Lab 3: Qualitative and Quantitative Tests for the Analysis of Carbohydrates Flashcards
What are two common qualitative tests for the presence of carbs?
Molisch and Anthrone
For the Molisch and Anthrone tests what is a positive result?
Molisch: purple = presence of a carb
Anthrone: greenish blue
L> they only tells us a carb is present not what type
Benedict test?
- qualitative test
- for reducing sugars
L> positive: yellow or brick red copper oxide
Seliwanoff test (Resorcinol) ?
- test for ketoses
-positive= red colour
L> aldohexoses= light yellow or faint pink
L> pentoses= greenish colour
Bial orcinol test?
- test for pentoses
- ketoses: yellow-green
- aldoses: yellow-brown
- green= pentose
Glucose-oxidase method?
- 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.
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.
- Glucose
- Sucrose
- Starch
- Xylose
- Lactose
- Sorbose
- Fructose
- mono. aldose, 6C
- disaccharide
- polysaccharide
- mono, aldose, 5C
- disaccharide
- mono, ketose, 6 C
- mono, ketose, 6C
What are the hydrolysis products of the following carbs?
- Starch
- Lactose
- Xylan
- Sucrose
- Glycogen
- which dehydrates to furfural?
- glucose
- glucose and lactose
- xylose
- glucose and fructose
- glucose
- xylose
What is a glycoprotein or mucoprotein?
- a conjugate protein in which carb molecules are covalently bound to
How do glycoprotein and mucoprotein react with the Molisch Test?
glycoproteins dehydrate to their monomers in the molisch test.
Benedict’s test is based on a redox reaction Identify:
- reactant oxidized?
- reactant reduced?
- Oxidizing agent?
- Reducing agent?
- aldehyde or ketone (carbonyl group of the sugar/carb
- Cupric ions ( Cu 2+)
- Cupric ions (Cu +)
- Aldehyde or ketone carbonyl group of the sugar / carb
What is a reducing sugar?
a monosaccharide that can be oxidized by a weak oxidizing agent
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?
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.
A positive test for Benedicts is a yellow or brick red colour but what other colours occur?
- Blue which indicates no glucose and then varying colours are green, yellow and colourless.
Would sucrose and starch give a positive test for Benedicts?
NO
What happens to oligo or polysaccharides when heated with a strong acid?
- cleave to their monosaccharides
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.
- 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.
List factors that prevent aldoses from interfering with the Seliwanoff resorcinol test for ketoses.
- 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.
Seliwanoff tests for monosaccharide ketohexoses yet sucrose (disaccharide), raffinose (trisaccharide) and inulin ( fructosan polysaccharide ) all give a + result. Why?
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 !
Calc mg of sugar present :
Benedicts test, 2mL of 1% sugar?
1%= 1g/100mL
2mL x 1g/100mL = 0.02g
1000mg= 1g
0.02g x 1000mg/1g = 20mg of sugar
Calc mg of sugar present :
Seliwanoff test, 3 drops of 1% sugar. (20 drops/mL) ?
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
Why is it necessary to use a small amount of sugar in the Seliwanoff test?
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.
Why is it necessary to diule solutions in the Bial orcinol test especially when ketoses are present?
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.
In glucose oxidase method…what is the purpose of running tube b, the reagent blank through the test with the standards and unknown?
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.
Calculations: Standards
Mass glucose/tube?
= volume of standard in mL x 0.1mg/ml glucose
Calculations: Standards
concentration of glucose, mg/mL?
mass glucose/tube for the standard x 1 tube/ total volume of the tube
Calculations: Unknowns under different standards for say U-1 and standard 1
[glucose], mg/mL?
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 .
Calculations: Unknowns
- compare the four individual [glucose] , mg/mL?
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]
