Chapter 13 Notes: Constants of Fats, Fatty Oils, Waxes, Balsams, Resins, Etc.

Constants of Fats, Fatty Oils, Waxes, Balsams, Resins, Etc.

• Analysis of these substances involves determining physical and chemical properties called constants.
• These constants, along with color, odor, taste, and identity tests, determine purity and quality.
• Specific gravity, solubility, melting point, congealing point, refractive index, and optical activity are determined using methods discussed elsewhere.
• Sample Preparation:
  • If oil is turbid due to separated stearin, warm it in a 50°C water bath until clear.
  • Mix clarified oil thoroughly before weighing.
  • If warming doesn't clarify, filter through dry filter paper in a hot water jacketed funnel.
  • Weigh portions needed for various tests at once, using a bottle with a pipette dropper or a weighing burette.
  • Keep the sample melted if it's solid at room temperature until samples are withdrawn.

Acid Value

• Definition: milligrams of potassium hydroxide (KOH) needed to neutralize free acids in 1 gram of substance.
• Expressed as tenths of 1% of KOH required or cubic centimeters of 0.1N NaOH needed to neutralize free acid in 10 grams.
• Determination: titrate a weighed sample in alcoholic or alcohol-ether solution with standard alkali, using phenolphthalein as indicator.
  • Use 0.5N, 0.1N, or 0.02N alkali, with 0.1N being most suitable.
  • For balsams/resins, alcohol-ether mix is advantageous as it dissolves coloring matter.
  • Solid fats/waxes are melted on a water bath and titrated hot.
• Free acids arise from hydrolysis of esters due to chemical treatment, bacterial action, or light/heat.
• Fresh substances have little to no free acids; acid value increases with aging, especially with light/air exposure.
• Official standards set maximum limits for acid values, exceeding which indicates hydrolytic decomposition during preparation/storage.
• High acid values don't necessarily indicate rancidity, which results from air/bacteria action on liberated fatty acids.
• Minimum or min/max acid values are set for balsamic/resinous substances based on free acid content.
  • Example: Tolu balsam's acid value should be 112-168; a lower value indicates low acid content/adulteration, while a higher value suggests adulteration with high-acid substances.

Experiment: Determining the Acid Value of Rosin

• Objective: Determine the acid value of rosin.
• Materials: Rosin (1g), 0.1N sodium hydroxide, alcohol-ether mixture (50cc).
• Procedure:
  1. Pulverize rosin and dissolve in 40-50 cc of neutral alcohol-ether mixture.
     • Powdering facilitates dissolution. Check the alcohol-ether mixture for acidity using phenolphthalein and neutralize if needed using standard alkali solution.
  2. Add 1 cc of phenolphthalein and titrate with 0.1N NaOH while agitating continuously.
     • For colored solutions, dilution with 100-200 cc alcohol may improve endpoint visibility.
     • Ensure thorough shaking for fatty substances to extract fatty acids from the immiscible oily layer.
     • The alkali reacts with abietic acid and other acids in rosin.
• $Acid Value = ( \frac{Volume \;of \;NaOH \;x \;5.610}{Weight \;of \;sample} )$
  • Example: If 1g rosin requires 30cc 0.1N NaOH, the acid value is $\frac{30 \times 5.610}{1} = 168.30$

Acidity Limits

• Listed substances have definite acid value limits in official standards.
• Maximum free fatty acid content is determined by the volume of standard alkali solution required.

Saponification Value

• Definition: milligrams of potassium hydroxide required to neutralize free acids and saponify esters in 1 gram of substance.
• Represents the amount of potassium hydroxide, expressed in tenths of 1%, required to neutralize the total free and combined acids in 1 gram of the substance after saponification.
• Saponification values do not vary greatly because fats and oils consist of mixtures of glyceryl esters of higher acids
• Aids in detecting glycerides of acids containing less than 16 or more than 18 carbon atoms.
• Can indicate adulteration with unsaponifiable matter, such as mineral oil.

Experiment: Determining Saponification Value of Cottonseed Oil

• Objective: Determine the saponification value of cottonseed oil.
• Materials: Cottonseed oil (2g), 0.5N alcoholic potassium hydroxide (50cc), 0.5N hydrochloric acid (50cc).
• Procedure:
  1. Place 1.5 - 2g of sample in a 200-250cc flask and add 25cc of alcoholic half-normal KOH. Attach an air condenser and heat on a water bath for 30 minutes, rotating frequently.
     • Filtration is not needed for clear samples. The long glass tube serves as an air condenser to prevent the escape of alcohol vapor. Mix by imparting a rotatory motion to the flask to accelerate saponification. The alcoholic KOH solution used should not be deeply colored since the color would interfere with the observation of the end point in the titration of the saponification mixture.
     • Alcoholic KOH, rather than an aqueous solution, is employed, because the oils are more soluble in alcohol than in water, and because the products of saponification are completely soluble in alcohol, whereas when aqueous solutions are used, the unsaponifiable matter remains insoluble.
  2. Add 1 cc of phenolphthalein and titrate excess potassium hydroxide with 0.5N hydrochloric acid.
  3. Conduct a blank test alongside, using the same amount of alcoholic 0.5N potassium hydroxide.
• Calculation:
  • $Saponification \;Value = \frac{(cc \;of \;HCl \;in \;blank - cc \;of \;HCl \;in \;sample) \times 28.05}{weight \;of \;sample}$
  • Example: 1.532g cottonseed oil saponified with 25cc 0.5N alcoholic KOH required 11.0cc of 0.5N HCl to back-titrate the excess alkali. In the blank test 21.5cc of 0.5N HCl were required to titrate the alkali. Therefore, the saponification value of the sample of cottonseed oil is $\frac{(21.5 - 11.0) \times 0.0561}{1.532} \times 1000 = 192.2$

Ester Value

• Definition: Milligrams of potassium hydroxide required to saponify esters in 1 gram of substance.
• For substances without free acids, ester value = saponification value.
• When free acids are present, ester value = saponification value - acid value.
• Important in beeswax analysis to detect adulterants like paraffin, rosin, and stearic acid.
• Determination Method:
  1. Shake sample (1.5-2g) with neutralized alcohol (20-30 cc).
  2. Add 1cc phenolphthalein, titrate with 0.5N alcoholic potassium hydroxide until neutralized.
  3. Add 25 cc 0.5N alcoholic potassium hydroxide, proceed as in saponification value determination (excluding phenolphthalein).
• $Ester \;Value = \frac{(cc \;of \;HCl \;in \;blank - cc \;of \;HCl \;in \;actual \;test) \times 28.05}{weight \;in \;grams \;of \;sample}$

Unsaponifiable Matter

• Definition: Substances in oils/fats not saponified by alkali hydroxides but soluble in fat solvents.
• Residue after saponification:
  • Phytosterol in vegetable oils/fats.
  • Cholesterol in animal oils/fats.
  • Adulterants added intentionally.
• A high content of unsaponifiable matter indicates adulteration.
• Determination method:
  1. Weigh 5g of oil/fat into a 250-cc Erlenmeyer flask, add 2g KOH in 40cc alcohol, and heat under reflux for 2 hours.
  2. Evaporate alcohol, dissolve residue in 50cc hot water, transfer to a separator, rinsing the flask with two 25-cc portions of hot water.
  3. Cool to room temperature, extract with two 50cc portions of ether, adding a few drops of alcohol to facilitate separation.
  4. Combine ether extracts, wash with sodium hydroxide solutions (4 in 1,000, then 8 in 1,000), and finally with 15-cc portions of water until the last washing is not reddened by the addition of 2 drops of phenolphthalein T.S.
  5. Transfer the ethereal solution to a tared beaker, rinse the separator with 10 cc of ether, and add the rinsings to the beaker. Evaporate the ether just to dryness on a water bath, and dry the residue for 30 minutes at 100°.
  6. Cool the beaker in a desiccator for 30 minutes, and weigh the residue of Unsaponifiable Matter.

Iodine Value

• Definition: grams of iodine absorbed by 100 grams of substance under specified conditions.
• Measures the proportion of unsaturated fatty acids present.
• Characterizes fats/oils and indicates purity.
• Drying oils (linseed oil) and fish oils (cod liver oil) have high iodine numbers (>120).
• Nondrying oils (olive oil, almond oil) have low numbers (<100).
• Semidrying oils (cottonseed oil, sesame oil) have intermediate numbers (100-120).
• Animal fats have relatively low iodine numbers (typically < 90).
• Used with saponification value to detect adulteration.
• Methods: Hubl, Hanus, Wijs (Pharmacopoeia uses Hanus).

Iodobromide Test Solution Preparation:

1. Dissolve 13.2 g iodine in 1000 cc glacial acetic acid (gentle heat if needed).
2. Cool to 25°C, determine iodine content in 20 cc by titration with 0.1N sodium thiosulfate.
3. Add bromine equivalent to iodine present.

• $X : 12.05 :: 79.92 : 126.92$
  $X = \frac{12.05 \times 79.92}{126.92} = 7.59 \;grams \;of \;bromine$

Experiment: Determining Iodine Value of Olive Oil

• Objective: Determine the iodine value of olive oil.
• Materials: Olive oil (1g), Chloroform (20 cc), Iodobromide test solution (50 cc), Potassium iodide test solution (60 cc), 0.1N sodium thiosulfate solution (100 cc), Starch test solution.
• Procedure:
  1. Dissolve 0.3g olive oil in 10cc chloroform in a glass-stoppered flask, add 25cc iodobromide T.S., stopper securely, and let stand for 30 minutes in a cool, dark place.
     • The oil, upon standing in contact with the iodobromide test solution, absorbs iodine. Iodine, itself, is absorbed very slowly by the oil, but iodine is absorbed readily from bromine-containing solutions, probably through a reaction of iodine bromide, IBr, wherein the bromine functions as a catalytic agent. The unsaturated acids of the oleic and linoleic series present in the olive oil, as well as their glyceryl esters, absorb iodine to form addition products. Thus oleic acid $C<em>{17}H</em>{33}COOH$ takes up 2 atoms of iodine and forms the addition product diiodostearic acid $C<em>{17}H</em>{33}I_2COOH$.
  2. Add 30cc potassium iodide T.S. and 100cc water, then titrate with 0.1N sodium thiosulfate, shaking thoroughly after each addition. Add 1cc starch T.S. when the color is pale, and continue until blue color disappears.
  3. Run a blank test alongside with the same quantities of reagents.
• $Iodine \;Value = \frac{(cc \;of \;thiosulfate \;in \;blank - cc \;of \;thiosulfate \;in \;sample) \times 1.269}{weight \;in \;grams \;of \;sample}$

Hydroxyl Value

• Definition: Milligrams of potassium hydroxide equivalent to hydroxyl content of 1 gram of substance.
• Indicates identity and purity of fatty substances with alcoholic hydroxyl groups.
• Inversely proportional to molecular weight, low value indicates adulteration.

Experiment: Determining Hydroxyl Number of Cetyl Alcohol

• Objective: Determine the hydroxyl number of cetyl alcohol.
• Materials: Cetyl alcohol (2g), Pyridine (2 cc), Toluene (10 cc), 1.5M acetyl chloride in toluene (10 cc), 1N sodium hydroxide (30 cc).
• Procedure:
  1. Add 2 cc pyridine and 10 cc toluene to 2g cetyl alcohol in a dry 250-cc iodine flask. Add 10 cc of approximately 1.5M acetyl chloride in toluene, stopper and immerse in a water bath at 60° to 65° for 20 minutes.
     • Toluene serves as a mutual solvent for the solid cetyl alcohol, the acetyl chloride, and the cetyl acetate formed. Pyridine acts as a condensing agent by reacting with the hydrogen chloride formed during the acetylation, which proceeds according to the following equation:
       $CH<em>3(CH</em>2)<em>{14}CH</em>2OH + CH<em>3COCl \rightarrow CH</em>3(CH<em>2)</em>{14}CH<em>2O</em>2CCH_3 + HCl$
  2. Add 25 cc distilled water, stopper and shake vigorously for 1-2 minutes, then let stand for 5 minutes to decompose the excess acetyl chloride. Titrate to a permanent pink endpoint with 1N sodium hydroxide, using 1 cc phenolphthalein as an indicator.
     • The excess acetyl chloride is hydrolyzed to acetic acid by shaking vigorously with water to ensure contact between the water and the acetyl chloride that is dissolved in the oily layer. During the titration of the acetic acid with 1N sodium hydroxide, it is again necessary to shake vigorously to ensure complete neutralization of the acetic acid.
  3. Run a blank under identical conditions.
• $Hydroxyl \;Number = \frac{(cc \;of \;1N \;NaOH \;in \;blank - cc \;of \;1N \;NaOH \;in \;sample) \times 56.10}{weight \;of \;sample}$
  • The amount of acetyl chloride that has reacted with the fatty alcohol can be determined from the difference between the amount of 1N sodium hydroxide consumed by the excess acetyl chloride in the blank and in the sample. Each cubic centimeter of 1N sodium hydroxide is equivalent to $0.0001$ mole or $56.1 \;mg$ of potassium hydroxide.

Acetyl Value of Fatty Acids

• Corresponds closely to hydroxyl value of fatty alcohols.
• Found by acetylating hydroxy fatty acids and determining the saponification value of the product.

$A = \frac{S - F}{1 - 0.00075S}$

• Where:
  • A = acetyl value of free fatty acids.
  • S = saponification value of acetylated fatty acids.
  • F = acid value of original fatty acids.

Water and Sediment in Fatty Oils

• Certain unrefined fatty oils may contain moisture and nonfatty tissue residues.
• Official limits are set for these.
• Nondestearinated cod liver oil must contain less than 0.5% water/sediment by volume.
• Determination:
  1. Mix 50cc benzene with 50cc oil in centrifuge tubes.
  2. Stopper tightly, shake vigorously, immerse in a 50°C water bath for 10 minutes.
  3. Whirl for 10 minutes and read the combined volume of water and sediment.
  4. Repeat until the volume remains constant for three consecutive readings.
• The sum of the volumes of combined water and sediment in the two tubes represents the percentage, by volume, of water and sediment in the oil.