Theory:
In many organisms the Fats and oils are the principle stored forms of energy. These stored forms of energy molecules are extremely reduced compounds and are also the derivatives of fatty acids. Fatty acids actually consists of the carboxylic acids with hydrocarbon chains of 4 to 36 carbons, also these fatty acids can be saturated type or unsaturated type. The basic ,simple unit of lipids ,which is formulated from fatty acids are called the triacylglycerols or triglycerides. Triacylglycerols are composed or made up of three fatty acids each of which are in linkage with ester along a single glycerol molecule. These triacylglycerols molecules are nonpolar, hydrophobic molecules i.e.water repellant, which is not soluble in water. The Saponification process is defined as the hydrolysis of fats or oils under alkali environmental conditions to achieve value of glycerol and the salt of the respective fatty acid.
In literal meaning Saponification is known as “soap making”. This process or test has quite an industrial significance because it helps to know the quantity or value of free fatty acid present, since this value or quantity obtained only determines the refining loss in large measure.
This quantity or amount of free fatty acid is calculated and estimated by determining the quantity or amount of alkali (KOH) that should be added to the given fat sample to make it become neutral. This whole Determination is produced by heating an exact known quantity of the fat with a strong aqueous caustic soda solution, which will readily convert the free fatty acid into soap.
After that , This soap is then eliminated from the products and the remaining amount of fat is then determined. The loss in the form of soap is estimated and calculated by subtracting this remaining amount from the amount of fat sample taken for the test originally.
The saponification number or value is defined as the number or value of potassium hydroxide KOH in milligrams actually required to neutralize the total fatty acids, which were yielded from the complete hydrolysis of 1g of fat sample.
This test for saponification provides the relevant information regarding the characteristics attributes of the fatty acids of the fat sample – that means the longer the carbon chain is , the less amount of acid is liberated or released per gram of fat hydrolysed .
This is also taken into account as an Estimation of the average molecular weight (or the chain length) of all the fatty acids present in the fat sample after hydrolysis.
Principle:
When a fat sample is allowed to react with an alkali it leads to the fat hydrolysis that is breakdown of fats ,and because of the hydrolyzation there is formation of salts of the fatty acids which are also known as or called the soap and glycerol. This process is called saponification.
By knowing the exact amount of potassium hydroxide which is utilised during the hydrolysis of fats, then the saponification value of a given fat sample can be calculated. The saponification value or number is defined as mg of KOH required to saponify 1 g of fat sample.
In the chemical reaction of saponification ,3 molecules of potassium hydroxide KOH are consumed for saponification of each molecule of triacylglycerol irrespective of the chain length of fatty acid.
Apparently, in the fat sample each gram of a triacylglycerol having shorter chains of fatty acids will have a larger number of molecules of the triacylglycerol and l therefore , will need much more amount of potassium hydroxide KOH. The saponification value is thus an indication of average molecular weight of the fatty acid in an acylglycerol.
The procedure of this saponification value determination does involve the refluxing of the exact known amount of fat or oil with a fixed amount but an excess of alcoholic or ethanolic KOH. After hydrolysis the remaining amount of potassium hydroxide KOH is determined by back titration technique with the standardized 0.5N HCl and then the amount of KOH Utilised for saponification can thus be calculated.
Requirements for Saponification Value:
Apparatus required:
- Relux or Bulb condensers
- Conical flasks or Erlenmeyers flasks
- Boiling Water bath
- Pipettes
- Burette
Reagents required:
- a) 0.5N HCl solution and dilute exactly two time
- b) 0.5N Ethanolic potassium hydroxide standard solution
Prepare 0.5N solution of KOH , weigh accurately 28.05 g of KOH, then dissolve it in 20 ml of water, and then making the volume to 1000 ml or 1L with 95% (v/v) ethanol or absolute alcohol. - Indicator Phenolphthalein:
Take 1 g of phenolphthalein and make up to 100 ml with 95% ethanol. - d) Fat solvent: A mixture of 95% ethanol and ether (1:1 ,v/v)
Test sample:the following test compounds can be used to perform the experiment-Coconut oil , corn oil ,butter ,ghee , tristearin etc
Procedure:
- Weigh accurately one gram of the fat sample in a conical flask and dissolve it in 3 ml of the fat solvent
- Add 25 ml of 0.5N alcoholic KOH to this solution.
- Attach a reflux condenser to it ,and then reflux the contents on the boiling water bath for 30 min.
- Cool it to room temperature and add a few drops of phenolphthalein indicator into the flask.it will provide pink colour to the solution.
- Titrate the content on the flask 0.5N HCl till the pink colour disappears.
- Similarly, run a blank By refluxing 25ml of 0.5N alcoholic KOH without any fat or Lipids sample.
Calculations of Saponification Value:
| The amount of 0.5N KOH in blank = x ml | |
| The amount of 0.5N KOH in test sample = y ml | |
| Titre value of the examined test sample= | (x-y) ml |
| Saponification value = 28.05 Ă—titre value / | weight of Sample in g |
| Since 1ml solution of 0.5N KOH Contains 28.05 mg of KOH. Hence, | |
| It is used as the multiplication factor which | is incorporated in the above equation. |
Results of Saponification Value:
On observing the values obtained ,one can put the specific values in the given calculation equation ,and the saponification value can be calculated for a given fat or Lipids sample.
The calculated saponification value of the given fat sample is ——— mg/ g .
Precautions:
- Electric heaters should be used during refluxing because alcohol is Highly inflammable.
- During refluxing of the content in the conical flask,the efficacious cooling of condenser is required so that alcohol or ethanol does not get evaporated during the saponification reaction.
References –
