Determination of Saponification and Iodine Numbers of Some Lipids

 Biochemistry Laboratory (BCM 362L), Experiment No. 9, © March 9, 2006 3nd Quarter A.Y. 2005-2006

Determination of Saponification and Iodine Numbers of Some Lipids Mr. *****1, *****2, *****2 1 2

Professor, School of CHE-Chm, Mapua Institute of Technology  Student, BCM 362L/ A31, School of CHE-Chm, Mapua Institute of Technology 

 ______________________________________________________________________________________  ABSTRACT

Olive oil was used as a source of triacylglycerol one with the sample and one as a blank. The anal analys ysis is.. The The expe experi rime ment nt focu focuse sess on the the iodine number was 3.55348 g for the sample, and determ determina inatio tion n of iodine iodine and saponi saponific ficati ation on 1.2691 g for the blank solution. number number.. The saponi saponific ficati ation on of olive olive oil was was  Keywords: Olive oil, triacylg 0.672 which matches with the theoretical range. triacylglyc lycerol, erol, iodine, iodine, Two trials were made for the Iodine experiment, saponification.  ______________________________________________________________________________________  INTRODUCTION

Acylg Acylgly lycer cerols ols can be hydrol hydrolyz yzed ed by heating with acid or base or by treatment with lipa lipase ses. s. Hydr Hydrol olys ysis is with with alka alkali li is call called ed saponification and yields salts of free fatty acids ad glyce glycerol rol.. This This is how our ancestor ancestorss made made soap. soap. One method method used used potass potassium ium hydrox hydroxide ide leached from wood ashes to hydrolyze animal fat. When the fatty acids esterified at the first and third carbons of glycerol are different, the second carbon is asymmetric. The various acylglycerols are normally normally soluble soluble in benzene, benzene, chlorofor chloroform, m, ether, and hot ethanol. Saponification value of a fat or oil is defined as the number of milligrams of KOH needed to saponify 1g of fat. It basically involves the the heat heatin ing g of fat fat in alka alkali li lead leadin ing g to the the formation of glycerol and potassium of the fatty acids.The saponification value is an indication of  the sized or nature nature of the the fatty fatty acid chanin chaninss esterified to glycerol. METHODOLOGY

 A. Samponification Number  Two clean and dry round bottom flasks were were prepar prepared. ed. 1 g sample sample of olive olive oil were   placed into a round bottom flask. The second round bottom flask was served as a blank. 25 mL of 5% alcoholic KOH was delivered into each of  the two flasks flasks from a burett burette. e. A few boiling boiling chips were added to each flask, and was fitted

with with a waterwater-coo cooled led reflux reflux conden condenser ser.. Each Each sample was refluxed for an hour in a water bath on an electr electric ic mantle mantle.. The flasks flasks were were then then cool cooled ed to room room temp temper erat atur ure. e. The The refl reflux uxed ed solutions were transferred into Erlenmeyer flasks using small amounts of hexane hexane to thoroughl thoroughly y rinse off the round bottom flask. To each flask, 3 drop drop of 1% phenol phenolpht phthal halein ein and was titra titrated ted with standard 0.5 M HCl to colorless endpoint. The saponific saponificatio ation n number number and molecular molecular mass of the cooking oil was then computed.  B. Iodine Number  Two clean and dry 250 mL Erlenmeyer  flasks were prepared. About 0.1 g of cooking oil was weighed into a flask; the other flask was served as a blank. 5 mL ether and 15 mL Hanus reagent was added to each flask. The flasks were allowed to stand for 30 minutes in the dark and were occasionally shaked. After 30 minutes, the flasks were brought out and 5 mL of 15% KI was adde dded to each. ach. The mixt ixture ure was sha shaked ked thoroughly to extract any remaining free iodine into the KI solution. 12.5 mL of distilled water  water  was added to each flask and was titrated with standard 0.1 N Na 2S2O3 until the color of the solution was pale yellow. 1 mL of 1% starch solution was added to both flasks. The titration was continued with 0.1 N Na2S2O3 until the blueviolet color just disappears. The iodine number  was then calculated. RESULTS AND DISCUSSION

By heatin heating g a trigly triglycer ceride ide in aqueou aqueouss  potassium hydroxide (KOH) the fatty acyl esters can be cleaved off (hydrolysis) leaving behind glycerol and the potassium salt of the fatty acid. For For sapo saponi nifi fica cati tion on,, the the data data are are as follows: Titrant volume (mL) Blank titrant volume (mL) Net titrant volume (mL) Moles KOH consumed Saponification Number Molecular Mass

24 30.8 6.8 0.012 0.672 250 g/mole

The comput computati ations ons for saponi saponific ficati ation on number are:

For trial 1, I.N. = (0.014 L Na 2S2O3 x 0.09918 mol mol Na2S2O3/1L) x (1 mol I 2/0.099 /0.09918 18 mol  Na2S2O3 x 253. 82 g/ 1 mol) = 3.55348 g For blank, blank, I.N. I.N. = (0.005 (0.005 L Na2S2O3 x 0.09918 mol mol Na2S2O3/1L) x (1 mol I 2/0.099 /0.09918 18 mol  Na2S2O3 x 253. 82 g/ 1 mol) = 1.2691 g CONCLUSION

The smaller smaller the length length of fatty fatty acid acid chains the higher the saponification number. The valu alue of sap saponif onific icat atio ion n is a measur measureme ement nt of the ml of KOH KOH requir required ed to complete the hydrolysis of one gram of fat or oil. The iodine iodine mumbe mumberr is the number number of  grams of iodine absorbed by 100 g of fat or oil.

Moles KOH = (0.5 M HCl)(24 mL HCl)(1 L / 1000 mL)( 1 mole KOH / 1 mole HCl) = 0.012 mole.

LITERATURE CITED

SN = (0.012 mol x 56 g/mol) / 1 g = 0.672

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MM = (3 x 1 g) / 0.012 mol = 250 g /mol Fatty acids (component of triglycerides) triglycerides) can can be satu satura rate ted d (no (no C=C C=C doub double le bond bonds) s) or  unsatu unsaturat rated ed (C=C (C=C double double bonds bonds are presen present). t). Treatment of a triglyceride (or fatty acid) with a halogen will result in the addition of the halogen at the C=C double bond site: HC=CH + HI  HIC-CH2 The result is simple, more double bonds mean more iodine (or other halogens) will be absorb absorbed. ed. The The data data for the determ determina inatio tion n of  iodine number is located below: Trial 1 Titrant vol. Net Titrant vol. Meq I2 Consumed

Iodine No.

Blank  

35.5

47 11.5

0.00138852 moles

0.0004559 moles

3.55348 g

1.2691 g

The computation for iodine number are as follows: I.N. = 100 x (eq. I 2 x E.W. of I 2) / 1 g sample

1. Biochemistry, Garrett and Grisham, 3 rd Edition