STANDARD TEST METHODS

LAB.STM.OFF.01 Issue 1 Date: 02.10.2022

Title: Estimation of Acid Value

1.1 Principle
The free fatty acid in oil estimated by titrating against KOH in the presence of
phenolphthalein indicator. The acid value is the mg of KOH required to neutralise the free
fatty acids present in 1 mg sample.

1.2 Reagents
 1% phenolphthalein in 95% ethyl alcohol
 0.1N NaOH/KOH
 Neutral solvent- 25 ml ether +25 ml 95% ethyl alcohol. Add 1 ml 1% phenolphthalein
and neutralize with N/10.

1.3 Procedure
 5 g oil was dissolved in 50ml neutral solvent.
 Add few drops of phenolphthalein.
 The mixture was titrated against 0.1N KOH with constant stirring.
 Shake the mixture constantly until a pink colour, with persists for 15secs, was obtained.

1.4 Calculation

Acid value (mg KOH/g) = Titration value × N of KOH × 56.1

Weight of sample (g)

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 STANDARD TEST METHODS

LAB.STM.OFF.02 Issue 1 Date: 02.10.2022

Title: Estimation of Saponification Value

1.0 Principle
A known quality of oil is refluxed with an excess amount of alcoholic KOH. After
saponification, remaining KOH is estimated by titrating against standard acid.

2.0 Reagents
 0.5N alcoholic KOH.
 Standard 0.5N HCL (standardize against 0.5N Na2CO3).
 1% phenolphthalein in alcohol.

3.0 Procedure
 Accurately weigh 2 g of oil was taken in the flask and add 25 ml alcoholic KOH.
Dissolved the oil completely.
 The flask was connected to air condenser and boil for 30 minutes on boiling water both.
 Cool the mixture and add 3 drops of phenolphthalein as an indicator.
 The mixture was titrated against std. 0.5N HCL until the pink colour disappears.
 Similarly, the blank was titrated in the absence of oil.

4.0 Calculation

Saponification value of oil = (Blank – Titration value) ×28.06

Weight of sample (g)

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 STANDARD TEST METHODS

LAB.STM.OFF.03 Issue 1 Date: 02.10.2022

Title: Estimation of Peroxide Value

3.0 Principle
Peroxide value is a measure of the peroxide contained in the oil. The peroxide present are
determined by titration against thiosulphate in the presence of KI. Starch is used as an
indicator.
3.1 Reagents

 CH3COOH:CHCL3(3:1) V/V
 Saturated potassium iodide (KI) – dissolve excess amount of KI in fresh boiled water.
Excess solid should remain. Kept in dark.
 Standard 0.01N Na2S2O3 5H2O (standardize against 0.01N K2Cr2O7).
 1% Starch – 1gm starch dissolved in 10 ml water. Pour the suspension into 90 ml of
boiling water for 2 minutes.

3.2 Procedure
 5 gm of oil was dissolved in 30 ml CH3COOH: CHCL3
 Mix and dissolved the oil completely.
 0.5ml of saturated KI was added in to above mixture. Mix well and allowed stand for 1
minute.
 In the mixture add 430 ml water and 3-4 drops of starch and mix.
 The mixture was titrated against std. 0.01N Na2S2O3 5H2O until the blue colour just
disappears.
 The blank was titrated similarly in the absence of oil.
3.3 Calculation
A×N×1000
Peroxide value =
Weight of the oil (g)

Where, A= Test – blank N= Normality of Na2S2O3

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 STANDARD TEST METHODS

LAB.STM.OFF.04 Issue 1 Date: 02.10.2022

Title: Estimation of Iodine Value

4.0 Principle
The oil contains both saturated and unsaturated fatty acids. Halogen add across the double
bond of unsaturated fatty acids to form addition compounds. Iodine monochloride is allowed
to react fat in the dark. Iodine get incorporated into fatty acid chain wherever the double
bound exist. The amount of iodine consumed is then determined by titrating amount of iodine
(after adding of KI) with standard sodium thiosulphate and comparing with blank in which
the oil omitted. Hence, the measure of iodine absorbed by oil gives the degree of
unsaturation.
4.1 Reagents
 Hanus iodine solution: Dissolve 8 gm iodine in 500 ml of glacial acetic acid and heat to
get dissolved. Cool and add 1.5ml of bromine.
 15% of potassium iodide (KI) in water.
 Standard 0.1N Na2 S2 O3. 5H2O (standardize against 0.1N K2 Cr2 O7).
 1% Starch.
 Chloroform AR.
4.2 Procedure
 2 g oil was dissolved in 20 ml of chloroform and dissolved it completely.
 25mi of hanus iodine solution was added in the mixture. Mix well and kept the mixture in
the dark for 30 minutes.
 20ml KI was added in the mixture and mix well.
 The mixture was titrated against standard 0.1N Na2 S2 O3. 5H2O using starch as an
indicator with constant stirring to extract the iodine from the chloroform layer.
 The blank was treated similarly without oil

4.3 Calculation
A×N×0.1269×100
Iodine value = gl2 /100g of oil
Weight of oil(g)
Where,

A=Blank – test (ml of Na2 S2 O3)

N= normality of sodium thiosulphate

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 STANDARD TEST METHODS

LAB.STM.OFF.05 Issue 1 Date: 02.10.2022

Title: Determination of Unsaponifiable Matter

5.0 Principle
The oil is completely saponified with alcoholic potassium hydroxide solution and extract
with petroleum ether. The petroleum extract is washed with aqueous then again with water.
The washed ether extract is evaporated and the residue weight. Unsaponifiable matter is this
residue minus the fatty acid present in it, which is determined by titration with sodium
hydroxide solution in alcoholic medium.

5.1 Reagents
 Alcoholic potassium hydroxide solution- dissolved 70g KOH in 90 ml water and add
equal amount of ethyl alcohol. Make up to 1 litter. Allow the mixture to stand overnight,
decant the clear liquid.
 95%ethyl alcohol
 1%phenolphthalein
 Petroleum ether
 Aqueous alcohol
 0.02 standard sodium hydroxide (NaOH) solutions.
 Acetone AR

5.2 Procedure
 5 gm of oil was dissolved in 50ml of alcoholic KOH.
 The mixture was boiled gently but steadily under reflux condenser for 1 hour or until the
saponification was to be completed.
 The condenser was washed with about 10ml of ethyl alcohol.
 The mixture was kept for cooling and transferred it in to separating funnel.
 The transfer was completed by washing the flask first with ethyl alcohol and then with
cold water.
 50 ml of water was added in to the separating funnel followed by an addition of 50 ml of
petroleum ether.
 Insert the stopper and shake vigorously for at least one minute allow settling until both
layer clear.
 The lower layer was transferred and repeat the ether extraction for at least six time more
using 50 ml of petroleum ether for each extraction.
 The ether extracts were collected in separating funnel and it was washed for three time by
25 ml aqueous alcohol.
 Again, the ether layer was washed with 20 ml water until the wash water no longer turn
into pink on edition of a few drops of phenolphthalein solution. Do not remove any ether
layer.

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  The ether layer was transferred to a flask containing a few pieces of pumice stone.
 The mixture was kept on a water bath for the evaporation. The last traces of ether were
removed by placing the flask in air oven at 800 c for about one hour.
 The last traces of moisture were removed by the addition of few mi of acetone and try at
500 c for 30 minutes.

5.3 Calculation

Weigh in g of the free fatty acids in the extract as oleic acid =0.28 V×N

Where,
V= volume in mL of standard sodium hydroxide solution.
N=normality of standard sodium hydroxide solution.

100×(A-B)
Unsaponifiable matter percentage =
W

Where,
A= weight of the residue in g
B=weight of free fatty acids in the extract in g
W=weight of the sample in g

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 STANDARD TEST METHODS

LAB.STM.OFF.06 Issue 1 Date: 02.10.2022

Title: Determination of fatty acids profile

6.0 Principle
Fatty acids are made volatile by convert them in to methyl esters. The conversion of fatty
acids in methyl ester is carried out directly by trans esterification i.e. from glycerol ester to
methyl esters. Esterification can also have done after saponification. The esters are identified
and quantified by injecting into GC – FID and comparing with a set of standard esters.
6.1 Reagents
Petroleum ether (40-600c) AR – Merck Co.
Methanol AR – Merck Co.
Potassium hydroxide AR – Merck Co
6.2 Materials
Borosil stopper glass test tube.
100µl glass syringe
6.3 Apparatus
Gas chromatography (Shimadzu GC – 2014) with FID.

6.4 Condition of the apparatus

 Injector: maintain the injector of the vaporizing type at least 2100C.
 Oven temperature: 2100C.
 Column: 2.1 stainless seal column packed by 15% DEGS (dietylase glycol succinate)
with 2mm ×3mm ID and WAW 80/100 mesh. (Maximum temperature -2000C.)
 Column temperature: 195 C.
 Detector: flame ionization detector (FID)
 Detector temperature: 220C.
 Carrier gas: nitrogen.
 Other gas: 0% Air and Hydrogen.

6.5 Procedure
 The three valves of hydrogen gas, nitrogen gas and air were opened and adjusted the
flow rate at 3kg/cm2, 6kg/cm2 and 6kg/cm2 respectively.
 The instrument was stated and the temperature of the column (oven), injector block and
detector block were adjusted at 1950 C, 2100C and 2200C. respectively.
 The condition instrument was kept for 30 minutes.
 1µl petroleum ether was injected before injecting the sample or standard for washing.
 Then 1 µl sample (FAME) was injected.

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6.6 Preparation of FAME
 8 to 10 drops of oil were taken in clean glass stopper test tube.
 In the test tubes 10 ml petroleum ether (40-600 C) was added.
 In the mixture 0.5 ml 4% methanolic KOH was added.
 The mixture was gently shacked for 30 min.
 The mixture was kept steady for 20 minutes.
 1µl was injected from the upper layer (FAME) in to GC – FID.

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 STANDARD TEST METHODS

LAB.STM.OFF.07 Issue 1 Date: 02.10.2022

Title: Determination of Glucosinolate content

7.0 principle
Brassica oil seed species (Rapeseed – mustard) are important animal and human food
source. mainly because of its high-quality oil and high protein seed meal. however, the high
glucosinolate content in meal make it unacceptable for animal consumption. there is a need
to eliminate antinutritive glucosinolate present in the meal. Determination of induvial or
total glucosinolate content in rapeseed- mustard meal is carried out by advance HPLC
method.
The suggested protocol to determine (quantity) individual or total glucosinolate was faster
(reduction of extraction number and desulfatation time). It was very useful for the analysis a
high number of sample. Rate on other hand analysis costs were reduced (manpower, delay
of desalfatation, costs of HPLC grade solvent). This was more precise and advance method.

7.1 Reagents and materials

1. Rapeseed mustard meal
2. Methanol (HPLC grade)
3. Acetonitrile (HPLC grade)
4. Water (HPLC grade)
5. Sinigrin (internal std., sigma)
6. Helix pomatia sulfafase (tyoe h-1, sigma)
7. DEAE sephadex A – 25 (sigma)
8. 0.2 micro m filters (Biored Co.)
9. biored ion exchange columns
10. Micropipettes and tips HPLC system (agilent 1100) with VWD and data recorder.
11. 50 micro liter syringes.
12. Epperndofs

7.2 Procedure
 200 mg of rapeseed meal was homogenized with 10ml of 750 C methanol/ water (70/30)
and with an internal standard (sinigrin) in mortor pestle.
 The mixture was permanently agitated with a magnetic stirrer for 10 minutes.
 Then centrifuge the mixture for 15 minutes at 5000 rpm.

 1-5 ml of centrifuged extraction of samples was put on the top of an ion exchange
column containing dry deaf sephadex A-25 (25mg) and the ads 10 micro litters of helix
pomatia sulfatase.
 The prepared ion exchange column was kept for minimum 11 hours. it was necessary for
the complete desulfatation in operating condition.

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  The desulfo glucosinolates were eluted and released with 4 × 0.5 ml ultra-pure water
(milli Q form mille pore).
 All the fractions were collected in high quality sterile vials for the analysis.
 Desulfo – glucosinolates were separated by HPLC using an intertsil 3ODS-3 column
(100 ×3 mm, 3 micro m) with water / acetonitrile gradient (from 2% to25% in 35 min).
 Variable wavelength detector (VWD) was used for analysis at 229nm.
 The data were recorded for the results and for the calculation.

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 STANDARD TEST METHODS

LAB.STM.OFF.08 Issue 1 Date: 02.10.2022

Title: Bleaching ability

8.0 Principle
The bleaching of edible oil and fats is a part of refining process of crude oils and fats. Which
removes contaminants that adversely impact the appearance and performance of these
triglyceride based materials. Proceeded generally by degumming and refining process.
Bleaching is required to remove specific detrimental contaminants that are not effectively
removed by these processes before the oil progresses through deodorization.
8.1 Reagents
 Phosphoric acid.
 Caustic soda.
 1%phenolphthalein.
 1.5% Galloon earth
 0.5% Bajaj earth
 0.1%Bajaj carbon

8.2 Procedure
 250 ml beakers taken add 200 ml of crude oil.
 Kept in magnetic stirrer adding 30 ml of water. Mixing thoroughly then stop the stirrer.
 The taken sample heat 450 C reached add 1 ml of phosphoric acid then again heating 55 0
C reached.
 Add caustic soda (approximately 10ml) then the solution neutral checking it will take
cotton to dip phenolphthalein indicate again the cotton to dip the sample pink colour is
formed. The sample is neutralized.
 The solution kept in separate place in 1 hour not distributed.
 The beaker showing two layers is found one layer is oil bottom layer is soap oil. Soap oil
is decanted.
 Balance oil is taken from 100ml oil to heat 1000 C reached temperature cut off. Solution
kept in stir add these three martial 1.5% Galloon earth,0.5% Bajaj earth and 0.1%Bajaj
carbon
 Filter the solution with help of filter paper.
 To take 50 ml of oil to check visible colour

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 STANDARD TEST METHODS

LAB.STM.OFF.09 issue 1 Date: 25.04.2022

Title: Mineral test

5.0 Principle
Method A (Holde's Test) - The presence of mineral oil is indicated by the development of
turbidity when hot distilled water is added to a freshly made alcoholic solution of the soap
formed by the oil.

6.0 Reagents
 0.5N alcoholic KOH
 Ethanol
 Iodine flask 250ml
 DM water

7.0 Procedure
 250 ml of iodine flask taken 1 to 2 drops of oil sample.
 To prepare 0.5N alcoholic KOH solution. 2.5 g in 25 ml of ethanol.
 The heat it with heating mantle the 250ml flask top side air condenser fitted and
releasing the halogens 45mintues heated at maintained 250 C TO 450 C.
 After remove the condenser the iodine flask added 100 ml boiled hot water.
 Turbidity (or) precipitate formed mineral are presented.
 No Turbidity (or) precipitate formed mineral are absence.

8.0 Calculation

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 STANDARD TEST METHODS

LAB.STM.OFF.10 issue 1 Date: 22.04.2022

Title: Determination of moisture content- air over method

9.0 Principle
Moisture content of oils and fats is the loss in mass of the sample on the heating oat 105 to
106 o C under operating conditions specified.

10.0 Reagents
Phosphorus pentoxide.

11.0 Materials
Oil /fats

12.0 Apparatus
Air oven
13.0 Procedure
 Weigh previously dried and tared dish about 5-10 g of oil or fat. Which has been
thoroughly mixed by stirring.
 Loosen the lid of the dish and heat, in an oven at 105 to 106 oC for 1 hour.
 Remove the dish from the oven and close the lid.
 Cool in a desiccator containing phosphorus pentoxide or equivalent desiccant and
weigh.
 Heat in the oven for a further period of 1 h cool and weigh.
 Repeat this process until change in weight between two successive observations does
not exceed 1 mg.
 Carry out the determination in duplicate.

14.0 Calculation

Moisture and volatile matter percentage = W1×100

W
Where,
W1 loss in weigh (g) of the material on drying
W weigh in g of the material taken for test

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 STANDARD TEST METHODS

LAB.STM.OFF.11 issue 1 Date: 22.04.2022

Title: Determination of specific gravity

15.0 Principle
Specific gravity is the radio of density of a substance to the density of a reference substance
(water) equivalently; it is the radio of the mass of the substance to the mass of a reference
substance (water) for the same given volume.

16.0 Reagents
Oil/fats

17.0 Apparatus
Glassware
0
Pycnometer fitted with thermometer of suitable range (with 0.1 or 0.2 C subdivision) or a
density bottle.
Water bath maintained at 30 ± 2.00 C

18.0 Preparation of reagent

 The thermometer should be checked against a standard thermometer calibrated and certified
by nation physical laboratory, new Delhi.
 Carefully clean the pycnometer by filling with chromic acid cleaning solution and letting it
stand for several hours.
 Empty pycnometer and rinse thoroughly with water, fill with recently boiled water,
previously cooled to about 20 0 C and place constant temperature water bath held at 30 0 C.
 After 30 min adjust water level to prepare point on pycnometer and stopper, remove from
bath, wipe dry with chem wipes/clean cloth or towel and weigh.

19.0 Sample preparation

 Melt sample if necessary. filter through a filter paper to remove any impurities and the last
traces of moisture.
 Make sure that the sample is completely dry.
 Cool the sample to 30 0 C or ambient temperature desired for determination.

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20.0 Procedure
 Fill the dry pycnometer with their prepared sample in such a manor to prevent entrapment
of air bubbles after removing the cap of the side arm.
 Insert the stopper, immerse in water bath at 30± 20 C and hold for 30 min.
 Carefully wipe off any oil that has come out of the capillary opening.
 Remove the bottle from the both, clean and try it thoroughly.

 Remove the cap of the side arm and quickly weigh ensuring that the temperature dost not
fall below 30 0 C.

21.0 Calculation

Specific gravity at 30 0 C (g/mL) = A-B

C-B
WHERE,
A-weight in g of specific gravity bottle with oil at 30 0 C.
B- weight in g specific gravity bottle at 30 0 C.
C- weight in g of specific gravity bottle with water at 30 0 C.

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 STANDARD TEST METHODS

LAB.STM.OFF.12 issue 1 Date: 20.04.2022

Title: DETERMINATION OF SPECIFIC GRAVITY

22.0 Preparation of sample :

Melt sample if necessary. Filter through a filter paper to remove any impurities and the last
trace of moisture. Make sure that the sample is completely dry. cool the sample to 300 C or ambient
temperature desired for determination
23.0 Apparatus:
 Pycnometer fitted with a thermometer of suitable range,with 0.1 or 0.20C subdivision or a
density bottle.
 Balance
 Water both maintained at 30 ±2.00C.
 The thermometer should be checked against a standard thermometer calibrated and certified
by national physical laboratory, New Delhi or any other NABL approved institution.
24.0 Standardisation of pycnometer:
Carefully clean the pycnometer by filling with chronic acid cleaning solution and
letting it stand for several hours. empty h water, pycnometer and rinse thoroughly with water,
fill with recently boiled water, previously cooled to about 20 0C and place in constant
temperature water bath held at 300C. after 30 minutes adjust water level to praper point on
pycnometer and stopper, remove from bath, wipe dry with chemwipes/clean cloth or towel and
weight.
25.0 Procedure:
Fill the pycometer with the prepared sample in such a manner to prevent entrapment of
air bubbles after removing the cap of the side arm. Insert the stopper, immerse in water bath at
30 ±2.0 0C and hold for 30 minutes.
Carefully wipe off any oil that has come out of the capillary opening remove the bottle from
the both, clean and dry it thoroughly. Remove the cap of the side arm and quickly weigh weigh
ensuring that the temperature does not fall below 300C.

A-B
0
Specific gravity at 30 C = C-B

Where,
A= weight in gm of specific gravity bottle with oil at 300c.
B= weight in gm of specific gravity bottle at 300c.
C=weight in gm specific gravity bottle with water at 300c.

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 STANDARD TEST METHODS

LAB.STM.OFF.13 Issue 1 Date: 20.04.2022

Title: Determination of Azadirachtin content (A+B)

26.0 Principle
Total Azadirachtin content is extracted from sample using 90%Methanol and detected at 215 nm
by HPLC-UV.
27.0 Reagents

 Methanol – gradient grade HPLC

 Water -HPLC grade
 Acetonitrile – HPLC grade
 Reference standard – working standard of known purity
 Volumetric flasks – 5ml,10ml
28.0 Method of HPLC-programme

 Column – C18,25cm × 4.6 mm stainless steel 5µ particle size or equivalent

 Flow rate - 1.0 ml/min approx.
 Injection volume - 20µl
 Column temperature – 30 degree celcius
 Detector – ultraviolet (UV) – 215nm
 Mobile phase – Acetonitrile :Water (35/65) (v/v)
 Retention time - Azadirachtin A-10-12 minutes (approx.)
 Retention time – Azadirachtin B- 11-13 minutes (approx.)
 Microsyringe - 25µl capacity.

29.0 Preparation of reference standard solution

 Weigh accurately the 2.0 mg working std of known purity into 50 ml volumetric flask and
dissolve in methanol: water (90:10).
 Make up to the mark and shake well.
 Take 1 ml of this solution to make up to 5 ml or 10 ml volumetric flask with 90:10 Methanol:
water using solvent solution.
 Using microsyringe to take 20µl of solution to inject to HPLC system to observe the STD
peak of Azadirachtin (A+B) A-10-12, B-11-13 minutes will found the peaks.
 Note down the peak area of value.

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30.0 Sample Prepararion

 Appreoximately 5 g Neem oil is weighed in a 100ml volumetric flask.

 All the solvents are vacuum filter and sonicator 15 mins after using testing purpose.
 Make up the flask with 90:10 Methanol:Water and keep it sonicator for 15 mintes.
 Keep the flask for 1hour to reach room temperature and shake well.
 Filter the sample using Syringe filter then to take 20µl of sample and inject to HPLC-UV.
 Find out the graph in Azadirachtin content (A+B) peak area

31.0 Calculation

 A1 = Peak area of Azadirachtin in sample solution (Aza A+B);

 A2 = peak area of Azadirachtin in working standard (Aza A+B);
 M1 = mass in g of the sample taken for test;
 M2 = mass, in g of the working standard Azadirachtin; and
 P = purity of working standard.

Azadirachtin content A1 × M2 × p
Percent by mass = A2 × M1

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 STANDARD TEST METHODS

LAB.STM.OFF.13 issue 1 Date: 22.04.2022

Title: Determination of colour.

32.0 Principle:
The method determines the colour of oils by comparison with lovibond glasses of
known colour characteristics. The colour is expressed as the sum total of the yellow and red
slides used to match the colour of the oil in a cell of the specified size in the lovibond tintometer.

33.0 Apparatus:
 Lovibond titometer
 Glass cells
34.0 Procedure:
Melt the sample if it is not already liquid and filter the oil through a filter paper to
remove any impurities and traces of moisture. Make sure sample is absolutely clear and free
turbidity. clean the glass cell desired size with carbon terachoride and allow it to dry. Fill it with
the oil and place the oil in position in the tintometer. Match the colour with sliding red, yellow
and blue colours.

Report the colour of the oil in terms of lovibond units as follows:

Colour reading =(ay+5bR)
Where,
A= sum total of the various yellow slides(Y) used
B= sum total of the various red( R ) slides used
Y=5R is the mode of expressing the colour of light coloured oils; and Y + 10R is
for dark –coloured oils.
Although the yellow and red slide required to match the colour shade of an oil in a tintometer are
assessed separately, it is found that to a certain extent these slide are mutually compensatory.
Consequently different workers may report different values for the yellow and red units for the
same oil and the same works may report different values for the yellow and red units for the oil
examined at different times. To obviate such personal errors a composite factor is used for
checking the colour comprising the sum total of the yellow units and 5 or 10 times the total of
red units as specified for the oil or fat.

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 STANDARD TEST METHODS

LAB.STM.OFF.13 issue 1 Date: 22.04.2022

Title: Determination of melting point of fat

Oil and fats are chiefly mixtures of triglycerides. They do not exhibit either a definite
or sharp melting point. Therefore the melting point does not imply the same characteristics that it
does with pure crystalline substance. Fats pass through a stage of gradual softing before they
become completely liquid. The melting point is therefore defined by the specific conditions of
the method by which it is determined.
35.0 Open – tube capillary method
36.0 Principle:
The melting point is 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.

37.0 Apparatus:
A) Melting point tubes –thin walled with uniform bore capillary glass tube open at both ends
with following dimensisons.

 Length 50 to 80 mm
 Inside diameter 1.0 mm
 Outside diameter 2.0 mm

B) Thermometer with 0.20C subs divisions with a suitable range. The thermometer should be
checked against a standard thermometer that has been calibrated and certified by national
physical laboratory, New Delhi or any other laboratory approved for calibration of
instruments.
C) Beaker with a side tube heating arrangement – thiele melting point tube may be used.
Alternatively a melting point apparatus may also use.

38.0 Procedure:
 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 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 tube.
 Dip at least 3 clean capillary tubes in the completely liquid sample so that sample rises
about 10 mm high in tubes.
 Chill the sample at once by holding the ends of the tubes that contain the sample against
a piece of ice until the fat solidifies. Place the tube in a small beaker and hold it in a
refrigerator at 40C to 100c for 16 hours.

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  Remove the tube from the refrigerator and attach with rubber band to the thermometer
bulb, so that the lower end of the capillary tube and the thermometer bulb are at same
level.
 Suspend the thermometer in 600 ml beaker of cler distilled water. The bottom of
thermometer is immersed in the water to immersion mark.
 Take water at 100C 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 thielr tube at the
rate of 2 0c per min, till the temperature reaches 250c, and thereafter at the rate of
0.50cper 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 determination of as the melting point, provided
that the reading does not differ by more than 0.50C.

Prepared by Approved by

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