A PROJECT REPORT ON

A BRIEF STUDY ON ASSESSMENT OF NUTRITIONAL PARAMETERS

ON VARIOUS FOOD STUFFS

SUBMITTED TO

THE HR

FARE LABS Pvt. Ltd.,

GURUGRAM,

HARYANA

IN PARTIAL FULFILMENT FOR THE

TRAINING OF ASSESSMENT OF NUTRIONAL

PARAMETERS IN FOOD DEPARTMENT

BY

POOJA KASHYAP

UNDER THE SUPERVISION OF

Dr. Meenakshi Tripathi

(Quality Manager)

FARE Labs Pvt. Ltd., Gurugram

DECLARATION FORM
I, POOJA KASHYAP, a student of PhD (Food Science and Nutrition), Banasthali Vidyapith
have completed my one month training work entitled “A BRIEF STUDY ON

ASSESSMENT OF NUTRITIONAL PARAMETERS ON VARIOUS FOOD

STUFFS” successfully from the Chemical Department, FARE Labs under the able
guidance of Dr. Meenakshi Tripathi, Quality Manager, FARE Labs Food Analysis &
Research Laboratory, Gurugram.

I, hereby affirm that the work has been done by me in all aspects. I have sincerely prepared
this project report and the results reputed in this study are genuine and authentic.

POOJA KASHYAP

Name and Signature of Course Coordinator with Date

 TO WHOM IT MAY CONCERN

This is to certify that the dissertation entitled “A Brief Study on Assessment of Nutritional
Parameters on Various Food Stuffs” herewith submitted by Pooja Kashyap, in partial
fulfilment of the requirements for the training of assessment of nutritional parameters in food
department is an authentic record of the training work carried out by her under our
supervision. She has successfully completed her one month project from August to
September 2021.

We wish her good luck and a bright future.

FARE Labs Food Analysis & Research Laboratory

Gurugram
 CERTIFICATE

This is to certify that Pooja Kashyap, a student of PhD (Food Science and Nutrition),
Banasthali University has completed her one-month training work entitled “A Brief Study
on Assessment of Nutritional Parameters on Various Food Stuffs” successfully. She has
completed this work from Chemical Department of FARE Labs Food Analysis &
Research Laboratory, Gurugram under the guidance of Dr. Meenakshi Tripathi, Quality
Manager, FARE Labs Food Analysis & Research Laboratory, Gurugram.

I wish her good luck and a bright future.

Piyush Jai Mallick

 ACKNOWLEDGEMENT

I would like to thank Almighty God for blessing me with His wisdom, understanding and
knowledge. God’s guidance and strength helped me to achieve success in every area of my
life. This thesis appears in its current form due to assistance and guidance of several people.
It gives me great pleasure to express my gratitude to all those who supported me and have
contributed in making this thesis possible.

I express my deep gratitude Mr. D. Mathur (Director), Mr. C.S. Joshi (Director), Dr.
Meenakshi Tripathi (Quality Manager), FARE Labs Food Analysis & Research Laboratory,
Gurugram for providing me the opportunity to work in his research laboratory. I am thankful
to my guide Mr. Piyush Jai Mallick and Miss Chhaya Shakya who had shown his keen
interest in guiding me to carry out my work and above all their overwhelming attitude to help
their students.

I would like to express my sincere gratitude to my senior Mrs. Shalini, Miss Faiqua Firdous,
for their constant guidance, cooperation and support during my training work.

I thank all my friends for their valuable support which helped me to finish my work within
the stipulated period and also thank all the people who are directly or indirectly associated
with the successful completion of my work.

Last but the most important, I would like to express my sincere gratitude from the bottom of
my heart to my father, Mr. R.C. Kashyap, mother, Mrs. Rekha Devi, for their love, support,
encouragement, guidance, motivation and support to my decision that helped me to get
success in every area of my life.

Pooja Kashyap

Dated:

Place: Gurugram
 CONTENTS

S.NO. PARTICULARS

1. ACKNOWLEDGEMENT

2. CONTENTS

3. COMPANY DETAILS

4. ACTIVITIES AND RESPONSIBILITIES

5. LISTS OF EQUIPMENTS

6. LISTS OF PARAMETERS

7. PARAMETERS

8. CONCLUSION
 COMPANY DETAILS

● Founded in 1999, promoted by Mr. Dwijendra Mathur and Mr. Chandra

Shekhar Joshi
● Accredited by NABL and AERB (ATomic Energy Regulatory Board)
● APEDA, Tea board, IOPEPC
● · Recognized by BIS (Bureau of Indian Standards), DSIR (Department of Scientific
and Industrial Research), MOEF & CC (Ministry of Environment Forest and Climate
Change), HSPCB (Haryana State Pollution Control Board), PPCB (Punjab Pollution
Control Board)
● Authorized by FSSAI (Food Safety and Standards Authority of India)
● Certified by OHSAS 18001, ISO9001, ISO27001, ISO14001
 ACTIVITIES AND RESPONSIBILITIES

There are many activities or projects that should be involved and be responsible towards the
works given. The following table is a list of the types of parameters which I was involved in
during the course of my training and the responsibilities and extent of my involvement in
each. From this course I learned how to handle many instruments, how to use apparatus in the
laboratory and do the experiment accurately. All of the parameters were tested, data were
collected, then recorded and analyzed by using the method depending on what type of our
substances.
 LIST OF INSTRUMENTS

INSTRUMENTS FUNCTIONS
WEIGHING BALANCE A weighing balance is a device used to
measure mass or weight. It was used to
measure all samples required for parameters.
The maximum limit of the balance was 200g.

pH METER pH is the measurement of H+ ions activity; It

measures active acidity; pH may be
determined by measuring the electrode
potential between glass and reference
electrode. pH meter is standardized using
standard pH buffers.

CENTRIFUGE Centrifuge is a device that uses

centrifugal force to separate various
components of a fluid. This is achieved by
spinning the fluid at high speed within a
container separating fluids of different
densities. Revolutions Per Minute (RPM) in
regards to centrifugation is simply a
measurement of how fast the centrifuge rotor
does a full rotation in one minute.

VORTEX Vortex is a simple device used commonly in

laboratories have fairly simple mechanism to
 agitate samples and encourage reactions or
homogenization with high degrees of
accuracy.

SPECTROPHOTOMETER A spectrophotometer is an instrument that

measures the amount of photons (the intensity
of light) absorbed after it passes through
sample solution. The basic principle is that
each compound absorbs or transmits light
over a certain range of wavelength, by this
different wavelengths of light can be
analyzed.

SONICATOR “The process in which sound waves are used

for agitating the particles in the solutions.”
These disruptions are used for mixing of the
solutions, to increase the speed of dissolution
of a solid into a liquid, and for the removal of
dissolved gases from the liquids. The
equipment used for sonication is known as a
sonicator.
 LIST OF PARAMETERS

S.NO. PARAMETERS

1. TOTAL REDUCING SUGAR

2. SUCROSE

3. STARCH

4. VITAMIN C

5. VITAMIN C (USP METHOD)

6. PROLINE CONTENT

7. HMF CONTENT

8. PHOSPHORUS
 9. SULPHUR DIOXIDE

10. MOISTURE CONTENT

11. ASH CONTENT

12.
BULK DENSITY

13. pH VALUE

14. PROTEIN

ICUMSA
15.

16. SALT

POLYPHENOL
17.

PARAMETER 1: Determination of total sugar content in food stuffs

Reference: IS 6287, FSSAI Manual 4, Lane Eynon Method

Principle: Lane and Eynon method is based on the principle of reduction of Fehling’s solution
by reducing sugars. Fehling’s solution is a mixture of copper sulphate and alkaline Rochelle
salt (sodium potassium tartrate). Rochelle salt complexes with the cupric hydroxide formed in
alkaline solution and prevents it from precipitation. Reducing sugars reduces the complexed
cupric hydroxide to red, insoluble cuprous oxide under the experimental conditions.

Chemicals and Reagents:

● Ammonia (10%)
● Acetic Acid (10%)
 ● Zinc Acetate (1N)
● Potassium Ferrocyanide (1N)
● Hydrochloric Acid (HCl)
● Fehling’s solution A: Dissolve 69.28 g Copper Sulphate Pentahydrate (CuSO4.5H2O)
and 1ml H2SO4 in distilled water and dilute to 1000 ml.
● Fehling’s solution B: Dissolve 346 g Rochelle salt (Potassium sodium
tartrate: KNaC4H4O6. 4H2O) and 100 g NaOH in distilled water. Dilute to
1000 ml.
● Methylene Blue (2%)
● Phenolphthalein
● 40% NaOH

Procedure:

Weigh the sample (between 5 to 35gm depending on the food product)

Mix well with 100ml distilled water

Add 5ml ammonia of 10% for fat separation. Keep the sample on hold for 15 minutes

Add 5ml Acetic acid of 10%

Add 10ml Zinc Acetate (1N) and 10 ml Potassium Ferrocyanide (1N)

Makeup the volume upto 250ml using distilled water

Filter the sample using filter paper (Whatman Grade 1)

 Take 25-50ml aliquot from the filtrate sample in 100ml volumetric flask

Add 5ml of HCl and Mix well

Heat the sample and keep it overnight for inversion

Add few drops of Phenolphthalein and neutralize the sample using 40% NaOH till color
converts to pink

Makeup the volume upto 100ml using distilled water

Fill the burette with sample and Titrate it against 5ml Fehling A and 5ml Fehling B

End point is dark brick red color

𝐹𝑒ℎ𝑙𝑖𝑛𝑔 𝐹𝑎𝑐𝑡𝑜𝑟 × 𝑉𝑜𝑙𝑢𝑚𝑒 𝑚𝑎𝑘𝑒𝑢𝑝 × 100 × 100

𝑇𝑜𝑡𝑎𝑙 𝑅𝑒𝑑𝑢𝑐𝑖𝑛𝑔 𝑆𝑢𝑔𝑎𝑟𝑠 (𝐴𝐼) =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 × 𝐵𝑢𝑟𝑒𝑡𝑡𝑒 𝑅𝑒𝑎𝑑𝑖𝑛𝑔 × 𝐴𝑙𝑖𝑞𝑢𝑜𝑡
PARAMETER 2: Determination of sucrose in food stuffs

Reference: IS 6287, FSSAI Manual 4, Lane Eynon Method

Principle: Lane and Eynon method is based on the principle of reduction of Fehling’s
solution by reducing sugars. Fehling’s solution is a mixture of copper sulphate and alkaline
Rochelle salt (sodium potassium tartarate). Rochelle salt complexes with the cupric
hydroxide formed in alkaline solution and prevents it from precipitation. Reducing sugars
reduces the complexed cupric hydroxide to red, insoluble cuprous oxide under the
experimental conditions.

Chemicals and Reagents:

● Ammonia (10%)
● Acetic Acid (10%)
● Zinc Acetate (1N)
● Potassium Ferrocyanide (1N)
● Hydrochloric Acid (HCl)
● Fehling’s solution A: Dissolve 69.28 g Copper Sulphate Pentahydrate (CuSO4.5H2O)
and 1ml H2SO4 in distilled water and dilute to 1000 ml.
 ● Fehling’s solution B: Dissolve 346 g Rochelle salt (Potassium sodium tartrate:
KNaC4H4O6. 4H2O) and 100 g NaOH in distilled water. Dilute to 1000 ml.
● Methylene Blue (2%)

Procedure:

Weigh the sample (between 5 to 35gm depending on the food product)

Mix well with 100ml distilled water

Add 5ml ammonia of 10% for fat separation. Keep the sample on hold for 15 minutes

Add 5ml Acetic acid of 10%

Add 10ml Zinc Acetate (1N) and 10 ml Potassium Ferrocyanide (1N)

Makeup the volume upto 250ml using distilled water

Filter the sample using filter paper (Whatman Grade 1)

Fill the burette with filtered sample and Titrate it against 5ml Fehling A and 5ml Fehling B

End point is dark brick red color

 𝐹𝑒ℎ𝑙𝑖𝑛𝑔 𝐹𝑎𝑐𝑡𝑜𝑟 × 𝑉𝑜𝑙𝑢𝑚𝑒 𝑚𝑎𝑘𝑒𝑢𝑝 × 100
𝐵𝑒𝑓𝑜𝑟𝑒 𝐼𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 (𝐵𝐼) =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 × 𝐵𝑢𝑟𝑒𝑡𝑡𝑒 𝑅𝑒𝑎𝑑𝑖𝑛𝑔

𝐴𝑑𝑑𝑒𝑑 𝑆𝑢𝑔𝑎𝑟 𝑜𝑟 𝑆𝑢𝑐𝑟𝑜𝑠𝑒 = [𝐴𝑓𝑡𝑒𝑟 𝐼𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 (𝐴𝐼) − 𝐵𝑒𝑓𝑜𝑟𝑒 𝐼𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 (𝐵𝐼)] × 0.95

PARAMETER 3: Determination of starch content in food stuffs

Reference: IS 4706

Chemicals and Reagents:

● Diethyl Alcohol
● 10% Ethyl Alcohol
● 2.5% diluted Hydrochloric Acid
● Sodium Carbonate
● Fehling’s solution A: Dissolve 69.28 g Copper Sulphate Pentahydrate (CuSO4.5H2O)
and 1ml H2SO4 in distilled water and dilute to 1000 ml.
● Fehling’s solution B: Dissolve 346 g Rochelle salt (Potassium sodium
tartrate: KNaC4H4O6. 4H2O) and 100 g NaOH in distilled water. Dilute to
1000 ml.
● Methylene Blue (2%)
Procedure:

Weigh 0.5gm sample

Wash the sample with 40ml diethyl alcohol using filter paper

Again wash the sample with 150ml ethyl alcohol of 10%

Wash of residue with 200ml distilled water

Heat the residue after adding 200ml of 2.5% diluted HCl in bottom flask equipped with
reflux condenser for 2.5 hours

Cool and neutralize with Sodium carbonate

Filter and transfer to 250ml volumetric flask and make upto volume

Fill the burette with filtered sample and Titrate it against 5ml Fehling A and 5ml Fehling B

End point is dark brick red color

𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 × 𝑣𝑜𝑙𝑢𝑚𝑒 𝑚𝑎𝑘𝑒𝑢𝑝 × 𝑓𝑎𝑐𝑡𝑜𝑟

𝑆𝑡𝑎𝑟𝑐ℎ 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 × 𝐵𝑢𝑟𝑒𝑡𝑡𝑒 𝑟𝑒𝑎𝑑𝑖𝑛𝑔 × (100 − 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒)
PARAMETER 4: Determination of Vitamin C content in food stuffs

Reference: IS 5838, 2,6-dichlorophenol indophenols method

Principle: The hydrogen atoms of the two enolic groups of ascorbic acid (Vitamin C) can be
oxidized readily, as this compound is a strong reducing substance. Measurement of this
reducing property of ascorbic acid under appropriate conditions is the basis of several
methods for determining the quantity of ascorbic acid.

Chemicals and Reagents:

● Trichloroacetic Acid (TCA) Reagent- 10 percent. Dissolve 10gof TCA in 100ml

of water.
● Standard Indophenol Solution — Dissolve 50 mg of sodium 2,6- dichlorobenzenone
indophenol, that has been stored in a desiccator over soda lime, in .50 ml of water
to
 which 42 mg of sodium bicarbonate has been added. Shake vigorously and, when the
indophenol has completely dissolved, dilute it to 200 ml with water. Filter the solution
through a fluted filter paper into an amber glass-stoppered bottle

Procedure:

Weigh 5g of sample in a glass beaker

Add TCA and mix it well

Put the sample in 100ml volumetric flask and make up the volume using TCA

Filter and collect 10ml of filtrate in a conical

Titrate it against blue dye

End point is light rosy color

𝑓𝑎𝑐𝑡𝑜𝑟 × 1000 × (𝐵𝑙𝑎𝑛𝑘 − 𝐵𝑢𝑟𝑒𝑡𝑡𝑒 𝑅𝑒𝑎𝑑𝑖𝑛𝑔)

𝑉𝑖𝑡𝑎𝑚𝑖𝑛 𝐶 (𝑚𝑔) =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡
PARAMETER 5: Determination of Vitamin C content in food stuffs

Reference: USP Method

Chemicals and Reagents:

● Concentrated H2SO4
● Starch Solution
● Iodine Solution

Procedure:

Weigh 500mg of sample in a IV flask

 Dissolve 100ml distilled water

Add 25ml H2SO4

Add few drops of starch

Titrate it against iodine solution

End point is black color

8.806 × 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑𝑖𝑧𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑖𝑜𝑑𝑖𝑛𝑒 × 𝐵𝑢𝑟𝑒𝑡𝑡𝑒 𝑅𝑒𝑎𝑑𝑖𝑛𝑔) × 100

𝑉𝑖𝑡𝑎𝑚𝑖𝑛 𝐶 (𝑚𝑔) =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡

PARAMETER 6: Determination of Proline content

Proline is the dominant amino acid in honey, and has been considered an indicator of honey
quality. The proline content in honey depends on the time the nectar is processed by the bees.
Indirectly, proline levels also reflect botanical origin.

Reference: AOAC 979.20

Chemicals and Reagents:

● 3 percent ninhydrin
● formic acid
● Isopropyl alcohol

2.5-gram sample was weighed in a centrifuge tube

 volume made up to 50 ml in distilled water

Prepared 2 test tube (sample and blank)

0.5 ml sample +0.25 ml formic acid + 1ml of 3 percent ninhydrin was added to sample test
tube

Test tube was put on boiling water bath for 15

minutes Sample was allowed to cool at room

temperature

1.25ml distilled water + 5ml isopropyl alcohol was added in sample test tube and blank test
tube

Sample was vortexed

Readings were taken at 520 nm on Spectrophotometer

Formula used:

Proline content =
𝐶×𝑉 𝑠.𝑤𝑡

Where,

C = final concentration, Cabs - Cblank/ slope of graph

V = volume

S.wt = sample weight

 Samples after boiling on water bath

PARAMETER 7: Determination of Hydroxymethyl furfural content

Reference: AOAC 980.23

HMF is a cyclic aldehyde produced by sugar degradation through the Maillard reaction (a
non- enzymatic browning reaction) during food processing or long storage of honey. The
presence of simple sugars (glucose and fructose) and many acids, as well as minerals, in
honey can further enhance the production of this substance.

Chemicals and Reagents:

● Zinc Acetate 1N
● Potassium ferrocyanide 1N
● Sodium bisulphite 0.2%

Procedure:
 5g sample was weighed in a glass beaker

Dissolved and volume made up to 50 ml (49ml+0.5ml potassium ferro cyanide+ 0.5ml zinc
acetate) in volumetric flask

Filtered the sample using 1 number whatman filter paper into a test tube

Initial 20ml was discarded to rinse 2 test tubes (sample and blank)

5ml Filtrate was transferred in sample test tube and blank test tube

5ml distilled water was added in sample test tube and 5ml sodium bisulphite of 0.2 percent

Readings were taken at 284nm and 336 nm on spectrophotometer

Formula used:

HMF mg/100 g = 𝐴𝑏(284)−𝐴𝑏(336)×14.97×5×10

𝑠𝑤

Where,

Ab= absorbance

Sw= weight of sample

 Samples with Blanks

PARAMETER 8: Determination of Phosphorus content

Reference: AOAC 986.24

Principle: Phosphorus is determined by spectrophotometry on the ashed test portion by

complexing with molybdovanadate reagent.

Chemicals and Reagents:

● Hydrochloric Acid (1:3) – Add 250ml HCl to 750ml water

● Molybdovanadate reagent: Dissolve 20g ammonium molybdate in 200ml hot water
and cool. Dissolve 1.0g ammonium metavanadate in 125ml hot water, cool and add
160ml HCl. Gradually add with stirring, molybdate solution to vanadate solution
and dilute with water to 1L.
● Nitric Acid (70%)

Procedure:
 Weigh 5g of sample and keep in digester for charring

Chearing in muffle furnace for 2 hours

Cool at room temperature

Add 30ml HCl:Water (1:3)

Add 1-2 drops of nitric acid (70%)

Keep it in hot plate

Filter it through filter paper (Whatman grade 1) and make up the volume to 100ml

Pipette out 10ml Ammonium Molybdovanadate salt in two 50ml volumetric flask

Add drops of sample from 10ml pipette to detect color change and if not 10 ml of sample and
make up the volume

Take reading at 400nm

𝐶 × 100 × 𝑑
𝑃ℎ𝑜𝑠𝑝ℎ𝑜𝑟𝑢𝑠 (%) =
𝑆𝑊 × 𝑠𝑎𝑚𝑝𝑙𝑒 𝑓𝑜𝑟 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛

Where,
C = Concentration obtained from the graph

D = dilution

SW = Weight of the sample

PARAMETER 9: Determination of Sulphites

Reference: AOAC 990.28

Principle: Method measures free sulfite plus reproducible portion of bound sulfites, such as
carbonyl addition products, in foods. Test portion is heated with refluxing HCl (ca IM) to
convert sulfite to SO2. Stream of Nitrogen introduced below surface of refluxing solution
sweeps SO2 through water-cooled condenser and, via bubbler attached to condenser, with 3%
H2O2 solution, where SO2 is oxidized to H2SO4 sulfite content is directly related to
generated H,SO, which is determined by titration with standardized NaOH solution. For
verification, sulfate can be determined gravimetrically as BasO4

Chemicals and Reagents:

· Aqueous hydrochloric acid

· Methyl red indicator

· Standardized titrant.-0.010M NaOH.

· Hydrogen peroxide solution

Procedure:

Weigh 50g of sample

Prepare receiver, add 30ml Hydrogen Peroxide (3%) and add few drops of methyl red
indicator

Add (0.01N) NaOH few drops till the receiver becomes yellow in color

Add 400ml of water in the sample in SO2 apparatus

Add 100ml of ethanol (5%) in the sample and 4N HCl 90ml

Connect the assembly of SO2, open the nitrogen valve and put on heating mantle at 70 ० C

Keep it for one and half hour

If SO2 is present, light pink color appears in the receiver

Take the receiver solution and titrate it against 0.01N NaOH

End point is yellow, note the burette reading

Calculation:
 32.03 × 𝑉(𝑁𝑎𝑂𝐻) × 𝑀 × 1000
𝑆𝑂2 (𝑝𝑝𝑚) =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡

PARAMETER 10: Determination of Moisture

Reference: IS 1155, Hot air oven method

Procedure:

Weigh accurately about 5 g of the material in a dish made of porcelain, silica or platinum,
previously dried in an electric oven and weighed.

Place the dish in an electric oven maintained at 105° ± 1°C, for 3 to 4 hours.

Cool the dish in a desiccator and weigh with the lid on.
 Repeat the process of heating, cooling and weighing at half-hour intervals until the loss in
weight between two successive weighing is less than one milligram.

Record the lowest mass obtained.

WEIGHING BALANCE

PARAMETER 11: Determination of Ash content

Weighed accurately 5 g of the sample in a crucible

Put in digester for an hour at 100℃

Transfer it to muffle furnace at 500℃ for 2 hours

Cool the dish in a desiccator and weigh

Again put the dish in Muffle furnace for constant reading and weigh

Formula used:

100
(𝑀2−𝑀)
𝑀1−𝑀
Where

M2 = mass, in g, of the dish with the ash.

M = mass, in g, of the empty dish.

M1 = mass, in g, of the dish with the material taken for the test.

PARAMETER 12: Determination of Bulk Density

Fill the sample upto 50ml level mark of a measuring cylinder

Note down the weight

𝑀𝑎𝑠𝑠
𝐵𝑢𝑙𝑘 𝐷𝑒𝑛𝑠𝑖𝑡𝑦 =
𝑉𝑜𝑙𝑢𝑚𝑒

PARAMETER 13: Determination of pH value

Weigh 1g of sample
 Dissolve 10ml of distilled water

Measure using pH meter and note down the reading

pH METER

PARAMETER 14: Determination of Protein

Total protein, by the Kjeldahl method, is defined as the amount of nitrogen experimentally
found and multiplied by an appropriate conversion factor.

Reference: IS 7219

Principle: The sample is oxidized in the presence of Sulphuric acid and nitrogenous
compounds are converted into ammonium sulphate. Mercury is added to the digestion
mixture as a catalyst and alkali sulphate as a boiling-point elevator. Ammonia is liberated by
adding an excess of alkali and is quantitatively distilled into a measured volume of standard
hydrochloric or Sulphuric acid. The acid not neutralized by ammonia is back-titrated with
standard alkali.

Procedure:

DIGESTION
 Weigh the sample (0.3-1g) according to the type of sample

Weigh catalyst mixture of 0.5g CuSO4 and 10g Na2SO4

Add 25ml conc. H2SO4

Keep Kjeldahl flask in heating mantle at 100℃

End point of digestion is light green color

Cool the sample

DISTILLATION

Add some glass beads

Add 400ml distilled water and mix well

Prepare receiver with 50ml 0.1N H2SO4 and add 8-10 drops of methyl red indicator

Connect distillation assembly and wax all connections

Add 90ml NaOH(40%) in Kjeldahl flask

 Switch on the flame

Collect nearly 250ml distillate

TITRATION

Remove receiver

Titrate the receiver with 0.1N NaOH

End point is yellow color

Note burette reading

Calculate N% and P% according to the following formula

𝑁% =
(𝐵𝑙𝑎𝑛𝑘 − 𝑇𝑉) × 1.4 × 𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦
Where,
𝑆𝑊
N = Nitrogen content

TV = Sample titrate value

𝑃% = 𝐹𝑎𝑐𝑡𝑜𝑟 × 𝑁%

Where, P = Protein content

PARAMETER 15: Estimation of color in sugar (ICUMSA)

Reference: IS 15279

Introduction: The value of the absorbance index multiplied by 1000 is reported as ICUMSA
Colour. The resulting values are designated as ICUMSA Units (IU).

Principle: White sugar is dissolved in distilled water to give a 50 percent sugar solution. The
solution is filtered through a membrane filter to remove turbidity. The absorbance of the
filtered solution is measured at a wavelength of 420 nm and the solution colour is calculated.

Reagents: Only distilled water

Procedure:

Weigh 50g of sugar sample

Add 50ml of water and dissolve it

 Filter the sample

Calculate the TSS of sample using Refractometer (at 20℃)

Calculate absorbance at 420nm using UV-Vis Spectrometer

𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑦 𝐼𝑛𝑑𝑒𝑥 × 1000 = 𝐼𝐶𝑈𝑀𝑆𝐴 𝐶𝑜𝑙𝑜𝑢𝑟

1000 × 𝐴
𝐼𝐶𝑈𝑀𝑆𝐴 =
𝑏×𝑐

Where,

b = cell length (4-10cm)

c = concentration according to TSS

PARAMETER 16: Determination of Salt content

Weigh 0.1-1g of sample depending on the salt content

Dissolve 100ml distilled water

Add few drops of potassium chromate

Titrate against silver nitrate

End point is brick red

PARAMETER 17: Determination of Total Polyphenols

Reference: ISO 14502

Principle: The reagent contains phosphor-tungstic acids as oxidants, which on reduction by

readily oxidized phenolic hydroxy groups yield a blue color with a broad maximum
absorption at 765nm. This is due to the formation of so-called tungsten and molybdenum
blues. The Folin-Ciocalteu phenol reagent reacts with a wide range of polyphenol
compounds and, although the response can vary with the individual components, selection of
gallic acid as a calibration standard enables useful total polyphenol data to be obtained.

Reagents & Chemicals:

● Methanol
● Folin-Ciocalteu reagent (10 %-volume fraction)-Using a pipette, transfer 20 ml of
Folin-Ciocalteu phenol reagent to a 200 ml volumetric flask. Dilute to the mark
with water and mix. Prepare fresh reagent solution daily.
 ● Methanol/Water mixture (70 % methanol-volume fraction)
● Sodium Bicarbonate (7.5 %)

Procedure
Weigh 0.2 g of sample into test tubes.

Add 5ml of hot methanol/water mixture into the test tube.

Then vortex the mixture.

Place the test tubes in the water bath at 70 °C.

Continue heating test tubes for 10 minutes in water

bath.

Remove the extraction tubes from water bath.

Allow to cool at room temperature.

Again mix on the vortex after 10 minutes.

Centrifuge the test tubes at 3500 rpm for 10

minutes. Then carefully supernatant into a graduated

tube. Repeat the extraction process from 1 to 10

steps.

Combine the extracts.

Then make up the extract to 10ml with cold methanol/water extraction mix and mix the
contents.
Using a pipette, transfer 1ml of the sample extract into 100 ml volumetric flask.
 Dilute to the mark with distilled water and then mix

Using a pipette, transfer 1 ml of water in duplicate into separate tubes. These are reagent
blanks.

Using a pipette, transfer 1 ml of diluted sample extract in duplicate, into separate tubes.

Using a pipette, add 5 ml of dilute Folin-Ciocalteu phenol reagent into each tube and mix

Within 3 min to 8 min after the addition of the dilute Folin-Ciocalteu phenol reagent, pipette
4 ml of sodium carbonate solution into each tube. Stopper and mix.

Allow to stand at room temperature for 60 minutes.

Measure the optical densities at 765 nm.

Calculation

The total polyphenol content, wT, expressed as a percentage by mass on a sample dry matter
basis, is given by the formula:

𝐶 × 100
𝑇𝑜𝑡𝑎𝑙 𝑝𝑜𝑙𝑦𝑝ℎ𝑒𝑛𝑜𝑙 (𝑝𝑝𝑚) =
1×𝑊

Where,
C= concentration obtained from the graph
W= mass in gram of the material taken for the test
 CONCLUSION

The experience and knowledge during the training in the chemical department at FARE Labs
was great. This department has a superb work culture, great minds and very high quality of
work. At the laboratory, they provide many instruments and their trainee will conduct the
entire instrument on their own. During the internship, I was introduced and learned to handle
equipment such as UV-Vis Spectrophotometer. The knowledge and skills get from the
training will be used to apply for further work. Working with different people was a rare
chance and it was another opportunity to make friends and share ideas. During the internship
I saw that the faculty people are really hardworking and very helpful. They will help us until
the problem is solved. Besides that, when trainees do the training at the laboratory they not
only get the knowledge during working but they also will learn how to be independent and
survive with different types of people, their culture, lifestyle and language.