Scribd
Upload
3K views4 pages

Principle / Theory:: Experiment 5: Quantitative Estimation of Reducing Sugars by DNS Method

1) This experiment aims to quantify reducing sugars using the DNS colorimetric method. DNS reagent changes color from yellow to orange/brown upon reacting with reducing sugars under alkaline conditions. 2) Absorbance readings at 540nm are taken for sugar standards and unknown samples after boiling with DNS reagent. A standard curve of absorbance versus concentration is plotted. 3) The concentration of sugars in unknowns is determined using the standard curve equation. Calculations are shown to account for dilution and determine molar concentration and extinction coefficient.

Uploaded by

Ankush Roy
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
3K views4 pages

Principle / Theory:: Experiment 5: Quantitative Estimation of Reducing Sugars by DNS Method

1) This experiment aims to quantify reducing sugars using the DNS colorimetric method. DNS reagent changes color from yellow to orange/brown upon reacting with reducing sugars under alkaline conditions. 2) Absorbance readings at 540nm are taken for sugar standards and unknown samples after boiling with DNS reagent. A standard curve of absorbance versus concentration is plotted. 3) The concentration of sugars in unknowns is determined using the standard curve equation. Calculations are shown to account for dilution and determine molar concentration and extinction coefficient.

Uploaded by

Ankush Roy
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 4

Experiment 5: Quantitative estimation of reducing sugars by DNS method.

Aim:
To determine the amount of sugar present in a given sample using DNS (dinitrosalicylic)
colorimetric method.

Principle / Theory:

Reducing sugars having free carbonyl groups (free aldehyde group or a free ketone group)
act as reducing agents and reduce some of the organic reagents like 3,5-dinitrosalicylic
acid (DNS) under alkaline conditions upon boiling.

In this process DNS reagent reduced to 3 amino five nitro salicylic acid. Upon reaction
with reducing sugars, DNS changes color from yellow to orange/brick red or brown.

Colour absorbance maximum at 540nm are directly proportional to the amount of


reducing sugars present in a given solution.

What are reducing sugars, remember?

Sucrose in non- reducing sugar glucose and fructose is reducing sugar

Points to wonder:

1. What is the unit of absorbance? What is transmittance?


2. How does spectrophotometer work?
3. What kind of light is used in spectrophotometer?
4. Why do we need a blank spectra or blank reading?
Materials & Methods:

Equipment and materials required:


1. Spectrophotometer and cuvettes
2. Water bath
3. Reagent requirement:
4. Dextrose sugar
5. DNS reagent
6. Distilled water

Procedure:

1. Take clean and dry test tubes (8 Numbers)


2. Prepare sugar standard solution as concentration mentioned below table
3. Unknown solution given to you before you start
4. Add 1 ml of DNS reagent to all the tubes
5. Place the tubes in boiling water bath for 5 minutes.
6. Take out the tubes

Stock solution for standard curve: sugar stock 0.1M or 18mg/ml

Observation table:

Sugar Sol Makeup DNS Distilled Absorbance (λ 540nm)


in µl water Reagent water Group Mean
(mg/ml) (µl) (ml) added readings
after
boiling in
a water
bath (ml)
Blank 0 (0) 1000 2 5 Blank it
(make spec
zero)
1 10 ( mg/ml) 990 2 5 a)
b)
2 20 ( mg/ml) 980 2 5 a)
b)
3 30 ( mg/ml) 970 2 5 Repeat as
4 40 ( mg/ml) 960 2 5 above for all
5 50 ( mg/ml) 950 2 5 solutions
6 60 ( mg/ml) 940 2 5 from 3 to 7,
7 70 ( mg/ml) 930 2 5 as well as
Unknown1 50 950 2 5 for
Unknown2 70 930 2 5 Unknown 1
and
Unknown 2
Calculations:
Plotting the standard curve for sugar estimation:

1. Plot the known concentration of sugars (mg/ml) on ‘X’ axis and their respective
absorbance (540nm) readings on ‘Y’ axis. (See figure at the end)
2. Draw a trendline that connects these observations and identify the intercept value on X-
axis. (See figure at the end)
3. Manually calculate slope. (See figure at the end)
4. Plot the unknown values now and calculate mg/ml of sugar present in the unknown
samples using the trendline.

The concentration of unknown samples can be calculated as:


!"#$%"&'()
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = !"#$%
mg/ml …………………………………….. (i)

5. Take the dilution factor into consideration and calculate the total amount of sugar
present in the given solution as follows:

(Here, 1 ml of unknown sugar is present in 7 ml of total solution)

𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = 𝑅𝑒𝑠𝑢𝑙𝑡 𝑓𝑟𝑜𝑚 (𝑖) ∗ 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 (ℎ𝑒𝑟𝑒 7) ………….. (ii)

Example: If λ of unknown or food sample is 0.45 and the slope of the trendline is 0.55
then:
!.!"
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = !.!! = 0.818 ∗ 7 (𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟) = 5.72𝑚𝑔/𝑚𝑙

(Dilution factor = 2ml of DNS and 5ml of distilled water = 7)

6. Calculate the molar concentration of the sugar present as follows:

!"#$%#&'(&)"# !"#$ (!!)


!"#$%&#'( !"#$!! !" !"#$%&' (!!"! !"#)
moles/L or M ….…………………………..(iii)

This represents the number of moles of glucose present per liter of solution.

The slope and trendline can also be calculated using MS-XL as follows:

Using MS-XL plot a standard curve taking (mg/ml or mM) standard sugar
concentration on the ‘X’ axis and absorbance (λ 540nm) on the ‘Y’ axis. Using
algebraic equation y=mx+c obtain the slope of the curve and calculate the unknown
sugar concentration.
7. Calculation of “molar extinction coefficient” or ℇ (epsilon).

The molar extinction coefficient is a measure of how strongly a chemical species or


substance absorbs light at a particular wavelength. Its unit is M-1cm-1

According to Beer-Lambert law:

𝐴 = ℇ ∗ l ∗ C ……………………………………………………..(iv)

Where A = absorbance (no unit)


ℇ = Molar extinction coefficient (in Lmol-1cm-1 or M-1cm-1)
C = concentration (in M or mol/L)
L = path length (in cm)

In essence the law states that the quantity of light absorbed by a substance dissolved
in a fully transmitting solvent is directly proportional to the concentration of the
substance and the path length of the light through the solution.

Note that the equation: 𝐴 = ℇ ∗ l ∗ C is of the form

𝑌 = 𝑚 𝑥 + 𝑐 ……………………………………………….(v)
(Assume line goes through origin so c= zero)

In equation (v) above m = Slope


Therefore, Slope = ℇ ∗ l
Here, l = 1cm which is the length of the cuvette.

∆!
And, from graph we know that 𝑆𝑙𝑜𝑝𝑒 = ∆!
= ℇ ∗ l ………………………………(vi)
Therefore, solve for ℇ.

Dotted line on the trendline shows that the


line passes through origin, this need not be the
Absorbance

∆A case in your graph. This is a simplified graph


for your understanding of relation between ℇ
∆C and the slope and how to calculate ℇ from slope.
Note that concentration is an “independent variable”
and absorbance is the “dependent variable”.

Concentration (mol/L)

NOTE: You are expected to show following 4 calculations:


1) Concentration of unknown sugar (equation i)
2) Concentration of unknown sugar with dilution factor (equation ii).
3) Molar concentration of unknown sugar with dilution factor (equation iii)
4) Molar extinction coefficient (ℇ) (equation vi).
All results should contain the correct units.

You might also like