Helmig D (1999) Review: air analysis by gas chro-

matography. Journal of Chromatography, A 843:
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In Townshend A (ed.), Encyclopedia of Analytical
Science, 1st ed. London: Academic Press.

Molina MJ and Rowland FS (1974) Stratospheric sink for

chlorofluoromethanes: chlorine atom catalysed destruc-
tion of ozone. Nature 249: 810–812.

Montzka SA, Butler JH, Myers RC, Thompson TM,

Swanson TH, Clarke AD, Lock LT, and Elkins JW
(1996) Decline in the tropospheric abundance of halogen
from halocarbons: implications for stratospheric ozone
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Oram et al. (1995) Geophysics Research Letters 22:

2741–2744.

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Prinn RG, Weiss RF, Fraser PJ, Simmonds PG, Cunnold

DM, Alyea FN, O’Doherty S, Salameh P, Miller BR,
Huang J, Wang RHJ, Hartley DE, Harth C, Steele LP,
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history of chemically and radiatively important gases in
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CHROMATOGRAPHY

Contents

Overview

Principles

Multidimensional Techniques

Overview

V R Meyer

, EMPA St Gallen, St Gallen, Switzerland

& 2005, Elsevier Ltd. All Rights Reserved.

This article is reproduced from the first edition, pp. 720–729,
& 1995, Elsevier Ltd., with revisions made by the Author.

Introduction

Chromatography can be used to solve a very broad
range of analytical problems. This versatility is re-
flected in the large number of chromatographic tech-
niques that are successfully applied today. They can
be classified according to a number of criteria, the
most important of which is the type of mobile phase
used. Subsequently the shape of the chromatographic
bed and the properties of the stationary phase
expand the possibilities offered by chromatography.
Once a particular method is chosen, it is possible to
influence the separation using programmed elution.
Finally special techniques can be used to perform
difficult analyses or to obtain short separation times.
The individual methods are discussed in detail in

their respective articles, so here only a short intro-
duction is given.

Classification of Chromatographic
Techniques

The Type of Mobile Phase

Chromatographic techniques can be classified on the
basis of a number of different criteria. Nevertheless,
a logical hierarchy is given by first specifying the type
of mobile phase, second the shape of the chro-
matographic bed and third the type of stationary
phase. In Table 1 the chromatographic methods are
listed according to this scheme. The type of mobile
phase is the most important criterion because it de-
termines the class of samples that can be analyzed
with one of the associated techniques. (From a the-
oretical point of view, each type of mobile phase also
governs a certain range of diffusion coefficients,
which determines and limits the speed of analysis
and the efficiency of the method.) Therefore a dis-
tinction can be made between gas, supercritical fluid,
and liquid chromatography (GC, SFC, and LC,
respectively).

CHROMATOGRAPHY

/ Overview

89

SFC is less important than GC or LC. In many

cases, it is not difficult to choose between the latter
two methods: the prerequisite for GC is that the
analyte is volatile and thermally stable (although
derivatization in order to obtain these properties is
possible in many cases).

The Shape of the Chromatographic Bed

If the mobile phase is a gas or a supercritical fluid, it
is necessary to let it flow through a tube, a so-called
column, that contains the stationary phase. In the
case of liquid chromatography one can choose
between a column or planar geometry because the
mobile phase can move through a sheet of paper or a
thin layer by capillary action. If a column is used, the
mobile phase is forced through it by pressure gene-
rated by a pump or by a gas stored in a pressurized
cylinder. (As a preparative laboratory technique, liq-
uid chromatography is also performed in columns
packed with coarse stationary phases; in this case
simple hydrostatic pressure may be sufficient.)

The column can be an open capillary or a packed

tube. In the first case the mobile phase is coated as a
thin film on the inner wall of the capillary. If the
mobile phase has a certain solvating power, as in
SFC, it is necessary to cross-link this liquid film,
whereas in GC linear polymers are used in many
cases because the usual carrier gases, helium and
hydrogen, cannot dissolve any stationary phase.
(Owing to problems with manufacturing and

instrumentation, liquid chromatography with open
capillaries is only of theoretical interest.) If the column
contains packing, many possibilities are offered by
contemporary technology. The stationary phase can be
an inorganic adsorbent, a cross-linked and thereby
rigid organic polymer, an inorganic or organic material
with chemically modified surface, or even a liquid film
coated on a granular carrier material.

Figure 1 shows the three possibilities: packed col-

umn, open capillary, and plane.

Terminology of the Methods

Taking the type of chromatographic bed and sta-
tionary phase into account, GC, SFC, and LC can

Type of mobile phase

Shape of chromatographic

bed

Type of stationary phase

Method

Abbreviation

Type of method

Gas

Column

Open tubular

Open tubular

Packed

Packed

Liquid,
cross-linked
liquid

Cross-linked
liquid

Liquid

Solid

Gas-liquid
chromatography

Column liquid
chromatography

Paper
chromatography

Thin-layer
chromatography

Gas-solid
chromatography

Capillary
supercritical
fluid
chromatography

Packed column
supercritical fluid
chromatography

Column

Supercritical fluid

GC.
GLC

GC.
GSC

SFC

Adsorption,
molecular sieve,
porous polymer

Adsorption,
bonded phase

Adsorption,
reversed phase,
bonded phase,
ion exchange,
affinity,
size exclusion

Adsorption,
reversed phase,
bonded phase

LC
HPLC

a

TLC
HPTLC

b

PC

Solid

Liquid

Column

Plane

(Open tubular)

Packed

Paper

Thin-layer

Solid

Solid

Liquid

a

High-performance liquid chromatography.

b

High-performance thin-layer chromatography.

The high-performance methods use stationary phases of very small particle diameter.

Table 1

Classification of chromatographic methods

(A)

(B)

(C)

Figure 1

Three possibilities for creating a chromatographic

bed: (A) packed column, (B) open capillary, and (C) plane. The
third technique can only be used with a liquid mobile phase; here
a thin-layer plate is drawn but the plane can also consist of a
sheet of paper. In all three cases the stationary phase is shown in
gray. The particles of the packed column can be round, as in the
figure, or irregular.

90

CHROMATOGRAPHY

/ Overview

now be subdivided, although the usual terms do not
follow the same criteria in all cases. Table 1 uses the
expressions gas–liquid, gas–solid, capillary supercrit-
ical fluid, packed-column supercritical fluid, column
liquid, paper, and thin-layer chromatography. The
following abbreviations are used: GLC, GSC, but in
most cases the type of stationary phase is omitted
and both techniques are termed GC; SFC; LC (usu-
ally only used for column techniques, although thin-
layer and paper chromatography are also ‘LC’); PC
(sometimes also used for ‘preparative chromatogra-
phy’); and TLC. For LC and TLC, which both use a
granular stationary phase, a special term was intro-
duced to distinguish the more recent instrumental
methods based on very fine stationary phases from
the classical ones: HPLC and HPTLC where HP is
for ‘high performance’. Here the particle diameter is
not larger than

B10 mm, which is the key to obtain-

ing high plate numbers per unit length.

Especially in LC, and also in other fields, it is usual

to distinguish in more detail between very different
types of method. This will be discussed below.

Comparison of the Methods

Table 2 lists some characteristic features of GC, SFC,
and LC. In most cases GC is used as open-tubular
GLC, and LC is performed in packed columns. As
can be seen from the physical parameters of density,
viscosity, and diffusion coefficient, SFC lies between
GC and LC and it is no surprise that it can be used
equally well with open capillaries and packed col-
umns. The values in the table are to some extent
arbitrary but are typical. The values of the three basic
physical parameters are not only of theoretical
interest but are linked directly to some of the main

properties of the methods: the density governs the
solvating power of the mobile phase and thereby de-
termines whether the separation can be influenced by
a particular choice of eluent; the viscosity influences
the pressure needed to force the mobile phase
through the chromatographic bed and sets the
upper limit of the solute diffusion coefficient. This
latter should be high because low diffusivity means
slow mass transfer and therefore slow chromatography.
If the sample diffusion coefficient is low, it is neces-
sary to keep low the characteristic chromatographic
dimension, i.e., the capillary or particle diameter.
Therefore, LC with 5 mm particles is more efficient
than with a 100 mm packing.

Practical aspects of the three methods are listed

under Variables and Sample Prerequisites at the end
of Table 2.

Gas Chromatography

If the mobile phase is a gas, the sample needs to be
volatile. Its boiling point at atmospheric pressure
should not be higher than

B3601C. If the tempera-

ture of the GC column or capillary is adequate, the
sample molecules will be transported by the gas
owing to their volatility. Retention is governed by
both vapor pressure and affinity to the stationary
phase of a given compound. The gaseous mobile
phase has no direct influence on the separation.
GC can be a simple and rapid technique and is the
method of choice for the investigation of volatile and
even very complex samples. An example is given in
Figure 2.

The most frequently used mobile phases for GC

are hydrogen and helium. The lower the molecular

Table 2

Comparison of analytical column-type chromatographic methods

GC (open-tubular GLC)

SFC

LC

Density of mobile phase (g ml

1

)

10

3

0.5

1

Viscosity of mobile phase (poise)

10

4

10

3

10

2

Diffusion coefficient of solute in mobile phase (m

2

s

1

)

10

5

10

7

10

9

Diameter of capillary (mm)

320

100

Diameter of packing (mm)

5

5

Length of column (m)

25

25, 0.25

a

0.1

Number of theoretical plates (m

1

)

3 000

3 000, 50 000

a

50 000

Number of theoretical plates per column

75 000

75 000, 12 000

a

5 000

Pressure drop (bar)

1

Variable

b

100

Variables

Stationary phase

Stationary phase

Stationary phase

Temperature

Mobile phase

Mobile phase

Temperature

(Temperature)

Pressure

Sample prerequisites

Volatility

Solubility

Solubility

Thermal stability

(Thermal stability)

a

For open tubular and packed columns, respectively.

b

In SFC the pressure drop over the column can be chosen.

CHROMATOGRAPHY

/ Overview

91

mass of a gas, the lower its own diffusivity as well as
the diffusivity of the sample molecules and the faster
the chromatography. Therefore, hydrogen would be
the favored carrier gas but it is often barred on safety
grounds. Sometimes nitrogen is used because it is
cheap but this can only be recommended for simple
analytical problems because the separation perform-
ance is poorer than with gases of low molecular
mass. The fact that some detectors demand the use of
a certain gas must also be taken into consideration.

A typical stationary phase for GC is a viscous liq-

uid with low vapor pressure (at the temperature re-
quired for a given range of application). The two
most important types of stationary phases are sili-
cones and polyglycols; their structures are given in
Table 3. The silicones especially can be substantially
chemically modified in order to obtain a wide range
of polarities and specialized functionalities (including
chiral groups). The stationary phase is coated as a
thin film (typically 0.25 mm) on the inner wall of the
open capillary or on the surface of a granular,
porous, inert packing material, in this case called a
solid support. For special types of analyses the sta-
tionary phase is not a liquid but a porous–solid
packing. Adsorbents (silica), molecular sieves and
porous polymers are used for the GSC of highly
volatile samples such as mixtures of permanent gases
or low-molecular-mass hydrocarbons.

In GC, the eluted compounds are most often de-

tected with a flame-type detector that generates ions,
the so-called flame ionization detector, FID; for
special purposes nitrogen- and phosphorus-sensitive
FIDs, electron capture, or thermal conductive detec-
tors and mass spectrometers are used.

If a sample is not volatile, several derivatization

techniques are known that allow reduction in the
boiling points of certain classes of compounds.

Alcohols, amines, amino acids, carboxylic acids, car-
bohydrates, and steroids can be trimethylsilylated;
amines, phenols, carbohydrates, and steroids can
be acylated with trifluoroacetic acid or a higher
homolog; carbonic acids and phenols can be alkylated.

Liquid Chromatography

Liquid chromatography has a number of different
configurations with regard to technical (instrumen-
tal) as well as separation modes. Paper, thin-layer,
and classical column techniques all belong to liquid
chromatography and the ‘high performance’ tech-
nique especially (though to a lesser extent the other
methods also) offers a great variety of separation
principles.

Paper Chromatography

The simplest and cheapest technique is paper chro-
matography, where the chromatographic bed con-
sists of a sheet of paper, i.e., cellulose. The stationary
phase consists of water adsorbed to the cellulose as
well as of the polymer itself, although ion exchange
and complexation processes may play an important
role. The sample solution is applied as a spot near
one end of the paper. A few centimeters of the sheet
are dipped into the mobile phase which then ascends
(or descends, as descending mode is also possible)
into the stationary phase. When the mobile phase has
almost reached the other end of the sheet the paper is
removed from the developing tank and dried. If the
analytes are not visible because they are not colored,
the sheet is treated with a reagent to visualize the
spots.

0

10

20

30

40

50

60

70

Time (min)

1

2

3

4

7

5

6

10

9

8

12

13

11

14

15

19

18

16

21

20 22 25

23

24

26

2730

31

28 29

33

32

34

36

41

45

49

51

43
42

38

3740

39

46

48

44

47

50 52

53

55

56

54

57

58

60

59

61

64

67

70

69

68

65

65

66

62

63

71

72

7374

75

78

79

76 77

80

81

82

84

83

85

86 87

88

89

90

94

91
92

9396

97

98

95

99

104

103

105

106

108

109

112

111

110

115

107

Figure 2

Gas chromatographic separation of hydrocarbons found in an urban air sample. Open capillary, 0.32 mm i.d.

 60 m length;

stationary phase, DB-1 (dimethyl polysiloxane); film thickness, 0.25 mm; carrier gas, helium; temperature programme, 5

1C isothermal

for 3 min, 5–50

1C at a rate of 31C min

1

, 50–220

1C at a rate of 51C min

1

; detector, flame ionization. With this method, a total of 142

hydrocarbons could be separated and identified; 128 of them were found in the urban air sample. (After Ciccioli P, Cecinato A,
Brancaleoni E, Frattoni M, and Liberti A (1992) Use of carbon adsorption traps combined with high resolution GC–MS for the analysis
of polar and nonpolar C4–C14 hydrocarbons involved in photochemical smog formation. Journal of High Resolution Chromatography
15: 75.)

92

CHROMATOGRAPHY

/ Overview

Thin-Layer Chromatography

Thin-layer chromatography is more versatile than
paper chromatography since a number of different
stationary phases are available such as silica,
derivatized silica, or cellulose (the analogue to pa-
per); also, developing times are much shorter. An
immense number of spray reagents have been pub-
lished that allow detection of any type of analyte.
HPTLC is the ‘high-performance’ version of TLC
and uses 10 mm or 5 mm stationary phase particles.
The separation performance of these plates is higher,
but to take full advantage it is necessary to use in-
strumentation for sample application, development,
and detection. As an example of TLC, Figure 3
presents the separation of ten rare earths.

Liquid Chromatography

Whereas chromatography in open columns is mainly
used for preparative purposes, the analytical technique

is LC using microparticulate packings. Under
these circumstances it is necessary to use a pump
for mobile phase transport and a detector for the
observation of the fractions (usually the concentra-
tion of the analytes in the eluate is low), e.g. UV
absorbance, fluorescence, refractive index, or elect-
rochemical detectors according to the properties of
the analytes. It is also possible to derivatize the sam-
ple prior to or after the separation. Precolumn
derivatization can be performed offline or online;
postcolumn derivatization is usually carried out on-
line. An example of LC is given in Figure 4, which
shows the separation of the three stereoisomers of
mivacurium, a neuromuscular blocking agent, in a
plasma extract.

Liquid Chromatographic Separation Principles

Liquid chromatography can be performed in a
variety of modes; the most important ones are

Table 3

Important stationary phases for GLC and (HP)LC

GC

Silicones

Polyglycols

R

R'

Si O

O

n

n

CH

2

CH

2

OH

(

(

With the proper choice of R and R' a
wide range of polarities and special
functionalities is available

Polar stationary phase:
n ranges from 4 to 800

LC
Silica

Octadecyl silica

Octyl silica

Diol silica

Nitrile silica

Amino silica

Polystyrene

Strong cation exchanger
Weak cation exchanger
Strong anion exchanger
Weak anion exchanger

(SiO

2

)

n

(SiO

2

)

n

(SiO

2

)

n

(SiO

2

)

n

CH

2

CHOH

CH

2

OH

C

18

H

37

C

8

H

17

Si

Si

Si

CH

2

CH

2

CN

Si

(SiO

2

)

n

CH

2

CH

2

NH

2

Si

(SiO

2

)

n

Si

OH

SO

3

−

H

+

NR

3

+

OH

−

NH

3

+

OH

−

COO

−

H

+

Three-dimensional network

Reversed phases

Polar bonded phases

Three-dimensional network due to
cross-linking with divinylbenzene

Can be silica or polystyrene

}

}

}

(CH-CH

2

)

n

CHROMATOGRAPHY

/ Overview

93

presented briefly. Schematic drawings are shown in
Figure 5, and Table 3 also lists some stationary
phases used in LC.

Adsorption chromatography

The stationary phase

is a polar adsorbent, in most cases silica. The mobile
phase is nonpolar (usually a solvent with polarity
within the range from hexane to esters). It competes
with the sample molecules for adsorption at the
active sites of the stationary phase. Nonpolar com-
pounds are eluted first, followed by solutes of inc-
reasing polarity. Steric properties of the sample
compounds can play an important role and there-
fore adsorption chromatography is the method of
choice for the separation of many classes of isomers.

Reversed-phase

chromatography

The stationary

phase here is nonpolar; in most cases it is derivati-
zed silica that carries C

18

(i.e., C

18

H

37

) or C

8

(i.e.,

C

8

H

17

) groups. The mobile phase is polar, in most

cases a mixture of water (or buffer solution) with
methanol, acetonitrile, or tetrahydrofuran. Such an
eluent cannot wet the surface of the stationary phase
and the solutes are retained owing to an energy gain

that comes from the decrease in contact area between
the two phases as long as the sample molecules ad-
here to the hydrocarbon chains. Polar analytes are
eluted first and homologs will be retained more
strongly the longer their chain length. Ionic com-
pounds can be separated on reversed phases if a
neutral ion-pair is formed by the addition of a coun-
ter-ion to the eluent. This was carried out in the sep-
aration shown in Figure 4: mivacurium is a
quaternary amine, and therefore a sulfonic acid was
added as an agent to mask its charge.

Other bonded phases on silica (not illustrated in
Figure 5)

Besides the nonpolar hydrocarbons, other

functional groups can also be bonded to silica. Im-
portant stationary phases are diol, nitrile, amino (see
Table 3), and a great number of special functionali-
ties including chiral groups. The retention mecha-
nisms are as variable as the stationary phases and are
not known in some cases.

Ion

exchange

chromatography

Ion

exchange

groups can be bonded to silica or to polystyrene.
‘Classical’ ion exchange is based on ionic equilibria
between solute, buffer and stationary phase ions and
counter-ions. Besides this ion exclusion mechanisms
can also be utilized and special types of ion ex-
changers have been developed for the separation of
the ions of strong acids and bases.

Size exclusion chromatography

If the mobile phase

has a good affinity for both the sample molecules and
the stationary phase and if the latter has a well-de-
fined pore structure, such a chromatographic system
will separate the solutes according to their size. They
will not be retained by the column packing but will
enter the pores where the mobile phase is stagnant.
Large molecules can utilize a smaller fraction of the
pore volume than small ones and will be eluted ear-
lier. Molecules that are too large to enter the pores
are excluded and will appear as the first fraction at
the column end.

Affinity chromatography

The stationary-phase ma-

trix can be loaded with chemically bonded, biologic-
ally active groups such as enzymes or antibodies. If a
complex sample is injected into an affinity column,
only those molecules will be retained that bind to the
ligands; in the cases mentioned above these will be
substrates or antigens. All other compounds will be
swept away by the mobile phase. Afterwards the re-
tained molecules can be eluted by switching to a
specially designed mobile phase (e.g., change of pH
or ionic strength). Affinity chromatography is a

Er

Ho

Tb

Gd

Eu

Nd

Sm

Pr
Ce

La

Figure 3

High-performance thin-layer chromatographic sepa-

ration of ten rare earths (as nitrates). Sample, 1 mg each of rare
earth; layer, silica, impregnated with ammonium nitrate prior to
the separation; mobile phase, 4-methyl-2-pentanone/terahydrofu-
ran/nitric acid/2-ethylhexylphosphonic acid mono-2-ethyl hexyl-
ester 3:1.5:0.46:0.46; developing distance, 5 cm; detection
reagent, (1) spray of saturated alizarin solution in ethanol, (2)
ammonia vapour, (3) gentle heating. (After Wang QS and Fan DP
(1991) Journal of Chromatography 587: 359.)

94

CHROMATOGRAPHY

/ Overview

highly selective method and works by an ‘on–off
switching’ mechanism.

Supercritical Fluid Chromatography

The phase diagram of a pure compound shows not
only regions of the solid, liquid, and gaseous states,
the equilibrium lines and the triple point, but also the
critical point. If pressure and temperature exceed the
critical values, the compound will be neither a liquid
nor a gas, nor will the two phases coexist, but a
supercritical fluid exists. This phase is denser and
more viscous than a gas without attaining the prop-
erties of a liquid as shown in Table 2. The advantage
of SFC over LC lies in the higher diffusion coefficient,
which allows faster separations; in comparison to
GC the mobile phase has a large solvating power and
thus influences selectivity.

In most SFC separations carbon dioxide is used as

the mobile phase; often a modifier (of polarity) such
as methanol, other alcohols or water is added. The
critical data for CO

2

are 31.3

1C and 72.9 bar, values

that can easily be handled by instrumental chro-
matography. To keep the column outlet under criti-
cal conditions, a restrictor (a device with a high

resistance to the eluent flow) needs to be installed
after or at the outlet of the column.

Owing to its intermediate position between GC

and LC, SFC can be performed equally well in open
capillaries and packed columns. The separation can
be influenced by the type of stationary phase and of
modifier, by pressure, pressure drop, and tempera-
ture. In contrast to GC, SFC can also be used for the
separation of nonvolatile or thermally labile com-
pounds (although some temperature compatibility is
necessary). The separation of enantiomers on chiral
stationary phases can be very attractive because the
temperature is lower than in GC, which increases the
separation factors. SFC is an alternative to normal-
phase LC because it is fast and carbon dioxide is
ecologically sound. An example of an SFC separation
can be found in the previous article, Principles, where
Figure 2 shows the separation of orange oil compo-
nents.

Special Chromatographic Techniques

Preparative Methods

Chromatography can equally well be used for ana-
lytical and preparative purposes. ‘Preparative’ is not

+

+

CH

3

O

CH

3

O

OCH

3

OCH

3

CH

2

CH

3

CH

3

O

O

1 2

O

O

O

O

2CI

−

H

3

C

CH

3

O

CH

2

OCH

3

OCH

3

OCH

3

OCH

3

N

0

5

10

Time (min)

15

2

1

3

4

Figure 4

Liquid chromatographic separation of mivacurium stereoisomers in human plasma extract. The drug is a mixture of three

isomers; the structure is drawn without stereochemical preference. Column, 4.6 mm i.d.

 12.5 cm length; stationary phase,

LiChrospher 60 RP (reversed-phase) select B, 5 mm; mobile phase, acetonitrile/water 40:60 with 0.005 mol l

1

octanesulfonic acid (as

ion-pair reagent), 1 ml min

1

; detector, fluorescence 202/320 nm. Peaks: (1) is the trans–trans isomer (1R, 1

0

R, 2S, 2

0

S); (2) is the cis–

trans isomer (1R, 1

0

R, 2R, 2

0

S); (3) is the cis–cis isomer (1R, 1

0

R, 2R, 2

0

R); (4) is the internal standard, the trans–trans analog of

mivacurium with a benzene ring instead of the double bond in the middle of the molecule. (After Brown AR, James CD, Welch RM, and
Harrelson JC (1992) Stereoselective HPLC assay with fluorometric detection for the isomers of mivacurium in human plasma. Journal
of Chromatography 578: 302.)

CHROMATOGRAPHY

/ Overview

95

reserved to the fractionation of large samples, but
indicates that the separated compounds are collected
and used for a subsequent purpose: identification or
structure elucidation, chemical modification by syn-
thetic methods, use as a reference material, determi-
nation of chemical or biological properties, or for
sale. If only small amounts of material are needed,
the only difference from analytical chromatography
lies in the use of a fraction collector; for routine sep-
arations it should be computer controlled.

If the sample size is increased, the shape of the

peaks changes to rectangular (in the case of volume
overload) or triangular (with mass overload); mixed
forms and distorted peak shapes are also observed.
Displacement effects can occur where a compound is
‘pushed’ and concentrated by a following one that
has a stronger affinity to the stationary phase.

If large samples need to be separated, the diameter,

and also often the length, of the column are in-
creased. (Obviously, open capillaries cannot be used
for this purpose.) Preparative GC is an attractive
approach (though the fraction collector needs to be
cooled) but few commercial instruments are avail-
able. Preparative LC is the most important techni-
que in organic synthesis, biochemical research,
downstream processing in biotechnology, and for
the commercial preparation of certain chemicals or
drugs.

Programmed Elution

In a complex sample the individual analytes often
have very different retention factors in a given chro-
matographic system. It is therefore not possible to

Figure 5

Separation principles of liquid chromatography. (A) Adsorption chromatography: the adsorptive sites of the stationary

phase are symbolized by A; the solute molecules interact with their polar groups X or Y; the mobile phase drawn is hexane, which can
also interact weakly with A. (B) Reversed-phase chromatography: the solute molecules interact via their nonpolar groups with the
nonpolar stationary phase. (C) Ion-exchange chromatography: a styrene-divinylbenzene type cation exchanger is shown; sample ions
S

þ

and buffer cations K

þ

compete for interaction with the exchange sites. (D) Size exclusion chromatography: sample molecules can

occupy the pore volume according to their size, therefore the macromolecule will spend less time in the pores and elute first. (E) Affinity
chromatography: only certain sample molecules can fit to the ligands of the stationary phase, the others are washed out.

96

CHROMATOGRAPHY

/ Overview

separate and elute them efficiently without changing
the properties of the system, i.e. under so-called iso-
thermal (GC) or isocratic (LC) conditions. In this
case a GC separation is started at relatively low
temperature, an LC separation at low eluting power
of the mobile phase. Subsequently the temperature or
mobile phase strength is increased in order to elute
compounds that were strongly retained under the
initial conditions. In GC this technique is called a
temperature program (see Figure 2); the correspond-
ing LC term is gradient elution. Note that in normal-
phase LC the polarity of the mobile phase needs to be
increased (however, gradient elution on silica is al-
most never performed because steep gradients are
not possible and it takes a long time to re-equilibrate
the column after the separation), whereas in
reversed-phase LC the eluent polarity is decreased.
A gradient from 10 to 100% acetonitrile in water can
separate a very broad range of compounds on a
reversed-phase column; pH or ionic strength gradi-
ents are also possible. In SFC, mobile phase, pres-
sure, and temperature gradients are of equal
importance.

Column Switching

An alternative to programmed elution can be the
coupling of two (or even more) columns with differ-
ent stationary phases. This technique is known as
multidimensional chromatography. The first column,
for example, will separate the sample according to
polarity groups. Then selected fractions are switched
online to the second column, where the fine separa-
tion into chemically pure compounds takes place.

It is even possible to couple LC and GC; here, LC

plays the role of a sample preparation technique that
eliminates compounds that would affect the gas
chromatographic separation. Because GC cannot tol-
erate high volumes of liquid, it is necessary to use
narrow-bore LC columns, to split the eluate, or to
use a special interface that eliminates most of the
liquid.

Special GC Techniques

Headspace analysis

For the investigation of the

volatile ingredients of complex mixtures, e.g., of
olfactory principles, the sample is stored in a closed
vial and perhaps gently heated. A portion of the
vapor that fills the space over the solid or liquid
sample is collected by a syringe and injected into the
gas chromatograph. To obtain reproducible results it
is necessary to control storage temperature and time
strictly.

Thermal desorption

Volatile compounds in gases

such as pollutants in air can be trapped in a small
adsorption tube, either by pumping the gas through
or by passive diffusion. The packing in the trap can
be chosen from a wide variety of adsorbents (molec-
ular sieves, graphitized carbon blacks, organic poly-
mers). After sample collection the adsorption tube is
rapidly heated in a stream of purge gas which trans-
ports the released analytes to the GC column where
the separation runs.

Pyrolysis chromatography

For the GC analysis of

high-molecular-mass samples such as plastics or
wood, the sample can be pyrolyzed (heated until
breakdown into smaller molecules occurs) online
prior to injection. A ‘fingerprint’ of the material is
obtained that can be used for quality control or
identification purposes.

See also: Chromatography: Principles. Gas Chro-
matography: Column Technology; Pyrolysis; Detectors.
Headspace Analysis: Static; Purge and Trap. Ion
Exchange: Overview. Liquid Chromatography: Over-
view; Ion Pair; Size-Exclusion. Supercritical Fluid
Chromatography: Overview; Applications. Thin-Layer
Chromatography: Overview.

Further Reading

Anton K and Berger C (eds.) (1997) Supercritical Fluid

Chromatography with Packed Columns: Techniques and
Applications. New York and Basel: Dekker.

Fowlis IA (1995) Gas Chromatography. Chichester: Wiley.
Fried B and Sherma J (1999) Thin-Layer Chromatography.

New York and Basel: Dekker.

Grant DW (1996) Capillary Gas Chromatography. Chich-

ester: Wiley.

Grob RL and Barry EF (1995) Modern Practice of Gas

Chromatography, 4th edn. Chichester: Wiley.

Guiochon G and Guillemin GL (1988) Quantitative Gas

Chromatography. Amsterdam: Elsevier.

Hahn-Deinstrop E (2000) Applied Thin-Layer Chro-

matography: Best Practice and Avoidance of Mistakes.
Weinheim: Wiley-VCH.

Jennings W, Mittlefehldt E, and Stremple P (1997)

Analytical Gas Chromatography, 2nd edn. New York:
Academic Press.

Katz E, Eksteen R, Schoenmakers P, and Miller N (eds.)

(1998) Handbook of HPLC. New York and Basel:
Dekker.

Lindsay S and Barnes J (eds.) (1992) High Performance

Liquid Chromatography, 2nd edn. Chichester: Wiley.

Lough WJ and Wainer IW (eds.) (1995) High Performance

Liquid Chromatography. Fundamental Principles and
Practice. London: Blackie.

CHROMATOGRAPHY

/ Overview

97

McMaster MC (1994) HPLC – A Practical Users Guide.

Chichester: Wiley.

McNair HM and Miller JM (1997) Basic Gas Chro-

matography. Chichester: Wiley.

Meyer VR (1999) Practical High-Performance Liquid

Chromatography, 3rd edn. Chichester: Wiley.

Smith RM (1999) Supercritical fluids in separation science

– the dreams, the reality and the future. Journal of
Chromatography A 856: 83–115.

Wells PS, Zhou S, and Parcher JF (2003) Unified chro-

matography with CO

2

-based binary mobile phases.

Analytical Chemistry 75: 18A–24A.

Principles

V R Meyer

, EMPA St Gallen, St Gallen, Switzerland

& 2005, Elsevier Ltd. All Rights Reserved.

Introduction

Chromatography is one of the most important ana-
lytical techniques. It allows the separation and sub-
sequently the qualitative and quantitative analysis of
complex mixtures, as long as the samples are volatile
or soluble in a suitable solvent. Since chromatogra-
phy is based on the partition of the sample compo-
nents between two phases, one stationary and one
moving, it is necessary to distinguish between gas,
liquid, and supercritical fluid chromatography,
according to the type of mobile phase used. Chro-
matography is versatile and can be highly efficient;
full automation is possible. Some basic principles of
its theory are presented here as knowledge of the
underlying phenomena is necessary to take real
advantage of all the possibilities offered by chrom-
atographic techniques.

The Chromatographic Process

In order to obtain a chromatographic separation,
two phases are needed – a moving or mobile phase
and a fixed or stationary phase. The stationary phase
can be either a solid or a liquid, the mobile phase is a
liquid, a gas, or a supercritical fluid. Both phases
must be able to interact physically or chemically with
the sample molecules; chromatography is based on
transport, solvation, and ‘adsorption’ (in a very
broad sense) phenomena. When the mobile phase is
flowing through or over the stationary phase the
analytes in the sample mixture undergo characteristic
partition between the two phases. The mobile phase
transports, the stationary phase retains. A mixture
can be separated if its compounds are retained to
varying degrees.

Figure 1 is a simple representation of the process.

In (A) a mixture of seven molecules each of



and

m

is introduced into the chromatographic system. In (B)
they are distributed between the upper mobile and
lower stationary phase, and in (C) the mobile phase
has transported the dissolved molecules over a small
distance: new equilibria between the phases are es-
tablished. When this process has been repeated many
times, as in (D), the compounds



and

m are sep-

arated because their preference for one of the two
phases differs strongly.

In practice, chromatography takes place on a plane

or in a tube. The plane can be a sheet of paper (paper

(A)

Stationary phase

Mobile phase

(B)

(C)

(D)

Figure 1

Schematic representation of the process of a chro-

matographic separation. (A) Sample injection; (B) partition be-
tween the two phases; (C) progression of the mobile phase and
new equilibrium; and (D) separation of the two compounds after a
number of partition processes.

98

CHROMATOGRAPHY

/ Principles