(11) Carbohydrate Characterization

Question 1

use + principle of polarimetery

Answer 1

-polarimetry is the interaction of polarised light with asymmetric groups (chiral) like carbohydrates

To identify a sugar in pure solution and give quantitative data

• unpolarized light (light waves which have different planes of oscillation) passes through a filter and limit the waves of light to one plane of movement
• we assess the interaction based on difference in the degree of rotation of polarised light by various sugars (rotation is also temperature dependent)
• amount of optical rotation is determined by the molecular structure and concentration of chiral molecules in the substance
• can also classify sugars based on the direction of rotation (L (right): +ve angle and D (left): -ve angle)
• According to Biots law, the degree of rotation of that optical compound will be inversely proportional to the wavelength of polarised light (shorter wavelength, greater rotation)

Question 2

Application of polarimetry

Answer 2

quantitate and qualitate sugar

• used in quality control
• process control
• research in pharmaceutical, chemical, essential oil, flavour and food industry
• established in the US pharmacopoeia and FDA specifications
• ensures product quality by measuring the concentration and purity of sugar based foods

Question 3

Describe the glucose oxidase method of carb determination

Answer 3

• Since glucose is a reducing sugar, it can be oxidized
• the enzyme glucose oxidase catalyzes the oxidation of beta-D-glucose to D-gluconic acid.
• at the same time, oxygen in the presence of water is converted to hydrogen peroxide
• all these compounds are colourless and for meaningful determination, peroxidase is used to produce a brown colour
• the colour intensity produced is proportional to the concentration of glucose present

Question 4

describe the hexokinase method

Answer 4

It is a highly specific method for glucose concentration determination by spectrometrically measuring the NADP formed from a hexokinase catalysed transformation of gluocse and various intermediates

Glucose + ATP&raquo_space; Hexokinase» G-6-P + ADP

G-6-P + NADP+ >(G-6-PDH)&raquo_space; gluconate-6-phosphate + NADPH + H+
The amount of NADPH formed in this reaction is stoichiometric with the amount of glucose
NADPH is measured by its absorbance at 334nm

Question 5

Discuss the principles of the refractometer

Answer 5

Increase in carbohydrate concentration proportionally increases the degree of light refraction; can be used to measure the amount of cho present

-The higher the sugar concentration, the more light bends (higher refractive index). The refractive index is increasing because the solution is getting #thicker# creating a denser medium with a higher refractive index.

Question 6

Factors that affect refractive index

Answer 6

1. Wavelength of incident light
2. temperature
3. refractive index is expressed as nt
   where n = refractive index measurement
   T = temperature
   D = D-ray of sodium

Question 7

Discuss the Brix scale (%) of refractive index

Answer 7

• normally used in sucrose quantitation
• corresponds to the percentage by weight of sucrose in water solution

Food and beverages usually contain various other dissolved ingredients such as salt and protein other than sugar
- the brix scale merely indicates the percentage of water soluble solids

Question 8

Quantitative assay for polysaccharides

Answer 8

1. Depends on chemical or enzymatic hydrolyse
2. Analysis of monomers
   - example starch
3. Amylose + I&raquo_space; Blue
   spec reading for determining the amylose (measure blue colour)
4. amylopectin + I&raquo_space; Red

Question 9

What are optically active compounds?

Answer 9

substances that interact with light and have the ability to rotate the plane of polarized light

Question 10

compare between refractometry and polarimetry

Answer 10

Polarimetry filters unpolarized light to ensure the waves of light are limited to one plane of movement. We assess the interaction of polarised light with asymmetric groups (like carbohydrates) based on the difference in degree of rotation by various sugars. The amount of optical rotation is dertemined by both the molecular structure and concentration of chiral molecules in the substance. The degree of rotation will be inversely proportional to the wavelength of polarised light, according to biots law.

Refractometry on the other hand measures the degree of regular light refraction, relative to the incident angle (refractie index). An increase in carbohydrae concentration proportionally increases the degree of light refracted hence sample has a higher refractive index. Factors that affect refractive index are the wavelength of incident light and the temperature of the surroundings.

Question 11

saccharides

Answer 11

1. monosaccarides (hexoses 6c, furanoses 5c)
   - glucose (obtained from starch, legumes, animal tissue, liver
2. disaccharides (anhydrides of 2 monosaccharides)
   - maltose
   - cellobiose
   - sucrose (made of amylose and amylopectin)
   - lactose
3. oligosaccharides (strings of mixed mono; any compound that contains up to 10 saccahrides)
4. poly ( >10 saccahrides)
   - starch: made of amylose and amylopectin
   - cellulose

Question 12

isomers

Answer 12

substances that have the same MF but with the atoms connected differently
- monosaccharides are commonly isomers of each other (glucose, galactose, fructose) but have different properties

Question 13

reducing vs non reducing sugar

Answer 13

• sucrose is non reducing because it is made from fructose+glucose (1,2 linkage where the aldehyde (c1 glucose) and ketone (c2 fructose) are eliminated; those are the reducing groups)
• maltose on the other hand which is formed by a 1,4 linkage is a reducing sugar)

Question 14

pectin

Answer 14

• connected to form branched structures: amylopectin

Question 15

analysing monosaccahrides and oligosaccharides(4)

Answer 15

1. polarimetry
2. enzymatic methods
   - glucose oxidase
   - hexokinase
3. refractometer
4. chromatography methods
   - paper/TLC
   - GC
   - liquid column chromatography

Question 16

anomer meaning

Answer 16

same chemical structure and formula, only difference is location of hydroxyl group on C1 (below or above)
below = alpha
above = beta

Question 17

mutarotation of monosaccharides

Answer 17

the change in degree of optical rotation when glucose is dissolved in solution
- when dissolves, forms 2 compounds, each with different degree of rotation compared to the initial

Question 18

So many enzymes are able to be used to quantitate monosacharides like hexokinase and glucose oxidase; can you use the same principle for polysaccharides(starch, hemicelulose, pectin)?

Answer 18

yes, but polysacharides have to be hydrolysed first into monosaccharides using enzymes (cellulose use cellulase, amylose use amylase)/heat/acid then use same methodology as monosaccharide

Question 19

L and D sugar

Answer 19

distinguished by the location of hydroxyl group on c5

left D, right L

Question 20

TLC for cho

Answer 20

Stationary: silica gel

Question 21

GLC for cho

Answer 21

although GC mainly designed for lipid analysis, can also use it to analyse sugar

• but first, have to derive sugar and make it heat resistant (compounds must be volatile)
• sugars can form trimethylsilyl ether (for comparison, fats are changed to fatty acid methyl ester using transmethyl esterification)
• this replaces every hydrogen with TMC and then inject into GC column

Question 22

HPLC

Answer 22

best for sugar analysis: separate sugars based on hydrophobic/philic phases and also based on MW and size

Question 23

difference between monosaccharide and disaccharide when dissolved in water

Answer 23

monosaccaride: mutarotation
disaccharide: inverted sugar (breakdown of sucrose to glucose and fructose)
- reduces crystallisation (saccharisation), increases degree of sweetness bc fructose is much sweeter than sucrose on the same unit basis
- can be done with either enzyme (invertase) or boil in acidic conditions