Analysis of Carbohydrate
Kimia Bahan Makanan (CHM342) Nelson Gaspersz
Source of Carbohydrate
Processed Food of Carbohydrate Source
I. Qualitative Analysis
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.
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
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.
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.
furfural fructose resorcinol
red-colored dye
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.
anthrone
furfural blue green-colored dye
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.
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.
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
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.
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+
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.
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).
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.
phenylhydrazine D-glucose
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.
Tollens' reagent is a chemical reagent used to determine
the presence of an aldehyde, aromatic aldehyde and alphahydroxy 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.
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
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 iodidethe triiodide anion (I3−) complexes with starch, producing an intense blue/purple colour. To put it simply, when the iodine solution comes into with starch, it turns dark blue/purple. Otherwise, it will remain brown in color.
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
II. Quantitative Analysis
Determination of carbohydrates from polysaccharides and oligosaccharides groups, need pretreatment, i.e. hydrolysis to obtain monosaccharides.
Hidrolysis Oligo/polysaccharides (Starch)
acid/enzyme
monosaccharides (glucose)
Monosaccharides Determination: - Chemical Method - Physic/Optic Method - Enzymatic Method - Chromatography Method
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
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
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
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
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
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 O R C H+ 2 C u S O 4
O R C O N a+ C u O+ 3 H O 2 2
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-5nitrosalicyllic 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.
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 (Dglucosamine, D-galactosamine, or N-acetyl-Dglucosamine) through the calibration curve using a spectrophotometer at 530 nm (amino sugar) and 544 or 585 nm (N-acetyl sugar).
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-6phosphate (F6P) with hexokinase enzyme aid and Adenosine–5-triphosphate (ATP)
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
G-6-P + NADP gluconate-6-P + NADPH + H+
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)
Paper Chromatography/Thin Layer Chromatography: measured Rf value for each carbohydrate component 𝐦𝐢𝐠𝐫𝐚𝐭𝐢𝐨𝐧 𝐝𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐨𝐟 𝐬𝐮𝐛𝐬𝐭𝐚𝐧𝐜𝐞 𝐑𝐟 = 𝐦𝐢𝐠𝐫𝐚𝐭𝐢𝐨𝐧 𝐝𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐨𝐟 𝐬𝐨𝐥𝐯𝐞𝐧𝐭 𝐟𝐫𝐨𝐧𝐭 Rf value for each sugar types for same treatment affected by: o Various solvent o Chamber size o Temperature o Type of stationary phase o The properties of the analyzed substance
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.
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)
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)
- 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)
Determination of refractive index with refractometer each type of sugar has a specific refractive Benefit: • large interval scale refractive index 1.30-1.75 • very few samples (few drops) • precision : 0,0002
denoted by
n
20 D
= measured at t = 20ºC with sodium ray as a source of monochromatic rays.
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
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
D t
[] t D C I
100 lC
: specific rotation : temperature measurement (ºC) : Na ray (589 nm) : rotate angle observed : concentration (g sample/100 mL solvent) : tube length (dm)
The effect of concentration on () is very small ignored Temperature effect need correction:
D t
1 0,000184t 20 D t
Determination of Crude Fiber
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.
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 predigestion with proteolytic enzymes Residue = crude fiber containing 97% cellulose and lignin + unidentified compounds
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 48,4 100 lactose/100 mL milk = A x x 100 25
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 342 CF = = 2. MW reducing sugar 360
Starch hydrolised by acid/enzyme reducing sugar calculated m[C6H10O25] + mH2O mC6H12O6 pati glukosa MW = m.162 MW = m.180 Starch = 0,9 x reducing sugar CF
MW starch = MW reducing sugar m x 162 = = 0,9 m x 180
Monosaccharide
Reductive group
Alpha D-glucose Beta- D-fructose
Opened Glucose
Pyran Ring
Furan Ring
Disaccharide Reductive group
1-4 linkage
Disaccharide Glucose+ Fructose= Sucrose Glucose+ Galactose=Lactose
Reductive group close each other (non-reductive)
Starch/Amylum
Reductive group
amylopectin
amylose
Glykogen in animal body glucose polymer with (alpha-) bond