Testing methods Manual

OILS & FATS

Table of Contents

	Table of Content of Oils and Fats
S.No.	Parameters	Reference No.	Page No.
1	Appearance	Food/Staple/Oil & Fats/TM-01/Vr.-02	5
2	Odor/Taste	Food/Staple/Oil & Fats/TM-02/Vr.-02	6
3	Colour (Tinto meter)	Food/Staple/Oil & Fats/TM-03/Vr.-02	8
3.1	Color (Physically)	Food/Staple/Oil & Fats/TM-03.1/Vr.-02	9
4	Sediments	Food/Staple/Oil & Fats/TM-04/Vr.-02	10
5	Cold Test	Food/Staple/Oil & Fats/TM-05/Vr.-02	11
6	MIV/Moisture	Food/Staple/Oil & Fats/TM-06/Vr.-02	13
7	FFA	Food/Staple/Oil & Fats/TM-07/Vr.-02	15
8	Butyro refractometer reading at 40°C	Food/Staple/Oil & Fats/TM-08/Vr.-02	17
9	Refractive index at 40°C	Food/Staple/Oil & Fats/TM-09/Vr.-02	20
10	Saponification Value	Food/Staple/Oil & Fats/TM-010/Vr.-02	22
11	Unsaponifiable Matter	Food/Staple/Oil & Fats/TM-11/Vr.-02	24
12	Peroxide Value	Food/Staple/Oil & Fats/TM-12/Vr.-02	26
13	Acid Value	Food/Staple/Oil & Fats/TM-13/Vr.-02	28
14	Iodine value	Food/Staple/Oil & Fats/TM-14/Vr.-02	30
15	TBHQ	Food/Staple/Oil & Fats/TM-15/Vr.-02	33
16	Hexane	Food/Staple/Oil & Fats/TM-16/Vr.-02	35
17	Flash Point deg C (min.)	Food/Staple/Oil & Fats/TM-17/Vr.-02	40
18	Phosphorous content as Phosphatides	Food/Staple/Oil & Fats/TM-18/Vr.-02	41
19	Insoluble Bromide Test	Food/Staple/Oil & Fats/TM-19/Vr.-02	44
20	Milk Fat	Food/Staple/Oil & Fats/TM-20/Vr.-02	46
21	Melting point	Food/Staple/Oil & Fats/TM-21/Vr.-02	48
22	Allyl isothiocynate	Food/Staple/Oil & Fats/TM-22/Vr.-02	50
23	Argemone Oil Test	Food/Staple/Oil & Fats/TM-23/Vr.-02	53
24	Mineral Oil Test	Food/Staple/Oil & Fats/TM-24/Vr.-02	55
25	Castor Oil Test	Food/Staple/Oil & Fats/TM-25/Vr.-02	56
26	Cottonseed Oil Test (Halphen’s test)	Food/Staple/Oil & Fats/TM-26/Vr.-02	58
27	Rancidity	Food/Staple/Oil & Fats/TM-27/Vr.-02	59
28	Hydrocyanic acid	Food/Staple/Oil & Fats/TM-28/Vr.-02	61
29	Separated water	Food/Staple/Oil & Fats/TM-29/Vr.-02	63
30	Suspended or foreign matter	Food/Staple/Oil & Fats/TM-30/Vr.-02	64
31	Hexabromide (Linseed oil)	Food/Staple/Oil & Fats/TM-31/Vr.-02	65
32	Karanja oil	Food/Staple/Oil & Fats/TM-32/Vr.-02	67
33	Tricresyl phosphate	Food/Staple/Oil & Fats/TM-33/Vr.-02	69
34	Baudouin test	Food/Staple/Oil & Fats/TM-34/Vr.-02	71
35	Polybromide	Food/Staple/Oil & Fats/TM-35/Vr.-02	72
36	Added Colouring matter	Food/Staple/Oil & Fats/TM-36/Vr.-02	73
37	Bellier test (acetic acid method)	Food/Staple/Oil & Fats/TM-37/Vr.-02	77
38	Added flavoring substances	Food/Staple/Oil & Fats/TM-38/Vr.-02	79
39	Phytosterol Acetate Test	Food/Staple/Oil & Fats/TM-39/Vr.-02	81
40	Animal Fat Test	Food/Staple/Oil & Fats/TM-40/Vr.-02	82
41	Coat tar dye	Food/Staple/Oil & Fats/TM-41/Vr.-02	83
42	Mashed Potato, sweet potatoes and other starches	Food/Staple/Oil & Fats/TM-42/Vr.-02	84
43	Fatty Acid profile	Food/Staple/Oil & Fats/TM-43/Vr.-02	85

Physical Parameters

TM-01 Evaluation of Appearance

Title:

Evaluation of Appearance.

Principle:

Appearance includes the clarity, color, physical state of samples and compared with reference samples.

Reagents:

· No

Apparatus

4.1. Wide mouth bottle (Transparent)

Procedure

5.1.Melt the sample if solid and pour the sample in wide mouth transparent bottle.

5.2.Place the sample in proper light condition.

5.3.Observed the Clarity- record as clear, very slightly hazy, slightly hazy, hazy or cloudy.

5.4.Observe the color- Record as water white (colorless), off white, light straw, light yellow, yellow, light amber, amber, and dark amber, light brown, brown and dark brown.

5.5.Observe the physical state- record as liquid, semi-solid, soft solid or solid.

5.6.If in the solid state, record color as white for water white for water white cream colored or off- white for light straw.

5.7.All the above parameters should be compared with the standard samples.

Calculation-

6.1. No

7. Reference/Protocol

7.1. ASTM F 1037

7.2. Fem Analytika Lab Manual

TM-02 Evaluation of Odor /Taste

1. Title

Sensory Evaluation Odor/Taste.

2. Principle

The test involves organoleptic evaluation, usually at constant temperature in a well ventilated, odour-free room.

3. Reagents

No reagents

4. Apparatus

4.1.Wide mouth transparent closed bottle.

4.2.Spoons

5. Procedure

5.1. Prepare homogeneous and representative sample. All control or reference samples are to be prepared in the same way as test samples.

5.2. Fill the sample in a closed small container.

5.3. Left for two minute then open the lid of container in a place there is no aroma of any other material.

5.4. In heal the aroma and compare with standard sample.

5.5. Make the aroma with standard aroma sample.

5.6. Place a small amount of control or reference sample into the mouth by using a spoon.

5.7. Hold the sample in the mouth for a few seconds and slowly draw air through the mouth to aerate the sample and carry the aroma to the olfactory cell region.

5.8. Expectorate the sample, and draw more air through the mouth. Particular attention should be given to any aftertaste noted.

5.9. Rinse the mouth thoroughly with warm water before tasting another sample.

5.10. Repeat steps 5.2, 5.3, and 5.4 with the test sample.

5.11. Evaluate for any flavor differences noted between the test sample and the reference control for the stated attributes in the specification.

5.12. Evaluate reference control for the stated attributes in the specification.

5.13. Evaluate the flavor noted while sample was held in the month and also any aftertaste noted.

5.14. If no differences or off-flavors are noted, report as "normal" or "matches standard" or ‘Characteristics’.

5.15. Any off-flavor is to be characterized. Be as specific as possible.

5.12. Off-flavors noted should be confirmed by other tasters.

6. Calculation-

6.1. No calculation

7. Reference/Protocol

7.1. Fem Analytika Lab Manual

TM-03 Determination of Color (Tinto meter).

1. Title

Determination of colour.

2. Principle

The method determination the colour of oils or fat by comparison with Lovibond glasse of known colour characteristics .The colour is expressed as the sun total of the Yellow and red slides Used to match the colour of oil in a cell of the specified size in the Lovibond Tintometer.

3. Reagents

No reagents

4. Apparatus

4.1. Lovibond Tintometer.

Glass Cells- Presently, the Lovibond call designations generally employed in the laboratories in the country, mainly ¼ .1/2, 1 & 5 ¼, inch cell in are recommended.

5. Procedure

5.1. The sample, if is not already liquid and filter through a filter paper to Remove impurities and the last Traces of moisture .Make sure that the sample is absolutely clear and free from turbidity.

5.2. Clean the glass cell of the desired size with carbon tetrachloride and allow it to dry. Fill it with the clear filtered sample and place cell in position in the tintometer.

5.3. Place along side of it such red, yellow, blue lovibond glass sides or any combinations of these as are necessary to the colour shade of the oil, observing the colour of the oil.

6. Calculation –

Report the color of the oil in terms of Lovibond units as follows.

Y + 5 R or Y +10 R

Where - Y= The sum of the Various Yellow slides used

R- The sum of the various Red slides used.

7. Reference/Protocol

7.1.BIS-IS 548( PART-1)-1964

TM-3.1 Determination of Color.

Title:

Determination of Color in food grade samples by Visual Inspection.

Principle:

Color is a critical parameter for food products. By attractive color the food products is

labile to eat and it’s determined by sensory evaluation.

Reagents:

· No

Apparatus

· Wide mouth bottle (Transparent)

Procedure

5.1.Pour the sample in transparent wide mouth bottle.

5.2.Observe the color- Record as water white (colorless), off white, light straw, light yellow, yellow, light amber, amber, and dark amber, light brown, brown and dark brown.

5.3.If in the solid state, record color as white for water white for water white cream colored or off- white for light straw.

5.4. All the above parameters should be compared with the standard samples.

Calculation-

6.1.No

Reference/Protocol

7.1.ASTM F 1037

7.2.Fem Analytika Manual

TM-04 Determination of Sediments.

1. Title

Determination of sediments.

Principle

The large particles present in liquid medium are settle down at the bottom of container due to heaver in their weight.

Reagents

3.1.No

Apparatus

4.1.taste tube

Procedure

5.1. Properly homogenized the sample by mixing.

5.2. Pour the sample in to 50 ml transparent test tube.

5.3. Place the sample in proper light condition.

5.4. And left for 5 minute.

5.5. Observe the bottom of the test tube and report if any sediment particle observed.

6. Calculation-

No.

7. Reference

7.1. Fem Analytika Lab Manual.

TM-05 Determination of Cold Test

1. Title

Determination of Cold test in oils.

2. Principle

Generally some sample on room temperature give clear solution but we the temperature will down then they have crystals and non clear.

3. Reagents

4. Apparatus

4.1. Volumetric flask.

4.2. Measuring cylinder.

4.3. Water bath.

5. Procedure

5.1. Accurately take 500ml of sample.

5.2. Heated to 130°C to remove moisture and crystals that may be present, and then filtered in 500ml capacity bottles.

5.3. Place in a 25 °C water bath, followed by an ice-water bath for 5.5 h.

5.4. Oils that remained visually clear after 5.5 h passed the cold test.

6- Calculation-

7. Reference/Protocol:

7.1. Cold-test methods. The AOCS Official Method Cc 11-53,

Chemical Parameters

TM-06 Determination of Moisture/MIV

1. Title

Determination of Moisture.

2. Principle

Moisture is defined as the total volatile components which are lost at 105°c within 1 hour.

3. Reagents

3.1.No Reagents

Apparatus

4.1.Moisture dish.

4.2.Porcelain, silica, glass or Aluminium. (7.5 x 2.5 cm.),

4.3.Oven: electric maintained at 105 ±1 ⁰C,

4.4.Dedicator

Procedure

5.1. Weigh accurately about 5 gm of the sample in the moisture dish previously dried in an oven and weighed.

5.2. Place the dish in the oven maintained at 105±1°C for four hours.

5.3. Cool in the desiccator and weigh.

5.4. Repeat the process of drying, cooling, and weighing at 30 minutes interval until the difference in two consecutive weighings is less than 1 mg.

5.6. Record the lowest mass.

6. Calculation-

Moisture percent by weight = 100 X (M1-M2)

M1 - M

Where,

M1 = mass, in gm, of the dish with material before drying.

M2 = mass, in gm, of dish with material after drying to constant mass; and

M = mass in gm of the empty dish.

7. Reference/Protocol

71. FSSAI Methods Manual – Oils and Fats.

TM-07 Determination of FFA/Acid Value

TITLE

Determination of Acid value or Free Fatty Acid

PRINCIPLE

The number of milligrams of potassium hydroxide required to neutralize the free fatty acid present in one gram of the oil or fat under the prescribed condition.

REAGENTS

3.1. Ethyl Alcohol – 95 % (by volume) or Rectified spirit (Confirming to IS: 323-1959) specification for rectified spirit (Revised) neural to phenolphthalein indicator.

3.2. Phenolphthalein indicator solution- dissolve 1 gram phenolphthalein indicator in 100 ml of ethyl alcohol.

3.3. Std. aqueous Potassium Hydroxide or Sodium hydroxide – 0.1 N or 0.5 N

Apparatus

4.1.Hot plate

4.2.Glass ware

PROCERDURE

5.1. Mix the oil or melted fat thoroughly before weighing weigh accurately a suitable quantity of the cooled oil or fat in a conical flask.

5.2. The weight of the oil or fat taken for the test the strength of the alkali used for the titration shall that the volume of alkali required for the titration does not exceed 10 ml. Add 50- 100 ml of freshly neutralized hot ethyl alcohol and one ml of phenolphthalein indicator solution. Boil the mixture for about five minutes and titrate while as hot as possible with standard aqueous alkali solution, shaking vigorously during titration.

CALCULATION

Acid Value - 56.1 X N XV

i. W

Where- V= Volume of standard alkali solution

N= Normality of Std. solution
W= weigh of sample.

Free Fatty Acid-

The acidity is frequently as the percentage of free fatty acid present in the sample .The percentage of free fatty acids in most of the oils and fats is calculated on the basis of oleic acid.

The free fatty acids in term of different fatty acids are as below.

Free fatty Acid in term of oleic acid %- 28.2XVXN

Free fatty acid in terms of lauric acid %- 20.0xVxN

Free fatty acid, in terms of Ricinoleic acid % 29.8 xVxN

Free fatty acid, in term of palmatic acid % 25.6 xVxN

Reference:

7.1.BIS- IS: 548 ( Part-1)1964

TM-08 Butyro refractometer reading at 40°C

1. Title

Determination of Butyrorefractometer reading.

2. Principle

Measurement of the refractive index of the sample is done by means of a suitable refractometer.

3. Reagents

No reagents

4. Apparatus

4.1. Refractometer – Butyro Refractometer

5. Procedure

5.1. Place 1-2 drops of sample on the lower prism. Close prisms and adjust mirror until it gives sharpest reading.

5.2. If reading is indistinct after running constant temperature water through instrument for sometime, test sample is unevenly distributed on prism surfaces. As refractive index is greatly affected by temperature, use care to keep temperature constant.

5.3. The temperature of the refractometer should be controlled to within + 0.1 ºC and for this purpose it should be provided with a thermostatically controlled water bath and a motor driven pump to circulate water through the instrument.

5.4. When butyro refractometer is used its reading can be converted to refractive index with the help of the table

5.5. Calibration of the Instrument:

5.5.1. The instrument is calibrated with a glass prism of known refractive index (an optical contact with the prism being made by a drop of a bromonapthalene) or by using distilled water which has refractive index of 1.3330 at 20.0 ºC and 1.3306 at 40.0 ºC, the usual temperature of taking readings.

5.5.2. Light Source:

If the refractometer is equipped with a compensator, a tungsten lamp or day light may be used. Otherwise a monochromatic light such as sodium vapour lamp (589.3 nm) may be used.

5.6. Melt the sample if it is not already liquid and filter through a filter paper to remove impurities and traces of moisture. Make sure sample is completly dry. Circulate stream of water through the instrument. Adjust the temperature of the refractometer to the desired temperature. Ensure that the prisms are clean and dry. Place a few drops of the sample on the prism. Close the prisms and allow standing for 1-2 min. Adjust the instrument and lighting to obtain the most distinct reading possible and determining the refractive index or butyro-refractometer number as the case may be.

5.7. Temperature correction: - Determine refractive index at the specified temperature. If temperature correction is necessary use following formula

R = R 1 + K 1 (T1 – T)

Where

R = Reading of the refractomter reduced to the specified temperature T 0 C

R1 = Reading at T 1 C

K = constant 0.000365 for fats and 0.000385 for oils (If Abbe Refractometer is used) or

= 0.55 for fats and 0.58 for oils (if butyro refractometer is used)

T1 = temperature at which the reading R1 is taken and

T = specified temperature (generally 40 ⁰C.)

Significance

Refractive index of oils increases with the increase in unsaturation and also chain length of fatty acids.

6. Calculation

7. Reference/Protocol

7.1. A.O.A.C 17th edn, 2000, Official method 921.08 – Index of refraction of oils and fats / I.S.I Handbook of Food analysis (Part XIII) – 1984, page 70)

7.2. Table for conversion of B.R. readings to Refractive Index

Annexure

TM- 09 Determination of Refractive index at 40°C

Title

Determination of Refractive Index at 40°C.

Principle

Measurement of the refractive index of the sample is done by means of a suitable refractometer.

Reagents

No reagents

Apparatus

4.1. Refractometer — Abbe or Butyro refractometer. The temperature of the refractometer should be controlled to within ±0.1°C and for this purpose it should be provided with a thermostatically controlled water-bath and a motor-driven pump to circulate water through the instrument. The instrument should be standardized, following the

4.2. Manufacturer’s instructions, with a liquid of known purity and refractive index or with a glass prism of known refractive index. Distilled water, which has a refractive index of 1.333 0 at 20.0°C, is a satisfactory liquid for standardization.

4.3. Light Source — If the refractometer is equipped with a compensator, a tungsten lamp or a daylight bulb may be used. Otherwise, a monochromatic light, such as an electric sodium vapor lamp, should be used

5. Procedure

5.1. Melt the sample, if it is not already liquid, and filter through a filter paper to remove any impurities and the last traces of moisture. Make sure that the sample is completely dry. Adjust the temperature of the refractometer to 40.0 ± 0.1°C or to any other desired temperature. Ensure that the prisms are clean and completely dry, and then place a few drops of the sample on the lower prism. Close the prisms, tighten firmly with the screw-head, and allow standing for one or two minutes. Adjust the instrument and light to obtain the most distinct reading possible, and determine the refractive index.

5.2. Temperature Corrections: Unless the correction factors are specified in the detailed specification, approximate corrections shall be made using the following equation:

R = R' + K ( T' – T )

where

R = the reading of the refracto meter reduced to the specified

temperature, T°C;

R' = the reading at T'°C;

K = constant, 0.000 365 for fats, and 0.000 385 for oils ( if

Abbe refractometer is used ), or

= 0.55 for fats and 0.58 for oils ( if Butyro refractometer is used ); and

T' = the temperature at which the reading R' is taken;

T = the specified temperature (generally 40.0°C).

SUPPLIED BY BOOK SUPPLY BUREAU TO CSIR/DST CONSORTIUM

FOR INTERNAL USE BY SUBSCRIBING MEMBER ONLY.

6. Calculation-
No

7. Reference/Protocol

7.1 A.O.A.C 17th edn, 2000, Official method 921.08 – Index of refraction of oils and fats / I.S.I Handbook of Food analysis (Part XIII) – 1984, page 70) Table for conversion of B.R. readings to Refractive Index

7.2. IS : 548 (Part I) - 1964

TM- 10 Determination of Saponification Value

1. Title

Determination of Saponification Value of edible oil.

2. Principle

The oil sample is saponified by refluxing with a known excess of alcoholic potassium hydroxide solution. The alkali required for saponification is determined by titration of the excess potassium hydroxide with standard hydrochloric acid.

3. Reagents

3.1. Alcoholic potassium hydroxide solution - Reflux 1.2 litre alcohol 30 minutes with 10 gm KOH and 6 gm granulated Aluminium or Al foil. Distill and collect 1 litre after discarding first 50 ml.

3.2. Dissolve 40 g of potassium hydroxide in this 1 litre alcohol keeping temperature below 15 0 C while dissolving alkali. Allow to stand overnight, decant the clear liquid and keep in a bottle closed tightly with a cork or rubber stopper.

3.3. Phenolphthalein indicator solution - Dissolve 1.0 g of phenolphthalein in 100 ml rectified spirit.

3.4. Standard hydrochloric acid: approximately 0.5N

4. Apparatus

4.1. 250 ml capacity conical flask with ground glass joints.

4.2. 1 m long air condenser, or reflux condenser (65 cm minimum in length) to fit the flask (a).

4.3. Hot water bath or electric hot plate fitted with thermostat.

5. Procedure

5.1. Melt the sample if it is not already liquid and filter through a filter paper to remove any impurities and the last traces of moisture.

5.2. Make sure that the sample is completely dry. Mix the sample thoroughly and weigh about 1.5 to 2.0 g of dry sample into a 250 ml Erlenmeyer flask. Pipette 25 ml of the alcoholic potassium hydroxide solution into the flask.

5.3. Conduct a blank determination along with the sample. Connect the sample flasks and the blank flask with air condensers, keep on the water bath, boil gently but steadily until saponification is complete, as indicated by absence of any oily matter and appearance of clear solution. Clarity may be achieved within one hour of boiling.

5.4. After the flask and condenser have cooled somewhat wash down the inside of the condenser with about 10 ml of hot ethyl alcohol neutral to phenolphthalein. Titrate the excess potassium hydroxide with 0.5N hydrochloric acid, using about 1.0 ml phenolphthalein indicator.

6. Calculation

Saponification Value = 56.1 (B-S) N

Where,

B = Volume in ml of standard hydrochloric acid required for the blank.

S = Volume in ml of standard hydrochloric acid required for the sample

N = Normality of the standard hydrochloric acid and

W = Weight in gm of the oil/fat taken for the test.

Note:- When titrating oils and fats which give dark coloured soap solution the observation of the end point of titration may be facilitated either (a) by using thymolpthalein or alkali blue 6B in place of phenolphthalein or (b) by shaking 1 ml of 0.1 % (w/v) solution of methylene blue in water to each 100 ml of phenolphthalein indicator solution before the titration.

7. Reference/Protocol

7.1.A.O.A.C 17th edn, 2000, Official method 920.160 Saponification number of oils and fats / IUPAC 2 . 202 / I.S.I Handbook of Food Analysis (Part XIII) 1984, page 78

7.2.IS : 548 (Part I) - 1964

TM-11 Determination of Unsaponifiable Matter

1. Title

Determination of Unsaponifiable Matter

2. Principle

The unsaponifiable matter is defined as the substances soluble in oil which after saponification are insoluble in water but soluble in the solvent used for the determination. It includes lipids of natural origin such as sterols, higher aliphatic alcohols, pigments, vitamins and hydrocarbons as well as any foreign organic matter non volatile at 100 ⁰C e.g. ( mineral oil) which may be present. Light Petroleum or diethyl ether is used as a solvent but in most cases results will differ according to the solvent selected and generally the use of diethyl ether will give a higher result.( Ref:- F.A.O Manual of Food quality control 14/8, page 261)

3. Reagents

3.1. Alcoholic potassium hydroxide solution: Dissolve 7 to 8 g of potassium hydroxide in an equal quantity of distilled water and add sufficient aldehyde free ethyl alcohol and make up to 100 ml.

3.2. Ethyl alcohol: Ninety-five per cent

3.3. Phenolphthalein indicator solution: Dissolve one gram of phenolphthalein in 100 ml of ethyl alcohol.

3.4. Petroleum ether (40-60 ^oC): Analytical reagent grade

3.5. Aqueous alcohol: 10 percent of ethyl alcohol in water

3.6. Standard sodium hydroxide solution: Approximately 0.02N

3.7. Acetone: Analytical reagent grade

3.8. Anhydrous sodium sulphate.

4. Apparatus

4.1. Flat bottom flask or conical flask with a ground glass joint, 250 ml capacity

4.2. Air condenser 1 metre long to fit the flask

4.3. Separating funnel, 500 ml capacity

5. Procedure

5.1. Weigh accurately 5 gm of well mixed oil / fat sample into a 250ml conical flask. Add 50ml of alcoholic potassium hydroxide solution. Boil the content under reflux air condenser for one hour or until the saponification is complete (complete saponification gives a homogeneous and transparent medium).

5.2. Take care to avoid loss of ethyl alcohol during the saponification. Wash the condenser with about 10 ml of ethyl alcohol.

5.3. Transfer the saponified mixture while still warm to a separating funnel, wash the saponification flask first with some ethyl alcohol and then with cold water, using a total of 50 ml of water to rinse the flask. Cool to 20 to 25ºC. Add to the flask 50 ml of petroleum ether, shake vigorously, and allow the layers to separate.

5.4. Transfer the lower soap layer into another separating funnel and repeat the ether extraction for another 3 times using 50 ml portions of petroleum ether. Some oils high in unsaponifiable matter, e.g., marine oils, may require more than three extractions to completely remove unsaponifiable matter.

5.5. Wash the combined ether extract three times with 25 ml portions of aqueous alcohol followed by washing with 25 ml portions of distilled water to ensure ether extract is free of alkali (washing are no longer alkaline to phenolphthalein). Transfer ether solutions to 250 ml beaker, rinse separator with ether, add rinsings to main solution. Evaporate to about 5ml and transfer quantitatively using several portions of ether to 50ml Erlenmeyer flask previously dried and weighed.

5.6. Evaporate ether. When all ether has been removed add 2-3 ml acetone and while heating on steam or water bath completely remove solvent under a gentle air. To remove last traces of ether, dry at 100 0 C for 30 minutes till constant weight is obtained Dissolve residue in 50 ml of warm ethanol which has been neutralized to a phenolphthalein end point. Titrate with 0.02N NaOH.

6. Calculation-

Weight in g of the free fatty acids in the extract as oleic acid = 0.282 VN

Where,

V = Volume in ml of standard sodium hydroxide solution

N = Normality of standard sodium hydroxide solution

Unsaponifiable matter = 100 (A-B)

Where,

A = Weight in g of the residue

B = Weight in g of the free fatty acids in the extract

W = Weight in g of the sample

7. Reference/Protocol

7.1. I. S. I. Handbook of Food Analysis (Part XIII)-1984, page 67 / A.O.A.C 17th edn, 2000, Official method 933.08, Residue (unsaponifiable) of oils and fats.

TM- 12 Determination of Peroxide Value.

1. Title

Determination of Peroxide Value

2. Principle

The peroxide value is a measure of the peroxides contained in a sample of fat, expressed as milli-equivalents per 1000 gm s of the material.

3. Reagents

3.1. Acetic acid - chloroform solvent mixture (3: 2). Mix 3 volumes of glacial acetic acid with 2 volumes of chloroform.

3.2. Freshly prepared saturated potassium iodide solution.

3.3. 0.I N and 0.0I N sodium thiosulphate solutions. Weigh 25 g of sodium thiosulphate and dissolve in 1 L of distilled water. Boil and cool, filter if necessary, Standardize against standard potassium dichromate solution.

3.4. Starch solution - 1% water-soluble starch solution

4. Apparatus

4.1. Pipette ,-Graduated ,1 ml capacity .

4.2. Conical flask –Glass –stopperd ,250 mal capacity

5. Procedure

5.1. Weigh 5 g (+ 50 mg) sample into a 250 ml stoppered conical flask. Add 30 ml acetic acid chloroform solvent mixture and swirl to dissolve.

5.2. Add 0.5 ml saturated potassium iodide solution with a Mohr pipette let stand for 1min in dark with occasional shaking, and then add about 30 ml of water.

5.3. Slowly titrate the liberated iodine with 0.1 N sodium thiosulphate solutions, with vigorous shaking until yellow colour is almost gone. using Add about 0.5 ml starch solution as indicator and continue titration shaking vigorously to release all I 2 from CHCl3 layer until blue colour disappears.

5.4. If less than 0.5 ml of 0.1 N Na2S2O3 is used repeat using 0.01 N Na2S2O 3.Conduct blank determination ( must be less than 0.1 ml 0.1 N Na2S2O3)

6. Calculation-
Peroxide value expressed as milli equivalent of peroxide oxygen per kg sample (meq/kg)

Peroxide value = (S-B ) XNx1000

Wt of the sample

Where Titre = ml of Sodium Thiosulphate used (blank corrected)

N = Normality of sodium thiosulphate solution.

Fresh oils usually have peroxide values well below 10 meq/kg, A rancid taste often begins to be noticeable when the peroxide value is above 20 meq/kg (between 20 – 40 meq / Kg). In interpreting such figures, however, it is necessary to take into account the particular oil or fat.

7. Reference/Protocol

7.1. A.O.A.C. 17th edn, 2000,Official Method 965.33 Peroxide Value in Oils and Fats / Pearsons Composition and Analysis of Foods 9th edn page 641

7.2. IS-548(part-1)

TM- 13 Determination of Acid Value

1. TITLE

Determination of Acid value or Free Fatty Acid

2. PRINCIPLE

The number of milligrams of potassium hydroxide required to neutralize the free fatty acid present in one gram of the oil or fat under the prescribed condition.

3. REAGENTS

3.1. Ehtyl Alcohol – 95 % ( by volume ) or Rectified spirit ( Confirming to IS: 323-1959 specification for rectified spirit ( Revised) neural to phenolphthalein indicator.

3.2. Phenolphthalein indicator solution- dissolve 1 gram phenolphthalein indicator in 100 ml of ethyl alcohol .

3.3. Std. aqueous Potassium Hydroxide or Sodium hydroxide – 0.1 N or 0.5 N

4. APPARATUS

4.1.Hot plate

4.2.Glass ware

5. PROCERDURE

5.1. Mix the oil or melted fat thoroughly before weighing weigh accurately a suitable quantity of the cooled oil or fat in a conical flask.

5.2. The weight of the oil or fat taken for the test the strength of the alkali used for the titration shall that the volume of alkali required for the titration does not exceed 10 ml.

5.3. Add 50- 100 ml of freshly neutralized hot ethyl alcohol and one ml of phenolphthalein indicator solution. Boil the mixture for about five minutes and titrate while as hot as possible with standard aqueous alkali solution, shaking vigorously during titration.

6. CALCULATION

Acid Value - 56.1 X N XV

Where- V= Volume of standard alkali solution

N= Normality of Std. solution

W= weigh of sample.

Free Fatty Acid-

The acidity is frequently as the percentage of free fatty acid present in the sample .The percentage of free fatty acids in most of the oils and fats is calculated on the basis of oleic acid .

The free fatty acid in term of different fatty acids are as below.

A- Free fatty Acid in term of oleic acid %- 28.2XVXN

B- Free fatty acid in terms of lauric acid %- 20.0xVxN

C- Free fatty acid ,in terms of Ricinoleic acid % 29.8 xVxN

D- Free fatty acid ,in term of palmatic acid % 25.6 xVxN

7. REFERENCE

7.1.BIS- IS: 548 ( Part-1)1964

TM-14 Determination of Iodine Value

1. Title

Determination of Iodine Value of edible oil .

2. Principle

The oil/fat sample taken in carbon-tetrachloride is treated with a known excess of iodine monochloride solution in glacial acetic (Wijs solution). The excess of iodine monochloride is treated with potassium iodide and the liberated iodine estimated by titration with sodium thiosulfate solution.

3. Reagents

3.1.Potassium dichromate AR

3.2.Concentrated hydrochloric acid AR

3.3.Glacial acetic acid, free from ethanol

3.4.Carbon tetrachloride, analytical reagent grade

3.5.Iodine mono-chloride (ICl)

3.6.Potassium iodide (free from potassium iodate) - 10% solution prepared fresh

3.7. Starch solution - Mix 5 g of starch and 0.01 g of the mercuric iodide with 30 ml of cold water and slowly pour it with stirring into one litre of boiling water. Boil for three minutes. Allow to cool and decant off the supernatant clear liquid.

3.8. Wij’s Iodine monochloride solution - Dissolve 10 ml of iodine monochloride in about 1800 ml of glacial acetic acid and shake vigorously. Pipette 5 ml of Wij's solution, add 10 ml of potassium iodide solution and titrate with 0.1N standard sodium thiosulphate solution using starch as indicator. Adjust the volume of the solution till it is approximately 0.2 N or prepare Wij’s iodine solution by dissolving 13 gm resublimed Iodine in 1 litre acetic acid and pass in dried chlorine (dried through H2SO4.) until original Sod thiosulphate titre of the solution is not quite doubled. (Characteristic color change at the end point indicates proper amount of Chlorine. Convenient method is to reserve some amount of original I solution, add slight excess of Cl to bulk of solution and bring to desired titre by re additions of reserved portion). Store in an amber bottle sealed with paraffin until ready for use. Wij’s solutions are sensitive to temp, moisture and light. Store in dark at less than 30⁰ C. Determine I / Cl ratio as follows.

Iodine Content – Pipette 5 ml Wij Solution into 500 ml Erlenmeyer flask containing 150 ml saturated Cl – water and some glass beads. Shake heat to boiling point and boil briskly 10 minutes. Cool, add 30 ml H2SO4 (1 + 49) and 15 ml 15 % KI solution and titrate immediately with 0.1 N Na2S2O3.

Total Halogen content – Pipette 20 ml Wij’s solution into 500 erlenmeyer flask containing 150 ml recently boiled and cooled water and 15 ml 15 % KI solution. Titrate immediately with 0.1 N Na2S2O3. I / Cl = 2 X / (3B – 2 X) where X = ml of 0.1 Na2S2O3 required for I content and B = ml required for total halogen content. I / Cl ratio must be 1.10±0.1

3.9. Standard sodium thiosulphate solution (0.1N) - Dissolve approximately 24.8 g of sodium thiosulphate crystals (Na2S2O3.5H2O) in distilled water and make up to 1000 ml. Standardize this solution by the following procedure:

Weigh accurately about 5.0 g of finely powdered potassium dichromate which has been previously dried at 105ºC + 2ºC for one hour, dissolve it in distilled water and make up to 1 L. For standardization of sodium thiosulphate, pipette 25 ml of this solution into a 250 ml conical flask. Add 5 ml of concentrated hydrochloric acid and 15 ml of a 10 percent potassium iodide solution. Allow to stand in dark for 5 min and titrate the content with sodium thiosulphate solution using starch as indicator at the end. End point is change of blue color to green.

N = 25W

49.03 V

Where,

N = Normality of the sodium thiosulphate

W = Weight in g of the potassium dichromate, and

V = Volume in ml of sodium thiosulphate solution required for titration

4. Apparatus

4.1.500 ml Erlenmeyer flask

5. Procedure

5.1. Melt the sample if it is not already liquid and filter through a filter paper to remove any impurities and the last traces of moisture. Make sure that the sample is completely dry.

5.2. Mix the sample thoroughly and weigh about 1.5 to 2.0 g of dry sample into a 250 ml Erlenmeyer flask. Pipette 25 ml of the alcoholic potassium hydroxide solution into the flask. Conduct a blank determination along with the sample.

5.3. Connect the sample flasks and the blank flask with air condensers, keep on the water bath, boil gently but steadily until saponification is complete, as indicated by absence of any oily matter and appearance of clear solution.

5.4. Clarity may be achieved within one hour of boiling. After the flask and condenser have cooled somewhat wash down the inside of the condenser with about 10 ml of hot ethyl alcohol neutral to phenolphthalein. Titrate the excess potassium hydroxide with 0.5N hydrochloric acid, using about 1.0 ml phenolphthalein indicator.

6. Calculation-

Iodine value =

12.69 (B – S) N

Where,

B = volume in ml of standard sodium thiosulphate solution required for the blank.

S = volume in ml of standard sodium thiosulphate solution required for the sample.

N = normality of the standard sodium thiosulphate solution.

W = weight in g of the sample.

7. Reference/Protocol

7.1.A.O.A.C 17th edn, 2000, Official method 920.160 Saponification number of oils and fats / IUPAC 2 . 202 / I.S.I Handbook of Food Analysis ( Part XIII) 1984, page 78

7.2.IS : 548 (Part I) - 1964

TM-15 Determination of TBHQ

1. Title

Determination of TBHQ.

2. Principle

This Standard is applicable for Tert-butylhydroquinone (TBHQ for short), a kind of food additive using hydroquinone as raw material and produced through alkylation reaction.

3. Reagents

3.1. Acetone

3.2. Hydroquinone standard with known purity

3.3. TBHQ standard: Purity≥99％

3.4. T-butyl-benzoquinone standard: Purity≥99％

3.5. 2,5－di-t-butyl-hydroquinone standard: Purity≥99％

4. Apparatus

4.1. Gas Chromatograph: Equipped with hydrogen flame ionization detector and automatic integrator.

4.2. Reference Chromatographic Conditions

Chromatographic Column: HP-5 elastic quartz capillary column, 30 m long, which inner diameter is 0.32 mm, and coating thickness is 0.25 μm; or other equivalent chromatographic columns.

Flow Rate: Carrier gas refers to high-purity nitrogen, which line speed is 30 cm/s.

Temperature: Column temperature is 220℃, sample inlet temperature is 250℃, and detector temperature is 300℃, Splitting Ratio: 20:1, Sample Size: 1 μL.

4.3. Preparation of Standard Solutions

Take 10mg of hydroquinone, TBHQ, T-butyl-benzoquinone, and 2,5－di-t-butyl-hydroquinone standards respectively.

Dissolve with acetone, and transfer to 10mL measuring flasks respectively. Dilute till the solution meets a certain scale, and shake up.

5. Procedure

5.1. Preparing Sample Solution:

Take 0.2g samples and dissolve with acetone. Transfer to a 10mL measuring flask. Dilute till the solution meets a certain scale, and then shake it up.

5.2. Determination

Conduct meteorological and chromatographic analyses on all standard

Solutions, determine the retention time of all standards, and then feed 1μL sample solution for chromatographic analysis.

6. Calculation

Calculate the contents of TBHQ, T-butyl-benzoquinone, 2,5－di-t-butyl-hydroquinone, and hydroquinone respectively with area normalization method. The test result is subject to arithmetic mean of parallel determination results. Relative deviation of the determination result of TBHQ is no more than 0.2%, and relative deviation of determination results of other substances is no more than 2%.

7. Reference/Protocol

7.1. National food safety standard Food Additive Tertiary Butylhydroquinone (TBHQ) GB26403-2011.

TM-16 Determination of Hexane

1. TITLE

Determination of Hexane

2. PRINCIPLE

The residual hexane content is the quantity of volatile hydrocarbons remaining in the fats and oils following processing involving the use of solvents. The volatile hydrocarbons are desorbed by heating the sample at 80°C in a closed vessel after addition of an internal standard. After determination of a calibration factor, hydrocarbons in the head space are determined by gas chromatography using packed or capillary columns. Results are expressed as hexane in mg / kilogram (mg / kg, or ppm). The method is applicable to the determination of „free‟ volatile hydrocarbons expressed in terms of hexane remaining in animal and vegetable fats and oils after extraction with hydrocarbon based solvents. It is suitable for determination of quantities of hexane between 10 and 1500 mg / kg in fats and oils.

3. REAGENTS

3.1. Gases

3.2. Carrier - Helium (preferred for better resolution) or Nitrogen 99.99 % pure, dried and containing a maximum of 10 mg O2 / kg

3.3. Flame Ionization Detector - Hydrogen, minimum purity 99.95 %, Air or Oxygen , dry, hydrocarbon free ( less than 2 ppm hydrocarbon equivalent to CH4)

3.4. Technical Hexane or light petroleum with a composition similar to that used in industrial extraction or failing these n-hexane. For calibration, technical extraction hexane is preferred.

3.5. n- Heptane (internal standard) analytical reagent grade

3.6. Vegetable Oil -, solvent free, freshly refined and deodorized. The oil is to be used for calibration and should be of a similar nature as the sample. It should be free from extraction solvent (less than 0. 01%)

4. APPARATUS

4.1. Gas Chromatograph having (a) Thermostatic column capable of maintaining the desired column temperature within ±1°C ,

4.2. Sample inlet system, separately thermostated which can be maintained at a minimum temperature of 100°C. If a capillary column is used, the inlet system must be capable of a 1/100 split injection. For serial analysis a headspace gas chromatograph with automatic sample injection and tempering bath is satisfactory.

4.3. Flame ionization detector which can be separately thermostated and maintained at a minimum of 100°C

4.4. Recorder - If a recorder trace is to be used for calculating the composition of the samples analysed, an electronic recorder of high precision is required or Electronic Integrator (preferred) which permits rapid and accurate calculations.

4.5. Chromatographic Column - Either packed or capillary column with the following minimum requirements. Packed Column - stainless steel or glass, approx 2 metres long and 1 / 8 inch internal diameter with acid washed and silanised diatomaceous earth,150- 180 mu particle size ( 80- 100 mesh Chromosorb WAW is suitable), stationery phase - squalene consisting of 10 % of packing. Capillary column - glass or fused silica approx 30 metres long and 0.3 mm internal diameter.

4.6. Stationery phase - Methyl polysiloxane (film thickness 0. 2 mu). (5) Syringe - 1ml , 10 ml , 1000ml capacity, gas tight.

4.7. Septum vial -20 ml capacity

4.8. Septa and Aluminium caps suitable for septum vials together with crimping pliers. The septa must be resistant to oils and solvents (butyl rubber or red rubber is recommended.)

4.9. Tongs suitable for holding septum vials

4.10. Heating bath with clamps for holding septum vials, thermostatically regulated and capable of maintaining a temperature of 80°C. For continuous operation glycerol is recommended as heating liquid (10) Shaking machine.

4.11. GC Operating Conditions

Carrier gas flow depends on the carrier gas and the type of column being used for analysis and should be optimized accordingly. The flow of hydrogen and air or oxygen to the FID should be optimized according to the manufacturer‟s recommendation. Injector and detector temperatures should be set at about 120 degree C. The column should be maintained at 400C.

5. PROCERDURE

51. Determination of the calibration factor - Weigh to the nearest 0.01 gm, 5 gm of solvent free vegetable oil (reagent 4) into each of the 7 septum vials. Seal each vial with a septum and cap. By means of a syringe add technical Hexane to 6 of the seven vials (the vial with no added solvent is the blank) according to the following table:

ml / 5 gm 0.5 1 2 4 7 10

mg / 100gm 67 134 268 536 938 1340

One vial remains without the addition of solvent. If n - hexane is used for calibration the following table applies

ml / 5 gm 0.5 1 2 4 7 10

mg /100 gm 66 132 264 528 924 1320

5.1.Shake the 6 vials containing the solvent in the shaking machine vigorously for 1 hour. Using the syringe add 5 ml of internal standard (reagent 3) to each of the 7 vials. Successively immerse the vials upto the neck in the heating bath at 80°C at intervals of approx 15 minutes. This time interval depends on the duration of the GC analysis which is complete on the elution of the internal standard (n - heptane ). The samples must be placed in the heating at intervals such that each sample is tempered for exactly 60 minutes.

5.2.Warm the gas tight syringe to 60°C. After tempering at 80°C for exactly 60 minutes and without removing the vial from the heating bath, use the gas tight syringe and withdraw through the septum 1000 µl ( 1 ml) of the head space above the oil. Inject immediately into the gas chromatograph. For each of the vial containing added solvent a calibration factor F may be determined by the formula. F=CSxA1 (A H - A B - A 1) x C 1

Where,

A H = Total peak area of solvent hydrocarbons including the area of internal standard present in the spiked oil. For identification purposes a typical chromatogram of solvent composition should be obtained. Hydrocarbons which usually make up the technical hexane are 2 Methyl pentane, 3 Methyl pentane. Methyl cyclo pentane, cyclohexane etc. Do not include peaks due to oxidation products which may be present in significant amounts.

AB = Peak area of the solvent hydrocarbons present in the oil to which solvent has not been added (blank) less the peak area of the internal standard.

A 1 = Peak area corresponding to the internal standard in the spiked samples

C 1 = Quantity of the internal standard added expressed in mg / kg of the oil

CS= Quantity of technical hexane added to the oil present in the vial expressed in mg / kg of the oil. Express the results to the third decimal place.

Calibration factors of the six standards should be approx the same.

The mean calibration factor should be 0.45 if n - heptane is used and 0.57 if cyclohexane is used.

The factor (F) so evaluated can be used for determining vial quantities of hexane less than 60 mg / kg. If the value of F found for the vial containing 0.5 mL of hexane is significantly below the mean value, this deviation is probably due to difficulty in introducing exactly 0.5 mL and this determination must be either eliminated or repeated. For quantities of hexane between 10 and 20 mg / kg it is better to prepare calibration standards by adding 2 ml of internal standard instead of 0.5 ml.

5.3.Sample Analysis

5.3.1. Weigh to the nearest 0.01 gm, 5 gm of the test sample into a septum vial as quickly as possible and close immediately with a septum and cap. Using a syringe add through the septum exactly 5 ml of the internal standard. Shake vigorously by hand for about 1 minute and then immerse the vial upto the neck in the heating bath. At 80 degree C for exactly 60 minutes. Warm the gas tight syringe to 60°C. After tempering at 80°C for exactly 60 minutes use the gas tight syringe and take from the vial without removing it from the bath 1000 ml (1 ml) of the head space above the sample. Immediately inject into the gas chromatograph. Carry out two determinations in rapid succession on each sample

6. CALCULATION

The residual solvent expressed in mg / kg (ppm) is given by the formula:

W = (A H - A1) x F x C1 A1

Where,

A H = Total peak area of solvent hydrocarbons including the area of internal

standard. Hydrocarbons which usually make up the technical solvents are 2

methyl pentane, 3 methyl pentane, methyl cyclopentane, cyclohexane etc. Do not include peaks due to the oxidation products. Some of these products may be present in significant amount.

A1 = Peak area corresponding to internal standard in the sample

C1 = Quantity of the internal standard added in mg / kg

Note: - For an addition of 5 mL of heptane / 5 gm of sample C1 = 680 mg /

kg and C1 = 750 mg / kg if cyclohexane is used.

F = Calibration factor obtained in procedure

Report as the final result the mean of the results of two determinations.

7. Reference:

7.1.A.O.C.S (1989) Official Method Ca 3b.- 87)

TM-17 Determination of Flash Point deg C (min.)

1. Title

Determination of Flash point - Pensky Marten (Closed Cup) method

2. Principle

The method determines the temperature at which the sample will flash when a test flame is applied under the conditions specified for the test.

3. Reagents

No reagents

4. Apparatus

4.1. Pensky-Martens closed cup apparatus with thermometer.

5. Procedure

5.1. Thoroughly clean and dry all parts of the cup and its accessories before starting the test, being sure to remove any solvent which had been used to clean the apparatus. Support the tester on a level steady table.

5.2. Fill the cup with the oil to be tested up to the level indicated by the filling mark. Place the lid on the cup and properly engage the heating devices. Insert the thermometer, light the test flame and adjust it to 4.0 mm in diameter. Heat the sample so that the temperature increase is about 5 to 6 ⁰C per min. During the heating, turn the stirring device from one to two revolutions per second.

5.3. Apply the test flame when the temperature of the sample is a whole number not higher than 17 ⁰C below the flash point At every 5 ⁰C rise in temperature, discontinue stirring and apply the test flame by opening the device which controls the shutter and lowers the test flame into the shutter opening.

5.4. Lower the test flame in for 0.5 second and quickly return to the raised position. Do not stir the sample while applying the test flame. As soon as the test flame has been returned to the raised position, resume stirring.

5.5. The flash point is the temperature indicated by the thermometer at the time of the flame application that causes a distinct flash in the interior of the cup.

6. Calculation-
No

7. Reference

7.1. I.S 1448 – 1970 Methods of test for petroleum and its products ( P: 21) Flash Point( Closed) by Pensky Martin apparatus.)

TM-18 Determination of Phosphorous content as Phosphatides

1. Title

Determination of Phosphorus content as Phosphotides.

2. Principle

The method determines Phosphorous or the equivalent phosphatide by ashing in the presence of zinc oxide followed by spectrophotometer measurement of phosphorous as blue phosphomolybdic acid

3. Reagents

3.1. Hydrochloric acid, conc sp. gr 1.18

3.2. Zinc Oxide, reagent grade

3.3. Pot. Hydroxide, reagent grade

3.4. Sulphuric acid, conc , sp .gr 1.84

3.5. Sodium molybdate , reagent grade (6) Hydrazine sulphate , reagent grade

3.6. Potassium dihydrogen phosphate, reagent grade dried for 2 hrs at 101°C

Solutions

3.7. Sodium molybdate Carefully add 140 ml of conc sulphuric acid to 300 ml distilled water. Cool to room temperature and add 12.5 gm of Sodium molybdate. Dilute to 500 ml with distilled water. Mix thoroughly and allow to stand for 24 hrs before use.

3.8. Hydrazine sulphate - 0.015% Dissolve 0.150 gm hydrazine sulphate in 1 litre water.

3.9. Potassium Hydroxide - 50% solution Dissolve 50 gm KOH in 50 ml distilled water

3.10. Standard Phosphate solution

(a)Stock solution - Dissolve 1.0967 gm of dry Potassium dihydrogen

phosphate in distilled water and make upto 250 ml in a volumetric flask.

The solution contains 1 mg phosphorous per ml

(b) Working Solution - Dilute 5 ml of standard stock solution with distilled water to 500 ml in a volumetric flask. This solution contains 0.01 mg phosphorous per ml.

Preparation of standard curve

Pipette 0.0, 1.0, 2.0, 4.0 , 6.0, 8.0 and 10.0 ml of standard working

solution into 50 ml volumetric flasks. Dilute each to 10 ml with distilled water using a measuring pipette. Add hydrazine sulphate and sodium molybdate as above. Plot the absorbance of each standard against its phosphorous content in mg on a linear graph paper.

4. Apparatus

4.1. Platinum basins or crucibles suitable to withstand temperature of 600°C

4.2. Electric hot plate and muffle furnace

4.3. Watch glass 75 mm dia and Funnel short stem 50 mm diameter (4) Filter paper - ashless, Whatman No 42 , 90 mm diameter.

4.4. Wash bottle - 1 litre for use with hot water

4.5. Volumetric flasks - 50ml, 100 ml, 250 ml and 500 ml with glass stoppers.

4.6. Pipette - Mohr type 10 ml with 0.1 ml subdivision.

4.7. Spectrophotometer with 1.0 cm cuvettes. For use in the visible region

5. Procedure

5.1. Weigh accurately 3 - 4 gm of sample in a crucible or Pt basin, add 0.5 gm Zinc oxide and heat slowly on the hot plate until the sample thickens, then gradually increase the heat until the mass is completely charred. Place in a muffle furnace at 550 - 600°C and hold for 2 hrs. Remove and cool to room temperature.

5.2. Add 5 ml distilled water and 5 ml HCl to the ash. Cover the crucible with a watch glass and heat gently to boiling for 5 minutes. Filter the solution in a 100ml volumetric flask. Wash the inside of the watch glass and the crucible with about 5 ml of hot water using a wash bottle with a fine stream of water.

5.3. Wash the crucible and filter paper with 4 additional portions of hot distilled water. Cool the solution to room temperature and neutralise to a faint turbidity by dropwise addition of 50 % KOH solution. Add conc. HCl dropwise until the zinc acetate precipitate is just dissolved, then add 2 additional drops. Dilute to volume with water and mix thoroughly.

5.4. Pipette 10 ml of this solution into a clean , dry 50 ml volumetric flask. Add 8 ml of hydrazine sulphate solution and 2 ml of sodium molybdate solution in this order. Stopper and invert 3 - 4 times.

5.5. Loosen the stopper and heat for 10±0.5 minutes in a vigorously boiling water bath. Remove from bath , cool to 25±5°C in a water bath, dilute to volume and mix thoroughly Transfer the solution to a clean dry cuvette and measure the absorbance at 650 nm in a spectrophotometer adjusted to read 0 % absorbance (100 % transmittance) for distilled water.

5.6. Prepare a reagent blank without the oil test sample. Measure the phosphorus content of the sample and the blank by comparison with the standard curve.

6. Calculation-

Phosphorous % = 10 x (a - B) WxV

Where

A = Phosphorous content of sample aliquot in mg

B = Phosphorous content of the blank aliquot in mg

W = Weight of sample in gm

V = Volume of solution taken for colour development

Note:- Phosphorous content can also be determined by Atomic Absoption Spectrophotometer at a wave length of 213 nm following other instrumental parameters.

7. Reference/Protocol

7.1. (Ref:- A.O.C.S ( 1989) Official Method Ca 12 -55, Phosphorous)

TM-19 Determination of Insoluble Bromide.

TM-20 Determination of Milk Fat

1. Title

Determination of Milk Fat.

2. Principle

Milk fat is defined as the % of Milk fat present in sample and not contain moisture.

3. Reagents

No Reagents

Apparatus

4.1.Moisture dish –

4.2.Porcelain, silica, glass or Aluminium. (7.5 x 2.5 cm.),

4.3.Oven: electric maintained at 105 ±1 ⁰C,

4.4.Dedicator

5. Procedure

5.1. Weigh accurately about 5 gm of the sample in the moisture dish previously dried in an oven and weighed.

5.2.Place the dish in the oven maintained at 105±1°C for four hours.

5.3. Cool in the desiccators and weigh.

5.4. Repeat the process of drying, cooling, and weighing at 30 minutes interval until the difference in two consecutive weighing is less than 1 mg.

5.5. Record the lowest mass.

5.6. After obtain the value of moisture substrate the value of moisture from 100%.

6. Calculation-

Moisture percent by weight = 100 (M1-M2)

M1 – M

100-Result of Moisture = Milk Fat

Where,

M1 = mass, in gm, of the dish with material before drying.

M2 = mass, in gm, of dish with material after drying to constant mass; and

M = mass in gm of the empty dish.

7. Reference/Protocol

7.1. Fem Analytika Lab Manual.

TM-21 Determination of Melting Point

1. Title

Determination of Melting Point of edible oil & Vanaspati Ghee.

2. Principle

The temperature at which the oil or fat softens or becomes sufficiently fluid to slip or run as determined by the open-tube capillary-slip method

3. Reagents

No reagents

4. Apparatus

4.1.Melting point tubes -thin walled with uniform bore capillary glass tubes open at both ends with following dimensions:

Length 50 to 60 mm

Inside dia 0.8 to 1.1 mm

Outside dia 1.2 to 1.5 mm

4.2. Thermometer with 0.2ºC sub-divisions and a suitable range. The thermometer should be checked against a standard thermometer which has been calibrated and certified by National Physical Laboratory New Delhi or any other laboratory approved for calibration of instruments

4.3. Beaker with a side tube heating arrangement – Thiele melting point tube may be used

4.4. Heat source: Gas burner or Spirit Lamp

5. Procedure

5.1.Melt the sample and filter it through a filter paper to remove any impurities and last traces of moisture. Make sure that the sample is absolutely dry.

5.2.Mix the sample thoroughly. Introduce a capillary tube into the molten sample, so that a column of the sample, about 10 mm long, is sucked into the tube. Chill the tube containing the sample immediately by touching the tube, against a piece of ice until the fat solidifies.

5.3.Place the tube in a small beaker and hold it for one hour either in a refrigerator or in water maintained at 4ºC to 10ºC. Remove the tube and attach with a rubber band to the thermometer bulb, so that the lower end of the capillary tube and the thermometer bulb are at the same level.

5.4.Take water at 10ºC in the ‘Thiele’ tube and immerse the thermometer with the capillary tube containing the sample of fat. Gradually increase the temperature by heating at the side-tube of the Thiel Tube at the rate of 2ºC per min, till the temperature reaches 25ºC, and thereafter at the rate of 0.5ºC per min. Note the temperature of the water when the sample column begins to rise in the capillary tube. Report the average of two such separate determinations as the melting point, provided that the readings do not differ by more than 0.5ºC.

6. Calculation

7. Reference/Protocol

7.1.I.S.I. Handbook of Food Analysis (Part XIII) – 1984 , page 68 / I.S : 548 ( Part 1 ) – 1964, v Methods of Sampling and test for Oils and Fats page 33

7.2.IS : 548 (Part I) - 1964

TM-22 Determination of Allyl isothiocynate

1. Title

Determination of Allyl isothiocynate

2. Principle

Every Volatile compound has to evaporate on heating and separate by column on their boiling point.

3. Reagents

Allyl Isothiocynate standard solution – 30.5 mg / 100 ml Measure 30 μl of allyl isothiocynate in 50 μl syringe with 0.5 % accuracy. Add to 50 ml 10 % alcohol in 100 ml volumetric flask and shake intermittently until dissolved. Dilute to volume with water.

4. Apparatus

4.1. Gas Chromatograph – with Flame Ionization Detector Approx operating conditions - Temp – Column 145 ⁰C, Detector 200 ⁰C Injector 160 ⁰C, N2 Flow rate 100 ml / min. Optimum conditions are obtained when not less than 10 cm peak is obtained for 8 μl standard injection solution

4.2.Column and packing – 3.7 m x 4 mm i.d, Carbowax 4000 on floropak 80, 20- 40 mesh

5. Procedure

5.1.Grind more than 5 gm sample to pass through No 20 sieve. Immediately weigh 6 gm into 300 ml Erlenmeyer flask, add 150 ml 5 % alcohol, stopper tightly and stir magnetically 90±5 minutes in 37 ⁰C water bath

5.2. Gas Chromatography Method

Distill about 70 ml into 100 ml volumetric flask containing 20 ml 5% alcohol (v /v) taking care that the end of the condenser dips below surface of solution. Dilute to volume with water. Inject 4 – 10 μl into gas chromatograph. Compare peak height of sample with that from same volume of standard solution.

5.3. Titration Method

Distill about 60 ml into100 ml volumetric flask containing 10 ml NH4OH (1+ 2) taking care that the end of the condenser dips below surface of solution. Add 20 ml 0.1 M AgNO3 to distillate and let stand overnight, heat to boiling point on water bath (boil behind safety barrier) to agglomerate Ag 2S , cool dilute to 100 ml with water and filter. Acidify 50 ml filtrate with about 5 ml HNO 3 and titrate with 0.1 M NH4SCN using 5 ml Ferrous ammonium sulphate as indicator 1 ml 0.1M AgNO3 = 0.004958 gm allyl isothiocyanate

Note 1:- Before discarding Ag 2 S and filter paper, treat with 25 ml 0.5 M Sodium Thiosulphate in 1 M Sodium Hydroxide

Note 2: - During storage mustard becomes moist – conditions which encourage production of allyl isothiocynate, which tends to be lost by volatilization

6. Calculation

7. Reference/Protocol

7.1.A.O.A.C 17th edn , 2000 Official Method 970.55 Volatile oil in Mustard Seed) / Pearson’s Composition and Analysis of Foods 9th edn. 1991 Page417.

Adulteration Parameters

TM-23 Determination of Argemone Oil

1. Title

Determination of Argemone Oil Test, TLC

2. Principle

The hydrochloric acid extract of the oil sample containing argemone oil when subjected to TLC for separation of alkaloid gives fluorescent spot under UV light.

3. Reagents

3.1. Solvent mixture (mobile phase)

3.1.1. Butanol: Acetic acid: water 70:20:10 (v/v)

3.1.2. Hexane or Heptane: Acetone 60:40 (v/v)

3.2. Diethyl ether

3.3. Hydrochloric acid, cons. Sp. Gr. 1.19

3.4. Chloroform: Acetic acid (90:10 v/v) mixture

3.5. Aqueous sodium hydroxide solution 20 %

3.6. Standard Argemone oil extract

4. Apparatus

4.1. TLC plates coated with silica gel G or pre-coated ready made plates cut to suitable size.

4.2. Ultraviolet lamp (long wave – 366 nm) in a visualization chamber.

4.3. Pear-shaped flask.

4.4. Hot water bath

4.5. Separating funnel – 50 ml capacity

4.6. Glass beaker – 10 ml capacity

5. Procedure

5.1. Take 10 ml sample in a separating funnel and dissolve in 15 ml Diethyl ether. Add 5 ml conc. HCL and shake vigorously for 2 – 3 minutes.

5.2. Allow to separate. Contents of the separatory funnel may be heated cautiously over the vent of heating water bath for some time for quick separation.

5.3. Transfer the acid layer to a 25 ml beaker. Place the beaker into a boiling water bath and evaporate till dryness. Dissolve the residue obtained after evaporation of hydrochloric acid in 1 ml of a mixture of chloroform and acetic acid 9:1) and spot on TLC plate with the help of spotting capillary.

5.4. Spot side by side standard Argemone oil extracts (0.1 % in Ether). Develop the plate in (a) Butanol: Acetic acid: water; or (b) Hexane: Acetone mixture. Allow the solvent front to move up a distance of 10 cm and allow the plate to dry. Place the plate under UV light in the visualization chamber.

5.5. Bright yellow or orange yellow fluorescent spots having Rf similar to the standard argemone oil will confirm presence of argemone oil.

5.6. The spot gives blue florescence under UV-light if plate is sprayed with 20% aqueous sodium hydroxide solution.The method is very sensitive and can detect argemone oil up to 50 ppm level.

6. Calculation-

7. Reference/Protocol

7.1. Manual methods of Analysis for Adulterants and Contaminants in Foods I. C. M. R (1990) page 12.

TM-24 Determination of Mineral Oil.

1. Title

Test for presence of Mineral Oil - TLC test

2. Principle

Being non-polar, mineral oils give faster moving spots on thin layer chromatographic plates, than the triglycerides.

3. Reagents

3.1. Silica-gel ‘G’ with calcium sulphate as binder (commercially available)

3.2. Petroleum ether

3.3. Spray reagent: 0.2 percent solution of 2’, 7’-dichloro-fluoresein in 95 percent ethanol.

4. Apparatus

4.1. Glass slides (7.6 x 2.5 cm) or glass plates of 20 x 5 cm or 20 x 10 cm may be used.

4.2. Developing tank

4.3. Ultra-violet lamp (365 nm). This should be placed in a darkened enclosure.

5. Procedure

5.1. Hold two slides together face to face and dip them in slurry of silica gel G (45g) in a mixture of chloroform and methanol (80 + 20 ml).

5.2. Withdraw the slides, separate them and allow to dry in air and activate at 110 ⁰C for 15 minutes and cool in desiccators. Apply 10 ml of a 10 percent solution of oil in chloroform on the glass slide/glass plate using a capillary tube.

5.3. Allow to dry and place the slide in a developing tank containing petroleum ether. Cover the tank and allow the solvent to travel for 6 cm from the origin (about 4 min). Remove the plate from the tank, dry in air, spray with the fluorescein solution and view under UV light.

5.4. Appearance of a yellow fluorescent spot on the solvent front indicates the presence of mineral oil. The vegetable oil forms a yellow streak about 2-3 cm long from the point of spotting.

Note:

If desired, a standard sample containing 1 percent by mass of liquid paraffin in a sample of pure oil under test may be prepared and tested simultaneously as reference sample.

6. Calculation

7. Reference/Protocol

7.1. I.S.I. Handbook of Food Analysis (Part XIII)-1984 Page 89

TM-25 Castor Oil Test

1. Title

Castor Oil Test (by TLC in edible oil).

2. Principle

Castor oil sample under test is applied to silica gel coated TLC plate and eluted with a solvent system. Pure castor oil gives two spot whereas castor oil adulterated with any other oil gives three or more prominent spots.

3. Reagents

3.1.Silica Gel G Plates — 20 × 20 cm having 250 nm thick layers.

3.2.Silver Nitrate Solution — 5 percent solution in 50 percent

3.3.Chloroform

3.4.Solvent System — benzene: 95 percent ethanol: acetic acid: 98.8: 1.6: 0.5.

3.5. Alcoholic Ortho phosphoric Acid Solution — 50 percent (v/v) solution of Ortho phosphoric acid (85-88 percent) in 95 percent ethanol.

3.6. Reference Oil — Sample of uncontaminated and undamaged castor seeds shall be extracted with n-hexane and the extract obtained evaporated under vacuum until the residual oil is completely free from solvent

4. Apparatus

4.1.TLC Applicator

4.2.Drying Oven

4.3. Desiccators

4.4. TLC Pipettes or Capillaries — approximately 50 ml capacity.

4.5. Glass Plates — 20 × 20 cm.

5. Procedure

5.1.Preparation of the TLC Plate — prepare a slurry of 30 g of silica gel G in 60 ml of water and spread evenly on five 20 × 20 cm glass plates using TLC equipment adjusted to obtain a 250 μm thick coating.Dry the silica gel G plates for 1 h at 110°C and cool to room temperature. Spray uniformly the silver nitrate solution until the plates appear wet. Dry the plates again at 110°C for 15 minutes. Cool to room temperature and store the plates over silica gel in desiccators.

5.2.Preparation of the Test Solution — Prepare the following solutions in chloroform :

5.2.1. Mark the starting point on the prepared TLC plate.

5.2.2. Apply a known volume of up to 50μl of the sample solution to one starting point on the TLC plate.

5.2.2.1.One percent solution of the oil to be tested.

5.2.2.2.One percent solution of the reference oil, and

5.2.2.3. One percent solution of mixture (the reference oil containing 3 percent of the other oil

5.3. TLC Procedure

5.3.1. Mark the starting point on the prepared TLC plate.

5.3.2. Apply a known volume of up to 50μl of the sample solution to one starting point on the TLC plate.

5.3.3. Apply the same volume of the test solution containing the reference Oil to one starting point on the TLC plate.

5.3.4. Apply the same volume of the test solution containing the reference oil/other oil mixture to one starting point on the plate.

5.4.Elution of Oils

5.4.1. Allow the solvent to evaporate. Place the TLC plate in a developing vessel containing solvent system benzene: ethanol (95 percent v/v): acetic acid :: 98.8 : 1.6 : 0.5. Running time is about 35 to 40 minutes to run 18 cm.

5.4.2. Dry the plates in an oven at 110°C for 5 minutes.

5.4.3. Spray the plates with the alcoholic ortho phosphoric acid solution and char the plates in the oven at 150°C for 15 minutes.

5.4.4. The oil spots would then appear. Pure castor oil gives two brown spots. A sample adulterated with 3 percent groundnut oil would give two brown and one grey prominent spots. Castor oil adulterated with other oils gives three or more prominent spots.

5.5.Applicability — Peroxides and free fatty acids do not interfere in the test.

5.6. Sensitivity — this test is sensitive to detect the presence of other oils in castor oil to b the extent of 3 percent.

6. Calculation-
No

7. Reference/Protocol

7.1.I.S.I. Handbook of Food Analysis (Part XIII) –1984 Modified test for presence of Castor oil, page 91

7.2.IS : 548 (Part II) - 1976

TM-26 Cottonseed Oil Test

1. Title

Test for Presence of Cottonseed Oil (Halphen's Test)

2. Principle

The development of red colour on heating the oil with a solution of sulphur in carbon disulphide indicates the presence of cottonseed oil. The test is also given by Hempseed oil, Kapok seed oil / oils and fats containing cyclopropenoid fatty acids (such as sterculic and malvalic acid). Hydrogenation and deodorization wholly or partially destroy the cromogens and react with diminished intensity. A positive reaction is not given by oil heated to 250 ⁰C or above. The fat of animals fed on cottonseed meal (butter, lard) or other cottonseed products may give faint positive reaction by this test.

3. Reagents

3.1. Sulphur solution:

Prepare a one percent (w/v) solution of sulphur in carbon disulphide and then add an equal volume of amyl alcohol.

4. Apparatus

4.1. Test tubes

4.2. Water bath

4.3. Oil bath or Brine bath maintained at 110 ⁰C– 115 ⁰C

5. Procedure

5.1. Take about 5 ml of the oil or melted fat in a test tube and add to it an equal volume of the sulphur solution.

5.2. Mix thoroughly by shaking and heat gently on a water bath (70º to 80ºC) for a few minutes with occasional shaking until the carbon disulphide has boiled off and the sample stops foaming.

5.3. Place the tube in an oil bath or a saturated brine-bath maintained at 110-115ºC and hold for 2.5 hours. A red colour at the end of this period indicates the presence of cottonseed oil. The test is sensitive to the extent of 0.5 % cottonseed oil in other oils.

6. Calculation

7. Reference/Protocol

7.1. I.S.I. Handbook of Food Analysis of (Part XIII)-1984 Page 86 / A.O.A.C 17th edn, 2000, Official method 197.02-oil (cottonseed) in oils and fats / F.A.O. Manuals of Food Quality Control 14 / 8 Page 271

TM-27 Determination of Rancidity

1. Title

Determination of Rancidity

2. Principle

This is an indication of the extent of oxidation suffered by oil.

3. Reagents

3.1.Acetic acid - chloroform solvent mixture (3: 2) Mix 3 volumes of glacial acetic acid with 2 volumes of chloroform.

3.2.Freshly prepared saturated potassium iodide solution.

3.3.0. I N and 0.0I N sodium thiosulphate solutions. Weigh 25 g of sodium thiosulphate and dissolve in 1 L of distilled water. Boil and cool, filter if necessary, Standardise against standard potassium dichromate solution.

3.4.Starch solution - 1% water-soluble starch solution

4. Apparatus

4.1.Beaker

4.2.Weighing balance

4.3.Burette

4.4.Pipette

5. Procedure

5.1. Weigh 5 g (± 50 mg) sample into a 250 ml stoppered conical flask. Add 30 ml acetic acid chloroform solvent mixture and swirl to dissolve.

5.2. Add 0.5 ml saturated potassium iodide solution with a mohr pipette let stand for 1min in dark with occasional shaking, and then add about 30 ml of water. Slowly titrate the liberated iodine with 0.1 N sodium thiosulphate solutions with vigorous shaking until yellow colour is almost gone.

5.3. Using Add about 0.5 ml starch solution as indicator and continue titration shaking vigorously to release all I₂ from CHCl ₃ layer until blue colour disappears. If less than 0.5 ml of 0.1 N Na₂S₂O₃ is used repeat using 0.01 N Na₂S₂O₃.Conduct blank determination ( must be less than 0.1 ml 0.1 N Na₂S₂O₃)

6. Calculation-

Peroxide value expressed as milli equivalent of peroxide oxygen per kg sample (meq/kg)

Peroxide value = Titre X N X 100

Weight of the sample

Where Titre = ml of Sodium Thiosulphate used (blank corrected)

N = Normality of sodium thiosulphate solution.

Fresh oils usually have peroxide values well below 10 meq/kg. A rancid taste often begins to be noticeable when the peroxide value is above 20 meq/kg. (Between 20 – 40 meq / Kg). In interpreting such figures, however, it is necessary to take into account the particular oil or fat.

7. Reference/Protocol

7.1. A.O.A.C. 17th edn, 2000, Official Method 965.33 Peroxide Value in Oils and Fats/ Pearsons Composition and Analysis of Foods 9th edn

TM-28 Hydrocyanic acid

1. Title

Determination of Hydrocyanic Acid Test

2. Principle

Hydrocyanic acid is sometimes present as an impurity in synthetic allyl isothiocyanate which is commonly used as an adulterant to enhance the flavour of poor quality mustard oil. Two methods have been prescribed for the purpose of this test. Method A shall be used as referee method and method B as routine method.The hydrocyanic acid in the oil when heated over water bath is displaced by bubbling air and is absorbed in potassium hydroxide solution. The cyanide is then tested with ferric chloride solution.

3. Reagents

3.1. Potassium hydroxide solution - approximately 2N

3.2. Lead acetate solution - approximately 2 N

3.3. Ferrous sulphate solution - approximately 2%

3.4. Hydrochloric acid

3.5. Ferric chloride solution - 20 percent (w/v) in water to which sufficient hydrochloric acid has been added to prevent hydrolysis.

4. Apparatus

4.1. Separating funnel

4.2. Slides: microscopic slides (7.6 X 1.5 cm) or glass plates of 20 X 5 cm or 20 X10 cm may be used.

4.3. Developing tank: a tall beaker of at least 10 cm height/TLC developing chamber

4.4. Visualization tank (Iodine chamber): A dry beaker or developing tank saturated with iodine vapor by placing a few crystal at the bottom and leaving for an hour.

4.5. Beaker: 25 ml

5. Procedure

5.1. Heat about 50 ml of the oil in a distillation flask by placing it on a water bath. During heating pass through the oil for about 30 min, air which has been purified by scrubbing through solution of potassium hydroxide and lead acetate.

5.2. Connect the distillation flask to an absorption tube containing 5 ml of potassium hydroxide solution. The air bubbling through the oil carry with it the hydrocyanic acid and this is absorbed by the potassium hydroxide solution.

5.3. Shake the solution with few drops of ferrous sulphate solution, acidify with few drops of hydrochloric acid and warm gently for 5 min. Filter and add a few drop of ferric chloride solution. A blue or bluish-green colour or precipitate in the solution indicates the presence of cyanide.

Calculation

Reference/Protocol

7.1. I.S.I Handbook of Food Analysis ( Part XIII) – 1984, page 88

7.2. IS : 548 (Part II) - 1976

TM-29 Separated Water

Title

Evaluation of Separated Water.

Principle:

It includes the no separation of samples to other liquid materials and compared with reference samples.

Reagents:

Apparatus

4.1.Test Tube (Transparent)

Procedure

5.1. Melt the sample if solid and pour the sample in to transparent Test tube.

5.2. Place the sample in proper light condition.

5.3. Observed the Separation- Observed any other layer present in oil sample if any other layer observed it’s may be water.

6. Calculation

7. Reference/Protocol

7.1. Fem Analytika Lab Manual

TM-30 Suspended or foreign matter

1. Title

Determination of Suspended or foreign matter.

Principle

The large particles present in liquid medium are settle down at the bottom of container due to heaver in their weight.

Reagents

Apparatus

4.1.taste tube

Procedure

5.1.Properly homogenized the sample by mixing.

5.2.Pour the sample in to 50 ml transparent test tube.

5.3. Place the sample in proper light condition.

5.4. And left for 5 minute.

5.5. Observe the bottom of the test tube and report if any Suspended or foreign matter.

6. Calculation-

No.

7. Reference

7.1. Fem Analytika Lab Manual.

TM-31 Determination of Hexa bromide (Linseed oil)

1. Title

Test for presence of Linseed Oil (Hexabromide Test)

2. Principle

The formation of a precipitate of hexabromide, when the oil in chloroform is treated with bromine and then with alcohol and ether in cold condition indicates the presence of linseed oil.

3. Reagents

3.1. Chloroform – A.R

3.2. Liquid bromine – A.R

3.3 Ethyl alcohol

3.4. Diethyl ether

4. Apparatus

4.1. Boiling tubes

4.2. Ice water bath

5. Procedure

5.1.Pipette one ml of the oil into a boiling tube (wide-mouthed 100 ml capacity). Add 5 ml of chloroform and about one milliliter of bromine drop-wise till the mixture becomes deep red in colour and cool the test-tube in an ice water-bath.

5.2.Add about 1.5 ml of rectified spirit drop-wise while shaking the mixture until the precipitate which was first formed just dissolves and then add 10 ml of diethyl ether. Mix the contents and place the tube with in the ice water-bath for 20 minutes. Appearance of precipitate indicates the presence of linseed oil.

Note

This test is not applicable for detecting linseed oil in mahua oil.

The use of safe and suitable pipette i.e. Lunge-Ray pipette is suggested for the handling and addition of bromine.

The test is also given by fish oils and fats containing highly unsaturated fatty acids. It has been observed that in low erucic rapeseed oil, and mahua oil having linolenic acid content greater than 12.0% may also give positive test. The results obtained in such cases have to be viewed with caution. Experiments conducted with these oils with or without added linseed oil have shown that, if linseed oil is present even at 1% level, hexabromide insoluble in cold ether are formed within 20 min. Any hexabromides insoluble in cold ether formed after 20 min need not be taken for the presence of linseed oil.

An explanation for this behavior of these oils can be given on the basis of glyceride structure. Oils and fats are mixed tri acyl glycerols i.e. different fatty acid are present in each of the positions of the glycerol molecule. Exception to this rule is the oil /fat containing a particular fatty acid in amounts greater than 50 % where such a fatty acid may take all the three positions of the glycerol molecule giving rise to simple tri acyl glycerols. Linseed oil is such an example containing greater than 50 % Linolenic acid.

6. Calculation

7. Reference/Protocol

7.2. Manual of Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R (1990) Page 5 / I.S.I. Handbook of Food Anaysis Part ( XIII) – 1984 page 86

TM-32 Determination of Karanja oil

1. Title

Determination of Karanja (Pongamia glabra) Oil

2. Principle

Extraction of glabrin, karanjin, karanjone, pongaglabrone and pongamol using concentrated hydrochloric and their detection on TLC under ultra-violet light.

3. Reagents

3.1. Hydrochloric acid AR Sp. Gr. 1.18

3.2. Solvent Mixture as mobile phase, petroleum ether: diethyl Ether: acetic acid 60: 40: 1 (v/v).

3.3. Standard Karanja oil extract (1.0 % oil in any other oil extracted simultaneously with the sample)

Apparatus

4.1. All-glass separating funnel (100 ml capacity)

4.2. Glass beaker (50 ml capacity)

4.3. Measuring cylinder for separating funnel.

4.4. Wooden stand for separating funnel.

4.5. Hot water-bath f) Capillary tubes.

4.6. TLC plates (0.25 mm). Prepared by coating slurry of silica gel G on glass plate of 10 x 20 cm diameter, activated at 110 0C for 1 hour and stored in a desiccators.

4.7. Ultra-violet lamp long wave (366 nm) in a visualization chamber.

Procedure

5.1. Take 20 ml of the suspected oil in a 100 ml capacity separating funnel and add to it 10 ml concentrated hydrochloric acid. Shake the content gently, but consistently for 15 min. Keep the separating funnel on a wooden stand for about 30 min to allow the separation of acid layer.

5.2. Draw out the acid layer in a glass beaker. Keep the beaker on a boiling water bath and evaporate the hydrochloric acid till dryness.

5.3. Dissolve the residue in 0.5 ml of chloroform. Spot the chloroform solution on a pre-activated TLC plate with the aid of capillary tube. Spot standard Karanja oil extract side by side. Develop the plate in solvent system petroleum ether: diethyl ether: acetic acid 60: 40: 1 v/v for 20 min.

5.4. Remove the plate, dry at room temperature and view under ultra- Violet lamp. Appearance of three bluish green spots at Rf 0.34, 0.22 and 0.17 confirms the presence of Karanja oil.

Note: - The test is sensitive to the extent of 0.01 % Karanga oil.

Calculation

Reference/Protocol

7.1. Manual Methods of Analysis for Adulterants and Contaminants I.C.M.R.(1990) page 12.

TM-33 Determination of Tricresyl Phosphate

1. Title

Determination of Tricresyl Phosphate, TLC

Principle

Tricresyl phosphate in contaminated edible oils is extracted using acetonitrile and detected by thin-layer chromatography as well as gas liquid chromatography.

Reagents

3.1. Developing solvent - Iso-octane-ethyl acetate (90: 10). Line developing chamber with filter paper.

3.2. Spray reagent: 0.5% solution of 2, 6-dichloro-quinone chlorimide. A.R in absolute ethyl alcohol (Gibbs reagent). Store reagent at 10ºC and use within 5 days.

3.3. Standard Tricresyl phosphate (TCP) and tri-O-cresyl phosphate (TOCP)

Apparatus

4.1. Separatory funnels - 250 ml capacity.

4.2. TLC Plates - Prepare slurry of silica gel G with water (1: 2 w/v) and spread over glass plates (25mm layer on 20 x 20 cm plates) with applicator. Let the plates set at room temperature. Activate at 110 ⁰C for 1 hour, cool and store in a desiccator.

4.3. Gas Chromatograph - Fitted with flame ionization detector; stainless steel column (10' x 1/8') packed with 10% OV - 101 on 60 to 80 mesh Chromosorb-AW-DMCS; nitrogen carrier gas 30 ml/min, column temperature 250 º C, detector and injector temperature 300 ⁰C; chart speed 1 cm/min.

Procedure

5.1. Take 10 ml oil sample containing Ca. 50 μg TCP or TOCP into separatory funnels; add 50 ml petroleum ether (40 to 60ºC ) to dissolve the oil followed by 10 ml acetonitrile previously saturated with petroleum ether.

5.2. Shake contents vigorously and let stand 10 min. Collect lower acetonitrile layer in beaker and evaporate solvent on hot water bath. Dissolve residue in Ca. 1 ml ethyl or methyl alcohol.

5.3. Thin layer chromatography: Spot ca 0.1 ml (Ca. 5 mg TOCP) of solution on TLC plate. Develop plate in glass chamber containing iso-octane ethyl acetate (90:10) ca. 45 min to a height of 10 cm. Remove plate and dry in air.

5.4. Spray plate with Gibbs reagent and heat in 100ºC oven Ca. 15 min. Observe for characteristic blueviolet spot at Rf 0.27 corresponding to standard TCP or TOCP.

Inject about 1 mg (2.5 mg TOCP) of acetonitrile extract of the oil sample into GC apparatus; compare retention time and peak area of sample with that of standard T.C.P or T.O.C.P for quantization.

Calculation

Reference/Protocol

7.1. Manual Methods of Analysis for Adulterants and Contaminants in Foods I.C.M.R (1990) page 14

TM-34 Test for Sesame oil (Baudouin test).

Title

Test for Sesame Oil (Baudouins Test)

Principle

The development of pink colour with furfural solution in the presence of hydrochloric acid indicates the presence of sesame oil. The color is produced on account of reaction with sesamolin present in sesame oil.

Reagents

3.1. Hydrochloric acid (concentrated) Sp. Gr. 1.19

3.2. Furfural solution (2 per cent furfural–freshly distilled in ethyl alcohol)

Apparatus

4.1. Glass stopper test tubes / measuring cylinders

Procedure

5.1. Take 5 ml of the oil or melted fat in a 25 ml measuring cylinder (or test tube) provided with a glass stopper, and add 5 ml of conc. hydrochloric acid and 0.4 ml of furfural solution. Insert the glass stopper and shake vigorously for two minutes. Let it stand and allow the mixture to separate.

5.2. The development of a pink or red colour in the lower acid layer indicates presence of sesame oil. Confirm by adding 5 ml of water and shaking again. If the colour in acid layer persists, sesame oil is present, if the colour disappears it is absent. (As furfural gives violet tint with HCl, it is necessary to use the dilute solution specified.)

Note: Test the sample for the presence of colouring matter which are cromogenic in presence of Hydrochloric acid. For this purpose, take 5 ml of the sample in a 25ml measuring cylinder provided with a glass stopper and shake with 5ml of concentrated HCL. If there is no development of pink or red color in the aqueous layer apply the test as above. If pink or red color develops in the aqueous layer, remove the red acid layer which collects at the bottom and repeat the procedure until no further coloration takes place. After complete removal of HCl layer perform the test as prescribed above.

Calculation-

Reference/Protocol

7.1.I.S.I. Handbook of Food Analysis (Part XIII)-1984 Page 86 / A.O.A.C 17th edn, 2000, Official method 893.01-Oil (sesame ) in Oils and Fats Modified Villavecchia Test.

TM-35 Determination of Polybromide test

1. Title

Determination of Polybromide.

2. Principle

An ethereal solution of the fat or fatty acid is treated with bromine. The formation of a precipitate gives a qualitative indication of the presence of fatty acids with three or more non conjugated double bonds

3. Reagents

3.1. Diethyl ether

3.2. Bromine

4. Apparatus

4.1. Conical Flask 100 ml capacity

4.2. Burette with a finely drawn out jet

5. Procedure

5.1.Dissolve approximately 3 gm of clear fat in 25 ml diethyl ether in the conical flask.

5.2.Place the flask in a melting ice bath for 15 minutes and then slowly add 1 ml bromine dropwise from burette with continuos swirling and cooling (the first half ml in 20 minutes and the remainder in 10 minutes). Cool the flask and keep it in the ice bath for a further 3 hours. If a precipitate forms, the reaction is considered positive.

6. Calculation-

7. Reference/Protocol:

7.1.Laboratory Handbook for Oil and Fat Analysis, Cocks and Reid, page 147- 148.

TM- 36 Determination of Added Color

Title

Determination of Added color

Principle

Paper Chromatographic Separation of Synthetic Food Colours

The general scheme for identifying synthetic food colours present in foods normally involve preliminary treatment of the food, extraction of the colour from the prepared solution of the food, separation of colours in case of mixtures and identification of the separated colours.

Reagents

3.1. White knitting wool: - Extract pure white wool in a soxhlet extractot with petroleum ether for 2-3 hrs to remove fat. Boil in very dilute solution of sodium hydroxide and then in water to free it from alkali.

3.2. Paper: Whatman No. 1 chromatographic paper.

3.3. Solvents:

3.3.1. 1 ml (0.88 sp. gr) ammonia + 99 ml water.

3.3.2. 2.5% aqueous sodium chloride.

3.3.3. 2% sodium chloride in 50% ethanol.

3.3.4. Acetic acid solution in water (1:3).

3.3.5. Iso-butanol-ethanol-water (1: 2 : 1, v/v).

3.3.6. n-butanol-water-glacial acetic acid (20 : 12 : 5, v/v).

3.3.7. Iso-butanol-ethanol-water (3 : 2 : 2, v/v). to 99 ml of this add 1ml of (0.88 sp gr.) ammonia.

3.3.8. 80 gm phenol in 20 gm water

Apparatus

4.1. Pipette.

4.2. Beaker.

4.3. Flask.

Procedure

5.1. Preliminary treatment of food: Assuming that an acidic colour is present, the preliminary treatment involves removing interfering substances and obtaining the dye in acid solution prior to boiling with wool.

5.1.1. Non-alcoholic beverages e.g. soft drinks: As most foods in this group are acidic they can be usually treated directly with wool, otherwise, slightly acidify the food with acetic acid.

5.1.2. Alcoholic liquids (e.g. Wine): Boil to remove alcohol and acidify if necessary as in (i).

5.1.3. Starch based foods (e.g. cakes, custard powder etc): Grind 10 gms of sample thoroughly with 50 ml of 2 % ammonia in 70% alcohol, and allow it to stand for an hour and centrifuge. Pour the separated liquid into a dish and evaporate on water bath. Take up the residue in 30 ml dilute acetic acid.

5.1.4. Candied fruits: Treat as in (iii).

5.1.5. Products with high fat content (e.g. Sausages, meat, fish paste): De-fat the sample with light petroleum and extract the colour with hot water (acidify etc. as usual). Note that oil soluble colours tend to give coloured solutions in organic solvents. If the extraction is difficult treat with warm 50-90% acetone or alcohol (which precipitates starch) containing 2% ammonia. The organic solvent should be removed before acidifying as in (iii).

5.2. Extraction of the colour from the food:

5.2.1. Introduce about 20 cm length of woollen thread into a beaker containing about 35 ml of the prepared acidified solution of the sample and boil for a few minutes till the woolen thread is dyed. Take out the woollen thread and wash it with tap water.

5.2.2. Transfer the washed woolen thread to a small beaker containing dilute ammonia and heat again. If the colour is stripped by the alkali, the presence of an acid coal-tar dye is indicated. Remove the woollen thread.

5.2.3. Make the liquid slightly acidic and boil with a fresh piece of woollen thread. Continue boiling until the colour is taken by the woollen thread. Extract the dye from the woolen thread again with a small volume of dilute ammonia, filter through a small plug of cotton and concentrate the filtrate over a hot water bath.

5.2.4. This double stripping technique usually gives a pure colour extract. Natural colours may also dye the wool during the first treatment, but the colour is not usually removed by ammonia.

5.2.5. Basic dyes can be extracted by making the food alkaline. with ammonia, boiling with wool and then stripping with dilute acetic-acid. At present, all the permitted water soluble coal-tar dyes are acidic, hence an indication of the presence of a basic dye suggests that an unpermitted colour is present

5.3. Identification of the separated food colours by paper chromatography: Draw a pencil-line parallel to the bottom edge of the paper (Whatman No.1) at about 2 cm distance. Spot the concentrated solution of the unknown dye on the line together with a series of spots (about 2 cm apart) of aqueous solutions of standard permitted dyes of similar colour and dry. Run the chromatogram, by ascending technique, using a selected solvent. Solvent No.5 is often helpful for general purposes. Identify the colour in the sample by matching its spot with the spot of the standard colour and confirm by co spotting

5.4. Determination of Synthetic food colours in food products:

5.5. For samples containing single colour

5.5.1. Preparation of standard curve: Stock solution: Weigh 0.1 g m of each reference colour and dissolve in 0.1N HCl in separate 100 ml volumetric flasks and make up the volume with 0.1N HCl in each case.

5.5.2. Working standard: Pipette 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 ml of stock solution of each of the reference colours into series of clean and dry 100 ml volumetric flasks and dilute to volume with 0.1N HCl. Determine the optical densities of each of the reference colours at the respective wave length of maximum absorption (refer table) Obtain the standard curve for each colour by plotting optical density against concentration.

5.6. Determination in sample by column chromatography:

5.6.1. Transfer a known weight of the sample (approximately 5 - 10 gm) into a glass stoppered separatory funnel. Extract the colour with 70% acetone. Shake acetone extract with petroleum ether (40-60ºC) in order to remove carotenoids and other natural pigments, if any.

5.6.2. Continue extraction with petroleum ether until petroleum ether extract is colourless. Pass the acetone extract containing only coal-tar food colours through a column (2.1 x 45 cm) containing aluminium oxide acidified with 1% HCl. Elute the adsorbed colour with 1% ammonia.

5.6.3. Evaporate the eluate to dryness on a hot waterbath, dissolve the residue with 0.1 N HCl, transfer quantitatively to a 100 ml volumetric flask and make up the volume with 0/1N HCl.

5.6.4. Determine the optical density of the dye solution at the wavelength of maximum absorption. Calculate the dye concentration from the standard curve.

Calculation-

TABLE SHOWING ABSORPTION MAXIMA OF PERMITTED

FOOR COLOURS

_____________________________________________________________

Sl. No. Name of the Colour Absorption maxima (nm)

_____________________________________________________________

1. Carmosine - 516

2. Ponceau4 R - 507

3. Erythrosine - 527

4. Green FCF - 624

5. Indigo Carmine - 609

6. Brilliant Blue FCF - 630

7. Tartrazine - 427

8. Sunset yellow FCF - 482

_____________________________________________________________

Reference/Protocol

7.1. Manual Methods of Analysis for Adulterants and Contaminants in Food, I.C.M.R 1990 Page 56.

TM-37 Bellier test (acetic acid method)

1. Title

Bellier Test (Turbidity Temperature) Acetic Acid Method

2. Principle

Oils containing long chain saturated fatty acids give a precipitate at a particular temperature which is specific for the oil when their alcoholic soap solution is treated with dilute acetic acid solution and 70% ethyl alcohol.

3. Reagents

3.1. Purified / Rectified spirit: Reflux 1.2 liters of rectified spirit for 30 minutes in a distillation flask with 10g of caustic potash and 6g of granulated aluminium (or aluminium foil pieces). Distil and collect one liter after discarding the first 50 ml. Use this purified rectified spirit for preparation of all the reagents.

3.2. Alcohol 70 percent (by volume): Dilute 700ml of alcohol to 950ml with distilled water and check the strength by specific gravity determination and adjust if necessary. The specific gravity of 70 percent alcohol at 15.5ºC is 0.8898 and at 30 ⁰C is 0.8807. The final strength should be checked accurately.

3.3. Alcoholic potash (1.5 N): Dissolve 8.5 g potassium hydroxide in 100 ml purified rectified spirit. It is preferable to keep this solution in a dark colour bottle.

3.4. Dilute acetic acid Mix one volume of glacial acetic acid with two volumes of distilled water.

3.5. Phenolphthalein indicator: Dissolve 0.5 g of phenolpthalein in 50 ml of purified rectified spirit and mix the solution with 50 ml of distilled water.

4. Apparatus

4.1. Conical flask - 100 ml capacity with cork

4.2. Thermometer (0 - 60ºC calibrated to read 0.5ºC)

4.3. Water bath

5. Procedure

5.1. Measure with the aid of a pipette one mili-litre of the filtered sample of oil in a flat-bottom 100 ml conical flask (preferably with a long neck), add 5 ml of 1.5N alcoholic potash and saponify completely by heating over a boiling water-bath using an air condenser (about 1.3 meters long) to avoid loss of alcohol as far as possible.Complete saponification usually takes about 10 minutes.

5.2. During saponification swirls the flask several times. Cool, add 0.1 ml of phenolpthalein indicator, neutralise exactly by adding carefully dilute acetic acid and then add an extra amount of 0.4 ml (accurately measured). Add 50 ml of 70 percent alcohol and mix. Fit a thermometer (0º to 60ºC reading to 0.5ºC, accurately calibrated) into the flask, with the aid of a velvet cork in such a way that the bulb of the thermometer is immersed in the liquid but does not touch the bottom of the flask.

5.3. Heat the flask gently over the water-bath until the temperature reaches 50ºC and the solution is clear. Allow the flask to cool in air with frequent shaking until the temperature falls gradually to 40ºC (in case of pure groundnut oil turbidity appears at 39 ºC to 41ºC). Then, cool the flask with constant shaking by occasional immersion in a cooling bath maintained at 15 ºC (±1ºC) so that the temperature drops roughly at the rate of 2ºC per minute.

5.4. Note the temperature at which the first distinct turbidity appears which is the turbidity temperature. This turbidity temperature is confirmed by a little further cooling which would result in deposition of the precipitate.

5.5. Dissolve the precipitate by gently heating the contents to 50ºC over water bath, again cool as described above and make a duplicate determination of the turbidity temperature. The mean of the two values is taken as the true turbidity temperature. Duplicate shall agree within ±0.5ºC.

Note: It is essential that stirring is continuous and moderate while the contents are being cooled in the cooling bath. Violent shaking or agitation would be avoided as it will affect the result adversely.

6. Calculation

7. Reference/Protocol

7.1. I.S.I. Handbook of Food Analysis (Part XIII) 1984 - page 90

7.2. S : 548 (Part II) - 1976

TM-38 Added flavoring substances

1. Title

Sensory Evaluation of Added Flavoring substances.

2. Principle

The test involves organoleptic evaluation, usually at constant temperature in a well-ventilated, odour-free room.

3. Reagents

No reagents

4. Apparatus

4.1.Glass container with cap.

4.2.Glass/steel Plates

4.3.Glasses for Drinking water

4.4.Spoons

5. Procedure

5.1. Prepare homogeneous and representative sample. All control or reference samples are to be prepared in the same way as test samples.

5.2. Pour the liquid sample and standard in a container and left for 10 minute.

5.3. After 10 minute open the cap of container and inhale the smell of standard and sample and compare them.

5.4. If any differences found between standard and sample then reported as added flavor.

5.5. If the sample eatable…

5.6. Place a small amount of control or reference sample into the mouth by using a spoon.

5.7. Hold the sample in the mouth for a few seconds and slowly draw air through the mouth to aerate the sample and carry the aroma to the olfactory cell region.

5.8. Expectorate the sample, and draw more air through the mouth. Particular attention should be given to any aftertaste noted.

5.9. Rinse the mouth thoroughly with warm water before tasting another sample.

5.10. Repeat steps 2, 3, and 4 with the test sample.

5.11. Evaluate for any flavor differences noted between the test sample and the reference control for the stated attributes in the specification.

5.12. Evaluate reference control for the stated attributes in the specification.

5.13. Evaluate the flavor noted while sample was held in the month and also any aftertaste noted.

5.14. If no differences or off-flavors are noted, report as "normal" or "matches standard" or ‘Characteristics’.

5.15. Any off-flavor is to be characterized. Be as specific as possible.

5.16. Off-flavors noted should be confirmed by other tasters.

6. Calculation-

7. Reference/Protocol

7.1.Fem Analytika Lab Manual.

TM-39 Phytosterol Acetate Test

1. Title

Test for the presence of Animal Fat in Vegetable oils (Phytosterol Acetate Melting Point Test).

2. Principle

The sterols present in oil or fat are first converted into digitonides by treatment with an alcoholic solution of digitonin. The digitonides are then converted into acetates by boiling with acetic anhydride. The acetate of the sterols is purified by repeated crystallization in alcohol and melting point determined.

3. Reagents

3.1.Digitonin Solution: 1 % solution in 95% ethyl alcohol.

3.2.Chloroform, Ether, Absolute alcohol, Sulphuric acid, Glycerine.

4. Apparatus

4.1.Water- Jacketed Filtering Apparatus.

4.2.Witt’s Filtering Apparatus.

4.3.Melting point Apparatus

5. Procedure

5.1. Liquefy the fat and filter it free from impurities, moisture and sediment and take about 25 g of the filtered sample of fat, 10 ml of chloroform and 15 ml of digitonin solution in a 250-ml conical flask and shake, by giving a rotator motion by hand over a water bathe maintained at 60 °C for 15 minutes.

5.2. Heat the 100ml sintered glass filter to 60 °C for some time, drawing air through the filter bed with gentle suction to dry it. Disconnect suction and pour into the filter the contents of the conical flask after wiping the outside of the flask. Start the vacuum pump and allow the contents to filter keeping the mass well stirred during filtration and maintaining the bath at 60 °C. Use gentle suction. Just before the filtration is over and the precipitate is dry, wash it with about 5 ml of chloroform five or six times, each addition of chloroform being made just before the previous addition is almost filtered and the precipitate has a chance to dry.

5.3. When washing with chloroform is complete, draw air through the filter by means of the vacuum pump till the precipitate appears dry and the place the filter in the vacuum desiccator for not less than half an hour. Remove the dried digitonide from the filter bed with the help of a mounted needle or a pointed glass rod.

5.4. Transfer the digitonide to the stoke’s tube. Add 5 ml of acetic anhydride and heat over sand-bath by a low flame till it dissolves completely. If necessary, filter the solution while hot in the 100ml sintered glass filter, rinsing the tube with 0.5 ml of acetic anhydride. To this solution, add 20 ml of 50% alcohol, when a milky white precipitate will appear; mix through a 30ml filter over Witt’s filtering apparatus, wash thoroughly fie to six times with cooled 95% alcohol and dry by drawing air through the filter with the help of vacuum for about 10 minutes.

5.5. Repeat the process given in 5.4 to effect a second crystallization and dry the filter with the acetate crystals, overnight, in a vacuum desiccator.

5.6. After taking the acetate crystals in 3 or 4 melting point capillary tubes, seal the ends of the tubes. Attach one of the capillary tubes to the bulbs of thermometer by means of rubber bands and adjust the melting point apparatus. Heat the apparatus by a micro-burner slowly so that the temperature rises very gradually and steadily. From 110°C onwards, the heating should be so regulated that the rise in temperature is at a rate of not more than 0.5°C per minute.

5.7. The temperature at which complete fusion takes place is taken as the melting point of the sterol acetate. Determine the melting point of two more capillary tubes as described in 5.6 for conformation.

5.8. The Melting points of sterol acetates from various oils and fats as also their major sterols are as follows.

Fat/Oil	Major Sterol	Melting Points of Sterol Acetate
Animal fat	Cholesterol	114.6°C
Groundnut oil, Hydrogenated groundnut oil, Cottonseed oil	Β-sitosterol	125 to 127°C
Coconut oil	Stigmasterol	144°C
Mustard oil	Brassicasterol	158°C

6. Calculation-

7. Reference/Protocol

7.1.IS: 548 (Part II)-1976, Page No-19 to 25.

TM-40 Animal Fat Test

Title

Determination of Animal Fat.

Principle

Animal body fats such as beef tallow and lard have been shown to contain trisaturated glycerides. On crystallization these glycerides exhibit a characteristic crystalline appearance when viewed under microscope. The procedure recommended by Williams Sutton for the microscopy of fat crystals have been suitably modified and given.

Reagents

3.1.Ether and ethyl alcohol (1:1)

3.2.Glycerine

Apparatus

4.1.Weighing balance

4.2.Microscope

Procedure

5.1. Take about 2 g of melted fat samples in test tubes and mix with 10 ml diethyl ether. Plug the tubes with cotton and allow standing for 30 min in ice water for 24 hrs at 20ºC (slow crystallization gives bigger crystals).

5.2. In certain cases it is preferable to first crystallize with a stronger solution of fat from a mixture of ether and ethyl alcohol (1:1). In such cases separate the crystals by filtration and recrystallise in ether. Place the crystals on a drop of glycerine previously taken on a microscopic slide.

5.3. Cover the crystals immediately with cover glass. Examine the crystals under x 160 and finally x 400 magnifications. The typical appearance of beef tallow crystallized into characteristic fan like tufts, the ends of which are more or less pointed can be seen. Lard crystals are of chisel shaped.

5.4. Hydrogenated fats deposit smaller size crystals. The size and shape of the crystals depend upon the strength of solution, amount of fat taken and the time allowed for crystallization.

Calculation

Reference/Protocol

7.1.Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R ( 1990)

TM-41 Determination of Coal tar dye.

Title

Determination of Coal tar dye by Hydrochloric acid method.

Principle

The petroleum ether solution of oil sample gives different shades of colour with different concentrations of hydrochloric acid in presence of coal tar synthetic oil soluble colour in the oil. /fat.

Reagents

3.1.Concentrated hydrochloric acid - Prepare 4:1, 3 : 1, 2 : 1 and 1 : 1

3.2.Hydrochloric acid : water mixture ii) Petroleum ether

Apparatus

4.1.Test tube

4.2.Pipette

Procedure

5.1.To 5 ml of oil sample in separate test tubes add 15 ml of petroleum ether followed by 5 ml of hydrochloric acid of different concentrations to different tubes.

5.2.Observe for the change in the colour indicating the presence of synthetic oil soluble colour in the sample.

Calculation

Reference/Protocol

7.1. Manual Methods of Analysis for adulterants and contaminants in Foods I.C.M.R. ( 1990 ) page 16.

TM-42 Determination of Mashed Potato, sweet potatoes and other starches.

Title

Determination of Mashed Potato, sweet potatoes and other starches.

Principle

The presence of Mashed potato or sweet potatoes in sample of Ghee can easily be detected by adding of few drops of Iodine, which is brownish in color turns to blue if mashed potatoes/sweet potatoes/ other starch are presents.

Reagents

3.1.Iodine

Apparatus

4.1.Test tube

4.2.Pipette

Procedure

5.1.Take 5 ml of sample in a test tube.

5.2.Add few drops of Iodine solution.

5.3.The sample turn to blue in color that’s indicate the presence of mashed potatoes/ sweet potatoes/ other starch.

Calculation

Reference/Protocol

7.1.FSSAI manual on adulteration test-Quick test for some adulterants in food- Instruction Manual- Part 1.

TM-43 Determination of Fatty acid Composition

Title Determination of Fatty acid composition
Principle

The methyl esters are formed using boron trifloride or methanol and alkali and separated by gas – liquid chromatography using a flame ionization detector. The pattern of methyl esters can be compared with authentic oils for identification

Reagents

3.1. Carrier Gas – Inert gas (nitrogen, helium, argon) thoroughly dried and containing less than 10 mg / kg of oxygen Auxillary gas Hydrogen 99.9 % minimum purity. Free from organic impurities, air or oxygen.

3.2. Reference standards – a mixture of methyl esters or methyl esters of oils of known purity preferably similar to the fatty matter being analysed.

3.3. Methanolic Sodium hydroxide solution - approx 0.5 N. Dissolve 2 gm of Sod. Hydroxide in 100 ml methanol containing not more than 0.5 % m /m water. When the solution has to be stored for considerable time, a small amount of white ppt of Sod. Carbonate may be formed. This has no effect on the preparation of the methyl esters.

3.4. Methanolic solution of Boron trifloride – 12- 15 % m /m, 14 and 50 % solutions is commercially available. The metahnolic solution of boron trifloride should be stored in a refrigerator.

3.5. Heptane- Chromatographic quality, Redistilled pet. Ether 40 – 60 ⁰C

3.6. Anhydrous Sodium sulphate, saturated solution of Sodium chloride.

3.7. Methyl red - 1gm / litre in 60 % alcohol

Apparatus

4.1. Gas liquid chromatograph with the following characteristics

4.2. Injection system heated to a temperature of 20 – 50⁰ C higher than the column.

4.3. Oven – capable of heating the column to at least 220⁰ C and maintaining the temperature to within 1⁰ C. If temperature programming is to be employed, twin columns are recommended.

4.4. Packed column - may be glass or stainless steel. However glass is preferred as steel may decompose polyunsaturated fatty acids having more than 3 double bonds. Some successful column packings with column length, internal diameter and operating temperature are as follows. 12- 15 % ethylene glycol succinate on 100 / 120 mesh gas chrom P(2mx 4 mm, at180⁰ C ). 2- 10 % Apizon –L on 80/ 100 mesh Chromosorb W or Celite ( 2 m x 4 mm at 220 ⁰ C ).

4.5. Detector – Flame ionization detector – capable of being heated to a temperature above that of the column.

4.6. Recorder – electronic with high precision with rate of response below 1.5 second, width of paper 25cm, paper speed 25-150 cm / hr

4.7. Integrator or calculator for rapid and accurate calculations.

4.8. 50 and 100 ml boiling flasks.)

4.9. Reflux condenser

4.10. Graduated pipette – 10 ml

4.11. Test tubes with ground stoppers.

4.12. 250 ml Separating funnels.

Procedure

5.1. Prepare the methyl esters of the fatty acids. The method using boron trifloride gives good results and is preferable to alternative methods which may be used when boron trifloride is not available. Because of the toxic character of boron trifloride various operations must be performed under a ventilated hood.

5.2. All glass ware must be washed with water immediately after use. If the oil or fatty acids include fatty acids containing more than 2 double bonds it is advisable to purge the air from the methanol and the flask by passing a stream of nitrogen into the methanol for a few minutes.

5.3. Transfer about 350 mg of clear oil to a 50 ml conical flask; add 6 ml of 0.5 N methanolic sod. hydroxide solution, 7 ml of boron trifloride solution and a boiling chip. Fit the condenser to the flask. Boil under reflux until the droplets of oil disappear (5- 10 minutes). Add the appropriate amount of boron trifloride solution with a bulb or automatic pipette through the top of the condenser.

5.4. Continue boiling for 2 minutes. Add 2- 5 ml of heptane to the boiling mixture through the top of the condenser. Continue boiling for 1 minute. Withdraw the source of heat and then remove the condenser.

5.5. Add a small amount of saturated Sod. Chloride solution to the flask in order to bring the level of liquid into the neck of the flask.. Transfer about 1 ml of the upper layer (Heptane solution) into a test tube with a ground glass neck and add a little anhydrous Sod. Sulphate to remove any trace of water,. This solution will contain about 5 – 10 % of methyl esters and may be injected directly into the column of gas liquid chromatograph.

Calculation

Fatty acid % = AT/AS x DS/DTx100

Where AT = Area of Test.

AS = Area of Standard

DS= Dilution of Standard

DT= Dilution of Test

Reference/Protocol

7.1. IUPAC 2.301 , 2.302 ( 1979) / F.A.O Manuals of Food quality Control 14 / 8 , pages 274 – 281 / A.O.A.C 17th edn , 2000 Official method 969.33 and 969.22 Fatty acids in oils and fats Preparation of methyl esters / Gas chromatographic method.

FEM ANALYTIKA LAB

The test involves organoleptic evaluation, usually at constant temperature in a well ventilated, odour-free room.

The test involves organoleptic evaluation, usually at constant temperature in a well-ventilated, odour-free room.

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Analytical science