1. Analysis of Carbohydrate
Kimia Bahan Makanan (CHM342)
Nelson Gaspersz

2. Source of Carbohydrate

3. Processed Food of Carbohydrate Source

4. I. Qualitative Analysis

5. 1. Molisch’s Test
Molisch's test (named after Austrian botanist Hans Molisch) is a sensitive chemical test for the presence of carbohydrates, based on the dehydration of the carbohydrate by sulfuric acid or hydrochloric acid to produce an aldehyde, which condenses with two molecules of phenol (usually α-naphthol, though other phenols (e.g. resorcinol, thymol) also give colored products), resulting in a red- or purple-colored compound.
The test solution is combined with a small amount of Molisch's reagent (α-naphthol dissolved in ethanol) in a test tube. After mixing, a small amount of concentrated sulfuric acid is slowly added down the sides of the sloping test-tube, without mixing, to form a layer. A positive reaction is indicated by appearance of a purple ring at the interface between the acid and test layers.

6. 1. Molisch’s Test
All carbohydrates – monosaccharides, disaccharides, and polysaccharides – should give a positive reaction, and nucleic acids and glycoproteins also give a positive reaction, as all these compounds are eventually hydrolyzed to monosaccharides by strong mineral acids. 
Pentoses are then dehydrated to furfural, while hexoses are dehydrated to 5-hydroxymethylfurfural.
Either of these aldehydes, if present, will condense with two molecules of naphthol to form a purple-colored product, as illustrated below by the example of glucose

7. 1. Molisch’s Test

8. 2. Seliwanoff’s Test
Seliwanoff’s test is a chemical test which distinguishes between aldose and ketose sugars. Ketoses are distinguished from aldoses via their ketone/aldehyde functionality.
If the sugar contains a ketone group, it is a ketose. If a sugar contains an aldehyde group, it is an aldose. When added to a solution containing ketoses, a red color is formed rapidly indicating a positive test. When added to a solution containing aldoses, a slower forming light pink is observed instead.

9. 2. Seliwanoff’s Test
The reagents consist of resorcinol and concentrated hydrochloric acid (or H2SO4 & CH3COOH):
The acid hydrolysis of polysaccharide and oligosaccharide ketoses yields simpler sugars followed by furfural.
The dehydrated ketose then reacts with two equivalents of resorcinol in a series of condensation reactions to produce a molecule with a deep cherry red color.
Aldoses may react slightly to produce a faint pink color.
Fructose and sucrose are two common sugars which give a positive test. Sucrose gives a positive test as it is a disaccharide consisting of fructose and glucose.

10. 2. Seliwanoff’s Test
fructose
furfural
red-colored dye
resorcinol

11. 3. Anthrone’s Test
Anthrone’s test is a tricyclic sweet-smelling ketone. It is utilized for a well-known cellulose measure and in the colorimetric determination of carbohydrates. The anthrones are utilized as a part of drug store as diuretic. They improve the movement of the colon and are in charge of less water reabsorption. 
Starches are dried out with concentrated H2SO4 to frame “Furfural”, which gathers with anthrone to shape a blue-green shading complex which can be measured by utilizing colorimetrically at 620 nm.
Anthrone reacts with dextrins, monosaccharide, disaccharides, polysaccharides, starch, gums and glycosides. If this happens, the yield of shading is where is to frame sugar to starch.

12. blue green-colored dye
3. Anthrone’s Test
anthrone
blue green-colored dye
furfural

13. 4. Benedict’s Test
Benedict's reagent (often told as Benedict's Qualitative Solution or Benedict's Solution) is a chemical reagent named after an American chemist, Stanley Rossiter Benedict.
It is a complex mixture of sodium carbonate, sodium citrate and copper(II) sulfate pentahydrate. Benedict's reagent is a chemical reagent commonly used to detect the presence of reducing sugars, however other reducing substances also give a positive reaction. This includes all monosaccharides and many disaccharides, including lactose and maltose. Such tests that use this reagent are called the Benedict's tests.

14. 4. Benedict’s Test
Generally, Benedict's test detects the presence of aldehydes, alpha-hydroxy-ketones, also by hemiacetal, including those that occur in certain ketoses. Thus, although the ketose fructose is not strictly a reducing sugar, it is an alpha-hydroxy-ketone, and gives a positive test because it is converted to the aldoses glucose and mannose by the base in the reagent.
A positive test with Benedict's reagent is shown by a colour change from clear blue to a brick-red precipitate.

15. 4. Benedict’s Test
The principle of Benedict's test is that when reducing sugars are heated in the presence of an alkali they get converted to powerful reducing species known as enediols.
RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+
Brick-red
precipitate

16. 4. Benedict’s Test
The color of the obtained precipitate gives an idea about the quantity of sugar present in the solution, hence the test is semi-quantitative. A greenish precipitate indicates about 1 g% concentration; yellow precipitate indicates 1.5 g% concentration; orange indicates 2.5 g% and red indicates 3,5 g% or higher concentration.

17. 5. Barfoed’s Test
Barfoed's test is a chemical test used for detecting the presence of monosaccharides. It is based on the reduction of copper(II) acetate to copper(I) oxide (Cu2O), which forms a brick-red precipitate.
(Disaccharides may also react, but the reaction is much slower.) The aldehyde group of the monosaccharide which normally forms a cyclic hemiacetal is oxidized to the carboxylate.
RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+
A number of other substances, including sodium chloride, may interfere. It was invented by Danish chemist Christen Thomsen Barfoed and is primarily used in botany.
Barfoed's reagent consists of a 0.33 molar solution of neutral copper acetate in 1% acetic acid solution. The reagent does not keep well and it is therefore advisable to make it up when it is actually required. May store indefinitely according to several MSDS's.

18. 6. Fehlings’s Test
In this test the presence of aldehydes but not ketones is detected by reduction of the deep blue solution of copper(II) to a red precipitate of insoluble copper oxide (Cu2O). The test is commonly used for reducing sugars but is known to be NOT specific for aldehydes. For example, fructose gives a positive test with Fehling's solution as does acetoin.
Two solutions are required: Fehling's "A" uses 7 g CuSO4.5H2O dissolved in distilled water containing 2 drops of dilute sulfuric acid. Fehling's "B" uses 35 g of potassium tartrate and 12 g of NaOH in 100 ml of distilled water.

19. 7. Osazone’s Test
The famous German chemist Emil Fischer developed and used the reaction to identify sugars whose stereochemistry differed by only one chiral carbon. Glucosazone and fructosazone are identical. Osazones formation test involves the reaction of a reducing sugar (free carbonyl group) with excess of phenylhydrazine when kept at boiling temperature.
All reducing sugars form osazones. Therefore, sucrose, for example, does not form osazone crystals because it is a non reducing sugar as it has no free carbonyl group. The reaction involves formation of a pair of phenylhydrazone functionalities, concomitant with the oxidation of the hydroxymethylgroup in alpha carbon (carbon atom adjacent to the carbonyl center).

20. 7. Osazone’s Test
The reaction can be used to identify monosaccharides. It involves two reactions. Firstly glucose with phenylhydrazine gives glucosephenylhydrazone by elimination of a water molecule from the functional group. The next step involves reaction of one equivalent of glucosephenylhydrazone with two equivalents of phenylhydrazine (excess).
First phenylhydrazine is involved in oxidizing the alpha carbon to a carbonyl group, and the second phenylhydrazine involves in removal of one water molecule with the new-formed carbonyl group of that oxidized carbon and forming the similar carbon nitrogen bond. The alpha carbon is attacked here because its more reactive than the others.

21. 7. Osazone’s Test
D-glucose
phenylhydrazine
glucose osazone
Osazones are highly coloured and crystalline compounds and can be easily detected. Each sugar has a characteristic crystal form of osazones.
Maltose forms petal-shaped/sun flower-shaped crystals
Lactose forms powder puff-shaped crystals
Galactose forms rhombic-plate shaped crystals
Glucose, fructose and mannose form broomstick or needle-shaped crystals.

22. 7. Osazone’s Test

23. 8. Tollens’s Test
Tollens' reagent is a chemical reagent used to determine the presence of an aldehyde, aromatic aldehyde and alpha-hydroxy ketone functional groups.
The reagent consists of a solution of silver nitrate and ammonia. It was named after its discoverer, the German chemist Bernhard Tollens. 
A positive test with Tollens' reagent is indicated by the precipitation of elemental silver, often producing a characteristic "silver mirror" on the inner surface of the reaction vessel.

24. 8. Tollens’s Test
This reagent is not commercially available due to its short shelf life, so it must be freshly prepared in the laboratory. One common preparation involves two steps. First a few drops of dilute sodium hydroxide are added to some aqueous silver nitrate. The OH−ions convert the silver aquo complex form into silver oxide, Ag2O, which precipitate from the solution as a brown solid:
2 AgNO3 + 2 NaOH → Ag2O (s) + 2 NaNO3 + H2O
In the next step, sufficient aqueous ammonia is added to dissolve the brown silver(I) oxide. The resulting solution contains the [Ag(NH3)2]+ complexes in the mixture, which is the main component of Tollens' reagent. Sodium hydroxide is reformed:
Ag2O (s) + 4 NH3 + 2 NaNO3 + H2O → 2 [Ag(NH3)2]NO3 + 2 NaOH
diamminesilver(I) complex

25. 8. Tollens’s Test

26. 9. Iodine Test
The iodine test is used to test for the presence of starch. When treated with KI solution-iodine dissolved in an aqueous solution of potassium iodide- the triiodide anion (I3−) complexes with starch, producing an intense blue/purple colour. To put it simply, when the iodine solution comes into contact with starch, it turns dark blue/purple. Otherwise, it will remain brown in color.
However, the intensity of the color decreases with increasing temperature and with the presence of water-miscible organic solvents such as ethanol. The test cannot be performed at very low pH due to the hydrolysis of the starch under these conditions.

27. 9. Iodine Test
However, the intensity of the color decreases with increasing temperature and with the presence of water-miscible organic solvents such as ethanol. The test cannot be performed at very low pH due to the hydrolysis of the starch under these conditions.

28. 9. Iodine Test
Left to right : Iodine solution, starch solution, starch solution with iodine
Amylose, a linier chain component of starch, gives a deep blue color.
Amylopectin, a branched chain component of starch, gives a purple color.
Glycogen, gives a reddish brown color.
Dextrins, Amylo, Eryhthro and Achrodextrins, form as intermediates during hydrolysis of starch gives violet, red, and no color with iodine

29. II. Quantitative Analysis

30. Monosaccharides Determination: - Chemical Method - Physic/Optic Method
Determination of carbohydrates from polysaccharides and oligosaccharides groups, need pretreatment, i.e. hydrolysis to obtain monosaccharides.
Hidrolysis
Oligo/polysaccharides  monosaccharides
(Starch) acid/enzyme (glucose)
Monosaccharides Determination:
- Chemical Method
- Physic/Optic Method
- Enzymatic Method
- Chromatography Method

31. A. Chemical Method Cupri oxide is reduced by reducing sugar Reagent :
1. Oxidation Method with Cupri
Cupri oxide is reduced by reducing sugar
Reagent :
Luff Reagent (mixed CuSO4, Na2CO3, and citric acid)
Soxhlet Reagent (mixed CuSO4with K – Na –tartrate). K-Na-tartrate prevent CuSO4 precipitated in reagents

32. Cupri oxide : - oxidator
- reduced by reducing sugar
to formed cupro oxide
(Brick-red precipitate)
Detemination of cupro oxide formed:
Weigh after dried
Dissolve again, then titrated
Determined of cupri oxide difference before and after reacting with reducing sugar
Determination of reducing sugar in solution:
Luff Schoorl Method
Munson Walker Method
Lane-Eynon Method

33. Luff Schoorl Method
For Lufff Schoorl method CuO was determined before and after reacting with reducing sugar
= (mL blanko titration – mL sample titration)
Reaction :
R – COH + CuO  Cu2O + R – COOH
H2SO4 + CuO  CuSO4 + H2O
CuSO4 + 2KI  CuI2 + K2SO4
2CuI2  Cu2I2 + I2
I2 + Na2S2O3  Na2S4O6 + NaI
I2 + starch : blue

34. Munson Walker Method

35. Lane-Eynon Method

36. 2. Oxidation Method with Alkaline Ferricyanide
Reduction of ferricyanide  ferrocyianide by reducing sugar.
2K3Fe(CN)6 + 2KI  2K4Fe(CN)6 + I2
2K4Fe(CN)6 + 3 ZnSO4  K2Zn2[Fe(CN)6]2 + 3 K2SO4
Reducing can determined :
 Based on  I2
 Based on  Na2S2O3 for titration
Indicator : starch (blue colour disappear)
K4Fe(CN)6 formed is calculated from difference between
K3Fe(CN)6 before and after reduction reaction.
Standardization experiments are required

37. Excess I2 is titrated with Na2S2O3
3. Iodometri Method
Excess I2 is titrated with Na2S2O3
Specific for aldose, but ketose slightly oxidized
Must be removed substances that can react with Iodine: ethanol, aceton, mannitol, glycerin, Na lactate, Na formate and Urea
Sample Titration :
R–COH + I2 + 3NaOH  R–COONa + 3NaI + 2H2O
I2(sisa) + 2Na2S2O3  Na2S4O6 + 2NaI
I2 + starch  I2-starch (blue)
Blanko Titration :
I2(total) + 2Na2S2O3  Na2S4O6 + 2NaI

38. 4. Somogyi-Nelson Method (1954)
Somogyi-Nelson method is based on the reduction of Cu2+ ions to Cu+ ions in the presence of reducing sugars. Cu+ ions further reduced the arsenomolibdate complex.
Reduction of arsenomolibdate complex produced a stable blue-colored dye and can be measured spectrophotometrically at 500 nm.
The arsenomolibdate complex is prepared by reacting ammonium molybdate and sodium arsenate in sulfuric acid

39. 5. DNS Method (1959)
The determination of total reducing sugar by using the DNS method (3.5-dinitrosalicyl Acid) (Miller, 1959). This method can be used to determined the sugar content of reducing glucose, fructose, maltose.
The DNS will be reduced by sugar to 3-amino-5- nitrosalicyllic acid, which gives red brick or brown red colour with maximum absorption wavelength of 540 nm.
To determined the total amount of reducing sugar, a series of standard solutions such as glucose or maltose are required.

40. 6. Morgan-Elson Method
In the Morgan-Elson method, the amino sugars or N-acetyl sugars are heated in alkaline solution to formed chromogen, which is produced red/purple compound when reacted with N,N-dimethyl-p-aminobenzaldehyde in acid solution.
The sugars contained in the sample were determined by comparing their absorbance with absorbance of the standard sugar solution (D- glucosamine, D-galactosamine, or N-acetyl-D- glucosamine) through the calibration curve using a spectrophotometer at 530 nm (amino sugar) and 544 or 585 nm (N-acetyl sugar).

41. 6. Morgan-Elson Method

42. B. Enzymatic Method Especially for sugar determination in mixture
 because enzyme is specific
Example: glucose and fructose determination
Principle :
glucose and fructose phosphorylated form glucose–6-phosphate (G6P) and fructose-6-phosphate (F6P) with hexokinase enzyme aid and Adenosine–5-triphosphate (ATP)
1. Glucose and Fructose Determination

43. Glucose + ATP  G-6-P + ADP Fructose + ATP  F-6-P + ADP
G-6-P-DH
G-6-P + NADP  gluconate-6-P + NADPH + H+
G-6-P oxidazed by NADP formed gluconate-6-phosphate by aid of glucose-6-phosphate dehidrogenase (G-6-P-DH).
NADPH which formed equivalent with glucose that react  measured by spectrophotometer ( = 334, 340, 365 nm).
F-6-P need to converted into G-6-P by aid of phosphoglucose isomerase (PGI).
PGI
F-6-P  G-6-P
( = 334, 340, 365 nm)
G-6P-DH

44. 2. Lactose and Galactose Determination
Principle :
Lactose can hydrolized formed glucose and -galactose by -galactosidase and water. Then, -galactose oxidazed by NAD formed galatonate acid and NADH with aid of galatose dehydrogenase (GAL-DH). NADH which formed equivalent with lactose that react  measured by spectrophotometer ( = 334, 340, 365 nm).
-galactosidase
Lactose + H2O  Glucose + -galactose + H2O
GAL- DH
-galactose + NAD  galatonate acid + NADH + H+ 
(= 334, 340, 365 nm)

45. C. Chromatography Method
Paper Chromatography/Thin Layer Chromatography: measured Rf value for each carbohydrate component
𝐑𝐟= 𝐦𝐢𝐠𝐫𝐚𝐭𝐢𝐨𝐧 𝐝𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐨𝐟 𝐬𝐮𝐛𝐬𝐭𝐚𝐧𝐜𝐞 𝐦𝐢𝐠𝐫𝐚𝐭𝐢𝐨𝐧 𝐝𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐨𝐟 𝐬𝐨𝐥𝐯𝐞𝐧𝐭 𝐟𝐫𝐨𝐧𝐭
Rf value for each sugar types for same treatment affected by:
Various solvent
Chamber size
Temperature
Type of stationary phase
The properties of the analyzed substance

46. 1. Paper Chromatography
Paper chromatography is an analytical method used to separate colored chemicals or substances. It is primarily used as a teaching tool, having been replaced by other chromatography methods, such as thin-layer chromatography.
The paper chromatography variant, two-dimensional chromatography involves using two solvents and rotating the paper 90º in between. This is useful for separating complex mixtures of compounds having similar polarity, for example, amino acids.
The setup has three components. The mobile phase is a solution that travels up the stationary phase, due to capillary action. The mobile phase is generally an alcohol solvent mixture, while the stationary phase is a strip of chromatography paper, also called a chromatogram.
The chromatographic method is called adsorption chromatography if the stationary phase is solid.

47. 1. Paper Chromatography
Stationary phase: paper composed of pure cellulose (e.g. whatman paper no.1 medium velocity).
Drops sample (as small as possible), drops 3-4 x (big droplets  tailings/ separation are not perfect)
Insert the paper into a chamber containing the solvent  the solvent migrated on the paper until the mark (Solvent front)

48. 1. Paper Chromatography Identification :
Physic : irradiate the paper with UV light at
 = 254 – 370 nm
Chemical : spray with chemical solution e.g.
- reducing sugar : Anilinphthalate, AgNO3
- non reducing sugar: Naphthoresorcinol in
Phosphoric acid
- Chloroglusinol and HCl can be used for: `
Aldose pentose (violet)
Ketose pentose (dark green)
Ketoheksose (brown yellow)
Methyl pentose (green)

49. - Iodine Steam
Spray on the paper in the acid room
After dry, the colored stains will be appear
Calculate Rf sample, compare with Rf standard
Solvent:
Solvent: pure substance or mixture
For simple sugar determination:
Mixed butanol : acetic : water or
acetic acid: pyridine : water (4:1:5)

50. D. Physic/Optic Method
Determination of refractive index with refractometer  each type of sugar has a specific refractive
Benefit:
large interval scale refractive index
very few samples (few drops)
precision :  0,0002
denoted by
= measured at t = 20ºC with sodium ray as a source of monochromatic rays.

51. Determination of carbohydrates with polarimeter
Principle:
Carbohydrates are active optical (capable to rotated the polarized light field), because it has C asymmetric.
Benefit:
The sample is not damaged
Can be done quickly
In order for the results accurated, hence:
The solution should be clear and colorless
The solution does not contain an active optical others material
The optimum sample concentration: not too concentrated or aqueous

52. Determination of carbohydrates with polarimeter
Biot Law : the rotational capacity of each individual sugar is proportional to the concentration of the solution and the length of the liquid in the tube
[] : specific rotation
t : temperature measurement (ºC)
D : Na ray (589 nm)
 : rotate angle observed
C : concentration (g sample/100 mL solvent)
I : tube length (dm)

53. The effect of concentration on () is very small  ignored
Temperature effect  need correction:

54. Determination of Crude Fiber
Compounds that can not be digested in human or animal digestive organs
In the analysis:
calculated the amount of substance which is not soluble in acid/alkaline under certain conditions.

55. Steps for Crude Fiber Determination
1. Defatting : remove fat in the sample with fat solvent
2. Digestion :
- dissolved with acid
- dissolved with alkaline
 in a closed state at a controlled temperature (boiling)
 immediate filtering to prevent further damage
Protein complicates filtration  needs pre-digestion with proteolytic enzymes
Residue = crude fiber containing 97% cellulose and lignin + unidentified compounds

56. Lactose Determination
25 mL milk + reagent (hydrolizing)  filtrate
5 mL filtrate + reagent  titrated with Na2S2O3
100
A = (Tb – Ts) x N x 0,171 x  5
A = Lactose content (g/100 mL)
Tb = mL blanko titration
Ts = mL sample titration
Milk with protein content = 3,2%, fat = 3,5% from 100 mL milk  48,4 mL filtrate
48,
lactose/100 mL milk = A x  x 

57. Directly with Polarimeter/refractometer
Sucrose Determination
Directly with Polarimeter/refractometer
Chemical : hydrolisis (detemined of total reducing sugar)
C6H12O11 + H2O  C6H12O6 + C6H12O6
Sucrose fructose glucose
(342) (180) (180)
 Sucrose = 0,95 x  reducing sugar
MW Sucrose
CF =  = 
2. MW reducing sugar

58. Starch hydrolised by acid/enzyme  reducing sugar  calculated
Starch Determination
Starch hydrolised by acid/enzyme 
reducing sugar  calculated
m[C6H10O25] + mH2O  mC6H12O6
pati glukosa
MW = m MW = m.180
Starch = 0,9 x  reducing sugar
MW starch
CF = 
MW reducing sugar
m x 162
=  = 0,9
m x 180

59. Additional Reductive Groups in Carbohydrate (Reducing Sugar)

60. Monosaccharide Alpha D-glucose Beta- D-fructose Opened Glucose
Reductive group
Alpha D-glucose
Beta- D-fructose
Opened Glucose
Pyran Ring
Furan Ring

61. Disaccharide
Reductive group
a1-4 linkage

62. Reductive group close each other (non-reductive)
Disaccharide
aGlucose+ Fructose= Sucrose
aGlucose+ Galactose=Lactose
Reductive group close each other (non-reductive)

63. glucose polymer with  (alpha-) bond
Starch/Amylum
Reductive group
amylopectin
amylose
Glykogen in animal body
glucose polymer with  (alpha-) bond