Paper (b)

BY G . H A L L Grubb Parsons, Walkergate, Newcastle upon Tyne NE6 2Y B

The paper describes methods of analyses in direct determination of fat, protein and lactose. The Infrared Milk Analyser (IRMA) its fundamental component a double- beam infrared spectrophotometer which compares the absorption of a milk sample at specific wavelengths is described in detail. Calibration of the IRMA is discussed and the comparison of infrared and chemical analyses shown. (Editor’s summary)

INTRODUCTION In a paper by Kroger (1973), an American professor of food science pointed out that almost 80 years had been spent in refining milk fat determinations to their present sophistication and that milk fat, along with weight and certain hygienic data, has traditionally been the only milk-related criterion used in establishing the price of milk.

In recent years the Western world has begun to discriminate against food fats and there is much publicity throughout the rest of the world about looming food protein shortages. Almost suddenly it appears that milk protein has been discovered as a resource that needs to be measured and developed. There is, of course, a substantial amount of litera- ture describing milk protein research, but it is considerably less than that of fat research and fat testing.

The classical methods of analysis of milk usually attempted to estimate specific substances by separation, purification and weighing. Examples are the Rose-Gottlieb butterfat determination and the gravimetric total solids procedure. The approach of isolating and weighing milk protein is virtually impossible and certainly not feasible in the dairy laboratory.

The recognized, official method for protein determinations (the Kjeldahl method) is time- consuming, complicated and therefore costly. In order to meet today’s requirements for speed with accuracy, some attempts have been made to mechanize this and other older, direct methods and there has been some success in this direction, but we are moving away from the classical principle of direct analysis and we think in terms of indirect estimations in the development of instrumentation. Parameters such as transmission of light, infrared absorption, nuclear magnetic resonance and micro- wave attenuation are now being measured to give protein values along with fat, lactose and other estimations.

The Infrared Milk Analyser (IRMA) Infrared analysis is the only method which allows direct determination of fat, protein and lactose (and thence total solids or solids-not-fat) from a single, low-volume sample with one basic technique on one instrument. The infrared method of analysis is as rapid as other practical methods now being used, requires no pre-treatment chemicals and in terms of real cost/sample is extremely competitive.

The infrared method originated from fundamental research work conducted by Goulden (1964) and his colleagues at the National Institute for Research in Dairying, Reading. This work commenced in the early 1960s and is based on the fact that three components of milk (fat, protein and lactose) have specific absorptions in the infrared region of the spectrum.

The fundamental component of an Infrared Milk Analyser (IRMA) is therefore a double-beam infrared spectrophotometer which compares the absorption of a milk sample, at these three specific wavelengths, with the absorption of pure water. The fat absorption at 5.73 pm arises from the carbonyl group of the triglyceride structure. The protein absorption at 6-46 pm is due to the amide group in the peptide linkage of intact protein and the absorption at 9.60 pm is due to lactose (Fig. 1).

These absorptions are readily measured by placing a cell containing milk in one beam of the spectrophotometer and a similar cell containing water in the other beam of the instrument. After warming to 4045°C to melt globular fat and remove dissolved gases, the milk sample is homogenized with an ultrasonic homogenizer before introduction into the milk cell. This is to ensure that there is no scattering of the incident infrared energy by large fat globules. It is not essential for the homogenizer to produce fat globules of fixed size, but only globules at or below the optimum level.

Once the sample has been pumped through the homogenizer and into the milk cell, the infrared

192 Journal of the Society of Dairy Technology, Vol. 28, No. 4, October, 1975

Fig. 1 . Grubb Parsons automatic infrared milk analyser (IRMA)

spectrophotometer is set automatically to the first wavelength to be measured, normally the 5.73 pm fat wavelength. The amount of infrared energy absorbed by the milk at this wavelength is then measured and, by means of a fast electronic system, multiplied by an appropriate factor to give a direct read-out of the percentage of fat present. Having completed the measurement at the first wavelength, the instrument automatically sets to the 6.46 pm protein wavelength, a further absorption reading is taken and the percentage protein displayed. The total time taken from injecting the milk sample until displaying both fat and protein percentages is 27 s with the standard Mark I1 IRMA and only 18 s with the faster Mark 111 instrument. By using the autosampler facility, up to 200 samples/h can be analysed for fat and protein.

The method is specific for the components being measured and naturally the best results will be obtained with samples in which these components have not been partially destroyed by bacterial or chemical deterioration. Such deteriorations can be minimized during pre-analysis storage periods by the use of potassium dichromate or mercuric chloride preservative tablets, neither of which will affect the instrument readings. It is important that samples to be analysed should be in optimum physical condition and should be representative of the bulk from which they were obtained.

Calibrating Let us now turn to the question of calibrating the

IRMA. It is standard practice in the dairy industry to use the Kjeldahl method, with a suitable recovery characteristic, for the determination of protein in milk. As with any instrumental method, we must therefore correlate the protein values given by IRMA to those given by the chosen version or

modification of the Kjeldahl procedure. When the infrared results for a series of milks are plotted against the chemical results for the same milks, equal values for each milk will give a 45" line passing through the origin. In practice, when such a graph is plotted, it is unlikely that the line obtained will pass through the origin and have the desired slope. For the calibration of protein readings on IRMA there is the slight complication that there is a small, but significant, interference due to fat at the protein wavelength in the infrared measurement. This is due to a water displacement effect and a correction has to be made to the protein reading for a varying fat content. This correction is carried out automatically by the instrument during the measuring sequence.

To produce the required IRMA versus Kjeldahl correlation for protein, it is only necessary to adjust two potentiometers, which individually control the slope and intercept of the plotted line, to give the desired 45" slope. The use of trial and error methods for making these adjustments is avoided by using two optical test filters built into the instrument. Once a satisfactory calibration has been obtained, instrument readings can be taken for standard chemical solutions which have absorption maxima at the same wavelengths in the infrared as the components being measured and these readings may then be used for subsequent calibration checks.

When calibrating IRMA by comparing infrared and chemical analyses, it should be recognized that deviations from the desired calibration line are a combination of both methods. As with any instru- ment requiring calibration against a standard analytical method, the accuracy of the results obtained with IRMA is highly dependent on the accuracy of the analyses by means of which cali- bration is achieved. Even with optimum instrument performance and perfect calibration, the accuracy of the infrared, as indeed any other method, cannot exceed that of the reference chemical method. For IRMA, the protein accuracy specification is &0.045 per cent compared to the semi-micro Kjeldahl procedure. The corresponding figure for precision of measurement is that the standard deviation of the duplicate samples is better than f0.03 for all components (Biggs, 1972).

CONCLUSIONS IRMA provides a fast, accurate and direct method of fluid milk analyses without any chemical pre- treatment. Fat and protein (plus lactose, if required) are measured automatically from a single sample and presented directly as percentages. By summing fat, protein and lactose values and adding a constant corresponding to minerals content, total solids values may be readily obtained.

Milk samples may be loaded manually or may be

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presented to IRMA in racks (of 20 samples), from which each sample is automatically taken up and analysed. The results are presented sequentially on an illuminated digital display and simultaneously logged on a numerical printer to eliminate trans- cription errors. Additional data handling facilities, such as punched paper tape, punched card pro- cessing, optical character recognition systems, etc. can be incorporated as required.

IRMA will analyse both fresh milk and homo- genized milk without any changes in operational procedure or calibration. Seasonal effects on individual proteins do not give rise to any significant error since the infrared method measures total proteins.

IRMA is in use throughout the world in both dairy herd improvement and quality payment schemes. The infrared method is officially approved in Europe by the International Committee for Recording the Productivity of Milk Animals (ICRPMA) and in the USA by the Association of Official Analytical Chemists (AOAC).

REFERENCES Biggs, D. A. (1972) Journal of the Association of Oficial

Goulden. J. D. S. (1964) Journal o f Dairv Research. 31.273. Analytical Chemists, 55 (3) .

Kroger, M. (1973)>Americun Dairj Reviiw, September; 18.

DISCUSSION Mr. F. W i g : I am rather pleased that I had a ringside seat for the heavy-weight contest today, and the scoring was one blow below the belt each. We, in the Milk Marketing Board, are major users of both types of equipment and we have a lot of visitors from abroad, who regularly ask us which is the best type of equipment to use, and the answer is clear from today’s papers - that both manufacturers have the best type of equipment.

There are just two points which I would like to take up, really they are observations. One is that as an analytical chemist I was interested in Mr. Hall’s point on comparing the accuracy of the instrument with the accuracy of the reference method. I accept that we have, in calibrating instruments, to take into account any inaccursies of the reference method. I think however, that we have to be. very careful not to be in danger of moving too far the other way and having the tail wagging the dog, by having the instrumental method there to check the reference method. When we test by the Kjeldahl method we have a result which is, by definition, protein, and provided we ensure that the greatest accuracy is put into the reference method then we have to accept that it is correct, and that differences between it and the instrument, are due to the instrument and not to the reference test.

The second point I wish to make is that both suppliers have expressed their desire to give the users what they want, and I must compliment both gentlemen on this. An instrument comes out and is modified and improved over the years, and it could be, that three years hence, the equipment which is available is not at all like that which was originally evaluated. And I think there is a need for regular and continued contact between the manufacturers and the users, to ensure that as equipment is modified and changed it does, in fact, meet the industry’s re uirements, particularly where quality payment testing is inv&ed.

Mr. E. C. Armitt: I would like to take up Mr. Harding’s second point, on modifications. Obviously we can’t sit still and we do have this interaction between the large users and ourselves as to what is required on the instrument. May I reverse it though, and throw out something which is being widely circulated at the present moment, and suggest that probably technology is going so fast that investment can’t keep up with it. Mr. S. G. Coton: Mr. President, I wonder if Mr. Hall could give US the limits to which his instrument might be used in terms ofpercentages of thevariousconstituents. Mr. G. Hall: Certainly. The normal ranges specified in our catalogue are, for fats, up to 9 per cent, protein up to 8 per cent,lactoseup to53percent. Mr. S. G. Coton: I take it that nought is the lower limit in all three cases ? Mr. G. Hall: Yes. Dr.T.C.A.McGann:I think thelastpoint israther important. If that is so. then I think that the infrared analyser should have great potential in measuring filtrates from milks to determine casein, whey protein and whey protein denatura- tion. I wonder if anything has been done by Grubb Parsons on whey protein fractions? Mr. G. Hall: The answer is, by Grubb Parsons, no, not at the present time. I see no basic reason why they could not be looked at as you suggest. Obviously different calibration settings would be needed on the equipment to cope with the types of material you describe. Dr. T. C. A. McGann: 1 think this is an important point,.it opens up the whole area of process control and as Mr. Armitt mentioned - 1 work rather extensively in connection with dye binding. I would like to say that the Pro-Milk instrument happened to be used because it was convenient and many in the Irish industry have the Pro-Milk, but any dye-binding instrument or dye-binding method will do; the pfinciple involves measuring the total protein then measuring the filtrate, we usually express the result as a ratio, the whey divided by the total protein, and both the ratio and the total protein are of interest, for instance in powder. I also feel. that the whole infrared area seems to be opening up very widely and one of the limiting factors will be the degree of reproduci- bility. We can get dye binding to reproduce in terms of r t e i n f 0.002. Now I notice you give figures for reproduci-

ility of all three constituents of less than 0.03. Now that is not good enough for whey proteins. We want to go down a tenth of that, which may mean greater stabilization, it may mean more work. In the area of process control which is opening up much more, infrared has great potential for on-line control. As well as that, I think you should not limit yourself to fat protein and lactose. Probably one of the most important constituents in milk is water - in products I mean. The control of the water content in milk powder, in .casein powders and caseinates, especially when linked to in-line control, where the feedback mechanism can be to the control of heat in the evaporator or into the spray drier. This is an area where I think infrared analysis could contribute in a major way to process control especially of protein-based foods. The President: Thank you, Dr. McGann. This opens up a whole new line of discussion, with the possibility of moving out of the laboratory into the process area much as has already been tried with the photometric systems of fat analysis tentatively being used for on-line standardization. of milk and butterfats. One is in terms of, possibly on-line protein analysis, for reasons of production and on-line moisture anal sis for, say, spray driers. This is a very interesting andrattractive area for the industry. It has, I am sure, rich financial rewards attached to it. I am sufe both companies are themselves pursuing this idea, behind the scenes. We shall not hear of that today - that is asking too much, but I am sure we would like to hear from the authors

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what advantages could be seen for this approach in their own industries. Would youlike tocomment, Mr. Armitt? Mr. E. G. Armitt: Yes, from a personal point of view, I think that the future for us lies in process control, not only in the estimation of fats, standardization of fat, but also possibly in ratio control of fat to protein, fat to salt, or whatever is required. This takes the instruments right out of the lab- oratory and probably those who work in the laboratory are saying ‘Now we are out of business’, but they are not, they are very much in business, because these instruments will require careful control. But I am certain that the future will hold some weird and wonderful things. The President: And they will not only require careful control, they will require special engineering to suit them to the environment in which they find themselves and I take it that this is being considered. Mr. G. Hall: Yes, I would echo the comments Mr. Armitt has made, my company is also active in fact, in some of the things to which Dr. McGann referred, and indeed we certainly regard the on-line equipment business potential as good for the milk.industry in general, and therefore good for both our companies. Dr. E. W. Evans: Mr. Chairman, thinking of the time scale that some of these instruments have, they can come to fruition and to sell well, and that implies looking at IRMA for example, started in the early sixties, and perhaps now starting to pay back for the costs of R & D. This is a period of 15 years. If one were thinking of this at the time the research was started, one might say on any cash flow basis that this sort of research and development should never be started. I wonder, really, whether these two gentlemen would care to comment on the philosophy behind the development of such new instruments from the financial aspect. Mr. G. Hall: I could make a comment, to start with, that your organization, Dr. Evans, pioneered the principle; the problem often is putting the principle into really useful practice, and that can take a lot of time and money as you point out. But the germ of the idea is often many years ahead of the practical solution, to produce an instrument which people would be willing to purchase, and would find suitable for their needs. You are quite right in that both companies, I would think, have spent a lot of money on research and development, and it is only possibly in recent years that some of this money is now being actively recovered and, that profits, that dirty word, are being made. Obviously we have to continue, because there is a large market for automatic milk analysis equipment. That is why there are two companies represented here today doing this work. One or two others - and some major companies I believe, have dabbled and tried, and perhaps have not succeeded. The market potential for an instrument maker is pretty big, SO he has to be prepared to invest money in the long term. Mr. E. G. Armitt: Yes, I would agree with that, Dr. Evans, and would also try and give you some figures. Foss Electric try to invest 20 per cent of turnover every year in R & D. This is an approximate figure and is probably going down now as sales increase, but three years ago this was the figure put forward. We would expect it to take about five years from an idea to a finished product on the market, that is a normal situation Then when you have the idea there is the problem of making i t to suit the customer’s requirements. In other words, it is no use coming up with a brilliant idea which doesn’t meet with the testing capacity or capabilities of the customer. The instrument must be tailored to meet the customer’s requirements. Then of course there is the economic situation - when the instrument is ready to be put on the market - are people willing to invest in this? Then there are the great risks when trying to forecast what the market will require. Mr. G. Hall: I would agree very much with the last part of that statement. If someone had a good crystal ball and could tell Mr. Foss and Mr. Grubb Parsons what everyone here

would like for the next ten years, and the maximum they wished to pay for it, we would be delighted to know. Mr. E. G. Armitt: May I give another example? We recently had an enquiry for a piece of equipment from Japan. The enquiry was in November, and they would start investing in June. That is a fantastic speed, but in Japan they were not interested in investment for profit, they invest to keep the money going round in a big circle, but sometimes someone has their hand on the model and keeps passing it on - it is a completely different policy. Dr. J. C. D. White: Is it theoretically impossible to produce an instrument which does not require calibration against the reference vessel ? Mr. G. Hall: I would suggest possibly yes, because all the instrumental methods mentioned in my talk are indirect and the dairy field is rather traditional in its outlook. For example, many people still today regard the Gerber result as the right one, and I think it is fair to say that the Gerber is not necessarily the right result in the fat test. So I think that traditionally, the people who are producing and using milk like to use their accepted standard procedures. Any new form of measurement developed is always referenced back to that before it is acceptable to the industry as a whole. Mi. E. G. Armitt: To a certain degree. it is a question of faith. Mr. Harding could probably help me here on the number of times we used to check calibration. We used to check every 10 or was it every 20samples? Mr. F. Harding: In milk records? I think we check about every 60. I agree it is a question of faith, and equally, of course, how much faith one has in the reference tests. I saw Alan Lakin here today - you asked about an instrumental method which could be referenced. Of course the Amido Black method could be referenced if one had pure.+mido Black as an analar reagent. I know that many universities are working on this. I gather they have had some problems in purifying the Amido Black but that would be a method which, in theory, would produce a standard reference instrumental method. Mr. A. Lakin: There is no problem in getting a pure Amido Black. but the interesting thing is that there seems to be no outside interest in this commodity being made available as long as it comes from Germany with a reasonably high degree of purity. The Amido Black used in milk testing equipment is of a purity between about 95 and 96 per cent, and as long as the equipment is standard with reference to Kjeldahl, everyone seems to be happy. I don’t know how one could determine protein absolutely. I think this would be the thing that would worry me, ultimately. I don’t know what method you have. It is just that everyone agrees on using the Kjeldahl. But it is an arbitrary assessment no matter how you look at it, because it is almost impossible to get a pure protein and know that it is absolutelypure. At least that is my opinion. Mr. S. G. Coton: Mr. President, I don’t have a question, but I was interested in the 20 per cent as a research investment which Mr. Armitt mentioned - interested, and perhaps not very surprised, because looking at industries they do vary enormously, from the aircraft industry and electronics, which spend the greatest proportion of their turnover on research and development - it amounts in fact to about one-third, or rather more than this, and it goes down to something like the food industry where I think the figure is 0.3 per cent. My comment is simply: do we spend enough? Mr. E. G. Armitt: It is reflected, by the way, in the end price, hence some of the accusations I get. The President: Everyone has been very tactful about price, so far. I take it there is no major price difference between the two basic systems ? Significant enough to cause market movement one way or the other? Mr. E. G. Annitt: I would think not. The President: Perhaps the technologists are tending to converge in some areas.

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Dr. E. W. Evans: May I ask Mr. Armitt whether they are developing any instruments for determining individual proteins in milk, such as the individual whey proteins, or any other selective analysis of proteins? Mr. E. G. Armitt: Yes, a lot of interest is being shown in the possibility of using particular dyes in dye binding for par- ticular proteins, and a lot of research is being done in this area. It may be possible to select dyes for particular proteins. Mr. J. G. Clark: There are two observations that I would like to make, the first relates to the accuracy of the instruments described by Mr. Armitt and Mr. Hall, I am not a chemist or a physicist, but it seems to me that it is, at the moment, only of academic interest that these instruments can produce results more accurate than can be obtained by conventional methods. In quality payment schemes for milk the methods to be used for the determination of, for example, fat or solids - not -fat content will be laid down and the important point is that if instruments are used for these determinations the results they give should agree, or be adjusted to agree with those obtained by the prescribed methods.

My second observation is in connection with on-plant applications, I was interested to read recently in our sister journal, The New Zealand Journal of Dairy Science and Technology, a paper covering the development of the dairy industry in that country over the past four years. They have gone to a great extent over to the production of milk powder - some 22 spray-drying plants having been installed or placed on order. I was particularly interested to read that powders are being made to 56 different specifications - of which nearly halfreiate to skim milk powders -to meet customers' end use requirements.

In New Zealand milk production for manufacture is highly seasonal. The cows all calve in the spring, leading to a common lactation period during the summer and all dry off for the winter. This means that changes that take place in milk during the period of lactation are more pronounced in New Zealand than, for instance, in this country where there is all the year round production. This means that the seasons in which powders of certain specifications can be made are limited (although techniques for their extension are being developed). It seems to me that this is an ideal situation for practical application of the instruments that have been described.

For example the installation cf an instrument before or after the evaporator of a spray-drying plant, that could measure, at frequent intervals, the undenatured whey protein content of the milk, would enable the plant operator, perhaps by adjustments to preheating conditions, to ensure that powder of the required specification was being produced and inform him, when the time arrives, of the point in the season when, in spite of changes in preheating conditions, powders of a certain specification can no longer be made. The President: Thank you Mr. Clark. You are thinking particularly of the whey protein lysine contents of milk. 1 think we would agree generally that process control is the way ahead and this is where we ought to look for our next ten years of profits as a dairy industry, through greater control, greater accuracy, better measurement of what we sell and buy. The money has to come from the dairy industry in the first place.

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