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

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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

0016The pump is the system for delivering the solvent

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.

0017Constant-pressure pumps are less expensive and

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.

0018Constant-flow-rate pumps afford the advantage of

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.

0019Reciprocating pumps, also a type of constant-flow-

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

0020Introducing the sample on to the column is one of the

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.

0021Several methods are employed to deliver the

sample on to the column. In on-column injectors the

1276 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.

0026The length of both conventional and microbore

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.

0027Open capillary columns similar to those employed

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 mminsizeand

subsequently heating and drawing the tube to an

internal diameter of 50–125 mm.

0028In addition to the column on which the separation

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.

0029Chromatography is customarily performed at am-

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

0030In order to be suitable for use in HPLC, detectors

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.

0031The detectors most frequently employed are the

refractive index detector, the photometric detector,

and the fluorescence detector.

CHROMATOGRAPHY/High-performance Liquid Chromatography 1277

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

0038The use of HPLC in food analysis is growing daily,

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

0039Nearly all chromatographic modes may be used in

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

0040A variety of chromatographic modes are also applied

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

0041Most separations of amino acids and amines are per-

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

0042Diverse techniques are used to separate peptides on

account of the broad range of molecular weights

1278 CHROMATOGRAPHY/High-performance Liquid Chromatography

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

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-

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

0044In addition to the major groups of food components

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.)

Related Techniques

0045FPLC designates a fast chromatographic method, de-

veloped by Pharmacia, which is similar to HPLC and

yields high resolution. Since FPLC needs only a rela-

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.

Moreover, the mechanical components are resistant

to corrosive buffers, and there is no contamination or

inactivation or the components of interest.

0046Given the range of columns available in the market,

a variety of separation modes can be applied using

this technique: size exclusion, hydrophobic inter-

action, chromatofocusing, ion exchange, and re-

versed-phase chromatography.

0047This method was developed to separate and purify

biomolecules and is very useful in separating isoen-

zymes and molecular species with similar charge char-

acteristics. It is also used to distinguish between

different types of meat or grains.

0048SFC is another technique, related to HPLC, which

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

fluid at a pressure and temperature above the critical

point. The properties of supercritical fluids are inter-

mediate between those of gases and those of liquid.

Thanks to their higher diffusivity and lower viscosity

as compared to liquids, high efficiencies are achiev-

able with shorter analysis times than those customar-

ily employed using HPLC.

0049The basic advantage of SFC with respect to gas

chromatography (GC) is the possibility of analyzing

Absorbance at 214 nm

0.00

0.05

0.10

0.15

10 20

Time (min)(a)

(b)

Time

(

min

)

30 40 50

0.00

0.05

0.10

0.15

10 20 30 40 50

fig0001 Figure 1 Chromatogram of peptides with molecular weights (a)

higher and (b) lower than 700 Da from a sparkling wine. Column:

Nova Pak C

. Solvents: (a) TFA/water; (b) TFA/acetonitrile.

Gradient elution. Detection at 214 nm. Reproduced from Moreno-

Arribas MV, Bartolome

B, Pueyo E and Polo MC (1998) Isolation

and characterization of individual peptides from wine. Journal of

Agriculture and Food Chemistry 46: 3422–3425, with permission.

CHROMATOGRAPHY/High-performance Liquid Chromatography 1279

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 Ibe

rica.

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, Bartolome

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 Martı

nez-Castro, J Sanz and M V Dabrio,

Instituto de Quı

mica Orga

nica General (CSIC), Madrid,

Spain

Background

0001Gas chromatography was born in 1951, when James

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

0002The analytical instrument consists of a mobile phase

supply (usually a gas cylinder) regulated by a con-

trol system, a sample introduction system (usually

called injector), a thermostatically controlled oven

1280 CHROMATOGRAPHY/Gas Chromatography

containing the column, a detector, and a data-acqui-

sition system (Figure 1).

Mobile Phase

0003 The separation process is mainly based on the inter-

action of the analytes with the stationary phase; the

mobile phase is called the ‘carrier gas,’ since its role is

merely to transport the analytes through the column.

Helium, nitrogen, hydrogen, and argon are usually

employed as mobile phases. High-purity gases are

necessary because traces of impurities, such as oxygen

or water, can spoil the stationary phase or disturb the

detector response. Helium is recommended to achieve

faster separations, and nitrogen is a less expensive

alternative, whereas the use of hydrogen requires

special care.

0004 Carrier gas pressure is between 5 and 700 kPa at

the inlet (p

), while the outlet pressure (p

) is usually

ambient pressure. The density, pressure, and velocity

(u) of the carrier gas vary nonlinearly through the

column, and it is necessary to specify at which column

point they are measured or calculated. A factor that

corrects for the mobile phase compressibility in the

column can be calculated as:

j ¼

ðp

 1

ðp

 1

: ð 1Þ

The average linear carrier gas velocity can be

obtained by dividing the column length (L) by the

retention time of an unretained compound:

u ¼ L=t

ð2Þ

is called the ‘hold-up time.’

0005 The flow rate at the column outlet (F

) can be easily

measured and related to the average flow rate

through

F ¼ jF

. It is also related to linear velocity:

F ¼ ku

¼ k

: ð3Þ

The retention time of a compound is defined as the

length of time between sample injection and the emer-

gence of the compound peak maximum. It is related

to the volume of mobile phase entering the column

during this time (V

, retention volume) by the

following equation:

¼ V

: ð4Þ

The net retention volume, V

, takes into account the

compression correction factor and the hold-up time:

¼ jF

ðt

 t

Þ: ð5Þ

The specific retention volume at the column tempera-

ture (V

) is the net retention volume per gram of

stationary phase (W

¼ V

: ð6Þ

The specific retention volume at 0



C is:

273:17

: ð7Þ

The variation of efficiency with the carrier-gas vel-

ocity is described by the Van Deemter equation:

H ¼ A þ

þðC

þ C

Þu; ð8Þ

where C

and C

, respectively, represent the contri-

bution of the stationary and mobile phases to the zone

spreading caused by the resistance to mass transfer

and other diffusion phenomena. When the column is

an open capillary, Golay’s equation can be used:

H ¼

þðC

þ C

Þu: ð9Þ

In most cases, C

is negligible when compared with

Stationary Phase

0006Stationary phase type (solid or liquid) determines

the two main gas chromatographic modes: gas–solid

chromatography (GSC) and gas–liquid chromatog-

raphy (GLC). System selectivity, which depends on

the compounds to be separated and on the stationary

phase, is estimated by the separation factor, a:

 ¼ V

¼ðt

 t

Þ=ðt

 t

Þ: ð10Þ

Solid Phases

0007These consist of adsorbent particles; the separation

mechanism is mainly physical adsorption. Since

fig0001 Figure 1 Scheme of a gas chromatograph. 1, gas cylinder;

2, pressure control system; 3, injector; 4, oven; 5, column; 6,

detector; 7, recorder/data system.

CHROMATOGRAPHY/Gas Chromatography 1281

sorption/desorption processes are usually faster than

those of solution/vaporization (occurring in GLC),

transfer kinetic constants are higher, and C

values

are lower in GSC, allowing better efficiencies. Selec-

tivities can be high enough to separate stereoisomers

and isotopes. Other characteristics of GSC are a wide

temperature operation range, isotherms with a nar-

row linearity range, and high retention values. These

characteristics make GSC the technique of choice for

the separation of permanent gases and small polar

molecules. Solid phases are formed by small particles

of uniform size, with a high specific surface area.

Commercial solid phases are based on silica

(Spherosil, Porasil), graphitized carbon (Carbopack,

Graphpac), or porous organic polymers (Porapak,

Chromosorb Century series, Tenax).

Liquid Phases

0008 These are usually inert and thermally stable poly-

meric molecules. When the stationary phase is a

liquid, the main separation mechanism is partition,

although other mechanisms like chirality, mesomer-

ism, and complex formation may also contribute.

0009 Partition Compounds dissolved in the liquid phase

are in equilibrium (Henry’s law) with the vapor; the

elution order may be proportional to the boiling

points or very different, depending on the activity

coefficients.

0010 Chiral interactions Enantiomer pairs always coelute

when chromatographed on achiral phases. However,

liquid phases possessing chiral groups can show a

greater interaction with one of the pair members,

which is then relatively more retained. As chiral inter-

action energies are small, a values are usually close

to 1, and a high efficiency is required, such as that

provided by capillary columns. Chiral phases are

based on peptides, amide-substituted polysiloxanes,

or substituted cyclodextrins.

0011 Interactions with mesophases (liquid crystals) These

are formed by elongated or planar molecules. In a

temperature range from the melting point to the an-

isotropic transition, they are liquids ordered in a

mono- or bidimensional orientation. These phases

present a high selectivity for long or planar isomers.

0012 Complex formation Organometallic compounds

possessing metal ions are able to interact selectively

with solutes having electron-donor groups.

0013 Characteristics of liquid phases The liquids used as

stationary phases have to be thermally stable over a

wide temperature range. The lower limit is marked by

the melting point, and the so-called maximum allow-

able operation temperature (MAOT) is determined

by the increase in the detector signal caused either

by an appreciable value of vapor pressure or by ther-

mal degradation of the phase: in both cases, some

molecules from the phase arrive continuously at the

detector. The most important characteristic of a sta-

tionary phase is its polarity, which is usually defined

by various empirical scales, the most popular being

that of McReynolds, which uses as probes 10 solutes

with different polar groups and a series of saturated,

linear hydrocarbons. The molecular interactions are

supposed to be additive and are described by several

parameters (McReynolds constants) deduced from

the retention observed in the studied phase and in

squalane, which was defined as the zero on this

polarity scale. Five McReynolds constants, which

appear in most commercial catalogs, are directly

related to phase polarity. Cross-linking improves

the phase stability.

0014Table 1 shows some characteristics of the most

commonly used liquid phases. Hydrocarbons (like

squalane or apolane) are used to obtain reference

data. Polysiloxanes (with methyl, phenyl, trifluoro-

propyl, or cyanoalkyl substituents, showing a wide

polarity range) are commonly called ‘silicones’ and

are nowadays the most popular liquid phases, since

they have a good thermal stability and high permea-

bility to solutes. Polyethylene glycols with different

chain lengths (from a molecular weight of 150 to

around 4.1 10

) are another set of highly used

phases.

Columns

0015Columns are usually classified as open or packed.

Figure 2 shows a cross-section of the main types of

columns.

0016In open columns, the stationary phase is distributed

on the inner wall, and the mobile phase circulates

through a central channel. Packed columns consist

of a glass or metal tube packed with solid particles

(100–250 mm), whose surface adsorbs the analyzed

solutes (GSC) or is covered with a thin film of liquid

stationary phase (GLC). Table 2 lists the main

characteristics of the analytical columns.

0017Packed columns are mainly used for permanent

gases analysis by GSC; they are cheap, robust, and

easy to handle. The most frequently used GLC

columns are open tubular and are usually referred to

as ‘capillary’ since their diameter is very small. Since

the system efficiency depends on the dimensions of

the analytical columns, they should be chosen in

order to achieve the desired resolution in the min-

imum time. Efficiency is always directly related to

1282 CHROMATOGRAPHY/Gas Chromatography

column length, as shown in eqn (2). The efficiency of

a packed column is mainly related to the particle

diameter, which appears in two terms (A and C

)of

the van Deemter equation (eqn (8)). Column diameter

has a large influence on the efficiency of capillary

columns, as Golay’s equation (eqn (9)) shows. Open

tubular capillary columns require more sophisticated

equipment than packed columns, but their efficiency

is very high, since it is possible to use very long

columns with a moderate pressure drop (between

0.1 and 7 kg cm

2

0018Capillary columns with the inner wall covered by a

porous layer open tubular (PLOT, Figure 2)orbya

support-coated open tubular layer (SCOT, Figure 2),

where the stationary phase is coated, have both a high

sample load capacity and a high efficiency.

Operating Conditions

0019The flow rate and column temperature can be easily

modified, enabling the best conditions to be selected

for an adequate separation.

0020The flow rate is usually controlled through either a

pressure regulator or a flow controller. According to

eqn (4), t

is inversely proportional to the flow rate

for a given compound. A linear velocity of carrier gas

close to the van Deemter optimum (eqn (8)) affords

the best efficiency, but higher flow rates reduce the

analysis time.

0021Temperature has a decisive influence on retention.

When the temperature increases, V

and t

decrease

(eqn (7)). The temperature operation should be

selected in such a way that the retention factor (k)

values lie between 1 and 15. When a mixture contains

WCOTPLOT

Packed

SCOTPacked capillary

fig00 02 Figure 2 Cross-sectionofcolumns.PLOT,porouslayeropen

tubular;SCOT,support-coatedopentubular;WCOT,wall-coated

opentubular.AdaptedfromDabrioetal.(2000)Cromatografiay

electroforesisencolumna,p.122.Barcelona:Springerwithper-

mission.

tbl00 01 Table 1 Commonliquidstationaryphases

CompositionCommercialnamesMcReynoldsconstants(120



C)Operating

temperature

range(



XYZUS

2,6,10,15,19,23-Hexamethyl-

tetracosane

Squalane0000020/120

24,24-Diethyl-19,29-dioctadecyl-

heptatetracontane

Apolane-8721103122535/260

Poly(dimethylsiloxane)100%OV-1,SE-30,OV-101,SP-2100,CP-Sil5CB,

DB-1,SPB-1,BP-1

165544654230/330

Poly(94%methyl,5%phenyl,1%

vinylsiloxane)

SE-54,DB-5,CP-Sil8CB,SPB-5,BP-5,AT-5337266986750/300

Poly(86%methyl,7%phenyl,7%

cyanopropylsiloxane)

OV-1701,CP-Sil19CB,BP-10,DB-1701,

HP-17

8217015723616030/250

Poly(50%methyl,50%

phenylsiloxane)

OV-17,DB-17,SP-2250,HP-50,SPB-5011915816224320220/350

Poly(50%methyl,50%

trifluoropropylsiloxane)

OV-210,SP-2401,DB-21014623835846831020/275

Poly(50%methyl,25%phenyl,

25%cyanopropylsiloxane)

OV-225,XE-60,DB-225,CP-Sil43CB,BP-1522836933849238620/250

Poly (ethylene glycol) Carbowax, Superox, Supelcowax, DB-Wax,

CP-Wax 52CB, BP-20

322 536 368 572 510 60/225

Poly (ethylene glycol) modified

with nitroterephthalic acid

FFAP, SP-1000, AT-1000, OV-351, Nukol,

CP-Wax 58CB, BP-21

340 580 597 602 627 50/250

Poly(biscyanopropyl siloxane) OV-275, CP-Sil 88, SP-2340, Silar 10C 629 872 763 1106 849 30/250

CHROMATOGRAPHY/Gas Chromatography 1283

compounds with very different volatilities, it is not

possible to find an optimum temperature for separat-

ing all components. Since the oven temperature is

easily controlled, it can be changed during the

chromatographic run (‘programmed’) in such a way

that each compound traverses the column within an

optimum temperature range.

Sample Introduction

0022 The objective of injection is to introduce the sample

as a narrow band, whose composition should corres-

pond closely to that of the original mixture. No uni-

versal system is available to handle all the different

samples encountered. Permanent gases may be intro-

duced with gas-tight syringes or using multiport

valves. Liquid samples and solutions are usually

introduced by microsyringe at the injection port,

which is a heated chamber where the carrier gas

enters under controlled flow or pressure. The sample

is vaporized and transferred to the column inlet by the

carrier gas stream. A schematic diagram of an injector

is shown in Figure 3.

0023The chamber is provided with an inert glass or

quartz liner to minimize sample contact with hot

metallic surfaces. It is closed with a mechanical seal

or, more usually, with an elastomer membrane called

an septum, with can be pierced with a microsyringe to

introduce the sample. Depending on the column

dimensions, the quantity of sample can be varied

from nanoliters to a few microliters, containing nano-

grams or micrograms of analytes. There are special

devices capable of transferring several hundreds of

microliters of very diluted samples, the solvent excess

being discarded before it enters the analytical column.

Injection Modes

0024There are several different ways of injecting samples.

The more common modes are detailed below.

0025Direct injection The sample is introduced through

the septum in the hot injector, where the liquid

Septum

(a)

Liner

Carrier gas

Heater

Column

(b)

Carrier gas

Septum

purge

Splitter

Column

fig0003 Figure 3 Scheme of an injector. a) for direct injection; b) for split/splitless injection. Adapted from Dabrio MV et al. (2000) Cromato-

grafia y electroforesis en columna, p. 130. Barcelona: Springer with permission.

tbl0002 Table 2 Some characteristics of GC columns

Type Name Internaldiameter (mm) Length (m) d

(mm) Particle size (mm)

Open WCOT (Wall-coated open tubular) 0.05–0.75 10–100 0.1–0.5 –

PLOT (Porous layer open tubular) 0.5–1 10–100 0.1–5

–

SCOT (Support-coated open tubular) 0.5–1 10–100 0.1–5

–

Packed Micropacked 0.8–1 2–7 5–15

80–200

Packed 2–6 2–7 5–15

80–500

Bed thickness (particles þliquid phase).

percentage of liquid phase (referred to support þphase).

1284 CHROMATOGRAPHY/Gas Chromatography

rapidly vaporizes; carrier gas sweeps the vapor into

the column inlet.

0026 Split injection This was designed for the introduc-

tion of samples into capillary columns. The basic

design used for direct injection was modified as

shown in Figure 3. The carrier gas, controlled by a

pressure regulator or a combination of a flow con-

troller and a back-pressure regulator, is divided into

two streams: one is used to eliminate the possible

impurities from the septum membrane; the other

enters the vaporization chamber. This gas stream,

after sweeping the vaporized sample, is split before

it enters the column inlet to attain a high flow rate.

The sample is also proportionally divided, so only a

small part reaches the column. Since quantitative

analysis presents some problems related to the split

process, the use of internal standards is recom-

mended.

0027 Splitless injection When very dilute solutions need

to be injected, the amount of sample entering the

column can be increased, stopping the split process

by using a solenoid valve. The split valve is closed, the

sample is introduced, and after a controlled delay

(usually 15–60 s), the split valve is opened again.

Most of the sample enters the column while the split

is closed, while the rest is purged out when the valve

opens. Since the sample enters the column as a broad

band, it is a necessary focusing step. Usually, the

column is kept at a low temperature during injection,

and sample vapors condense at the column inlet in a

narrow band (cryofocusing); next, the temperature is

raised in order to elute the solutes correctly. Sample

solvent condensation can also be used (solvent effect)

to retain the solutes as a narrow band at the column

inlet.

0028 Programmed temperature vaporizer (PTV) With

the same basic design of a split/splitless detector, it

can be heated and cooled very fast. The injector is

cold when the sample is introduced and deposited on

inert packing; then, the injector is heated to the de-

sired temperature so that the vaporization is homo-

geneous, and sample discrimination is reduced.

0029 On-column injection A liquid sample is deposited

directly at the column head, without previous vapor-

ization. The injectors are very simple, having a

mechanical stop valve instead of a septum, and the

microsyringes usually have very thin needles (less

than 0.3 mm outer diameter). This system reduces

the discrimination, thermal decomposition, and deg-

radation of labile compounds. Its main drawback is

possible column contamination with nonvolatile

sample impurities.

External Sampling Devices

0030Pyrolysis A solid sample is thermally decomposed;

the volatile fragments resulting from its breakdown

can be chromatographically separated and detected,

and their chromatographic pattern related to that of

the original sample.

0031Headspace sampling The sample is thermostatized

in a closed vial, and the vapor is sampled after equi-

librium is attained. Usually, the process is automatic,

under either atmospheric or high-pressure conditions,

and the technique is frequently used for aroma

analysis.

0032Purge and trap A stream of inert gas passes over or

through the sample, and the stripped volatiles are

trapped on a solid sorbent. These can be recovered

either by a solvent or by thermal desorption (see

below). The enrichment factors obtained are very

high; matrix interferences are largely reduced. This

technique is useful when very low concentrations of

volatile substances have to be analyzed.

0033Thermal desorption The injector chamber can be

ballistically heated to the desired temperature while

it is purged with carrier gas. When a solid matrix is

placed in this chamber, the volatile substances present

are released and stripped towards the column; they

can then be cryofocused before arriving at the column

inlet. In the direct mode (DTD, direct thermal desorp-

tion), the solid matrix is a sample (e.g., a spice or a

herb). In the indirect mode, volatiles have been previ-

ously stripped from the sample and trapped on an

adsorbent.

Multidimensional Gas Chromatography

0034This usually refers to a combination of columns with

different selectivity, where a small fraction from a

complex sample is preseparated on the first column

and introduced into the second column for further

separation. Whereas the first column may be packed

or may be a capillary, the second column is always a

capillary and more selective. A special pneumatic

switching arrangement is necessary in order to

provide a sharp sample band.

Detectors

0035A high number of detectors have been designed

for GC. The most frequently used detectors can be

classified into four types.

CHROMATOGRAPHY/Gas Chromatography 1285