estimation of reducing sugars by dinitrosalicylic...
TRANSCRIPT
![Page 1: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/1.jpg)
Estimation of reducing sugars
by dinitrosalicylic acid method
BCH445 [Practical] 1
![Page 2: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/2.jpg)
• The major constituents of milk are lactose, fats and proteins .
• Other free carbohydrates found in milk but at low concentrations, including
glucose , galactose and others.
• As lactose is the main carbohydrate in commercial milk, its determination is a
basic indicator of
2
![Page 3: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/3.jpg)
• Human milk contain more lactose than cow’s milk.
3
![Page 4: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/4.jpg)
• Lactose is a disaccharide sugar derived from galactose and glucose.
• It is a reducing sugar (why?).
• Some of methods for lactose detection in milk are based on the assumption that
lactose is the only reducing sugars in milk.
• In this experiment, dinitrosalicylic acid (DNS) method will be used, which based
on the detection of reducing sugar ( which will give a general estimation for
lactose not an accurate one, because in milk there are also other reducing sugars).
4
![Page 5: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/5.jpg)
5
What is reducing sugar ?
![Page 6: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/6.jpg)
Practical Part 6
![Page 7: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/7.jpg)
• Estimation of reducing sugars by dinitrosalicylic acid method in milk sample.
7
![Page 8: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/8.jpg)
• The dinitrosalicylic acid (DNS) method for estimating the concentration of
reducing sugars in a sample.
• Not specific.
• Reducing sugars contain free carbonyl group, have the property to
many of the reagents.
• All monosaccaride and some disaccaride are reducing sugars (sucrose?).
8
![Page 9: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/9.jpg)
9
v
Free
carbonyl
group
Reducing Non-reducing
![Page 10: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/10.jpg)
• When alkaline solution of 3,5-dinitrosalicylic acid reacts with reducing sugars (eg. Glucose,
lactose..) it is converted into 3-amino-5-nitrosalicylic acid with orange color.
• Intensity of the colour is an index of reducing sugar.
10
![Page 11: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/11.jpg)
11
Glucose solution
(100mg/dl)
(ml)
Sample
(ml)
Water
(ml)
DNS reagent
(ml)
Sodium potassium
tartrate
(ml)
B -- -- 1 3 Cover the
tubes (with
aluminuim
foil)
And heat for 5
min. in a
boiling water
bath
1
1 0.1 -- 0.9 3 1
2 0.2 -- 0.8 3 1
3 0.3 -- 0.7 3 1
4 0.4 -- 0.6 3 1
5 0.5 -- 0.5 3 1
6 0.6 -- 0.4 3 1
7 0.7 -- 0.3 3 1
8 0.8 -- 0.2 3 1
9 0.9 -- 0.1 3 1
10 1 -- -- 3 1
S1 -- 1 --- 3 1
S2 -- 0.6 0.4 3 1
![Page 12: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/12.jpg)
• Mix the contents.
• Cool by immersing in cold water and read at 510 nm.
• Plot the standard curve and calculate the amount in the sample from standard
curve and calculate the contents.
12
![Page 13: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/13.jpg)
13
Tube Absorbance at 510nm CHO content (mg/dl)
B -- --
1
2
3
4
5
6
7
8
9
10
S1
S2
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 10 20 30 40 50 60 70 80 90 100
Ab
sorb
ance
at
51
0nm
reducing sugar concentration (mg/dl)
Estimation of reducing sugars by dinitrosalicylic
acid method
![Page 14: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/14.jpg)
• Dilution factor (DF) = final volume / aliquot volume
• The amount of carbohydrate in the sample= ………….mg/dl x DF
14
![Page 15: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/15.jpg)
• Is there any other more specific method? If yes name one?
15
![Page 16: Estimation of reducing sugars by dinitrosalicylic …fac.ksu.edu.sa/sites/default/files/2_estimation_of...•The dinitrosalicylic acid (DNS) method for estimating the concentration](https://reader030.vdocuments.us/reader030/viewer/2022040222/5e48fcadcf60696d0b1511f2/html5/thumbnails/16.jpg)
• Meurant G. Handbook of Milk Composition. Academic Press, 1995.
• Leo M.L. Nollet, Toldra F. Handbook of Dairy Foods Analysis. CRC Press,
2009.
• Allan C. Somersall. The Healing Power of 8 Sugars: An Amazing
Breakthrough in Nutrition, Sciences and Medicine. Natural Wellness Group,
2005.
• BCH 445- practical note
16