analyticalmethods2-hafta.ppt

Oils and FatsOils and Fats

Major Component (%95-99)

Triglycerides

Minor Components (%1-5)

Triglyceride Derivatives

Glycerol

Free Fatty Acids

Mono- and Diglycerides

Non-Triglyceride Derivatives

Phospholipids

Sterols

Pigments

Vitamins

Antioxidants

Oxidation Products

Trace Metals

Hydrocarbons

Triglyceride Structure

HO -C

O

- R1

3 fatty acids

+

C

O

- R3HO -

HO - C

O

- R2

OH

OH

OHglycerol

HC

H2C

H2C

O

R

–One chiral carbon with 1-3 acyl groups–simpler stereochemistry than sugars - C –more possible substituents acyl

O

C

C

OC

OO

O

O

- R1

triacylglycerol

= - R2

- R3

HC

H2C

H2C

+3H2

0

Triglycerides differ from each Triglycerides differ from each other in regard toother in regard to

• Number of Carbon Atoms in fatty acid chains

• Number of double bonds

• Isomerization

• Distribution of FA on glycerol backbone

Analytical MethodsAnalytical Methods

• Saponification Value

• Iodine Value

• Gas Chromatographic Analysis for

Fatty Acids

• Liquid Chromatography

Saponification Value

Saponification - hydrolysis of ester under alkaline condition.

The saponification value of an oil or fat is defined as the number of mg of potassium hydroxide (KOH) required to neutralize the fatty acids resulting from the complete hydrolysis of 1 g of the sample.

Saponification Value Determination

Saponification # --mgs of KOH required to saponify 1 g of fat.

1. 5 g in 250 ml Erlenmeyer.

2. 50 ml KOH (0.5 N) in Erlenmeyer.

3. Boil for saponification.

4. Titrate with HCl (0.5 N) using phenolphthalein.

5. Conduct blank determination.

B - ml of HCl required by Blank.

S - ml of HCl required by Sample.

N- Factor of 0.5 N HCL.

SP# = 56.1(B -S) x N of HCl

Gram of Sample

Saponification ValueO

C

C

OC

OO

O

O

- R

triacylglycerol

- R

- R

HC

H2C

H2C

+ 3

K+OH -

OH

OH

OHglycerol

HC

H2C

H2CPotassium salt

+ C

O

- RKO -3

Similarly;Similarly;

RCOOH + KOH RCOO-K+ + Glycerol

MG + KOH RCOOK + Glycerol

DG + 2KOH 2RCOOK + Glycerol


1 mol TG 3 mol KOH required

1 g TG X mol KOH required

MWKOH: 56 g = 56000 mg

1 g TG : 1 g / MWTG (g/mol) mol

1 mol TG 3x 56000 mg KOH required

1 g TG / MWTG X mg KOH required

TGMW

168000SNX


• What is the MWTG ?

TGMW

168000SVX

O

C

C

OC

OO

O

O

- R

- R

- R

HC

H2C

H2C

O

C

C

OC

OO

O

O

- R1

- R1

- R2

HC

H2C

H2C

O

C

C

OC

OO

O

O

- R1

- R2

- R3

HC

H2C

H2C


• Which one’s MW should be taken?

TGMW

168000SVX

O

C

C

OC

OO

O

O

- R

- R

- R

HC

H2C

H2C

O

C

C

OC

OO

O

O

- R1

- R1

- R2

HC

H2C

H2C

O

C

C

OC

OO

O

O

- R1

- R2

- R3

HC

H2C

H2C


• The Answer is the Weighted Average MW

TGAMW

168000SVX

O

C

C

OC

OO

O

O

- R

- R

- R

HC

H2C

H2C

O

C

C

OC

OO

O

O

- R1

- R1

- R2

HC

H2C

H2C

O

C

C

OC

OO

O

O

- R1

- R2

- R3

HC

H2C

H2C


Calculation of AMWTG Oil consists of only type Simple Triglyceride

O

C

C

OC

OO

O

O

- R

- R

- R

HC

H2C

H2C

1)-(MWFA341 AMW RTG


Calculation of AMWTG Oil consists of Simple and Mixed type Triglyceride

with two fatty acids R1 (%90 w/w) and R2 (%10 w/w)

O

C

C

OC

OO

O

O

- R1

- R1

- R1

HC

H2C

H2C

1]-)MWFAxMWFA(x[341 AMW2211 RRRRTG

O

C

C

OC

OO

O

O

- R2

- R2

- R2

HC

H2C

H2C

O

C

C

OC

OO

O

O

- R1

- R1

- R2

HC

H2C

H2C

O

C

C

OC

OO

O

O

- R1

- R2

- R2

HC

H2C

H2C

Average Moleculer Weight of FAs in Oil (AMWFA)


Generalized Calculation of AMWTG Oil consists of Simple and Mixed type Triglyceride

with N fatty acids

1]-)MWFAx([341 AMWN

1i RRTG ii

FA xi

R1 x1

R2 x2

. .

. .RN xN


1)]MWFAx([341

168000

AMW

168000SV

N

1i RRTGii

Milk Fat 210-233

Coconut Oil 250-264

Cotton Seed Oil 189-198

Soybean Oil 189-195

Fat

SV

Lard 190-202

GC Analysis for Fatty Acids

1. Extract fat.

2. Saponify (hydrolysis under basic condition).

3. Prepare methyl ester (CH3ONa).

4. Chromatography methyl ester.

5. Determine peak areas of fatty acids.

Fatty acids are identified by retention time.

6. Compare with response curve of standard.

Fatty Acids Methyl Esters:

14

18:1

18:2 2018:3

22

21:1 2416 18

Time

Response

GC condition: 10% DEGS Column (from supelco)

Column temperature 200C.

TRIGLYCERIDE ANALYSIS BY LIQUID CHROMATOGRAPHY

Soybean Oil

Solvent CH3CN/HF

Column ODS- Octadesilsilan (C:18)

RESPONSE

RETENTION TIME

Iodine Number• The iodine value of an oil or fat is defined as the mass of iodine

absorbed by 100 g of the sample.

• The unsaturated fatty acid residues of the glycerides react with iodine, and thus the iodine value indicates the degree of unsaturation of the fatty acid residues of the glycerides.

• It is constant for a particular oil or fat, but depends on the method used. Animal fats (butter, dripping, lard) 30 - 70 Iodine Value

• Non-drying oils (olive, almond) 80 - 110 Iodine Value• Semi-drying oils (cottonseed, sesame, soya) 80 - 140 Iodine Value• Drying oils (linseed, sunflower) 120 - 200 Iodine Value

• The iodine value is often most useful in identifying the source of an oil. Generally, the higher iodine values indicate oils and the lower values fats. Iodine values are normally determined using Wigs or Hanus methods.

Determination of Iodine Number

Iodine Value = (ml of Na2S2O3 volume for blank - ml of Na2S2O3 volume for sample) N of Na2S2O3 0.127g/meq 100

Weight of Sample (g) CH CH CH CH

Cl I

ICl

Iodine chloride

+ ICl KI KCl

I2

I2

Na2S2O3 Na2S4O6 NaI

+

+ 2 2+

+

Excess unreacted ICl

Theoretical Iodine Value

• Monoene + I2 Saturated

• Diene + 2*I2 Saturated

• Triene +3* I2 Saturated

Sample: 100 g basis

Assumption: Oil =TG

FA xi

C16:0 5C18:0 15C18:1 15C18:2 40C18:3 1C20:0 3


1 mol C18:1 1 mol I2 (254 g)

15 g C18:1 X (g) I2

1 mol C18:2 2 mol I2 (2x254 g)

40 g C18:2 X (g) I2

Assumption: Oil =TG

FA xi

C16:0 5C18:0 15C18:1 15C18:2 40C18:3 1C20:0 3

282

254151:18

CIV

280

2542402:18

CIV


1 mol C18:3 3 mol I2 (3x254 g)

1 g C18:3 X (g) I2

276

25431IV 3:C18

Theoritical IV= IV C18:1 + IV C18:2+ IV C18:2

Real IV= 0.95xTheoricital Value

analyticalmethods2-hafta.ppt

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