Analysis of Food Revision

8/2/2019 Analysis of Food Revision

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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:

Triglycerides Wax

Simple Mixed

One type of fatty acid > One type of fatty acid

2- Compound lipids or conjugate lipids:

1. Phospholipids 2. Glycolipids 3. Sphingomilines

Consists of

Fatty acids + glycerol

+ phosphoric acid +

nitrogenous base

Ex.: Lecithin present

in egg yolk

Consists of fatty acids

+ carbohydrates +

nitrogenous base as

called: Glucolipids

cerebrozides

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.


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Q.1.Compare between fats &oils?

Answer: Both fats and oils have the same chemical properties (esters offatty 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 Unsaponifiable matter

(98%) (2%)

Glyceride part (98%), solvent Non- glyceride part (2%)

Liable to alkaline hydrolysis

(Saponification): giving glycerol &

K salt of fatty acid (Soap)

O

O

O

C

O

C

O

O

R

C

R

R

KOHAlkaline

HydrolysisC

O

OK

OH

OH

OH

GlycerolK salt of fatty acid (soap)

+ 3 3 R +

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.

The physical and chemical

properties of lipids vary with

variations of the glyceride part.

Determine whether the oil or fat is

genuine or not

Types:

TG DG MG

Important Notes:

1. C4 is present only in butter fat.


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2. Butter fat contains C4, C6, C8, C10 which are all short chain

fatty acids of which:

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

4. C8 and C10 are steam volatile but water insoluble and

alcohol soluble.

5. C6, C8, C10 are present in butter fat and coconut oil.

6. C12 20 are long chain fatty acids.

7. 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.

8. 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.

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


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General Characteristics of naturally occurring fatty acids:

1. 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.

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. Naturally present fatty acids are cis-non conjugated.

5. 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


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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

1- Highly

unsaturated

(5 d.b. or more)

Synthetic

(not naturally occurring)

Ex. oleic, linoleic, linolenic a 2- Omega f.a are present

only in marines

not in plants.

3- Very

strong

antioxidants.

(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 presentin 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.


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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 inthe lipid

2. Ester linkage

of the glycerides

3.Hydroxyl

groups of thehydroxy acids

4. Double bonds

of hydrocarbonchain 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 orenzymes.

CHR2OCO

CH2

CH2

OCOR1

OCOR3

H O HOCH

CH2

OH

CH2OH

R1

COOH

R2

COOH

R3

COOH+ 3 2 +

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).


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Procedure:

End point = colourless 1st pink.

Some remarks:

1. Why n-alcohol

To remove its acidity (since alcohol is liable to oxidation yieldingacid).

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/ lipidscontain more than one fatty acid and so since we don't know the mol. wt.

3 10 drops ph ph5 ml neutral alcohol

1 g lipid

0.1 N KOH


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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 themol. wt. of this main f.a. and this is called:

Percent activity:

% acidity = sampleofwt.100xFxstandardofmls.

1000

Nwt xEq.

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

A- Saponification 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"

St. acid (HCl)

E.P.: Pink colourless

Saponifiable part = Known Xss residual KOH

Known Xss mls of HCl (E.P.)

Known xss of KOH

Lipid Residual (Pink)


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Procedure:

Important Questions

(1) Why 0.5 N KOH not 0.1 N?

Answer: 1) To reduce the volume taken of KOH

2) To accelerate the reactions by providing more drastic

conditions by conc. of alkali used.

(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

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


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bicarbonate step (0.5 CO32- = 0.5 KOH) in presence of phenol

phthalin as indicator.

CO32- + H+ HCO3

+ H+ H2O + CO2

pH 11 8.3 3-8

ph.ph pink Colourless Colourless

M.O Red Red Yellow

(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.

Residual KOH + K salt of f.a. H C l K salt of f.a. H C l 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.


St. acid (HCl)

E.P.: Pink colourless

Saponifiable part = Known XSS residual KOH

Known xss of KOH

Lipid Residual (Pink)


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Known XSS mls of HCL (E.P.)

Significance:

Gives an indication about the mean molecular weight and the chainlength of f.a. comprising the lipid

Sap value wt.mol.mean

1

lengthchain

1

In: Oils Butter

T.Gs: Long chain f.a. 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.


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Principle:


+ H2O

Liberation of acetic acid

titration against alcoholic KOH using

ph.ph. as indicator.

E.P.: Colorless 1st

pink.Procedure:

Equations:

OCO

OH

OCO

anhdride

CH3OCO

OCO

OCOCH3

OCO

HO

+ Acetic

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 againstalcoholic KOH.

Known xss of acetic anhydride

Lipid Residual

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)


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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 Rancid

If normal Castor oil.

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.

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)


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OCO

OCOCH3

OCO

HO

OCO

OH

OCO

CH3OCO KOH

KOH

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

+ 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 ofrancidity 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.


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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 Composition Time of addition

Hanus 0.2 N IBr in glacial aceticacid

60-120 min

Bromine dioxane reagent 0.2 N Br2 + Dioxane in

chloroform

O OBr

Br+

Br

Br+

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:

+ KI

I2 Na2S2O3 using starch as indicator

I2 + 2 S2 O32- 2 I- + S4 O6

2-

Procedures:

25 ml Hanus reagent5 ml CHCl3

0.2 g lipid

Leave to stand in dark for 1 hr

In G.S.C.F.

Known xss of Hanus reagent

Lipid Residual IBr


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4. I.V. 1/ Melting point of lipid.

5. I.V Liability to oxidative rancidity.

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

Group I.V. Main F.a. Examples

1. non drying 80-100 Oleic acid Olive, almond, arachis

2. Semidrying 100-140 Linoleic acid Cotton seed, sesame

3. Drying 140-200 Linolenic acid 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).


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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 forlinolenic, 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 I.V. (SCN)2 V

Oleic 90 90

Linoleic 180 90

Linolenic 270 180

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


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If Br2 V = I2 v. No hydrogenation

If Br2 V. > I2 V hydrogenation.

Disadvantages:

1. Can't differentiate between conjugated and unconjugated f.a.

2. 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).

2. 3 g oil + 25 ml maleic anhydride reagent hrs3forReflux

Residual maleic anhydride dilute with H2O and extract with

ether aqeous solu. # 0.1 N NaOH using ph.ph. as indicator.

E.P: colorless 1st pink.

Significance:

Used for detection of conjugated double bonds only therefore usedas an indication for hydrogenation.

Equations:


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CHCH

CHCH

2

CHCO

CHCO

OCH

CHCH

CH

CHCH

CO

CO

O+

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 oNiRaney partially hydrogenated lipids + isoacids.

Temp.

Pressure

170

o

C4 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 Niformate.

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.


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3. Compare between complete & partial hydrogenation ?

Complete hydrogenation Partial hydrogenation

1. All unsaturated

fatty acid are

converted into stearic

acid.

1. Part of the unsaturated fatty

acids undergo hydrogenation

2. Accompanied by isomerism

which could be :

Geometrical Positional

(Trans acids) Cis but conjugated

(Isoacids)

3. Give hard fat

(used in waxes,

candles, soap).

3.Give lipids which has M.P. close

to that of mouth temperature

(prefered).

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


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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) Addition of Pb acetate to form the Pb salt of f.a.

d) Addition of ether.

Soluble Insoluble

Natural unsaturated f.a. Saturated Iso-acid

Then measure I2 value, if +ve Iso-acid


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Analysis of Food Revision