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NMR oilseed analysis in the plant and lab

Understanding the amount of oilwhich may be extracted fromoil seeds is fundamental to the

industry for a number of reasons.Depending on the location and theseed type, the producers may be paidon the basis of the oil content of theseeds they deliver, or the extractionprocess may be in some way con-trolled on the basis of oil content tomaximize processing efficiency. Ineither case, the time delay betweentaking a representative sample andhaving an analytical result is of greatimportance.

The traditional commercial methodis by extraction. Most methods todetermine oil content involve the useof solvents on a laboratory scale, how-ever, there is growing environmentalpressure, and a tendency toward leg-islative pressure, which are makingsuch techniques less attractive. Per-haps more fundamentally, the labora-tory solvent extraction process is time-consuming and generally performedremotely from the plant, where theresults are needed.

From as early as the 1960s, instru-mentation manufacturers have seenbusiness opportunities to provide sim-ple devices to measure oil content inseeds. Early on, Newport Instrumentsdeveloped a continuous wave NMRinstrument which gave nondestructivebulk measurement of oil content. Inmore recent times, near infraredinstrumentation and supercnticat fluidextraction techniques also have begunto be developed, but these technolo-gies have not yet been proven to pro-vide the precision, reliability androbustness required of industrial anal-ysis. It is NMR to which agenciessuch as the Federal Grain InspectionService of the U.S. Department ofAgriculture (USDA) look as the "goldstandard" for oil content analysis. andwhich has been the most widely usedinstrumental technique.

Three generations01 NMR analytlcallnstru·menta. At upper tett Is anearty continuous wave

system; at upper right Is aputsed wave machine; tothe right Is a more recent

unit, the MQA6005.

The history of the involvement ofNMR in cilseeds analysis has pro-ceeded in three stages. The first instru-ments, and still the most widely usedtoday, are the continuous wave NMRunits developed by Newport. whichlater became part of the Oxford lnsuu-ments group. These instruments couldmeasure oil content according to theISO 5511 procedure, the FOSFA offi-cial NMR method, or, in the UnitedStates, according to the USDA NMRHandbook. The system uses a large150-mL sample which allows a morerepresentative sample from the seedbatch to be analyzed-particularlyimportant in the case of larger seedsor samples, such as olives. The maindrawback of the continuous wavemethod is the requirement for a time-consuming drying stage before analy-sis can take place, since the oil contentmeasurement is affected by the con-flicting NMR signal from the water

TIUs repon was pf'I!PQf'I!d by And'mv CrooMll of Orfon! Instruments, Ana-lytical Syslems Division, 130 A. /JQker Ave. Extension, Concord. MA 01742and acknowledges the experimental work of Dr. EliuJbelh Faord and Mr.CltristopMr Dunn.

present in the sample. The USDAmethod, for example, indicates a dry-ing time at l300e for three hours fol-lowed by a cooling stage of two tothree hours before analysis.

The second stage of the evolutionof NMR for oilseeds analysis involvedthe transition from continuous waveNMR to pulsed NMR. Instrumentswere produced by the mainstreammanufacturers, Bruker and Oxford(the Minispec and QP20+ respective-ly), and later from a few other instru-ment manufacturers. All of these sys-tems analyzed oil content, but wererestricted in sample size to volumes of40 mL or smaller. This loss in repre-sentative sampling volume was offsetsomewhat by the stronger magneticfields used in these instruments; how-ever, much of the reason for makingthe transition was based around thedesire to measure moisture and oilcontent simultaneously. The ISO10565 standard of 1993 (equivalent toAOeS Ak 4-95) was written based ona series of field tests of the Brukerinstrument, selling standards forrepeatability, typical examples ofwhich are shown in Table l.

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Table 1can be considered as an alignment of

Precision (standard deviation, 0) Targets for different seed types the spin properties of these nuclei withthe magnetic field. Subjecting the

Rapeseed Sunflower Soybean Linseed sample \0 radio frequency (RF) elec-tromagnetic energy will cause this

Mean oil content 41.12% 47.90% 19.70% 40.02% alignment to be disturbed. but onlywhen the frequency of the energy

Std. deviation 0.20 0.30 0.15 0.13 matches an absorption frequency char-Mean moisture 6.5% 5.0% 9.4% 5.7% acteristic of each distinct atomicStd. deviation 0.035 0.Q1 0.12 0.05 nucleus in the presence of the magnet-• ic field. For example, in a 0.47 Tesla

tcominuedfmm page 515)magnetic field. hydrogen nuclei willabsorb energy at 20 mHz, and fluorine

The third development has been the in an industrial environment. such as nuclei will absorb energy at 18.9recent introduction of NMR instru- grain elevators or extraction facilties. mHz. These are known as the reso-mentation with the sampling capacity requires a level of robustness and min- nance frequencies for these nuclei.of the older continuous wave instru- imal scheduled maintenance that is We can thus picture two distinctments, but with advanced digital data rarely seen in instruments designed for types of NMR experiments. In theacquisition and processing capabilities laboratory use. Expectations of the continuous wave experiment, the sam-that allow the precision requirements simplicity and intuitive nature of the pie is subjected to a single varying RFof ISO 10565 to be exceeded for both user interface have grown as the world frequency which is swept through themoisture and oil content measure- of home computing and graphical user resonance frequency, exciting themem. Instrumentation of this type. the interfaces has opened up. Worldwide sample. The absorption of energy isOxford MQA6005. is being used by high-speed support has become a pre- recorded and measured by the instru-the USDA as part of a feasibility study requisite for instruments to be used as mern. the amount of energy absorbedleading toward endorsement of the integral parts of critical processes. A being proportional to the number ofpulsed NMR technique as a replace- set of criteria against which potential nuclei present in the sample whichrnent for the continuous wave NMR providers of instrumentation for undergo resonance at that frequency.techniques. The continuous wave and oilseed analysis may be assessed could and the width of the absorption peakpulsed NMR techniques are drawn include (a) performance conforms to corresponding to the physical environ-together in the new device, providing recognized standards; (b) sample vel- ment in the sample-nuclei in solid-an ability to analyze oil content ume is suitable for larger seed types like environments give a very broadaccording to the official methods. and existing standards; (c) robustness continuous wave NMR signal, and liq-such as that endorsed by the USDA. is suitable for plant or laboratory use; uid components give a much narroweror to eliminate the drying stage and (d) global support and service are signal (Figure I).analyze both oil and moisture content available; (e) user interface is suitable In the pulsed NMR experiment, thein accordance with the newer ISO for use by untrained operators in plant sample is subjected to a pulse of RF10565/AOCS Ak 4-95 method. Table environment; and (f) rapid results energy containing frequencies at and2 shows results obtained using the match business requirements around the resonance frequency. TheMQA6005 which can be compared to nuclei are capable of absorbing energythe targets in Table I, An overview of NMR do so. releasing the energy theyIt is clear from the results shown in When samples are subjected to a mag- absorbed after the pulse is complete.

Table 2 that the MQA6005 pulsed netic field, an interaction between the The instrument detects and measuresNMR method is capable of analyzing electromagnetic properties of certain the energy that is released after thesamples with a wide range of oil con- atomic nuclei in the sample and the pulse. this energy being proportionaltent, from sunflower (ranging from magnetic field takes place. The effect to the number of nuclei that werearound 35-55% oil) to soybean meal{defatted, typically 0.5-1% oil) withexcellent precision. The samples werenot prepared in any way, being mea- Table 2sured at room temperature as pre- 011and moIsture content. Instrument: MQA6005scribed by ISO 10565. so the totalanalysis time comprised filling and Rapeseed Linseed Soya meal Soya flakeweighing the sample tube plus mea-

Mean oil content 39.8<J, 34.6% 1.21<J, 0.97%suring using the NMR instrument.In parallel with developments in Std. deviation 0.06% 0.02 0.017 0.025

instrumentation. the expectations of Mean moisture 6.0% - 12.0% 9.13%instrumentation users also have Std. oevteuon 0.02 - 0.058 0.035increased and been refined. Utilization •

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Liquid resonance

NMR signals, and the effect on thepulsed NMR experiment is for theNMR signal to decay more quicklythan the relaxation characteristics ofthe sample would dictate. In the lattercase, however, magnet inhomogeneityeffects can be compensated for by theuse of additional pulses which aredesigned to "refocus" the NMR signal.Solid resonance

Figure 1. Conllnuous wave NMR IJlgnaJ

excited by the pulse. The energyabsorbed by the nuclei is released in adecay curve (the process is known asrelaxation) which is characteristic ofthe physical environment in the sam-ple experienced by the nuclei. Thosein a solid-like environment relax rela-tively quickly, whereas signals fromliquid components relax much moreslowly (Figure 2).

Characteristics orNMR equipmentThe conditions required for resonanceare expressed in a relationshipbetween magnet field strength andexcitation energy. Thus a 0.47 testamagnet may sometimes be referred toas a 20-mHz (proton) magnet. As fieldstrength increases, so the resonancefrequency increases proportionally.

The NMR signal strength is related(in a somewhat complex fashion) tothe field strength (and therefore theresonance frequency). Thus, signaland signal-to-noise are improved athigher frequencies/stronger fields.

The NMR signal strength also isinversely proponional to the absolutetemperature of the sample. As thetemperature of the sample increases,

Solid decay

Field

How oil and moisture content aremeasured by pulsed NMRA seed sample can be considered insimple terms as consisting of threecomponents that contain hydrogen aspan of their molecular structures: (a)the solid matrix of the seed material.(b) the oil component, and (c) themoisture component. At relatively lowmoisture contents, e.g., up to around15% in the case of rapeseed, the mois-ture is bound to the seed matrix insome way and behaves in terms of itsNMR signal as if it were a semisolid.The oil component of the seed samplebehaves much more like a liquid.Residual moisture content above thatwhich is tightly bound to the matrixalso behaves in a liquid manner, and iseffectively indistinguishable from theoil. For similar reasons, seeds whichhave a solid oil component at roomtemperature (e.g.. coconut) are likelyto prove difficult since the oil is likelyto be indistinguishable from the mois-ture signal, making such samples dif-ficult to analyze without drying ormeasuring at elevated temperatures.

The pulse sequence used to mea-sure oil and moisture in seeds isshown in Figure 3. The sample is sub-jected to an initial excitation pulse,and the NMR signal is sampled assoon as possible after that pulse. Bythe time this first data block is sam-

(continued on page 520)

the NMR signal decreases. Variationsin temperature during NMR experi-ments may account for some of theinaccuracies and drift effects some-times seen.

The signal strength is related to thevolume of the sample. Large samplevolumes give larger signals and thusassist in improving signal-to-noisecharacteristics. There is a more impor-tant issue related to sample volume,however, when the sample is nonho-mogeneous. Here the larger the vol-ume, the more representative the sam-ple. This is the case with seeds, andmore so with olives.

Magnet homogeneity is an impor-tant characteristic. No permanent mag-net system gives a totally unifonn fieldat all points inside the sample, due tosmall random variations in the materi-als from which the magnet is con-structed and to imperfections in themagnet-charging process. As a result.the field strength is slightly different atdifferent points in the sample. andtherefore the resonance frequency isslightly different at different pointsalso. The effect on the continuouswave experiment is to broaden the

Liquid decay CombinJItion decay

tNFORM,Vol. 8. no. 5 (May 1997)

Agure 2. Schematic repf'esentatlon of putse NMR slgns.s

520

e-e oil

For information circle' 162

51 • moistur52 • oil51- 52 · moisru

putse Pulse

~

52

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Measure at SOmicroseconds (5 1)

nme~ Measure DI7.000microseconds (S2)

Agure 3. Pulse sequen<:e used 10meuure all and moIsture In ollseeds

nals [or samples of known oil andmoisture content and performing a lin-ear least squares fit. The requirementsfor calibration vary from method tomethod. Oilseed analysis using thecontinuous wave NMR instrumentsoriginally was performed using a sin-gle-point calibration corresponding toa 100% oil sample (a zero interceptwas used): however, current methodsfor continuous wave instruments use atwo-point calibration based on tworeference samples. The oil-and-mois-ture calibrations for pulsed NMR aremultipoint calibrations. It is recom-mended that at least six samples beused to form the basis of the calibra-tion line, although it is possible to usedifferent standards to create each ofthe oil and the moisture calibrations ifboth values are not known for theavailable standards. As with any refer-ence technique where calibrations areused (true of most spectroscopicmethods), the analytical results willonly be as good as the calibration theyare obtained from, and the calibrationin turn will only be as accurate as thereference values used for the stan-dards. It is thus of prime importancethat accurate reference values are usedfor calibration, and that the errors lim-its of the technique used to assign thereference values are understood andapplied. Results with an accuracy of ±O. [% cannot be obtained from aninstrument calibrated with sampleswhose oil and moisture contents areonly known to. say, ± 0.5%! •

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pled, the signal from the solid seedmatrix has completely relaxed and isnot detected. The signal which isobserved here contains contributionsfrom the oil component and all themoisture present. After a delay in theorder of a few milliseconds, the sam-ple is subjected to a second pulsewhich is used to refocus the signal.compensating for magnetic inhomo-geneity. Following the second pulse.data are again collected by which limethe signal from the tightly boundmoisture has decayed away complete-ly, and the second block of data corre-sponds only to the oil component (andany residual moisture which is in aliquid-like environment).

The signals from these two datablocks are digitized, filtered, and pro-cessed to obtain a single value repre-sentative of oil + moisture from thefirst data block and representative ofoil only from the second data block. Aprocess of subtraction allows a valuerepresentative of moisture only to beobtained.

Transforming these representativeNMR values into % oil and % mois-ture values is performed by relatingthe values to calibration lines. In prin-ciple. a single calibration is used foreach seed type. although often differ-ent seeds can be combined into singlecalibrations without significantlydegrading the standard error of thecombined calibration. The calibrationsare formed by measuring NMR sig-

INFORM. Vol. 8. no. 5(Moy 199n