EXPERIMENT-1
STANDARD MALTOSE CURVE
AIM
To construct the calibration curve for standard maltose. Principle
Maltose is a reducible disaccharide. It reduces pale yellow coloured alkaline 3, 5-dinitro salicylic
acid to orange coloured 3 amino, 5 nitro salicylic acid. The intensity of the colour is proportional
to the concentration of maltose whose absorbance is lead calorimetrically at 540nm.
Apparatus
Test Tube, Pipettes, Colorimeter
REAGENTS
3, 5-DNS: 1 g of DNA was dissolved in 50 ml of distilled water. Slowly add a small amount of 30
g of potassium sodium tartrate tetrahydrate. The solution will turn yellow (milky, not clear yellow).
After adding 20 mL of 2N NaOH, the solution turned clear orange-yellow. The final volume was
made up to 100 ml with distilled water. The solution was stored in an amber bottle. 1 g of DNA
was dissolved in 50 ml of distilled water. Slowly add a small amount of 30 g of potassium sodium
tartrate tetrahydrate. The solution will turn yellow (milky, not clear yellow). After adding 20 mL
of 2N NaOH, the solution turned clear orange-yellow. The final volume was made up to 100 ml
with distilled water. The solution was stored in an amber bottle.
Maltose working solution: 180mg of maltose was weighed and dissolved in 100ml of
distilled water.
Procedure
1. Standard maltose solution was pipette out in the range of 0.2, 0.4, 0.6, 0.8 and 1ml in 5 separate
test tubes.
2. The volume in each test tube was made up to 2ml with distilled water.
3. Including blank tube with 2 ml of distilled water.
4. 1.0 ml of DNS reagent was added to each tube and mixed well.
5. The tubes were covered with aluminium foil and placed in boiling water bath for 5 minutes
and cooled to room temperature.
6. 9.0ml of the distilled water was added to each of the tubes and mixed well.
7. The absorbance was read at 540nm against the blank.
8. The calibration curve was constructed on a graph sheet by plotting the amount of maltose in g
on X-axis and absorbance values at 540 nm.
1
Observation
Standard
O.D
g
Blank
[0]
0.2
Amount
of Amount of D.H2O
DNS(ml)
Incubate
D.H2O (ml)
at Maltose
Maltose
in Maltose in (ml)
it
540nm Solution
mol
at 1000C in (ml) for
5
0
0
2
1
minutes
9
0
360
1
1.8
1
9
0.04
0.4
720
2
1.6
1
9
0.08
0.6
1080
3
1.4
1
9
0.120
0.8
1440
4
1.2
1
9
0.160
1
1800
5
1
1
9
0.20
CALCULATION
AMOUNT OF MALTOSE IN g
1.8mg
180mg 100ml
1ml
0.2ml
0.2
?
1.8= 0.36 × 10ˆ3 = 360µ𝑔
0.4× 1.8 = 0.72 × 10ˆ3 = 720 µ𝑔
0.6× 1.8 = 1.08 × 10ˆ3 = 1080 µ𝑔
0.8× 1.8 = 1.44 × 10ˆ3 = 1440 µ𝑔
1
1.8 = 1.8
10ˆ3 = 1800 µ𝑔
AMOUNT OF MALTOSE IN mole
0.2ml 360
360 = 1 𝜇 𝑚𝑜𝑙𝑒
0.4ml 720
360 = 2 𝜇 𝑚𝑜𝑙𝑒
0.6ml 1080
360 = 3 𝜇 𝑚𝑜𝑙𝑒
0.8ml 1440
360 = 4 𝜇 𝑚𝑜𝑙𝑒
1ml 1800
2
360 = 5 𝜇 𝑚𝑜𝑙𝑒
3