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Chapter 3 Extraction and mechanism

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3.1 Extraction and Mechanism

The Extraction process is divided into 5 stages. The stages are explained with mechanism as

follows with orange peels as example.

3.1.1 Stage 1

Figure 19: Extraction Process Stage 1


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3.1.1.1 Preliminary treatment

Biodegradable materials are more prone to aerial oxidation and thus samples after selection

were sliced, dried under vacuum at 60 ºC for 48 h and blended well using blender to get fine

powder. This is done to increase the surface area which facilitates the reaction rate in the faster

mode. Powdered sample was used for further analysis.

3.1.1.2 Water extraction

100g of the raw material was weighed using the digital balance and was taken in 500mL

beaker with 75mL of double distilled water and 0.1N sulphuric acid 15mL and kept at 60 ºC for

about 5 h. Contents were cooled and stirred with magnetic stirrer for 30 min. All water soluble

components get dissolved at this stage and this steps leads to the acid hydrolysis which breaks high

molecular weight carbohydrates to lower molecular weight carbohydrates.

The extract was treated with calcium carbonate or barium hydroxide to remove excess of

acid and precipitated calcium sulphate or barium sulphate respectively is filtered off. The resulting

syrup was stored at 4 ºC in an amble coloured flask to prevent further degradations and thus

freezed.

3.1.1.3 Hydrolysis

Hydrolysis of starch is a chemical reaction during which one or more water molecules are

split into hydrogen and hydroxide ions which may go on to participate in further reactions. It is a

type of reaction that is used to break down certain polymers especially those made by the step

growth polymerization. Such polymer degradation is usually carried out in presence of acid, base

or enzymes. Often the yield increases with increase in strength of acid or pH.


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Figure 20: Hydrolysis of Polysaccharides to Monosaccharide

This process can be carried out by the following methods,

Enzyme hydrolysis

Acid hydrolysis

Base hydrolysis

Enzymatic hydrolysis is the major method, which is been followed in industries because

this is highly specific in nature. Acid hydrolysis is done by using sulphuric acid and hydrochloric

acid, which is more adapted. Base hydrolysis is the least adapted. After hydrolysis the excess of

acid is neutralized using barium hydroxide or calcium carbonate and resulting syrup was stored in

refrigerator.

H2SO4 (aq) + Ba(OH)2(aq) --> BaSO4(s) + 2H2O (l)


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3.1.2 Stage 2


3.1.2.1 Decolourisation

Charcoal has been used industrially for the purification of sugars for many years. But

studies revealed that charcoal is used separation of sugars with the displacing agent, because its

use leads to improved workability of the charcoal technique for the separation of sugars. Thus

preliminary orientation experiments with known sugars indicated that commercial activated

charcoal adsorb more because of its surface are approximately 1g=100m2 of surface area. Thus

experiments were conducted with the residue of coir pith which is burnt in presence of air. It has

been previously analyzed that this procedure removes only negligible amounts of sugars from


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solution. The syrup was treated with charcoal (coir pith) and agitated using shaker for 30 min and

filtrate was collected.

Charcoal is normally useful, because it acts as an adsorbent, and can effectively remove

particles and organics and heavy metals from water. Defined, adsorption is "the collection of a

substance onto the surface of adsorbent solids." It is a removal process where certain particles are

bound to an adsorbent particle surface by either chemical or physical attraction. Adsorption is

often confused with Absorption, where the substance being collected or removed actually

penetrates into the other solid.

Figure 22: close-ups of carbon surface and pores magnification increases left to right

The reason that carbon is such an effective adsorbent materia l is due to its large number of

cavernous pores. These provide a large surface area relative to the size of the actual carbon particle

and its visible exterior surface. An approximate ratio is 1 g = 100 m2 of surface area.

Figure 23: Scanning Electron Microscope Images of Pores

http://www.cee.vt.edu/ewr/environmental/teach/wtprimer/carbon/sketglos.html



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Charcoal Carbon uses the physical adsorption process whereby attractive vander Waals

forces pull the solute out of solution and onto its surface. Once the solute is bound to the carbon is

it considered "removed" from the water. The picture below illustrates this process where the

organics are drawn toward the activated carbon by these forces. Activated carbon adsorption

proceeds through three basic steps.

Figure 24: Adsorption of Organics into the Pores

Substances adsorb to the exterior of the carbon granules

Substances move into the carbon pores

Substances adsorb to the interior walls of the carbon

3.1.2.2 Removal of Inorganic Moieties

Silica gel (230-400mesh) was packed in a sintered crucible to about 2cm and the filtrate

was poured into the packed fraction in minimal quantity connected to suction pump which brings

high vacuum. As the higher mesh value smaller particles (say inorganic ions etc) get adhere to the

silica gel by flash chromatographic technique. The filtrate was collected and solvent was removed

in rotating evaporator.

3.1.3 Stage 3





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3.1.3.1 Ethanol extraction

The residue was placed in air tight glass container covered with 200mL of boiling 80%

ethanol. After simmering for several h in a steam bath, the container was sealed and stored at room

temperature. For the analysis, the sample was homogenized in a blender for 3-5min at high speed

and then suction filtered. After extraction with 80% ethanol (2x50mL) the whole syrup was

concentrated and further extracted in a separator funnel using methanol-dichloromethane-water

(0.3:4:1 v/v/v). The organic phase containing the organic impurities was discarded and the

methanol-water phase containing sugars was evaporated. The residue was oven dried at 50 ºC

overnight to remove the residual solvent and stored at -2 ºC for chromatographic analysis.

3.1.4 Stage 4



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3.1.4.1 Methanol- Dichloromethane - water (MDW mixture)

MDW in the ratio (0.3:4:1 v/v/v), Sample tubes fed with the mixture were loosely capped,

placed in a water bath for 5s, and left at room temperature for 10 min and taken in separating

funnel, agitated vigorously by occasional release of pressure, two phases separated. The organic

phase was discarded which removes the organic impurities and the methanol: water phase was

assayed for sugar.

3.1.5 Stage 5


3.1.5.1 Drying of Solvent

The aqueous layer was oven-dried at 50 ºC overnight to remove the residual solvent, and

stored in refrigerator for analysis.

Chapter 3 Instrumentation: separation and purification

82

3.2 Seperation and Purification

3.2.1 Reverse Phase Chromatography (Preparative HPLC)

Reverse phase liquid chromatography is the separation of molecules based upon their

interaction with a hydrophobic matrix which is largely based on their polarity. Molecules are

bound to the hydrophobic matrix in an aqueous buffer (polar) and eluted from the matrix using a

gradient of organic solvent (non-polar). The matrix usually consists of spherical silica beads

(3-5 micron) which has linear octadecane groups (C18) attached to the surface via covalent

bonds. These beads are usually porous in order to increase the surface area of the beads available

for binding. The C18 groups are very hydrophobic (non-polar) and can bind quite polar

molecules such as charged peptides in a highly polar solvent such as water. The name "reversed

phase" is derived from the opposite technique of "normal phase" chromatography which involves

the separation of molecules based upon their interaction with a polar matrix (silica beads without

octadecane groups attached) in the presence of a non-polar solvent. If the polar sites on silica or

alumina are capped with non-polar groups, they interact strongly with non-polar molecules

Figure 28: Normal Phase Polarity


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Figure 29: Reverse Phase Polarity

Figure 30: Separation of individual components from mixture


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Figure 31: Solvents and Column Used For Reverse Phase Separation

The mixture was separated in 26 min by reversed phase HPLC on an

Adsorbosphere column-NH2, (250 x 4.6 mm column) using both isocratic and gradient elution

with acetonitrile/water and detected using Waters ELSD 2420. In ELSD, the mobile phase is

first evaporated. Solid particles remaining from the sample are then carried in the form of a mist

into a cell where they are detected by a laser.

Figure 32: Reverse Phase HPLC Arrangement

Chapter 3 Qualitative analysis

85

3.3 Instrumentation

Sugars, quantitatively the largest organic compound group on earth, are widely

distributed among both flora and fauna. Higher classes of vegetation and algae contain large

quantities of sugars, and the shells of arthropods, represented by crabs and shrimp, are made of

chitin, which are polysaccharides. Although sugars represent a huge biomass; they also exist in

very small amounts within individual living organisms. Various kinds of sugars and compound

sugars are involved in bodily functions, and as sources of energy. Sugars are used as raw

materials within the textile, food processing and pharmaceutical industries.

Sugars have been analyzed by various methods:

1. Gas chromatorgraphy/mass spectrometry (GC/MS) methods require preliminary

derivatization to increase sugar's volatility.

2. High-performance liquid chromatography (HPLC) methods have limitations, including

detector sensitivity.

Commonly, one of two different detectors is used. Differential refraction detector,

Ultraviolet (UV)/fluorescence detector, following application of pre- or post-column

derivatization; without derivatization, sugars are not UV detectable.

3. Liquid chromatography/mass spectrometry (LC/MS) methods using electrospray ionization

(ESI) also require pre- or post-column derivatization to obtain a high level of sensitivity.

3.3.1 Liquid Chromatography-Mass Spectrometry (LC-MS)

Liquid chromatography-mass spectrometry (LC-MS) is an analytical chemistry technique

that combines the physical separation capabilities of liquid chromatography with the mass

analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many

applications which has very high sensitivity and specificity. Generally its application is oriented

towards the specific detection and potential identification of chemicals in the presence of other

chemicals (in a complex mixture). LC separates the samples and introduce them to the MS. MS

creates and detects the charged ions. LC-MS data can be used to provide information about the

molecular weight, structure, identity and quantity of the specific sample components. LC-MS

can also be used as a highly selective and sensitive tunable detector.


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Figure 33: Separation and LC/MS analysis of individual components

3.3.2 Liquid chromatography

3.3.2.1 Scale

A major difference between traditional HPLC and the chromatography used in LC-MS is

that in the latter case the scale is usually much smaller, both with respect to the internal diameter

of the column and even more so with respect to flow rate since it scales as the square of the

diameter. For a long time, 1mm columns were typical for LC-MS work (as opposed to 4.6 mm

for HPLC). More recently 300μm and even 75μm capillary columns have become more

prevalent. At the low end of these column diameters the flow rates approach 100nL/min and are

generally used with nanospray sources.

3.3.2.2 Flow splitting

When standard bore (4.6mm) columns are used the flow is often sp lit ~10:1. This can be

beneficial by allowing the use of other techniques in tandem such as MS and UV. However

splitting the flow to UV will decrease the sensitivity at flow rates of 200Μl/MIN or less. This is

because the analyte ions have to be vaporized (nebulized) in order to become charged.


87

3.3.2.3 Mass spectrometry (Mass analyser)

There are a lot of mass analyzers that can be used in LC/MS. Single Quadruple, triple

Quadrupole, Ion Trap, TOF(time of flight) and Quadrupole-time of flight (Q-TOF).

3.3.2.4 Interface

Understandably the interface between a liquid phase technique which continuously flows

liquid, and a gas phase technique carried out in a vacuum was difficult for a long time. The

advent of electrospray ionization changed this. The interface is most often an electrospray ion

source or variant such as a nanospray source; however fast atom bombardment, thermo spray and

atmospheric pressure chemical ionization interfaces are also used. Various deposition and drying

techniques have also been used such as using moving belts; however the most common of these

is off- line MALDI deposition.

3.3.2.5 Sample types

LCMS systems facilitate the analysis of samples that have been difficult to analyse. It

significantly expands the effective analytical use of mass spectrometry to a large number of

organic compounds. The sample types range from small pharmaceutical compounds to large

proteins. LC-MS is suitable for the analysis of large, polar, ionic, thermally stable and involatile

compounds.

3.3.2.6 Selectivity and Sensitivity

The MS combined with a liquid chromatogram can detect masses characteristic of a

compound or of a class of compounds. The system can selectively detect compounds of interest

in a selective matrix, thus making it easy to find and identify the suspected impurities at trace

levels.

LC-MS sensitivity can be comparable with that of a Diode Array Detector (DAD). Far

greater sensitivity is possible when the LC-MS is configured to detect only those masses

characteristic of the compound being monitored.


88

3.3.2.7 Interfacing LC and MS

There has been a major focus on improving the interface between LC and MS. LC uses

high pressure to separate a liquid phase and produces a high gas load. But an MS requires

vacuum and a limited gas load. LC operates at near ambient temperature and an MS requires

elevated temperature. There is no mass range limitation of samples analyzed by LC but there are

limitations for an MS analyser. Finally, LC can use inorganic buffers and an MS prefers volatile

buffers. Recent developments in atmospheric pressure ionization sources have expanded the

molecular weight, sample polarity and flow rate limitation of LC-MS techniques.

3.3.2.8 Atomic pressure ionization (API)

API techniques are soft ionization processes for the analysis of large and small, polar and

non polar, labile compounds. These techniques can be used to rapidly confirm the identity of a

wide range of volatile and non volatile compounds by providing sensitive and accurate molecular

weight and fragmentation information. This technique can also be used in metabolite

confirmation analysis of most of the pharmaceutical compounds and other applications.

3.3.2.9 API – Electrospray

API-ES is a process of ionization followed by evaporation. It occurs in three basic steps:

1. Nebulization and charging

2. Desolvation

3. Ion evaporation

API-ES is useful in analyzing samples that become multiply charged such as proteins,

peptides and oligonucleotides and also singly charged samples like benzodiazepines and sulfated

conjugates.

3.3.2.10 Atmospheric Pressure Chemical Ionization (APCI)

APCI is a process of evaporation followed by ionization. It is complementary to API-ES.

It is applicable to a wide range of polar and non polar analytes that have moderate molecular

weight.


89

3.3.2.11 Scan and SIM Mode

MS can be operated in either;

1. Scan mode: instrument detects signals over a mass range during a short period of time.

2. SIM or Selected Ion Monitoring: instrument can be set to monitor only a few mass-to-

charge (m/z) ratios.

SIM is more often used for target compound analysis.

LC-MS analysis was performed with Agilent LC-MSD/ Trap system (Agilent company)

equipped with an electrospray interface. The MS spectra were acquired in positive ion mode.

N2 was used as both drying gas with a flow rate of 10L/min and as nebulising gas with a pressure

of 60psi. The nebulizer temperature was set at 350 ºC and the capillary voltage was set at

3500V. The mass spectra were recorded in the range of 400-1500µm. A fragment amplification

of 1.5V was selected for MS2 analysis LC-MS is suitable for many applications from

pharmaceutical development to environmental analysis.


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3.3.3 Thin Layer Chromatographic Analysis

Paper chromatography has been replaced by thin- layer chromatography in the analysis of

most groups of compounds because of the greater efficiency and rapidity of the thin- layer

method. The exception has been the carbohydrates, mainly because inorganic adsorbents are less

efficient in differentiation of carbohydrates than in the separation of other groups of compounds.

Cellulose has been used as adsorbent for carbohydrate separation in only a few cases, and

even then the authors have either investigated only a small number of sugars, or have taken so

few mixture of sugars that the results are insufficient for use in general analysis.

This work describes the separation of a wider group of simple sugars on cellulose and

starch by one-dimensional chromatography. A solution of a mixture is applied as a spot/band at

the bottom of the plate and allowed to travel with the solvent up the plate.

Figure 34: Identification of Compounds by thin Layer Chromatographic Analysis with

Standard and unknown


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3.3.3.1 Sample & TLC plates

For the present study Commercial TLC plates (Cellulose MN 300G) were cut down into

small plates and marked using scale and pencil. The following pure sugar samples are used:

Monosaccharides Oligosaccharides

D(-) Arabinose (Ar) D(+)Mannose (Man) D(+) Sucrose (Su)

D(-) Ribose (Ri) L(-) Sorbose (So) D(+) Maltose (Mal)

D(+) Xylose (Xy) D(-) Fructose (Fr) D(+) Lactose (La)

D(+) Galactose (Gal) L(+) Rhamnose (Rh)

D(+) Glucose (Gl)

The theory of the method has been dealt with in some detail by who give the definition249,

Rf = Distance distance moved by solute

Distance moved by advancing front of liquid

10 mg of each standard sugar and the separated fractions were dissolved in 1mL of

deionised water. 1µL of each sugar solution was applied to the chromatoplate with the

micropipette in the usual manner. The chromatoplate was placed in the chamber containing the

developing solvent. The plates were developed in an almost vertical position at room

temperature, covered with lid. The two different solvent systems follow as below:

a) n-butanol - acetone - diethyl amine - water (10:10:2:6 v/v/v/v).

b) n-butanol – acetone – pyridine - water (10:10:5:5, v/v/v/v).

The chromatoplate was then placed in the chamber as for one-dimensional separation.

After the first development, the chromatoplate was dried for 30 min in an over at 80°C.

3.3.3.2 Detection of Spots

After development the chromatoplate was dried with a stream of warm air. The dry

chromatoplate was then sprayed with a freshly prepared solution of 5% diphenylamine in

ethanol, 4% aniline in ethanol and 85% phosphoric acid, mixed in the ratio 5:5:1. After being


92

dried for a few min at room temperature, the chromatoplate was heated for about 10 min 105°C.

Well defined coloured spots appear against a white back ground. The white back ground of

cellulose layer rapidly darkens after heating, but the white background of starch layer does not.

The Rf values relative to the solvent are reported 250-253.

Figure 35: Pictorial Representation of TLC Analysis


93

3.3.3.3 Results and discussion

With the one-dimensional technique on cellulose layer by means of the solvents used,

standard and separated sugar fractions could be clearly resolved. These solvents completely

separated the oligosaccharides, aldohexoses and pentoses examined. A larger number of sugars

could be separated by two-dimensional chromatography. The best resolution of the sugars

examined was obtain with solvent system, which permits the identification of 12 sugars at a time.

Starch was found not to be as suitable as cellulose. It gave the same separation sequence

of sugars but the Rf values were lower and closer, so that the seperations were poorer. Rf values

shown in parentheses are for starch thin- layers

Table 6: Rf values of standard sugars with solvent mixture a & b

Sugar Rf =100

a b

Lactose 18(10) 17(11)

Maltose 24(11) 26(11)

Sucrose 35(20) 44(20)

Galactose 36(16) 38(15)

Glucose 41(22) 44(18)

Mannose 47(24) 47(21)

Sorbose 46(21) 54(20)

Fructose 46(21) 51(21)

Arabinose 46(21) 52(22)

Xylose 53(26) 66(27)

Ribose 63(30) 69(33)

Rhamnose 70(36) 74(40)


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We can conclude that either sugar has a stronger affinity for starch than for cellulose, or

that the composition of both the mobile and stationary phases in starch and cellulose layers is

different. The explanation for this must be looked for in the different molecular structure of

starch and cellulose.

Although one-dimensional chromatography on cellulose layer seperates more sugars than

inorganic adsorbents do, two-dimensional chromatography offers still greater possibilities. It is

surprising that only papers using this technique for separation of sugars could be found in the

literature. Lato obtained excellent seperations on silicagel impregnated with boric acid. The

results show that two-dimensional thin- layer chromatography is a promising technique in

carbohydrate research.


95

3.3.4 Qualitative analysis of carbohydrates 255 -261

Flowchart 4: Qualitative analysis of carbohydrates


96

3.3.4.1 Molisch’s test

Reagent: A 5% solution of alpha naphthol in alcohol. Add 2 drops of Molisch reagent to

2mL of sugar solution in a test tube. Mix thoroughly. Add 2mL of concentrated sulphuric acid

by the side of the test tube slanting the tube. Then eruct the test tube slowly. The formation of

the reddish violet ring at the junction of the two liquids indicates the presence of carbohydrates.

Figure 36: Molisch’s Test

Discussion: The aldehyde is then subsequently reacted with a compound called a phenol.

The phenol reacts with the aldehyde to form a colored compound. The color of the compound

(adduct) depends on the nature of the phenol used in the test. A general test for pentose and

hexose carbohydrates utilizes α-napthol and is called Molisch’s test. In other words,

carbohydrates lose water molecules in the presence of acid to form furfural. The violet ring is

due to the condensation product of α- naphthol with furfural.

Scheme 23: Mechanism involved in Molisch’s test


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3.3.4.2 Iodine test

Reagent: Iodine solution is prepared by mixing 0.33g of Iodine crystal with 0.38g of

KI and it is made upto 25mL, 1 or 2 drops of Iodine solution is added to the Sugar solution.

Figure 37: Iodine test for starch

It forms an intense blue at room temperature and disappears on heating and reappears

again on cooling. The active formation of starch is amylase, a polymer of the sugar α-D-glucose,

with the repeating unit. The polymer exists as a coiled helix into which small molecules can fit.

In the presence of starch and I-, Iodine molecules form I6 chains that occupy the centre of the

amylose helix.

Figure 38: The starch/ triiodide complex reaction


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O

O

O

O

OO

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

I

I

I

I

I

I

O

Figure 39: Starch – Iodine complex

Table 7: Iodine test with some polysaccharides

3.3.4.3 Fehling test

Reagent: 6.93g of copper sulphate pentahydrate in 100mL of water.

(Fehling A) 20g of NaOH and 34.6g of Sodium potassium tartarate (Rochelle salt) in 100mL of

water (Fehling B). To few drops of sugar solution 1:1 ratio of Fehling A and B solutions are

added and the mixture is heated.

Colour Substance present

Blue Starch

Red Dextrin

Brown Glycogen

No change Absence of polysaccharide


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Figure 40: Fehling solution (left) and Reduced Copper (I) Oxide (Right)

Discussion: Aldoses and Ketoses can be oxidized readily by very weak oxidizing agents

such as Ag+, Cu2+, Bi2+ etc.

3.3.4.4 Benedict’s test

Reagent: 173g of Sodium citrate, 100g of anhydrous Sodium carbonate in 600mL of

water in a beaker. Into this with constant stirring, run slowly copper sulphate pentahydrate

containing 17.3g dissolved in 100mL of water. Cool and transfer to a litre flask and make to the

mark with water. To 8 drops of sugar solution add 5mL of Benedict qualitative reagent. Boil

vigorously for 2 min or placed in water for 3 min. Allow to cool spontaneously. A red, yellow

or green precipitate develops depending on the concentration of the sugar present.

Figure 41: Benedict’s Qualitative Test


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Table 8: Benedict’s method for the crude estimation of reducing sugar

Colour Approximate amount of reducing sugar

No change of blue colour Absence of reducing sugar

Blue colour changes to green precipitate 0.1 to 0.5g of reducing sugar

Blue colour changes to yellow precipitate 0.5 to 1.0g of reducing sugar

Blue colour changes to orange-red

precipitate 1.0 to 2.0g of reducing sugar

Blue colour changes to brick red precipitate Over 2.0g of reducing sugar

Discussion: Copper is getting reduced and sugar is getting oxidized.

3.3.4.5 Hydrolysis test

Sucrose on hydrolysis by HCl is converted into glucose and fructose. The presence of

these two monosaccharide are detected by the below tests.

Figure 42: Hydrolysis Test

Add 2 drops of HCl and 1 drop of thymol blue to 5mL of sucrose solution. The

pink colour indicates the solution acidic. Divide into two equal parts. Boil one portion

for about one minute and then cool it under the tap. Neutralize both portion by adding

2% sodium carbonate drop by drop. Formation of blue colour indicates the

neutralization. The sucrose in the boiled portion has been hydrolysed to form glucose


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and fructose which can be detected by Benedict’s test and Seliwanoff’s test. But the

unboiled sucrose does not answer for Benedict’s test.

3.3.4.6 Barfoed’s test

Reagent: 24g of copper acetate in 450mL of boiling water. If a precipitate is formed, do

not filter. Immediately add 25mL of 8.5% acetic acid to the hot solution. Shake, cool and dilute

to 500mL and filter of the impurities.

Figure 43: Barfoed’s Test

To 8 drops of sugar solution add 5mL of Barfoed’s reagent. Heat for 30 sec. Red

precipitate is formed indicating the presence of monosaccharide.

3.3.4.7 Bial’s test

Reagent: 300mg Orcinol. 100mL Con. HCl, Add 5 drops of 10% Ferric chloride solution.

Mix 5mL of Bial’s reagent, 0.5mL of sugar solution, Heat to boiling. A green compound results

showing positive test for pentoses.

Scheme 24: Mechanism involved in Bial’s test


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Dehydration of carbohydrates to form furfural and 5-hydroxymethylfurfural

Figure 44: Bial’s test

Discussion: HCl with pentoses link Orcinol give green compound.

Scheme 25: Structure of Orcinol

3.3.4.8 Seliwanoff’s test

Reagent: 50mg of Resorcinol in 33mL of Con. HCl. Dilute it to 100mL by adding water.

Figure 45: Seliwanoff’s test


103

Discussion: Fructose on HCl to form a derivative of furfuraldehyde which gives a red colour

compound when linked with resorcinol.

Scheme 26: Structure of Resorcinol

3.3.4.9 Methylamine test

Reagent: Add 1mL of 0.2% methylamine hydrochloride in water followed by 0.2mL of

10% sodium hydroxide to about 5mL of sugar solution.

Figure 46: Positive Test for Methylamine test

Cover heat 56ºC for 30 min. Remove bath and allow standing at room temperature. The

solution will show a red colour before the end of the heating if much lactose present colour

increases on standing.

3.3.4.10 Distinguishing glucose, mannose and Galactose

Three naturally occurring aldohexose say glucose, mannose and galactose can readily be

differentiated by the following method.


104

Add 2 mg of the unknown sugar material to a solution of pyrocatechol at a concentration

of 0.2 per cent in 85 % phosphoric acid syrup. Heat for 15 min. On a boiling water bath, shaking

vigorously at the end of the first minute of heating to effect solution of the sugar. In these

conditions, glucose produces a lilac colour, mannose produces a brown colour and galactose

produces a red colour intermediate in quality between the colours afforded by glucose and

mannose. The test is applicable equally to free and polymerized aldohexose 262.

Figure 47: Distinguishing test for hexose (Glucose- Lilac Colour,

Mannose - Brown Colour and Galactose - Red Colour)

Chapter 3 Quantitative studies

105

3.4 Quantitative analysis of carbohydrates

3.4.1 Reagents used for the study

Fehling A: 17.32g of CuSO4.5H2O dissolved in 250ml distilled water.

Fehling B: Sodium potassium tartarate (86.5g), NaOH (25g) are dissolved in

water and diluted to 250ml with distilled water.

Methylene blue indicator: 1g dissolved in 100ml and made upto the mark in

100ml standard flask.

Benedict quantitative reagent: In about 600ml of hot water dissolve 200g of sodium

citrate, 75g of sodium carbonate, 125 g of potassium thiocyanate. In about 100ml

of water dissolve 18g of copper sulphate pentahydrate. When the solutions have

been cooled, they are mixed together with constant stirring. Add 5ml of 5%

potassium ferrocyanide. The resulting solution is made upto 1 litre.

Ferric alum: 50g of ferric alum (ferric ammonium sulphate) is dissolved in 54.5ml

of concentrated sulphuric acid and then diluted to 500ml.

KMnO4: 0.1N solution.

AR Oxalic acid.

Estimation of Reducing Sugars (Volumetric Methods)

Bertrand’s

Method

Benedict’s Method

Qualitative

method

Quantitative

Method

Lane-Eynon

Method


106

Standard glucose solution (of various range from 0.2-0.8g made up in 100ml).

Unknown reducing sugar (of various range from 0.2-0.8g made up in 100ml).

3.4.1.1 Lane-Eynon method

Fehling's solution is a chemical test used to differentiate between

water-soluble carbohydrate and ketone functional groups, and as a test for

monosaccharides. The test was developed by German chemist Hermann von

Fehling in 1849.

Fehling's solution is always prepared fresh in the laboratory. It is made

initially as two separate solutions, known as Fehling's A and Fehling's B. Fehling's A is a blue

aqueous solution of copper(II) sulfate, while Fehling's B is a clear solution of aqueous potassium

sodium tartrate (also known as Rochelle salt) and a strong alkali (commonly sodium hydroxide).

Equal volumes of the two mixtures are mixed together to get the final Fehling's solution, which

is a deep blue colour. In this final mixture, aqueous tartrate ions from the dissolved Rochelle salt

chelate to Cu2+ (aq) ions from the dissolved copper(II) sulfate, as bidentate ligands giving the

bistartratocuprate(II)4- complex as shown below. The tartarate ions, by complexing copper

prevent the formation of Cu(OH)2 from the reaction of CuSO4.2H2O and NaOH present in the

solution 263-264.

Scheme 27: Formation of Copper Tartarate Complex

http://en.wikipedia.org/wiki/Chemical_test

http://en.wikipedia.org/wiki/Carbohydrate

http://en.wikipedia.org/wiki/Ketone

http://en.wikipedia.org/wiki/Functional_group

http://en.wikipedia.org/wiki/Hermann_von_Fehling

http://en.wikipedia.org/wiki/Hermann_von_Fehling

http://en.wikipedia.org/wiki/Copper%28II%29_sulfate

http://en.wikipedia.org/wiki/Potassium_sodium_tartrate

http://en.wikipedia.org/wiki/Potassium_sodium_tartrate

http://en.wikipedia.org/wiki/Sodium_hydroxide

http://en.wikipedia.org/wiki/Chelation

http://en.wikipedia.org/wiki/Denticity

http://en.wikipedia.org/wiki/Ligands

http://en.wikipedia.org/wiki/Cuprate#Inorganic_cuprates

http://en.wikipedia.org/wiki/File:Fehling_reaction.jpg


107

In Lane-Eynon method sugar solution is taken in the burette and known volume of

Fehling solution is taken in conical flask. This is titrated maintaining 65-70°C temperature.

Titration is continued till it acquires a very faint blue colour. At this stage 3 drops of methylene

blue indicator is added. The dye is reduced to a colourless compound immediately and the end

point is change of colour from blue to red. The result depends on the precise reaction time,

temperature and concentration of reagent used. In this method it is susceptible for interference

from other types of molecules that act as reducing agents.

A known volume of the Fehling solution (blue coloured solution of cupric ions) is

reduced to cuprous ions (precipitated as red cuprous oxide) by reducing sugar solution. The

strength of reducing sugar solution is then determined from the volume of reducing sugar

solution consumed in the reaction.

C6H12O6 + 2Cu2+ Cu2O + C6H12O7

Copper tartarate

Complex

Figure 48: Lane-Eynon Titration (End point)


108

C16H18Cl N3S

Methyl thionium chloride or methylene blue

IUPAC Name: 3, 7- bis(dimethyl amino)-phenothiazin-5-ium chloride

(Reduced form called Leuco methylene blue)

Scheme 28: Redox mechanism of sugar with copper solution and methylene blue indicator

The titration method of Lane and Eynon is used extensively in the cane sugar industry to

determine the reducing sugars content of a wide range of sugar products. The method is

especially suited to solutions containing between 0.15 and 2.35% reducing sugars and products

such as juice, syrup, massecuites and molasses can easily be analysed. There are, however,

certain aspects regarding this method which have a significant influence on the values obtained,

viz standardisation of Fehling's solution, rate and temperature of boiling.

Procedure

5ml of Fehling A and 5ml of Fehling B is pipetted out in a clean conical flask. Unknown

reducing sugar solution was filled in the burette. And the titration was carried under hot

condition, every 30 seconds 1/2 ml of the glucose solution was added from the burette till it


109

acquires a very faint blue colour. At this stage 3-5 drops of methylene blue indicator was added.

The dye is reduced to a colourless compound immediately.

Calculation

1ml of Fehling solution is equivalent to 0.005g of glucose

Unknown value = amount of reducing sugar consumed = titre value

Amount of Fehling solution taken = _______ ml

Standard value = _________ml x 0.005

3.4.1.2 Benedict method

The famous scientist, Benedict's goal was to improve the conventional

method to make the reagent less corrosive and more stable. He

accomplished this by substituting carbonate for hydroxide as the alkali

component, to reduce the corrosiveness, and by substituting citrate for

tartrate as the agent to chelate the Cu2+, to make the reagent more stable.

Benedict's Solution, or one of the many variants that evolved over the years,

was used as the reagent of choice for measuring sugar content for more than 50 years. It was the

most common test for diabetes and was the standard procedure for virtually all clinical

laboratories. Saul Roseman remembers that all inductees into the army during World War II had

their urine tested for sugar with Benedict's Solution. Although Benedict's assay was the method

of choice for more than 50 years, it suffered from lack of sugar specificity and was eventually

supplanted by the use of enzymatic methods such as glucose oxidase.

Benedict quantitative reagent overcomes many drawbacks of the above methods. For

instance end point is blue to white by using potassium thiocyanate which converts the red

cuprous oxide to white crystals of cuprous thiocyanate, it helps in visual view but here also the


110

condition plays the integral role which may lead to error. Benedict reagent contains potassium

thiocyanate, potassium ferrocyanide and cupric citrate, sodium carbonate. The function of the

excess alkali present in the Benedict reagent is to enolise the sugar, thereby causing them to be

strong reducing agent and also to liberate cabon dioxide which prevent atmospheric oxidation of

glucose. The role of copper citrate is to reduce them to cuprous ions and glucose oxidized to

gluconic acid 265.

Figure 49: Benedict’s Method Titration (End Point)

C6H12O6 + 2Cu2+ Cu2O + C6H12O7

Ferrocyanide helps to prevent the deposition of cuprous oxide and dissolve them. Finally

thiocyanate helps to convert red cuprous oxide to white crystals of cuprous thiocyanate.

Cu+ CuSCN

(Red precipitate) (White precipitate)

Scheme 29: Formation of copper thiocyanate complex in Benedict Method

The advantage of this reagent is that the end point can be easily detected and is more

stable.


111

Procedure

Titration I

Standardisation of Benedict’s quantitative reagent

Accurately pipette out 5ml of Benedict’s quantitative reagent into a clean conical flask.

2g of Sodium carbonate was added into the same conical flask. Few pieces of porcelain beads

were added in order to avoid bumping. The contents were heated to a temperature of 60-70ºC.

Then it is titrated against standard glucose with regular shaking until the blue colour disappears.

The end point is the appearance of chalky white precipitate.

Titration II

Estimation of reducing sugar

The given unknown sample solution was made up to 100ml with distilled water in a

standard flask. It was shaken well for uniform concentration. The burette was filled with this

unknown sample solution and the titration was performed as given in the above procedure till the

appearance of chalky white precipitate.

Calculation

Standard value = The amount of glucose taken in 100ml (A g in 100ml)

1 mlA

100g

The titre value obtained for standard glucose = _________ ml

Standard value = (X) x (Y) g

Unknown value is the tire value obtained for unknown reducing sugar

= ______ g


112

3.4.1.3 Bertrand’s method

Betrand’s method 266-267 is based on the reducing action of sugar

on the alkaline solution of tartarate complex with cupric ion; the

cuprous oxide formed is dissolved in warm acid solution of ferric alum.

The ferric alum is reduced to FeSO4 which is titrated against

standardized KMnO4; Cu equivalence is correlated with the table to get

the amount of glucose. Reducing sugar can be estimated by using

Bertrand’s method. This is based on the reducing action of sugar on

the alkaline solution of tartarate complex of cupric ion.

R-CHO + Cu2+ + 5OH- Cu2O + {different species of oxidized sugar}

The cuprous oxide thus formed is dissolved in warm acid solution of ferric alum. The

ferric alum id reduced to FeSO4 and the liberated FeSO4 is titrated against standard KMnO4.

Cu2O + Fe2(SO4)3 + H2SO4 2CuSO4 + 2FeSO4 + H2O

10FeSO4 + 2 KMnO4 + 8 H2SO4 K2SO4 + 2MnSO4 + 5Fe2(SO4)3 + 8 H2O

Scheme 30: Redox reaction between cuprous oxide, ferric alum and potassium

permanganate

It is clear that 2g equivalent of copper is equivalent to 2g equivalent of FeSO4. We know

that 1g equivalent of KMnO4 is equivalent to 1g equivalent of FeSO4.

1g equivalent of copper = 1g equivalent of KMnO4

1g equivalence of copper = 63.54

1g equivalence of KMnO4 = 1000mL of 0.1N KMnO4


113

Figure 50: Bertrand’s Method (Experimental Procedure and end point)

Thus 1ml of 0.1N KMnO4 is equivalent to 0.006354g of copper or 6.35mg of copper. Hence

we can calculate the weight of copper corresponding to reducing sugar employed. Once, the

weight of the copper is obtained from the table which is standard in which, copper equivalence

of different weight of glucose.


114

Procedure

Titration I

Standardization of KMnO4 using Oxalic acid

Weigh about 1.6 to 1.7g of oxalic acid crystals accurately. Transfer it into a 250ml

standard flask and it is made upto the mark.

Weight of oxalic acid crystals = x g

= (x) N

20ml of the made up oxalic acid is pipetted out in a clean conical flask and 10ml of 1M

H2SO4 was added and the contents are boiled for 2 min. The hot solution is titrated against

KMnO4. End point is the appearance of permanent pale pink colour. Titration is repeated to get

concordant value.

Calculations

Volume of oxalic acid taken (V1) = 20ml

Volume of KMnO4 consumed (V2) = Titre value in ml

Normality of Oxalic acid (N1) = (x) N

= (y) N

The unknown reducing sugar solution was made upto 100ml in a standard flask. From

which 20ml of the solution is pipetted out into a clean beaker followed by 20ml of Fehling A and

20ml of Fehling B. The solution was heated gently for 15 min over a hot plate and the contents

were cooled, so that cuprous oxide formed is allowed to settle. It is then filtered through a


115

Whatmann 40 filter paper. Take care to see that all the cuprous oxide is been transferred into the

funnel and always kept covered with solution. After repeated washings with water, the filter

paper with cuprous oxide is transferred into a conical flask. Then it is treated with enough ferric

alum (20mL) to completely dissolve cuprous oxide after which it is titrated against the

standardized KMnO4.

1ml of N/10 or 0.1N KMnO4 = 6.35mg of copper

= X mg

X mg of copper is equivalent to Y mg of glucose factor, which can be read from table

given below.

Amount of reducing sugar inthe whole of given solution

Y mg of glucose factor X 100

1000 X unknown reducing sugar (ml)

= Z g of glucose.


116

Table 9: Copper and Reducing Sugar (glucose) equivalence

Reducing sugar (mg)

Copper (mg)

Reducing sugar (mg)

Copper (mg)

Reducing sugar (mg)

Copper (mg)

Reducing sugar (mg)

Copper (mg)

10 20.4 33 64.6 56 105.8 79 144.5

11 22.4 34 66.5 57 107.8 80 146.1

12 24.3 35 68.3 58 109.3 81 147.7

13 26.3 36 70.1 59 111.1 82 149.3

14 28.3 37 72.0 60 112.8 83 150.9

15 30.2 38 73.8 61 114.5 84 152.5

16 32.2 39 75.7 62 116.2 85 154.0

17 34.2 40 77.5 63 117.9 86 155.6

18 36.2 41 79.3 64 119.6 87 157.2

19 38.1 42 81.1 65 121.3 88 158.8

20 40.1 43 82.9 66 123.0 89 160.4

21 42.0 44 84.7 67 124.7 90 162.0

22 43.9 45 86.4 68 126.4 91 163.6

23 45.8 46 88.2 69 128.1 92 165.2

24 47.7 47 90.0 70 129.8 93 166.7

25 49.6 48 91.8 71 131.4 94 168.3

26 51.5 49 93.6 72 133.1 95 169.9

27 53.4 50 95.4 73 134.7 96 171.5

28 55.3 51 97.1 74 136.3 97 173.1

29 57.2 52 98.9 75 137.9 98 174.6

30 59.1 53 100.6 76 139.6 99 176.2

31 60.9 54 102.3 77 141.9 100 177.8

32 62.8 55 104.1 78 142.8

Chapter 3 Novel method

117

3.5 Novel Spectrophotometric Method

3.5.1 Reagents

All reagents were analytical grade and used as purchased. These reagent include

Fehling A: 6.9280g of Cupric Sulphate in 100mL of deionised water, Fehling B: Sodium

potassium tartarate (34.6g), NaOH (10.33g) are diluted to 100mL, 10% Potassium ferrocyanide,

Wheat husk, 1M Sulphuric acid, Barium Hydroxide octa hydrate. All the solutions were stored at

room temperature.

3.5.2 Sample Solution Preparation

About 20mL of Fehling A and 20mL of Fehling B was taken in two 250mL beakers each under

identical conditions. The solution in the first beaker is transferred quantitatively into a 100mL

volumetric flask and diluted with deionised water followed by 5 times dilution (Stock solution).

To the other beaker standard glucose solution 250mg in 100mL was added and heated around

60- 65○C over hot plate for about 10-15 minutes. Red cuprous oxide formed was cooled to room

temperature, carefully filtered and the filtrate is collected for further use. The filtrate is

transferred into a 100mL volumetric flask quantitatively using deionised water followed by

5 times dilution as in the previous case (Unknown solution).

3.5.3 UV-Vis Spectroscopy

Aliquots, say, 2, 4, 6, 8, 10 mL etc are pipetted out from the stock and transferred into a 25mL

volumetric flask. To this 8mL of 10 % potassium ferrocyanide was added and made up to the

mark using deionised water. Keeping 10% potassium ferrocyanide as the blank, the absorbance

of the complex is measured spectrophotometrically at 670nm (Table 10) and the λmax value is

depicted in (Figure 51). The same procedure is repeated for the unknown by taking anyone of

aliquot as in the case of stock (Table 11). The values obtained are plotted in the graph by taking

concentration in the x axis and absorbance in the y axis which obeys Beer’s law the obta ined

absorbance value for unknown is interpolated to get the concentration.


118

3.5.4 UV-Vis spectrum

Copper (Cu2+) in aqueous solution exhibits a strong absorbance in the spectral range of

460 – 800nm, which corresponds to the yellow-orange-red region of the Electro-Magnetic

spectrum. The absorbance around 300 – 450nm is almost negligible. Hence it appears blue in

colour. Copper tartarate, deep blue complex appears at λmax 620nm. The spectrum of complex

read using Perkin Elmer UV/VIS Spectrometer. Copper forms an intense green coloured

complex with Sodium potassium tartarate and Potassium ferrocyanide which shows a strong

absorbance at 670nm. The absorbance from 400 – 800nm is intense when compared to that of

free Cu2+ and tartarate complexed ion in aqueous solution. The colour of the complex is

attributed to the Metal-Ligand charge transfer or vice-versa. This may be due that copper is

complexed with tartarate and cyano ligands.

Figure 51: Strong Absorbance at 670nm of Alkaline Copper Tartarate

Ferrocyanide Complex


119

3.5.5 Mathematical Analysis

Weight of CuSO4.5H2O taken = 6.9280g

249.68g of CuSO4.5H2O contains 63.54g of copper

6.9280g of CuSO4.5H2O Contains63.54 X 6.9280

249.68

g

= 1.7631 g of copper

100mL of solution contains 1.7631 g of copper

Concentration of Copper in mg/ml1.7631 X 1000

100

mg/ml

= 17.631 mg/mL

Concentration of Copper after 5 times dilution 5

mg/ml17.631

= 3.5262 mg/mL

Table 10: Concentration of Copper and Corresponding Absorbance Measurements

Amount of

Copper (A) mL

Concentration (mg)

C x A

Absorbance

2 7.0524 0.172

4 14.1048 0.270

6 21.1572 0.398

8 28.2096 0.524

10 35.2620 0.652

Unknown (6) ? 0.284


120

Table 11: Extrapolated Unknown concentration from Beer’s Plot

Stock Copper21.1572 X 100

6

Unknown Copper15.4812 X 100

6

Amount of copper reacted = 352.62 - 258.02 = 94.6 mg

The amount of reduced copper which is directly proportional to the amount of glucose

reacted. The equivalent glucose amount can be obtained from the reference table.

Amount of Sugar present inthe 100ml of the given solution

49.6 x 100

20

= 248 mg

The same amount of glucose solution is used up and quantitative values of various

standard methods with the error factor are tabulated below. And the data gives supporting

evidence that this Spectrophotometric method is having minimum error.

Amount of

Copper (A)

Concentration (mg)

6mL (Stock) 21.1572

6mL (Unknown) 15.4392


121

3.5.6 Comparative analysis

Table 12: Estimated Glucose Amounts and Percentage Error of the

currently taken methods

S.No Name of the method

Estimated

glucose

(mg)

Percentage

Error

(%)

1 Betrand’s method 233 6.8

2 Lane Eynon method 237 5.2

3 Benedict’s method 241 3.6

4 Novel Spectrophotometric

method 248 0.8

Figure 52: Comparative Error Percentages of Various Methods

0 1 2 3 4 5 6 7

Betrand’s method

Lane Eynon method

Benedict’s method

Spectrophotometric method

Percentage error (%)

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