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

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using appropriate software (for example, library

search or quantification routines).

0006 In the special case of SIM, preselected masses

are scanned in preference to the entire spectrum.

This yields much greater sensitivity and is used for

quantifying analytes. Figure 3 shows an LC-MS SIM

chromatogram of the molecular anions of the gluco-

sinolates glucoraphanin, glucoerucin, glucobrassicin,

4-hydoxyglucobrassicin, 4-methoxyglucobrassicin,

and neoglucobrassicin extracted from broccoli.

0007 Sinigrin (a glucosinolate not present in broccoli) is

used as a quantitative spike. Peak areas are integrated

and the ratios of these areas against that of the sini-

grin internal standard are used to generate quantita-

tive data by comparison with a calibration curve.

Gas Chromatography–Mass Spectrometry

0008 GC-MS was the first combined chromatography–

mass spectrometry technique to enjoy widespread ac-

ceptance in food science (one of the earliest published

applications of GC-MS was in flavor analysis). GC-

MS is still used widely in food science, particularly in

flavor analysis. User-friendly and cheap benchtop in-

struments are now available for conducting these

types of analysis. Despite their compactness and sim-

plicity, these instruments are sensitive and equipped

with powerful data systems that are used not only to

control and acquire data from the gas chromatograph

and mass spectrometer, but also to identify flavor

components by automated matching against reference

libraries of spectra of known flavorants. More sophis-

ticated GC-MS instruments are also used in many

other areas of food analysis, especially in the identifi-

cation of toxic residues (e.g., dioxins). Advances in

column technology (particularly the widespread use

of flexible fused silica columns) and improved mass

spectrometer vacuum pumps mean that coupling a gas

chromatograph to a mass spectrometer is now routine

and does not require any special form of interface.

Capillary columns generally operate at helium gas

flows of a few milliliters per minute, and modern

turbomolecular vacuum pumps are easily able to

cope with these flow rates and still maintain ion

source pressures (for electron ionization (EI) ) of

<10

3

Pa. One of the main recent advances in GC-

MS technology has been the introduction of ‘fast’ GC-

MS techniques in which the speed of analysis available

from some types of modern GC capillary columns

have been complemented by fast-scanning mass spec-

trometers (time-of-flight instruments, for example).

GC-MS techniques are now routine and will not be

dwelt on at any length here. LC-MS is now providing

some of the most interesting and versatile new meth-

odologies in food analysis and food science generally

and this article will focus mainly on this technology.

Liquid Chromatography–Mass

Spectrometry

0009LC-MS presents far more technical problems than

GC-MS because of the incompatibility of flowing

100

−2

100

−1

m/z 463.05

m/z 447.05

m/z 436.04

m/z 420.05

m/z 358.03

m/z 477.06

11.0010.009.008.007.006.005.00 12.00

13.00 14.00 15.00 16.00 17.00

18.00 19.00

18.33

17.85

17.75

16.54

7.16

9.40

11.57

12.17 12.65 13.45 13.94

14.57 15.91

19.14

19.55

16.37

17.09

Neoglucobrassicin

4-Methoxyglucobrassicin

4-Hydroxyglucobrassicin

14.35

Glucobrassicin

Glucoerucin

Glucoraphanin

7.34

5.00

6.70

7.33

7.77

9.08

9.41

13.12

16.96

17.23

19.32

Time

7.77

Sinigrin

fig0003 Figure 3 Negative-ion electrospray liquid chromatography–mass spectrometry (LC-MS) selected ion-monitoring (SIM) chromato-

gram of intact glucosinolates extracted from broccoli. Copyright Institute of Food Research, reproduced with permission.

1296 CHROMATOGRAPHY/Combined Chromotography and Mass Spectrometry

liquids with the high vacuum necessary for mass spec-

trometers to function. A typical liquid chromatograph

eluent flow of 1 ml min

1

becomes over 1000 times this

flow of gas when converted to a vapor, far in excess of

the gas flow that a conventional mass spectrometer ion

source can cope with and still maintain vacuum. Fur-

thermore, the expansion from liquid to gas results in

dilution of analyte molecules entrained in the solvent,

potentially reducing sensitivity by a very large factor.

Consequently, special interfaces and ion sources have

been developed to remove excess solvent vapor and

insure that the analytes are not diluted excessively.

LC-MS Interfaces and Ion Sources

0010 Several different techniques, or interfaces, for com-

bining liquid chromatography and mass spectrometry

were developed in the 1970s and 1980s. These in-

cluded the direct liquid introduction (DLI) interface,

the moving belt interface, dynamic FAB (fast atom

bombardment) or dynamic LSIMS (liquid secondary

ion mass spectrometry), and thermospray ion source.

All of these techniques had some disadvantages in

terms of the range of compounds amenable to analy-

sis, robustness and reliability, sensitivity and, in some

cases, limited flow rates.

Electrospray and Atmospheric Pressure Chemical

Ionization

0011 The interfaces mentioned above have been super-

seded by electrospray (ESI) and atmospheric pressure

chemical ionization (APCI) ion sources. Both of these

ion sources can be coupled directly to liquid chro-

matographs, with very few restrictions on flow rate

or solvent composition (See Mass Spectrometry: Prin-

ciples and Instrumentation). Both techniques have

been used very widely in food and nutrition studies.

Examples of SIM chromatograms of 1 and 20 pmol of

b-carotene (Figure 4) and a mass chromatogram

(Figure 5a) and spectrum (Figure 5b) of quercetin 3

glucoside are shown. These types of data are useful in

quantitative and qualitative metabolic studies.

The Particle Beam Interface

0012A third type of interface, the particle beam, completes

the trio of modern LC-MS technology, providing a

suite of techniques capable of handling nearly all types

of analysis. A schematic diagram of a particle beam

interface is shown in Figure 6.

0013The incoming liquid stream (i.e., the high-perform-

ance liquid chromatography (HPLC) eluent) enters

the device on the left of the diagram. The liquid is

nebulized by helium gas flowing, forming a fast-

moving spray of droplets that enter the heated deso-

lvation chamber. Solvent begins to evaporate from the

spray droplets which shrink, thus concentrating any

dissolved sample molecules. The partially evaporated

spray then passes through a narrow orifice (skimmer

1 in the diagram) and across a region that is main-

tained under vacuum by a rotary pump. A supersonic

molecular beam is formed in this region and, because

100

20 pmol beta-carotene

1 pmol beta-carotene

Peak area 61625

Peak area 5292

11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

Time

fig0004 Figure 4 Positive-ion atmospheric pressure chemical ionization liquid chromatography–mass spectrometry selected ion-

monitoring chromatogram of the molecular ion of b-carotene (1 and 20 pmol injections, respectively). The slight retention time shift

between the two traces is caused by temperature drift affecting the chromatography. These types of data are useful for quantifying

carotenoids in food extracts and in metabolic experiments. Copyright Institute of Food Research, reproduced with permission.

CHROMATOGRAPHY/Combined Chromotography and Mass Spectrometry 1297

the solvent molecules and helium atoms are signifi-

cantly lighter than sample molecules, they diffuse

away more rapidly and are pumped out of the system

preferentially. This leaves the heavier solute mol-

ecules concentrated in the beam. A second skimmer

removes yet more solvent molecules and allows par-

ticles of sample to enter the EI or chemical ionization

(CI) ion source. The interface can handle flows of

between 0.1 and 1 ml min

1

, depending on the

volatility of the solvent.

0014The great advantage of the particle beam system is

that it can generate CI and, especially, EI spectra that

are highly reproducible and comparable with spectra

obtained by other types of EI and CI inlet systems.

These spectra are therefore searchable against data-

bases of known spectra. The major difference be-

tween the particle beam interface and other LC-MS

systems is that the device processes the HPLC eluent

in such a way that sample molecules can be ionized by

the conventional mass spectrometric techniques of EI

100

−2

Sum of intensities of m/z 300−800

Quercetin 3' glucoside

Diode array trace (190−450 nm)

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

Time

100

155.2

160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520

m/z

480.2

481.2

520.2

501.1

482.2

468.5

463.2

443.2

426.7

414.9

403.0

376.0

368.0

357.1

344.1

322.1

305.0

304.1

172.2

190.1

208.5

222.1

229.2

247.2

261.6

274.2

287.1

303.0

479.2

fig0005 Figure 5 Positive-ion electrospray ionization liquid chromatography–mass spectrometry (a) chromatogram and (b) mass spectrum

of the important food flavonoid quercetin 3

glucoside. Copyright Institute of Food Research, reproduced with permission.

1298 CHROMATOGRAPHY/Combined Chromotography and Mass Spectrometry

and CI. APCI and ESI are effectively ionization

techniques that generate anions or protonated mol-

ecules with few fragment ions under normal operat-

ing conditions. The particle beam method does not

have the versatility and sensitivity of APCI and ESI

but is useful for analyzing nonthermally labile

molecules that are insufficiently volatile for GC-MS

analysis.

Capillary Electrochromatography–Mass

Spectrometry

0015 CEC is a high-resolution separation technique that

has been described as a hybrid of HPLC and CE.

The high efficiency available from CEC makes it an

ideal partner for coupling to mass spectrometry as the

narrow chromatographic peaks result in increased

mass flow per unit time into the mass spectrometer

ion source, and thus greater sensitivity as well as

selectivity. Many of the technical problems associated

with coupling CEC to mass spectrometry (principally

Joule heating and consequent bubble formation) are

being tackled successfully, to the extent that auto-

mated sample introduction systems may be used

with CEC-MS systems. Although the technique

has not yet caught on in food science and nutrition,

CEC-MS has great potential in studies requiring

separation of complex mixtures and high-sensitivity/

high-selectivity analysis.

Capillary Electrophoresis–Mass

Spectrometry

0016Many of the difficulties associated with combining

the ultrahigh-resolution separation technique of CE

with MS have been overcome. However, a significant

number of publications devoted to interfacing CE

with MS still appear annually, indicating that an

ideal solution is still not available. Despite this, sev-

eral mass spectrometer manufacturers now supply

special probes for interfacing CE with MS, indicating

that the technology is now commercially viable. The

ionization technique most compatible with CE is elec-

trospray and the vast majority of CE-MS applications

reported make use of this ionization technique. Re-

cently the use of microfabricated devices (‘CE on a

chip’) with mass spectrometers has been reported and

promises significant advances in sensitivity and repro-

ducibility. As with CEC, there are few reported appli-

cations of CE-MS in the food and nutrition sciences;

nevertheless, the potential of technique in these fields

is very high.

Supercritical Fluid Chromatography–

Mass Spectrometry

0017Techniques for coupling SFC to MS were first

described in the late 1970s. Although a number of

different SFC-MS interfaces were developed in the

Quadrupole

analyzer

Vacuum pump 2

Vacuum pump 1

GC transfer line

EI/CI source

Skimmer 2

Skimmer 1

Desolvation chamber

Helium

Nebulizer

fig0006 Figure 6 Schematic diagram of a particle beam liquid chromatography–mass spectrometry interface. LC, eluent exiting liquid

chromatograph; EI/CI, electron impact/chemical ionization; GC, gas chromatography. Reproduced from Mellon FA, Self R and Startin

JR (2000) Mass Spectrometry of Natural Substances in Foods. London: Royal Society of Chemistry, with permission.

CHROMATOGRAPHY/Combined Chromotography and Mass Spectrometry 1299

intervening years, APCI, with specially adapted SFC

probes, is currently the main method of choice. The

APCI source is particularly suited to SFC because it is

unaffected by the increasing flow rates resulting from

pressure programming of the supercritical mobile

phase and because of the ease of connection with

E+06

1,739

100

Relative abundance (%)

Time (min:s)

26:40 30:00 33:20 36:40 40:00 43:20 46:40

fig0007 Figure 7 Reconstructed ion chromatogram of milk fat triacylglycerols analyzed by capillary supercritical fluid chromatography–

mass spectrometry. Samples ionized by atmospheric pressure chemical ionization with ammonia as reactant ion. Reproduced from

Laakso P and Manninen P (1997) Identification of milk fat triacylglycerols by capillary supercritical fluid chromotagraphy atmospheric

pressure chemical ionization mass spectrometry. Lipids 32: 1285–1295, with permission.

100

350

400 450 500 550 600 650 700 750

m/z

[M-C

]

411.4

{

[M-C

]

439.3

{

[M-C

]

467.5

{

[M-C

]

495.5

{

[M-C

]

523.5

{

[M-8:0]

551.6

{

[M-6:0]

577.6

{

[M-4:0]

605.7

710.7(40:1)

712.7(40:0)

708.7(40:2)

{

[M+18]

{

fig0008 Figure 8 Mass spectrum of peak 9 in Figure 7. Samples ionized by atmospheric pressure chemical ionization with ammonia as

reactant ion. Reproduced from Laakso P and Manninen P (1997) Identification of milk fat triacylglycerols by capillary supercritical fluid

chromotagraphy atmosphere pressure chemical ionization mass spectrometry. Lipids 32: 1285–1295, with permission.

1300 CHROMATOGRAPHY/Combined Chromotography and Mass Spectrometry

SFC systems. Although SFC-MS is much less popular

than LC-MS in the food sciences, it is particularly

suited to certain types of analysis. A good example

is provided by the identification of milk fat triacylgly-

cerols by capillary SFC-MS. Samples were ionized, in

APCI mode, by admitting methanol or 0.5% ammo-

nia in methanol as reagent gas. A reconstructed ion

chromatogram of the milk fat triacylglycerols is

shown in Figure 7.

0018 Individual mass spectra yielded abundant [M þ

18]

and [M RCOOH]

ions when ammonia was

present in the reagent gas, thus defining molecular

weight and the nature of the fatty acid constituents

of the triacylglycerols. As can be seen in Figure 7,

complete chromatographic resolution of all triacyl-

glycerols was not achieved, However, the specificity

of the mass spectrometric data allowed identification

of different components of unresolved chromato-

graphic peaks where fatty acid chain lengths yielded

characteristic fragment ions. The mass spectrum of

peak number 9 in Figure 7 is shown in Figure 8.

0019 Three different chromatographically unresolved

triacylglycerols, 40:0, 40:1, and 40:2, are present.

This study demonstrated the value of SFC-MS in the

analysis of both saturated and unsaturated triacylgly-

cerols.

0020 The interpretation of the mass spectrum in Figure 8

was complicated by the occurrence of several differ-

ent-molecular-weight species in a single chromato-

graphic peak. However, the [M RCOO]

ion

region contained ion clusters that indicated the pres-

ence of C

and C

fatty acid moieties in the triacyl-

glycerols.

See also: Chromatography: High-performance Liquid

Chromatography; Gas Chromatography; Supercritical

Fluid Chromatography; Mass Spectrometry: Principles

and Instrumentation; Applications

Further Reading

Chapman JR (1995) Practical Organic Mass Spectrometry:

A Guide for Chemical and Biochemical Analysis, 2nd

edn. Chichester: John Wiley.

Cole RB (1997) Electrospray Ionization Mass Spectrom-

etry. New York: John Wiley.

James P (2000) Proteome Research: Mass Spectrometry

(Principles and Practice). Berlin: Springer Verlag.

Johnstone RAW and Rose ME (1996) Mass Spectrometry

for Chemists and Biochemists, 2nd edn. Cambridge:

Cambridge University Press.

McMaster MC and McMaster C (1998) GC/MS: A

Practical User’s Guide. New York: John Wiley.

Mellon FA, Self R and Startin JR (2000) Mass Spectrometry

of Natural Substances in Food. London: Royal Society

of Chemistry.

Siuzdak G (1996) Mass Spectrometry for Biotechnology.

San Diego: Academic Press.

Willoughby R, Sheehan E and Mitrovich S (1998) A Global

View of LC/MS: How to Solve Your Most Challenging

Analytical Problems. Pittsburgh: Global View.

CHROMIUM

Contents

Properties and Determination

Physiology

Properties and Determination

R Farre

and M J Lagarda, University of Valencia,

Burjassot, Spain

Physical and Chemical Properties

0001 Chromium is a hard, brittle, white metal of the first

transition series, with an atomic number of 24, a

relative atomic mass of 51.996 amu, an electronic

configuration [Ar]3d

, and a relative density of

7.19. The melting and boiling points are 1857



and 2672



C, respectively.

0002The resistance of chromium to attack from a var-

iety of chemicals at normal temperature makes it

useful in protecting other more reactive metals, e.g.,

it is a component of stainless steel. However, at high

temperatures it reacts with many chemicals.

0003As a typical transition metal element, chromium

forms many compounds that are colored and para-

magnetic. It has oxidation states from 2toþ6, but

CHROMIUM/Properties and Determination 1301

the most common and stable oxidation states are

þ2, þ3, and þ6. Since Cr(II) is a very strong redu-

cing agent, it is not found in biological systems. All

Cr(VI) compounds, except the hexafluoride (CrF

are oxo compounds; chromium occurs predomin-

antly as either chromate (CrO

2

) or dichromate

(Cr

2

). Cr(VI) compounds are strong oxidizing

agents and are therefore readily reduced to Cr(III) in

acidic solutions. The most stable and important oxi-

dation state is þ3.

0004 One of the outstanding features of Cr(III) che-

mistry is the ability of trivalent chromium to form

coordination complexes, most of which are hexacoor-

dinated. These coordination complexes are relatively

inert. In aqueous solution the hexahydrate ion

[Cr(H

]

3þ

exists in an octahedral configuration.

In aqueous solution the ligand and displacement

reactions have half-lives of several hours.

0005 Chromium can form bridges with hydroxyl groups

(olation). Olation is enhanced by alkalis and tempera-

tures up to 120



C, but can be prevented and reversed

by oxalate ions and other strong ligands, whereas

weaker ligands can only prevent the reaction. In bio-

logical systems pyrophosphate, methionine, serine,

glycine, leucine, lysine, and proline therefore inhibit

the olation of Cr(III). In such systems chromium is

able to react because its solubility is maintained by

weaker organic and inorganic ligands.

Determination in Foods and Beverages

0006 There are a wide range of techniques available for

the analysis of chromium and other trace elements.

Chromium is one of the most difficult elements to

determine accurately because of the very low levels

in foods.

0007 The determination of chromium content in bio-

logical matrixes has long been a controversial prob-

lem because of the many errors that can arise and

invalidate the data. Therefore, much of the earlier

work should be viewed critically. Reasons for this

skepticism arise from a number of problem areas:

0008 The low chromium content of normal (nonconta-

minated) biological samples. In food samples the

concentration is on the order of nanograms per

gram and, therefore, it is essential to apply very

sensitive measurement techniques.

0009 Chromium losses by volatilization during sample

dry ashing above 450



0010 Contamination at any point through sample collec-

tion and handling, before the content is measured.

0011 Errors inherent in the applied analytical method,

such as, for instance, matrix interferences in

electrothermal atomic absorption spectrometry

(ETA-AAS) and spectral in inductively coupled

plasma–atomic emission spectrometry (ICP-AES).

0012Studies should report precise data on all steps of the

analytical procedure, including sample collection,

homogenization, and matrix reduction. An accurate

determination is a challenge because of problems that

originate from the risk of chromium losses by volatil-

ization or adsorption and/or enrichment by contam-

ination. Interlaboratory assays therefore give rise,

in the case of chromium analysis, to very variable

results.

0013Of all of the proposed analytical methods, AAS is

the one most often used for chromium determination

in biological samples. When applied with a graphite

furnace (ETA-AAS) it is one of the most sensitive

techniques, but it is open to a wide range of interfer-

ences, which depend on the matrix and can lead to

erroneous results. The advances in background cor-

rection procedures have contributed to overcome

these limitations.

0014ICP-AES is also applied to determine chromium in

foods and beverages. Although the detection limits

are higher than those obtained with ETA-AAS, it

offers the advantages of simpler sample preparation

and the possibility of carrying out multielemental

analysis.

0015Determination of total chromium content may not

be a valid indicator of the nutritional benefit of a food

because not all chromium in foods has activity as a

glucose tolerance factor. Some authors point out that

the alcohol-extractable fraction of chromium seems

to be a more reliable index. The speciation of chro-

mium is of great interest because of the differences

in bioavailability, essentiality, and toxicity between

Cr(III) and Cr(VI).

Precautions to be Taken in Chromium

Determination in Foods

0016As mentioned above, contamination is one of the

main problems encountered in chromium determin-

ation in foods. Indeed, it is the explanation why

food chromium contents reported some years ago

are higher than those obtained recently, as better

measures have now been adopted to avoid contam-

ination.

0017The possibility that small amounts of chromium

can be retained by the surface of vessels makes it

necessary to decontaminate the material used,

whether glass or plastic. In order to decontaminate

the containers, all material used has to be soaked in

diluted nitric acid (10–20% v/v) for 12–24 h and then

rinsed several times with deionized water. A mixture

of 4 mol l

1

nitric acid and 4 mol l

1

perchloric acid

1302 CHROMIUM/Properties and Determination

(1:1, v/v) or a solution of 2,4-pentanedione or dithi-

zone/carbon tetrachloride has also been proposed.

0018 To avoid losses by adsorption on to container

walls, it is advisable to store digested samples and to

prepare standard solutions in diluted acid (nitric or

hydrochloric acid) below pH 1.5. However in acidic

conditions reduction of Cr(VI) to Cr(III) is favored.

0019 Since the acids used in chromium determination,

especially nitric acid, are a source of contamination, it

is advisable to use reagents of high quality or to distil

them previously. Reagent blanks must be used to

monitor such contamination.

0020 Contact with materials that can give up chromium,

i.e., metallic surfaces such as stainless steel, should be

avoided. Therefore, knives, mixers, homogenators, or

mills which are made from stainless steel should be

avoided and replaced by others made from polymeric

materials, quartz, or titanium. Colored polymeric

materials such as screw-caps for bottles, dispensers,

and pipette tips can contain chromium and should be

controlled for contamination.

0021 Chromium determinations below the 1 mgl

1

level

require strict contamination control and clean-room

facilities.

Sample Digestion

0022 The choice of the digestion method is important in

trace chromium determination. The use of dry

methods carries the risk of the formation of acid-insol-

uble Cr(III) oxide and can lead to chromium losses by

adsorption on the walls of the crucible or by volatil-

ization after formation of chromyl chloride at tem-

peratures higher than 550



C. These losses are

minimized when the temperature is increased grad-

ually and does not exceed 450–500



C. Nevertheless,

some authors have reported losses of chromium by

volatilization at 450



C and obtained better results at

lower temperatures together with oxygen plasma. In

the latter procedure, the amount of sample is limited

and relatively large amounts of hydrogen peroxide are

needed as an ashing aid. A dry-ashing in Pt crucibles

with a temperature ramp at a rate of 50



1

up to

450



C is the digestion method adopted by the Nordic

Committee on Food Analysis as its official method-

ology for chromium determination in foodstuffs.

0023 In wet digestion procedures, mixtures of acids and

oxidizing agents are used, the most commonly used

ones being nitric, sulfuric, and perchloric acids with

hydrogen peroxide or vanadium pentoxide. When

foods have a high fat content, mixtures of nitric,

perchloric, and sulfuric acids, or nitric and sulfuric

acids, are recommended.

0024 The high boiling point of sulfuric acid helps the

activity of oxidizing agents and, with the addition of

nitric acid, the disadvantage of forming sulfate com-

pounds of low solubility is overcome. Digestion,

however, with nitric and sulfuric acids requires good

refluxing to prevent losses.

0025Significant losses of chromium during wet ashing

occur when perchloric is present and the sample is

concentrated close to dryness because of chromyl

chloride (CrO

) formation, which boils at

117



C. Formation of chromyl chloride can be

minimized by the addition of sulfuric acid.

0026The main risk of wet digestion is contamination

through the reagents, especially when high volumes

are used. Digestions in closed systems and/or in micro-

wave systems require less time and reagents. There-

fore, the risk of contamination decreases and losses by

volatilization are also reduced to a minimum.

0027Microwave digestion is now widely used to meas-

ure chromium contents in foods, because it is faster,

safer, and provides more reproducible conditions

than conventional methods, resulting in better analyt-

ical precision.

0028In some vegetable species chromium is retained by

an insoluble residue of silica. When this occurs, treat-

ment with hydrofluoric acid is necessary to overcome

the problem of low recovery values.

0029Extreme care must be taken in all wet ashing

procedures where strong acids and strong oxidizing

agents are used. The formation of explosive nitro

compounds has been reported when fat-rich food

samples have been digested with nitric acid.

Determination

Molecular Absorption Spectroscopy

0030The traditional spectrophotometric method of

520 nm, based on the violet complex formed with

1,5-diphenyl-carbazide (DPC), is still widely used

for determining chromium in water, because of

its high sensitivity. Chromium must be present as

Cr(VI) and the diphenylcarbazide is oxidized to the

diphenylcarbazone. Iron, copper, molybdenum, and

vanadium can interfere. To remove interference and

to increase the sensitivity of the method, both cation

exchange resins (iron and copper) and cupferron

(iron, copper, molybdenum, and vanadium) have

been used.

0031This method has been applied to chromium deter-

mination in foods after destroying the organic matter

and removing the interferences. Detection limits of

10 ng g

1

have been reported.

0032The complex chromium hematoxiline, with a

maximum absorbance between 360 and 390 nm, has

also been applied to foods, although less often than

the diphenylcarbazide method.

CHROMIUM/Properties and Determination 1303

Flame Atomic Absorption Spectrophotometry

(FAAS)

0033 The low levels of chromium in foods and the com-

plexity of the matrix make it difficult to measure

chromium directly in digested samples by FAAS.

With an air-acetylene flame and at a wavelength of

357.9 nm it is possible to obtain detection limits of

0.1 mgg

1

. It has been well established that chromium

atomization is dependent on flame stoichiometry,

with higher sensitivity with reducing flames (rich in

acetylene). A 10-fold difference in sensitivity between

fuel-lean and fuel-rich flames has been reported.

However, the composition of the flame affects the

behavior of the interfering compounds, and often

the conditions for maximum signal-to-background

are quite different from those required for minimizing

interferences.

0034 This determination is not free of interelement

interferences. Iron depresses chromium absorbance

in fuel-rich air-acetylene flames. It can be separated

from the analyte by extraction with acetylacetone/

chloroform (1:1, v/v) at pH 1–2, or by extraction, as

Fe(III), from 5 mol l

1

hydrochloric acid into isobutyl

acetate or 4-methyl-2-pentanone (MIBK).

0035 Direct chromium determination by FAAS in

digested samples is not often applied, owing to inter-

ferences and the low concentration of chromium. For

this reason it is more usual to enrich and to remove

interference by chelating chromium and then

extracting with an organic solvent.

0036 Chromium can be chelated as Cr(III) with 2,4-

pentanedione at pH 7.5 and heating to nearly 80



The chelate is then extracted with chloroform or

MIBK. With the former, a backextraction to an aque-

ous solution should be carried out before measuring

by FAAS. To remove metal interferences the same

chelating reagent or ammonium pyrrolidine dithio-

carbamate (APDC) is added at a lower pH value

and then extraction with chloroform is carried out.

0037 Alternatively, chromium can be chelated with

APDC as Cr(III) (pH 5 at 80–90



C) or as Cr(VI)

(pH 3–9 at room temperature). Therefore, to work

under less strict conditions it is advisable to oxidize

chromium to Cr(VI), and to eliminate the excess oxi-

dizing agent. The procedure is not free of interfer-

ences, because manganese and tin are also chelated.

In order to remove interfering substances and to

increase the stability of the element in solution,

some authors have proposed that backextraction

should be carried out.

0038 Another method that has been applied to chro-

mium determination in foods is based on the forma-

tion of a compound by ionic association between

Cr(VI) and the system hydrochloric acid/MIBK



C). Prior to this, chromium is oxidized to Cr(VI)

with potassium permanganate. This procedure offers

the advantage that it is not necessary to remove the

excess oxidizing agent.

Electrothermal Atomization Atomic Absorption

Spectrophotometry

0039ETA-AAS is considered one of the techniques of

choice for chromium determination in biological

matrices and it is the technique most often used to

determine chromium in foods. It is not free of inter-

ferences, but it is simpler than FAAS solvent-extrac-

tion methods. In foods, detection limits of 1 mgkg

1

have been reported.

0040If organic matter is present, chromium is found

mainly in the form of Cr(III), and in this oxidation

state it easily forms refractory oxides during heating

in graphite tubes and then reacts with graphite to

form a carbide. To avoid the resulting decrease in

the signal, pyrolytically or specially coated graphite

tubes should be used. Pyrolytically coated tubes

increase sensitivity threefold with respect to uncoated

ones.

0041When the furnace temperature reaches 450



breakdown products of chromium complexes are

formed. Some of these are volatile and, therefore,

there is a risk of losses of the element. Alternatively,

other breakdown products can prevent complete

atomization of chromium, because of the strong

bonding with oxygen, nitrogen, or carbon. To achieve

complete atomization of chromium, temperatures

higher than 2300



C should be used. The usual ranges

of temperatures are: drying (100–200



C), ashing

(600–1600



C), atomization (2300–2700



C).

0042Diammonium hydrogen phosphate, magnesium

nitrate, and palladium have been used successfully

as matrix modifiers. The use of magnesium nitrate

as a matrix modifier extends the possible charring

temperature to 1600



C, thus insuring a more com-

plete remove of inorganic salts.

0043Since chromium is measured at a wavelength of

357.9 nm, it is better to correct background absorp-

tion with the Zeeman effect or a tungsten lamp rather

than with a deuterium lamp. The emission intensity of

the deuterium arc is low at the chromium resonance

line of 357.9 nm. The hollow cathode lamp current

has to be reduced in order to balance the source and

background beams. The Zeeman effect can compen-

sate for high absorbances, such as those produced by

biological matrices.

0044The standard addition method is generally applied,

even when prior digestion of organic matter is carried

out. In the case of liquid samples, e.g., beer or wine, it

is possible to determine chromium by injecting the

1304 CHROMIUM/Properties and Determination

diluted sample directly into the graphite furnace and

then applying the standard addition method. This

direct procedure has also been applied to suspensions

of vegetables and milk samples (whole, low-fat, skim,

condensed, evaporated, and powdered). In these cases

slurries of the samples were prepared with Triton

X-100. Chromium contents below 0.2 mgg

1

are

measurable.

Inductively Coupled Plasma Spectrometry

0045 The detection limits of ICP with atomic emission

(ICP-AES) are lower than those obtained by direct

FAAS, but not commensurate with ETA-AAS. More-

over, they can be seriously affected by spectral inter-

ferences, which are much more usual in ICP-AES than

in AAS. A wavelength of 267.716 nm is usually

chosen, though some overlap with emission lines of

manganese and phosphorus can be observed. Other

emission lines used are 283–563, 284–325, and

357–869 nm, but all are prone to spectral interfer-

ence. Algorithms are generally used to correct for

such interferences.

0046 One of the main advantages of ICP is that sample

preparation is simpler than in ETA-AAS, because

elements can be measured when organic residues are

present and several elements can be measured simul-

taneously. This explains why it is used in chromium

determination in foods.

0047 Online coupling mass spectrometry (MS) with ICP

improves the detection limits 10–100-fold, obtaining,

in optimized conditions, values below 0.1 mg1

1

and

therefore close to those of ETA-AAS. ICP-MS has the

advantage of multielement capability. An ICP-MS

method was proposed as the Association of Official

Analytical Chemists (AOAC) official method for

chromium determination in drinking, groundwater

and waste waters. On the other hand, the lower back-

ground and greater sensitivity of the double-focusing

instruments at low-resolution mode make it possible

to obtain better detection limits than with quadru-

pole-based instruments.

0048 Chromium determination in food samples by flow-

injection ICP-MS has been described. The digests

obtained in a wet high-pressure microwave system

are delivered at a previously established flow rate to

the ICP-MS instrument. FIA gives good recovery

values and these are more reproducible than when a

conventional method is used.

Electrochemical Methods (Polarography,

Voltammetry)

0049 Differential pulse polarography (DPP) is used to

measure chromium in water and biological materials

(including foods). Chromium is only detectable as

Cr(VI). A reduction peak is observed at 0.80 V,

when sodium citrate is used as a buffer (pH 9–10.5)

and ammonia (ammonium chloride) as a support

electrolyte. Detection limits as low as 0.12 mgl

1

can

be obtained.

0050DPP is mainly used for chromium determination in

water. Preparation of the sample depends on its com-

plexity and the expected chromium concentration.

0051Nitric acid should not be used to acidify the

sample, since the formation of nitrous acid can par-

tially reduce Cr(VI) to Cr(III). If water contains sul-

fites, these can also reduce Cr(VI) to Cr(III), so Cr(VI)

must be chelated with sodium diethyldithiocarba-

mate (pH 4, diluted sulfuric acid/ potassium acid

phthalate) and extracted with MIBK. Sulfuric acid is

added and the solution is heated to remove the solv-

ent. Hydrogen peroxide and sulfuric acid are added to

insure that chromium is present entirely as Cr(VI).

0052In the case of samples with a high chloride or

organic matter content, sodium sulfite is added to

prevent the formation of chromyl chloride.

0053Samples having chromium levels lower than 1 mg

1

can be enriched by passing the solution through

an ion exchange resin.

0054Voltammetric chromium measurements have been

applied to wet digested biological samples after a

controlled adsorptive preconcentration step on a

hanging-mercury-drop electrode. Optimal conditions

were a preconcentration potential of 1 V, a precon-

centration period of 60 s, and a solution pH of 6.3. A

current peak was recorded at 1.1 V.

0055Electrochemical determinations can also be useful

to differentiate between chromium species.

Gas-Liquid Chromatography (GLC)

0056The great sensitivity of GLC allows the detection of

extremely small amounts of chromium. GLC has

been applied mainly to chromium determination in

biological fluids, such as blood and urine, and to

chromium determination in water, when speciation

is wanted. It is applied less frequently to foods, be-

cause this method requires prior destruction of the

organic matter and chromium levels are higher in

foods than in biological fluids.

0057Since GLC can only be applied to volatile com-

pounds, volatile chromium chelates are formed

with fluorine substitutes of acetylacetone derivatives.

Chromium complexes with b-diketones are volatile

and stable, and trifluoroacetyllactone (TFA) is the

chelating agent most frequently reported in practical

applications, because of the enhanced sensitivity of

halides using electron capture detection (ECD). This

determination requires a purification step to remove

the excess chelating reagent and other interfering

CHROMIUM/Properties and Determination 1305