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On-line Applications in Food Science
On-line Applications in Food Science CHARLES TELLIER Laboratoire de RMN et Rkactivite' Chimique, CNRS URA 472,2 rue de la Houssinidre, 44072 Nantes Cedex 03, France
FRANCOIS MARIETTE CEMAGREF, Division Technologie, 17 rue de Cucille',35044 Rennes Cedex, France
1. Introduction 2. On-line NMR: theoretical background 2.1. Equilibrium conditions 2.2. Effect of flow on NMR parameters 2.2.1. Flow continuous-wave NMR 2.2.2. Flow Fourier transform NMR 2.3. Continuous-flow or stopped-flow NMR? 3. Technical requirements 3.1. Sampling 3.2. Instrumentation 3.3. Signal processing 4. Potential applications 4.1. High-resolution NMR 4.2. Low-resolution NMR 5. Future prospects References
105 106 106 107 107 107 109 110 110 111 114 114 115 117 119 121
1. INTRODUCTION
On-line process control can often improve productivity and quality in the food industry by allowing a rapid identification of the raw materials used as ingredients and by adjusting the blending to keep the final composition of the product constant. For these reasons on-line process control is a rapidly growing branch of analytical chemistry. On-line sensors must be able to collect chemical or physical information from a sample and convert them into an interpretable signal for regulation within a short time according to the downstream process. On-line sensors for use in the food industry must meet special requirements such as food compatibility of the material in contact with the food sample and the ability to be easily cleaned and sterilized. For these reasons, the on-line use of sensors which are sensitive to the chemical composition of ANNUAL REPORTS ON NMR SPECTROSCOPY
VOLUME 31 ISBN 0-12505331-2
Copyright 0 1995 by Academic Press Limited AII rights of reproduction in any form reserved
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the product is poorly developed in the food industry. Biosensors, despite their high sensitivity and selectivity,' are often unsuitable for industrial purposes as they are unsterilizable, unreliable and invasive. Among physicochemical sensors, only infrared techniques have been really introduced in industrial production* and until now very few examples describe the use of an NMR sensor in industry and particularly in the food i n d ~ s t r y . ~ The great contrast between the wide applications of NMR techniques in food science and their virtual non-existence in industrial production may originate from several factors: NMR techniques are complicated and measure strange parameters that do not mean much to non-NMR specialists; NMR machines are very expensive; NMR is not as sensitive as other techniques such as IR spectroscopy and may be equally sensitive to extraneous factors like the industrial environment. However, the noninvasive and non-destructive character of NMR, coupled with the ease of cleaning and even sterilizing the probe, emphasize the usefulness of this technique for on-line monitoring in food process engineering. The objectives of this review are to discuss the theoretical and technical requirements of on-line NMR monitoring and to present examples illustrating various on-line applications of NMR which have already been tested industrially or which have potential interest for the future.
2. ON-LINE NMR: THEORETICAL BACKGROUND The use of NMR as an on-line sensor implies that the instrument should ideally be located in the production line or on a side-stream line. Therefore, on-line measurements suggest the existence of a continuous or a stoppedflow through the NMR sensor, which may perturb the quantitative response of the technique. In this section, the significant changes in the NMR signal induced by the flow will be presented and suggestions are given to choose the parameters that govern the optimization of the NMR response. 2.1. Equilibrium conditions
An NMR signal only appears when a sample is in a uniform magnetic field so that the individual nuclei align themselves with the external magnetic field and precess at the Larmor frequency. The resulting magnetization (Ma) of the sample is achieved in an exponential manner, over a time period dependent upon the spin-lattice relaxation time T I :
This equilibrium condition must be achieved prior to observation for
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quantitative NMR measurements. In a continuous or a stopped-flow experiment premagnetization of the sample occurs as the sample flows through the magnet before the detection probe. Assuming ideal flow behaviour, one can estimate the lifetime in the premagnetization region Tp from the rate (Q) and the premagnetization volume (V,): Tp = V&Q. For quantitative measurements, all sample nuclei must be completely premagnetized prior to observation. According to equation (l),this condition is met when: Tp> 5T1 where Tl corresponds to the maximum longitudinal relaxation time within the sample to be analysed. 2.2. Effect of Bow on NMR parameters
2.2.1. Flow continuous-wave NMR Analysis of the flow continuous-wave NMR experiment indicated two fundamental changes in the flowing system absorptive NMR signal measurement, both related to relaxation phenomena. If complete premagnetization of sample nuclei is assumed, the effects of flow on spin-lattice, TI, and spin-spin, T2, relaxation can be understood in terms of equations (2) and (3): 1 1 1 Tstatic + 7-pF=
1 -pF=-
1 1 Tstatic + -7
(3)
where r is the mean lifetime of the nucleus in the detection region (T = Vd/Q, v d is the detection volume), Tstaticare the conventional relaxation times and T O b s are the apparent relaxation times of the flowing sample.’ These equations suggest that at faster flow rates both apparent Tl and T2 should decrease. Shorter effective Tl is explained by the replacement of saturated nuclei with an influx of polarized nuclei which induces a signal enhancement in the flowing system. The shorter T2 is responsible for a broadening of the Lorentzian lineshape because of the decrease in observation time. 2.2.2. Flow Fourier transform NMR Equations (2) and (3) are equally applicable to the signal enhancement and line-broadening phenomena associated with the discrete flow FT-NMR
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0.4
-
-
I
I
I
I
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Fig. 1. Intensity of the NMR signal as a function of the flow rate. Individual contributions from premagnetization and saturation are calculated assuming V , = 7 ml, V , = 1ml, T, = 0.5 s and T , = 1 s.
signal. For example, saturation effects observed in static FT-NMR at high recycle times (T,) are avoided in flowing system as long as the lifetime T of the nuclei in the detection region ( V d ) is equal to or shorter than the minimal delay between multiple scan (Tc). However, as flow rate increases and T decreases, the constant flux of excited nuclei leaving the detection region during data acquisition is responsible for the degradation in spectral linewidth. As the sample moves across the NMR probe, the two basic factors that determine the NMR signal intensity are the premagnetization time and the saturation effect. Figure 1 represents their individual contributions to the NMR signal and the composite result as a function of the flow rate ( Q ) .The premagnetization effect, dependent on V,,, induces significant decay from Mo when V,lQ is shorter than 5T1.On the other hand, saturation effects decrease as the flow rate increases until elimination at T, > 7. The composite signal thus produces a maximum for which minimized saturation effects at faster flow must effectively offset improved premagnetization at slower flow.
ON-LINE APPLICATIONS IN FOOD SCIENCE 00
109 0
non quantitative conditions
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., \
Flow
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4
,
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\
non quantitative conditions
... ,, .,'.
6
z
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0 0 0
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0
0
2
4
6
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00
Tc Fig. 2. The conditions for quantitative flow as a function of flow rate and recycle time T, in a pulse NMR experiment. Tp and 7 are respectively the lifetime in the premagnetization and the observation regions. (Adapted from ref. 6, used by permission.)
Figure 2 graphically describes the conditions that ensure quantitative flow FT-NMR measurements as a function of the flow rate and the recycle time, assuming ideal flow behaviour.6 These conditions must be defined for the nuclei with the longest Tl in the sample.
2.3. Continuous-flowor stopped-flow NMR? In the earliest days, flow NMR was used to improve the sensitivity of NMR detection before the first Fourier transform spectrometer^.^ A further application of the flow experiment was the measurement of the flow rate. In particular, a variety of methods were developed for the analysis of flow rate inside pipes,' blood vessels' or plants." Until now, very few process controls have utilized flow NMR methods. However, flow NMR presents an advantage over off-line or stopped-flow measurements as the NMR result represents the average signal of a large amount of sample due to the flow across the probe during the time of the measurement. Therefore, flow NMR partially resolves the problem of sampling in heterogeneous materials and could avoid the necessity to
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construct large sample probes, which also require costly magnet modifications. 3. TECHNICAL REQUIREMENTS NMR is traditionally a laboratory-based technique. In order to use this technique as an on-line sensor in industry, a number of technical hurdles need to be overcome. Problems arise from scaling, which implies that sampling devices have to be adapted to the product conveyors. Moreover, there are additional problems associated with the factory environment such as variable temperature, nearby machinery and electrical noise. Finally, the NMR sensor should provide measurements in real-time, be robust enough to withstand a variety of industrial environments, have high reliability, and low operating costs, and needs a suitable pricelperformance ratio so that it is economically attractive to industrial users to implement this technology. 3.1. Sampling
With most NMR applications, sample preparation is generally minimal. The sample is loaded directly into the sample tube with no preparation at all. However, the first condition for reliable on-line NMR measurements is that the sample is in equilibrium with its environment. These equilibrium conditions involve a regular laminar flow for liquid, no thermal or concentration gradient, and a constant packing for solid samples. Sampling requirements also depend on the location of the sensor (fully encased in the line or on a side-stream of a fraction of the line) and on the loading solution in the NMR instrument, with a continuous flow or with a removeheplace system. A system where the NMR sensor completely encloses the production line avoids special sampling devices and offers the best NMR performance, but is not suitable for physically large samples which would require a large and expensive magnet. With a side-stream solution, the sample is diverted from the main product stream through a smaller sensor. After measurement, the sample is redirected back into the main product stream. This approach, which assumes that the sample has the same characteristics as in the main stream, is probably valid for liquids or solids. For viscous samples like meat or cheese paste, this assumption may be invalid because of the lamination of the sample into small pipes. Lamination can induce changes in the sample texturation or a destabilization of the food emulsion and may affect the NMR signal. Such effects have been observed" by continuously recycling a
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minced-meat paste into a small pipe (diameter 15mm) at low flow rate (1.2 1 min-I). The resulting water/fat ratio determined by on-line highresolution NMR measurement increases continuously as the meat paste is recycled. For solid samples, a variety of sampling techniques has been suggested for the side-stream solution. For on-line measurements of wheat moisture content, an air drive device4 has been used with a vibrator to fill the NMR probe immediately downstream from the wheat tempering screw. Nicholls and De Los SantosI2 have proposed a configuration where the sensor is suspended beneath a hole in the transport chute (Fig. 3). The NMR sensor is filled with the sample by lowering a piston which normally seals the aperture in the transport system. At the end of the measurement sequence the piston is driven back up the sample tube, returning the measured product to the process stream. Interestingly, cavities containing reference samples can be integrated within the piston, thus allowing the calibration of the sensor after each measurement. However, the system supposes that the conveyor material and the movement of the product do not perturb the NMR signal. Differences in the packing of solid samples is a problem frequently encountered which affects the volume-sensitive NMR response. For laboratory applications, the sample is usually weighed before measurement and signal intensity/weight ratio is used to compare the samples. This preweighing technique is unsuitable for on-line applications and must be replaced by new calibration methods. For the determination of moisture content in foodstuffs, the density problem can be solved by using the intensity of the rapid portion of the signal decay as a measure of how much solid is in the detector ~ 0 i l . l ~ 3.2. Instrumentation
Previously, modified commercially available NMR spectrometers were used for on-line process control, but only with partial success. Plant operators found them difficult to adjust and calibrate because they were laboratory instruments which had been repackaged for on-line use and were difficult to maintain. They did demonstrate, however, that NMR could be used to make accurate measurements on Any magnetic resonance spectrometer contains several electronic circuits which must be kept adjusted and calibrated. An industrial magnetic resonance process control analyser can be used by plant operators who have little or no familiarity with magnetic resonance. To minimize the time required to train operators to use the analyser, several self-calibrating and self-adjusting features are necessary. For example, all setting-up and monitoring of the instrument’s performance should be made automatic, e.g.
PRODUCT STREAM
-
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RF /
CAVITIES IN PISTON CONTAINING CALIBRATIO MATERIALS
PISTON
Fig. 3. Sampling from a conveyor belt using a piston containing calibration samples. (Adapted from ref. 12, used with permission.)
ON-LINE APPLICATIONS IN FOOD SCIENCE
\
113
CORE
Fig. 4. One-sided access NMR sensor configuration. (Adapted from ref. 12, used with permission.)
receiver gain, field adjustment, pulse width. Permanent magnets are usually preferred because of their low operating cost and their long-term stability. In addition, adopting NMR for on-line process/quality control depends on the following factors: suitable data reduction scheme, strong pulsed excitation to excite the solid-phase spins (i.e. excitation pulse width < solid TZ), automated sample handler, ability to control the effect of temperature in the magnet, and robust measurement protocol to cope with non-equilibrium structures. Because the CW NMR technique detects only the liquid-phase signal and uses saturation conditions in order to improve signal-to-noise levels, it is probably unsuitable for on-line applications. In order to apply pulsed N M R in circumstances of an industrial process, new magnets and coils should be developed that can be configured for different applications. For example, to use NMR as a sensor adapted to a food conveyor belt, an assembly is required in which the sample is external to both the magnet and the r.f. coil (Fig. 4). Innovations in NMR technology have resulted in the development of a one-sided configuration and several authors14>15have proposed N M R
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instruments where the sample is external to the instrument package. By using an r.f. sensor which resembles one half of a coaxial cable that is split along its axis, an NMR signal can be detected from a vertically elongated region that is about 10 cm long, 1cm by 1cm in cross-section, and situated 2 cm deep in the sample.I6 A tractor-mounted pulsed NMR instrument has even been developed" in which the sensor, consisting of an electromagnet, detection coil and tuning capacitor, provides a continuous readout of the volumetric soil water content at selected depths to 63mm. However, a disadvantage of the one-sided or flat surface technique is that it is much more difficult to obtain a homogeneous static field at the point of measurement. This can result in reduced measurement efficiency and decreased accuracy. Another important requirement for an on-line sensor is simple (ideally automatic) cleaning, and eventually sterilizing, especially in the food industry. As NMR is a non-contacting sensor, automatic cleaning devices are easy to set up by circulating strong base solution through the probe'' or water vapour at high temperature without affecting the quality of the NMR sensor. The development of a one-sided and non-contacting NMR sensor is advantageous as these sensors should not require costly cleaning systems. 3.3. Signal processing
'H NMR works by obtaining a signal which is proportional to the number of protons in the sample. To convert this signal into a meaningful number for responsive feedback, it needs to be compared with a signal from a known reference sample. On-line NMR detection requires automatic calibration devices which should be quickly re-standardized using artificial standards. These procedures need to be particularly robust in industry where the physical environment, electrical noise level and temperature are not stable. 4. POTENTIAL APPLICATIONS
Examples of the introduction of an NMR sensor for on-line process control are still very sparse, especially in food sciences. Nevertheless, with the growing number of laboratory applications of NMR in food science, it is possible that a few applications may eventually be adapted for on-line food analysis in the near future. We will now describe existing on-line applications of NMR and potential applications that could be easily developed in the future providing that the technical problems described above will be solved. On-line NMR applications can be divided in two groups, depending on the NMR instrumentation: (1) On-line applications of high-resolution NMR, which relies on instruments with high and homogeneous magnetic field.
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(2) On-line applications of low-resolution NMR, for which low-cost and bench-top NMR instruments have been developed. 4.1. High-resolution NMR
The laboratory applications of high-resolution solution-state NMR spectroscopy are mainly concerned with the elucidation of chemical and biochemical structures. In food science, this field of structural analysis is also useful but has no evident potential on-line application. However, purely analytical applications of high-resolution NMR (e.g. analysis of the content of food fluids like wine, beer or milk) could be developed for on-line use. Examples of such on-line analytical applications are found in the chemical industry3 and could be easily transferred to food science. The first on-line development of NMR was proposed by coupling high-performance liquid chromatography (HPLC) with high-resolution NMR. In 1978, Watanabe” obtained the first coupled HPLC-NMR stoppedflow measurement by using a 60MHz spectrometer. Later, with the development of the NMR sensitivity using high-field superconducting magnets, several authorszO,zlproposed a continuous-flow analysis of chromatographic eluents. Special technical devices were then developed to interface NMR with gas chromatography or liquid chromatography (Fig. 5 ) in order to allow the liquid to reside a certain time in the magnetic field and in the r.f. Many reactions in chemical or food process engineering are run continuously or in semi-batch mode so it has become useful to monitor the process on line. Recently, a 300 MHz NMR spectrometer has been coupled to a chemical reactor.23 It has been shown that this technique allows the detection of some intermediates with short lifetimes, and chemical equilibria that are normally influenced by sampling. On-line NMR analysis of liquid foodstuffs is generally complicated by the strong signal arising from the solvent, mainly water. This signal may saturate the memory of a computer used for data acquisition and thus prevent the observation of the weak signal from the solute. There are different solutions to avoid these problems such as the use of non-protonated solvents or selective solvent peak suppression. The use of non-protonated solvents is obviously impractical for food science because of their toxicity. A wide variety of solvent suppression techniques have been adapted for flow NMR. Classical gate homonuclear decoupling techniques require a relatively lengthy saturation periodz4 and are not suitable for flow measurement because of the renewal of the liquid in the detection coil. Binomial pulse sequences such as the 1-1 and the 1-3-3-1 sequence can suppress two solvents resonances sim~ltaneously.~~ In the same way, Curran and Williams z6 have compared both of these “hard pulsed” sequences and have
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CHARLES TELLIER AND FRANCOIS MARIElTE TO VACUUM
INLET
PREMAGNETIZATION REGION
5 mm OD
SAMPLE TUBE
15 m m OD QUARTZ TUBE
--- -
1.5 mm OD PYREX TUBING
Fig. 5. Example of a flow LC-NMR insert allowing complete premagnetization of the sample. (From ref. 21, used by permission.)
concluded that the 1-3-3-1 sequence is an acceptable choice for high concentrations of the solute but for low concentrations the 1-1 sequence gives the best results. The recent development of new solvent suppression techniques using pulsed field gradients2' should greatly enhance the analytical application of high-resolution NMR. In less dilute samples, like food paste from meat or dairy origin, high-resolution NMR can be used for rapid determination of the water and fat content. In these products, protons from water and lipids have different chemical shifts and give resolved signals even in a low-field high-resolution spectrometer.28 Integration of each NMR line provides an accurate and rapid estimation of the lipid and fat content. Recently Tellier et d." demonstrated that reliable NMR determination of water and fat in minced meat can also be obtained in flowing conditions, a finding which suggests future on-line applications. Small variation of the water content (<4%) can be detected with a time response of less than 1min. However, the reliability of these measurements depends on maintaining a constant temperature of
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the sample. As high-resolution NMR detects only liquid signals, temperature variations, which affect the solid/liquid ratio of the fat, induce signal fluctuation. Despite the potential utility of high-resolution NMR, uncertainties still remain on the long-term stability of a homogeneous field in an industrial environment. 4.2. Low-resolution NMR
Low-resolution NMR is the technique that has been most extensively applied to the study of food systems. The success of this technique is partly due to the development by the NMR manufacturers of dedicated, low-cost bench-top instruments. Consequently, it was also low-resolution NMR techniques that were initially adapted for on-line control in food industry. The first on-line applications have relied on available laboratory instruments which have been modified for industrial use. In the future, on-line development of pulsed low-resolution NMR will essentially depend on new NMR instruments specifically tailored to a particular on-line industrial application. Many off-line measurements that are routinely employed in the laboratory could then be transformed for on-line food process control. Until now, the few on-line NMR applications were essentially devoted to the determination of product moisture content. Real-time measurement of the moisture content in industrial drying processes are particularly important in order to optimize drying efficiency and so to reduce energy consumption. Pulsed low-resolution NMR presents a number of advantages over other current moisture monitoring techniques such as dielectric constant measurements or absorption of microwaves: it is insensitive to the sample packing density and to sample inhomogeneity; the NMR signal provides quantitative information on all of the water, both bound and free; and it is applicable to a broad range of materials. Pearson et d 4 , 1 3 have described an on-line moisture analysis in freshly tempered wheat. Their NMR sensor, initially designed for the on-line measurement of moisture in aluminium oxide, was adapted with an appropriate sampling device for flour. The measurement of the moisture content of wheat was accomplished without weighing the sample by using the hydrogen NMR signal from the non-moisture portion of the NMR signal as a measure of the amount of sample in the NMR coil. Good calibration curves were generated by plotting the ratio of the slow to the fast portions of the decay as a function of known moisture content of the sample (Fig. 6). This method is rapid and insensitive to the packing density of the sample volume but is only suitable for low moisture content (<30%). At higher moisture content, the water relaxation time ( T2) increases and the decaying liquid signal is primarily affected by the magnet inhomogeneity. To
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113 114115 16 1%
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Fig. 6. Free induction decay for moist wheat. Insert: calibration curve for wheat obtained by plotting the slow decay/ fast decay ratio versus the gravimetric moisture content. (Data from ref. 4, used by permission.)
overcome this limitation, the use of the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence has been suggested which produces a series of echoes with an amplitude unaffected by magnet imperfections. l2 Measurement of the ratio of the height of one of the echoes to the amplitude of the fast decaying component of the FID is then correlated with moisture content up to 60% moisture. Such measurements could be easily extended for on-line control of moisture in other foodstuffs such as margarine, crackers or pet food. In the food industry, fermentation processes are widely exploited and extensive research has been undertaken to develop suitable sensors for carrying out rapid and on-line analysis. It has been shown recently2* that low-resolution low-field pulsed NMR techniques allow the simultaneous determination of ethanol and sugars (Fig. 7) in an oenological fermentation
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Fig. 7. On-line monitoring of alcoholic fermentation by pulsed low-resolution NMR: variation in sugar and alcohol content as a function of time.
medium within a short time (3.5 min). The alcoholic concentration was measured from the echo modulation in the Carr-Purcell sequence29 and the sugar concentration was determined after attenuation of the strong water signal by the addition of a paramagnetic reagent. Sample preparation, including filtration followed by the addition of the relaxation reagent, was incorporated into an automated system for on-line monitoring of alcoholic fermentati~n.~' This laboratory-scale on-line application of NMR demonstrates that long-term processes can be efficiently followed even if the NMR technique utilizes a complicated pulse scheme. 5. FUTURE PROSPECTS
Given the number of applications of low-resolution NMR which have been developed recently for routine analysis and quality control in the food industry, the introduction of the corresponding on-line measurements can be anticipated in the very near future. Low-resolution NMR, which utilizes
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simple bench-top instruments, offers great prospects for on-line control. Several instrument manufacturers have already started to propose simple dedicated low-cost NMR sensors for specific food application^.^^ NMR sensors could be introduced in large-scale industrial drying installations where the resultant energy savings should rapidly compensate the investment of the NMR sensor. Other future on-line applications could include the simultaneous determination of fat and moisture in food products. Conventional methods for the determination of fat by Soxhlet extraction and moisture by oven-drying or Karl-Fischer methods are unsuitable for on-line control. By developing NMR sensors and protocols which allow the simultaneous determination of several food constituents, one could increase their potential utility in industrial processes. Characterizing the effect of a process on the food product quality is also an important field of potential applications for on-line NMR. The food industry uses increasingly sophisticated technological processes such as thermal treatment (Pasteurization or sterilization), freeze-drying, extrusion and now high pressure. All these treatments may affect the food products so that a rigorous control of the time or of the intensity of the treatment is necessary. Low-resolution NMR studies have shown that relaxation measurements could reveal critical information about the food product during a drying process3* or the acid coagulation of milk,33or could be used to predict the pelleting behaviour of animal feeds.34 It is also possible to determine qualitative information about the firmness and ripeness of fruits3' and also to reveal details about the heterogeneity of the structure of gels or emulsions.36These new NMR applications could be adapted on an industrial site in the near future. The future of high-resolution NMR as an on-line sensor is more doubtful due to the high cost of the spectrometers and their unknown long-term stability in an industrial environment. However, high-resolution studies provide frequency information on a spectrum which can be used to discriminate between molecular species when low-resolution studies based on different relaxation properties fail. Although only 'H NMR has been mentioned, the use of other sensitive nuclei such as I9F or 31P should be considered for very specific applications. 19FNMR offers an advantage for complex food media which are difficult to analyse with proton spectra; it can also be used as a label to provide specific chemical information. From a nutritional point of view, it is important to estimate both the content and the chemical form of fluorine in foods. For example in tea infusion, Horie et have shown that the main chemical form of fluorine was F- ions, which are known to have the highest bioavailability among the various fluorinated compounds. Fluorine could also be used as a probe to detect some fluorinated pesticides in Despite the great potential of the NMR technique, developments of on-line NMR sensors in the food industry are mainly prevented by the lack
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of dedicated, simple instruments. New NMR technology specifically tailored towards on-line control has to be developed by the NMR companies in collaboration with research institutions and the food industry. One must keep in mind that a scientifically acceptable solution must also be economically acceptable. This means that the economy benefits and savings must be much greater than the installation, maintenance and operating costs involved in the introduction of an on-line NMR sensor in food process control. REFERENCES 1. G. Geppert and L. Asperger, Bioelect. Bioenerg., 1987, 17, 399. 2. E. Lama and B. W. Li, J . Food Sci.,1984, 49, 995. 3. F. A. Nelson, C. A. Reilly and W. E. Savage, Ind. Eng. Chem., 1960, 52, 488. 4. R. M. Pearson, L. R. Ream, C. Job and J. Adams, Cereal Food World, 1987, 32, 822. 5 . D. W. Jones and T. F. Child, Advances in Magnetic Resonance (ed. J. S . Waugh), p. 123. Academic Press, New York, 1976. 6. D. A. Laude Jr. and C. L. Wilkins, Macromolecules, 1986, 19, 2295. 7. G. Suryan, Proc. Indian Acad. Sci. A , 1951, 33, 107. 8. K. Fukuda, A. Inouye, Y. Kawabe and A. Hirai, J. Phys. SOC.Jap., 1985, 54, 4555. 9. J. R. Singer, Science, 1959, 130, 1652. 10. H. Van As and T. J. Schaafsma, Biophys. J . , 1984, 45, 469. 11. C. Tellier, M. Trierweiler, J. Lejot and G. J. Martin, Analusis, 1990, 18, 67. 12. C. I. Nicholls and A. De Los Santos, Drying Technol., 1991, 9, 849. 13. R. M. Pearson and C . Job, Proc. ASAE Conf., 1991, 40. 14. R. K. Cooper and J. A. Jackson, J. Magn. Reson., 1980, 41, 400. 15. R. L. Kleinberg, A. Sezginer, D. D. Griffin and M. Fukuhara, J . Magn. Reson., 1992, 97, 466. 16. A. Sezginer, D. D. Griffin, R. L. Kleinberg, M. Fukuhara and D. G. Dudley, J . Elect. Waves and Appl., 1993, 7, 13. 17. R. F. Paetzold, G. A. Matzkanin and A. De Los Santos, Soil Sci. SOC.A m . J . , 1985, 49, 537. 18. C. Tellier, M. Guillou-Charpin and D. J. Le Botlan, Analusis, 1991, 19, M45. 19. N. Watanabe and E. Niki, Proc. Jap. Acad. B., 1978, 54, 194. 20. J. Buddrus and H. Herzog, Org. Magn. Reson., 1980, 13, 153. 21. E. Bayer, K. Albert, M. Nieder, E. Gross, G. Wolff and M. Rindlisbacher, Anal. Chem., 1982, 54, 1747. 22. J . F. Haw, T. E. Glass, D. W. Hausler, E. Motell and H. C. Dorn, Anal. Chem., 1980,52, 1135. 23. R. Neudert, E. Strofer and W. Bremser, Magn. Reson. Chem., 1986, 24, 1089. 24. J . Buddrus, H. Herzog and J. Cooper, J . Magn. Reson., 1981, 42, 453. 25. D. A. Laude Jr., R. W.-K. Lee and C . L. Wilkins, Anal. Chem., 1985, 57, 1464. 26. S. A. Curran and D. E. Williams, Appl. Spect., 1987, 41, 1450. 27. R. E. Hurd, J . Magn. Reson., 1990, 87, 422. 28. J. P. Renou, A. Briguet, P. Gateliier and J. Kopp, Int. J. Food Sci. Technol., 1987, 22, 169. 29. C. Tellier, M. Guillou-Charpin, P. Grenier and D. Le Botlan, J. Agric. Food Chem., 1989, 37, 988. 30. M. Guillou and C. Tellier, Anal. Chem., 1988, 60,2182.
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31. R. L. Dechene and A. Roy, Proc. ASAE Conf., 1990, 174. 32. J. Monteiro Marques, D. N. Rutledge and C. J. Ducauze, Lebensm. Wiss. Technol., 1991, 24, 93. 33. F. Mariette, C. Tellier, G. Brult and P. Marchal, J . Dairy Res., 1993, 60,175. 34. A. Davenel and P. Marchal, Trans. ASAE, 1992, 35, 1891. 35. R. L. Stroshine, S. I. Cho, W. K. Wai, G. W. Krutz and I. C. Baianu, Proc. ASAE Conf., 1991, 6565. 36. C. Tellier, F. Mariette, J.-P. Guillement and P. Marchal, J. Agric. Food Chem., 1993, 41, 2259. 37. H. Horie, T. Nagata, T. Mukai and T. Goto, Biosci. Biotech. Biochem., 1992, 56, 1474. 38. R. D. Mortimer and B. A. Dawson, J . Agric. Food Chem., 1991, 39, 1781.
FRANCOIS MARIETTE CEMAGREF, Division Technologie, 17 rue de Cucille',35044 Rennes Cedex, France
1. Introduction 2. On-line NMR: theoretical background 2.1. Equilibrium conditions 2.2. Effect of flow on NMR parameters 2.2.1. Flow continuous-wave NMR 2.2.2. Flow Fourier transform NMR 2.3. Continuous-flow or stopped-flow NMR? 3. Technical requirements 3.1. Sampling 3.2. Instrumentation 3.3. Signal processing 4. Potential applications 4.1. High-resolution NMR 4.2. Low-resolution NMR 5. Future prospects References
105 106 106 107 107 107 109 110 110 111 114 114 115 117 119 121
1. INTRODUCTION
On-line process control can often improve productivity and quality in the food industry by allowing a rapid identification of the raw materials used as ingredients and by adjusting the blending to keep the final composition of the product constant. For these reasons on-line process control is a rapidly growing branch of analytical chemistry. On-line sensors must be able to collect chemical or physical information from a sample and convert them into an interpretable signal for regulation within a short time according to the downstream process. On-line sensors for use in the food industry must meet special requirements such as food compatibility of the material in contact with the food sample and the ability to be easily cleaned and sterilized. For these reasons, the on-line use of sensors which are sensitive to the chemical composition of ANNUAL REPORTS ON NMR SPECTROSCOPY
VOLUME 31 ISBN 0-12505331-2
Copyright 0 1995 by Academic Press Limited AII rights of reproduction in any form reserved
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the product is poorly developed in the food industry. Biosensors, despite their high sensitivity and selectivity,' are often unsuitable for industrial purposes as they are unsterilizable, unreliable and invasive. Among physicochemical sensors, only infrared techniques have been really introduced in industrial production* and until now very few examples describe the use of an NMR sensor in industry and particularly in the food i n d ~ s t r y . ~ The great contrast between the wide applications of NMR techniques in food science and their virtual non-existence in industrial production may originate from several factors: NMR techniques are complicated and measure strange parameters that do not mean much to non-NMR specialists; NMR machines are very expensive; NMR is not as sensitive as other techniques such as IR spectroscopy and may be equally sensitive to extraneous factors like the industrial environment. However, the noninvasive and non-destructive character of NMR, coupled with the ease of cleaning and even sterilizing the probe, emphasize the usefulness of this technique for on-line monitoring in food process engineering. The objectives of this review are to discuss the theoretical and technical requirements of on-line NMR monitoring and to present examples illustrating various on-line applications of NMR which have already been tested industrially or which have potential interest for the future.
2. ON-LINE NMR: THEORETICAL BACKGROUND The use of NMR as an on-line sensor implies that the instrument should ideally be located in the production line or on a side-stream line. Therefore, on-line measurements suggest the existence of a continuous or a stoppedflow through the NMR sensor, which may perturb the quantitative response of the technique. In this section, the significant changes in the NMR signal induced by the flow will be presented and suggestions are given to choose the parameters that govern the optimization of the NMR response. 2.1. Equilibrium conditions
An NMR signal only appears when a sample is in a uniform magnetic field so that the individual nuclei align themselves with the external magnetic field and precess at the Larmor frequency. The resulting magnetization (Ma) of the sample is achieved in an exponential manner, over a time period dependent upon the spin-lattice relaxation time T I :
This equilibrium condition must be achieved prior to observation for
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quantitative NMR measurements. In a continuous or a stopped-flow experiment premagnetization of the sample occurs as the sample flows through the magnet before the detection probe. Assuming ideal flow behaviour, one can estimate the lifetime in the premagnetization region Tp from the rate (Q) and the premagnetization volume (V,): Tp = V&Q. For quantitative measurements, all sample nuclei must be completely premagnetized prior to observation. According to equation (l),this condition is met when: Tp> 5T1 where Tl corresponds to the maximum longitudinal relaxation time within the sample to be analysed. 2.2. Effect of Bow on NMR parameters
2.2.1. Flow continuous-wave NMR Analysis of the flow continuous-wave NMR experiment indicated two fundamental changes in the flowing system absorptive NMR signal measurement, both related to relaxation phenomena. If complete premagnetization of sample nuclei is assumed, the effects of flow on spin-lattice, TI, and spin-spin, T2, relaxation can be understood in terms of equations (2) and (3): 1 1 1 Tstatic + 7-pF=
1 -pF=-
1 1 Tstatic + -7
(3)
where r is the mean lifetime of the nucleus in the detection region (T = Vd/Q, v d is the detection volume), Tstaticare the conventional relaxation times and T O b s are the apparent relaxation times of the flowing sample.’ These equations suggest that at faster flow rates both apparent Tl and T2 should decrease. Shorter effective Tl is explained by the replacement of saturated nuclei with an influx of polarized nuclei which induces a signal enhancement in the flowing system. The shorter T2 is responsible for a broadening of the Lorentzian lineshape because of the decrease in observation time. 2.2.2. Flow Fourier transform NMR Equations (2) and (3) are equally applicable to the signal enhancement and line-broadening phenomena associated with the discrete flow FT-NMR
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0.4
-
-
I
I
I
I
I
Fig. 1. Intensity of the NMR signal as a function of the flow rate. Individual contributions from premagnetization and saturation are calculated assuming V , = 7 ml, V , = 1ml, T, = 0.5 s and T , = 1 s.
signal. For example, saturation effects observed in static FT-NMR at high recycle times (T,) are avoided in flowing system as long as the lifetime T of the nuclei in the detection region ( V d ) is equal to or shorter than the minimal delay between multiple scan (Tc). However, as flow rate increases and T decreases, the constant flux of excited nuclei leaving the detection region during data acquisition is responsible for the degradation in spectral linewidth. As the sample moves across the NMR probe, the two basic factors that determine the NMR signal intensity are the premagnetization time and the saturation effect. Figure 1 represents their individual contributions to the NMR signal and the composite result as a function of the flow rate ( Q ) .The premagnetization effect, dependent on V,,, induces significant decay from Mo when V,lQ is shorter than 5T1.On the other hand, saturation effects decrease as the flow rate increases until elimination at T, > 7. The composite signal thus produces a maximum for which minimized saturation effects at faster flow must effectively offset improved premagnetization at slower flow.
ON-LINE APPLICATIONS IN FOOD SCIENCE 00
109 0
non quantitative conditions
T,> 5 Ti
., \
Flow
2
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4
,
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\
non quantitative conditions
... ,, .,'.
6
z
\
%>T,
0 0 0
W
0
0
2
4
6
0 0 0
00
Tc Fig. 2. The conditions for quantitative flow as a function of flow rate and recycle time T, in a pulse NMR experiment. Tp and 7 are respectively the lifetime in the premagnetization and the observation regions. (Adapted from ref. 6, used by permission.)
Figure 2 graphically describes the conditions that ensure quantitative flow FT-NMR measurements as a function of the flow rate and the recycle time, assuming ideal flow behaviour.6 These conditions must be defined for the nuclei with the longest Tl in the sample.
2.3. Continuous-flowor stopped-flow NMR? In the earliest days, flow NMR was used to improve the sensitivity of NMR detection before the first Fourier transform spectrometer^.^ A further application of the flow experiment was the measurement of the flow rate. In particular, a variety of methods were developed for the analysis of flow rate inside pipes,' blood vessels' or plants." Until now, very few process controls have utilized flow NMR methods. However, flow NMR presents an advantage over off-line or stopped-flow measurements as the NMR result represents the average signal of a large amount of sample due to the flow across the probe during the time of the measurement. Therefore, flow NMR partially resolves the problem of sampling in heterogeneous materials and could avoid the necessity to
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construct large sample probes, which also require costly magnet modifications. 3. TECHNICAL REQUIREMENTS NMR is traditionally a laboratory-based technique. In order to use this technique as an on-line sensor in industry, a number of technical hurdles need to be overcome. Problems arise from scaling, which implies that sampling devices have to be adapted to the product conveyors. Moreover, there are additional problems associated with the factory environment such as variable temperature, nearby machinery and electrical noise. Finally, the NMR sensor should provide measurements in real-time, be robust enough to withstand a variety of industrial environments, have high reliability, and low operating costs, and needs a suitable pricelperformance ratio so that it is economically attractive to industrial users to implement this technology. 3.1. Sampling
With most NMR applications, sample preparation is generally minimal. The sample is loaded directly into the sample tube with no preparation at all. However, the first condition for reliable on-line NMR measurements is that the sample is in equilibrium with its environment. These equilibrium conditions involve a regular laminar flow for liquid, no thermal or concentration gradient, and a constant packing for solid samples. Sampling requirements also depend on the location of the sensor (fully encased in the line or on a side-stream of a fraction of the line) and on the loading solution in the NMR instrument, with a continuous flow or with a removeheplace system. A system where the NMR sensor completely encloses the production line avoids special sampling devices and offers the best NMR performance, but is not suitable for physically large samples which would require a large and expensive magnet. With a side-stream solution, the sample is diverted from the main product stream through a smaller sensor. After measurement, the sample is redirected back into the main product stream. This approach, which assumes that the sample has the same characteristics as in the main stream, is probably valid for liquids or solids. For viscous samples like meat or cheese paste, this assumption may be invalid because of the lamination of the sample into small pipes. Lamination can induce changes in the sample texturation or a destabilization of the food emulsion and may affect the NMR signal. Such effects have been observed" by continuously recycling a
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minced-meat paste into a small pipe (diameter 15mm) at low flow rate (1.2 1 min-I). The resulting water/fat ratio determined by on-line highresolution NMR measurement increases continuously as the meat paste is recycled. For solid samples, a variety of sampling techniques has been suggested for the side-stream solution. For on-line measurements of wheat moisture content, an air drive device4 has been used with a vibrator to fill the NMR probe immediately downstream from the wheat tempering screw. Nicholls and De Los SantosI2 have proposed a configuration where the sensor is suspended beneath a hole in the transport chute (Fig. 3). The NMR sensor is filled with the sample by lowering a piston which normally seals the aperture in the transport system. At the end of the measurement sequence the piston is driven back up the sample tube, returning the measured product to the process stream. Interestingly, cavities containing reference samples can be integrated within the piston, thus allowing the calibration of the sensor after each measurement. However, the system supposes that the conveyor material and the movement of the product do not perturb the NMR signal. Differences in the packing of solid samples is a problem frequently encountered which affects the volume-sensitive NMR response. For laboratory applications, the sample is usually weighed before measurement and signal intensity/weight ratio is used to compare the samples. This preweighing technique is unsuitable for on-line applications and must be replaced by new calibration methods. For the determination of moisture content in foodstuffs, the density problem can be solved by using the intensity of the rapid portion of the signal decay as a measure of how much solid is in the detector ~ 0 i l . l ~ 3.2. Instrumentation
Previously, modified commercially available NMR spectrometers were used for on-line process control, but only with partial success. Plant operators found them difficult to adjust and calibrate because they were laboratory instruments which had been repackaged for on-line use and were difficult to maintain. They did demonstrate, however, that NMR could be used to make accurate measurements on Any magnetic resonance spectrometer contains several electronic circuits which must be kept adjusted and calibrated. An industrial magnetic resonance process control analyser can be used by plant operators who have little or no familiarity with magnetic resonance. To minimize the time required to train operators to use the analyser, several self-calibrating and self-adjusting features are necessary. For example, all setting-up and monitoring of the instrument’s performance should be made automatic, e.g.
PRODUCT STREAM
-
I ! L SAMPLE TUBE NMR COIL
RF /
CAVITIES IN PISTON CONTAINING CALIBRATIO MATERIALS
PISTON
Fig. 3. Sampling from a conveyor belt using a piston containing calibration samples. (Adapted from ref. 12, used with permission.)
ON-LINE APPLICATIONS IN FOOD SCIENCE
\
113
CORE
Fig. 4. One-sided access NMR sensor configuration. (Adapted from ref. 12, used with permission.)
receiver gain, field adjustment, pulse width. Permanent magnets are usually preferred because of their low operating cost and their long-term stability. In addition, adopting NMR for on-line process/quality control depends on the following factors: suitable data reduction scheme, strong pulsed excitation to excite the solid-phase spins (i.e. excitation pulse width < solid TZ), automated sample handler, ability to control the effect of temperature in the magnet, and robust measurement protocol to cope with non-equilibrium structures. Because the CW NMR technique detects only the liquid-phase signal and uses saturation conditions in order to improve signal-to-noise levels, it is probably unsuitable for on-line applications. In order to apply pulsed N M R in circumstances of an industrial process, new magnets and coils should be developed that can be configured for different applications. For example, to use NMR as a sensor adapted to a food conveyor belt, an assembly is required in which the sample is external to both the magnet and the r.f. coil (Fig. 4). Innovations in NMR technology have resulted in the development of a one-sided configuration and several authors14>15have proposed N M R
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instruments where the sample is external to the instrument package. By using an r.f. sensor which resembles one half of a coaxial cable that is split along its axis, an NMR signal can be detected from a vertically elongated region that is about 10 cm long, 1cm by 1cm in cross-section, and situated 2 cm deep in the sample.I6 A tractor-mounted pulsed NMR instrument has even been developed" in which the sensor, consisting of an electromagnet, detection coil and tuning capacitor, provides a continuous readout of the volumetric soil water content at selected depths to 63mm. However, a disadvantage of the one-sided or flat surface technique is that it is much more difficult to obtain a homogeneous static field at the point of measurement. This can result in reduced measurement efficiency and decreased accuracy. Another important requirement for an on-line sensor is simple (ideally automatic) cleaning, and eventually sterilizing, especially in the food industry. As NMR is a non-contacting sensor, automatic cleaning devices are easy to set up by circulating strong base solution through the probe'' or water vapour at high temperature without affecting the quality of the NMR sensor. The development of a one-sided and non-contacting NMR sensor is advantageous as these sensors should not require costly cleaning systems. 3.3. Signal processing
'H NMR works by obtaining a signal which is proportional to the number of protons in the sample. To convert this signal into a meaningful number for responsive feedback, it needs to be compared with a signal from a known reference sample. On-line NMR detection requires automatic calibration devices which should be quickly re-standardized using artificial standards. These procedures need to be particularly robust in industry where the physical environment, electrical noise level and temperature are not stable. 4. POTENTIAL APPLICATIONS
Examples of the introduction of an NMR sensor for on-line process control are still very sparse, especially in food sciences. Nevertheless, with the growing number of laboratory applications of NMR in food science, it is possible that a few applications may eventually be adapted for on-line food analysis in the near future. We will now describe existing on-line applications of NMR and potential applications that could be easily developed in the future providing that the technical problems described above will be solved. On-line NMR applications can be divided in two groups, depending on the NMR instrumentation: (1) On-line applications of high-resolution NMR, which relies on instruments with high and homogeneous magnetic field.
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(2) On-line applications of low-resolution NMR, for which low-cost and bench-top NMR instruments have been developed. 4.1. High-resolution NMR
The laboratory applications of high-resolution solution-state NMR spectroscopy are mainly concerned with the elucidation of chemical and biochemical structures. In food science, this field of structural analysis is also useful but has no evident potential on-line application. However, purely analytical applications of high-resolution NMR (e.g. analysis of the content of food fluids like wine, beer or milk) could be developed for on-line use. Examples of such on-line analytical applications are found in the chemical industry3 and could be easily transferred to food science. The first on-line development of NMR was proposed by coupling high-performance liquid chromatography (HPLC) with high-resolution NMR. In 1978, Watanabe” obtained the first coupled HPLC-NMR stoppedflow measurement by using a 60MHz spectrometer. Later, with the development of the NMR sensitivity using high-field superconducting magnets, several authorszO,zlproposed a continuous-flow analysis of chromatographic eluents. Special technical devices were then developed to interface NMR with gas chromatography or liquid chromatography (Fig. 5 ) in order to allow the liquid to reside a certain time in the magnetic field and in the r.f. Many reactions in chemical or food process engineering are run continuously or in semi-batch mode so it has become useful to monitor the process on line. Recently, a 300 MHz NMR spectrometer has been coupled to a chemical reactor.23 It has been shown that this technique allows the detection of some intermediates with short lifetimes, and chemical equilibria that are normally influenced by sampling. On-line NMR analysis of liquid foodstuffs is generally complicated by the strong signal arising from the solvent, mainly water. This signal may saturate the memory of a computer used for data acquisition and thus prevent the observation of the weak signal from the solute. There are different solutions to avoid these problems such as the use of non-protonated solvents or selective solvent peak suppression. The use of non-protonated solvents is obviously impractical for food science because of their toxicity. A wide variety of solvent suppression techniques have been adapted for flow NMR. Classical gate homonuclear decoupling techniques require a relatively lengthy saturation periodz4 and are not suitable for flow measurement because of the renewal of the liquid in the detection coil. Binomial pulse sequences such as the 1-1 and the 1-3-3-1 sequence can suppress two solvents resonances sim~ltaneously.~~ In the same way, Curran and Williams z6 have compared both of these “hard pulsed” sequences and have
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CHARLES TELLIER AND FRANCOIS MARIElTE TO VACUUM
INLET
PREMAGNETIZATION REGION
5 mm OD
SAMPLE TUBE
15 m m OD QUARTZ TUBE
--- -
1.5 mm OD PYREX TUBING
Fig. 5. Example of a flow LC-NMR insert allowing complete premagnetization of the sample. (From ref. 21, used by permission.)
concluded that the 1-3-3-1 sequence is an acceptable choice for high concentrations of the solute but for low concentrations the 1-1 sequence gives the best results. The recent development of new solvent suppression techniques using pulsed field gradients2' should greatly enhance the analytical application of high-resolution NMR. In less dilute samples, like food paste from meat or dairy origin, high-resolution NMR can be used for rapid determination of the water and fat content. In these products, protons from water and lipids have different chemical shifts and give resolved signals even in a low-field high-resolution spectrometer.28 Integration of each NMR line provides an accurate and rapid estimation of the lipid and fat content. Recently Tellier et d." demonstrated that reliable NMR determination of water and fat in minced meat can also be obtained in flowing conditions, a finding which suggests future on-line applications. Small variation of the water content (<4%) can be detected with a time response of less than 1min. However, the reliability of these measurements depends on maintaining a constant temperature of
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the sample. As high-resolution NMR detects only liquid signals, temperature variations, which affect the solid/liquid ratio of the fat, induce signal fluctuation. Despite the potential utility of high-resolution NMR, uncertainties still remain on the long-term stability of a homogeneous field in an industrial environment. 4.2. Low-resolution NMR
Low-resolution NMR is the technique that has been most extensively applied to the study of food systems. The success of this technique is partly due to the development by the NMR manufacturers of dedicated, low-cost bench-top instruments. Consequently, it was also low-resolution NMR techniques that were initially adapted for on-line control in food industry. The first on-line applications have relied on available laboratory instruments which have been modified for industrial use. In the future, on-line development of pulsed low-resolution NMR will essentially depend on new NMR instruments specifically tailored to a particular on-line industrial application. Many off-line measurements that are routinely employed in the laboratory could then be transformed for on-line food process control. Until now, the few on-line NMR applications were essentially devoted to the determination of product moisture content. Real-time measurement of the moisture content in industrial drying processes are particularly important in order to optimize drying efficiency and so to reduce energy consumption. Pulsed low-resolution NMR presents a number of advantages over other current moisture monitoring techniques such as dielectric constant measurements or absorption of microwaves: it is insensitive to the sample packing density and to sample inhomogeneity; the NMR signal provides quantitative information on all of the water, both bound and free; and it is applicable to a broad range of materials. Pearson et d 4 , 1 3 have described an on-line moisture analysis in freshly tempered wheat. Their NMR sensor, initially designed for the on-line measurement of moisture in aluminium oxide, was adapted with an appropriate sampling device for flour. The measurement of the moisture content of wheat was accomplished without weighing the sample by using the hydrogen NMR signal from the non-moisture portion of the NMR signal as a measure of the amount of sample in the NMR coil. Good calibration curves were generated by plotting the ratio of the slow to the fast portions of the decay as a function of known moisture content of the sample (Fig. 6). This method is rapid and insensitive to the packing density of the sample volume but is only suitable for low moisture content (<30%). At higher moisture content, the water relaxation time ( T2) increases and the decaying liquid signal is primarily affected by the magnet inhomogeneity. To
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3 0.5-
CK
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0.2-
i
0.1
-
O<
;a 5
;
lb 111 1: Moisture (YO)
113 114115 16 1%
1 ;
2b
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.-c
Fig. 6. Free induction decay for moist wheat. Insert: calibration curve for wheat obtained by plotting the slow decay/ fast decay ratio versus the gravimetric moisture content. (Data from ref. 4, used by permission.)
overcome this limitation, the use of the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence has been suggested which produces a series of echoes with an amplitude unaffected by magnet imperfections. l2 Measurement of the ratio of the height of one of the echoes to the amplitude of the fast decaying component of the FID is then correlated with moisture content up to 60% moisture. Such measurements could be easily extended for on-line control of moisture in other foodstuffs such as margarine, crackers or pet food. In the food industry, fermentation processes are widely exploited and extensive research has been undertaken to develop suitable sensors for carrying out rapid and on-line analysis. It has been shown recently2* that low-resolution low-field pulsed NMR techniques allow the simultaneous determination of ethanol and sugars (Fig. 7) in an oenological fermentation
ON-LINE APPLICATIONS IN FOOD SCIENCE
-
119
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Fig. 7. On-line monitoring of alcoholic fermentation by pulsed low-resolution NMR: variation in sugar and alcohol content as a function of time.
medium within a short time (3.5 min). The alcoholic concentration was measured from the echo modulation in the Carr-Purcell sequence29 and the sugar concentration was determined after attenuation of the strong water signal by the addition of a paramagnetic reagent. Sample preparation, including filtration followed by the addition of the relaxation reagent, was incorporated into an automated system for on-line monitoring of alcoholic fermentati~n.~' This laboratory-scale on-line application of NMR demonstrates that long-term processes can be efficiently followed even if the NMR technique utilizes a complicated pulse scheme. 5. FUTURE PROSPECTS
Given the number of applications of low-resolution NMR which have been developed recently for routine analysis and quality control in the food industry, the introduction of the corresponding on-line measurements can be anticipated in the very near future. Low-resolution NMR, which utilizes
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CHARLES TELLIER AND FRANCOIS MARIE'ITE
simple bench-top instruments, offers great prospects for on-line control. Several instrument manufacturers have already started to propose simple dedicated low-cost NMR sensors for specific food application^.^^ NMR sensors could be introduced in large-scale industrial drying installations where the resultant energy savings should rapidly compensate the investment of the NMR sensor. Other future on-line applications could include the simultaneous determination of fat and moisture in food products. Conventional methods for the determination of fat by Soxhlet extraction and moisture by oven-drying or Karl-Fischer methods are unsuitable for on-line control. By developing NMR sensors and protocols which allow the simultaneous determination of several food constituents, one could increase their potential utility in industrial processes. Characterizing the effect of a process on the food product quality is also an important field of potential applications for on-line NMR. The food industry uses increasingly sophisticated technological processes such as thermal treatment (Pasteurization or sterilization), freeze-drying, extrusion and now high pressure. All these treatments may affect the food products so that a rigorous control of the time or of the intensity of the treatment is necessary. Low-resolution NMR studies have shown that relaxation measurements could reveal critical information about the food product during a drying process3* or the acid coagulation of milk,33or could be used to predict the pelleting behaviour of animal feeds.34 It is also possible to determine qualitative information about the firmness and ripeness of fruits3' and also to reveal details about the heterogeneity of the structure of gels or emulsions.36These new NMR applications could be adapted on an industrial site in the near future. The future of high-resolution NMR as an on-line sensor is more doubtful due to the high cost of the spectrometers and their unknown long-term stability in an industrial environment. However, high-resolution studies provide frequency information on a spectrum which can be used to discriminate between molecular species when low-resolution studies based on different relaxation properties fail. Although only 'H NMR has been mentioned, the use of other sensitive nuclei such as I9F or 31P should be considered for very specific applications. 19FNMR offers an advantage for complex food media which are difficult to analyse with proton spectra; it can also be used as a label to provide specific chemical information. From a nutritional point of view, it is important to estimate both the content and the chemical form of fluorine in foods. For example in tea infusion, Horie et have shown that the main chemical form of fluorine was F- ions, which are known to have the highest bioavailability among the various fluorinated compounds. Fluorine could also be used as a probe to detect some fluorinated pesticides in Despite the great potential of the NMR technique, developments of on-line NMR sensors in the food industry are mainly prevented by the lack
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of dedicated, simple instruments. New NMR technology specifically tailored towards on-line control has to be developed by the NMR companies in collaboration with research institutions and the food industry. One must keep in mind that a scientifically acceptable solution must also be economically acceptable. This means that the economy benefits and savings must be much greater than the installation, maintenance and operating costs involved in the introduction of an on-line NMR sensor in food process control. REFERENCES 1. G. Geppert and L. Asperger, Bioelect. Bioenerg., 1987, 17, 399. 2. E. Lama and B. W. Li, J . Food Sci.,1984, 49, 995. 3. F. A. Nelson, C. A. Reilly and W. E. Savage, Ind. Eng. Chem., 1960, 52, 488. 4. R. M. Pearson, L. R. Ream, C. Job and J. Adams, Cereal Food World, 1987, 32, 822. 5 . D. W. Jones and T. F. Child, Advances in Magnetic Resonance (ed. J. S . Waugh), p. 123. Academic Press, New York, 1976. 6. D. A. Laude Jr. and C. L. Wilkins, Macromolecules, 1986, 19, 2295. 7. G. Suryan, Proc. Indian Acad. Sci. A , 1951, 33, 107. 8. K. Fukuda, A. Inouye, Y. Kawabe and A. Hirai, J. Phys. SOC.Jap., 1985, 54, 4555. 9. J. R. Singer, Science, 1959, 130, 1652. 10. H. Van As and T. J. Schaafsma, Biophys. J . , 1984, 45, 469. 11. C. Tellier, M. Trierweiler, J. Lejot and G. J. Martin, Analusis, 1990, 18, 67. 12. C. I. Nicholls and A. De Los Santos, Drying Technol., 1991, 9, 849. 13. R. M. Pearson and C . Job, Proc. ASAE Conf., 1991, 40. 14. R. K. Cooper and J. A. Jackson, J. Magn. Reson., 1980, 41, 400. 15. R. L. Kleinberg, A. Sezginer, D. D. Griffin and M. Fukuhara, J . Magn. Reson., 1992, 97, 466. 16. A. Sezginer, D. D. Griffin, R. L. Kleinberg, M. Fukuhara and D. G. Dudley, J . Elect. Waves and Appl., 1993, 7, 13. 17. R. F. Paetzold, G. A. Matzkanin and A. De Los Santos, Soil Sci. SOC.A m . J . , 1985, 49, 537. 18. C. Tellier, M. Guillou-Charpin and D. J. Le Botlan, Analusis, 1991, 19, M45. 19. N. Watanabe and E. Niki, Proc. Jap. Acad. B., 1978, 54, 194. 20. J. Buddrus and H. Herzog, Org. Magn. Reson., 1980, 13, 153. 21. E. Bayer, K. Albert, M. Nieder, E. Gross, G. Wolff and M. Rindlisbacher, Anal. Chem., 1982, 54, 1747. 22. J . F. Haw, T. E. Glass, D. W. Hausler, E. Motell and H. C. Dorn, Anal. Chem., 1980,52, 1135. 23. R. Neudert, E. Strofer and W. Bremser, Magn. Reson. Chem., 1986, 24, 1089. 24. J . Buddrus, H. Herzog and J. Cooper, J . Magn. Reson., 1981, 42, 453. 25. D. A. Laude Jr., R. W.-K. Lee and C . L. Wilkins, Anal. Chem., 1985, 57, 1464. 26. S. A. Curran and D. E. Williams, Appl. Spect., 1987, 41, 1450. 27. R. E. Hurd, J . Magn. Reson., 1990, 87, 422. 28. J. P. Renou, A. Briguet, P. Gateliier and J. Kopp, Int. J. Food Sci. Technol., 1987, 22, 169. 29. C. Tellier, M. Guillou-Charpin, P. Grenier and D. Le Botlan, J. Agric. Food Chem., 1989, 37, 988. 30. M. Guillou and C. Tellier, Anal. Chem., 1988, 60,2182.
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31. R. L. Dechene and A. Roy, Proc. ASAE Conf., 1990, 174. 32. J. Monteiro Marques, D. N. Rutledge and C. J. Ducauze, Lebensm. Wiss. Technol., 1991, 24, 93. 33. F. Mariette, C. Tellier, G. Brult and P. Marchal, J . Dairy Res., 1993, 60,175. 34. A. Davenel and P. Marchal, Trans. ASAE, 1992, 35, 1891. 35. R. L. Stroshine, S. I. Cho, W. K. Wai, G. W. Krutz and I. C. Baianu, Proc. ASAE Conf., 1991, 6565. 36. C. Tellier, F. Mariette, J.-P. Guillement and P. Marchal, J. Agric. Food Chem., 1993, 41, 2259. 37. H. Horie, T. Nagata, T. Mukai and T. Goto, Biosci. Biotech. Biochem., 1992, 56, 1474. 38. R. D. Mortimer and B. A. Dawson, J . Agric. Food Chem., 1991, 39, 1781.