106 LIQUID CHROMATOGRAPHY / Overview
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polymyxin B-selective adsorption and subsequent high- Xiao Y, Chen Y, Kennedy AW, Belinson J, and Xu Y
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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
purification of more than simple mixtures. Chro-
Overview
matography is a more efficient method in this re-
spect. Any chromatographic system consists of three
components: a stationary phase, a mobile phase, and
M D Palamareva, University of Sofia, Sofia, Bulgaria
the sample. Liquid chromatography (LC) operates
& 2005, Elsevier Ltd. All Rights Reserved.
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
Introduction
establish the presence and quantity of specific
The classical methods of recrystallization and compounds in physiological materials, etc. Thus,
distillation fail to provide isolation and proper this chromatographic method is daily applied in
LIQUID CHROMATOGRAPHY / Overview 107
laboratories such as in analytical chemistry, organic GC requires elevated temperatures. It is an indis-
synthesis, biochemistry, medicine, and ecology and in pensable method for the separation of volatile
production processes in industry. This article deals organic compounds that do not decompose at higher
briefly with the history, importance, and different temperatures.
classifications of LC. Moreover, the parameters con- LC is usually performed at room temperature.
nected with the retention and separation of analytes Thus, it is suitable for analysis of all kinds of com-
are discussed along with the correlations among pounds: organic and inorganic compounds, com-
them. These parameters are related to the structure pounds of low and high molecular mass, and labile
of analytes and the chromatographic conditions compounds such as explosives and stable com-
used. Their adjustment is crucial for obtaining good pounds. The conventional CC and planar techniques
or optimum separation. do not require the use of expensive instruments.
HPLC competes with GC in precision and ef-
fectiveness. However, the reproducibility is usually
LC versus GC
lower. If necessary, a high reproducibility is obtained
In 1903, a botanist Mikhail Tswett achieved unex- with more precautions.
pectedly the separation of chlorophyll and xantho-
phylls into their components. In the method he used,
Interaction of Analyte with
a petroleum ether solution of any of these mixtures is
passed through calcium carbonate placed in a glass
Both Phases
tube (column). This new separation method was
Let us consider that the analyte is composed of one
called  Chromatography ; i.e.  colored writing ,
compound. The analyte interacts in a specific way
owing to the development of separated colored zones
with both the stationary phase (s) and the mobile
within the length of the column. Any of these zones
phase (m). The interactions are usually weak (solvat-
corresponds to a specific component. This experi-
ion, adsorption, etc.) without formation of chemical
ment is the beginning of both chromatography and
bonds. An electrostatic interaction occurs in specific
LC. In this case, calcium carbonate was the solid
cases only. According to its structure, an analyte X
stationary phase and petroleum ether (a mixture of
interacts better with the stationary phase by sorption
hydrocarbons) is the liquid mobile phase. Since then,
or mobile phase by desorption. Equilibrium proces-
column liquid solid chromatography (LSC) has been
ses between (1) the analyte and stationary phase and
widely used. In the case of column chromatography
(2) the analyte and mobile phase take place. These
(CC), the stationary phase is a three-dimensional
processes are represented in a simpler way by a single
bed. The column contains the stationary phase and
equilibrium process:
the analyte. The mobile phase is added at the top of
the column under atmospheric pressure. Its move-
Xm$Xs ½1Š
ment results in different retention of the analyte
components according to their structure.
Within this equilibrium, the molecules of X are
The development of LC was rapid with the
sorbed and desorbed and thus move to some extent
appearance of several different methods and tech-
with the flow of the mobile phase. The equilibrium is
niques. Some trends are outlined. Liquid liquid chro-
characterized by a distribution coefficient KD:
matography (LLC) was introduced in 1941. In this
case, both the stationary phase and mobile phase are
½XŠs
KD ź ½2Š
immiscible liquids. The stationary phase is a porous
½XŠm
material (support) covered with a thin film of liquid.
A variation of LLC is paper chromatography (PC), where [X] is the molar concentration of compound X
where the stationary phase is paper with the water in the corresponding phase. Thus, KD is a measure of
included in its pores. PC was the first planar tech- the retention of X in the chromatographic system.
nique. The term  planar comes from the fact that the The greater KD is, the greater the retention of X and
stationary phase (paper) is a two-dimensional bed. A vice versa. The plot of ½XŠs versus ½XŠm is called a
subsequent planar technique is thin-layer chro- sorption isotherm. Its shape is different, but usually
matography (TLC). In this case, the stationary phase there is some part that is linear. Performance of LC in
is a thin layer of solid material, composed of small this part of the isotherm is most effective. The ca-
particles, spread on a glass plate or an aluminum pacity, and thus efficiency, of a chromatographic sys-
sheet. tem depends on the ratio of the masses of the analyte
High-performance liquid chromatography (HPLC) and the stationary phase. If the quantity of the
is the modern version of CC. analyte is greater, the chromatographic system is
108 LIQUID CHROMATOGRAPHY / Overview
overloaded and operates, with a decreased efficiency, 1 1
in the nonlinear part of the isotherm.
flow flow
Mechanisms of Interaction
Depending on the chromatographic system, the ana-
lyte interacts in a different way or through a different
1 + 2 1 + 2
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 3
3
4
in the stationary phase than in the mobile phase, its
KD is greater (Figure 1A).
Adsorption mechanism: The stationary phase con-
(A) (B)
tains, on its surface, active sites, and the analyte
adsorbs on them (Figure 1B). The mobile phase tries
to desorb them. KD is greater if adsorption dominates
1 1
over desorption.
Ion-exchange mechanism: The stationary phase
flow flow
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.
1 + 2 1 + 2
Such an analyte has a greater KD 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 KD. An
+ +
analyte larger than the pores moves with the mobile
3 3
5
phase outside the porous particles and has a smaller
KD (Figure 1D).
Other mechanisms: Affinity and ion-pair LC are
(C) (D)
based on modifications of the adsorption mechanism
and ion-exchange mechanism, respectively.
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
Classifications of Liquid
of liquid, 4 being the stationary phase itself); 5, an inner pore in a
Chromatography
stationary phase particle 3.
LC involves various methods and techniques. This
requires its classification from different points of
pointed. Methods 1, 2a, and 2b are of general ap-
view.
plicability. Methods 3 and 4 apply in specific cases.
Method 1 is suitable for separation of organic and
Classification by the Physical State of Phases
inorganic ionic compounds. Methods 2a and 2b are
indispensable and widely applied for analysis of
The mobile phase is always liquid. The stationary
nonionic organic compounds. Method 2a is better
phase is liquid or solid. Table 1 shows in detail this
for separation of isomers including stereoisomers. A
classification. There are two modifications of LSC:
normal phase (NP) and reversed-phase (RP), depen- modification of LSC is supercritical fluid chrom-
atography, where the mobile phase is a supercritical
ding on the relative polarity of the two phases. The
same is valid for LLC, but this subdivision is rarely fluid.
LIQUID CHROMATOGRAPHY / Overview 109
Table 1 Classification and mechanisms of LC methods
Liquid mobile phase flow
No. Method Stationary Main sorption
phase mechanism
1 LLC Thin film of Partition
liquid (on between two
solid) immiscible
liquids
Mobile phase
2 LSC Solid
2a NP LC Polar solid Adsorption
2b RP LC Nonpolar Adsorption
solid
3 Ion-exchange Ion- Ion-exchange
Stationary phase and analyte
chromatography exchange
zones separated
resin
4 Size-exclusion Solid Sieving
chromatography
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
No. 1 No. 2 No. 3
particle diameter (dp) is usually in the range 60
200 mm. Thus, atmospheric pressure is sufficient to
overcome the flow resistance of the packed column.
1 Fractions collected
This ensures a normal flow rate (1 3 ml min ) of
the mobile phase. HPLC runs at the higher pressure Figure 2 Schematic representation of conventional CC.
necessary to overcome the resistance of the smaller
particles (usually with dp 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
Mobile phase
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
Pump, high pressure
applied to the top of a glass column containing the
stationary phase. The mobile phase, called the eluent,
Injector
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-
Column
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)
Detector Recorder Chromatogram
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 Outcoming mobile phase
and analyte
composed of peak(s): any peak corresponds to a
specific compound if a complete separation is
Figure 3 Schematic representation of the components of
achieved (Figure 4). HPLC.
110 LIQUID CHROMATOGRAPHY / Overview
1,2
tR (VR)
3
to (Vo)
w
20
(A) Time (min)
3
1
Time (volume) 2
Figure 4 HPLC chromatogram of a compound.
Classification by the Composition
of the Mobile Phase
20
To tune the retention of the vast number of known
(B) Time (min)
compounds, the mobile phase in LC is usually a
mixture of two or more solvents. Figure 5 Comparison between (A) isocratic technique and
(B) gradient technique.
Isocratic technique. In this technique, the mobile
phase has a constant composition, for example ace-
tonitrile water 72:28.
the column when the stationary phase has smaller
Gradient technique. In this case, the composition
particles (dp 40 60 mm). To this end, the column is
of the mobile phase varies in a specific way, resulting
connected with a bottle of liquid nitrogen or helium.
in better separation. It is especially useful if there is a
This technique is called low-pressure CC or flash
greater difference in the retention of analyte compo-
chromatography.
nents. It reduces the peak broadening especially for
HPLC and TLC operate in both modes. In the case
compounds with greater retention times (Figure 5).
of preparative TLC, thick layers of the stationary
For instance, the gradient change of the mobile-phase
phase are used.
composition can be expressed in the following way:
mobile phase A ź hexane ethyl acetate 70:30, mo-
bile phase B ź hexane ethyl acetate 30:70, linear
Classification by the Possibility of
gradient from A to B in 16 min.
Structural Determination
LC itself does not give the possibility of determining
Classification by the Analyte Quantity
the structure of the individual analyte components in
Analytical technique. The quantity of the analyte is the course of their separation. However, the combi-
small (usually a few micrograms). This chromatogra- nation of LC with a specific spectral method enables
phic technique gives an analysis of the analyte com- such a determination. In such a case, after the de-
position. tector, the mobile phase passes online through the
Preparative technique. The quantity of the analyte relevant spectrometer. The spectrometer records the
is greater (usually B0.5 1 g). Thus, the individual spectrum of any peak. LC mass spectrometry and
components of the analyte are isolated in some quan- LC infrared spectrometry are the most popular tech-
tity. Conventional column LC operates in this mode niques. LC nuclear magnetic resonance spectrometry
only. The ratio between the mass of the analyte and is becoming increasingly important. If such instru-
stationary phase, i.e., the column capacity, varies ments are not available, structural determination is
usually from 1:50 to 1:100. Chromatographic filtra- performed in the classic way. This requires quanti-
tion is a technique where this ratio is smaller, for tative separation of the analyte and isolation of the
instance 1:20. It is applied when the analyte is individual components in milligram quantities. Spec-
composed of compounds with a greater difference in troscopic instruments that are not connected with the
retention. The efficiency of conventional CC is im- chromatographic system are used to elucidate the
proved by an increase in pressure (1.2 1.5 atm.) over structure of the separated compounds.
Detector signal
Detector signal
Detector signal
LIQUID CHROMATOGRAPHY / Overview 111
Miscellaneous Methods or Techniques
In the case of conventional CC, the retention is
approximately measured by VR. This technique is
Modification of the mobile phase or the stationary
preparative; the chromatographic system is over
phase leads to new methods or techniques. For in-
loaded by the analyte and thus VR varies from sep-
stance, inclusion of silver ions from silver nitrate
aration to separation.
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-
Chromatographic Parameters
ble bond forms a complex with the silver ion and this
Characterizing Analyte Separation
complex has greater retention, giving the possibility
Let us assume that the analyte is composed of two or
of differentiating the retention of compounds with
more compounds. The main goal in LC is to separate
double bond(s). Modification of the stationary phase
a pair of compounds.
with chiral compounds enables separation of the
Referring to compounds 1 and 2 analyzed under
chiral compounds, which are of primary importance
same conditions, the separation factor, a, is defined in
nowadays. This technique is called chiral separation.
the following way:
k2
Chromatographic Parameters
a ź or log a ź log k2 log k1 ź RMð2Þ RMð1Þ ½6Š
k1
Characterizing Analyte Retention
where the subscript specifies the compound. The
The distribution coefficient, KD, is difficult to deter-
greater a is, the better is the separation. No separa-
mine from the chromatogram. Thus, other chrom-
tion is achieved if a ź 0 or log a ź 1. Parameter a
atographic parameters are defined to characterize the
does not take into account the peak width or zone
analyte retention directly from the chromatogram.
width. However, this factor is important since the
In the case of HPLC, the retention volume (VR)
wider the peaks are, the poorer the separation. The
and retention time (tR) of a compound are equal to
resolution, R, depends on the retention and peak
the volume of the mobile phase and the time passed
width (Figure 6):
until its peak appears in the chromatogram, re-
2½tRð2Þ tRð1ÞŠ
spectively (see Figure 4). These two parameters are
R ź ½7Š
Wð1Þ þ Wð2Þ
not constant. Their values depend on the column
type being characterized by the parameters Vo
where W is the width of the corresponding peak. In
(breakthrough or dead volume) and the correspond-
analogy with a fractionation column, the chro-
ing time, to (breakthrough or dead time), for an
matographic system is considered to consist of a
unretained compound. The retention factor, k,
vast number of imaginary plates, called theoretical
as defined in eqn [3], is a constant measuring the
plates, N, being easily found from the HPLC chro-
retention.
matogram. The equilibrium between sorption and
desorption is assumed complete within any theoret-
tR to VR Vo
k ź or k ź ½3Š
ical plate. R is a function of N, the mean value of k
to Vo
for both compounds, and a:
The greater k is, the stronger the retention of the
pffiffiffiffi
ffi
N a 1 k
compound.
R ź ½8Š
4 a 1 þ k
In the case of planar chromatography, the first
defined parameter is RF:
S
tR(2)
RF ź ½4Š
So
tR(1)
where S and So are the distances from the start line to
the center of the zone and to the front line, re-
to
spectively. This parameter measures, in fact, the mo-
w(1) w(2)
bility of the analyte since the greater RF is, the
smaller the analyte retention. The retention factor in
TLC, RM, is related to k and RF:
Time (volume)
1
Figure 6 Parameters necessary for calculation of resolution, R,
RM ź log k ź log 1 ½5Š
RF
using eqn [7].
Detector signal
112 LIQUID CHROMATOGRAPHY / Principles
In LC, the value of N varies usually from 1000 to equation is
10 000. This factor depends mainly on the stationary
H ź A þ B=u þ Cs u þ Cm u ½9Š
phase particle size and is the main reason for the
where A represents the flow anisotropy created by
better separation achieved by LC compared with
the lack of homogeneity of the column packing, the
distillation. The values of k and a should adjust in
second term on the right side results from longit-
such a way as to give a greater value of R for a
udinal diffusion, and the third term from mass
reasonable analysis time. To this end, variation of the
transfer into and out of the stationary phase. A plot
mobile phase composition and its flow rate, F, is of
of H versus u gives a curve showing a minimum.
significant importance. According to eqn [8], R is
The use of the linear velocity, u (and the relevant
greater at better separation, a, and stronger retention
flow rate), corresponding to that minimum results in
(greater values of k). Reasonable values of k fall in
the best separation efficiency of the chromatogra-
the range 1 20. Greater values of k require a longer
phic system regarding diffusion and mass transfer.
analysis time, and this is an unfavorable factor.
Moreover, the peaks at greater k become diffuse and
See also: Chromatography: Principles. Gas Chro-
asymmetric. This phenomenon is known as peak matography: Overview. Liquid Chromatography: Prin-
ciples; Biotechnology Applications.
(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 Further Reading
are a compromise of various factors.
Bidlingmeyer BA (1992) Practical HPLC Methodology and
As mentioned, the flow rate of the mobile phase in
Applications. New York: Wiley.
the chromatographic system is an important factor
Giddings JC (1991) Unified Separation Science. New York:
for its separation selectivity. It is connected with
Wiley.
diffusion and mass transfer in the chromatographic
McMaster MC (1994) HPLC: A Practical User s Guide.
system since LC is a dynamic process. A small flow
New York: VCH Publishers.
rate results in greater retention times and strong Meyer VR (1999) Practical High-performance Liquid
Chromatography, 3rd edn. Chichester: John Wiley.
peak broadening due to diffusion effects. A high
Neue UD (1997) HPLC Columns: Theory, Technology,
flow rate leads to short retention times and peak
and Practice. New York: Wiley-VCH.
broadening because the mass transfer between the
Poole CF (2003) The Essence of Chromatography.
stationary and mobile phases needs some time.
Amsterdam: Elsevier.
Thus, an optimum flow rate is applied. It is estab-
Snyder LR, Glajch JL, and Kirkland JJ (1994) Practical
lished by the Van Deemter equation. The theoretical
HPLC Method Development, 2nd edn. New York: Wiley.
plate height, H (equal to the ratio of bed length, L,
Snyder LR and Kirkland JJ (1979) Introduction to Modern
to N), is expressed as a function of the linear velo-
Liquid Chromatography, 2nd edn. New York: Wiley-
city, u, of the mobile phase. The flow rate is
Interscience.
1
a product of u (in cm s ) and the column diam- Touchstone JC (1992) Practice of Thin Layer Chro-
eter. A recent modification of the Van Deemter matography, 3rd edn. New York: Wiley.
Principles
M D Palamareva, University of Sofia, Sofia, Bulgaria
polarity of both phases. Two other methods are
known: ion exchange and size exclusion. The chrom-
& 2005, Elsevier Ltd. All Rights Reserved.
atographic parameters k, RF, RM, a, and R measure
retention of analytes and their separation. The rela-
tionships among these parameters are important to
Introduction
obtain good separation for reasonable analysis time.
All methods are performed by the conventional
The process in liquid chromatography (LC) is the
result of the interaction among an analyte, a station- column chromatography (CC), modern column
ary phase, and a mobile phase. Moreover, LC is sub- chromatography, denoted as high-performance
divided into liquid liquid chromatography (LLC) liquid chromatography (HPLC), or planar thin-
and liquid solid chromatography (LSC). Both meth- layer chromotography (TLC).
ods are performed in normal-phase (NP) mode and This article deals with the essential theory of
reversed-phase (RP) mode depending on the relative LC. Attention is paid to the stationary-phase types,