High Performance Liquid

Chromatography

What is HPLC ?

H: High
P : Performance (Pressure)
L : Liquid
C : Chromatography

 High Performance Liquid Chromatography

(HPLC) is one mode of chromatography, the
most widely used analytical technique.
Chromatographic processes can be defined as
separation techniques involving mass-transfer
between stationary and mobile phases.

 HPLC utilizes a liquid mobile phase to separate

the components of a mixture. These components
(or analytes) are first dissolved in a solvent, and
then forced to flow through a chromatographic
column under high pressure. In the column, the
mixture is resolved into its components.

In HPLC, several instrument and column chemistry

parameters need to be optimized in order to generate a
satisfactory separation.
Each of the following parameters need to be optimized
in order to generate a chromatogram that is suitable
for qualitative or quantitative purposes.

Mobile phase composition

Bonded phase chemistry
Column and packing dimensions
Injection volume
Sample pre-treatment and concentration
Mobile phase flow rate
Column temperature
Detector parameters

Figure 1

1: The mobile phase composition (usually water and an

organic solvent plus other additives) needs to be
optimized in order to gain good separation.
2: Degassers are often used to remove air from the
mobile phase, leading to better chromatographic
baselines.
3: The detector conditions are chosen to give the best
response to the analytes of interest and to achieve good
sensitivity.

4: The column dimensions and stationary phase

chemistry are chosen and optimized to give separations
of the quality required.
5: The autosampler introduces a plug of sample into the
mobile phase flow which is then swept onto the column.
6: Dual reciprocating pumps are used to deliver the
mobile phase at back pressures of up to 400 bar. A
steady stream of liquid delivered at a constant flow rate
is important.

What is HPLC used for ?

1. Separation of mixed components
2. Qualitative analysis / Quantitative analysis
3. Preparation of interest components

The ability of HPLC to separate such a wide diversity of species

leads to its popularity in a wide range of industries. It is
important to note that HPLC is capable of separating analytes in
the following categories:
High molecular weight (>2000)
Low molecular weight (<2000)
Polar
Non-polar
Ionizable
Cationic
Anionic

Selection guide for all of the different chromatographic

techniques with liquid mobile phases

Normal Phase Chromatography

In NP chromatography (also called adsorption or liquid

solid chromatography), the stationary phase is more
polar than the mobile phase. The retention increases as
the polarity of the mobile phase decreases, and polar
analytes are more strongly retained than non-polar
ones.

Retention in NPC is best described by a displacement

process, based on the fact that the silica surface is
covered by a monolayer of solvent molecules that
are adsorbed from the mobile phase. Consequently,
for a solute molecule to be retained in NPC, one or
more previously adsorbed solvent molecules must be
displaced from (leave) the silica surface in order to
make room for the adsorbing solute.

Stationary Phases
Normal-phase (NP) chromatography generally uses
the same types of mobile phase as for the adsorption
mode. The difference, however, is the nature of the
stationary phase. In NP chromatography, the packing is
silica gel that has been bonded with a polar phase. The
usual polar phases widely available from many
manufacturers include cyano, amino, nitro, and diol
phases.

Mobile Phases
A variety of solvent can be used for adsorption
chromatography, ranging non-polar to polar.
Adsorption chromatography functions very well
for the separation of non-polar and moderately
polar compounds using moderately polar
eluents. It does not function well with high
polarity eluents.

In normal phase chromatography the mobile phase

is more nonpolar than the stationary phase and
therefore made of organic solvents. The intention of
the mobile phase is to:
Keep the analytes in solution;
Transport the analytes through the bed of
stationary phase;
Contribute to the separation;
Compete with the analytes for the adsorption
sites on the stationary phase.

The strength of the mobile phase is decided by the

polarity of the solvent used, and solvents can be ranked
according to their solvent strength, (see Table).
Solvent strength increases with increasing polarity.
Increased solvent strength of mobile phase decreases
the retention of the substances.

 Control of sample retention in adsorption

chromatography is achieved almost by modifying the
composition of the mobile phase. Minor change in the
eluent strength have dramatic effect on k and values.
The elution power of a solvent is measured by its
solvent strength parameter 0.
Often a single solvent is unable to effect resolution of
sample components and binary and ternary mixtures
must be employed.

 A measure of solvent strength of these mixtures may

be determined from the solvent polarity indices
The polarity index, P, is a numerical measure of the
relative polarity of various solvents as determined from
their solubility in some specific solvents.
The polarity index PAB for a mixture can then be
determined from the polarity indices of the pure
components and their respective volume fractions
(A ,B)

The following equation can be used:

where PA and PB are the polarity indexes of the

two solvents and A and B are the volume
fractions of solvents A and B.

Any desired polarity index can be obtained by mixing

the appropriate amounts of solvents. An increase in the
polarity of the solvent mixture means a stronger eluent
and hence smaller k values. This expressed in the
following relation:

Thus , a two unit change in polarity index results

in a 10-fold change in k.

example
In a normal-phase partition column, a solute
was found to have a retention time of 29.1 min, and
an unretained sample had a retention time of 1.05
min when the mobile phase was 50% by volume
chloroform and 50% n-hexane.
Calculate (i) k for the solute and (ii) a solvent
composition that would bring k down to a value of
about 10.

Reversed-phase
chromatography
Neutral Compounds

RPC is usually a first choice for the separation of

both neutral and ionic samples, using a column
packing that contains a less polar bonded phase
such as C8 or C18.
The mobile phase in RP chromatography is
normally more polar than the stationary phase.

Stationary Phases
Historically, silica gel was the most common material
used in the early development of column liquid
chromatography (LC).However, silica is a polar material
that contains hydroxyl groups (silanols) that are both
acidic and strongly hydrogen-bonding in character.

These properties make it unsuitable as a stationary

phase for many typical organic molecules that are
predominantly hydrophobic compounds. In addition,
the silanols interact strongly with basic compounds
leading to poor chromatographic results (peak tailing).

In order to overcome these undesirable effects of silica

and to have a medium more suitable for the separation
of a large variety of organic compounds, modification
of the surface is necessary to provide a more non-polar
(hydrophobic) material.

Therefore, chemical modification is the only practical

approach to modifying the silica surface in order to
create a stationary phase that is compatible with the
types of solutes to be separated. The most common
method for modifying silica in order to produce a
hydrophobic surface is organosilanization.

To minimize unwanted interactions with residual

silanol groups column packings for RPC are usually
endcapped, by a further reaction of the bonded
phase with a small silane such as
trimethylchlorosilane or dimethyldichlorosilane .

Imad Abu Reid

38

This procedure decreases the concentration of

unreacted silanols, as well as their interaction
with retained solute moleculesbut does not
totally eliminate silanol-solute interaction (endcapping increases the percentage of reacted
silanols by only 2030% ,corresponding to a
somewhat smaller decrease of unreacted
silanols).

Reversed-phase stationary phases are more or

less hydrophobic, and the degree of this
property is characterized by their hydrophobicity
H. As a general rule, retention times are longer
the more Carbon atoms the bonded stationary
phase contains.

Within this context, the predominant factors in

determining the hydrophobicity are the length of the
alkyl chain or the total number of carbon atoms as well
as the bonding density.

Effect of chain length on retention

Mobile Phase
The mobile phase generally consist of mixtures of water or
aqueous buffer solutions with various water-miscible
solvents.
The strength of binary mixtures is not a well defined
function of %B but depends on analyte and stationary
phase properties. Nevertheless, it is possible to give
numerical data which allow to obtain a good
approximation of mobile phase composition when it is
necessary to try more than one solvent in order to find the
best selectivity.

The following equation can be used:

where PA and PB are the polarity indexes of the

two solvents and A and B are the volume
fractions of solvents A and B.

Example
In a reversed-phase column, a solute was found to have
a retention time of 31.3 min, and an unretained species
required 0.48 min for elution when the mobile phase
was 30% (by volume) methanol and 70% water. Calculate (a) k and (b) a water-methanol composition that
should bring k to a value of about 5.

Solution

Alternatively, a nomogram can be used. It is based on

numerous experimental data determined with small
organic molecules

Solvent strength of binary mixtures for reversed-phase

chromatography

Problem
If a separation with 70% of methanol gives
adequate retention but poor selectivity, which
other solvent mixtures could be tried?

IONIC SAMPLES:
REVERSED-PHASE &
ION-PAIR
CHROMATOGRAPHY

Imad Abu Reid

55

Ionizable Compounds
analysis by RP

ACIDBASE EQUILIBRIA AND REVERSED-PHASE

RETENTION
The RPC retention of neutral samples decreases
for less hydrophobic (more polar)molecules.
When an acid (HA) or base (B) undergoes ionization
(i.e., is converted from an uncharged to a charged
species), the compound becomes much more polar
or hydrophilic. As a result its retention factor k in
RPC can be reduced 10-fold or more:

Imad Abu Reid

57

Imad Abu Reid

58

Knowing the pka a substance, its percent

ionization at certain pH can be calculated from
the equation:

Imad Abu Reid

59

Effect of pH of the retention of basic

analyte

Imad Abu Reid

61

The retention dependence of basic components on

the pH of mobile phase could be subdivided into three
regions:
A. Fully protonated analyte (cationic form), which
shows the lowest retention. The analyte is in the most
hydrophilic form. Its interactions with the
hydrophobic stationary phase are suppressed.

Imad Abu Reid

62

B. Partial protonation region. Coexistence of two

analyte forms (protonated and deprotonated) in the
mobile phase in equilibrium may cause poor peak
shape and unstable retention.
C. Analyte in its neutral form (the most
hydrophobic), which shows the longest retention.

Effect of pH of the retention of acidic

analyte

Imad Abu Reid

64

Similar retention curves can be obtained for

acidic components, but obviously their retention
dependence will be the mirror image of that for
basic analytes

Imad Abu Reid

65

Knowledge of the pKa of the analytes in the mixture

is very important. Significant changes in retention
and even reversals in elution order can be observed.
Take, for example, two analytes: one basic and one
acidic, both with pKa values of approximately 4.

If a mixture of these two analytes were analyzed

at pH 2.3 and pH 6.0, the base would show lower
retention at pH 2.3 and higher retention at pH
6.0, and the acidic component would show higher
retention at pH 2.3 and lower retention at pH 6.0

Choice of Buffers
Whenever acids or bases are separated, it is necessary
to buffer the mobile phase in order to maintain a
constant pH and reproducible retention during the
separation.
In selecting a buffer for RPC separation, several buffer
properties may prove relevant:

Imad Abu Reid

69

Factors to be considered
pKa and buffer capacity
solubility
UV absorbance (when UV detection is used)
volatility (when mass-spectrometric or
evaporative light-scattering detection is used)
ion-pairing properties
stability and compatibility with the equipment
Imad Abu Reid

70

Imad Abu Reid

71

Imad Abu Reid

72

Imad Abu Reid

73

ION-PAIR CHROMATOGRAPHY
(IPC)

Imad Abu Reid

74

Ion-pair chromatography (IPC) can be regarded

as a modification of RPC for the separation of
ionic samples. The only difference in conditions
for IPC is the addition of an ion-pairing reagent
R+ or R to the mobile phase, which can then
interact with ionized acids A or bases BH+ in an
equilibrium process:
Imad Abu Reid

75

Imad Abu Reid

76

The use of IPC can thus create similar changes in sample

retention as by a change in mobile-phase pH ,but with greater
control over the retention of either acidic or basic solutes, and
without the need for extreme values of mobile-phase pH (e.g.,
pH < 2.5 or > 8).
Typical ion-pairing reagents include alkylsulfonates RSO-3( R)
and tetraalkylammonium salts R4N+ (R+), as well as strong
(normally ionized) carboxylic acids (trifluoroacetic acid, TFA;
heptafluorobutyric acid, HFBA [R]),and so-called chaotropes
(BF4 , ClO 4 , PF 6 ).
Imad Abu Reid

77

Imad Abu Reid

78

Basis of Retention
Two possible retention processes or
mechanisms exist for separation by IPC.As an
example, we will use the ion-pairing of an ionized
acidic solute A by a tetraalkylammonium IPC
reagent R+. The ion-pairing of a protonated basic
Solute B+ by an alkylsulfonate IPC reagent R can
be described similarly.
Imad Abu Reid

79

The analytes form hydrophobic ion-pairs in the eluent,

and the paired ions are retained on a hydrophobic
stationary phase surface by their hydrophobicity.
Usually less than 0.1 wt% of the counter-ion and buffer
concentration is employed. The selection of a stationary
phase material is simple and a variety of hydrophobic
stationary phase materials for reversed-phase liquid
chromatography can be used.
Imad Abu Reid

80

The organic modifier concentration depends on

the hydrophobicity of stationary phase materials
used and the ion pair reagent. Increasing the
size of the counter-ion increases the retention
time. The maximum retention time is reached
when the counter-ion concentration reaches the
micelle condition.
Imad Abu Reid

81

Relationship between analytes and counterions in ion-pair liquid chromatography

Imad Abu Reid

82

Example: ion-pair liquid chromatography of amino

acids. Amino acids are zwitterions. The amino group
can form an ion-pair with an alkanesulfonate ion (such
as octanesulfonate), and the carboxyl group can form
an ion-pair with a tetrabutylammonium ion, depending
on the pH of the solution.

Imad Abu Reid

83

In reversed-phase liquid chromatography, the

ionization of the solute decreases the retention.
The addition of counter-ion under these
conditions forces the formation of an ion-pair
between the ionized solute and counter-ion,
and then the retention of the analyte increases
as the paired-ion is retained.
Imad Abu Reid

84

Two mechanisms for retention in reversed-phase

ion-pair liquid chromatography have been
considered. One is the adsorption of the hydrophobic
paired ion on the hydrophobic surface of stationary
phase material. In the second mechanism, the
hydrophobic counter-ion is held on the surface of the
hydrophobic stationary phase, and the analyte ion is
retained by ion-ion interactions.
Imad Abu Reid

85

Imad Abu Reid

86

The separation conditions available for the control of

selectivity in IPC include:
pH
IPC reagent type (sulfonate, quaternary ammonium
salt, chaotrope)
IPC reagent concentration
solvent strength (%B)
solvent type (ACN, MeOH, etc.)
temperature
column type
buffer type and concentration
Imad Abu Reid

87

DETECTION

characteristics of an ideal HPLC

detector
have high sensitivity and predictable response
respond to all solutes, or else have predictable
specificity
be unaffected by changes in temperature and carrier
flow
respond independently of the mobile phase
not contribute to extra-column peak broadening

Imad Abu Reid

90

 be reliable and convenient to use

have a response that increases linearly with
the amount of solute
be nondestructive of the solute
provide qualitative information on the
detected peak

4.6.2 Detection Techniques

There are four general techniques that are used
for HPLC detection:
bulk property or differential measurement
sample specific
hyphenated techniques

Imad Abu Reid

92

1. Bulk Property Detectors

A bulk property detector can be considered a universal
detector as it measures a property that is common to all
compounds. The detector measures a change in this
property as a differential measurement between the
mobile phase containing the sample and that without the
sample. The most familiar of the bulk property
detectors is the refractive index detector .Bulk property
detectors have the advantage that they detect all
compounds.
Imad Abu Reid

93

2. Sample-Specific Detectors
Some characteristic of a sample is unique to that sample,
or at least is not common to all analytes, and the samplespecific detector responds to that characteristic. The UV
detector is the most commonly used sample-specific
detector. It responds to compounds that absorb UV light
at a specific wavelength. Other common sample-specific
detectors rely on the ability of an analyte to fluoresce
(fluorescence), conduct electricity (conductivity), or react
under specific conditions (electrochemical).
Imad Abu Reid

94

3. Hyphenated Techniques
Hyphenated techniques refer to the coupling of an
independent analytical instrument to the HPLC system
to provide detection, and often are abbreviated with a
hyphenas LC-(plus the technique). The most common
hyphenated technique is LC-MS, where a mass
spectrometer is coupled with an HPLC system. Other
less widely used techniques are LC-IR or LC-FTIR and LCNMR.
Imad Abu Reid

95

I. UV Detector
UV-Vis detectors are the most commonly used detectors in
HPLC. Solutes which absorb UV or visible radiation (typically
190 - 600 nm) can be detected. The degree of absorption is a
function of the molar absorptivity of the sample molecule, the
path length of the detector flow cell and the solute
concentration. The solute concentration is directly
proportional to the absorbance allowing quantification. UVVis detectors can routinely achieve detection of only a few
nanograms. They have a large linear dynamic range and are
very robust.

Imad Abu Reid

96

Imad Abu Reid

97

Imad Abu Reid

98

variable wavelength UV detector

Imad Abu Reid

99

diode-array UV detector

Imad Abu Reid

100

Diode Array Spectral Capabilities

Imad Abu Reid

101

General UV-Detector Characteristics

Imad Abu Reid

102

II. Fluorescence detectors

The fluorescence detector is a highly sensitive and specific
detector for HPLC. A 1000 fold increase in sensitivity over
UV detection is possible. About 20% of compounds can
naturally absorb UV radiation becoming excited and
subsequently emitting radiation at a lower energy and
longer wavelength than the excitation energy. Many
others can be made to fluoresce through derivatization.
Radiation from a deuterium or xenon source is focused
onto the first grating.
Imad Abu Reid

103

Schematic of fluorescence detector

Imad Abu Reid

104

Imad Abu Reid

105

III. ELECTROCHEMICAL
(AMPEROMETRIC) DETECTORS
Many compounds that can be oxidized or reduced in the
presence of an electric potential can be detected at very low
concentrations by selective electrochemical (EC) measurements.
By this approach the current between polarizable and reference
electrodes is measured as a function of applied voltage. Because
a constant voltage normally is imposed between the electrodes,
and only the current varies as a result of solute reaction, EC
detectors are more accurately termed amperometric devices. EC
detectors can be made sensitive to a relatively wide variety of
compound types.
Imad Abu Reid

106

Imad Abu Reid

107

Schematic of electrochemical detector

Imad Abu Reid

108

IV. CONDUCTIVITY DETECTORS

Conductivity detectors are most commonly used
for detection of inorganic and organic ions
usually after ion exchange chromatography. This
detector measures the conductance of the
mobile phase. The sensitivity of the detector is
largely dependent upon the initial conductance
of the mobile phase.
Imad Abu Reid

109

Imad Abu Reid

110

V. REFRACTIVE INDEX DETECTORS

The refractive index detector is one of the most universal LC
detectors. Anything that changes the refractive index of the
mobile phase can be detected. It is also one of the least
sensitive LC detectors. Refractive index detectors must
always be thermostatically controlled as the refractive index
will change with temperature.
The most common type of refractive index detector is the
beam deflection device. The Fresnel prism can be used for
microbore work. The laser interferometer is the most
sensitive but can be the least reliable.
Imad Abu Reid

111

Schematic of refractive index detector

Imad Abu Reid

112

Imad Abu Reid

113