Chemical Quality Assurance

Module 3. Chemical analysis of milk and milk products

Lesson 10
INSTRUMENTAL METHODS OF ANALYSIS

10.1 Introduction

The technology of using instruments to measure and control the physical and chemical properties of material is called instrumentation. Investigations in food science and technology, whether in universities, governmental agencies, or the food industry, often require determination of food composition. The dairy scientists and technologists often determine the chemical composition as part of research on food product development or quality assurance activities. The chemical composition of foods is often determined to establish the acceptability or nutritive value of food product. The field of milk and milk product analysis involves a considerable amount of time spent learning principles, methods and instrument operations and perfecting various techniques. Also the relationship between the physical properties of milk and its chemical composition has been exploited in the dairy industry to develop various instrumental methods for the determination of chemical quality or composition of milk. A variety of methods are available to assay particular components of milk and milk products. Speed, precision and accuracy are often the key factors that determine the choice of method. Process control samples are usually analyzed by rapid methods, whereas for legal requirements generally requires the use of official reference methods.

10.2 Sophisticated Laboratory Instruments

The sophisticated laboratory instruments often used during analysis of milk and milk products are as follows

• Milko-Tester, Infra Red Milk Analyzer (Milko Scan), Milko-Scan 133-B
• Lactostar automatic milk analyzer
• Lactoscope
• Pro-Milk MK ΙΙ
• Butyro refractometer
• Rancimat
• Kjeldahl digestion and distillation apparatus
• Flame Photometer
• Atomic Absorption Spectrophotometer

10.2.1 Milko-tester

The Milko-Tester is a product of Danish ingenuity and has been used since 1964. The results with Milko-Tester are comparable to those with the reference method.

10.2.1.1 Principle

Fig. 10.1 Milko tester

The photometric method for determining fat in milk is based on the measurement of light scattering by a diluted sample. A calcium chelating agent, ethylenediaminetetraacetate (EDTA) eliminates the turbidity caused by the casein micelle, so the light scattering is due solely to the fat in the milk. The amount of light scattered is dependent on number and size of fat globules. If the size distribution is uniform the amount of light reaching the photocell will be proportional to the fat content. The results are read from a galvanometer scale graduated in percent fat.

10.2.1.2 Apparatus

There are three models- the Mark ΙΙ, the Mark ΙΙΙ and the automatic. The Mark ΙΙ model is the least expensive and least automatic. The sample is drawn into the machine by suction where it passes through a 60°C bath and then into a 4-stage homogenizer. The sample is mixed with ethylene di- amine tetra -acetate (EDTA) diluents in a funnel. From the funnel it passes into the flow through cuvette. The beam of light passes through the sample and the amount of light transmitted is detected by a photocell from which a signal is transmitted electrically to the readout meter. Automatic and Mark ΙΙΙ models differ from the Mark ΙΙ by having a digital read out or recorder, which replaces the readout meter, the EDTA diluent from the reservoir passes through a heated container which expels dissolved air, and milk and EDTA are mixed prior to homogenization thereby reducing the size of the sample, increasing the rate of analysis (80 verses 120/hr); minimizing plugging instrument with sour milk, and reducing energy for efficient homogenization. Mark ΙΙΙ is semiautomatic faster than Mark ΙΙ and uses 1.6 ml milk. Milko Tester Automatic (MTA) is fully automatic.

The Milko-Testers are calibrated at the factory against the Rose-Gottlieb method which is the accepted international reference method.

10.2.1.3 Procedure

Calibrate the instrument with milk samples of known fat and SNF content. Take unknown milk sample in the receptacle provided with the instrument. Bring the receptacle below the suction tube of the instrument and run the instrument. Incase of low- cost models sample is homogenized manually using a handle attached with the instrument. Incase of sophisticated instruments every thing is automatic. Record the reading fro Fat and SNF as displayed on the instrument display.

10.2.2 Lactostar automatic milk analyzer

Lactostar a new , versatile, easy to operate microprocessor based , fully automatic milk analyzer for the determination of fat, SNF, protein, lactose and freezing point of milk has been introduced in India recently by FUNKE GERBER, Germany through their sole distributors BENNY IMPEX PRIVATE LIMITED, NEW DELHI.

10.2.2.1 Principle

Fig. 10.2 Lactostar automatic milk analyzer

The Lactostar applies a combined thermo-optical method to analyze the constituents of milk. The device measures both the optical and the thermal properties of the milk. The optical measuring method (Turbidimetric) determines the sum of fat and protein and lactose contents by computational analysis. The freezing point of milk is predicted on the basis of protein content and fat-free dry matter.

10.2.2.2 Procedure

Calibration

For its successful use, it must be calibrated with milk samples of known composition. It needs (i) zero calibration i.e. with distilled water, (ii) A-calibration i.e. with milk of half dilution and (iii) B-calibration i.e. with pure milk. The lactostar has 20 channels and we can calibrate separate channel for each type of different milks. Thus we can have separate calibrated channels for cow milk, buffalo milk, vendors milk, skimmed milk, goat milk, etc.

Take milk sample in the receptacle provided with the instrument. Bring the receptacle below the suction tube of the Lactostar and run the program as stored in the channel of the instrument. Selection of the program depends upon the channel calibrated for a specific type of milk. Run the program and record the results as displayed on the screen or take the print out.

10.2.3 Pro-milk MK II

The Pro-Milk instrument is used for the determination of protein in milk by Dye-binding method. The basis of this method is that certain dyes react stoichiometrically with proteins in acid solutions. After mixing the dye in acid buffer solution with the milk sample, the precipitate is removed and the optical density of the filtrate containing unreacted dye solution is measured in a colorimeter. The first commercially available instrument for dye binding method was produced by Foss Electric Co. Denmark.

10.2.3.1 Principle

The amino groups of arginine, lysine and histidine of milk proteins are involved in binding certain dyes. An aqueous solution dye buffered to about pH 2.0 is added to a milk sample. The protein and the dye that it binds precipitate and are separated from the solution of unbound dye by centrifuging or filtration. The optical density of the filtrate is measured in a colorimeter fitted with a galvanometer type read out. Read out is given in direct percentage protein on a specially designed scale. Dyes used are Amido Black-10 B, Acid Orange-12 and Orange-G.

10.2.3.2 Apparatus

The Pro-Milk MK consists of essentially of a dye container (Dispensing Equipment) with a dispenser pump which discharges a predetermined volume of dye. This dye is mixed with a measured volume of milk in a specially designed mixing chamber (measuring unit). The contents of the chamber are then mixed, reaction being almost instantaneous and the solution filtered under pressure. The filtrate is passed through a colorimeter fitted with galvanometer type read out. Read out gives direct percentage protein on 2.5 – 5.0 scale.

10.2.3.3 Determination

One ml of milk sample is mixed with 20 ml of dye solution in the measuring unit. It is then forced through a filter into the cuvette of the calorimeter. The unreacted dye intensity is determined by measuring the light transmitted through the cuvette. The reading is taken on the Read out which gives directly the percentage of protein content in the sample. The whole process takes about sixty seconds to complete.

For individual milks, 50 % of the differences between the Kjeldahl values and the Amido-black determinations are due to the variations in the NPN fractions of the sample. The accuracy of the Pro-milk is dramatically improved when true protein is used as reference instead of total protein. Casein reacts at 100 % but non- protein Nitrogenous matter (NPN) practically does not bind dye. Whey proteins bind about 28 % more dye than casein.

10.2.4 Bactoscope/Soma scope

It is an accurate and reliable instrument with which the total bacterial count/ somatic cells in milk can be quantified very quickly and at low costs. The use of automatic settings means that the maximum number of analysis can be reached per hour. Results are given in CFU as per reference method. It also allows the counting of individual bacteria. The Bactoscope does not count directly without incubation. These instruments work on the principle of flow cytometry.In case of somascope a latest flow cytometry technology is used to produce a laminar flow of the somatic cells through the measuring flow cell. This laminar flow ensures that every somatic cell is counted. This results in a linear measurement up to 1*107 cells/ml.

Fig. 10.3 Somascope and bactoscope

System is total computer based and controlled by the company designed software. Take about 3 ml of sample of milk and heat it to the temperature of 40°± 2C. Put the sample container below the suction tube of the instrument and press the start button. The data is capture by software and saved in the file. It takes about 30 second to analyse one sample .

10.2.5 Rancimat

The Rancimat is a modern PC controlled instrument and allows determination of oxidative stability very comfortably. A rough estimation of the shelf life of a product is possible with unique temperature exploration. In this method the highly volatile organic acids produced by auto oxidation are absorbed in water and used to indicate the induction time.

Fig. 10.4 Rancimat

10.2.5.1 Principle

The Rancimat method developed as an automated version of extremely demanding AOM method (active oxygen method) for determination of induction time of fats and oils. Highly volatile organic acids produced by auto-oxidation are absorbed in water and used to indicate induction time.

Fig. 10.5 Reacton vessel

10.2.5.2 Procedure

Weigh 3 g sample of completely melted ghee accurately into each of the reaction vessels. Place the vessels in the heating block of the Rancimat apparatus. Then connect the reaction vessels to the measuring vessels via connecting tube. Add 60 ml of deionised water into each of the measuring vessels, containing the electrodes. Now, place the measuring vessels in the Rancimat apparatus. Connect all parts to the apparatus as per the operating instructions, and carry out the test until the induction period of all the samples ends, with a maximum allowable limit of 48 hours.

10.2.6 Butyro refractometer

Refractive index measurements have long been used for the qualitative identification of unknown compounds by comparing the RI of the unknown with literature values of various known substances. The butyro refractometer is widely used to measure the refractive index of fats and oils. An instrument is constructed that measure the critical angle of the sodium D line (589 nm) at 20°C.

Fig. 10.6 Butyro refractometer

10.2.6.1 Principle

Refractive index and butyro-refractometer (BR) readings, characteristic for the particular liquid or solid, are inter-convertible and are concerned with the degree of bending of light waves passing through a liquid or transparent solid . Generally, it is expressed as the ratio between the sine of the angle of incidence to the sine of the angle of refraction when a ray of light of a definite known wavelength (usually 589.3 nm, the mean of the D-lines of sodium) passes from air into oils and fats.

10.2.6.2 Procedure

Before determining the BR reading of a sample, the temperature of the refractometer was adjusted to 40.0 ± 0.1°C using circulatory water bath and the prisms were cleaned and dried completely. The refractometer was calibrated with the standard provided by the company before taking the reading of the different samples. A drop of the molten fat sample was placed on the lower prism of the refractometer and the prisms were closed and held for 2 minutes. After adjusting the instrument and light to get the most distinct reading possible and bringing the temperature to 40°C, the BR reading of the fat was recorded.

10.2.7 Foss automated Kjeltec

The Danish investigator Kjeldahl worked out in 1883 a method for determining organic nitrogen in his studies on protein changes in grain used in the brewing industry. Basically the sample is heated in sulphuric acid and digested until the carbon and hydrogen are oxidized and the protein nitrogen is reduced and transformed into ammonium sulphate. Then concentrated sodium hydroxide is added, and the digest heated to drive off the liberated ammonia into a known volume of a standard acid solution. The unreacted acid is determined and the results are transformed, by calculation, into a percentage of protein in the organic sample.

Fig. 10.7 Distillation unit

The FOSS automated Kjeltec™ instruments include systems for sample digestion and distillation. The Kjeltec series is designed to attain the best possible accuracy and precision. It also sets safety standards in Kjeldahl analysis. Also, the Kjeltec™ 2400/2460 is an autosampler system with automatic distillation and titration providing ease of use, speed and accuracy in routine analysis. Operational safety and convenience are enhanced by the unattended operation and features such as automatic reagent addition, tube emptying and a fully integrated safety system. Up to 60 samples can be analysed unattended. The system is truly modular where the automatic distillation/titration unit Kjeltec 2400 can be upgraded to a fully automatic system by the addition of the autosampler system Kjeltec 2460.

Fig. 10.8 Digestion unit

10.2.7.1 Procedure

a) Preparation of test sample

Warm the milk sample (for other milk products, follow the prescribed procedure for sample preparation) to between 38°C to 40°C in the water bath. Gently mix the test sample thoroughly by repeatedly inverting the sample bottle without causing frothing or churning. Cool the sample to room temperature immediately prior to weighing the test portion.

b) Test portion and pre-treatment

To a clean and dry digestion tube, add 12 g of potassium sulfate, 1.0 ml of the copper sulfate solution, approximately 5 g of the prepared test sample of milk and 20 ml of sulfuric acid. Use the sulfuric acid to wash down any copper sulfate solution, potassium sulfate or test portion left on the upper walls of the digestion tube. Gently mix the contents of the tube.

c) Digestion

• Set the digestion block at a low initial temperature so as to control foaming (approximately 180°C). Transfer the tubes to the digestion block and place the exhaust manifold which is itself connected to a water jet pump in the top of the tube. The suction rate of the water jet pump should be just sufficient to remove fumes.
• Digest the sample until white fumes develop. Then increase the temperature of digestion block to between 410°C and 430°C and continue digestion of the sample until the digest is clear.
• After the digest clears (clear with light blue-green colour), continue digestion at between 410°C and 430°C for at least 1 h. During this time the sulfuric acid should be boiling. If visible boiling of the clear liquid is not apparent as bubbles forming at the surface of the hot liquid around the perimeter of the tube, then the temperature of the block may be too low. The total digestion time will be between 1.75 h and 2.5 h.
• At the end of the digestion, the digest shall be clear and free from undigested material. Remove the tube from the block with the exhaust manifold in place.
• Allow the digest to cool to room temperature over a period of approximately 25 min. The cooled digest should be liquid with a few small crystals at the bottom of the tube. Excessive crystallization indicates too little residual sulfuric acid at the end of digestion and may cause a decrease in protein estimation results. To reduce acid loss during digestion, reduce aspiration rate.
• After the digest has cooled to room temperature in approx. 25 min, remove the exhaust manifold and carefully add 85 ml of water to each tube. Swirl to mix while ensuring that any separated out crystals are dissolved. Allow the contents of the tube to cool to room temperature again.

d) Distillation

• Transfer the digestion tube to the distillation unit and place a conical flask containing 50 ml of boric acid solution under the outlet of condenser in such a way that the delivery tube is below the surface of the excess boric acid solution.
• Adjust the distillation unit to dispense 65 ml of sodium hydroxide solution and distill off the ammonia liberated by the addition of sodium hydroxide solution.
• Following the manufacture’s instructions, operate the distillation unit in such a way as to steam distil the ammonia liberated by addition of sodium hydroxide solution, collecting the distillate in the boric acid solution. Continue with the distillation process until at least 150 ml of distillate is collected. Remove the conical flask from the distillation unit and completely drain the distillation tip. Rinse the inside and outside of the tip with water, collecting the rinsing in the conical flask. Always rinse the tip with water between samples. During the distillation, the ammonia solution turns to green.

e) Titration

Titrate the contents of the conical flask with the hydrochloric acid standard volumetric solution using a burette and read out the amount of titrant used. The end point is reached at the first appearance of violet colour in the contents.

f) Blank Test

Carry out a blank test following the procedure described above taking 5 ml of water and about 0.85 g of sucrose instead of test portion.

g) Calculation and expression of results

Calculate the nitrogen content, Wn, by using the following equation:

Where,

Wn = is the nitrogen content of the sample, expressed as percentage by mass
Vs = is the numerical value of hydrochloric acid standard volumetric solution used in determination in millilitres, expressed to the nearest 0.05 ml
Vb = is the numerical value of the volume of hydrochloric acid standard volumetric solution used in the blank test in millilitres, expressed to the nearest 0.05 ml

N= is the numerical value of the exact normality of the hydrochloric acid standard volumetric solution expressed to four decimal places.
m = is the numerical value of the mass of the test portion in grams, expressed to the nearest 1 mg,

h) Calculation of crude protein content

Calculate the crude protein content Wp using the following equation:

Wp = Wn x 6.38

Where,

Wp = is the crude protein content, expressed as a percentage by mass.
Wn = is the nitrogen content of the sample, expressed as a percentage by mass to four decimal places.

6.38 = is the generally accepted multiplication factor to express the nitrogen content as crude protein content

10.2.8 Flame photometer

Early studies during the nineteenth century by J.F.Herschel, D. Alter, and G. Kirchhoff and R. Bunsen laid the foundations for the qualitative differentiations of salts depending on their emission in a flame. Flame photometer is used for quantitative chemical analysis for the determination of alkali earth metals present in solution. Flame photometer is must where the concentration of the element is very low, say of the order of 1 ppm as ordinary methods such as gravimetric and volumetric will not respond.

Fig. 10.9 Flame photometer

A modern flame photometer consists essentially of an atomizer, a burner, some means of isolating the desired part of the spectrum, a photosensitive detector, sometime an amplifier, and finally a method of measuring the desired emission by a galvanometer. The instruments are used primarily to determine calcium, sodium and potassium from milk and milk products.

10.2.8.1 Principle

Flame analysis is based on the fact that when a metallic salt solution is drawn into a non-luminous flame, it emits light of characteristic wave length. This emitted light, isolated to the characteristic wave band by an optical filter, is allowed to fall on a photocell whose output is measured by a suitable deflection for instance electronic amplifier and a meter or a galvanometer.

10.2.8.2 Preparation of sample

Since a very fine capillary is used to draw the sample into the flame the sample must not contain big solid particles leading to the blockage of the capillary. In case of fluid milk or blood, diluted sample can be used. However, in case of solid products like cheese, khoa, chhana, etc., the sample must be ashed and the ash is then dissolved in dilute acid solution. The concentration of the metal in the diluted sample may be in the range from 5-8 ppm.

10.2.8.3 Preparation of standard solution

Standard solution of the metal or its salt must be prepared in such a way that the concentration of the metal is again 5-10 ppm. For determining a very low concentration of the metal in the unknown sample as well standard must be prepared in some organic solvent or in a mixture of water and organic solvent.

10.2.8.4 Determination of metals in the unknown

For determination of the metal in the sample first of all the instrument must be standardized with a standard solution. Once the instrument is adjusted then the unknown solution is fed to the flame and the reading is recorded in ppm.

10.2.9 Atomic absorption spectrophotometer

Atomic spectroscopy has played a major role in the development of our current database for mineral nutrients and toxicants in milk and milk products. Atomic absorption spectrophotometer (AAS) is widely used and accepted technique capable of determining trace (µg/ml) and ultratrace (sub- µg/ml) levels of elements or metals in a wide variety of samples, including biological, clinical, environmental, food, and geological samples, with good accuracy and acceptable precision.

Fig. 10.10 Atomic absorption spectrophotometer

10.2.9.1 Principle

Atomic absorption spectrophotometer measures absorption of characteristics radiation by atoms of a particular element to be determined which are thermally atomized either by flame or by graphite furnace. The element which is to be determined is dissolved in a suitable chemical (normally an acid) and this solution is fed into the flame through an aspiration chamber. In case of furnace atomization auto sampler or micro syringe is used for transforming the sample solution into the furnace. A hollow cathode lamp of the element to be determined is used as a source of radiation, which is absorbed by the atoms produced in flame or furnace of that element and absorption is directly proportional to the concentration of the analyte atom. This absorption is compared with the absorption of standard solution of that particular element and actual concentration of element is determined.

10.2.9.2 Sampling and processing of samples

Milk is not totally homogenous because different layers may have different concentrations of minerals. It is, therefore, utmost important that bulk sample be sufficiently homogenized to ensure that the aliquot/sub-sample which is taken for analysis must be representative of the whole. The size of the sample should be proportional to the bulk. Thorough mixing of sub-samples from a large bulk is preferred in representative sampling. When milk sample is taken from a cow, total milk drawn at each milking is mixed and about 100 ml sample is taken in pre-washed stoppered polyethylene bottles. For mineral element analyses, dry ashing or wet digestion can be followed.

(a) Dry Ashing

Generally, 2 to 10 g sample is taken in a silica crucible after preliminary drying of sample. For powdered milk, one gm sample is taken. Then, the sample is placed in the muffle furnace and the temperature is brought to 550°C and held for 4 hr. After cooling, the resulting ash is dissolved in dilute HCl (6M) and then made upto suitable volume. The resulting solution is used in mineral element determination.

10.2.9.3 Analysis of minerals on atomic absorption spectrophotometer

HCl extract or wet digested samples after suitable dilution are used for the estimation of major and trace elements. Major minerals can also be estimated by gravimetric or colorimetric methods apart from AAS, ICP or other techniques used preferably for the analysis of trace minerals.