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QUALITATIVE ANALYSIS OF CARBOHYDRATES Principles of tests !olisc"#s test $ Principle Con.

sulphuric acid hydrolyse lyophilic bonds which are then dehydrated to form furfural and its derivatives. These products combine with sulphonated - naphthol to give a purple colour. !et"o% Add 2 drops of - naphthol solution to 2ml of test solution then carefully pour about 1 ml of conc. H2S answer this test. Io%ine test Principle 'odine forms coloured absorption comple(es with polysaccharides. !et"o% The test Solution is acidified with con. HC) then 2 drops of 'odine Solution is added. Starch gives a blue colour where as de(trins and glycogen gives red colour. Sol&'ilit( Test !et"o% A pinch of sugar is dissolved in 2ml of water. #ono and dissaccharides are Soluble in water. &olysaccharides are insoluble in water. Fe"lin)s*s Test Principle *educing sugars under al+aline conditions tautomerise and in enediol forms reduce cupric ions to cuprous form. The cuprous hydro(yl formed which is not easily soluble +ept in solution by a metal chelator$ sodium potassium tartarate. The cuprous hydro(yl during the process of heating is converted red cuprous o(ide. !et"o% 1ml of sugar solution$ e,ual volume of -ehling.s solution A and / was added and +ept in boiling water bath for 2 minutes. All monosaccharides$ lactose and maltose give red precipitation. Starch and sucrose gives negative result.
!

down the sides of the test tube to form 2 layers. A purple

colour is formed at the "unction of the two layers. The #ono$ %i and &olysaccharides

Bene%ict#s Test $ Principle Carbohydrate with a potentially free CH or free C0 1 group have reducing

properties in al+aline Solution. /enedicts modified the original -ehling.s test to produce a single Solution$ which is more convenient for tests as well as more stable than -ehlings reagent. !et"o% - 1ml of /enedicts reagent is heated in boiling water bath. 1ml of test Solution is added to it and heated. A bric+ red colour indicates and which confirms the presence of reducing sugar. 2lucose$ fructose$ Arabinose$ mannose$ maltose and lactose gives red precipitation$ Starch and sucrose gives negative tests. %e(trins form light green colour. B+rfoe%#s test Principle This reagent is wea+ly acidic and is only reduced by monosaccharides. &rolonged boiling may hydroly3e dissaccharides to give false positive reactions. Colour of Cu2 is more bric+ red than the orange brown obtained with /enedicts test. !et"o% 1ml of test Solution is added with 2ml of /arfoed4s reagent boiled for 1min. /ric+ red colour is observed with monosaccharides and polysaccharides answers this test only after hydrolysis. Bi+l*s Test Principle 5hen pentoses are heated with con. HCl$ furfural is formed which condenses with orcinol in the presence of ferric ions to give a blue green colour. !et"o% To 1ml of test Solution$ add 2-6 ml of the reagent and heat it to boiling. A blue green colour indicates the presence of pentose. Seli,+noff#s Test Principle 7etose are dehydrated more rapidly than aldoses to give a furfural derivative which then condense with resorcinol to form a red comple($ prolonged heating must be avoided. !et"o% 1ml of test Solution is added with 2ml of this reagent the contents are boiled in a water bath for 1 min. The reaction is given by fructose$ Sucrose and other fructose containing carbohydrates.

Test for s&crose Sucrose is the only common non-reducing dissaccharide. So that it does not reduce on al+aline solution or form an osa3one. Therefore it is hydrolysed in acid Solution to glucose and fructose$ which are then tested. H(%rol(sis of S&crose 6ml of Sucrose is ta+en and 2ml of con. HCl is added heated for 6 min in boiling water. Then solid 8a2C
9

is added till the effervescence cases. Then

the reduction test and the seliwanoff4s test are performed to the hydrolysed Solution.

QUALITATIVE REACTIONS OF CARBOHYDRATES E- No D+te E-peri.ent 01!olisc"*s Test Add two drops of #olisch.s reagent :6; -naphthol in alcohol< to about 2ml of given sugar solution and mi( well. 'ncline the tube and add about 1ml of concentrated sulphuric acid along the sides of the tube. bserve the colour at the "unction of two li,uids. O'ser/+tion Inference

21 Io%ine Test Add a few drops of iodine solution to about 1ml of the given test solution.

31Fe"lin)*s Test a< To 1ml of -ehling.s solution A$ add 1ml of -ehling.s solution / and a few drops of the test solution containing either glucose$ fructose or maltose. /oil for few minutes

b< &erform the test with sucrose solution

41Bene%ict*s Test a< To 2 ml of /enedict.s reagent$ add five drops of the test solution containing any one of the reducing sugars. /oil for 6 minutes in a water bath. Cool the solution.

b<&erform the test with sucrose solution.

51 B+rfoe%*s test To 1ml of the test solution add about 2ml

of /arfoed.s reagent. /oil it for one minute and allow to stand for a few minutes.

61 Seli,+noff*s test To 2ml of Seliwanoff.s reagent :resorcinol in HCl< add 2 drops of the test solution and heat the mi(ture to "ust boiling.

71Bi+l*s test To 6ml of /ial.s reagent :orcinol in HCl< add 2-9 ml of sugar solution and warm it gently. 5hen bubbles rise to the surface$ cool under the tap.

81Test for s&crose a< %o /enedict.s test with the given sucrose solution

b<Add 6 drops of concentrated HCl to 6ml of sucrose solution in another test tube. Heat for 6min in a boiling water bath. Add 11; sodium hydro(ide to give a slightly al+aline medium:test with litmus paper<.8ow perform /enedict.s test with this hydrolysed sucrose solution. T+'&l+tion #ar+ :=< sign for positive reactions and :-< sign for negative reaction Io%ine test Bene%icts test Fe"lin)s test B+rfoe%#s test Seli,+noff#s test !olisc"#s test Bi+l#s test

C+r'o"(%r+te

2lucose -ructose >ylose )actose Starch Sucrose ?i<

/efore

hydrolysis ii< After Hydrolysis

ESTI!ATION OF REDUCIN9 SU9ARS BY DINITROSALICYLIC ACID !ETHOD E- No

D+te

Sugars with reducing property :arising out of the presence of a potential aldehyde or +eto group< are called reducing sugars. Some of the reducing sugars are glucose$ galactose$ lactose and maltose. -or sugar estimation the dinitrosalicylic acid method@simple$ sensitive and adoptable during handling of a large number of samples at a time. Principle 9$6-%initrosalicylic acid :%8S< is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 9-amino-6-nitrosalicylic acid$ which absorbs light strongly at 6!1 nm. !+teri+ls 1. %initrosalicylic Acid *eagent :%8S *eagent< %issolve by stirring 1 g dinitrosalicylic acid$ 211 mg crystalline phenol and 61 mg sodium sulphite in 111 m) 1; 8a H. Store at !AC. Since the reagent deteriorates due to sodium sulphite$ if long storage is re,uired$ sodium sulphite may be added at the time of use. 2. !1; *ochelle salt solution :&otassium sodium tartrate<. 9. Stoc+ glucose solution- 111 mg in 111 m) distilled water :1 mgBml<. !. 5or+ing standard glucose solution- 11 m) of stoc+ solution diluted to 111 m) with distilled water C111 DgBm)E. Proce%&re 1. 5eigh 111 mg of the sample and e(tract the sugars with hot F1; ethanol twice :6 #l each time<. 2. Collect the supernatant and evaporate it by +eeping it on a water bath at F1AC. 9. Add 11 m) water and dissolve the sugars. !. &ipette out 1.6 to 9 m) of the e(tract in test tubes and e,uali3e the volume to 9 m) with water in all the tubes. 6. Add 9 m) of %8S reagent. G. Heat the contents in a boiling water bath for 6 min. H. 5hen the contents of the tubes are still warm$ add 1 m) of !1; *ochelle salt solution. F. Cool and read the intensity of dar+ red colour at 611 nm. I. *un a series of standards using glucose :1J611 Dg< and plot a graph. Res&lt ESTI!ATION OF STARCH E- No D+te

Carbohydrates are classified as monosaccharides$ disaccharides and polysaccharides. ne of the important polysaccharide is starch. Starch is the most important storage carbohydrate of plant cells. 't is a mi(ture of two polysaccharides vi3.$ amylose and amylopectin. The amylose molecule is a long unbranched chain of 1-! alpha %-gluco pyranose units lin+ed by alpha 1-! lin+ages. The amylopectin is branched and is made of chains of 1-! lin+ed alpha %-glucose units$ with alpha 1-G lin+age at the branching points. Starch is hydrolysed into simple sugars by acid hydrolysis and the ,uantity of simple sugars are measured colorimetrically. Ai. To estimate the amount of starch present in the given sample Principle Starch is e(tracted with perchloric acid in hot acid medium. Starch is hydrolysed and dehydrated to hydro(ymethyl furfural. This compound forms green coloured product with anthrone which has absorption ma(imum at G21 nm. !+teri+ls 1. Anthrone ? 211 mg of anthrone is dissolved in 111 ml of cold I6 ; sulphuric acid. 2. F1; Kthanol 9. 62; perchloric acid !. Standard glucose solution. A. Stoc+ solution? %issolve 111 mg of glucose in 111 ml of distilled water. /. 5or+ing standard? %ilute 11 ml of the stoc+ solution to 111 ml with distilled water :1 ml contains 111 g of glucose<. Proce%&re Homogeni3e the given starch sample in hot F1; ethanol to e(tract sugars$ centrifuge and retain the residue. *epeat the washing with hot F1; ethanol 2 or 9 times till the solution does not give colour with anthrone reagent. %ry the residue well in a water bath. Add 6.1 ml of distilled water and G.6 ml of 62; perchloric acid to the

residue. K(tract for 21 minutes at e(traction using fresh perchloric acid.

C$ centrifuge and retain the e(tract. *epeat the

Centrifuge$ pool the e(tracts and ma+e upto 111 ml. &ipette 1.1 ml of the e(tract and ma+e upto 1.1 ml with distilled water in a tube. &repare standards by pipetting 1.2$ 1.!$ 1.G$ 1.F and 1.1 ml of the standard solution$ ma+e up the volume to 1.1 ml with distilled water in series of test tubes. Add !.1 ml of anthrone reagent to all test tubes and +eep for F minutes in a boiling water bath. Cool rapidly under running tap water and read the colour at G21 nm. Calculate the starch percentage by using the formula$ starch content 0 glucose content ( 1.I. %raw a standard graph with concentration of glucose on > a(is and % value on L a(is. -rom the standard graph read the concentration of un+nown glucose solution. O'ser/+tions +n% c+lc&l+tions S1No1 Vol&.e of ,or:in) st+n%+r% ;.l< 1. 2. 9. !. 6. G. H. F. /lan+ 1.2 1.! 1.G 1.F 1.1 1.1 :M< 1.1 :%< Concentr+tio n of )l&cose ; )< 21 !1 G1 F1 111 Vol&.e of ,+ter to 'e +%%e% ;.l< 1.1 1.F 1.G 1.! 1.2 1.I 1.I Vol&.e of Ant"rone re+)ent ;.l< !.1 !.1 !.1 !.1 !.1 !.1 !.1 !.1 OD +t 62= n.

-rom graph 1.1 ml of un+nown solution contains 111 ml of un+nown solution contains 0 0 > g of glucose g of glucose

111 ( > B 1.1 0 A

Starch content of the sample

0 2lucose content ( 1.I g 1111

mg

Res&lts The starch content of the sample is NNNNNNNNNmgB g

ESTI!ATION OF PROTEIN BY BIURET !ETHOD E- No D+te &rotein contain amino acids lin+ed by peptide bonds. They differ in the number of amino acids and the se,uence of amino acids$ resulting in different types of proteins.

There are more than three methods for the estimation of proteins. A colorimetric method is described here. Ai. To estimate the amount of protein present in the given solution by /iuret method. Principle Compounds containing two or more peptide bonds :e.g. protein< ta+e a characteristic purple colour when treated with dilute CuS
!

in al+aline Solution.

The colour is apparently caused by the co-ordination comple( of the copper atoms and four nitrogen atoms$ two from each of two peptide chains. proteins for colour formation. Re+)ents 1< &rotein standard :11mgBml< 2< /iuret reagent ? %issolved 1.6 g of CuS about 611 ml water.
!

't re,uires 1-21 mg

and G.1 gm of sodium potassium tartarate :*ochelle< in

Added with constant stirring 911ml freshly prepared

carbonate free 11; sodium hydro(ide. #ade upto 1 lit with water and stored in a brown polyethylene bottle. %iscard if a red or blac+ precipitate appears. Proce%&re &ipetted out 1.2$ 1.!$ 1.G$ 1.F and 1ml of the protein standard solution into a series of test tubes. The given un+nown solution was made upto the volume with distilled water and pipetted out 1.2ml into one tube. The volume was made up to 1ml in all the test tubes by adding re,uired amount of distilled water. Added !ml of /iuret reagent to each tube and mi(ed thoroughly. After 91 minutes at room temperature$ read the absorbance of the solution at 661 nm against the blan+. The standard graph was drawn and the amount of protein present in the given un+nown solution was calculated.

Res&lt The amount of protein present in the whole of the given solution was NNNNNNNmg.

ESTI!ATION OF ASCORBIC ACID BY DYE !ETHOD E- No D+te

Ascorbic acid$ commonly +nown as vitamin C$ is abundant in fresh fruits and vegetables. 't is water soluble and heat labile vitamin. Ascorbic acid is synthesi3ed from glucose in plants. Ai. To estimate the amount of vitamin C present in the given sample of foodstuff. Principle Oitamin C is a good reducing agent and it reduces the dye 2$G J dichlorophenol indophenol. 'n this reaction$ the ascorbic acid itself is o(idi3ed to dehydroascorbic acid. 'n the absence of interferring substance the capacity of an e(tract of the sample$ to reduce a standard solution of the dye as determined by titration is directly proportional to the vitamin C content. Though the dye is a blue colored compound$ the end product is the appearance of pin+ color. (alic acid is not only used to reduce the pH of the e(tracting medium thereby establishing vitamin C but also to form comple(es with metallic ions$ e(ample Cu==$ thereby preventing the catalytic o(idation by vitamin C. Re+)ents Re>&ire% 01 2? 6 %ic"lorop"enol in%op"enol %(e %issolve !2.1 mg of sodium bicarbonate and 62.1 mg of 2$G dichlorophenol indophenol dye in 61 ml of water. This was diluted to 211 ml$ filtered and stored in refrigerator. 21 =14@ o-+lic +ci% 31 Stoc: St+n%+r% sol&tion ;0.)A.l< %issolve 111 mg of pure ascorbic acid crystals in 111 ml of 1.!; o(alic acid. 41 Bor:in) st+n%+r% sol&tion ;=10.)A.l< 11 ml of stoc+ solution is made upto 111ml with 1.!; o(alic acid. Proce%&re

St+n%+r%iC+tion of t"e %(e &ipetted out 6.1 ml of the wor+ing standard ascorbic acid solution into a clean conical flas+ and titrated against the dye in the burette :O1ml<. The end point was the appearance of pale pin+ colour$ which persist for 91 seconds. The amount of dye consumed is e,uivalent to the amount of ascorbic acid. The given un+nown solution was made up to given volume with 1.!; o(alic acid. &ipetted out 6ml of this made up solution into a conical flas+ and follow the titration as above:O2ml<. *epeat the titration till concordant values obtained. &ipetted out 6ml of the diluted lime "uice:1?1< into a conical flas+$ added 6ml of 1.!; o(alic acid and follow the titration as above :O9ml<. *epeat the titration till concordant values was obtained. -rom the titer values$ calculate the amount of ascorbic acid in i< ii< the given un+nown solution and 111ml of fresh lime "uice.

C+lc&l+tion I1 A.o&nt of +scor'ic +ci% present in 5.l of ,or:in) st+n%+r% D =15.) Oolume of dye solution re,uired to o(idi3e 1.6mg ascorbic acid 0 O1 ml Amount of ascorbic acid re,uired to reduce O1ml of the dye solution 0 1.6mg Therefore$ Amount of ascorbic acid re,uired to reduce 1ml of dye solution:dye factor<

0 :1.6BO1< 0 ( mg

II1

A.o&nt of Ascor'ic +ci% re>&ire% to re%&ce 0.l of %(e sol&tion D E .) +scor'ic +ci%

Therefore$ Amount of ascorbic acid re,uired to reduce

O2 ml of dye solution 6ml of the un+nown solution contains Therefore$ ml of un+nown solution contains

P 0 : > ( O2<mg 0 L mg
0 L mg ascorbic acid 0L( 6 0 mg ascorbic acid

Res&lts i< ii< The ascorbic acid present in ml of the given solution is NNNNNNNmg

The ascorbic acid present in 111ml of fresh lime "uice is NNNNNNNNmg.

ESTI!ATION OF ASH AND ACID INSOLUBLE ASH E- No D+te

Ash is the inorganic residue from the incineration of organic matter. The amount and composition of ash in a food product depend on the nature of the food ignited and on the method of ashing. The most widely used methods are based on the fact that minerals are not destroyed by heating$ and that they have a low volatility compared to other food components. Ash contents of fresh foods rarely e(ceed 6;$ although some processed foods can have ash contents as high as 12;$ e.g.$ dried beef.
Acid insoluble ash is usually silicates primarily from soil$

Ai. To determine the ash content of the given food sample. Principle /y continuous heating the substance gets charred$ which can be used for the determination of minerals present. App+r+t&s nee%e% &orcelain crucible$ muffle furnace$ desiccator$ weighing balance. Proce%&re Tot+l As" About 6.1 gm of the sample was weighed accurately into a tarred platinum :or< porcelain crucible which had previously been heated to about G1 oC and cooled. The crucible was then placed on a clay pipe triangle and heated over a low flame till all the material gets charred followed by the heating in a muffle furnace for about 9-! hours at about G11 oC. The crucible was then cooled in a desiccator and weighed. To ensure completion of ashing$ the crucible was again heated in the muffle furnace for 1 Q hour$ cooled and weighed. This was repeated till two consecutive weights were the same and the ash was almost white :or< greyish in colour. Aci% insol&'le +s" 1. Added HCl 11; :26ml< to the crucible containing the total ash. 2. Covered with watchJglass. 9. /oiled gently for 6 minutes. !. *insed the watchglass with 6ml of hot water and added this li,uid to the crucible. 6. Collected the insoluble matter on an ashless filterpaper. G. 5ashed with hot water until the filtrate is neutral. H. Transferred the filter paper containing the insoluble matter to the original crucible

F. %ried on hotplate and ignite to constant weight. I. Allowed the residue to cool in desiccators for 91minutes. 11. 5eighed without delay. 11. Calculated the content of acid insoluble ash in mg per g of air dried material. C+lc&l+tion Tot+l +s" 5eight of the crucible 5eight of the crucible = sample 5eight of the ash Ash content :gm B 111 gm of the sample< ? A gm ? / gm ? C-A gm 0 5eight of the ash ------------------------ (1110 5eight of the sample ta+en

5eight of the crucible = sample after heating in a muffle furnace? C gm

gm.

Total ash :; on dry weight< 0 :52 J 5< 111 111 :51 J 5< :111 J #< 5here 51 0 5eight in gms of dish.= sample 52 0 5eight in gms of dish = ash 5 0 5eight in gms of empty dish. #0 #oisture ; of the sample. Aci% insol&'le +s" Acid insoluble ash ; 0 : 52 J 5 < ( 111 51 J 5

where 5 05eight in gm of empty dish 51 05eight in gm of the dish with the air dried material. 52 0 weight in gm of dish with the acid insoluble ash Res&lts The ash content of the given sample was NNNNNNNNNNgB 111g EETRACTION OF OIL AND ESTI!ATION OF OIL CONTENT E- No D+te

-ats are fatty acid esters of glycerol. -at as li,uid is called oil. Seeds li+e gingelly$ groundnut$ castor$ sunflower$ etc$ contain oil as reserve food materials for the embryo. Ai. To determine the oil content of the given food stuff. Principle The fat content is estimated by using so(hlet apparatus. Kther e(traction of crude fat in vegetable products is carried out in a continuous e(tractor$ that is an apparatus in which ether after dissolving a portion of fat of the material and discharging into the e(traction flas+ is volatili3ed$ condensed and again allowed to act on the material. The steps in the process are being repeated automatically and continuously until the e(traction is complete. The so(hlet e(traction used depends on the intermittent action of glass siphon. The ether gradually condenses into the e(traction tube containing the material until it rises to the top when it is discharged into the e(traction flas+. Materials required: 1. &etroleum ether :!1 J G1 C< 2. 5hatman filter paper 9. Absorbent cotton !. So(hlet apparatus S+.ple prep+r+tion Sample preparation varies depending upon the materials. -or oil seed such as sunflower and groundnut$ grinded 1-2g of the dry seeds in a mortar and pestle and transferred it to a piece of filter paper. -or other seeds having less oil percentage$ 6g of sample can be ta+en. Procedure The so(hlet flas+ was weighed for two consecutive concordant weights. -ive gram of the moisture free sample was pac+ed into an e(traction thimble and placed in an

e(tractor which was fi(ed into a so(hlet flas+. &our the sufficient amount of ether so as to permit siphon action. The thimble and contents were allowed to soa+ in the ether for 2! hrs. The entire set up was +ept over an electric water bath and the e(traction was connected to the condensor. The no33le of the condensor was always plugged with moistened cotton. The temperature was maintained at G1oC. The steady stream of water in the condensor was maintained. The evaporated ether would rise up but owing to the condensor arrangement$ it fall bac+ into the e(tractor. 5hen the e(tractor gets filled with ether$ it was siphoned bac+ into the flas+. This goes on till the ether collected in the e(tractor was free from any yellow colour indicating presence of fat. The so(hlet flas+ was then disconnected and ether was evaporated in a water bath maintaining at G1oC. 5hen ether in the flas+ was evaporated$ the flas+ was weighed again to get concordant values. -rom the difference in weight$ the fat content was calculated.

C+lc&l+tion 5eight of the so(hlet flas+ 0 a gm

5eight of the so(hlet flas+ = fat 5eight of the fat alone 111 gm of the sample contained 0 a-b 0 NNNgm of the given sample contained

0 b 0 c

gm gm

c gm of fat c ( 111 ------------gm of fat 0

Results Amount of oil present in 111g of the given seed material 0 NNNNNNN g.

DETER!INATION OF SAPONIFICATION NU!BER E- No D+te

Ai. To determine the saponification number of edible oil. Principle Saponification number is defined as the mg of potassium hydro(ide re,uired to saponify 1g of fat or oil. 't is a measure of the average molecular weight of the fatty acids present in a fat. This also gives the average chain length of a fatty acid. Saponification number is inversely proportional to the average chain length of the fatty acid present in the fat. Saponification number is higher for fats having short chain fatty acids and lower for fatty acid containing long chains. CH2-C CH-C *1 *2 =7 H *C 7 = CH2- H CH- H CH2- H 2lycerol

CH2-C *9 Triglyceride

&otassium salt of fatty acid

Re+)ents re>&ire% 1< Kthanolic potassium hydro(ide 1.68? 2.Fg of potassium hydro(ide was dissolved in 111ml of ethanol. 2< &henolphthalein 9< 1.68 Hydrochloric acid !< 1.6 8 8a H 6< 1.6 8 (alic acid Proce%&re St+n%+r%iC+tion of so%i&. "(%ro-i%e 11 ml of o(alic acid is titrated against sodium hydro(ide in burette. &henolphthalein is used as indicator. The end point is the appearance of pin+ colour. St+n%+r%iC+tion of H(%roc"loric +ci%

11 ml of hydrochloric acid is ta+en in conical flas+ and titrated against standardi3ed sodium hydro(ide with phenolphthalein as indicator. The end point is the appearance of pin+ colour. Deter.in+tion of S+ponific+tion n&.'er About 1g of oil was weighed and transferred into a clean conical flas+. To this 26ml of 1.68 alcoholic 7 H is added. 'n another conical flas+$ 26ml of 1.68 alcoholic 7 H was ta+en which serves as a blan+. A funnel was +ept on mouth of both the flas+s and +ept in boiling water bath for 91 minutes. After cooling$ a drop of phenolphthalein was added and titrated against HCl ta+en in the burette. The end point was the disappearance of pin+ colour. The volume of HCl consumed was noted. C+lc&l+tion Oolume of HCl consumed by blan+ 0 O1 ml Oolume of HCl consumed by oil 0 O2 ml

:O1 J O2< ( normality of HCl ( K,uivalent weight of Saponification number 0 &otassium hydro(ide 5eight of sample :1 g < 0 Res&lt The saponification number of the given edible oil NNNNNNN :O1 J O2< ( 1.6 ( 6G 1

DETER!INATION OF IODINE NU!BER E- No

D+te The iodine value is a measure of the degree of unsaturation in oil. 't is constant for particular oil or fat. 'odine value is a useful parameter in studying o(idative rancidity of oil since higher the unsaturation higher is the possibility of the oil to go rancid. Ai. To determine the 'odine number of given edible oil. Principle ils contain both saturated and unsaturated fatty acids. 'odine gets incorporated into the fatty acid chain wherever the double bonds e(ist. Hence the measure of iodine absorbed by oil gives the degree of unsaturation. 'odine value or 'odine number is defined as the grams of iodine absorbed by 111g of the oil. K(cess amount of Hanus iodine solution :'/r< is allowed to react with the oil for a definite period of time. '/r adds to the double bonds present in the unsaturated fatty acids. To the remaining '/r$ potassium iodide solution is added and the liberated iodine is titrated against the standard sodium thiosulphate. Re+)ents re>&ire% i< Hanus 'odine Solution? 9.91 gm of 'odine is dissolved in 211 ml of acetic acid by sha+ing continuouslyR 61 ml of CH9C H containing H6ml of /r2BH2o is added to '2 solution and mi(ed thoroughly. ii< 16; 7' iii< Starch as an indicator. iv< Chloroform :CHCl9 < v< 1.1 8 Sodium thiosulphate :8a2S2 9 < vi< 1.18 &otassium dichromate vii< 28 H2S !

Proce%&re St+n%+r%iC+tion of So%i&. t"ios&lp"+te

21ml of potassium dichromate is ta+en in the conical flas+. To this 6ml of 28 H 2S

11ml of 11; potassium iodide and few drops of 1; starch indicator are added. Then titrated against sodium thiosulphate solution until the colour changes from blue to light green. Deter.in+tion of io%ine n&.'er 1g of fat is weighed and transferred into an iodine flas+ and dissolved in 6ml of chloroform. Add 21ml Hanus iodine reagent using a pipette$ draining it in a definite time. #i( well and allow standing for e(actly 91 min$ with occasional sha+ing. Add 11ml of 16; 7' solution sha+e thoroughly and add 11ml of freshly boiled and cooled water$ washing down any free iodine on the stopper. Add 1 drop of starch as indicator and titrate it against thiosulphate till the blue colour disappears. Towards the end of the titration$ stopper the flas+ and sha+e vigorously so that any iodine remaining in solution in chloroform is ta+en up by potassium iodide solution. Similarly conduct the same procedure without oil for blan+. C+lc&l+tion The ,uantity of thiosulphate re,uired for blan+ minus the ,uantity re,uired for the sample gives thiosulphate e,uivalent of iodine absorbed by the fat or oil ta+en for analysis. 1 ml of 1.18 thiosulphate 0 12.GI mg of iodine. 'odine number 0 : /-S < ( 8 ( 12.GI 5eight of sample :1g< 5here$ / 0 ml sodium thiosulphate for blan+ S 0 ml sodium thiosulphate for sample 8 0 8ormality of thiosulphate Amount of fatBoil ta+en should be ad"usted such that the e(cess iodine in the added 21ml Hanus iodine solution has G1; of e(cess iodine of the amount added. i.e. if : /-S < is greater than /B2$ repeat with smaller amount of the sample. Res&lt The iodine value of the given fat NNNNNNNNNNN. ESTI!ATION OF IRON E- No

D+te Ai. To estimate amount of iron present in the given sample. Principle The iron content in the sample is determined by converting all the iron into ferric form using potassium persulphate as the o(idi3ing agent and thereafter developing colour with potassium thiocyanate to form the red ferric thiocyanate which is measured spectrophotometrically at 6!1nm. Re>&ire.ents 1. &otassium thiocyanate? H9 g of potassium thiocyanate was dissolved in 261ml of distilled water. 2. Saturated potassium persulfate. 9. 91; sulfuric acid. !. Stoc+ ferrous ammonium sulfate? H1.2mg of ferrous ammonium sulfate was dissolved in 111 ml of distilled water. Concentration - 111gBml. 6.wor+ing standard solution? 1 ml of stoc+ solution was made up to 11ml with distilled water. Concentration - 11gBml. Prep+r+tion of s+.ple +s" 01As"in) J 6 g of sample was ta+en in a crucible. The crucible was heated overflame till all the material was dried and incinerated completely. This was followed by ashing in a muffle furnace for 6 hours at 661C and cooled in a desiccator. 21 Prep+r+tion of .iner+l sol&tion J The ash was moistured with 1ml of distilled water and 6ml of con. HCl was added. The mi(ture was evaporated to dryness on a boiling water bath. Another 6ml of HCl was added again and the solution was evaporated to dryness as before. !ml of con.HCl and 6ml of water were added and the solution was warmed over a boiling water bath. 't was cooled and distilled through 5hatman 8o.!1 filter paper into 61ml volumetric flas+ and the final volume was made up with distilled water.

Proce%&re

1. Ta+en 1.2ml$ 1.!ml$ 1.Gml$ 1.Fml S 1ml of wor+ing standard in concentration range of 2-21 g in series of test tubes. 2. Ta+en 1ml of sample ali,uot in test tube. 9. Added 1ml of 91; H2S and blan+. !. The volumes of all tubes were made to F.6ml with distilled water. 6. Added 1.6ml of potassium thiocyanate to all test tubes and mi(ed. G. Allowed the color to develop for 21 mins and intensity of color is read at 6!1nm. H. Standard graph was ta+ing concentration on (-a(is and absorbance on y-a(is. -rom the graph the amount of iron is calculated. T+'&l+tion Test tubes Ool. of 5or +ing Std 1.2ml 1.!ml 1.Gml 1.Fml 1ml 1ml Conce ntratio n 2g !g Gg Fg 11g Ool.of 91; H2S ! 1ml 1ml 1ml 1ml 1ml 1ml 1ml Saturated potassium per sulfate 1ml 1ml 1ml 1ml 1ml 1ml 1ml 5ater &otassium thiocyanat e 1.6ml 1.6ml 1.6ml 1.6ml 1.6ml 1.6ml 1.6ml After 21 mins .% at 6!1nm
!

S 1ml of saturated potassium per sulfate in all test tubes

/lan+ S1 S2 S9 S! S6 Sampl e

G.6ml G.9ml G.1ml 6.Iml 6.Hml 6.6ml 6.6ml

Res&lt The amount of iron present in the 111g of sample 0

FON9U EN9INEERIN9 COLLE9E


PERUNDURAI$ERODE $638=52
;An A&tono.o&s Instit&tion +ffili+te% to Ann+ Uni/ersit(? Coi.'+tore1<

DEPART!ENT OF FOOD TECHNOLO9Y

Bioc"e.istr( L+'or+tor( !+n&+l


Re)&l+tion 2=00

Fno,le%)e

E-cellence Cre+ti/it(

INDEE
E-p1 No1 1 2 9 ! 6 G H F I 11 N+.e of t"e e-peri.ent Tualitative tests for monosaccharide$ disaccharides$ polysaccharides Kstimation of reducing sugar by dinitrosalicylic acid
method

Kstimation of starch %etermination of iodine number K(traction of oil and estimation of oil content %etermination of saponification number Kstimation of ash and acid insoluble ash Kstimation of vitamin C Kstimation of protein Kstimation of iron

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