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

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Thermal Conductivity Detectors (TCD)

0036 These are universal detectors, with a medium sensi-

tivity, and are concentration-dependent. The column

effluent passes through a chamber containing a hot

filament. When a compound appears in the effluent,

the thermal conductivity of the mixture solute/carrier

gas, and hence the heat dissipation velocity, varies;

this causes a temporal variation in filament tempera-

ture and a change in its electric resistance, which is

very easy to measure. The best carrier gases for this

detector are hydrogen and helium. TCD is appropri-

ate for substances such as permanent gases, and can

also be coupled in tandem with other detectors.

Ionization Detectors

0037 These induce sample ionization by using thermal,

chemical, electromagnetic, or radioactive energy.

Solute ions in the carrier gas produce changes in the

electric field between two electrodes. This is shown

schematically in Figure 4.

0038 Flame-ionization detectors These are the most com-

monly used detectors in GC and are almost universal,

with a high sensitivity to organic carbon-containing

compounds, a wide linear range, and an excellent

baseline stability. They are also very reliable, with a

negligible dead volume and a fast response time. The

ionization energy is supplied by a flame, obtained by

a hydrogen flow, which is mixed with the column

effluent. The organic compounds are burnt, produc-

ing H

O and CO

; the response is proportional to the

solute mass flow and is similar for most organic sub-

stances. Molecules with less C—H bonds than hydro-

carbons give a lower response than hydrocarbons.

These detectors can be used with all carrier gases

and are easy to calibrate.

0039 Electron capture detectors (ECD) These are very

specific and sensitive to compounds with a high elec-

tron affinity. The ionization source is a foil (usually

Ni or

H), which emits b-radiation. Nitrogen and

an argon/methane mixture are suitable carrier gases

for ECD. A low potential is established between

electrodes, producing a current that decreases when

a solute capable of accepting (‘capture’) electrons

appears in the effluent. The relative response is very

different for compounds that have groups with differ-

ent electronic affinities: e.g., a polychlorinated com-

pound has a response 10

times higher than that of

a saturated hydrocarbon. Thus, it is comparatively

easy to detect less than 1 pg of polyhalogenated com-

pounds in the presence of other molecules that have

only C, H, and O atoms. Since the ECD response is

dependent on the sample concentration and electron

affinity of each analyte, calibration using standards is

necessary for quantitative analysis.

Electrochemical Detectors

0040These are designed mainly for the environmental

analysis of substances with heteroatoms like S, N, or

halogens, and these detectors are very specific and

sensitive.

Spectroscopic Detectors

0041Several spectroscopic devices can be coupled with GC

in order to obtain structural information about the

eluting peaks. The most commonly used detectors are

those based on mass spectrometry (MS) (See Chroma-

tography: Combined Chromatography and Mass

Spectrometry; Mass Spectrometry: Applications;

Principles and Instrumentation); and Fourier trans-

form infrared (FTIR); See Spectroscopy: Infared and

Raman.) The former have a high selectivity and

sensitivity. The column effluent is continuously fed

into the ionization chamber of a mass spectrometer,

and a mass spectrum is recorded every few seconds.

GC–MS coupling can be used in two modes: scan, in

which all the ion fragments are collected and regis-

tered, giving structural information about all chroma-

tographic peaks, and selected ion monitoring (SIM),

in which only a few mass fragments are recorded,

improving the signal-to-noise ratio and significantly

reducing the detection limit.

Applications

0042GC has been used to analyze the main constituents of

foods, such as lipids, carbohydrates, or amino acids,

and it is especially useful in the study of minor con-

stituents such as aromas or pollutants.

Food Constituents

0043Lipids GC has been the technique of choice for the

study of fats. It is possible to study the degree of

lipolysis by analyzing the free fatty acid (FFA) profile

or the authenticity of fats and oils by determining the

Carrier gas

+ −

fig0004 Figure 4 Scheme of an ionization detector.

1286 CHROMATOGRAPHY/Gas Chromatography

glyceride fraction, the fatty acid composition, and the

unsaponifiable fraction. FFAs can be studied using

modified polar stationary phases, although fatty

acids are usually determined as fatty acid methyl

ester derivatives; these derivatives are volatile and

easy to resolve on polar phases such as polyesters,

polyethylene glycols and cyanoalkyl silicones. There

is considerable interest in the separation of geometric

isomers of unsaturated fatty acids: the use of very long

capillary columns coated with cyanoalkyl silicones is

the best choice. Direct analysis of triacylglycerols, ini-

tially problematic because of their low volatility, has

improved since the development of fused silica capil-

lary columns with a thin layer of stationary phase with

high MAOT values; to date, very good separations

have been reported, based on the number of carbon

atoms and even on unsaturations. The injection of

high-molecular-weight triacylglycerols requires the

use of a cold-on column or PTV injection.

0044 Carbohydrates GC is used to separate mono-

saccharides, disaccharides, and oligosaccharides.

Although the preparation of volatile derivatives

is necessary, the advantage that GC affords over

HPLC is its greater sensitivity and higher resolution.

The most popular derivatives are silyl ethers, oximes,

methyl ethers, and acetates. Silicones ranging from

methyl to cyanoalkyl are the preferred stationary

phases. These methods have been used for juices,

milk, molasses, honeys, and beverages.

0045 Amino acids These have to be derivatized to achieve

a sufficient volatility. The carboxylic group is usually

esterified, and the amino group can be acylated or

silylated.

0046 Minor constituents Sterols are determined in order

to assess food authenticity, especially with fats and

oils, since the sterol composition is characteristic of

each species. They can be analyzed, either as free

sterols or as trimethyl silyl ethers, using packed

columns (methyl or phenyl silicones), but they are

better resolved on capillary columns. Naturally oc-

curring wax esters (from C

to C

) have been ana-

lyzed using stable thin-film capillary columns.

0047 Volatiles Volatile components are difficult to ana-

lyze because they are usually present as very complex

mixtures at very low concentrations. Thus, the first

requirement is the use of high-resolution columns:

these are long, well-deactivated capillaries, coated

with nonpolar (methyl or methyl vinyl silicones)

or medium polar (polyethylene glycols) phases

in order to elute hydrocarbons, alcohols, esters, pyr-

azines, aldehydes, acids, etc. High-resolution gas

chromatography coupled with MS and sometimes

with other spectrometric detectors like FTIR detect-

ors allows the successful analysis of complex mix-

tures and the identification of many new

compounds. A data-acquisition system is suitable for

profile analysis and computer-based pattern recogni-

tion. A prior isolation and concentration step is usu-

ally necessary in order to suppress the matrix.

Different off-line or on-line techniques can be used:

these are based on extraction, static headspace, ther-

mal desorption, and purge-and-trap devices.

0048Chiral compounds GC affords excellent separations

of enantiomeric pairs, due to the high efficiency of

capillary columns. Commercial columns are prepared

with d-orl-valine tert-butylamide derivatives, metal

complexes, or substituted cyclodextrins. They have

been used to determine the racemization of amino

acids, to detect adulterations in several food products

by analyzing the enantiomeric purity of marker com-

pounds, and to assess the aging or thermal processing

of different foods.

0049Permanent gases CO

and SO

, present in certain

beverages or wines, can be determined using packed

or SCOT columns and TCDs.

Pollutants

0050GC is the basic method used to detect and analyze the

presence in foods of pollutant residues, such as pesti-

cides, toxaphenes, polychlorinated biphenyls (PCBs),

polycyclic aromatic hydrocarbons (PAHs), chlorin-

ated solvents, polychlorodibenzodioxins (PCDDs),

and polychlorodibenzofurans (PCDFs). Multiresidue

procedures are used more frequently than specific

methods. Analysis of these substances, which are pre-

sent at p.p.b. or p.p.t. levels, requires several extrac-

tion, clean-up, and purification steps. High-purity

solvents and reagents must be used, and glassware

has to be cleaned with special care to avoid introduc-

ing new residues. High-efficiency capillary columns

are recommended; this is especially true for PAHs,

which can be resolved efficiently using liquid crystal

phases, and for chiral PCBs. Selective detectors are

required for all of these compounds. Chlorinated

compounds are detected using ECDs; organophos-

phorus compounds are better detected using thermo-

ionic or flame-photometer detection. MS detectors

working in the SIM mode are useful for all pollutants.

Those present at very low levels, such as PCDDs

and PCDFs, require high-resolution MS or MS–MS

systems to be selectively characterized. (See Contam-

ination of Food; Pesticides and Herbicides: Residue

Determination; Pesticides and Herbicides: Types of

Pesticide.)

CHROMATOGRAPHY/Gas Chromatography 1287

Other

0051 GC allows analysis with very low detection limits of

different substances such as illegal drugs used in cattle

fattening, N-nitrosamines, residues in food deriving

from plastic packaging, additives like antioxidants,

preservatives, certain colorants, and other food

ingredients

See also: Chromatography: Combined Chromatography

and Mass Spectrometry; Contamination of Food; Mass

Spectrometry: Principles and Instrumentation;

Applications; Pesticides and Herbicides: Types of

Pesticide; Residue Determination; Spectroscopy:

Infrared and Raman

Further Reading

Biermann CJ and McGinnis GD (1989) Analysis of Carbo-

hydrates by GLC and MS. Boca Raton, FL: CRC Press.

Christie WW (1989) Gas Chromatography and Lipids. Ayr,

UK: The Oily Press.

Grob K (1986) Classical Split and Splitless Injection in

Capillary GC. Heidelberg: Hu

thig.

Guiochon G and Guillemin CL (1988) Quantitative Gas

Chromatography. Amsterdam: Elsevier.

IUPAC (1993) Nomenclature for chromatography. Pure

and Applied Chemistry 65: 819–872.

nig WA (1992) Gas Chromatographic Enantiomer

Separation with Modified Cyclodextrins. Heidelberg:

thig.

Marsili R (1997) Techniques for Analyzing Food Aroma.

New York: Marcel Dekker.

Rotzsche H (1991) Stationary Phases in Gas Chromatog-

raphy. Amsterdam: Elsevier.

Sandra P (1985) Sample Introduction in Capillary Gas

Chromatography, vol. 1. Heidelberg: Hu

thig.

Scott RPW (1996) Chromatographic Detectors. New York:

Marcel Dekker.

Shibamoto T (1998) Chromatographic Analysis of Envir-

onmental and Food Toxicants. Amsterdam: Elsevier.

Wittkowski R and Matissek R (1993) Capillary Gas Chro-

matography in Food Control and Research. Lancaster,

PA: Technomic.

Supercritical Fluid

Chromatography

J W King, Los Alamos National Laboratory,

Los Alamos, NM, USA

Background

0001 Supercritical fluids are unique substances that exhibit

physical properties that are intermediate between

those of a gas or liquid. Their density can be adjusted

by regulating the pressure or temperature of the fluid,

thereby allowing the fluid’s density to range from that

of a dilute gas to liquid-like values. Such a variation in

density accounts for the adjustable, liquid-like prop-

erties that these fluids exhibit when used for extrac-

tion and for separation purposes. Mass transport

properties of supercritical fluids (SFs) also exhibit

values that are in between those of gases and liquids,

but in general, properties like solute diffusion coeffi-

cients in SFs are more gas-like than liquid-like in

magnitude. These favorable characteristics allow SFs

to readily penetrate samples, thereby allowing for

rapid dissolution and high flux rates of solutes into

the SFs.

0002The supercritical fluid region is usually defined in

the upper right region of a pressure and temperature

phase diagram by a substance’s critical points, the

critical pressure (P

) and critical temperature (T

This is shown in Figure 1 for carbon dioxide (CO

the substance usually utilized in supercritical fluid

extraction (SFE) and supercritical fluid chromatog-

raphy (SFC). Above a pressure of 7.38 MPa (1070

psig) and a temperature of 31



C, carbon dioxide

will always be in its supercritical state (SC-CO

)no

matter what external pressure is applied to CO

. This

relatively low T

,CO

’s high nonideality, and its

relative environmentally benign nature, make CO

the fluid of choice for SFC and SFE.

0003The application of SFC to foods and nutritional

analysis came about naturally owing in part to the

early application of SC-CO

extraction in the food

industry, i.e., for the extraction of coffee, hops, and

similar food items used routinely by the consuming

public. As will be demonstrated, SFC is particularly

applicable for the analysis of lipid-containing

Triple point

gas

Pressure

Critical pressure

1070 psig

Liquid CO

Critical point

Solid CO

(Dry ice)

Supercritical

fluid

Temperature

Critical

temperature 31 ⬚C

fig0001Figure 1 Pressure and temperature phase diagram for carbon

dixoide.

1288 CHROMATOGRAPHY/Supercritical Fluid Chromatography

materials, owing to relative high solubilities exhibited

by these solutes (analytes) in SC-CO

. Analysis and

detection of ultratrace components in foodstuffs, e.g.,

pesticides or drugs, has not generally been successful

owing to the problems in routinely interfacing and

using sensitive detectors, such as the electron capture

detector with SFC, owing to the change in mobile

phase characteristics with respect to time during an-

alysis. However, the ability to routinely use the flame

ionization detector (FID) with SFC has provided the

analyst with a useful technique to detect an array of

components, or a specific moiety in a complex food

matrix.

0004 With respect to the chromatographic technique

utilized, it was initially the capillary mode of SFC

that was cited more often than packed column SFC

for the analysis of foods. This has changed somewhat

in recent years, however, particularly with the advent

of using packed column SFC for the analysis of chiral

compounds. This has created more opportunities for

applying SFC using packed columns in food analysis,

since a knowledge of the chirality of a compound can

be of importance even in the food industry (e.g.,

flavor esters). Figure 2 shows the basic schematic

for a capillary SFC (a) and a packed column SFC

(b) instrument. The components making up the

instrumentation for both modes of SFC are very

similar: dual pumps for the SFs and cosolvent (or

modifier), respectively; columns contained inside a

thermostatted oven and detectors external to the

oven. However, in the case of the capillary SFC, the

fluid is depressurized directly into the detector,

whereas in the packed column case, the detector is

usually maintained under pressure, before it is dis-

charged to waste (W). In the packed column mode,

the SFC can be interfaced with a ultraviolet detector

(UV), evaporative light-scattering detector (ELSD), or

a mass spectrometer. In general, the coupling of ana-

lytical supercritical fluid extraction (SFE), on-line

with SFC, has not been adopted to any considerable

extent by food analysts, owing to the lack of an

interface that permits routine coupling for use of the

SFE/SFC mode. However, preparative and produc-

tion-scale SFC have been utilized for specialized appli-

cations in the food-production industries and will

probably see increased use owing to the current interest

in producing high-value nutraceutical components, in

a ‘natural’ and environmentally benign manner.

0005SFC is perceived as a niche technique in the food

industry, so it is critical to recognize when and where

it can be used to advantage relative to what can be

achieved using other forms of chromatography, such

as gas chromatography (GC) and high-performance

liquid chromatography (HPLC). (See Chromatog-

raphy: High-performance Liquid Chromatography;

Gas Chromatography.) Some of these opportunities

are as follows:

0006reduction of the use of organic solvents relative to

HPLC;

0007direct analysis of samples avoiding sample prepar-

ation step;

0008deformulation of commercial food products;

0009detection of product adulteration or deterioration;

0010support of food engineering extraction/reaction

process development.

With respect to nonpolar solutes, pressure- or dens-

ity-programmed SFC provides the capability to

analyze compounds having molecular weights ap-

proaching 1200 Da using one chromatographic an-

alysis. Separation of these compounds by SFC is a

function of their solubility in the mobile phase, their

respective vapor pressures, and the miscibility pres-

sure/temperature of the solute in the fluid phase. For

example, in Figure 3, a diverse number of components

have been separated using the capillary SFC method,

that traditionally would have required the use of

either GC or HPLC, as well as derivatization of

some of the analytes. Utilizing SFC allows the analyst

to avoid using either of both GC or HPLC, and

to directly analyze the sample, obtaining a ‘snapshot’

of the entire molecular composition of the sample.

These characteristic elution patterns produced using a

SFC can be used as means of identifying the presence

SCF

Modifier

SCF

(a)

(b)

Modifier

Oven

Column

Detector

Injector

High

pressure

pumps

High

pressure

pumps

fig0002 Figure 2 Schematic diagram of (a) capillary SFC, and

(b) packed column SFC.

CHROMATOGRAPHY/Supercritical Fluid Chromatography 1289

or absence of a particular analyte in a food sample,

thereby providing valuable information for a food

product formulator, to match or alter, in developing

new and competitive food-related products.

0011 Increasing concerns about minimizing or eliminat-

ing the use of hazardous organic solvents in the

laboratory also bodes well for use in SFC. By incorp-

orating SFC for the separation and detection of food-

related solutes, one not only eliminates most of the

traditional solvent needs associated with HPLC, but

potentially solvents utilized in the extraction or

sample workup steps prior to analysis. In this regard,

SFC is an excellent tool for monitoring the end result

of an extraction or reaction of a food component

using supercritical fluid media. Also, by using SFC,

food-related analytes that are thermally labile or sus-

ceptible to degradation via oxidation are not exposed

to the harsh conditions that often accompany their

analysis by GC or HPLC. Such an advantage can be

attributed to the protective action of SC-CO

, which

excludes oxygen, and the low temperatures used when

separating components via SFC.

Selecting and Optimizing SFC Separation

Conditions for Food Components

0012 For the SFC analysis of food-related samples, the

analyst will undoubtly want to start with a general

fluid programming sequence to separate as many

components in the sample matrix as possible. These

programs are executed for an extended time to assure

optimum resolution and detection of the unknown or

target analyte(s). Run times of 1.5 h in length are not

unusual in this beginning stage of method develop-

ment for SFC separations. After the target analyses

have been identified by retention time matching with

standards, or via an independent method such as mass

spectrometry (MS), the original program can be

modified to reduce the analysis time or improve the

resolution within the chromatogram. Usually, changes

in the mobile phase program will be executed to

hasten the elution of early or late eluting components

that are of no importance for the analysis.

0013Because of the molecular complexity exhibited by

many food ingredients or compositions, it is not

unusual to have a temperature gradient with respect

to time, superimposed on the mobile phase pressure

program during the SFC run. For example, separation

of the like-carbon number triglycerides in soybean oil

is not possible by pressure or density programming

alone, but by superimposing a temperature gradient

during the analysis, these oil components can be sep-

arated quite well. SFC analysis under isobaric condi-

tions is limited in application when analyzing foods,

but it should not be overlooked, since it can yield

often the most precise and accurate results.

Time

FID response

Fatty acids

Squalene

δ-tocopherol

β-tocopherol

γ-tocopherols

α-tocopherol

Diglycerides

Phytosterols

Triglycerides

fig0003 Figure 3 Supercritical fluid chromatography analysis of deodorizer distillate with a SB-octyl-50 column using flame ionization

detection.

1290 CHROMATOGRAPHY/Supercritical Fluid Chromatography

0014 By far, the FID has been the most used detector

used to date in SFC. FID sensitivity to food compon-

ents is lower than that obtained using GC, since ex-

pansion of the mobile phase dilutes the detector signal

substantially. However, the FID signal can be ampli-

fied to permit analysis down to the p.p.m. level, pro-

vided that baseline shifts can be compensated for.

Analytes with chromaphoric properties are amenable

to analysis using the ultraviolet (UV) detector in

conjunction with SFC. The absorption maxima of

components shifts as a function of fluid density or

pressure, but most detector units constructed for op-

eration at these elevated pressures allow for stop flow

or in situ, on-the-fly scanning of peaks to determine

absorbance maxima shifts. Bathochromic shifts of

15–20 nm have been recorded for carotenoids in SC-

over a 25-MPa pressure interval. The mating of

the ELSD detector with SFC has been reported by

several investigators, but the day-to-day stability is

inferior to that obtained with HPLC–ELSD couplings

when applied to food analysis. Heteroelement spe-

cific detectors, such as the electron capture or flame

photometric detector, have been utilized mostly in

research studies using SFC and have not been adopted

for routine analysis. The detector sensitivity and

stability under SFC conditions limit their sensitivity

at best to the p.p.m. range.

0015 Many of the promising applications of capillary

SFC have utilized nonpolar bonded phases utilizing

siloxane functionalities as methyl, octyl, phenyl, and

biphenyl in a polymeric form. The weak elutropic

strength of neat SC-CO

has favored the use

of short-chain-length, monomeric silance-modified

columns having C

, phenyl, amino, and

diol phases for packed column SFC. The choice of

these phases is not so much related to their selective

interaction with food-related solutes, but to their sur-

face-modifying properties, which reduce peak tailing

and solute interaction with the base silica matrix.

Polymer resin columns have also been utilized, but

they are susceptible to voiding unless specifically

packed for use under supercritical fluid conditions.

Types of food Ingredients Analyzed by

SFC

0016 A myriad of food-related components and matrices

have been analyzed by SFC, as indicated by the partial

listing in Table 1. These include naturally occurring

ingredients such as fats/oils, spices, etc., minor un-

wanted constituents like pesticides, antibiotic drugs,

and mycotoxins, and specific food components, in-

cluding nutraceuticals and flavoring aids. Inspection

of Table 1 indicates a preponderance of applications

in the lipid analysis area. Indeed, SFC is tailor-made

for lipid analysis, although somewhat lacking in the

high-resolution capabilities demonstrated by high

temperature GC. The retention pattern for typical

lipid solutes in SFC as shown in Figure 4, follow a

distinct pattern governed approximately by the

solute’s molecular weight/volatility characteristics.

Elution of the following classes of lipids occurs in

the following order: fatty acid methyl esters, free

fatty acids, hydrocarbons, vitamins, sterols, wax

esters, mono- and then diglycerides, followed by tri-

glycerides and steryl esters. Although there is some

tbl0001Table 1 Food components separated and analyzed by SFC

Carbohydrates: Derivatized corn syrups, mannose glycans

Chiral compounds: Monoterpenes, pyrazines, clenbuterol

Drugs/antibiotics: Caffeine, erythromycin, polycyclic ether

antibiotics, sulfonamides, assorted steroids

Hydrocarbons: Sesquiterpenes, squalene, waxes and wax

esters

Lipids: Fatty acids, fatty acid esters, monoglycerides,

diglycerides, triglycerides, sterol esters, sterols (cholesterol),

fat-soluble vitamins, tocopherols, phospholipids (lecithin), lipid

hydroperoxides, glycolipids

Nutraceuticals: Valeriana, gingolides, sawtooth palmetto berry

Oils/fats: Celery oil, coconut, fish, soybean, wheat germ, palm

oil, rice oil, milk/cheese triglycerides

Packaging/film components: Polypropylene oligomers,

polyvinyl chloride, phenolic antioxidants, low-molecular-

weight polystyrene

Pesticides: Halogenated, organophosphorus, carbamate,

pyrethrins, acidic phenoxy herbicides, sulfonyl ureas

Pigments: Carotenoids, xyanthophyls

Speciality ingredients: Hops components

Spices/flavors: Capsicum, cardoman, coumarin, curry, garlic

components, marjoram, rosemary, vanillan

Terpenes/essential and fruit oils: Grapefruit oil, limonenes,

mint, lemon

Other toxicants: Mycotoxins, nitrosamines, polycyclic aromatic

hydrocarbons

Retention time

Waxesters

Cholesterol

Squalene

Triglycerides

Etherlipids

Cholesterolesters

Diglycerides

Free

fatty

acids

Vita-

mins

fig0004Figure 4 General elution order (retention time) of lipids in SFC

on nonpolar stationary phases.

CHROMATOGRAPHY/Supercritical Fluid Chromatography 1291

overlap between individual classes of the above

solutes, owing to the overlapping molecular weight

ranges (e.g., triglycerides and steryl esters), this solute

separation pattern has proven very useful in tracking

conversion of lipid species undergoing reactions, as

well as in the quality control of food ingredients.

0017 Triglyceride-based oils/fats are also readily amen-

able to analysis by SFC. Separation of the individual

components is once again governed by molecular

weight considerations, allowing SFC to facilitate

the separation of the major triglyceride species, i.e.,

, etc. For some oils, such as coconut oil,

picturesque chromatograms result (Figure 5), as

shown by the separation of the C

to C

saturated

triglycerides as equally spaced peaks. For other oils

such as soybean oil, there is some overlap between the

saturated and unsaturated triglyceride species, requir-

ing, as noted previously, a superimposition of a tem-

perature gradient along with the pressure gradient

program to achieve adequate resolution. However,

even without high resolution, the rapid SFC analysis

can be used to advantage in quality control, where

speed, rather than optimal separation, is often desired.

0018 Detection of minor components in foods is limited

by the detector sensitivity and stability as noted pre-

viously, but those components that can be detected by

using FID, UV, or ELSD, are often analyzed more

rapidly by SFC, owing to the time savings afforded

by avoiding elaborate preparation of the sample prior

to analysis. In addition, SFC analysis provides a more

detailed profile of the entire sample in addition to

detecting the target analyte. This allows an accurate

assessment of the total contribution of the minor

constituent to the entire ingredient profile, e.g., the

presence of sterol esters in sawtooth palmetto berry

extracts, where the fatty acids and triglycerides are

the major constituents.

0019Other food sample types that can be readily ana-

lyzed by SFC are the fat-soluble vitamins, essential

and flavor oil ingredients, spice extracts, hop com-

ponents, as well as functional food components, i.e.,

nutraceuticals. Some SFC-based separations require

the use of a cosolvent (usually 5–20 vol.%), in add-

ition to the SC-CO

, to modify the mobile phase. For

example, phospholipids are only sparingly-soluble in

neat SC-CO

, but these polar lipid compounds can

be chromatographed successfully on packed silica

columns by incorporating ethanol and/or water as a

modifier into the mobile phase. Likewise, carbohy-

drate moieties, which exhibit limited or no solubility

in SC-CO

or SC-CO

/cosolvent mobile phases, can

be derivatized so that they can be analyzed by SFC.

Selected Applications of SFC in Food and

Nutritional Analysis

0020In this section, several brief examples will be given to

illustrate the utility and potential of SFC for food and

nutritional analysis. As noted previously, both high

and low resolution of solutes are possible using SFC.

SFC separation and detection of a-tocopherol and

cholesterol in a fish oil capsule can be achieved on a

capillary SB-methyl column at 120



C, using a density

program with SC-CO

from 0.28 to 0.66 g ml

1

and a

density programming rate of 0.0006 g ml

1

. Al-

though the analysis took 90 min to perform, it

allowed the analyst to avoid any sample preparation,

other than diluting the oil from the capsule in small

quantity of solvent, and injecting it into the SFC.

Therefore, no derivatization of the sample was re-

quired, and there was sufficient resolution between

the a-tocopherol and cholesterol to quantify these

analytes. This was made possible by adjustment of

the elution conditions, i.e., the background compon-

ents (fish oil triglycerides), that were of no interest in

this analysis, and were programmed off the column

without resorting to a prefractionation of the sample

prior to SFC analysis.

0021Not all applications of SFC require the above high-

resolution separation. Packed column SFC (5 mm, C

– Deltabond) has been used to ‘clean up’ samples

prior to other types of chromatographic analysis

(GC). In this case, organochlorine- and phosphorus

pesticides extracted by SFE with SC-CO

from a meat

sample were separated from the coextracted fat moi-

eties using the packed SFC column. Hence, by ‘heart

cutting’ the appropriate elution fraction, a lipid-free,

pesticide-containing fraction was provided for

residue analysis by GC.

Time (density)

120 ⬚C

FID response

fig0005 Figure 5 Capillary supercritical fluid chromatogram of coconut

oil triglycerides.

1292 CHROMATOGRAPHY/Supercritical Fluid Chromatography

0022 SFC is an excellent technique to monitor reaction

chemistry between lipid species, since it avoids the

need to employ more than one analytical technique

or analyte derivatization. Further, it permits the

successful chromatography of all of the relevant

reactants and products using one chromatographic

analysis. Examples where SFC has been applied are

for the esterification or transesterification of lipids,

glycerolysis reactions, and randomization of fats/oils.

Figure 6 shows two SFC profiles for the enzymatic-

catalyzed transesterification reaction between the tri-

glycerides from a ground beef sample and methanol,

to produce the corresponding fatty acid methyl esters

(FAMEs) for nutritional labeling analysis. The 30-min

chromatograms show the effect of the moisture con-

tent of the ground beef sample on the yield of FAMEs,

(a) being the yield from the neat ground beef sample,

and (b) being the conversion attained by freeze-drying

the sample matrix prior to methanolysis. Note that

freeze-drying of the ground beef was crucial to

obtaining a quantitative yield of FAMEs for fat an-

alysis required in nutritional labeling. The transester-

ification reaction in this case was run in a flow

extractor-reactor in which the ground beef triglycer-

ides were extracted using SC-CO

and carried over a

bed of supported lipase in the presence of methanol to

produce the desired FAMEs.

0023Table 2 shows the analysis of the glyceride content

of a soybean oil glycerolysis mixture by both HPLC-

FID and SFC-FID. The information desired was the

relative amounts of free fatty acids (FFA), mono-

(MAG), di-(DAG), and triacylglycerols (TAG) in

the reaction mixture. A comparison of the SFC-FID

results with those obtained by HPLC-FID shows a

relatively good agreement between the two methods.

However, the SFC method does not require the time

and effort for sample preparation associated with the

alternative technique, and in addition saves on the

cost of solvents and chemical reagents.

0024Although mentioned previously, preparative or

production-scale SFC is now being used as a separ-

ation technique in the food industry. Fractionation

and isolation of food components, such as toco-

pherols and phospholipids, or the o-fatty acids/esters

from fish oils, have been cited in the literature.

Time (min)

FID response

FAMES

Fatty acids

Cholesterol

Triglycerides

Time (min)

(a) (b)

30 0 30

fig0006 Figure 6 SFC analyses of reaction products from the transesterification of ground beef lipids: (a) neat ground beef sample;

(b) freeze-dried ground beef sample.

tbl0002Table 2 Analysis of lipid species in a soybean oil glycerolysis

mixture

Methodofanalysis MAG DAG TAG FFA

SFC – FID 75.3 23.2 <0.1 1.5

GC – FID 76.2 21.8 <0.1 2.0

CHROMATOGRAPHY/Supercritical Fluid Chromatography 1293

Recently, a production plant for the separation of fish

oil ethyl esters has been constructed in Spain to pro-

duce 95% pure polyunsaturated fatty acids for the

nutraceutical market. The basic separation premise

upon which this production scale plant is designed

was based on chromatographic fractionations ini-

tially developed using analytical-scale, packed SFC

columns.

See also: Chromatography: Thin-layer Chromatography;

High-performance Liquid Chromatography; Gas

Chromatography