• Read absorbance at 540 nm