Liquid Chromatography Overview

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LIQUID CHROMATOGRAPHY

Contents

Overview

Principles

Column Technology

Packed Capillary

Mobile Phase Selection

Normal Phase

Reversed Phase

Ion Pair

Micellar

Size Exclusion

Chiral

Afﬁnity Chromatography

Multidimensional

Instrumentation

Liquid Chromatography–Mass Spectrometry

Liquid Chromatography–Fourier Transform Infrared

Liquid Chromatography–Nuclear Magnetic Resonance Spectrometry

Amino Acids

Chiral Analysis of Amino Acids

Biotechnology Applications

Clinical Applications

Food Applications

Pharmaceutical Applications

Isotope Separations

Overview

M D Palamareva

, University of Soﬁa, Soﬁa, Bulgaria

Introduction

The classical methods of recrystallization and
distillation fail to provide isolation and proper

puriﬁcation of more than simple mixtures. Chro-
matography is a more efﬁcient method in this re-
spect. Any chromatographic system consists of three
components: a stationary phase, a mobile phase, and
the sample. Liquid chromatography (LC) operates
with a liquid mobile phase as opposed to gas chro-
matography (GC). LC is widely used to follow the
course of reactions, to separate complex mixtures, to
establish the presence and quantity of specific
compounds in physiological materials, etc. Thus,
this chromatographic method is daily applied in

106

LIQUID CHROMATOGRAPHY

/ Overview

laboratories such as in analytical chemistry, organic
synthesis, biochemistry, medicine, and ecology and in
production processes in industry. This article deals
briefly with the history, importance, and different
classiﬁcations of LC. Moreover, the parameters con-
nected with the retention and separation of analytes
are discussed along with the correlations among
them. These parameters are related to the structure
of analytes and the chromatographic conditions
used. Their adjustment is crucial for obtaining good
or optimum separation.

LC versus GC

In 1903, a botanist Mikhail Tswett achieved unex-
pectedly the separation of chlorophyll and xantho-
phylls into their components. In the method he used,
a petroleum ether solution of any of these mixtures is
passed through calcium carbonate placed in a glass
tube (column). This new separation method was
called ‘Chromatography’; i.e. ‘colored writing’,
owing to the development of separated colored zones
within the length of the column. Any of these zones
corresponds to a specific component. This experi-
ment is the beginning of both chromatography and
LC. In this case, calcium carbonate was the solid
stationary phase and petroleum ether (a mixture of
hydrocarbons) is the liquid mobile phase. Since then,
column liquid–solid chromatography (LSC) has been
widely used. In the case of column chromatography
(CC), the stationary phase is a three-dimensional
bed. The column contains the stationary phase and
the analyte. The mobile phase is added at the top of
the column under atmospheric pressure. Its move-
ment results in different retention of the analyte
components according to their structure.

The development of LC was rapid with the

appearance of several different methods and tech-
niques. Some trends are outlined. Liquid–liquid chro-
matography (LLC) was introduced in 1941. In this
case, both the stationary phase and mobile phase are
immiscible liquids. The stationary phase is a porous
material (support) covered with a thin ﬁlm of liquid.
A variation of LLC is paper chromatography (PC),
where the stationary phase is paper with the water
included in its pores. PC was the ﬁrst planar tech-
nique. The term ‘planar’ comes from the fact that the
stationary phase (paper) is a two-dimensional bed. A
subsequent planar technique is thin-layer chro-
matography (TLC). In this case, the stationary phase
is a thin layer of solid material, composed of small
particles, spread on a glass plate or an aluminum
sheet.

High-performance liquid chromatography (HPLC)

is the modern version of CC.

GC requires elevated temperatures. It is an indis-

pensable method for the separation of volatile
organic compounds that do not decompose at higher
temperatures.

LC is usually performed at room temperature.

Thus, it is suitable for analysis of all kinds of com-
pounds: organic and inorganic compounds, com-
pounds of low and high molecular mass, and labile
compounds such as explosives and stable com-
pounds. The conventional CC and planar techniques
do not require the use of expensive instruments.
HPLC competes with GC in precision and ef-
fectiveness. However, the reproducibility is usually
lower. If necessary, a high reproducibility is obtained
with more precautions.

Interaction of Analyte with
Both Phases

Let us consider that the analyte is composed of one
compound. The analyte interacts in a specific way
with both the stationary phase (s) and the mobile
phase (m). The interactions are usually weak (solvat-
ion, adsorption, etc.) without formation of chemical
bonds. An electrostatic interaction occurs in specific
cases only. According to its structure, an analyte X
interacts better with the stationary phase by sorption
or mobile phase by desorption. Equilibrium proces-
ses between (1) the analyte and stationary phase and
(2) the analyte and mobile phase take place. These
processes are represented in a simpler way by a single
equilibrium process:

½1

Within this equilibrium, the molecules of X are

sorbed and desorbed and thus move to some extent
with the ﬂow of the mobile phase. The equilibrium is
characterized by a distribution coefﬁcient K

½X

½2

where [X] is the molar concentration of compound X
in the corresponding phase. Thus, K

is a measure of

the retention of X in the chromatographic system.
The greater K

is, the greater the retention of X and

vice versa. The plot of

½X

versus

½X

is called a

sorption isotherm. Its shape is different, but usually
there is some part that is linear. Performance of LC in
this part of the isotherm is most effective. The ca-
pacity, and thus efﬁciency, of a chromatographic sys-
tem depends on the ratio of the masses of the analyte
and the stationary phase. If the quantity of the
analyte is greater, the chromatographic system is

LIQUID CHROMATOGRAPHY

/ Overview

107

overloaded and operates, with a decreased efﬁciency,
in the nonlinear part of the isotherm.

Mechanisms of Interaction

Depending on the chromatographic system, the ana-
lyte interacts in a different way or through a different
mechanism with the stationary phase and mobile
phase. Four main mechanisms of interactions are
known for LC.

Partition mechanism: It concerns partition be-

tween two immiscible liquids. The analyte has dif-
ferent solubility in each phase. If it is better dissolved
in the stationary phase than in the mobile phase, its
K

is greater (Figure 1A).

Adsorption mechanism: The stationary phase con-

tains, on its surface, active sites, and the analyte
adsorbs on them (Figure 1B). The mobile phase tries
to desorb them. K

is greater if adsorption dominates

over desorption.

Ion-exchange mechanism: The stationary phase

contains, on its surface, ions (cations or anions), and
the analyte exchanges its own ions with the counter-
ions of the stationary phase. Figure 1C shows an
example of separation using an anion exchanger.
Such an analyte has a greater K

than does a com-

pound where such an exchange is not possible.

Size-exclusion mechanism: The stationary phase is

a solid material composed of porous particles with
specific inner pore diameters. An analyte with small-
er size goes into the pores and has a greater K

. An

analyte larger than the pores moves with the mobile
phase outside the porous particles and has a smaller
K

(Figure 1D).

Other mechanisms: Afﬁnity and ion-pair LC are

based on modiﬁcations of the adsorption mechanism
and ion-exchange mechanism, respectively.

Classiﬁcations of Liquid
Chromatography

LC involves various methods and techniques. This
requires its classiﬁcation from different points of
view.

Classiﬁcation by the Physical State of Phases

The mobile phase is always liquid. The stationary
phase is liquid or solid. Table 1 shows in detail this
classiﬁcation. There are two modiﬁcations of LSC:
normal phase (NP) and reversed-phase (RP), depen-
ding on the relative polarity of the two phases. The
same is valid for LLC, but this subdivision is rarely

pointed. Methods 1, 2a, and 2b are of general ap-
plicability. Methods 3 and 4 apply in specific cases.
Method 1 is suitable for separation of organic and
inorganic ionic compounds. Methods 2a and 2b are
indispensable and widely applied for analysis of
nonionic organic compounds. Method 2a is better
for separation of isomers including stereoisomers. A
modiﬁcation of LSC is supercritical ﬂuid chrom-
atography, where the mobile phase is a supercritical
ﬂuid.

1 + 2

(A)

(B)

(C)

(D)

flow

Figure 1

Retention mechanisms in LC: (A) partition; (B) ad-

sorption; (C) ion-exchange; and (D) size-exclusion. 1, liquid mo-
bile phase; 2, sample molecules or ions, shown as circles (the
latter and ‘2’ are connected by lines); 3, stationary phase (one
particle; in case (A), 3 is a support particle covered with thin ﬁlm
of liquid, 4 being the stationary phase itself); 5, an inner pore in a
stationary phase particle 3.

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LIQUID CHROMATOGRAPHY

/ Overview

Classiﬁcation by the Bed of the Stationary Phase

CC, three-dimensional bed. Conventional CC is per-
formed at atmospheric pressure. The stationary
phase particles are large compared with HPLC: the
particle diameter (d

) is usually in the range 60–

200 mm. Thus, atmospheric pressure is sufﬁcient to
overcome the ﬂow resistance of the packed column.
This ensures a normal ﬂow rate (1–3 ml min

) of

the mobile phase. HPLC runs at the higher pressure
necessary to overcome the resistance of the smaller
particles (usually with d

5 mm) of the stationary

phase. A normal ﬂow rate of the mobile phase is
obtained at a pressure of 10–20 MPa. The high ef-
ﬁciency in HPLC is due to the small and uniform size
of particles. Figures 2 and 3 illustrate the equipment
necessary for performing conventional CC and
HPLC, respectively. In the ﬁrst case, a unique instru-
ment (chromatograph) is not used. The analyte is
applied to the top of a glass column containing the
stationary phase. The mobile phase, called the eluent,
passes through the column, and this leads to sepa-
ration of the solute into its components. The outco-
ming solvent (eluate) from the column is collected in
separate fractions, and the compositions are fol-
lowed using another method. In the case of HPLC,
a chromatograph is used. It is composed of six
parts: (1) a mobile-phase delivery vessel(s), (2) a
pump for producing a high pressure, (3) an injector
for application of the analyte, (4) a column contain-
ing the stationary phase, (5) a detector (usually UV)
giving signals for the composition of the mobile
phase exiting from the column, and (6) a data
station and data processing unit: the record of the
separation is called a chromatogram. The latter is
composed of peak(s): any peak corresponds to a
specific compound if a complete separation is
achieved (Figure 4).

Mobile phase

Fractions collected

No. 3

No. 2

No. 1

Liquid mobile phase flow

Stationary phase and analyte

zones separated

Figure 2

Schematic representation of conventional CC.

Table 1

Classiﬁcation and mechanisms of LC methods

No.

Method

Stationary

phase

Main sorption

mechanism

LLC

Thin ﬁlm of

liquid (on
solid)

Partition

between two
immiscible
liquids

LSC

Solid

NP LC

Polar solid

Adsorption

RP LC

Nonpolar

solid

Adsorption

Ion-exchange

chromatography

Ion-

exchange
resin

Ion-exchange

Size-exclusion

chromatography

Solid

Sieving

Mobile phase

Pump, high pressure

Injector

Column

Detector

Recorder

Chromatogram

Outcoming mobile phase

and analyte

Figure 3

Schematic representation of the components of

HPLC.

LIQUID CHROMATOGRAPHY

/ Overview

109

Classiﬁcation by the Composition
of the Mobile Phase

To tune the retention of the vast number of known
compounds, the mobile phase in LC is usually a
mixture of two or more solvents.

Isocratic technique. In this technique, the mobile

phase has a constant composition, for example ace-
tonitrile–water 72:28.

Gradient technique. In this case, the composition

of the mobile phase varies in a specific way, resulting
in better separation. It is especially useful if there is a
greater difference in the retention of analyte compo-
nents. It reduces the peak broadening especially for
compounds with greater retention times (Figure 5).
For instance, the gradient change of the mobile-phase
composition can be expressed in the following way:
mobile phase A

¼ hexane–ethyl acetate 70:30, mo-

bile phase B

¼ hexane–ethyl acetate 30:70, linear

gradient from A to B in 16 min.

Classiﬁcation by the Analyte Quantity

Analytical technique. The quantity of the analyte is
small (usually a few micrograms). This chromatogra-
phic technique gives an analysis of the analyte com-
position.

Preparative technique. The quantity of the analyte

is greater (usually

B0.5–1 g). Thus, the individual

components of the analyte are isolated in some quan-
tity. Conventional column LC operates in this mode
only. The ratio between the mass of the analyte and
stationary phase, i.e., the column capacity, varies
usually from 1:50 to 1:100. Chromatographic ﬁltra-
tion is a technique where this ratio is smaller, for
instance 1:20. It is applied when the analyte is
composed of compounds with a greater difference in
retention. The efﬁciency of conventional CC is im-
proved by an increase in pressure (1.2–1.5 atm.) over

the column when the stationary phase has smaller
particles (d

40–60 mm). To this end, the column is

connected with a bottle of liquid nitrogen or helium.
This technique is called low-pressure CC or ﬂash
chromatography.

HPLC and TLC operate in both modes. In the case

of preparative TLC, thick layers of the stationary
phase are used.

Classiﬁcation by the Possibility of
Structural Determination

LC itself does not give the possibility of determining
the structure of the individual analyte components in
the course of their separation. However, the combi-
nation of LC with a specific spectral method enables
such a determination. In such a case, after the de-
tector, the mobile phase passes online through the
relevant spectrometer. The spectrometer records the
spectrum of any peak. LC–mass spectrometry and
LC–infrared spectrometry are the most popular tech-
niques. LC–nuclear magnetic resonance spectrometry
is becoming increasingly important. If such instru-
ments are not available, structural determination is
performed in the classic way. This requires quanti-
tative separation of the analyte and isolation of the
individual components in milligram quantities. Spec-
troscopic instruments that are not connected with the
chromatographic system are used to elucidate the
structure of the separated compounds.

Time (volume)

Detector signal

)

Figure 4

HPLC chromatogram of a compound.

1,2

Time (min)

Detector signal

Time (min)

(A)

(B)

Figure 5

Comparison between (A) isocratic technique and

(B) gradient technique.

110

LIQUID CHROMATOGRAPHY

/ Overview

Miscellaneous Methods or Techniques

Modiﬁcation of the mobile phase or the stationary
phase leads to new methods or techniques. For in-
stance, inclusion of silver ions from silver nitrate
mainly in the stationary phase is used in argentation
LC. This method enables a better separation of anal-
ytes containing one or more double bonds. The dou-
ble bond forms a complex with the silver ion and this
complex has greater retention, giving the possibility
of differentiating the retention of compounds with
double bond(s). Modiﬁcation of the stationary phase
with chiral compounds enables separation of the
chiral compounds, which are of primary importance
nowadays. This technique is called chiral separation.

Chromatographic Parameters
Characterizing Analyte Retention

The distribution coefﬁcient, K

is difﬁcult to deter-

mine from the chromatogram. Thus, other chrom-
atographic parameters are deﬁned to characterize the
analyte retention directly from the chromatogram.

In the case of HPLC, the retention volume (V

)

and retention time (t

) of a compound are equal to

the volume of the mobile phase and the time passed
until its peak appears in the chromatogram, re-
spectively (see Figure 4). These two parameters are
not constant. Their values depend on the column
type being characterized by the parameters V

(breakthrough or dead volume) and the correspond-
ing time, t

(breakthrough or dead time), for an

unretained compound. The retention factor, k,
as deﬁned in eqn [3], is a constant measuring the
retention.

½3

The greater k is, the stronger the retention of the

compound.

In the case of planar chromatography, the ﬁrst

deﬁned parameter is R

½4

where S and S

are the distances from the start line to

the center of the zone and to the front line, re-
spectively. This parameter measures, in fact, the mo-
bility of the analyte since the greater R

is, the

smaller the analyte retention. The retention factor in
TLC, R

, is related to k and R

¼ log k ¼ log

½5

In the case of conventional CC, the retention is

approximately measured by V

. This technique is

preparative; the chromatographic system is over
loaded by the analyte and thus V

varies from sep-

aration to separation.

Chromatographic Parameters
Characterizing Analyte Separation

Let us assume that the analyte is composed of two or
more compounds. The main goal in LC is to separate
a pair of compounds.

Referring to compounds 1 and 2 analyzed under

same conditions, the separation factor, a, is deﬁned in
the following way:

log a

¼ log k

log k

¼ R

ð2Þ

ð1Þ

½6

where the subscript speciﬁes the compound. The
greater a is, the better is the separation. No separa-
tion is achieved if a

¼ 0 or log a ¼ 1. Parameter a

does not take into account the peak width or zone
width. However, this factor is important since the
wider the peaks are, the poorer the separation. The
resolution, R, depends on the retention and peak
width (Figure 6):

½t

ð2Þ

ð1Þ

þ W

ð2Þ

½7

where W is the width of the corresponding peak. In
analogy with a fractionation column, the chro-
matographic system is considered to consist of a
vast number of imaginary plates, called theoretical
plates, N, being easily found from the HPLC chro-
matogram. The equilibrium between sorption and
desorption is assumed complete within any theoret-
ical plate. R is a function of N, the mean value of k
for both compounds, and a:

ﬃﬃﬃﬃﬃ

þ k

½8

Time (volume)

Detector signal

R(1)

R(2)

(1)

(2)

Figure 6

Parameters necessary for calculation of resolution, R,

using eqn [7].

LIQUID CHROMATOGRAPHY

/ Overview

111

In LC, the value of N varies usually from 1000 to

10 000. This factor depends mainly on the stationary
phase particle size and is the main reason for the
better separation achieved by LC compared with
distillation. The values of k and a should adjust in
such a way as to give a greater value of R for a
reasonable analysis time. To this end, variation of the
mobile phase composition and its ﬂow rate, F, is of
signiﬁcant importance. According to eqn [8], R is
greater at better separation, a, and stronger retention
(greater values of k). Reasonable values of k fall in
the range 1–20. Greater values of k require a longer
analysis time, and this is an unfavorable factor.
Moreover, the peaks at greater k become diffuse and
asymmetric. This phenomenon is known as peak
(band) broadening, being undesired in LC. It leads to
partial separation or overlap of two or more peaks.
Thus, the suitable conditions for performance of LC
are a compromise of various factors.

As mentioned, the ﬂow rate of the mobile phase in

the chromatographic system is an important factor
for its separation selectivity. It is connected with
diffusion and mass transfer in the chromatographic
system since LC is a dynamic process. A small ﬂow
rate results in greater retention times and strong
peak broadening due to diffusion effects. A high
ﬂow rate leads to short retention times and peak
broadening because the mass transfer between the
stationary and mobile phases needs some time.
Thus, an optimum ﬂow rate is applied. It is estab-
lished by the Van Deemter equation. The theoretical
plate height, H (equal to the ratio of bed length, L,
to N), is expressed as a function of the linear velo-
city, u, of the mobile phase. The ﬂow rate is
a product of u (in cm s

) and the column diam-

eter. A recent modiﬁcation of the Van Deemter

equation is

¼ A þ B=u þ C

u þ C

½9

where A represents the ﬂow anisotropy created by
the lack of homogeneity of the column packing, the
second term on the right side results from longit-
udinal diffusion, and the third term from mass
transfer into and out of the stationary phase. A plot
of H versus u gives a curve showing a minimum.
The use of the linear velocity, u (and the relevant
ﬂow rate), corresponding to that minimum results in
the best separation efﬁciency of the chromatogra-
phic system regarding diffusion and mass transfer.

See also: Chromatography: Principles. Gas Chro-
matography: Overview. Liquid Chromatography: Prin-
ciples; Biotechnology Applications.