Analysis of food

Lipids: Solution of unsaponifiable matter in saponifiable matter (glycerides). Classification of lipids: a) Simple b) Compound c) Derived

a) Simple lipids: Esters of fatty acids and glycerol fats and oils Esters of fatty acids & higher molecular weight monohydric alcohol wax.
1-

Simple lipids: are composed of: Wax Mixed > One type of fatty acid

Triglycerides Simple One type of fatty acid 2-

Compound lipids or conjugate lipids:

1. Phospholipids Consists of Fatty acids + glycerol + phosphoric acid + nitrogenous base Ex.: Lecithin present in egg yolk

2. Glycolipids Consists of fatty acids + carbohydrates + nitrogenous base as called: Glucolipids cerebrozides

3. Sphingomilines Consist of fatty acids + nitrogenous base + phosphoric acid but no glycerol

3. Derived Lipids: it is the hydrolysis product of the previous 2 types (e.g., fatty acids, glycerol, .)

N.B. 2: Phoshpolipids are also called phosphatides or phosphoglycerides.

Q.1.Compare between fats &oils? Answer: Both fats and oils have the same chemical properties (esters of fatty acids and glycerol) but they differ in their physical properties (Fats Solids or semisolids & Oils Liquids) Composition of lipids Lipids are solutions, its solvent is the glyceride part (saponifiable part 98%) and its solute is non glyceride (non saponifiable part 2%). Saponifiable matter (98%) Glyceride part (98%), solvent Liable to alkaline hydrolysis (Saponification): giving glycerol & K salt of fatty acid (Soap)
O C R O O O O C R O C R K salt of fatty acid (soap) OH Glycerol Alkaline Hydrolysis O 3 R C OK + OH OH

Unsaponifiable matter (2%) Non- glyceride part (2%) Does not undergo alkaline hydrolysis because they are composed of: 1. Fat soluble vitamins (A, D, E, K). 2. Chromolipids ex. carotene responsible for the yellow color. 3. Hydrocarbons ex. squalene. 4. Steroids or sterols. Determine whether the oil or fat is genuine or not

+ 3 KOH

The physical and chemical properties of lipids vary with variations of the glyceride part. Types: TG DG MG

Important Notes:
1.

C4 is present only in butter fat.

Butter fat contains C4, C6, C8, C10 which are all short chain fatty acids of which:
2. 3. 4.

C4 and C6 are water soluble fatty acid and steam volatile.

C8 and C10 are steam volatile but water insoluble and alcohol soluble.
5. 6. 7.

C6, C8, C10 are present in butter fat and coconut oil. C12 20 are long chain fatty acids.

C18 stearic acid is present in milk and animal fat and it is mostly used as a base in suppositories but commonly used as its salts because it is very irritant as an acid. C20 Arachidic acid present in arachids oil which is very close to olive oil and almond oil and therefore it can be used in adulteration of these oils because it is chapter but upon analysis when arachidic acid is found in these oils and normally it' not present except in arachis oil, this confirms adulteration.
8.

Q. Why Linoleic, linolenic, Arachidonic acids are called essential fatty acids? Answer: 1. They have more than are double bond and the body cannot synthesize them, so they have to be supplied externally. 2. Play an important role in vital activities as repairing and reproduction, normal growth, skin permeability. Fatty Acids 1. Saturated fatty acids 2. Unsaturated fatty acids 3. Cyclic fatty acids 4. Hydroxy fatty acids

Naturally occurring

Omega

Synthetic

General Characteristics of naturally occurring fatty acids: Carbon atoms in the fatty acids/ or the number of carbon atoms in the fatty acids chain is even no. (4-24) starting from the carbon atom of the carboxylic a group.
1.

2.

Hydrocarbon chain is not straight but zigzag.

3. They are either saturated or unsaturated. Unsaturated fatty acids may contain up to 6 double bonds. 4.
5.

Naturally present fatty acids are cis-non conjugated.

The main functional group is (-COOH), sometimes the fatty acid contains an additional functional group e.g., (-OH) as in case of hydroxy acids. General formula: CnH2n O2 Nomenclature of fatty acids: The name of fatty acid could be: 1. Generic name: 1. The name of the fatty acid is derived from the number of carbon atoms. 2. In case of SATURATED fatty acid the name has the suffix (noic) and this suffix is added after the number of carbon atoms. 3. In case of unsaturated fatty acids, they have the suffix (enoic) added after the number of carbon atoms (if there are 2 double bonds (dienoic), if three trienoic and so on. 2. Abbreviations: Position of double bond

C., . No. of No. of double Carbon atoms bonds 3. Trivial name: check from table of unsaturated fatty acids. . Unsaturated Fatty Acids

Naturally occurring unsaturated fatty acids

Omega fatty acid 1Highly unsaturated (5 d.b. or more) 2Omeg a f.a are present only in marines not in plants. 3Very strong antioxidants.

Synthetic (not naturally occurring)

Ex. oleic, linoleic, linolenic a

(Iso-acids) Characteristics of iso acids: 1. Trans 2. Conjugated 3.Formed during hydrogenation of oils. 4. Their presence indicates adulteration.

3. Hydroxy fatty acids: a- Those are fatty acids having hydroxyl groups and they could be present in saturated or unsaturated fatty acids. b- Present in castor oil has a hydroxyl value of 150. 4. Cyclic fatty acids: C16 Hydrocapric acid (chaulmoegra oil). C18 Chaulmoegric acid (chaulmoegra oil). Chaulmoegra oil = rapeseed oil.

Examination of Lipids It involves 2 steps: 1. Physical examination 2. Chemical examination II. Chemical Examination Chemical examination of lipids involves reactions with: 1. Free carboxylic acids present in the lipid 2. Ester linkage 3.Hydroxyl of the glycerides groups of the hydroxy acids 4. Double bonds of hydrocarbon chain of fatty acids.

1.

Reactions with carboxylic acid (-COOH) group:

Presence of free carboxylic acids in lipids is due to hydrolysis of lipids in presence of water. The hydrolysis is accelerated by acids, bases or enzymes.
CH2 R2OCO CH CH2 OCOR3 OCOR1 CH2 OH R1 COOH

+ 3 H2 O

HOCH CH2OH

R2 COOH R3 COOH

To determine the extent of hydrolysis or rancidity, we should determine the acid value of oil/fat (lipid).

Acid value:
Definition: It is the no. of mg. of KOH required to neutralize the free fatty acids present in 1 g oil or fat (lipid).

Procedure:

0.1 N KOH 3 10 drops ph ph 5 ml neutral alcohol 1 g lipid

End point = colourless 1st pink. Some remarks: 1. Why n-alcohol To remove its acidity (since alcohol is liable to oxidation yielding acid). 2. Why alcohol? For extraction of free fatty acids from lipid (dissolves the free f.a. and surface tension subdivides the oil into fine globules, S.A available for the reaction). 3. Why warming? To facilitate the reaction. 4. Why not use an organic solvent? Because the oil will completely be soluble in the organic solvent and the reaction will not take place. N.B.: Acid valve shouldn't exceed 5 if > 5 sample is rejected Significance: 1. Acid valve gives an indication about the hydrolytic rancidity of oils and edibility. N.B.: Acid valve can't differentiate between genuine and adulterated oils. 2. We use (mg KOH) in order to express acidity because oils/ lipids contain more than one fatty acid and so since we don't know the mol. wt.

of all fatty acids forming the oil so we depend on the fact that in the majority of oil the main f.a. is oleic acid so we use the mol. wt. of oleic acid in calculations. Except of roils in which the main f.a. is known in this case we use the mol. wt. of this main f.a. and this is called: Percent activity: % acidity =
m of standard x F x 100 ls. w of sam t. ple

Eq. wt x N 1000

In this case the mol. wt used in calculations: in case of palm oil is palmitic acid Coconut oil is : Lauric acid Significance: > 1 % acidity oil is rancid rejected

2.

Reactions of the ester linkage of the glycerides ASaponification value:

Definition: It's the number of mg KOH required to neutralize the free fatty acids and saponify the glycerides present in 1 g lipid (it's a measure of both free fatty acids and saponify the glycerides present in 1 g lipid (it's a measure of both free and combined fatty acids). Principle: Back titration"
Known xss of KOH Lipid Residual (Pink)

St. acid (HCl) E.P.: Pink colourless Saponifiable part = Known Xss residual KOH Known Xss mls of HCl (E.P.)

Procedure:

Air condenser 1. 1g lipid 2. 25 ml 0.5 N KOH (alcoholic KOH) 3. Reflux on boiling W.B for 30 min. 4. Add 10 drops ph.ph. (pink). 5. Titrate 0.5 N HCL E.P: Pink-colourless

Important Questions (1) Why 0.5 N KOH not 0.1 N?

Answer: 1) To reduce the volume taken of KOH To accelerate the reactions by providing more drastic conditions by conc. of alkali used.
2)

(2) Why alcoholic alkali is used? Answer: Because alcohol subdivides the lipid into fine globules, thus facilitates the interaction between the lipid and alkali during saponification. (3) Why KOH, not NaOH? KOH is more preferred since it produces soft soap, while NaOH produces hard soap (producing clumps). (4) Why should we do blank? Blank must be done as part of KOH may be converted into K2 CO3 by reaction with CO2 in air which reacts with HCl to the

bicarbonate step (0.5 CO32- = 0.5 KOH) in presence of phenol phthalin as indicator. CO32- + H+ HCO3 + H+ H2O + CO2 11 8.3 3-8 pink Colourless Colourless Red Red Yellow

pH ph.ph M.O

(5) Why ph.ph & not M.O? At the end of the Rx, the flask contains 3 substances: Residual KOH & Glycerol & K salt of f.a. (soap).

The end point should be used to determine only residual KOH and the ph.ph. changes its colour at pH suitable for determination of residual KOH (upon consumption of residual KOH) at pH range (8-10) while M.O. changes its color at pH range (3-4) upon complete consumption of residual KOH and K salt of f.a.) = total KOH.
H H Residual KOH + K salt of f.a. Cl K salt of f.a. Cl complete neutralize pH 8-10 (ph.ph. changes its color)

M.O. changes its colour (not suitable) pH. 3.4 and 1st Xss of HCl

6. Why air condenser? To avoid volatilization of alcohol. Principle: Back titration"

Known xss of KOH Lipid Residual (Pink)

St. acid (HCl) E.P.: Pink colourless Saponifiable part = Known XSS residual KOH

Known XSS mls of HCL (E.P.) Significance: Gives an indication about the mean molecular weight and the chain length of f.a. comprising the lipid Sap value In: T.Gs:
1 mean mol. wt.

1 chain length

Oils Long chain f.a.

Butter Short chain f.a.

N.B.: Sap. Value of vegetable oils = 180-195 Sap. Value of butter = 220-225 Sap. Value of mineral oil = zero

N.B.: Sap. value is not significant in detection of adulteration of oil by oil but can detect the adulteration of oil with liquid paraffin. B- Ester valve Definition: No. of mg. of KOH required to saponify the glyceride present in 1 g lipid. Ester value = Sap value - acid value

3. Reactions related to the hydroxyl (-OH) group: A) Hydroxyl value: Definition: it is the number of mg. KOH required to neutralize the acetic acid capable of combining by acetylation with 1 g lipid.

Principle: Principle: Back titration"

Known xss of acetic anhydride Lipid Residual

+ H2O Liberation of acetic acid titration against alcoholic KOH using ph.ph. as indicator. E.P.: Colorless 1st pink. Procedure:
Air condenser 1. 1g lipid 2. Acetic anhydride in pyridine 3. Reflux for 30 min on boiling WB. 4. Add H2O then boil 5. Back titrate the residual acetic acid KOH (alcoholic KOH)

Equations:
OH OCOCH3

OCO HO OCO

Acetic anhdride CH3OCO

OCO

OCO

Important Questions: 1. Why pyridine? Answer: Provide non aqueous conditions prevent conversion of acetic anhydride into acetic acid from the beginning. 2. Blanks are to be done, why? 1. Blank (B): is done due to the reaction conditions (heat and time) to find out the actual amt. of acetic anhydride available for the reaction. 2. Blank (A) ACID VALUE: Free fatty acids present in the sample react with KOH (leads to false +ve increase in E.P) can be determined by titration of lipid sample without acetic anhydride against alcoholic KOH.

Hydroxyl value = (B + A) (Sample + A) B + A S A So the error is cancelled. 3. Why H2O is added? 1. To decompose the residual acetic anhydride into acetic acid. 2. Stop the reaction. Significance: Used as a measure of rancidity and hydroxyl fatty acids (present in recinoleic acid present in castor oil). Hydroxyl value > 15 except for H.V. of castor oil = 150 Q: If the hydroxyl value was high, what should we suspect ? Answer: The oil is Castor oil or rancid Q. How to differentiate? Answer: Carry out acid value If If normal B) Acetyl value: Def: Number of mg of KOH required to neutralize the acetic acid resulting from 1 g of acetylated oil during soponification. Principle: 1. Saponification value of non acetylated oil is determined. Rancid Castor oil.

2. Acetylated oil prepared by reaction of oil sample with acetic anhydride. 3. Saponification value of acetylated oil is determined.

Acetyl value =
Saponification value of acetylated oil sap. value of non acetylated oil (Blank)

OCOCH3

OCO CH3OCO OCO

KOH

T.G. + D.G. + M.G. + hydroxy acids

OH

OCO HO OCO

KOH

T.G. = Sap value of non acetylated oil

By difference = acetyl value = D.G. + M.G. + Hydroxy acids. Significance: Hydroxyl value and acetyl value are the measure of rancidity and hydroxyl acids present in lipids. N.B.: The acetyl value of most lipids reaches 20 due to the presence of M.G, D.G. and sterols. 4. Reactions related to double bonds: 1. A measure of degree of unsaturation of lipids. 2. Halogenation is an addition reaction and it depends on: The rate of addition of halogens depends on:
1. The halogenating agent: halogens are arranged in the following

descending order according to their reactivity (F2 > Cl2 > Br2 > I2). 2. Type of unsaturation: No. of double bonds Distance between double bonds and carboxylic acid group Conjugation (inverse)

3. Conditions of halogenation: the reaction must be carried out in presence of an organic solvent, in a dark place.

The following chemical constants can be used to differentiate different lipids according to the degree of unsaturation. 1. Iodine value: Definition: Number of parts of iodine absorbed by 100 parts of lipid by weight.

I2 is usually accompanied by a carrier in order to facilitate the quantitative addition.

The following reagents can be used as a halogenating agent during determination of I.V.
Halogenating agent Hanus Composition 0.2 N IBr in glacial acetic acid Bromine dioxane reagent 0.2 N Br2 + Dioxane in chloroform Br Br O + O Br Br
+

Time of addition 60-120 min 1 minute

N.B. In bromine Dioxane reagent: loose coordination of Br2 enhances the addition py. Hanus reagent: consists of I2/ Br2 / glacial acetic acid Principle:
Known xss of Hanus reagent Lipid Residual IBr

+ KI I2 Na2S2O3 using starch as indicator I2 + 2 S2 O32- 2 I- + S4 O62-

Procedures:

Leave to stand in dark for 1 hr 25 ml Hanus reagent 5 ml CHCl3 0.2 g lipid

In G.S.C.F.

Add 10 ml KI Dilute with H2O Titrate the liberated I2 (equivalent to residual hanus

St. S2O32- (0.1 N) (Brown yellow straw yellow) 8 add 1 ml starch O Blue N a S colourless
2 4

Important Questions: 1. Why blank must be done ? Due to volatility of I2 2.Why starch is added after straw yellow color is obtained? Because from the beginning of the reaction, a large amount of I2 is present, forming a v. stable complex with iodine which cannot be broken by Na2S2O3. Significance: I.V. of fat or oil by increasing the degree of unsaturation of f. a., where F. a. with one double bond as oleic acid has I.V. = 90, while linoleic acid with 2 d.b. I.V. = 180 and linolenic acid with 3 d.b. I.V = 270.
1.

I.V. is used for detection of adulteration of fats or oils with liquid paraffin (I.V. = zero) so adulteration with liq. Paraffin I.V.
2.

3.

I.V. No. of double bonds.

4. 5.

I.V. 1/ Melting point of lipid. I.V Liability to oxidative rancidity.

6. I.V. classified oils into 3 groups:

Group 1. non drying 2. Semidrying 3. Drying I.V. 80-100 100-140 140-200 Main F.a. Oleic acid Linoleic acid Linolenic acid Examples Olive, almond, arachis Cotton seed, sesame Linseed, sunflower

Since olive and almond oils are both non drying oils I.V. can't detect adulteration of each one with the other but it can detect adulteration between oils of different groups. Ex. Adulteration of olive oil with sesame oil ( I.V.). In general: I.V. can differentiate between 2 oils if they are from different groups, but if they are from the same group I.V. is useless. 3. Why drying oils? Drying means when the oil is spread to a thin film and exposed to air, it will dry due to auto-oxidation of highly unsaturated f. a. with polymerization and formation of an elastic film. 4. I.V. and No. of double bonds Melting point of lipid Oxidative rancidity (Liability to)

2. Thiocyanogen value: Definition: It is the number of parts of thiocyanogen absorbed by 100 parts of lipid calculated as I2. Principle: As I2 value but the reagent used is thiocyanogen (SCN)2 or (SCN-SCN).

N.B.: We have to take in consideration that (SCN)2 leaves one double bond without addition when there is more than one double bond, in other words, (SCN)2: Reacts with only d.b. of oleic acid. Reacts with one d.b. of linoleic acid (2 d.b). Reacts with two d.b. of linoleic acid (3 d.b.) But why? Due to the steric hinderance caused by the bulky reagent, so it can react with one d.b. of oleic acid, and d.b. of the two d.b. of linoleic. As for linolenic, it reacts with 2 double bonds of the three double bonds (1st and 3rd) and this is confirmed through comparison between I.V and (SCN)2 v. of insaturated f.a. Fatty acid Oleic Linoleic Linolenic I.V. 90 180 270 (SCN)2 V 90 90 180

Significance: the proportions of saturated, oleic, linoleic linolenic acids can be calculated from I.V and (SCN)2 V. 3. Bromine value: Definition: it's the number of parts of Br2 absorbed by 100 parts of lipid. Significance: 1. Is useful for determination of total unsaturation as it measures both conjugated and unconjugated f.a. 2. Can detect hydrogenation of oil, how? Br2 value = Conjugated + Unconjugated. I2 value = conjugated only

If Br2 V = I2 v. No hydrogenation If Br2 V. > I2 V hydrogenation. Disadvantages: 1.

2.

Can't differentiate between conjugated and unconjugated f.a. Lack of stability of Br2 as a reagent.

3. Br2 V. can be calculated (through the increase in weight) gravimetrically. 4. Maleic anhydride value (Diene value): Definition: It's the number of parts of maleic anhydride absorbed by 100 parts of lipid (calculated as I2). Procedure: 1. Dissolve 60 g of maleic anhydride in toluene, warm then cool and dilute to I L (6% maleic anhydride reagent). 3 g oil + 25 ml maleic anhydride reagent Residual maleic anhydride dilute with H2O and extract with ether aqeous solu. # 0.1 N NaOH using ph.ph. as indicator.
2.
Reflux for 3 hrs

E.P: colorless 1st pink. Significance: Used for detection of conjugated double bonds only therefore used as an indication for hydrogenation.

Equations:

HC HC

CH

+
CH2

HC HC

CO O CO

HC HC

CH CH CH CH

CO CO

Conjugated diene (dienophile)

Maleic anhydride (diene)

Reaction between dienophile and dine occurs only in conjugated systems, and it's called Diene aldol or Dies Alder reaction. Hydrogenation of Oil: Definition: It is the addition of hydrogen to the double bonds in presence of a catalyst. This leads to an increase in M.P. of oils as M.P is inversely proportional to the no. of double bonds, oils which are liquids are converted to semisolid or solid lipids which melt at mouth temperature. Unsaturated lipid + H2
Raney Ni
o

partially hydrogenated lipids + isoacids.

Temp. Pressure 170oC 4 atm. press.

N.B.: Raney Ni or Pt can be used as catalyst in hydrogenation process. Important Questions: 1. What is the role of raney Ni in hydrogenation process ? Answer: Raney Ni can act as a catalyst by attraction of both H2 and double bonds by adsorption. It is prepared by reduction of NiCO3 or Ni formate. 2. Hydrogenation reaction is selective? Answer: Because the more unsaturated fatty acid undergoes saturation at first. To control the selectivity of the reaction: Temperature, pressure, catalyst conc.

3. Compare between complete & partial hydrogenation ? Complete hydrogenation 1. All unsaturated fatty acid are converted into stearic acid. Partial hydrogenation 1. Part of the unsaturated fatty acids undergo hydrogenation

2. Accompanied by isomerism which could be : Geometrical (Trans acids) (Isoacids) 3. Give hard fat (used in waxes, candles, soap). 3.Give lipids which has M.P. close to that of mouth temperature (prefered). Positional Cis but conjugated

4. What is the importance of hydrogenation? Answer: Hydrogenation is an industrial process used for production of edible fats from oils to simulate butter fat. The degree of hydrogenation is adjusted so that the oil is converted to semisolid product of suitable consistency for human consumption & melts easily at mouth temperature. 4. What are the advantages & disadvantages of hydrogenation? Answer:

Advantages

Disadvantages

1- offensive odour of marine oil is removed. 2- More faint in colour. 3- Less liable to rancidity. 4- Low price.

1- Rearrangement of the double bond so conversion of the cis configuration into trans- (which LDL conc. & HDL conc., risk of coronary artery disease, diabetes, obesity, cancer, liver dysfunction, infertility, Alzheimer's disease .

5-The product of hydrogenation has 2-Lack of the essential fatty acids improved stability & better keeping which cannot be synthesized by the quality. body & must be supplied from diet.

5. How to detect the adulteration of butter fat with hydrogenated oil? Answer: 1- Sterol acetate test. 1- Saponification by KOH: yielding a soluble part which is saponifiable part (98%) and insoluble partwhich is unsaponifiable part (2%). 2- Add alcoholic solution of digitonin to separate the unsaponifiable part. 3- Acetylation by acetic anhydride sterol acetate. 4- Check the melting point of the product. If M.P. =(114-114.8 oC) If M.P. =(125-127oC) Cholesterol acetate Ergosterol acetate

2- Test for Ni:

Butter fat or oil + H2SO4/HNO3 (to extract Ni traces) then Ni is oxidized into Ni2+ then the medium is made alkaline by ammonia dimethyl glyoxime, if +ve red gelatinous ppt. is formed. 3- Test for iso-acids (lead salt test): a) Saponification of the sample to liberate the K salt of f.a.

b) Acidification to liberate the free f.a. followed by extraction by organic solvent. c) d) Addition of Pb acetate to form the Pb salt of f.a. Addition of ether. Soluble Natural unsaturated f.a. Saturated Insoluble Iso-acid Iso-acid

Then measure I2 value, if +ve