Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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Lajunen LHJ (1992) Spectrochemical Analysis by Atomic

Absorption and Emission. Cambridge: Royal Society of

Chemistry.

Radziuk B and Schlemmer G (1999) Analytical Graphite

Furnace Atomic Absorption Spectroscopy: A Labora-

tory Guide. Basel: Birkhauser Verlag.

Sanz-Medel A (1999) Flow Analysis with Atomic Spectro-

metric Detectors. New York: Elsevier.

Sneddon J (2000) Advances in Atomic Spectroscopy.

Amsterdam: Elsevier.

Sperling M and Welz B (1999) Atomic Absorption Spec-

trometry Weinheim: Wiley-VCH.

Wilson RH (1994) Spectroscopic Techniques for Food

Analysis. New York: VCH.

Nuclear Magnetic Resonance

A M Gil, University of Aveiro, Aveiro, Portugal

Introduction

0001 The nuclear magnetic resonance (NMR) phenom-

enon was discovered at Stanford University in 1945

by Bloch, Hansen, and Packard, who detected a

proton signal from water and at Harvard University

by Purcell, Torrey, and Pound, who observed a signal

from the protons of paraffin wax. Bloch and Purcell

were jointly awarded the Nobel Prize for physics in

1952 for this discovery.

0002 The observation of an NMR signal from a nucleus

requires that the nuclear spin (I) is described by I 6¼0.

For instance,

C and

C have respectively I ¼0 and

I ¼1/2, hence only

C is observable by NMR. Asso-

ciated with the nuclear spin, there is a nuclear mag-

netic moment (m), which will interact with an applied

magnetic field B

. This interaction causes the mag-

netic moment of a nucleus of spin I to take 2I þ1

possible orientations, each corresponding to an

energy level. In the case of I ¼1/2 (e.g.,

N), two energy levels separated by DE ¼hgB

/2p

are generated, where g is the nuclear magnetogyric

ratio. However, the exact value of DE absorbed by a

nucleus is also very sensitive to its surrounding mo-

lecular environment; hence, by registering the

absorbed energy, NMR probes molecular structure

and molecular dynamics. Chemical shifts (d ), scalar

coupling constants (J) and relaxation times (T

, T

)

are NMR parameters which carry both structural and

dynamic molecular information.

0003 One of the earliest applications of NMR in food

science dates from 1957 and concerned the measure-

ment of moisture in foods. However, it is only from

the early 1980s that a consistent and widespread

growth in the use of NMR in food science has been

observed. This resulted from: (1) the increasing

sophistication and relative ease of use of NMR

instrumentation; (2) the increasing need of the food

industry to understand and innovate its products and

processes; and (3) the increasing pressure for new

methods to enforce legislation and control quality.

Many applications have grown in parallel to applica-

tions in the biological and biomedical fields, in which

the noninvasiveness of the technique is a requirement.

In foods, such requirement stems not from ethical

considerations but from the need to preserve and

examine food structure.

0004The first review of applications of NMR in food

science dates from 1993; the first international con-

ference on the subject was held in 1992 and has been

followed by meetings every 2 years since then.

The NMR Techniques for Food Analysis

0005Foods are generally very complex systems both chem-

ically (containing an enormous variety of compounds

differing in nature, size, and quantity) and physically

(often being multiphase systems). The choice of NMR

method depends primarily on the chemical and

physical complexity of the food. For liquid foods

like juices, wine, and oil, the method of choice is

usually liquid-state high-resolution one-dimensional

(1D) and two-dimensional (2D) NMR spectroscopy.

Total correlation spectroscopy (TOCSY), heteronuc-

lear correlation spectroscopy (e.g.,

Hor

and J-resolved spectroscopy are 2D experiments

which give, respectively, information on homonuclear

couplings, heteronuclear couplings, and J-values/

multiplicity. This information enables chemical iden-

tification and structural determination to be carried

out, provided the spectra are free from serious signal

overlap. In recent years, automated methods have

been developed to allow the handling of high numbers

of samples with little or no user intervention. Hy-

phenated techniques have also recently been de-

veloped, to handle the high chemical complexity of

many foods. These couple NMR with liquid chroma-

tography (LC) and mass spectrometry (MS) in the

forms of LC-NMR and LC-NMR/MS.

0006If the above NMR techniques are applied to solid

or semisolid foods, spectra with very broad lines

will result, from which structural information can

be very difficult to extract. Solid-like foods call for

specific methods which enable resolved spectra to be

obtained. Magic angle spinning (MAS) aids the

observation of dilute nuclei, e.g.,

N, when

coupled with cross-polarization (CP) and high-

power decoupling. The CPMAS experiment selects

the nuclei present in the solid phase of the food.

SPECTROSCOPY/Nuclear Magnetic Resonance 5447

Conversely, direct excitation methods select the nuclei

in the mobile phase of the food, therefore both CPMAS

and direct excitation methods can give complemen-

tary information on multiphase foods. But for solid

and heterogeneous foods, magnetic resonance im-

aging (MRI) has been one of the most recent and

popular methods. In this technique, the frequency of

the NMR signal is dependent on position and, there-

fore, putting together the information arising from

different parts of the sample gives rise to a 2D or 3D

representation. The contrast in such image may result

from changes in spin density (related to concentra-

tion), relaxation times, or diffusion coefficients. A

recent novel idea is the attempt to correlate molecular

properties, viewed by NMR, with macroscopic prop-

erties, e.g., rheological behavior. The method of

Rheo-NMR aims precisely at achieving this correl-

ation and its use for foods is now at its infancy. The

method registers NMR information (either using

imaging or spectroscopy) while stress (shear or ex-

tensional) is applied to the sample.

Water in Foods

0007 Water profoundly affects many aspects of food qual-

ity: texture, microbiological safety, nutritional status,

and digestibility. NMR has been used to probe water

activity and water translational mobility in foods.

Water relaxation times provide information on the

amount and mobility of water. Of the three NMR-

active nuclei available in water (

HinHOD,and

OinH

O),

O is the more direct probe since it is

not affected by the chemical exchange of protons

between solute and water. The different degrees of

‘water binding’ to solute or biopolymer molecules

have been measured in systems as varied as caseinate

solutions, potato and wheat starch suspensions, and

gelatin gels. Glass transitions in food ingredients like

casein, gluten, amylopectin, protein/sugar, and poly-

saccharide/sugar mixtures are strongly dependent on

water activity/content and have important conse-

quences in product quality. Again, the understanding

of molecular mobility of water and of the solute, by

NMR relaxation, has enabled structural models to be

proposed. For instance, relaxation data have sug-

gested a model of a maltose glass in which water

molecules undergo rapid rotational and translational

motion inside more rigid ‘cages’ or ‘channels’ formed

by maltose molecules. (See Water: Structures, Proper-

ties, and Determination.)

0008 The effect of food microstructure on water relax-

ation is important since a distribution of relaxation

times occurs and may be used to determine pore size

distributions in the food. Many studies have involved

model systems like water-saturated porous rocks,

sandstones, chalks, and cement pastes. Some studies

of actual food materials have been done, trying to

handle the additional complexity due to the chemical

nature of food components.

0009At a macroscopic scale, water located in different

types of tissue is characterized by different relaxation

properties and diffusion coefficients, which result

from distinct cell types and sizes. Water is distributed

among different subcellular compartments such as

vacuoles, starch granules, and the cytoplasm, and

again each compartment is characterized by a certain

distribution of water relaxation times. These NMR

measurements may be used to give information about

cell morphology and membrane water permeabilities.

Applied to fruits and vegetables, this approach is

useful to determine water/air/ice distribution, related

to overall quality changes, as the food is ripened,

dried, rehydrated, frozen, and stored.

0010The dependence of relaxation properties on tissue

type is the origin of contrast in relaxation or diffu-

sion-weighted MRI of intact fruits and vegetables.

MRI has long established its value and potential

in food science, not only enabling the qualitative

imaging of ‘static’ food structure but also, and more

importantly, following mass and heat transport in

foods, noninvasively, and in real time during process-

ing or storage. MRI may be used for temperature

mapping of foods, after a suitable calibration of the

relaxation times or diffusion coefficients against

temperature. The temperature profile of a whole

food may be used to deduce thermal diffusivity and

surface heat transfer coefficients. Subzero tempera-

ture MRI is very useful for the detection of freezing

effects, detection of unfrozen water, measurement of

freezing times, and of freeze-drying kinetics. MRI is

also used to monitor changes at food surfaces, an

important application since the efficiency of many

processing operations (e.g., baking, drying) depends

on the magnitude of heat and moisture transfer rates

at the food surface.

0011Besides providing the moisture and temperature

profile of a food, MRI is sensitive to several other

quality factors: lipid distribution, solute concentra-

tion, pH, solid–liquid ratios, protein and polysacchar-

ide aggregation. Table 1 gives some examples of the

applicability of MRI in foods, and a more compre-

hensive list can be found in the specialized manuals

indicated at the end of this text.

Food Biopolymers

Proteins

0012Both liquid-state and solid-state NMR methods have

been of value to elucidate aspects of structure and

5448 SPECTROSCOPY/Nuclear Magnetic Resonance

behavior of food proteins, such as the milk proteins

casein and a-lactalbumin and many cereal proteins.

Whereas the wide range of liquid-state NMR methods

has been applied to small proteins like a-lactalbumin,

seed and storage proteins generally have higher mo-

lecular weight and higher heterogeneity and are often

insoluble, which renders the usual high-resolution

methods rather ineffective. Hence,

Hand

C solid-

state NMR methods have been valuable in character-

izing the structure and behavior of large cereal pro-

teins of wheat, barley, maize, and sorghum. These

proteins have important functional and nutritional

properties which require an understanding at the

molecular level, in order to enable suitable quality

control. The Rheo-NMR method has already proved

promising in the attempt to correlate the molecular

structure of hydrated gluten (wheat protein) with

its characteristic viscoelasticity. Multinuclear NMR

(

Mg,

Ca) has also been used to study ion-

binding proteins, e.g., b-casein and a-lactalbumin, in

order to determine the characteristics of the binding

site and of metal competitivity under physiological

conditions (See Protein: Chemistry.)

Polysaccharides

0013The interest of the food scientist in polysaccharides

includes the issues of dietary components (starch,

plant cell wall polysaccharides) and functional ingre-

dients like gelling, thickening, and stabilizing agents.

Naturally, high-resolution NMR is a powerful tech-

nique for carbohydrate analysis in solution, whereas

CPMAS and related solid-state techniques have been

applied to solid polysaccharides, in gels and in hetero-

geneous samples. In solution, NMR can not only

provide a measurement of overall composition and

detailed conformational structure, but also be sensi-

tive to fine details such as degree of esterification,

degree and type of substitution, residue sequence,

and average block length. Table 2 shows some

examples of studies of several food polysaccharides

in solution (See Carbohydrates: Classification and

Properties.)

0014The knowledge of the physical properties of com-

plex carbohydrates is extremely useful for food

scientists. Such information is available in several

databases, including an NMR database. Processes

like gelation, crystallization, starch gelatinization,

and retrogradation are extremely important in deter-

mining the quality of a polysaccharide-containing

food. Cellulose and starch provide excellent examples

of solid polysaccharides from which extensive struc-

tural information may be obtained by solid-state

NMR.

C CPMAS NMR clearly distinguishes the

A (from cereals) and B (from tubers) starch poly-

morphs and can also measure the proportions of

crystalline and amorphous components. Starch

tbl0001 Table 1 Some examples of magnetic resonance imaging (MRI)

applications in foods

Food Study

Apple Water relaxation in parenchyma tissue;

drying; bruising; deterioration during

storage

Potato Water relaxation at subzero temperatures;

freeze-drying; drying

Carrot, potato MRI temperature mapping

Peach Freezing; bruising

Extruded pasta Rehydration, radial imaging

Cod Freezing (texture changes)

Peanut butter/bread Fat transport

Milk Cream separation

Food gels Temperature mapping; spatial

heterogeneity; drying

Cereal seeds Maltose and water distribution during

germination

Tomato Water distribution during ripening

Foams Water drainage

Biscuits Water and fat distribution during baking

Meat/fish Fat and water distribution

tbl0002 Table 2 Examples of food polysaccharides characterized by high-resolution nuclear magnetic resolution (NMR)

Polysaccharide Study

Pectins

H and

C NMR Degree of esterification; characterization of monomer sequences and average chain

lengths; galacturonic acid content; conformational analysis

Guar, locust bean gum

H and

C NMR Detection of substituted mannose residues; characterization of monomer sequences

Alginates

H and

C NMR Determination of average block lengths; effects of calcium binding

Amylopectins

H and

C NMR Ratio of a(1-4) and a(1-6) linkages

Wheat arabinoxylans

H and

C NMR Degree and type of substituted xylose rings; correlation between structure and gelling

capacity/thermal stability

Agarose, k, i, l,

b-carrageenans

Rb,

Na,

133

Cs NMR

Study of coil–helix transitions;strength of metal binding

Xanthan

H and

C NMR Acetate and pyruvate contents; study of coil–helix transition

Gellan gum

H and

C NMR Structural determination

Starch

H and

C NMR Amylose content; degree of amylopectin branching; interaction with metals

SPECTROSCOPY/Nuclear Magnetic Resonance 5449

gelatinization occurs upon heating up to 70



causing starch granules to absorb water and burst,

leading to the formation of a gel. On subsequent

cooling, starch crystallization or retrogradation

occurs. Retrogradation is the main cause for bread

staling and, therefore, it has attracted a lot of interest

from the food scientific community. A variety of

CPMAS,

H MAS, and relaxation times measure-

ments have been coupled to follow and understand

both the gelatinization and the retrogradation

processes, including the understanding of the role of

each component, amylose and amylopectin, in the

process.

P NMR can also be used to view phosphate

groups which are found in starch molecules, believed

to contribute to the clarity and viscosity of starch

pastes.

0015 Many gelling processes of a variety of food

carbohydrates (starch maltodextrins, carrageenans,

xanthan, pectins, scleroglucan, konjac glucomannan,

methyl and hydroxymethyl cellulose) have been char-

acterized, generally by

H, and in specific cases

127

Na,

Rb,

133

Cs NMR. Such studies give

information as varied as solid-to-liquid ratios, identi-

fication of different water populations, degrees of

metal binding, characteristics of coil–helix transi-

tions, and degree of aggregation. Plant cell wall poly-

saccharides, e.g., pectins, are a major source of fiber

and determine the texture of plant foods. Much NMR

work has been applied to acquire knowledge of the

structure, composition, and function of these poly-

mers, both in their natural environment – the plant –

and as food additives.

Analysis and Authentication of Foods by

NMR

0016 The high cost of NMR spectrometers and the rela-

tively low sensitivity of the technique compared to

competing methods has prevented the adoption of the

technique as a routine analytical tool in industry. A

very important exception to this is the low-resolution

‘benchtop’ NMR spectrometers which have found

numerous uses in food industry as well as in other

industries. However analytical applications of high-

resolution NMR to lipids, sugars, and many other

compounds still remain nonroutine. In the last few

years, high-resolution NMR has been coupled to

LC and to MS techniques, making way for a new

generation of hyphenated techniques with great

potential in rapidity, sensitivity, and precision. But,

one high-resolution NMR technique has alone justi-

fied the costs of sophisticated equipment enabling

the achievement of information of a unique nature:

the site-specific natural isotope fractionation NMR

(SNIF-NMR) technique.

Low-resolution Pulsed NMR

0017Low-resolution NMR methods involve the extraction

of information directly from the magnetization decay,

and not from the NMR spectrum. Usually the nucleus

observed is

H but others may be used, e.g.,

P. The rates of transverse magnetization decay (or

relaxation rates) following a 90



pulse are very

different for solid and liquid phases and a typical

determination which has been extensively explored

is the determination of solid fat content (SFC) of

semisolid fats, not requiring any sample preparation.

Absolute measurements of oil or moisture content

are based on the same principle but require sample

weighing and a calibration of the method with an

independent method. When species with similar re-

laxation times are present, e.g., oil and water in some

oil-seeds, the Carr–Purcell–Meiboom–Gill (CPMG)

experiment is used and appropriate software used to

obtain relaxation times and relative proportions of

the components of interest. These low-resolution

methods are noninvasive, involve short measuring

times (a few seconds), and require little sample prep-

aration. They are therefore ideally suited for quality

control of large numbers of samples and this has led

to their extensive industrial use, supported by the

development of dedicated, low-cost benchtop instru-

ments. Existing online applications include deter-

mination of oil and moisture contents in seeds,

determination of SFC of fats, use of relaxation times

to follow cake cooking, nondestructive detection of

fruit quality (as sugar content), and the determination

of phospholipid content in peas.

High-resolution NMR and Hyphenated Techniques

0018The applications of high-resolution NMR in food

science have been reviewed in 1996 and, since then,

the use of 2D NMR for structural analysis of complex

natural products, including minor food components,

has become widespread. The qualitative applications

are numerous and many quantitative determinations

have been explored.

0019One of the recognized applications of

C, and

P high-resolution NMR has been the study of lipid

structure and composition. The quantitative deter-

mination of lipids by NMR has been carried out

extensively. For example, for palm oil, composed of

triacylglycerol mainly of palmitic (P), oleic (O) and

linoleic (L) acids, the mole fractions of P, O, and L

may be obtained from integration of particular sig-

nals in the

C NMR spectrum and their positional

distribution may be determined. Applications have

included determination of saturated and unsaturated

fatty acids in edible oils and spreads, differentiation

of different grades of olive oils, detection of synthetic

5450 SPECTROSCOPY/Nuclear Magnetic Resonance

oils in mixtures of virgin olive oil, determination of

o-3 acids in fish samples, the study of the oxidative

deterioration of several oils, and the effects of storage

and thermal stressing, structural determination of

pigment compounds and antioxidants in oils. Inter-

estingly, the determination of oil in intact seeds has

been made possible by high-resolution MAS NMR,

and sunflower and rapeseeds are examples.

0020 Quantitative

C NMR has been applied to many

other systems, e.g., quantification of disaccharides in

honey and quantification of sugars and sugar deriva-

tives in wine. However,

H high-resolution NMR has

gradually proved its usefulness too. Up to the early

1990s

H NMR had received little attention as a tool

for food analysis, mainly because of the great com-

plexity of the spectra and reduced spectral dispersion,

as well as the need for efficient suppression of the very

high water signal. In recent years, water suppression

efficiency has improved largely and the use of higher

field instruments (> 400 MHz) has caused a renewed

interest for the analysis of complex food mixtures like

fruit juices (Figure 1), coffee, and wine by

H NMR.

Indeed, at higher fields a wealth of signals may be

detected in all regions of such spectra, besides the

main solute signals (sucrose, glucose, fructose, citrate,

etc.). Such spectra often show great spectral complex-

ity and signal overlap, even in 2D (Figure 2) and, in

some cases, multivariate analysis has proved useful

for spectral interpretation. It is in the disentanglement

of the information present in high-field

H NMR

spectra that hyphenated techniques play a determin-

ant role. Methods such as LC-NMR or LC-NMR/MS

enable NMR, LC, and MS data to be collected for the

same sample in relatively short experimental times

(min–1 h). The usual set-up of such experiments is

represented in Figure 3. These techniques present a

valuable aid in cases of metabolites which, because of

their low abundance or particular chemical nature,

cannot be identified using solely 2D NMR techniques.

Along with NMR parameters, the coupled techniques

add information about absolute or relative retention

times and molecular mass. These developments show

the potential for rapid detection and characterization

of minor compounds in foods, many with important

roles in aroma/flavor chemistry or with potential

interesting properties such as antioxidant or preser-

vative properties.

Site-Specific Natural Isotope Fractionation Nuclear

Magnetic Resonance

0021SNIF-NMR is certainly a success story in the use of

NMR for the practical needs of the food industry.

The technique has been developed based on the fact

that the percentage of heavy isotopes occurring for

a given element (hydrogen, carbon, oxygen) in nat-

ural products is not a fixed quantity but depends

on the history of the sample, e.g., geographical, cli-

matic, and biochemical factors. The differences arise

9.5 9.0 8.5 8.0 7.5 7.0 6.5 3.0 2.5 2.0 1.5

87654321

p.p.m.

fig0001 Figure 1

H nuclear magnetic resonance (NMR) spectrum of mango juice, at 600 MHz; 64 scans.

SPECTROSCOPY/Nuclear Magnetic Resonance 5451

from distinct isotopic composition of the starting

materials (e.g., rainwater at different latitudes) and

from fractionation effects occurring during biocon-

versions. Measurement of isotope ratios by MS is

widely used for assessment of the authenticity of

foods but this technique gives an average isotope

ratio and no information on the intramolecular distri-

bution of the isotopes. SNIF-NMR uses

H NMR to

measure the deuterium-to-hydrogen isotope ratios

at specific sites (i) in the molecule, (D/H)

. The analy-

sis and characterization of wines and other alco-

holic drinks were among the first applications of

SNIF-NMR. Such samples are examined as distillates

with about 95% ethanol. The proton-decoupled

NMR spectrum of an ethanolic distillate consists

of four resonances: from CH

DCH

OH (1),

CHDOH (2), CH

OD (3) and HDO (the

latter two collapse into one single resonance under

conditions of fast exchange). From the intensities

(or areas) of the peaks of 1 and 2 it is possible to

obtain the ratio (D/H)

/(D/H)

which varies for alco-

hols from different sources, reflecting the occurrence

of strong site-specific fractionation effects. (D/H)

is related to (D/H)

water

and may be obtained through

the measurement of (D/H)

water

by NMR or by

MS. Furthermore, absolute D/H ratios may be

obtained using a working standard with a known

isotope ratio.

0022Ethanols from different plants (sugar cane, corn,

wheat, barley, apple, grape, sugar beet, and potato)

have been distinguished using differences in the

(D/H)

ratio. These results have been used to detect

the origin of raw materials used to make commercial

spirits (whiskey, vodka, gin, rum, and fruit brandies).

The characterization of wines has involved additional

systematic experiments to relate the (D/H) ratios of

the starting materials (starting water and glucose)

with those of the end products, ethanol and water.

By showing the connection between isotope ratios of

starting and end products, it has been shown that the

ratios for ethanol and water can be used as ‘finger-

prints’ in the characterization of wines. SNIF-NMR

parameters in tandem with statistical analysis have

performed remarkably in distinguishing wines of

different origins, according to varieties, geographical

origins, and year of production. Distinction in terms

of country of origin reflects differences in tempera-

ture and rainfall figures. SNIF-NMR became the

most efficient technique to detect enrichment of

wines with exogenous sugars and watering of wines.

Other applications relate to the determination of

origin of flavors and fragrances (natural or synthetic),

ppm

3.54.04.55.0 ppm

EtOH

fig0002 Figure 2 Total correlation spectroscopy (TOCSY) spectrum of a beer sample, at 500 MHz.

5452 SPECTROSCOPY/Nuclear Magnetic Resonance

vinegars, fruit juices and jams (detection of beet sugar

addition), almond and cinnamon oils (detection of

synthetic benzaldehyde).

Other Foods

0023 Many additional studies have been devoted to other

foods, e.g., milk, meat, spices and phytochemicals,

tea, coffee, wine, fruits, and vegetables. These studies

often involve an interplay of high-resolution NMR,

solid-state NMR, and imaging methods. Additional

applications to those already mentioned are as varied

as predicting sugar content in intact fruits, analyzing

pesticide residues in vegetables, determining the

fluorine and aluminum contents in intact tea leaves,

and analysis of the insoluble deposits in bottled wine.

A comprehensive description of the enormous variety

of studies of foods by NMR may be found in many

reviews, some of which are indicated below. It was

the aim of this text to give the reader a flavor of the

versatility of NMR spectroscopy, adaptable to differ-

ent physical states and to the chemical complexities of

foods. Its applications now extend in new directions,

such as online applications and the study of nutri-

tional issues. Scientific growth is continuing in this

field and, increasingly, industrialists and scientists are

coming together to share practical problems and

scientific knowledge.

See also: Adulteration of Foods: Detection; Analysis of

Food; Carbohydrates: Interactions with Other Food

Components; Drying: Physical and Structural Changes;

Freeze-drying: Structural and Flavor (Flavour) Changes;

Freezing: Structural and Flavor (Flavour) Changes;

Protein: Determination and Characterization; Functional

Properties; Spectroscopy: Near-infrared; Infrared and

Raman; Starch: Structure, Properties, and Determination;

Water: Structures, Properties, and Determination; Water

Activity: Principles and Measurement

Further Reading

Ashurst PR and Dennis MJ (1998) Analytical Methods of

Food Authentication. London: Blackie.

Belton PS, Colquhoun IJ and Hills BP (1993) Applications

of NMR to food science. Annual Reports on NMR

Spectroscopy 26: 1–53.

Callaghan PT (1991) Principles of Nuclear Magnetic

Resonance Microscopy. Oxford: Oxford Science Publi-

cations.

Gidley MJ, McArthur AJ, Darke AH and Ablett S (1995)

High resolution nuclear magnetic resonance spectros-

copy of solid and semi-solid food components. In:

LC-NMR control unit

HyStar NT

Avance

NMR

LC-NMR/MS interface

Esquire LC

MS/MS

Fraction

collector

DAD

BSFU-O

BPSU-36

200−800 MHz

Serial and ethernet

communication

Bruker

fig0003 Figure 3 Set-up for the LC-NMR/MS technique. Courtesy of Bruker BioSpin, Germany.

SPECTROSCOPY/Nuclear Magnetic Resonance 5453

Dickson E (ed.) New Physico-chemical Techniques for

the Characterization of Complex Food Systems, pp.

296–318. Glasgow: Blackie.

Gil AM, Belton PS and Hills BP (1996) Applications of

NMR to food science. Annual Reports on NMR Spec-

troscopy 32: 1–49.

Gonzalez J, Jamin E, Remaud G, Martin YL, Martin GG

and Martin ML (1998) Authentication of lemon juices

and concentrates by a combined multi-isotope approach

using SNIF-NMR and IRMS. Journal of Agricultural

and Food Chemistry 46: 2200–2205.

Harris RK (1986) Nuclear Magnetic Resonance Spectros-

copy – A Physicochemical View. Essex: Longman Scien-

tific & Technical.

Hills B (1998) Magnetic Resonance Imaging in Food

Science. New York: Wiley.

Hills BP, Manning CE and Godward J (1999) A multistate

theory of water relations in biopolymer systems.

In: Belton PS, Hills BP and Webb GA (eds) Advances

in Magnetic Resonance in Food Science: Proceedings

of the Fourth International Conference on Applications

of Magnetic Resonance in Food Science. Cambridge:

RSC.

Kauten R and McCarthy M (1993) Applications of NMR

imaging in processing of foods. In: Food Processing:

Recent Developments, pp. 1–22. Amsterdam: Elsevier.

Linden G (1996) Analytical Techniques for Foods and Agri-

cultural Products, Cambridge: VCH.

McCarthy MJ (1994) Magnetic Resonance Imaging in

Foods. New York: Chapman and Hall.

McDonald PJ (1995) The use of nuclear magnetic reson-

ance for on line process control and quality assurance.

In: Gaonkar AG (ed.) Food Processing: Recent Develop-

ments, pp. 23–36. Amsterdam: Elsevier.

Mossoba MM (1998) Spectral Methods in Food Analysis:

Instrumentation and Applications. New York: Marcel

Dekker.

Ruan RR and Chen PL (1998) Water in Foods and

Biological Materials: A Nuclear Magnetic Resonance

Approach. Lancaster, PA: Technomic.

Sanders JKM and Hunter BK (1993) Modern NMR

Spectroscopy – A Guide for Chemists. Oxford: Oxford

University Press.

Visible Spectroscopy and

Colorimetry

T Chiba, Iwate Prefectural University, Takizawa, Japan

Background

0001 Visible spectroscopy and colorimetry are forms of

spectroscopic analytical methods. They are based on

the measurement of the absorption of visible light and

can provide qualitative and quantitative information

about a sample.

0002Visible light consists of electromagnetic radiation

in range wavelengths from 380 to 780 nm, to which

the human eye is sensitive. In this part of the spec-

trum, radiation with different wavelengths gives rise

to light with different colors, while a mixture of light

from these wavelengths forms white light. The color

of a substance depends on the wavelength of light

it absorbs. Thus, when a sample is exposed to white

light, certain wavelengths are absorbed, and the

remaining wavelengths are transmitted to the eye.

The color perceived by the eye is determined only

by the wavelength transmitted. In simple terms, the

color seen is the complementary color to the color(s)

absorbed (Table 1). The intensity of color relates to

the amount of light absorbed by a substance. The

wavelength and efficiency of absorption by a sub-

stance depend on the structure of the substance and

its environment, making it possible to measure the

presence or concentration of the substance.

0003Identification or determination of a substance

based on its color is probably one of the oldest

methods of examination in analytical chemistry.

Earlier work was entirely empirical and led to

qualitative or semiquantitative estimation rather

than quantitative determination. The hues and inten-

sity of colors were measured by visually comparing

the color of a sample with that of several permanent

color standards, which may have been samples con-

taining the same substance at known concentrations

or colored glass. These methods, with the human eye

as a detector, inherently include a subjective percep-

tion of color and are dependent on the reproducibility

of the observer’s eye. Moreover, even the best obser-

ver has difficulty comparing the intensity of two

colors with slightly different hues.

0004The development of detectors of radiation together

with the general advance of instrumentation has

resulted in improvements in measurement techniques.

Photometric techniques with a high level of sensitivity

and reliability are available. With these techniques,

an instrument called a photometer is used as a photo-

electric detector instead of the human eye and con-

verts the intensity of light to an electrical current.

Recent photometers are spectrophotometers capable

of splitting the incident light into different wave-

lengths and measuring the intensity of that radiation.

Spectrophotometric measurements are an important

tool for field testing, industrial applications, and re-

search departments. The term colorimetry describes

the measurement by the human eye, and colorimetric

methods have been largely replaced by spectrophoto-

metric methods.

5454 SPECTROSCOPY/Visible Spectroscopy and Colorimetry

0005 Colorimetric methods (visual methods) are still

utilized for fieldwork and simple determinations

such as monitoring the pH and chlorine levels of

swimming pools. The apparatus required for these

methods is simple and inexpensive. The sensitivity

and precision are often satisfactory for particular

determinations.

Principles

Beer–Lambert Law

0006 Electromagnetic radiation can be characterized as a

wave with frequency, n, and wavelength, l. This wave

has energy, E, is proportional to its frequency and is

given by the equation:

E ¼ hn ¼ hc=l,

where h is Plank’s constant, and c is the velocity of

light. If such a wave of radiation encounters a sub-

stance, and the energy is absorbed, a molecule of the

substance is promoted to an excited state. The ab-

sorption of visible light generally excites electrons of a

molecule from a ground electronic state to an excited

electronic state.

0007 The amount of light absorbed by a substance in a

solution is quantitatively related to its concentration.

This relationship is expressed in two fundamental

laws: the Bouguer–Lambert law, which is also

known as Lambert’s law, relates the amount of light

absorbed to the length of the path it travels through a

medium containing an absorbing substance; Beer’s

law relates light absorption to the concentration of

the absorbing substance. These two laws are com-

bined as the Beer–Lambert law, or simply called

Beer’s law, and are written as the equations (Figure 1):

log

ðI

=IÞ¼log

T ¼ abc ¼ A

=I ¼ 1=T ¼ 10

abc

where I

is the intensity of incident light, I is the

intensity of transmitted light, T is the transmittance,

b is the path length through an absorbing solution, c

is the concentration of the absorbing substance, and

A is the absorbance (extinction or optical density in

older literature).

0008The constant a, which is called the absorptivity or

the extinction coefficient, specifies a characteristic

property of the absorbing substance and is a function

of its wavelength. Its units depend on the units of

concentration and length of the path employed.

When c is expressed in moles per liter, and b is

expressed in centimeters, the constant is known as

the molar absorptivity, formerly called the molar ab-

sorption or molar extinction coefficient. The molar

absorptivity is used as a physical constant for

absorbing species under standard conditions. It is in

liters per mole per centimeter (l mol

1

) and is

given a symbol, e.

0009These expressions show that there is a linear rela-

tionship between the absorbance and the concentra-

tion of a given solution, if the length of the path

and the wavelength of light are kept constant. By

measuring the transmittance or absorbance, the

concentration of a substance in a solution can be

calculated.

Deviation from the Beer–Lambert Law and Errors in

Determination of Concentration

0010The Beer–Lambert law is not necessarily obeyed be-

cause the law applies strictly to instances when inci-

dent radiation is monochromatic, and when the

absorbing centers act independently of one another

regardless of the number and kind. In practice, there is

usually a deviation from a linear relationship between

the concentration and absorbance as with a concen-

trated solution (> 0.01 M) of most substances. These

deviations occur as the result of chemical and instru-

mental factors. Chemical factors can be caused by

chemical equilibria with the solution, intermolecular

tbl0001 Table 1 Colors of light

Absorbed

wavelength (nm)

Absorbed

color

Tr a n s m i t t e d c o l o r

(complement)

400–435 Violet Yellow–green

435–480 Blue Yellow

480–490 Blue–green Orange

490–500 Green–blue Red

500–560 Green Purple

560–580 Yellow–green Violet

580–595 Yellow Blue

595–620 Orange Blue–green

620–750 Red Green–blue

fig0001Figure 1 Absorption of radiation by a sample. I

, intensity of

incident light; I, intensity of transmitted light; b, path length of a

sample; c, concentration of a sample.

SPECTROSCOPY/Visible Spectroscopy and Colorimetry 5455

interaction, reagent and chromophore stability, and

impurities that fluoresce or absorb at the absorption

wavelength of incident light. Instrumental factors are

associated with instrumental limitations determined

by the quality of the instrument’s optical, mechanical,

and electrical systems. These deviations can produce

errors in application of the law for practical purposes,

but this problem can be overcome by preparing cali-

bration curves that show the experimental relation-

ship between absorbance and simple concentration

used for preparing a calibration curve, provided

this relationship is always followed under analytical

conditions.

0011 There are also several sources of error in measuring

transmittance and absorbance. These sources of error

include a faulty readout from a detector, incorrect

wavelength settings, improper handling of cells, and

faulty sample preparation. Any measurement of

transmittance or absorbance is more or less subject

to error.

0012 The magnitude of instrumental errors, in relation

to the real value of the concentration, changes with

the transmittance measured. The relative error is high

at both very high and very low values of transmit-

tance and lowest over the transmittance range of

20–65%. It is advisable to work in the transmittance

range 10–90% (absorbance 1.0–0.1) to prevent large

errors.

Methods of Operation and

Instrumentation

0013 It may be suitable to use a classification where the

two methods are distinguished by the means of

detecting light: the visual method, as judged by the

human eye, and the spectrophotometric method, as

detected by a photoelectric detector.

Visual Method

0014 This method is a visual comparison of samples with

standards of known concentration observed in ambi-

ent light by the human eye. Therefore, a successful

result requires that the sample be the same color as

the standard, with only the intensity of the color

differing.

0015 Dilution method (duplication method) This is per-

formed by matching color intensity by adding water

or a preferred diluent to a darker solution until the

two match in terms of color intensity. A standard and

a sample solution are placed in a colorless glass tube

of equal thickness with a flat bottom, called a Nessler

tube. Light from a source passes through each tube

and is compared. The more concentrated solution is

progressively diluted until the light emerging from

both tubes is equal in intensity when viewed crosswise

and protected from side illumination. At this point,

the concentrations of the two solutions are equal, and

the contents of the solutions are related to each other

by volume.

0016Series-of-standards method A sample solution is

compared with a series of standards consisting of

colored glasses or solutions of known concentration

prepared in the same way as the sample solution.

Nessler tubes or test tubes are used. A series of tubes

of the standard exhibiting a regular gradient of color

are placed in a rack. The tube of a sample solution is

compared side by side with the standard until a match

is found or until the concentration is determined to lie

between that of two standards.

Spectrophotometric Method

0017This is a more sophisticated method and uses a spec-

trophotometer, which is an instrument for measuring

the intensity of light of various wavelengths transmit-

ted by a sample. The intensity of light is determined

by photoelectric detectors, which convert radiation

energy to electrical energy and can eliminate the

need for subjective measurement by the human eye.

This method has a number of advantages over visual

methods: the limit of detection is lowered by measur-

ing the absorption of a solution at the maximal wave-

length; it is possible to avoid or minimize the effect

of foreign colored substances by working at a suit-

able wavelength; a greater precision can be obtained

with spectrophotometric methods than with visual

methods.

0018A spectrophotometer consists essentially of (1) a

light source; (2) a monochromator for furnishing

monochromatic light; (3) one or more sample con-

tainers; (4) a detector for measuring the intensity of

transmitted light; and (5) a signal processing and

readout device (Figure 2).

0019A common light source is a tungsten filament lamp.

This lamp produces continuous and intense radiation

in the range of 320–3000 nm and is adequate for

measurement over the visible region. Devices used as

monochromators include diffraction gratings and

glass prisms. A diffraction grating is a superior form

of general device, giving a constant and narrow radi-

ation bandwidth. Various types of optical filters may

be used as a monochromator, although they provide

only a limited wavelength while a different filter is

necessary for each wavelength required. Such an in-

strument is known as a filter photometer. Sample

containers are usually called cells or cuvets. They

must be transparent in the visible region. General

cells are commonly rectangular in shape with a path

length of 10.0 mm and are made of optical-quality

5456 SPECTROSCOPY/Visible Spectroscopy and Colorimetry