SUBMITTED BY :- SANYA VASHISTH

Btech Food Technology,

Lady Irwin College,

Delhi University.

TECHNIQUES USED IN CHEMICAL ANALYSIS OF FOODSUMMER INTERNSHIP REPORT

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PREFACE

This report document the worl done during the summer internship at SHRIRAM INSTITUTE FOR INDUSTRIAL RESEARCH (NEW DELHI).

The report first shall give an overview of the tasks completed during th e period of internship with technical details

I have tried my best to keep report simple and technically correct. I hope I have succeeded in my attempt

SANYA VASHISTH

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TO WHOSOEVER IT MAY CONCERN

This is to certify that Miss Sanya Vashisth, 2nd year, Btech Food Technology at Lady Irwin College, Delhi University has successfully completed her 15 days internship from 1st june 2015 to 15th june 2015 at SHRIRAM INSTITUTE FOR INDUSTRIAL RESEARCH (NEW DELHI ).

During the period of her internship program with us, we found her sincere, hard working, technically sound and result oriented. She worked well as part of team during her tenure.

We have given her the golden opportunity for her better future.

Dr. S.K. Nayak Dr. M.L. AggarwalSr. Scientist Dy. Director

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CONTENTS

I. Acknowledgement

II. Shriram Institute- At a Glance

III. Introduction

IV. Determination Of Moisture In Mustard Oil By Hot Air Oven Drying Method

V. Determination of moisture in cumin seeds by Dean And Stark method

VI. Determination of protein content by Kjeldahl method

VII. Determination of crude fat in animal feed by soxhlet extraction method

VIII. Determination of crude fibre in animal feed

IX. Determination of total ash content in animal feed

X. Determination of acid insoluble ash in animal feed

XI. Determination of volatile oil in spices

XII. Determination of free fatty acid and acid value in mustard oil

XIII. Determination of colour in mustard oil by Lovibond Tintometer

XIV. Determination of iodine value in mustard oil

XV. Determination of saponification value in mustard oil

XVI. Determination of unsaponificable matter in mustard oil

XVII. Determination of allyl isothiocyanate in mustard oil

XVIII. Determination of peroxide value in mustard oil

XIX. Determination of specific gravity of mustard oil

XX. Determination of copper, zinc, lead and cadmium in food products by

Atomic Absorption Spectroscopy

XXI. Determination of cholesterol content in ghee by GC

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ACKNOWLEDGEMENT

I am thankful to Lady Irwin College, Delhi University for providing me an opportunity and an open platform for learning

I would like to thank Dr. K.M. Chacko, Director, Shriram Institute for Industrial Research, New Delhi, for providing me this valuable opportunity to undergo this training programme.

I express my heartfelt gratitude to Dr. M.L. Aggarwal, Deputy Director (ASD/BIO) and Col. C.S. Ghosh, Deputy Director (Admin), SRI ,Delhi, for providing me an opportunity to undertake this training in Food And Farm Lab of SRI, Delhi.

I am deeply indebted to Dr. S.K. Nayak, Sr. Scientist, SRI ,Delhi, who guided me time to time in learning the skills for testing and analytical studies in the field of Food Science.

I would like to express my heartfelt gratitude to Dr. Sarabjeet kaur, Radhika Sharma, Neha Gupta, Dr. Anita Gaur , Dr. Veena Balyan , Pankaj And Shyam Singh Mehra, scientists of Food and Farm Lab, SRI, Delhi, for their valuable guidance and encouragement during the course of training programme.

I am thankful to all the staff members of SRI, Delhi for their help and knowledge they have instilled in me.

I would like to express my heartfelt thanks to my beloved parents for their blessings, love and support.

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Shriram Institute – At a Glance

ProfileShriram Institute for Industrial Research (SRI) is an independent, self sustaining, not-for-profit multidisciplinary contract research institute conducting research and development in the areas of special significance to industry, government agencies and other organizations. SRI is committed to develop, innovate, analyse and apply technology for products and processes.

SRI also brings its innovations to the marketplace by licensing its technologies and helps in establishing production units for the interested clients. 

SRI founded in 1947 by an illustrious founder Lala Shriram, started functioning in 1950. Lala Shriram believed that if India was to catch up with the rest of the world, it was necessary to understand existing technology and innovate it through research. 

SRI's strengths have been its staff , a knowledgeable, expert and experienced Governing Board and an innovative management. 

SRI is operational from Delhi and Bangalore. 

SRI's thrust areas are Materials Science, Analytical Science, Life Science, Irradiation of Medical and Surgical products and Quality Assurance. 

Recent thrust areas in material science includes Blood bags, Cactus Latex based products, Biomaterials, Materials for Aero Space Applications, Polymers for Electronics, Hightech Adhesives, Polymers, Composites, Specialty Chemicals, Renewable resources, Radiation based Technologies, Herbal Products, Waste Utilization, Technical Consultancy etc. 

The Analytical Sciences Division provides prompt, precise and dependable analytical services in the fields of metal and minerals, rubber and plastics, building materials, paper, leather and textiles, chemicals and agro chemicals, food and pharmaceuticals, petroleum products, home appliances and Microbiological studies. The Division standardises develop and validate new methods for analysis . It provides assistance to customers to ensure the quality of products laid down by various certifying agencies and statutory bodies. It also provides Calibration services. 

SRI's Life Sciences Division conducts research on various aspects of Environment including Biological Impact Studies, Rapid Environmental Impact Studies and Comprehensive Environmental Impact Studies. The Division undertakes

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toxicological studies to establish safety levels of chemicals, agrochemicals and herbal products 

Shriram Applied Radiation Centre, SARC, established with the technical assistance of BARC, conducts research in polymer modifications and undertakes irradiation of life saving medical and surgical products and spices. 

SRI's uncompromising commitment to excellence in research is underscored by its own Quality Assurance Division which ensures that the Institute itself adheres to the highest standards of scientific investigation and service.

Research & DevelopmentSince its inception, SRI has been maintaining its status as an independent, self-supporting contract research organization. It has been recognized as a pioneer contributor to Indian industry through its efforts with technologically innovative applied research approach.Over the years, the R&D activities of SRI have been expanded covering different areas of materials. The present activities of research involve:

Identification of new product, application, process and technology Development of product, process and technology Improvements in the existing product and process Scale up studies to take up development work from lab scale to

commercial scale Cost effective measures either by suggesting alternative route or process

of manufacturing of a particular product Rendering technical help in preparation of product, application and safety

data sheet for capturing market Providing opinion report on the existing product as well as process Product differentiation etc.

The areas in which R&D is being actively pursued are quite varied from polymers, health care, waste utilization to food, agricultural and herbal products

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About Food and Farm Laboratory

Thrust areas

» Quality evaluation of various raw and processed food products

» Quality evaluation of food additives and food packaging materials

» Method development and validation of analytical techniques

» Studies on product differentiation

» Shelf life / stability studies of products at both ambient and accelerated

conditions

» Inspection and certification of product quality

» Certification of packaging materials for food products

» Certification of materials used for serving food products

» Certification of organic food

» Evaluation of contaminants in foods

» Development of food products for special purpose

INTRODUCTION

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Food is any substance which, when consumed orally by humans is utilized by the body in its normal metabolic activities. Food contains various nutrients such as proteins, carbohydrates, fats, minerals, vitamins etc., essential for the growth and healthcare of our body. All the nutrients get digested in our body and as a result they contribute to the wellbeing of our body. In addition, food may contain several indigestible materials which aid in peristalsis. Water present in food serves as a vehicle for transporting food and its constituents throughout the process of digestion into different organs of the human body.

Water is the carrier for substances getting out of the food into the human blood, besides being the medium in which digestion process takes place. Further, water also carries the remaining of the digestion process i.e. waste materials from body and assists in the regulation of body temperature and other components such as flavouring agent, condiment, colour, etc., which aid in food acceptability. By the way, remember that the humans consume a significant amount of water for survival and since the water is also consumed orally, water is a food product. Food either raw or processed can be categorized into different groups: (a) Agricultural products (Cereals, pulses, fruits and vegetables),(b) Milk and dairy products, (c) Vegetable oils and fats, (d) Meat, fish and egg products, (e) Spices and condiments, (f) Sugar and confectionery (g) Alcoholic and non-alcoholic beverages.

It is often desired to analyze a given food product any of the purposes as stated below:(i) Nutritional labelling or proximate analysis which involves the determination of moisture, protein, fat, carbohydrates, fibre, etc.(ii) Determination of purity of a food products, and(iii) Estimation of impurities present in the raw or the processed food product.

In case of proximate analysis of foods, the methods may vary for different categories of food products. Therefore, the analytical methods need to be validated each time they are being used for different categories of food products. Analysis of food plays an important role not only for the assessment but also for the maintenance of food quality and safety, both for the industry manufacturing food products and for enforcement authorities engaged in ensuring the food safety and quality at the national and international levels. During earlier times, the food analysis was concerned more with the food adulteration but now-a-days there is an increasing tendency to examine food from a more positive view point. As a result, processed foods are produced within the limits of prescribed manufacturing formulations, set also to comply

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with legal or other requirements. In many food laboratories, most of the routine works is confined to proximate analysis and the analysis of additives and contaminants, which is required at different stages of food processing right from farm to the finalproduct. While most of the proximate analysis can be undertaken using wet chemical analysis, the analysis of additives and contaminants at very low level has necessitated the development of instrumental techniques, which are suitable for rapid assessment and control. The instrumental techniques are also required for the purpose of evaluating the food products from the point of view of contaminants and toxicants All sorts of analytical techniques are necessary in the development of food products and also in controlling the quality of food products. Knowledge of chemical composition of food products is important again not just for the purpose of the health and well being but also from the point of view of the health and safety of the consumers. Knowledge of the physico -chemical properties of foods is thus, important to ensure the health and health safety of the consumers. Besides this, the physico-chemical properties of food products are useful to manufacturers in understanding the importance of various nutritional constituents so that these may be maintained or improved during processing. The knowledge of principles of different food analysis techniques is useful to food analysts during select ion of appropriate techniques for analyzing a particular food. The compilation of physical and chemical techniques in this unit would be helpful to the graduate level students for understanding the basic principles of food analysis for the various physico –chemical parameters.

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Determination Of Moisture In Mustard Oil By Hot Air Oven Drying Method

Water, the simplest of all constituents of foods, is one of greatest concern to producer, manufacturer, processor, consumer and a food analyst. In cereals, pulses and flour, the moisture content usually ranges from 10 to 15%. In fruits and vegetables, the water content may be as high as 95%, and in starchy vegetables viz. potato and beans, it is normally 70 to 80%. Milk contains an average of 87% whereas lean meat and fish muscles contain about 50 to 70% moisture. Such a wide variation in moisture content may not a concern from the point of view of health safety of the raw and fresh food but it certainly is a matter of concern when one undertakes the processing of food products for value addition and for enhancement of the shelf-life. Understanding the variation of the moisture content with time during the period when the product is on the shelf, one can assess the quality and determine the shelf-life of the product. The accurate determination of moisture poses many challenges. One of the major problems is extracting the complete quantity of water from the food sample, thereby resulting in error in reporting the moisture content; lower than the actual moisture content. On the other hand, harsher conditions if applied to the food products although may result in removing all the moisture but simultaneously it would cause decompositionof the food product thereby resulting into total weight loss and thus again may giveinaccurate results. Most of the methods for the estimation of water in foods depend on the loss in weight on heating. This method is not suitable for the determination of moisture in foods like milk products or mineral mixture.

Principle

Determination of the loss in mass on drying of a given material under specified condition gives a measure of moisture present in oil.

Requirements

Apparatus

Moisture Dish - made of porcelain, silica, glass or aluminium.Oven - maintained at 105°C.Desiccator

Procedure

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Weigh accurately about 5 g of the prepared sample in the moisture dish, previously air dried in the oven and weighed. Place the dish in the oven maintained at 1051°C for 4 hours. Cool in the desiccator and weigh. Repeat the process of drying, cooling and weighing at 30 min intervals until the difference between the two consecutive weighing is less than 1 mg. Record the lowest weight.

Calculation

Moisture, % by mass = -100 (W1-W2) ---------------- W1-W

Where,W1 = weight, in g, of the dish with the material before drying,

W2 = weight, in g, of the dish with the material after drying, andW = weight, in g, of the empty dish.

Observation

Empty weight

Dish + sample Dish + dried sample

Moisture

W W1 W2 %70.7271 82.3912 82.3662 0.214273.9614 83.402 83.3782 0.2521

Result

The moisture content of mustard oil came out to be 0.23 %

Precautions

Use a calibrated analytical balance capable of weighing to an accuracy of 0.001g.

Use Grinding mill that is (a) made of material which does not absorb moisture; (b) easy to clean with as little dead space as possible; (c) able to grind rapidly and uniformly, without appreciable development of heat and, as far as possible, without contact with the outside air.

Use dish having an effective surface area enabling the test portion to be distributed so as to give a mass per unit area of not more than 0.3 g/cm2.

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Determination of moisture of cumin seeds by Dean & Stark method

The loss in weight on heating is not entirely due to the moisture content but may also result due to the loss of volatile substances which are present in most of the foods. Most of the spices contain notable quantities of volatile oil which pass off with the water. The moisture content of spices and oils/fats containing 2% or more of water may be determined by Dean & Stark distillation method using xylene, toluene or heptane.

Principle

This method is based on the principle that during heating, water and any immiscible solvent (toluene or xylene) distil off together at a constant ratio (azeotropic property) at a temperature lower than the boiling point of either component. As water is denser than toluene/xylene, the water is collected in the receiver measuring tube where it separates from the extracting solvent.

RequirementsApparatus

Dean & Stark apparatus

Heat source- an electric heater provided with a sliding rheostat or other means of heat control.

Copper wire: long enough to extend through the condenser, with one end twisted into a spiral. The diameter of the spiral should be such that it fits within the graduated portion of the receiver and yet may be moved up and down.

Reagents

Potassium dichromate- sulphuric acid cleaning solution (Chromic acid solution)

Xylene or toluene

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Figure 1. Dean & Stark Apparatus

Procedure

Clean the entire apparatus with potassium dichromate-sulphuric acidcleaning solution to minimize the adherence of water droplets to thesides of the condenser and the receiver. Rinse thoroughly with water anddry completely before using. Place the specified quantity of material,accurately weighed, in the distillation flask, add an equal volume of

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xylene or toluene, as desired, or at least 100 ml if less than 100 g of the material is used, and swirl to mix. Assemble the apparatus and fill the receiver with the solvent by pouring it through the condenser until it begins to overflow into the distillation flask. Insert a loose cotton plug in the top of the condenser to prevent condensation of atmospheric moisture within the tube. In order that the refluxing may be under control, wrap the flask and the tube leading to the receiver with asbestos cloth. Heat the flask so that the distillation rate is about 100 drops per minute. Purge the reflux condenser occasionally during the distillation with 5 ml portions of xylene or toluene to wash down any moisture adhering to the walls of the condenser. The water in the receiver may be made to separate from the xylene or toluene by moving the spiral copper wire up and down in the condenser and receiver occasionally, thus causing the water to settle at the bottom of the receiver. Reflux until the water-level in the receiver remains unchanged for 30 minutes and then shut off the source of heat. Flush the condenser with either xylene or toluene or cyclohexane, as required, making use of the spiral copper wire to discharge any moisture droplets. Immerse the receiver in water at about 27°C for at least 15 minutes or until the xylene or toluene or cyclohexane layer is clear, and then read the volume of water.

Calculation V 100Moisture, % (v/m) = ----------- W

Where,V = volume, in ml, of water collected, andW = weight, in g, of the test sample taken.

Observation

Water collected

weight of sample taken

moisture

V (ml) W (g) %0.5 10 4.9943

Results & Inference

The moisture content in cumin seeds was 4.99%

Precautions

Sample should be properly ground and passed through sieve.

Calibrated dean and stark apparatus should be used.

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Correction factor for volume in calculation should be used while reporting the results.

The apparatus should be properly cleaned and dried before use.

Use calibrated receiver tube for collection of water

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Determination of protein content by Kjeldahl method

The Kjeldahl method has a wide acceptance for the determination of protein in food products. The protein content of foods is usually calculated from total nitrogen by multiplying with a suitable conversion factor that is based upon the nitrogen % present in a particular protein.

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 to give a measure of the nitrogen content in the sample.

Equations

Carbohydrate + Protein + Fat (NH4)2SO4 + CO2+ SO2 + H2O

(NH4)2SO4 + 2NaOH 2NH4OH + Na2SO4

2NH4OH + H2SO4 (NH4)2SO4 + 2H2O

Requirements

Apparatus

For Digestion

Kjeldahl flasks (500 to 800 ml capacity)A heating device (heater/burner)

For Distillation(A) Round bottom flask (1 litre capacity)(B) Splash head

Neutralization(C) Condenser (Allihn type)(D) Trap(E) Beaker (500 ml capacity)

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(F) Receiving funnel

Reagents

Concentrated Sulphuric Acid, AR Grade

Potassium Sulphate or Anhydrous Sodium Sulphate, AR Grade

Sodium Hydroxide Solution - Dissolve about 450 g solid sodium hydroxidein distilled water, cool, and dilute for 1 litre. The specific gravity shouldbe at least 1.36 at 20°C.

Hydrochloric or Sulphuric Acid, Standard Solution: (0.1N or 0.5N).Prepare the standard solution as discussed in experiment No.1 (part-B).Standardize against sodium hydroxide standard solution.

Sodium Hydroxide Standard Solution - 0.1 N. Standardize against primarystandard and against standard acid solution.

Methyl Red Indicator - Dissolve 1 g methyl red in 200 ml alcohol.

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Figure 2. Nitrogen distillation assembly

Procedure

DigestionAccurately weigh 0.7 to 2.2 g of the sample into the digestion flask. Add 0.7g mercury oxide or 0.65 g mercury and 15 g powdered potassium sulphate or anhydrous sodium sulphate, and 25 ml sulphuric acid. Ratio of salt to acid (m/v) should be approximately 1: 1 at the end of digestion for proper temperature control. Digestion may be incomplete at a lower ratio and nitrogen may be lost at a higher ratio. Each gram of fat consumes 10 ml and each gram of carbohydrate consumes 4 ml sulphuric acid during digestion. Place the flask in an inclined position on a heater and heat gently until foaming ceases. A small amount of paraffin or silicon antifoam may be added to reduce foaming. Boil vigorously until the solution becomes clear and then continue boiling it for 1 to 2 hours.

DistillationCool, add about 200 ml distilled water, and in order to avoid complex formation, add 25 ml of the sulphide or thiosulphate solution. Mix to precipitate the mercury. Add a few zinc granules to prevent bumping, incline flask, and add

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without agitation 25 g of sodium hydroxide as solid or equivalent as solution, to make solution strongly alkaline (thiosulphate or sulphide solution may be mixed with the sodium hydroxide solution before addition to the flask). Immediately connect flask to distillation bulb or trap on condenser, and, with tip of the condenser immersed in a measured quantity standard acid (usually 50 ml, 0.5 N or an appropriate quantity of 0.1 N ) in the receiver, rotate flask to mix the contents thoroughly; then heat immediately until all ammonia has distilled over ( at least 150 ml distillate ). Lower the receiver before stopping distillation and wash tip of condenser with distilled water. Back titrate excess acid with standard 0.1 N sodium hydroxide, using methyl red as indicator. Correct for blank determination in reagents.

Blank - Conduct determinations using all reagents and 2 g of sugar.

Calculation (B - S) x N 1.4 KProtein, % by mass = ------------------------- W

where,B= volume, in ml, 0.1 N alkali used for titration for blank,S= volume, in ml 0.1 N alkali used for titration for sample,N= normality of alkali used for titration,K= Kjeldahl factor, andW= weight, in g, of sample taken for test.

Inference

Duplicate determinations of the nitrogen should agree within 0.05% nitrogen. Appropriate conversion factor for protein from nitrogen should be used for specific food samples. The following Kjeldahl factors are used for different food products in converting nitrogen to protein. S. No. Types of food material Kjeldahl factor 1. Rye, oat meal, whole wheat 5.83 2. Wheat flour & its products viz. bread, Macaroni,spaghetti, 5.70 3. Maize, rice polish, pulses, tea, cocoa, coffee, malt, beer, etc 6.25 4. Groundnut, brazil nut 5.46 5. Cashew, coconut & other tree nuts, sesame, safflower, sunflower, castor, cottonseed, linseed 5.30 6. Milk & milk products, margarine 6.38

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7. Egg whole, egg powder, 6.68

Precautions

Sample to be analyzed should be homogeneous.

Determine the strength of NaOH before use.

Sample should be checked for complete digestion through colour and there should not be any presence of carbon particles adhering to the neck of Kjeldahl flask.

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Determination of crude fat in Animal feed by Soxhlet extraction method

The crude fat content can be conveniently determined in foods by extracting the dried and ground material with petroleum ether or diethyl ether in Soxhlet extraction apparatus.

Principle

Extraction of the crude fat is carried out either with petroleum ether or diethyl ether in a Soxhlet unit followed by volatilization of the solvent after extraction and determination of the mass of the residue.

Requirements

ApparatusSoxhlet apparatus

Reagent

Diethyl Ether — anhydrous or Petroleum ether (bp. 60-80C)

Procedure

Extract 2 g of the ground material in a continuous extraction apparatus with ether for 18 hours. Remove the ether by distillation, followed by blowing with a stream of air, with the flask on a boiling water bath and dry in an oven at 110 ± 1°C till the loss in mass between two successive weighings is less than 2 mg. Shake the residue with 2 to 3 ml of ether at room temperature, allow to settle and decant the ether. Repeat the extraction until no more of the residue dissolves. Dry the flask again until the loss in mass between two successive weighing is less than 2 mg. Record the final mass.

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Figure 3. Soxhlet extraction apparatus.

Calculation

(M1-M2) 100Crude fat, % by mass = ------------------ M

Where,M1 = mass, in g, of the Soxhlet flask with the extracted fat,M2 = mass, in g, of the empty Soxhlet flask, andM = mass, in g, of the material taken for the test.

Observation

sample weight

empty weight of soxhlet

soxhlet + extracted flask

Fat

M (g ) M (g) M (g) %4.6746 112.4822 112.714 4.958

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74.5668 110.4556 110.6998 5.347

2

RESULT

The crude fat present in animal feed is 5.15 %

Precautions

The fat/oil obtained after drying should be clear and free from any particles. If the presence of particulate matter observed in the fat, the fat should be dissolved in petroleum ether again and filtered into other conical flask and dried.

If the charring of fat is observed during drying, discard the fat and repeat the experiment.

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Determination of crude fibre in animal feed

The seed coat of oilseeds, nuts and pulses, peels of fruits and vegetables, and bran of cereals contain considerably more fibre than the softer edible inner tissues. So, the fibre content can be employed for assessing the proportion of outer coating of plant materials. The digestibility of food varies inversely with the crude fibre content.

Principles

The crude fibre represents the cell wall material left after boiling with dilute acid and alkali. It contains a mixture of cellulose, lignin and pentosans, together with sand, silica and other mineral matter locked in the tissues and a little nitrogenous matter.

Requirements

Reagents

Sulphuric Acid — 0.255 N [1.25 percent ( m/v )], accurately prepared.

Sodium Hydroxide Solution — 0.313 N [1.25 percent ( m/v )], accurately prepared.

Procedure

Weigh accurately about 2 g of the dried material and extract the fat for about 8 hours with petroleum ether or hexane, food grade, using a Soxhlet or other suitable extractor or use the residue from the crude fat determination. Transfer the fat-free dry residue to a one-litre conical flask. Take 200 ml of dilute sulphuric acid in a beaker and bring to the boil. Transfer the whole of the boiling acid to the flask containing the fat free material and immediately connect the flask with a reflux water condenser and heat, so that the contents of the flask begin to boil within one minute. Rotate the flask frequently, taking care to keep the material from remaining on the sides of the flask out of contact with the acid. Continue boiling for exactly 30 minutes. Remove the flask and filter through fine linen (about 18 threads to the centimeter) held in a funnel, and wash with boiling water until the washings are no longer acid to litmus. Bring to the boil some quantity of sodium hydroxide solution under a reflux condenser. Wash the residue on the linen into the flask with 200 ml of the boiling sodium hydroxide solution. Immediately connect the flask with the reflux condenser and boil for exactly 30 minutes. Remove the flask and immediately filter through the

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filtering cloth. Thoroughly wash the residue with boiling water and transfer to a Gooch crucible prepared with a thin but compact layer of ignited asbestos. Wash the residue thoroughly first with hot water and then with about 15 ml of 95 percent (by volume) ethyl alcohol. Dry the Gooch crucible and contents at 105 ± 1°C in the air-oven to constant mass. Cool and weigh. Incinerate the contents of the Gooch crucible at 600 ± 20ºC in a muffle furnace until all the carbonaceous matter is burnt. Cool the Gooch crucible containing the ash in a desiccator and weigh.

Calculation

100 (M1-M2)(100-f)Crude fibre, % by mass = -----------------------------(on dry matter basis) M

Where,M1 = mass in g of Gooch crucible and contents before ashing,M2 = mass in g of Gooch crucible containing asbestos and ash,f = crude fat (on moisture-free basis), percent by mass, andM = mass in g of the dried material taken for the test.

Observation

sample weight

mass before ashing

mass after ashing

crude fibre

M (g ) M1 (g) M2 (g) %2.0204 31.9436 31.6997 12.07192.0405 29.889 29.443 11.9667

Result

The crude fibre present in animal feed is 12.02 %

Precautions

The fineness of the particles has an important bearing on the result accuracy. Hence, the sample should pass through a 1 mm sieve.

The concentration of sulphuric acid and sodium hydroxide is very important for the separation of other food constituents from crude fibre.

While dilution of sulphuric acid, always add acid to water but not water to acid.

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Determination of total ash contents In animal feed

Total ash refers to the inorganic residue remaining after total incineration of organic matter present in food. Because of its non variable nature, the ash content can be used for assessing the quality of food product with respect to the presence of inorganic substance in it.

Principle

Ash refers to the inorganic residue remaining after total incineration of organic matter. The ash content is determined from the loss of weight, which occurs from complete oxidation of sample at a high temperature 500 to 600°C through combustion and volatilization of organic materials.

Requirements

Apparatus

Flat-Bottom Dish - of stainless steel, porcelain, silica or platinum.

Muffle Furnace maintained at 55010°C.

Desiccator

Procedure

Weigh accurately about 3 g of the material in the dish, previously dried in an air-oven and weighed. Heat the dish gently on a flame at first and then strongly in a muffle furnace at 55010°C till grey ash results. Cool the dish in a desiccator and weigh. Heat the dish again at 55010°C for 30 minutes. Cool the dish in a desiccator and weigh. Repeat this process of heating for 30 minutes, cooling and weighing until the difference between two successive weighing is less than 1 mg. Record the lowest weight.

Observation

Calculation

100 (W2-W1)Total ash, % by mass = ----------------- W

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Where,W1 = weight, in g, of the empty crucible,W2 = weight, in g, of the crucible with ash, andW = weight, in g, of the test sample.

Observation

sample weight

weight of empty crucible

mass after ashing

ash

W (g ) W1 (g) W2 (g) %2.0001 33.7174 33.8912 8.689

6 Result

The total ash present in animal feed is 8.7%.

Precautions

The temperature of ashing is varied from product to product.

The use of higher temperature for ashing than the required temperature results low value of ash due to the loss of some inorganic matter like inorganic phosphate, sodium, etc.

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Determination of acid insoluble ash in animal feed

The acid insoluble ash is a measure of the sandy matter and plant body parts including calyx, leaves, etc, which contain higher content of noncombustible acid insoluble matter.

Principle

Acid insoluble ash is determined by dissolving ash in dilute hydrochloric acid (10% m/m), the liquid filtered through an ashless filter paper and thoroughly washed with hot water. The filter paper is then ignited in the original dish, cooled and weighed.

Requirements

Apparatus

Flat-Bottom Dish - of stainless steel, porcelain, silica or platinum.

Muffe Furnace - maintained at 55010°C.

Desiccator

Reagent

Dilute Hydrochloric Acid (5N)

Procedure

To the ash contained in the dish, add 25 ml of dilute hydrochloric acid, cover with a watch-glass and heat on a water-bath for 10 minutes. Allow to cool and filter the contents of the dish through a Whatman filter paper No. 42 or its equivalent. Wash the filter paper with water until the washings are free from the acid and return them to the dish. Keep it in an oven maintained at 100 f 2°C for about 3 hours. Ignite in a muffle furnace at 550 f 10°C for one hour. Cool the dish in a desiccator and weigh. Heat the dish again at 55010°C for 30 minutes, cool in a desiccator and weigh. Repeat this process of heating for 30 minutes, cooling and weighing until the difference between two successive weighing is less than 1 mg. Record the lowest weight.

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Calculation 100 (W2-W1)Acid insoluble ash, % by mass = ----------------- W

Where,W1 = weight, in g, of the empty dish,W2 = weight, in g, of the dish with acid insoluble ash, andW = weight, in g, of the sample.

Observation

sample weight

weight of empty crucible

weight with acid insoluble ash

ash

W (g ) W(g) W (g) %2.0001 33.7174 33.7352 0.89

Result

The percentage amount of acid insoluble ash present is 0.89% .

Precautions

Ashing should be proper.

Ashless filter paper should be used for the filtration.

31

Determination of volatile oil in spices

Volatile oil content is a measure of aroma strength of spices and condiments. The volatile oil content in different spices ranges from 1 to 12%. The volatile oil content varies with source, variety and seasons.

Principle

The method involves distilling the volatile oil over with boiling water, condensing and collecting the oil in a measured volume of xylene in a graduated tube, after cooling, direct reading from the volume of volatile oil separated from the distillate.

Requirements

Apparatus

Volatile Oil Traps — Clevenger-type with joints.

Flask with Magnetic Stirrer — 1 litre capacity round bottom and shortneck with standard joint and having egg-shaped magnetic stirrer bar.

Reagent

Xylene (A.R. grade)

32

Figure 5. Apparatus for determination of volatile oilProcedure

Transfer enough weighed sample to 1 litre flask to yield 2 to 4 ml volatile oil. Add water to fill flask to half-full. Insert stirring bar and place flask in heating mantle set over magnetic stirrer. Add antifoaming agent. Clean trap and condenser with chromic acid cleaning solution just before use and fill trap with water. Set the apparatus so that the condensate will not drop directly on surface of liquid in trap but will run down the sides. Start stirrer and heat mantle through variable transformer set at 90 Volts (63 Amp). If oil separates in graduated portion of trap or clings to walls, add several drops standard aqueous detergent solution through top of condenser. Repeat, if necessary (usually once is enough). Distil for 10 minutes after adding detergent to wash it out of trap. When density of oil is nearly 1 g/cc, as in cassia, or if oil separates into two fractions in trap, as in nutmeg and allspice, add 1 ml xylene, accurately measured, to lighter than water trap. Distil, until two consecutive readings taken at 1 hour intervals show no change in oil content (taken after 6 h); cool and read the volume of collected oil. If xylene was added, subtract its volume and report oil as ml per 100 g spice.

NOTE 1 — With the material containing volatile oils lighter than water and fixed oils heavier than water like nutmeg, discontinue distillation when the fraction of oil obtained during 1 hour is heavier than water.

NOTE 2 — To correct the unsatisfactory separation of oil and water, agitate the liquid in the trap with a copper wire through the condenser top. Measure the oil in the trap after allowing to stand until it is cooled. Report volatile oil in ml per 100 g of the material.

Calculation

a 100Volatile oil, % v/m = ----------- b

where,

a = volume, in ml, of steam volatile oil collected through steamdistillation; andb = mass, in g, of the sample taken.

Observation

33

extracted volatile oil

mass of sample taken

volatile oil

a (g) b (g) %0.5 25 2.001

Result

The percentage of volatile oil present id 2.001 %

Precautions

The apparatus should be cleaned before each distillation.

Clear separation of water and volatile oil is essential.

Distillation should be continued until successive readings of the volume of volatile oil are the same.

34

Determination of free fatty acids and acid value in mustard oil

The Acid value has wide implication in the oil refining industry. It conveys not only the quality of oil but also total quantity of alkali needed to neutralize the acidity in a particular batch for making it suitable for the purpose of hydrogenation or marketing of refined oil or fat of very low acidity. Acid value is a measure of the hydrolytic rancidity present in the sample.

Principle

The acid value is determined by directly titrating the material in an alcoholic medium with aqueous sodium or potassium hydroxide solution.Acid value is the number of mg of KOH required to neutralize the free fatty acids present in 1 g of the oil or fat. Free fatty acid is calculated as oleic, lauric, ricinoleic or palmitic acids.

Requirements

Reagents

Ethyl Alcohol — 95%v/v, neutral to phenolphthalein indicator.

Phenolphthalein Indicator Solution — Dissolve 1 g of phenolphthalein in 100 ml of ethyl alcohol.

NOTE — When testing oils or fats which give dark coloured soap solution, the observation of the end point of the titration may be facilitated either (a) by using thymolphthalein or alkali blue 6B in place of phenolphthalein, or (b) by adding 1 ml of a 0.1%, w/v solution of methylene blue in water to each 100 ml of phenolphthalein indicator solution before the titration.

Standard Aqueous Potassium Hydroxide or Sodium Hydroxide Solutions —0.1 N or 0.5 N.

Procedure

Mix the oil or melted fat thoroughly before weighing. Weigh accurately a suitable quantity of the cooled oil or fat in a 200-ml conical flask. The weight of the oil or fat taken for the test and the strength of the alkali used for the titration shall be such that the volume of alkali required for the titration does not exceed 10 ml. Add 50 to 100 ml of freshly neutralized hot ethyl alcohol, and about 1 ml of phenolphthalein indicator solution. Boil the mixture for about five minutes and

35

titrate while as hot as possible with standard aqueous alkali solution, shaking vigorously during titration.

Calculation

56.1 N VAcid value = ----------------- W

Where,V = volume in ml of standard KOH/NaOH solution used,N = normality of standard KOH/NaOH solution, andW = weight in g of the material taken for the test.

Observation

sample weight

sample titration

normality

ACID VALUE

W (g) V (ml ) N10.0554 1.9 0.01053 0.1116

Inference

The acid value of commonly used edible oils is given as below.

Type of oil Acid value Type of oil Acid valueCoconut oil 0.5 Safflower oil 2.0Cottonseed oil 0.3 Sunflower oil 0.5Groundnut oil 0.5 Soybean oil 0.5Mustard oil 0.5 Rice bran oil 0.5Sesame oil 0.5 Palm oil 0.5

Precautions

The formation of two layers should be avoided by vigorous shaking so that the free acids do not get transferred into the ethanolic layer.

The freshly neutralized alcohol must also be hot at the time of addition.

The weight of the oil or fat taken for acidity determination and the strength of NaOH should be such that the volume of alkali used does not exceed 10 ml.

36

Determination of colour of Mustard oil by Lovibond Tintometer

Colour measurement on Lovibond scale is useful for determining the quality of oils and in the refining processes. Measurement of colour serves to check the bleaching processes also.

Principles

This 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.

Requirements

Apparatus

Lovibond Tintometer or Colorimeter

Glass Cells —The Lovibond cell designations namely, ¼”, ½”, 1” and 5¼” are recommended.

Filter Paper

Procedure

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 absolutely clear and free from turbidity. 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 the cell in position in the tintometer. Place along side of it such red, yellow, blue or neutral Lovibond glass slides or any combinations of these as are necessary to match the colour shade of the oil, observing the colours of the oil and of the combination of the glass slides through an eyepiece.

Calculation

The dimensions of the cell used and the mode of expressing the colourreading for different oils shall be as follows:

Colour reading in (*) cell = (aY + 5 b R ) or ( a Y + 10 b R)

37

*Size designation of the cell used,

where,a = the sum total of the various yellow (Y) slides used, andb = the sum total of the various red (R) slides used.

Observation

colour result

1/4 inch cell Y+5R3R+11Y 26

Inference

The maximum colour intensity of commonly used edible refined oils in Lovibond scale is given as below.

Type of oil Y+5R units Type of oil Y+5R unitsCoconut oil 2 Safflower oil 15Cottonseed oil 10 Sunflower oil 5Groundnut oil 3 Soybean oil 20Mustard oil 15 Rice bran oil 20Sesame oil 2 Palm oil 50

Precautions

Fat should be melted and any oil or fat that is cloudy should be filtered at a temperature not more than 60C.

Colour readings need to be taken within a comparatively

Lamp should not be used continuously for long time.

During matching, the sample should be at room temperature for oils or not more than 10C above the melting point.

Do not strain your eyes for long time, allow to relax from time to time.

38

Determination of iodine value in Mustard oil

The glycerides of the unsaturated fatty acids unite with a definite amount of iodine and the iodine value is therefore a measure of the degree of unsaturation.

Principle

The material is treated, in carbon tetrachloride medium, with a known excess of iodine monochloride solution in glacial acetic acid (Wijs solution). The excess of iodine monochloride is treated with potassium iodide and the liberated iodine estimated by titration with sodium thiosulphate solution.

Requirements

Reagents

Potassium Dichromate.

Concentrated Hydrochloric Acid.

Potassium Iodide Solution — Prepare a fresh solution by dissolving 10 g of KI free from potassium iodate, in 90 ml of water.

Starch Solution — Triturate 5 g of starch and 0.01 g of 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.

Standard Sodium Thiosulphate Solution (0.1N).

Glacial acetic Acid.

Iodine Monochloride (ICl) — 98 %.

Wijs Iodine Monochloride SolutionDissolve 10 ml of iodine monochloride in about 1800 ml of glacial acetic acid (chemically pure) and shake vigorously. Pipette 5 ml of this, add 10 ml of KI solution and titrate with 0.1 N standard Na2S2O3 solution, using starch solution as indicator. Adjust the volume of the solution till it is approximately 0.2 N.

Carbon Tetrachloride or Chloroform — inert to Wijs solution.

39

Procedure

Melt the sample if it is not already completely liquid, and filter through a filter paper to remove any impurities and the last traces of moisture. Make sure that the sample as well as the glass apparatus used is absolutely clean and dry. Weigh accurately, by difference, an appropriate quantity of the oil or fat, into a clean dry 500-ml iodine flask or well ground glass-stoppered bottle to which 25 ml of carbon Wijs solution and replace the glass stopper after wetting with KI solution; swirl for intimate mixing, and allow to stand in the dark for 30 min in the case of nondrying and semi-drying oils and 1 h in the case of drying oils. Carry out a blank test simultaneously under similar experimental conditions. After standing, add 15 ml of KI solution and 100 ml of water, rinsing in the stopper also, and titrate the liberated iodine with standard Na2S2O3 solution, swirling the contents of the bottle continuously to avoid any local excess until the colour of the solution is straw yellow. Add 1 ml of the starch solution and continue the titration until the blue colour formed disappears after thorough shaking with the stopper on.

Calculation

12.69 (B-S) NIodine value = -------------------- W

Where,B = Volume, in ml, of Na2S2O3 solution required for the blank,S = volume, in ml, of Na2S2O3 solution required for the sample,N = normality of Na2S2O3 solution, andW = weight, in g, of the material taken for the test.

Observationsample weight

volume required for blank

volume required for sample

N of na2s2o3

iodine value

W (g) B (ml) S (ml) N 0.2743 46.8 23.5 0.1007 108.5478

Result

The iodine value is 108.54.

Inference

40

The range of iodine value for animal fats (30-70), non-drying oils ((80-110)), semi-drying oils (80-140) and drying oils (125- 200) and very small value for waxes. The iodine value of commonly used edible oils is given as below.Type of oil Iodine value Type of oil Iodine valueCoconut oil 7.5-10.5 Safflower oil 138-146Cottonseed oil 98-115 Sunflower oil 100-140Groundnut oil 87-98 Soybean oil 125-140Mustard oil 98-110 Rice bran oil 90-105Sesame oil 103-115 Palm oil 44-58

Precautions

As soon as Wij’s solution is added stopper the flask immediately.

Exactly for 30 minutes the flask should be kept in dark.

Exact and accurate weighing should be done according to the iodine value expected in the sample.

If B-S is greater than B/2, the test must be repeated using a lesser quantity of the sample.

41

Determination of saponification value in Mustard oil

The number of milligrams of potassium hydroxide required to saponify completely one gram of oil or fat. When fat is saponified by refluxing with a known excess of alcoholic potassium hydroxide solution, the triglycerides hydrolyze, glycerol and soap are formed. The alkali consumed for this hydrolysis is a measure of the saponification value, which is determined by titrating the excess alkali with standard hydrochloric acid.

Principle

The material is saponified by refluxing with a known excess of alcoholic potassium hydroxide solution. The alkali consumed for saponification is determined by titrating the excess alkali with standard hydrochloric acid.

Requirements

Apparatus

Conical Flasks — 250 to 300 ml capacity.

Reflux Condenser —at least 65 cm long.

Water-Bath or Electric Hot-Plate with Rheostat Control

Reagents

Alcoholic Potassium Hydroxide Solution — Dissolve 35 to 40 g of KOH in 20 ml of distilled water, and add sufficient aldehyde-free rectified spirit to make up to 1000 ml. Allow to stand overnight, decant the clear liquid and keep in a bottle closed tight with a cork or rubber stopper.

Aldehyde-Free Rectified Spirit Reflux 1.2 litres of rectified spirit for 30 min in a round-bottom flask with 10 g of KOH and 6 g of granulated aluminium (or aluminium foil). Distil and collect one litre after discarding the first 50 ml.

Phenolphthalein Indicator Solution — Dissolve 1.0 g of phenolphthalein in 100 ml of rectified spirit.

NOTE - When testing oils or fats which give dark-coloured soap solutions, the observation of the end point of the titration may be facilitated either (a) by using

42

thymolphthalein, or alkali blue 6B in place of phenolphthalein or (b) by adding 1 ml of a 0.1 % (w/v) solution of methylene blue in water to each 100 ml of phenolphthalein indicator solution before the titration.Standard Hydrochloric Acid (0.5 N).

Procedure

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. Mix the sample thoroughly, and weigh accurately by difference about 1.5 to 2.0 g of the sample in a conical flask. Add 25 ml of the alcoholic KOH solution and connect the reflux air condenser to the flask. Heat the flask on a water-bath or an electric hot plate for not more than 1 h. Boil gently but steadily until the sample is completely saponified as indicated by absence of any oilymatter and appearance of clear solution. 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. Add about 1 ml of phenolphthalein indicator solution, and titrate with standard hydrochloric acid. Prepare and conduct a blank determination at the same time.

Calculation

56.1 (B-S) NSaponification value = ----------------- W

Where,B = volume, in ml, of HCl required for the blank,S = volume, in ml, of HCl required for the sample,N = normality of HCl, andW = weight, in g, of the material taken for the test.

Observation

sample weight

volume required for blank

volume required for sample

N of Hcl

saponification value

W (g) B (ml) S (ml) N 1.9751 32.5 19.6 0.4614 169.06012.001 32.5 19.4 0.4614 169.2590

Result The saponification value of mustard oil is 169.26

Inference

43

The saponification value of commonly used edible oils is given as below.Type of oil Sap. value Type of oil Sap. value

Coconut oil 250-264 Safflower oil 189-195Cottonseed oil 190-198 Sunflower oil 188-194Groundnut oil 188-195 Soybean oil 189-195Mustard oil 169-177 Rice bran oil 180-195Sesame oil 188-193 Palm oil 195-205

Precautions

Alcoholic KOH should be prepared overnight.

Refluxing should not be done for more than one hour.

Condenser should be carefully washed with alcohol.

Blank determination should be carried out simultaneously along with the sample.

44

Determination of unsaponifiable matter in mustard oil

The unsaponifiable matter is that fraction of oil and fat, which is not saponified with caustic alkali but is soluble in non-polar solvents. The unsaponifiable matter in oil or fat consists of hydrocarbons, higher alcohols, oil-soluble vitamins and sterols, which are not soluble in water after esterification. Most oils and fats of normal purity contain less than 2% unsaponifiable matter. Higher value indicates the possibility of adulteration with mineral oil. Adulteration of oils and fats with paraffin hydrocarbons will appear in the unsaponifiable matter.

Principle

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

Requirements

Apparatus

Flat-Bottomed or Conical Flask — 250 to 300 ml capacity. An ordinary round, flat-bottomed flask, fitted with a long glass tube which acts as a condenser, may also be used.

Separating Funnels — 500-ml capacity.

Reagents

Alcoholic Potassium Hydroxide Solution — Dissolve 70 to 80 g of potassium hydroxide in an equal quantity of distilled water, and add sufficient aldehyde-free ethyl alcohol (95 %v/v by volume), to make up to 1 litre. Allow to stand overnight, decant the clear liquid and keep in a bottle closed tightly with a cork or rubber stopper.

Ethyl Alcohol — 95% v/v

Phenolphthalein Indicator Solution — Dissolve 1 g of phenolphthalein in 100 ml of ethyl alcohol.

45

Petroleum Ether —60-80C.

Aqueous Alcohol — containing 10 % v/v of ethyl alcohol.

Standard Sodium Hydroxide Solution — approximately 0.02 N.

Acetone — free from evaporation residue.

Procedure

Weigh accurately about 5 g of the well-mixed sample into the flask. Add 50 ml of alcoholic potassium hydroxide solution. Boil gently but steadily under a reflux condenser for one hour or until the saponification is complete. Wash the condenser with about 10 ml of ethyl alcohol. Cool the mixture and transfer it to a separating funnel. Complete the transfer by washing the flask first with some ethyl alcohol and then with cold water. Altogether, add 50 ml of water 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 and allow to settle until both the layers are clear. Transfer the lower layer containing the soap solution to another separating funnel, and repeat the ether extraction at least six times more using 50 ml of petroleum ether for each extraction. If any emulsion is formed, add a small quantity of ethyl alcohol or alcoholic potassium hydroxide solution.Collect all the ether extracts in a separating funnel. Wash the combined extracts in the funnel three times with 25-ml portions of aqueous alcohol shaking vigorously and drawing off the alcohol-water layer after each washing. Again wash the ether layer successively with 20-ml portions of water until the wash-water no longer turns pink on addition of a few drops of phenolphthalein indicator solution. Do not remove any of the ether layers. Transfer the ether layer to a tared flask containing a few pieces of pumice stone, and evaporate to dryness on a water-bath under a gentle stream of clean dry air. To remove the last traces of ether, place the flask in an air-oven at 80 to 90°C for about 1 h. To remove the last traces of moisture, add a few ml of acetone and pass a gentle stream of clean dry air over the surface of the material or evacuate using a water vacuum pump at about 50°C for about 15 min. Cool in a desiccator and weigh. Repeat the evacuating, cooling and weighing until a constant weight is obtained.After weighing, take up the residue in 50 ml of warm neutral ethyl alcohol, containing a few drops of phenolphthalein indicator solution and titrate with standard sodium hydroxide solution.

Calculation

(A-B) 100Unsaponifiable matter, % by mass = -----------------

46

W

Where,A = weight, in g, of the residue,B = weight, in g, of the fatty acids in the extract (B = 0.282VN),V = volume, in ml, of NaOH solution,N = normality of NaOH solution, andW = weight, in g, of the material taken for the test.

Observationsample weight

weight of flask +sample weight

residue

volume of Naoh used

fatty acids in extract

USM

W (g) A (g) V (ml) B %5.0282 140.2574 0.468 0.3 0.0084 0.7632

Result

The unsaponifiable matter present is 0.77% .

Inference

The unsaponifiable matter content in commonly used edible oils is given as below.

Type of oil Unsap. matter Type of oil Unsap. matterCoconut oil 0.2-0.5 Safflower oil 0.5-1.5Cottonseed oil 0.6-1.6 Sunflower oil 0.3-1.5Groundnut oil 0.2-1.0 Soybean oil 0.2-1.5Mustard oil 0.9-1.2 Rice bran oil 3-5Sesame oil 0.7-1.8 Palm oil 0.2-1.2

Precautions

The unsaponified portion must be washed free of alkali and this should be ascertained with phenolphthalein.

As a check residue is dissolved in 10 ml accurately neutralized alcohol and titrated with 0.1N NaOH solution using phenolphthalein as indicator. If more than 0.1 ml of alkali is consumed it demands repetition of the experiment.

Sufficient time should be given for the complete separation of ether and aqueous phases.

47

Formation of emulsion should be prevented by adding small quantities of alcohol or conc KOH and NaCl solution. Formation of emulsions which are difficult to break leading to a loss of quantitative accuracy.

Determination of allyl isothiocyanate in mustard oil

The oil obtained from mustard seeds and rape seeds contains sinigrin and myrosin (glucosinolates), which are sources of goitrogens. As such they are relatively non-toxic but their hydrolysis products include allyl isothiocyanates and oxazolidinethiones, which are powerful goitrogens, producing varying manifestations of toxicity in non-ruminants.

Principle

The allyl isothiocyanate in the oil is steam distilled into a known excess of silver nitrate solution, and the excess of silver nitrate solution is determined by titration with standard ammonium thiocyanate solution.

Requirements

Apparatus

Distillation Flask — 500 ml round-bottomed flask.

Any Efficient Reflux Condenser (~ 90 cm long).

Measuring Flask — 200 ml capacity.

Water-Bath

Reagents

Ethyl Alcohol — 95%, v/v neutral to phenolphthalein.

Silver Nitrate Solution —0.1 N.

Ammonium Hydroxide Solution — 10 % (w/v).

Conc. HNO3

Ferric ammonium sulphate Indicator — 0.1 % solution in water.

48

Standard Ammonium Thiocyanate (NH4SCN) Solution —0.1 N.

Procedure

Weigh accurately about 5 g of the material into a 500-ml distillation flask and add to it 25 ml of ethyl alcohol, 250 ml of water and a few pieces of pumice stone. Distil the mixture in steam and collect the distillate in a 200-ml measuring flask containing exactly 25 ml of silver nitrate solution and 10 ml of ammonium hydroxide solution. Collect as rapidly as possible about 150 ml of the distillate. Attach the reflux air condenser to the measuring flask and heat the mixture for about one hour on a boiling water-bath. Cool to room temperature, add water to make up to 200 ml and filter the contents after shaking. Take 100 ml of the filtrate, add 6 ml of HNO3 and a few drops of ferric ammonium sulphate indicator, and titrate with standard ammonium thiocyanate solution until a permanent red colour is obtained. Carry out a blank test simultaneously along with the sample.

Calculation 9.915 (B-S) NAllyl isothiocyanate, % by mass = ------------------- W

Where,B = Volume, in ml, of NH4SCN solution required for blank,S = Volume, in ml, of NH4SCN solution required for the sample,N = Normality of NH4SCN solution, andW = Weight, in g, of the sample taken for the test.

Observation

sample weight

titration value of blank

titration value of sample

normality of NH4HCN

essential oil

W (g) B (ml) S (g) N % (by mass)

5.1436 12.5 11 0.0936 0.27

Result

The allyl isothiocyanate content is 0.27% .

Inference

The allyl isothiocyanate content in different varieties if mustard varies from 0.1 to 0.7% by mass.

Precautions

49

Sample and blank test should be conducted simultaneously under similar conditions.Standardized ammonium thiocyanate should be used.

Determination of peroxide value of Mustard oil

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

Principle

The material in an acetic acid-chloroform medium, is treated with an aqueous solution of potassium iodide. The liberated iodine is titrated with standard sodium thiosulphate solution.

Requirements

Apparatus

Pipette — Graduated, 1 ml capacity.

Conical flask — Glass-stoppered, 250 ml capacity.

Reagents

Acetic Acid-Chloroform Solution — Mix three parts by volume of glacial acetic acid, with 2 parts by volume of chloroform.

KI Solution — Saturated. Prepare saturated solution of potassium iodide in recently boiled distilled water. Store in the dark.

Na2S2O3 Solution - 0.1 N, accurately standardized.

Na2S2O3 Solution - 0.01 N. This solution is prepared by diluting 100 ml of accurately standardized solution of 0.1 N Na2S2O3 to 1 litre with freshly boiled and cooled distilled water.

Starch Solution — 1 % by mass

Procedure

Weigh 5.00 ± 0.05 g of sample of fat in a 250-ml glass stoppered conical flask and then add 30 ml of the acetic acid-chloroform solution. Swirl the flask until

50

the sample is dissolved. Add 0.5 ml of saturated potassium iodide solution. Allow the solution to stand exactly one minute with occasional shaking and then add 30 ml of distilled water. Titrate with 0.1 N sodium thiosulphate solution with constant and vigorous shaking. Continue titration until the yellow colour almost disappears, Add 0.5 ml of starch solution and continue titration till the blue colour just disappears. If the titre value is less than 0.5 ml, repeat the determination using 0.01 N Na2S2O3 solution. Conduct a blank determination of the reagents in the same way. The titration in blank determination should not exceed 0.1 ml of the 0.1 N Na2S2O3 solution.

Calculation

1000 (S-B) NPeroxide value, meq./kg = ------------------- W

Where,S = Volume, in ml, of Na2S2O3 solution used up by the sample,B = Volume, in ml, of Na2S2O3 solution used in the blank,N = Normality of Na2S2O3 solution, andW = weight, in g, of the sample taken.

Observation

sample weight

titration value of blank

titration value of sample

normality of Na2S2O3

peroxide value

W (g) B (ml) S (g) N4.961 0 3.2 0.01007 6.54.521 0 3.2 0.01007 7.12

Result

The peroxide value is 6.81. Inference

The peroxide value of fresh edible oils is usually within 10 meq/kg.

Precautions

Standard solutions used should be properly standardized.

Maintain dark conditions during the experimentation should be properly maintained.

51

Determination of specific gravity of Mustard oil

Specific gravity is usually determined with a specific gravity bottle or pyknometer. Specific gravity alone is of limited value in locating the presence of other substances but in conjunction with other data it is of immense utility.

Principle

The specific gravity bottle method is a gravimetric method in which the weight of sample is divided with the weight of water of same volume at same temperature. This method is more accurate and gives quick result.

Requirements

Apparatus

Specific gravity bottle or pyknometer — with well-fitting ground glass joints. To calibrate, clean and dry the bottle or pyknometer thoroughly, weigh and then fill with recently boiled and cooled water at about 25°C after removing the cap of the side arm. Fill to overflowing by holding the bottle or pyknometer on its side in such a manner as to prevent the entrapment of air bubbles. Insert the stopper and immerse in a waterbath at the desired test temperature ±0.2°C. Keep the entire bulb completely covered with water and hold at that temperature for 30 minutes. Carefully remove any water, which has exuded from the capillary opening. Remove from the bath, wipe completely dry, replace the cap, cool to room temperature and weigh. Calculate the weight of water. This is a constant for the bottle or pyknometer, but should be checked periodically

Water-bath — maintained at 30.0 ± 0.2°C, or 95.0 ± 0.2°C as required.

Calibtrated Thermometer — any convenient thermometer of a suitable range with 0.1 or 0.2°C subdivisions.

Procedure

Melt the sample, if necessary, and 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°C or warm to the desired test temperature. Fill the bottle with the oil previously cooled to about 25°C or the melted fat to overflowing, holding the bottle on its side in such a manner as to prevent the entrapment of air bubbles after removing the cap of the side arm.

52

Insert the stopper, immerse in the water-bath at 30.0 ± 0.2°C and hold for 30 minutes. Carefully wipe off any oil, which has come through the capillary opening. Remove the bottle from the bath, clean and dry it thoroughly. Replace the cap of the side arm, cool to room temperature and weigh.

Calculation

(A-B)Specific gravity at 30°C/30°C = -------- (C-B)

where,A = weight, in g, of the specific gravity bottle with oil at 30°C,B = weight, in g, of the specific gravity bottle, andC = weight, in g, of the specific gravity bottle with water at 30°C.

Observation

weight of empty bottle

weight of bottle + water

weight of bottle + sample

specific gravity

B (g) C (g) A (g)22.9425 50.1479 47.6681 0.9088

Result

The specific gravity is 0.9088.

Inference

Specific gravity for most of the edible oils range between 0.90 to 0.93.

Precautions

Only a certified thermometer covering the range of specific gravity determination temperature should be employed.

There should be complete absence of air bubbles in oil body.

53

Determination of copper, zinc, lead and cadmium in food products by Atomic

Absorption Spectroscopy

Food products contain substantial quantity of organic matter which must be destroyed prior to the estimation of minerals. Dry ashing or wet digestion is generally used for the destruction of organic matter. The wet digestion method is more preferable to dry ashing method because some of minerals may loss during dry ashing at high temperature. Atomic absorption spectroscopy is an important analytical technique used for the detection and determination of metals in foods. Atomic absorption spectrophotometer consists of hollow cathode lamp or electrodeless discharge lamp, chopper, atomizer, monochromator, detector and electronics. AAS technique offers specificity and more accuracy as compared to the chemical methods.

Principle

The organic matter is removed by dry ashing or wet digestion. The residue is dissolved in dilute acid and sprayed into the flame of an atomic absorption spectrophotometer (AAS). The absorption or emission of the metal to be analysed is measured at a specific wavelength. AAS determines a wide range of elements based upon the principle of absorption of atoms. The liquid sample is aspirated and atomized in the flame where it converts into atomic vapour in their ground state. Atoms of metal of interest absorb the intensity of light from hollow cathode lamp. The amount of light absorbed in flame is proportional concentration of metal in solution.

Requirements

Apparatus

Silica crucible

Muffle furnace

Desiccator

Hot plate

AAS : Any instrument operating in absorption mode may be used providing it has facilities for the selection of required oxidant/fuel combination from a choice of air, nitrous oxide and acetylene and has wavelength ranges from 180 to 600 nm.

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Light source : Hollow cathode or electrodeless discharge lamps

Reagents

Nitric acid (sp gr 1.42)

Sulphuric acid-98%

Hydrochloric acid-sp gr 1.16 to 1.18)

Standard solutions

Copper (100 ppm): Dissolve 3.928 g of pure copper/copper sulphate (CuSO4.5H2O) in water, dilute to 1000 ml at 20C with water in a volumetric flask. Dilute 10 ml to 100 ml with water in a volumetric flask. One ml of solution contains 100 g Cu.

Zinc (100 ppm): Dissolve 1.000 g of pure zinc powder in 10 ml water, 5 ml of HCl and dilute to 1000 ml at 20C with water in a volumetric flask. Dilute 10 ml to 100 ml with water in a volumetric flask. One ml of solution contains 100 g Zn.

Lead (100 ppm): Dissolve 1.000g of pure lead in 10 ml HNO3 in 1 litre volumetric flask, dilute to 1000 ml at 20C with water in a volumetric flask. Dilute 10 ml to 100 ml with water in a volumetric flask. One ml of solution contains 100 g Pb.

Cadmium (100 ppm): Dissolve 2.282 g of CdSO4.8H2O in distilled water, dilute to 1000 ml at 20C with water in a volumetric flask. Dilute 10 ml to 100 ml with water in a volumetric flask. One ml of solution contains 100 g Cd.

Procedure

Sample preparation

By dry ashing : Accurately weigh 5-10 g of sample into a dried, tared crucible. Heat the dish on hot plate until fume cease and place the crucible in furnace maintained at 55010C till white ash results. To the ash, add 5-10 ml of 6N HCl, and heat to dryness at low temperature on a hot plate. Add 15 ml of 3N HCl and heat on a hot plate until the solution just boils. Cool and filter through ashless filter paper (Whatman 42) into 100 ml volumetric flask. Make up the volume with distilled water. Prepare a reagent blank along with sample.

By Wet digestion : In case of dried products, take 2 g of the sample. If the sample contains more moisture (>10%), take 5 g or more and transfer to a 500 ml Kjeldahl flask. Add 10 ml of conc sulphuric acid and shake vigorously. Ensure that there are no dry lumps. Add 5 ml of conc nitric acid and mix. Heat gently in a fume cupboard until the initial vigorous reaction has subsided. Thereafter, heat

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vigorously until most of the nitrous fume has ceased to evolve. Add nitric acid dropwise and continue heating until all the organic matter is destroyed and white fumes of sulphuric acid evolve. Cool the digest, add water again cool transfer to a volumetric flask and make up the volume with water. Carry out a blank through the operation using the same amount of reagents as in the case of sample.

Instrument conditionsSelect the wavelength and gases to be used for the particular elementunder consideration from table below:

Element Wavelength (nm) Gases

Zinc 213.9 Air/AcetyleneCopper 324.8 Air/AcetyleneLead 217.0 Air/AcetyleneCadmium 228.8 Air/Acetylene

The recommended settings for the various instrumental parameters vary from model to model and certain parameters require optimization at the time of use to obtain the best results. Instrument should therefore be adjusted as described in the manufacturer’s instructions using the type of flame and wavelength settings specified above. Set the AAS to appropriate condition. Measure the absorbance of the standards. Instrument automatically will plot a graph showing net absorbance against the concentration of element in standard solution.Now read the blank and the sample solutions.

Calculation

Conc of element (g/ml) DilutionElement in sample, mg/kg = ------------------------------------------ Weight of sample (g)

Precautions

Make sure that the sample is free from organic matter before reading in the AAS.

Digest sample always in fuming cupboard.

Use hand gloves, mask and guggles for protection from acid fumes while digesting the sample.

Thoroughly washed glassware should be used

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Prepare standard solutions accurately

Determination of cholesterol content in ghee by GC

Cholesterol is the most widely distributed sterol. It is found in animal body fat, eggs, milk fat and even in some vegetable oils. Due to the relationship between plasma cholesterol levels and the risk of coronary disease, the labeling of cholesterol in packaged food products is become mandatory as per new integrated food law.

Principle

The ghee sample is saponified using ethanolic KOH and the unsaponifiable matter is extracted with petroleum ether. The cholesterol is then derivatized to trimethylsilyl ether and quantified by GC. The separation of cholesterol from other sterols on GC is based on the distribution of species between two non-miscible phase in which the mobile phase is a carrier gas moving through or passing the stationary phase contained in a column. It is applicable to substances or their derivatives which are volatilized under the temperature employed.

Requirements

Apparatus

Gas chromatograph- Varian CP 3800 or any other model

Hot plate

Micropipette (20-200 l)

Vortex shaker

Weighing balance

Water bath

Rotary evaporator

Reagents

Dimethyl formamide (DMF)

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Hexamethyl disilane (HMDS)

Trimethylchlorosilane (TMCS)

5-Cholestane (internal standard solution-100 g/ml in n-heptane)

Cholesterol stock solution-2 mg/ml in dimethyl formamide

Cholesterol working solution- Dilute stock solution to obtain 3 solutions at concentration of 20, 100 and 200 g/ml.

Potassium hydroxide-50% w/w

Petroleum ether (40-60C)

Sodium sulphate (Anhydrous)

Glass wool

Procedure

Sample extraction

Accurately weigh 1 g of sample into a dried iodine flask. Add 40 ml of 95% ethanol and 8 ml of 50%KOH to the flask. Place flask on hot plate and attach condenser and reflux the sample for 7010 min. Rinse condenser with 95% ethanol. Remove the flask from the reflux assembly and close with stopper and cool solution to room temperature. Test solution is stable for 24 h. Pour the content into a 250 ml separating funnel. Add 50 ml petroleum ether to it and stopper the funnel and shake vigorously for 45 sec. Let the layer separate out and collect the aqueous layer into a beaker and ether layer into another separating funnel. Pour again the aqueous layer into separating funnel and extract with 50 ml of petroleum ether. Repeat the extraction once again. Wash the extracted ether with water till the aqueous layer becomes colourless with phenolphthalein. Filter ether into 250 ml flask through sodium sulphate. Evaporate ether in flask to dryness on rotary evaporator at 40C.

Sample derivatization

Pipette 1 ml aliquot of working standard solution and test solution into different test tubes. Add 0.2 ml HMDS and 0.1 ml of TMCS to test tube and keep the solutions undisturbed for 15 min. Stopper it and shake vigforously on vortex for 30 sec. Let stand solution undisturbed for 15 min and add 1 ml of 5-cholestane and 10 ml water to each test tube. Stopper the tubes and shake vigorously for 30 sec. The heptane layer separates from the aqueous layer. Derivatized solutions must be analyzed within 24 h.

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Instrument conditionsCapillary column – HP-5 (30 m 320 m 0.25 m)Detector- Hydrogen flame ionization detector (FID)/Temperature (300C)Injector - 280C/ Split mode (Split ratio-1:2)Oven - 270C hold 10 min, Increase 10C/min to 300C hold 5 min.Flow rate – Helium column about 2 ml/min Split vent about 30 ml/minPurge vent about 3 ml/minAuxillary make up gas about 20 ml/minHydrogen about 35 ml/minAir about 280 ml/min

Calculation

Area of sample conc of std DilutionpurityCholesterol, mg/kg = ------------------------------------------------------- Area of std sample weight (g)

Inference

The cholesterol content in some fat-riched food products are given in below.

Food products Total sterols, mg/kg Cholesterol, % sterolsButter 2843-2746 98.3Cod liver oil 4099 98.3Beef 1350 98.4Lard 1161 97.0Coconut oil 1550-7797 0.6-3.0Cottonseed oil 2690-6430 0.7-2.3Palm oil 376-4000 2.6-7.0Groundnut oil 901-2854 Trace-3.8

Precautions

Transfer the liquid during extraction carefully

Use dry glassware during derivatization.

For saponification, rinse the flask with ethanol after KOH addition in order to prevent joint of flask with condenser from freezing together.

Prepare standard solutions accurately.