1274 CHROMATOGRAPHY/High-performance Liquid Chromatography
Modified cellulose such as DEAE-cellulose can be
used for ion-exchange separations on TLC. Separ-
ation by size-exclusion chromatography can be
carried out with TLC plates coated with Sephadex
gel but is slower and significantly more difficult
than other forms of TLC. Polyamides such as
polyhexamethylenediaminoadipate can be used as
the adsorbent to separate components that interact
with it by hydrogen bonding. (See Chromatography:
Principles.)
Applications
0024 The actual method employed for preparing samples
from foodstuffs for analysis by TLC depends on the
nature of the substances being investigated. Many
methods involve the extraction of the food with a
suitable solvent followed by precipitation and filtra-
tion steps to remove classes of compounds that are
not of interest. (See Analysis of Food.)
0025 The choice of adsorbent and solvent system used in
TLC is dictated by the nature of the sample to be
analyzed. Examples of broad groups of substances
that have been successfully resolved by TLC methods
are shown in Table 1, along with suggested solvent
systems and detection reagents commonly used.
However, for most substances, there can be a variety
of different solvent systems and detection reagents
that are suitable, depending on the precise compos-
ition of the samples to be resolved.
See also: Chromatography: Principles; High-
performance Liquid Chromatography; Gas
Chromatography
Further Reading
Ackman RG (1982) Flame ionization detection applied to
thin-layer chromatography on coated quartz rods.
Methods in Enzymology 72: 205–252.
Henderson RJ and Tocher DR (1992) Thin-layer chroma-
tography. In: Hamilton RJ and Hamilton S (eds) Lipid
Analysis: A Practical Approach, pp. 65–111. Oxford:
Oxford University Press.
Jork J, Funk G, Fischer G and Wimmer CJ (1989) Thin-
layer Chromatography: Reagents and Detection
Methods. Cambridge: VCH.
Kirchner JG (1978) Thin-layer Chromatography. Tech-
niques of Chemistry, vol. XIV. New York: Wiley.
Sherma J (1996) Thin layer chromatography in food
analysis. Food Testing and Analysis 2: 12–15.
Touchstone JC (1988) Instrumentation for thin-layer chro-
matography: a review. Journal of Chromatographic
Science 26: 645–649.
Wilson ID (1996) Thin layer chromatography: a neglected
technique. Therapeutic Drug Monitoring 18: 484–492.
High-performance Liquid
Chromatography
M V Moreno-Arribas and M C Polo, Instituto de
Fermentaciones Industriales (CSIC), Madrid, Spain
Copyright 2003, Elsevier Science Ltd. All Rights Reserved.
Introduction
High-performance liquid chromatography (HPLC) is 0001
an instrumental form of liquid chromatography that
employs stationary phases consisting of small par-
ticles, thereby achieving more efficient separations
than those used in conventional liquid chromatog-
raphy. Since its origin in the late 1960s, it has been
known by several different names, including high-
pressure liquid chromatography, because of the
high pressures required to force the mobile phase or
solvent through the stationary phase, and high-
resolution liquid chromatography, because of the
good resolution achieved using this technique.
This article describes the equipment needed to 0002
carry out HPLC and summarizes some of the app-
lications of this technique in food analysis. Two
techniques related to HPLC, fast-protein liquid chro-
matography (FPLC) and supercritical fluid chroma-
tography (SFC), are also considered. The basic theory
of the chromatographic process and the factors
that affect separation efficiency are discussed in
the section on Chromatography. (See Chromatog-
raphy: Principles; Thin-layer Chromatography;
High-performance Liquid Chromatography; Gas
Chromatography; Supercritical Fluid Chromatog-
raphy; Combined Chromatography and Mass Spec-
trometry.)
Separation Modes
There are many different stationary and mobile 0003
phases that can be used in HPLC, and for this reason
there is a great variety of separation mechanisms.
The treatises on HPLC use different criteria in their
attempt to classify the modes of this technique:
type of stationary phase, predominant separation
mechanism, type of groups of compounds it is
aimed at. These criteria sometimes overlap; that is
to say, it is possible to work in several different chro-
matographic methods with the same stationary
phase, and the same group of compounds can be
separated using several different stationary phases,
and for this reason it is not easy to classify HPLC
techniques into specific groups. Generally speaking,
it can be said that there are three large groups
 CHROMATOGRAPHY/High-performance Liquid Chromatography
that dominate the field of chromatographic separ-
ations: reversed-phase chromatography, ion exchange
chromatography and size exclusion chromatography.
There are also other types of chromatography that
are rapidly gaining in importance and cannot be in-
cluded exactly in any of these three groups: ion pair
chromatography, affinity chromatography, and chiral
separations.
Reversed-Phase Chromatography
0004 This is the most commonly used method for chroma-
tographic separations, so it could in fact be called
‘normal-phase’ except that it was developed later
than the latter. In reversed-phase chromatography,
the stationary phase is of a nonpolar nature and the
mobile phase is more polar than the stationary one.
Among the most widely used stationary phases are
silica-based ones with cyano, phenyl, or alkyl func-
tionality and those of a polymeric nature, above all
polystyrene-divinylbenzene (PSDVB). The mobile
phase is usually water with an organic modifier
(methanol, acetonitrile, or tetrahydrofuran). Ternary
or quaternary mixtures of these components can be
used to improve selectivity.
0005 There is controversy over the mechanism that
controls reversed-phase separations. The three basic
models are the partitioning model, the solvophobic
effect, and the adsorption model. It is also necessary
to consider the possibility that a mixed mechanism
might exist.
0006 In the partitioning mechanism it is accepted that
the retention of a solute in reversed-phase liquid
chromatography is due to its partitioning between
the mobile phase and the stationary phase. This
mechanism assumes that the stationary phase behaves
in a similar way to a liquid, a situation that may occur
in certain conditions, such as when working with
monomeric phases and high percentages of water in
the mobile phase, and the size of the hydrocarbonated
chains is large enough for them to be associated. The
adsorption mechanism accepts that retention takes
place through the interaction of the solute to the
organic molecules bonded to the surface of the
packing matrix. The solvophobic effect theory
accepts that the solute is expelled from the mobile
phase towards the stationary phase.
Ion Exchange Chromatography
0007 This is used for the analysis of electrically charged
substances in working conditions. Stationary phases
with electrically charged functional groups are used
for this method of chromatography. The support can
be silica or a polymer. The mobile phase contains a
buffer which controls the loading of the solutes, and a
1275
salt that competes with the solutes in their interaction
with the charges of the stationary phase. The charges
permanently bonded to the chromatographic support
are compensated by counterions, that is to say, by free
ions with the opposite charge. Passage through the
column of a sample or of the mobile phase with ions
of the same sign as the counterions of the column may
result in the displacement of the counterions and the
retention of the ions of the sample or of the stationary
phase.
The ion exchangers can be classified as cationics 0008
and anionics. Within each group it is possible to
differentiate strong and weak exchangers. Strong
cation exchangers (SCX) usually contain sulfonic
groups, while weak ones have carboxylic groups.
Quaternary ammonium salts are the most usual func-
tional groups in strong anion exchangers (SAX), and
primary amines are used in weak anion exchangers.
Size Exclusion Chromatography
This separation method is based on the size of the 0009
molecules. For many years it was known as gel
permeation chromatography (GPC) or gel filtration
chromatography (GFC). The retention of the mol-
ecules depends on their size: the larger molecules
that do not enter the pores of the packing elute first,
while the smaller molecules, whose diameter makes it
possible for them to enter and exit the pores of the
chromatographic packing, take a longer time.
Ion Pair Chromatography
This chromatographic method enables the separation 0010
of solutes that are ionized in separation conditions.
On most occasions the separations are carried out on
the same columns as those used in reversed-phase
separations without the formation of ion pairs. The
mobile phase is made up of a water buffer and an
organic modifier, to which mixture is added a coun-
terion with an opposite charge to that of the solute.
Quaternary ammonium salts are often used as
counterions for the analysis of anion substances,
while n-alkyl sulfate salts are among the most com-
monly used for the analysis of cation substances.
Affinity Chromatography
This is based on the specific interaction between a 0011
molecule, or group of molecules, and a ligand,
which is a molecule that is attached to the station-
ary phase to interact with those of the solute. The
growing importance of affinity chromatography is
due to the development of bioaffinity chromatog-
raphy in which the interactions between ligands and
molecules are based on recognitions of a biological
type.
 1276 CHROMATOGRAPHY/High-performance Liquid Chromatography
Chiral Separations
0012 These can be approached mainly in two ways; by
transforming enantiomers into chromatographically
separable diasteroisomers, which does not necessitate
the use of chiral phases, or by preparing chiral phases.
Chiral phases can be obtained by covering the pack-
ings by making a solution pass through the column,
or by ionic or covalent bonding of the chiral reagent
with the column packing material.
Instrumental Configurations
0013 The basic equipment consists of a column packed
with a stationary phase, a driving force to propel the
solvent through the column (pump), a system (in-
jector) for introducing the sample on to the column,
a system (detector) for measuring a physical property
of the solutes being analyzed that differs from the
properties of the solvent of a property of the mobile
phase which is altered by the presence of the solute,
and a system for recording the detector signals and
converting them into graphic traces or chromato-
grams.
0014 A single solvent is often used to carry out the
separation (isocratic elution), but differing propor-
tions of various solvents are also often used (gradient
elution), in which case a gradient device is needed. A
variety of accessories, such as pressure controllers,
valves for switching solvents, valves for switching
column, and ovens for heating the columns, are also
commonly employed. Today most chromatographs
are controlled by a computer, which is also used for
data collection. This provides greater quality of quan-
titative data and enables automation of the system.
Solvents
0015 The nature of the solvent will depend upon the mode
of chromatographic separation employed, but a series
of precautions that are common to all types of HPLC
must be taken when preparing solvents. Because the
columns have frits at the ends to hold the packing in
place, the solvents must be devoid of particles and
consequently must be filtered through membranes
with a pore size of 0.5 mm or smaller, prior to use.
Bubble formation must also be avoided, since bubbles
may cause variations in the flow rate if they reach the
pump or perturbations in the chromatogram if they
reach or form in the detector cell. Solvents must
therefore be degassed by immersing the bottle con-
taining the solvent in an ultrasonic bath or by flushing
the solvent with a stream of helium or nitrogen before
delivering it to the chromatograph. A small stream of
helium is commonly bubbled through the solvent
during the chromatographic procedure to prevent
uptake of air. To prevent bubble formation in the
detector cell with depressurization, a restrictor is
sometimes attached to the outlet of the detector cell.
Pumps
The pump is the system for delivering the solvent 0016
from the solvent reservoir to the column through the
injector. Basically, two types of pumps are available:
constant-pressure pumps and constant-flow-rate
pumps. The latter are more frequently used in HPLC.
Constant-pressure pumps are less expensive and 0017
easy to operate, but the flow rate may vary with
changes in the viscosity of the mobile phase caused
by temperature fluctuations or by the accumulation
of undissolved sample components in the column.
These variations in flow rate affect retention times,
and they may also affect resolution, increasing
the difficulty of both qualitative and quantitative
analysis.
Constant-flow-rate pumps afford the advantage of 0018
maintaining retention times irrespective of changes in
solvent viscosity. This type of pump includes syringe
pumps, which consist of a cylinder containing the
mobile phase, which is expelled by a piston. The
piston is driven by a motor, so as to supply a constant
flow devoid of pulses. This type of pump can achieve
relatively high pressures, but maintenance and chang-
ing solvents are complicated.
Reciprocating pumps, also a type of constant-flow- 0019
rate pump, are most commonly employed. Their price
varies depending on their complexity of design, and
their main drawback is that they generate pulses that
may cause noise in the detector. Single-piston pumps
are the least expensive and have a rotating eccentric
cam, which drives the plunger, discharging the liquid
through a one-way valve. Duplex pumps have two
plungers driven by a single motor by means of a
shared cam. This arrangement means that, while
one of the plungers is in the discharge phase, the
other is in the intake phase, thereby superimposing
the two flow-rate profiles and considerably reducing
pulsation. In this type of pump, delivery from the
solvent reservoir is steepled and changing solvents is
rapid.
Injectors
Introducing the sample on to the column is one of the 0020
most critical steps in HPLC. Ideally, the sample
should reach the column in the form of a tiny droplet
that does not undergo diffusion, which would
broaden the chromatographic bandwidth and thus
lower resolution.
Several methods are employed to deliver the 0021
sample on to the column. In on-column injectors the
 CHROMATOGRAPHY/High-performance Liquid Chromatography
sample is introduced through a syringe which tra-
verses a septum and enables the desired quantity of
sample to be deposited at the column inlet. In this
method the mobile phase flows continuously through
the column. The stop-flow injector is a variation of
this type of injector. In this method the pump is
stopped before the syringe is inserted in the injector,
and injection is effected when column pressure has
dropped to atmospheric pressure. The advantage of
these injectors is that they are inexpensive and of
simple construction, yet they do not lower efficiency.
However, reproducibility is poor, they are not suitable
for high working pressures, and they are complicated
to operate.
0022 Valve injectors are most commonly used. In these
injectors the sample is delivered on to a pressurized
column with no appreciable interruption in flow. The
sample is deposited by a syringe into an external loop,
the valve selector is turned, and the mobile phase
passes through the loop on its way to the column.
The valve thus has two positions, a load position and
an injection position. High injection reproducibility
can be achieved with these injectors. The band-
broadening effect is comparable to or somewhat
higher than that obtained using on-column syringe
injectors. The drawback afforded by these injectors
is that there is a split-second interruption in mobile-
phase flow that may damage the column. To avoid
this problem, a bypass may be attached, such that
there is always a constant flow of mobile phase
from the pump to the column.
0023 Automatic injectors that are capable of running
analyses on up to 100 samples without operator at-
tention have been designed on the basis of the same
mechanism as valve injectors. There are also injectors
equipped with a system of valves connecting them to
several columns, which enables columns to be
switched without stopping the flow.
Columns
0024 The columns most commonly utilized in HPLC con-
sist of stainless-steel, plastic, or glass tubes, measuring
15–25 cm in length and packed with small-diameter
particles (3–20 mm). Internal column diameter is
normally between 2 and 5 mm.
0025 The use of small-internal-diameter (microbore)
columns has become increasingly important from
the early 1980s; they are similar to the columns de-
scribed above, but their internal diameter is between
0.5 and 2 mm, while they range in length from 10 to
25 cm. Packing particle size normally ranges from 3
to 5 mm. These columns are suitable for use when only
small samples are available or when low solvent con-
sumption is required.
1277
The length of both conventional and microbore 0026
columns is mainly limited by the pressure needed
to drive the solvent through the column, which is
inversely proportional to the size of the particles
used in the packing. Minimum column diameter is
limited by column-wall effects that cause solute mol-
ecules flowing next to the wall to move more slowly
than those flowing through the center of the column,
resulting in an increase in chromatographic band-
width.
Open capillary columns similar to those employed 0027
in gas chromatography are the most recent to have
come into use. The column tube is made of glass and
is 20–50 mm in diameter and several meters long. The
stationary phase is chemically bonded to the wall of
the tube. Capillary columns are also prepared by
packing a tube with particles 5–30 mm in size and
subsequently heating and drawing the tube to an
internal diameter of 50–125 mm.
In addition to the column on which the separation 0028
is carried out, two other types of column are also uti-
lized in HPLC to protect the analytical column. These
are precolumns and guard columns. Precolumns are
placed between the pump and the injector to saturate
the mobile phase with the stationary phase and thus
prevent dissolution of the stationary phase in the
analytical column. Guard columns are placed be-
tween the injector and the main column in order to
retain components in the sample that might otherwise
become permanently adsorbed on the analytical
column, thereby affecting column efficiency and per-
meability. Both precolumns and guard columns are
normally made of a material similar to that used in
the analytical column.
Chromatography is customarily performed at am- 0029
bient temperature, but it may be necessary to regulate
the column temperature or to carry out the separation
at a temperature other than room temperature.
In such cases thermostat-equipped compartments
(ovens) and systems for heating or cooling the
columns are required.
Detectors
In order to be suitable for use in HPLC, detectors 0030
must meet a number of requirements. First and
foremost, detector design must prevent broadening
of chromatographic bandwidth to insure that the
separations achieved on the column do not deterior-
ate in the detector. In addition, response time must be
short and the response must be linear over a suffi-
ciently broad range of concentrations.
The detectors most frequently employed are the 0031
refractive index detector, the photometric detector,
and the fluorescence detector.
 1278 CHROMATOGRAPHY/High-performance Liquid Chromatography
0032 Refractive index detectors measure the difference
between the refractive index of the mobile phase and
that of the column eluate. They are universal detect-
ors that are highly sensitive to small changes in the
mobile phase and even to small variations in tempera-
ture or pressure. This sensitivity means that to achieve
a suitable signal-to-noise ratio they are only capable
of detecting solute concentrations in the order of
micromoles. In addition, they are unsuitable for
working with gradient conditions.
0033 Photometric detectors measure absorbance in the
ultraviolet (UV) or visible regions of all the compon-
ents in the column eluate. They are less universal than
refractive index detectors but by the same token are
more specific. This type of detector is normally
capable of detecting nanomoles, provided that the
compound contains a strong chromophore. The three
types of photometric detector most frequently used
are fixed-wavelength detectors, variable-wavelength
detectors, and diode array detectors. This last type of
detector is capable of performing complete spectral
analysis of the column eluate on a continuous basis,
i.e., without stopping the flow.
0034 Fluorimeter detectors are more specific and more
sensitive than photometric detectors, but their linear
range is smaller. Detection limits are in the order of
picomoles for suitably fluorescent compounds, and
they are very useful in trace component analysis.
0035 Electrochemical detectors are also widely employed
in HPLC. These come in two types: amperometric
and conductometric detectors. Amperometric detect-
ors are highly sensitive but are only applicable to
analyses that can be oxidized or reduced; conducto-
metric detectors are moderately sensitive and are
applicable for detecting anions and cations. This
type of detector is the detector most commonly used
in ion exchange chromatography.
0036 Derivatization of the components being analyzed
may sometimes be employed to increase the detection
limit or specificity.
0037 Mass spectrometry (MS) is being used more and
more as an online detection system in HPLC. The use
of HPLC-MS coupling has spread since 1980 due to
the improvements introduced in the different types of
interface used. Several coupled MS-HPLC systems are
available commercially, and improvements are con-
tinually being introduced in existing equipment. At
the same time, new equipment is appearing all the
time. MS has been considered to be the ideal detector,
since it furnishes information on component struct-
ure. Microbore HPLC is particularly useful for online
HPLC-MS, which requires low sample volumes. MS
in combination with HPLC may become a standard
technique in a few years’ time. (See Mass Spectrom-
etry: Principles and Instrumentation.)
Selected Applications
The use of HPLC in food analysis is growing daily, 0038
and it is now routinely applied in many laboratories.
The many different types of columns and detectors
that are currently available commercially make it
possible to apply HPLC in analyzing nearly all the
nonvolatile components in foods, be they present
naturally or added artificially. The techniques
employed for certain groups of food components are
summarized below by way of example.
Carbohydrates
Nearly all chromatographic modes may be used in 0039
separating carbohydrates. For example, ion exchange
on strongly or weakly basic anionic resins or on
cationic resins, and partition chromatography on
ion exchange resins, on chemically bonded cyano,
amino, propyl-amino, or combined amino-cyano
phases, or on silica gel or gel permeation are all
possible. Differential refractometry is the conven-
tional detection system, although direct detection,
using short UV wavelengths or by forming derivatives
detectable at longer wavelengths or fluorescent
derivatives, is also employed. (See Carbohydrates:
Determination.)
Acids
A variety of chromatographic modes are also applied 0040
to this group of components. Certain workers have
employed ion exchange chromatography on strongly
acid cationic resins or strongly or weakly basic
anionic resins. Reversed-phase chromatography and
ion pair chromatography have also been used. Detec-
tion is carried out by refractometry, photometry using
UV, or visible wavelengths, as in the case of carbohy-
drates. (See Acids: Properties and Determination.)
Amino Acids and Amines
Most separations of amino acids and amines are per- 0041
formed using reversed-phase chromatography of dan-
syl chloride, orthophthaldialdehyde (OPA), or phenyl
dithioisocyanate derivatives. 9-Fluorenylmethyl
chloroformate (FMOC) is a suitable reagent for the
analysis of secondary amino acids. Detection is
carried out by means of fluorescence or UV absorp-
tion. The derivatizing, highly fluorescent, reagent
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
(AQC) reacts rapidly and easily with both primary
and secondary amines and its use is currently spread-
ing. (See Amines; Amino Acids: Determination.)
Peptides
Diverse techniques are used to separate peptides on 0042
account of the broad range of molecular weights
 CHROMATOGRAPHY/High-performance Liquid Chromatography 1279

of these components. Conventional reversed-phase
columns are applied for peptides with molecular
weights of less than 3 kDa, while reversed-phase
columns packed with particles with large pore sizes
or size exclusion columns are used for larger peptides.
Detection is performed at 214 nm. Figure 1 shows a
chromatogram of the peptides with molecular weights
ation of HPLC and MS analysis is commonly used for
the sequence analysis of glycoproteins. Detection is
carried out at 214 or 280 nm. (See Protein: Determin-
ation and Characterization.)
Other Compounds
In addition to the major groups of food components 0044

higher and lower than 700 from a sparkling wine, as an
example of this type of analysis. (See Peptides.)
Proteins
0043 Practically all known modes have been used in
the separation of proteins, e.g., separations based
on molecule size (gel permeation chromatography),
on charge (ion exchange chromatography), on
hydrophobicity (reversed-phase chromatography
and hydrophobic–interaction chromatography), and
even combinations of these mechanisms. The combin-
0.15
Absorbance at 214 nm
mentioned above, HPLC has found application in
many other areas of food analysis, and details
may be found in the relevant articles for the follow-
ing compounds or groups of compounds: lipid
components, phospholipids, triglycerides, vitamins,
colors, pesticides, drug residues, polycyclic aromatic
hydrocarbons, and nitrosamines. (See Colorants
(Colourants): Properties and Determination of
Natural Pigments; Properties and Determinants
of Synthetic Pigments; Fatty Acids: Analysis; Nitro-
samines; Pesticides and Herbicides: Types, Uses,
and Determination of Herbicides; Phospholipids:
Determination; Polycyclic Aromatic Hydrocarbons;
Triglycerides: Characterization and Determination;
Vitamins: Determination.)

0.10
Related Techniques
FPLC designates a fast chromatographic method, de- 0045

veloped by Pharmacia, which is similar to HPLC and

yields high resolution. Since FPLC needs only a rela-
0.05
tively low backpressure to drive the high flow rates at
which the separations are performed, the risk of de-
naturation caused by shearing forces is reduced.
0.00
Moreover, the mechanical components are resistant
0 10 20 30 40 50 to corrosive buffers, and there is no contamination or
(a) Time (min) inactivation or the components of interest.
Given the range of columns available in the market, 0046
0.15
a variety of separation modes can be applied using
this technique: size exclusion, hydrophobic inter-
action, chromatofocusing, ion exchange, and re-
Absorbance at 214 nm

0.10
versed-phase chromatography.
This method was developed to separate and purify 0047

biomolecules and is very useful in separating isoen-

zymes and molecular species with similar charge char-
0.05 acteristics. It is also used to distinguish between
different types of meat or grains.
SFC is another technique, related to HPLC, which 0048

uses as the mobile phase a supercritical fluid, i.e., a

0.00
0 10 20 30 40 50
fluid at a pressure and temperature above the critical
(b) Time (min) point. The properties of supercritical fluids are inter-
mediate between those of gases and those of liquid.
fig0001 Figure 1 Chromatogram of peptides with molecular weights (a) Thanks to their higher diffusivity and lower viscosity
higher and (b) lower than 700 Da from a sparkling wine. Column: as compared to liquids, high efficiencies are achiev-
Nova Pak C18. Solvents: (a) TFA/water; (b) TFA/acetonitrile.
able with shorter analysis times than those customar-
Gradient elution. Detection at 214 nm. Reproduced from Moreno-
Arribas MV, Bartolomé B, Pueyo E and Polo MC (1998) Isolation ily employed using HPLC.
and characterization of individual peptides from wine. Journal of The basic advantage of SFC with respect to gas 0049

Agriculture and Food Chemistry 46: 3422–3425, with permission. chromatography (GC) is the possibility of analyzing
 1280 CHROMATOGRAPHY/Gas Chromatography
components that span a broad range of volatilities as
well as heat-labile components. At the same time,
SFC is also compatible with many of the detectors
commonly used in GC or HPLC and SFC-MS
coupling is easy to carry out.
0050 The critical temperature of the supercritical fluids
employed as the mobile phases between 0
C and
200
C and the critical pressure of these fluids should
not be too high. Carbon dioxide, nitrous oxide,
alkanes (such as n-pentane), and xenon, all of which
are nonpolar, are most often used. Ammonia can be
used to elute polar solutes, but mixtures of phases,
e.g., a nonpolar mobile phase containing a small
quantity of a polar organic solvent, known as a
modifier are normally employed.
0051 SFC can be performed on capillary, packed, and
micropacked columns. Stationary phases should be
cross-linked; otherwise the supercritical fluids,
which are excellent solvents for polymers, could
extract the stationary phase. Enantiomers can be
resolved using chiral phases.
0052 The equipment used in SFC is similar to that used
in HPLC and basically consists of a high-pressure
syringe pump, an injector, and a restrictor or post-
column valve to keep the mobile phase in a supercrit-
ical condition inside the chromatographic column.
0053 Fluid density is commonly programmed to adjust
mobile-phase selectivity, in as much as the physico-
chemical properties of supercritical fluids (solvation
strength, viscosity, diffusion) are all dependent upon
density.
0054 This method has been applied in food analysis (oils,
cheeses, coffee, etc.) Some of the most interesting
applications include separations of acids, alcohols,
lipids, carbohydrates, vitamins, and terpenes. (See
Acids: Properties and Determination; Alcohol: Prop-
erties and Determination; Analysis of Food; Carbo-
hydrates: Determination; Coffee: Analysis of Coffee
Products; Vitamins: Determination.)
See also: Acids: Properties and Determination; Alcohol:
Properties and Determination; Amino Acids:
Determination; Chromatography: Principles; Coffee:
Analysis of Coffee Products; Colorants (Colourants):
Properties and Determination of Natural Pigments;
Properties and Determinants of Synthetic Pigments; Fatty
Acids: Analysis; Nitrosamines; Peptides;
Phospholipids: Determination; Polycyclic Aromatic
Hydrocarbons; Protein: Determination and
Characterization; Triglycerides: Characterization and
Determination; Vitamins: Determination
Further Reading
Charalambous G (1984) Analysis of Foods and Beverages.
Modern Techniques. Orlando: Academic Press.
Dabrio MV, Blanch GP, Cifuentes A et al. (2000) Cromato-
grafia y Electroforesis en Columna. Barcelona: Springer-
Verlag Ibérica.
Gonzalez-Llano D, Polo C and Ramos M (1990) Update on
HPLC and FPLC analysis of nitrogen compounds in
dairy products. Lait 70: 255–277.
Gruewedel D and Whitaker JR (1987) Food Analysis. Prin-
ciples and Techniques. New York: Marcel Dekker.
Henschen A, Hupe KP, Lottspeich F and Voelter W (1985)
High Performance Liquid Chromatography in Bio-
chemistry. Weinheim: VCH Press.
Macrae R (1987) HPLC in Food Analysis, 2nd edn.
London: Academic Press.
Moreno-Arribas MV, Bartolomé B, Pueyo E and Polo MC
(1998) Isolation and characterization of individual
peptides from wine. Journal of Agriculture and Food
Chemistry 46: 3422–3425.
Oliver RWA (1989) HPLC of Macromolecules. A Practical
Approach. Oxford: IRL Press.
Shaw LP (1988) Handbook of Sugar Separations in Foods
by HPLC. Boca Raton. FL: CRC Press.
Smith RM (1988) Supercritical Fluid Chromatography.
RSC Chromatography Monographs. Cambridge: Royal
Society of Chemistry.
Gas Chromatography
I Martı́nez-Castro, J Sanz and M V Dabrio,
Instituto de Quı́mica Orgánica General (CSIC), Madrid,
Spain
Copyright 2003, Elsevier Science Ltd. All Rights Reserved.
Background
Gas chromatography was born in 1951, when James 0001
and Martin developed a new separation technique in
order to analyze a mixture of 17 fatty acids. This
technique includes different chromatographic modes
that use a gas as the mobile phase: the separation
process takes place in a chromatographic column.
Like other separation techniques, gas chromatog-
raphy (GC) can be employed on the preparative
scale, but more frequently, it is used as a powerful
analytical technique.
Instrument
The analytical instrument consists of a mobile phase 0002
supply (usually a gas cylinder) regulated by a con-
trol system, a sample introduction system (usually
called injector), a thermostatically controlled oven