Lab 3: Qualitative and Quantitative Tests for the Analysis of Carbohydrates

Question 1

What are two common qualitative tests for the presence of carbs?

Answer 1

Molisch and Anthrone

Question 2

For the Molisch and Anthrone tests what is a positive result?

Answer 2

Molisch: purple = presence of a carb
Anthrone: greenish blue
L> they only tells us a carb is present not what type

Question 3

Benedict test?

Answer 3

• qualitative test
• for reducing sugars
  L> positive: yellow or brick red copper oxide

Question 4

Seliwanoff test (Resorcinol) ?

Answer 4

• test for ketoses
  -positive= red colour
  L> aldohexoses= light yellow or faint pink
  L> pentoses= greenish colour

Question 5

Bial orcinol test?

Answer 5

• test for pentoses
• ketoses: yellow-green
• aldoses: yellow-brown
• green= pentose

Question 6

Glucose-oxidase method?

Answer 6

• quantitative
  -quantity of a colour brown correlates directly with the amount of glucose in the original sample.
  L> enzyme has a high specifity for glucose but if given enough time it will oxidize 2-deoxy-glucose, d-mannose, d-fructose at slower rates interfering with the test.

Question 7

For each of the following carbs indicate if they are mono, di, oligo or polysaccharides. For monos indicate if an aldose or ketoses and number of carbons.

1. Glucose
2. Sucrose
3. Starch
4. Xylose
5. Lactose
6. Sorbose
7. Fructose

Answer 7

1. mono. aldose, 6C
2. disaccharide
3. polysaccharide
4. mono, aldose, 5C
5. disaccharide
6. mono, ketose, 6 C
7. mono, ketose, 6C

Question 8

What are the hydrolysis products of the following carbs?

1. Starch
2. Lactose
3. Xylan
4. Sucrose
5. Glycogen
   - which dehydrates to furfural?

Answer 8

1. glucose
2. glucose and lactose
3. xylose
4. glucose and fructose
5. glucose
   - xylose

Question 9

What is a glycoprotein or mucoprotein?

Answer 9

• a conjugate protein in which carb molecules are covalently bound to

Question 10

How do glycoprotein and mucoprotein react with the Molisch Test?

Answer 10

glycoproteins dehydrate to their monomers in the molisch test.

Question 11

Benedict’s test is based on a redox reaction Identify:

1. reactant oxidized?
2. reactant reduced?
3. Oxidizing agent?
4. Reducing agent?

Answer 11

1. aldehyde or ketone (carbonyl group of the sugar/carb
2. Cupric ions ( Cu 2+)
3. Cupric ions (Cu +)
4. Aldehyde or ketone carbonyl group of the sugar / carb

Question 12

What is a reducing sugar?

Answer 12

a monosaccharide that can be oxidized by a weak oxidizing agent

Question 13

In aqueous solution glucose occurs primarily as alpha and beta pyranose hemiacetal ring structures that do not have a functional group that would give a positive Benedict’s test. Why then is a positive test result observed?

Answer 13

A positive test is observed because glucose undergoes mutarotation causing there to be an equilibrium of the alpha and beta forms. There is an ope chain formed during this process which can participate in oxidation-reduction therefore allowing a positive test for a reducing sugar.

Question 14

A positive test for Benedicts is a yellow or brick red colour but what other colours occur?

Answer 14

• Blue which indicates no glucose and then varying colours are green, yellow and colourless.

Question 15

Would sucrose and starch give a positive test for Benedicts?

Answer 15

NO

Question 16

What happens to oligo or polysaccharides when heated with a strong acid?

Answer 16

• cleave to their monosaccharides

Question 17

Lactose and sucrose are both disaccharides but only one is a reducing sugar. Identify which is and explain why the other does not show reactivity.

Answer 17

• Lactose
• Sucrose is not a reducing sugar because the anaemic carbon in the sucrose ring does not have a free OH group and it cannot be reduced to an aldehyde or ketone. This is due to the fact that the glycosidic bond found in sucrose is formed between the reducing ends of both glucose and fructose instead of between the reducing end of one constituent and the nonreducing end of the other leaving a free reducing end open. Lactose exhibits the latter.

Question 18

List factors that prevent aldoses from interfering with the Seliwanoff resorcinol test for ketoses.

Answer 18

• it distinguishes ketoses from aldoses
• ketoses will dehydrate faster than aldoses and they will also yield larger amounts of hydroxymethylfurfural.
• aldoses produce a light yellow or pink colour whereas with ketoses they produce a higher concentration of hydroxymethylfurfural which will react with resorcinol forming a red condensation product.

Question 19

Seliwanoff tests for monosaccharide ketohexoses yet sucrose (disaccharide), raffinose (trisaccharide) and inulin ( fructosan polysaccharide ) all give a + result. Why?

Answer 19

They each have fructose consituents. Fructose is a ketoses so when the resorcinol reagent is added since it contains an acid component (HCl) it will hydrolyze the glycosidic bonds in these carbs yielding their monos and therefore will give a positive test !

Question 20

Calc mg of sugar present :

Benedicts test, 2mL of 1% sugar?

Answer 20

1%= 1g/100mL
2mL x 1g/100mL = 0.02g
1000mg= 1g
0.02g x 1000mg/1g = 20mg of sugar

Question 21

Calc mg of sugar present :

Seliwanoff test, 3 drops of 1% sugar. (20 drops/mL) ?

Answer 21

3/20= 0.15mL 
1%= 1g/100mL 
0.15mL x 1g/1000mL = 0.0015 g sugar
1000mg= 1 g 
0.0015g x 1000mg/1g = 1.5 mg sugar

Question 22

Why is it necessary to use a small amount of sugar in the Seliwanoff test?

Answer 22

bc if there is a high concentration of aldohexoses the hydroxymethylfurfural product from it undergoing dehydration rxns will also be in higher concentration meaning more of it will react with the resorcinol producing more of a red colour indicating ketoses presence instead of a pink indication of aldose presence. Keeping the sugar dilute avoids this.

Question 23

Why is it necessary to diule solutions in the Bial orcinol test especially when ketoses are present?

Answer 23

bc pentoses yield a green colour whereas ketoses yield a yellow-greenish colour…and if it is in high enough concentration it can lead to a false positive.

Question 24

In glucose oxidase method…what is the purpose of running tube b, the reagent blank through the test with the standards and unknown?

Answer 24

set the light absorbency to avoid interference from the reagent itself. This is important bc the amount of sugar/carb is directly correlated with the quantity of colures material; therefore, it is important accurate readings are made and the blank helps control for this.

Question 25

Calculations: Standards

Mass glucose/tube?

Answer 25

= volume of standard in mL x 0.1mg/ml glucose

Question 26

Calculations: Standards

concentration of glucose, mg/mL?

Answer 26

mass glucose/tube for the standard x 1 tube/ total volume of the tube

Question 27

Calculations: Unknowns under different standards for say U-1 and standard 1
[glucose], mg/mL?

Answer 27

absorbance U-1/ absorbance U-2 x [glucose mg/ml for S1)
* do this for each standard for each respected Unknown…… average values for U-1 ( S-1 and S-2) and U-2 ( S-1 and S-2).
then average U-1+U-2/2 for the final value of unknown .

Question 28

Calculations: Unknowns

- compare the four individual [glucose] , mg/mL?

Answer 28

U1: U2 =
U-1 S-1/S-2 : U2 S-1/S-2
statement: method is linear…values are measured accurately…..therefore U1 and U2 are equal in [glucose]