UNIT 8 HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC)
Structure
8.1

Introduction
Objectives

8.2
8.3

Principle
Instrumentation
Sample Injection System
Column
Packing Material or Stationary Phase
Solvent Supply System
Pumps
Detectors

8.4
8.5
8.6
8.7

Optimization of Separation
Advantages
Comparison with Gas Chromatography
Applications
Polyaromatic Hydrocarbons
Isomeric Compounds
Sugars in Popular Drinks
Drug Abuse
Separation of Nucleic Acids
Analysis of Amino Acids
Partition Chromatography
Ion Chromatography
Chiral Separation of Enantiomers
Ion-Exclusion Chromatography
Speciation Studies

8.8

Interfacing HPLC with Mass Spectrometry

Thermospray Method
Particle Beam Interface
Atmospheric Pressure Chemical Ionization
Electrospray Interface
Moving Belt Interface

8.9 Summary
8.10 Terminal Questions
8.11 Answers

8.1

INTRODUCTION

During early development period of column chromatography using a 50 - 100 cm long

and 1 - 5 cm diameter glass column packed with 100 - 200 m particle size material, it
was realized that column efficiency was very low taking long time for analysis.
Though, it could be increased by decreasing the column length and diameter and also
the particle size of the column material. This could be made possible only after 1960
when technology for producing packing material with particle size of 3 to 10 m was
developed. Further, the new technology required sophisticated instruments operating
at high pressure contrary to classical system where eluent flows under gravity. The
first instrument of liquid chromatograph was constructed by Csaba Horvath at Yale
University, USA in 1964 who describe it as high pressure liquid chromatograph
(HPLC). However, he later called the technique as high performance liquid
chromatography. Thus, the new technique was named as high pressure or high
performance liquid chromatography (HPLC) to distinguish it from the old procedure.
Modern HPLC has emerged from the confluence of need, the human desire to
minimize work, technological capability and the theory to guide development along
rational lines. In some cases, HPLC may detect nanogram or even picogram quantities.
47

It is now the most versatile and widely used technique by chemists for the separation,
qualitative identification and quantitative determination of species in a variety of
organic, inorganic, biological and other complex materials. It is a type of elution
chromatography where the sample, a mixture of solutes, is in a liquid solvent or
mobile phase. The technique is also known by other synonyms such as high speed
chromatography, high resolution chromatography and high efficiency
chromatography and is considered as the most sensitive method with continuous
major developments. HPLC is able to separate macromolecules and ionic species,
labile natural products, polymeric materials, and a wide variety of other high
molecular weight polyfunctional groups. HPLC separations are based on specific
interactions between sample molecules with both the stationary and mobile phases. A
large variety of stationary phases available in HPLC allow a great variety of selective
interactions causing better separations.

Objectives
After studying this Unit, you should be able to

explain the meaning of high performance liquid chromatography,

differentiate between classical liquid chromatography and HPLC,

discuss the basic principle and working of HPLC,

describe various components of instrumentation including stationary and

mobile phases,

describe characteristics of stationary phases in various modes including bonded

phase,

know the solvent delivery system, characteristics of mobile phase and elution
gradient,

understand various detectors used in HPLC,

learn about versatility and advantages of HPLC,

know about various interfaces while using mass spectrometer as detector, and

learn about applications of HPLC for the analysis of a variety of solutes.

8.2

PRINCIPLE

The basic principle of separation by high performance liquid chromatography is

similar to classical liquid or column chromatography (LC) though it differs with
regard to the size of the column and the sample. It differs from LC in terms of speed,
automation, elution time and individual manual assays of collected fractions. In case
of HPLC, microgram amounts of the sample is allowed to pass through a column
containing stationary solid inert phase coated with nonvolatile liquid phase by means
of pressurized flow of a liquid mobile phase where components migrate at different
rates due to different relative affinities. Comparison of column size, characteristics of
packing material and pressure requirements to force the mobility of mobile phase in
classical column chromatography and HPLC are illustrated in Fig. 8.1. According to
another version, HPLC may be considered as partition chromatography where
stationary phase is a second liquid coated on an inert surface and it is immiscible with
the liquid mobile phase. According to the stationary liquid phase, the technique may
be subdivided into two types; liquid-liquid and liquid-bonded phase chromatography.
These differ from each other in the way stationary phase is held on to the support
particles of the packing. In LLC, the polar liquid is physically adsorbed on to an inert
surface where it competes with the mobile phase. However, in case of bonded phase
chromatography, liquid is chemically bonded making it more stable.

48

(a)
Particle size:
>150 m
Column diameter: 10-50 mm
Column length:
50-200 cm
Pressure:
< 1 atm

(b)
40-70 m
1 3 mm
50-100 cm
30-50 atm

(c)
5-10 m
2 6 mm
10-50 cm
100-200 atm

Fig. 8.1: Comparison of characteristics of various forms of liquid chromatography:

(a) Classical column chromatography; (b) HPLC with pellicular packing; (c)
HPLC with microparticulate packing

In order to achieve the desired separation by HPLC, several operating conditions

including retention time, pressure and number of plates need to be optimized. A major
interest is short analysis time, or the plate count needed to accomplish a difficult
separation. First of all, a proper HPLC system such as adsorption, bonded-phase,
reverse phase, ion-exchange, exclusion, affinity or any other form of chromatography
must be selected. Then, all the parameters in the equations as mentioned in Unit 4 that
depend on the properties of the mobile and stationary phases are determined. As
already described in Unit 4, these are relative retention (), capacity factor (k) and
the plate count (N). It is desired that the compounds of interest should need at least ten
times longer time to travel the column length than the unretained peak. Further, the
viscosity of mobile phase and the diffusion coefficients of the solutes in the mobile
phase are also of concern besides other characteristics of column packing.
The plate height (H = L/N where, L is the column length and N the number of plates) is
reduced by the particle diameter (dp) and may be represented as
h = H/dp = L/N.dp

... (8.1)

It actually states the number of particle diameter (dp) that constitutes one plate height.
Thus, reduced velocity may be represented as
v = u.dp/ DM

(8.2)

where, DM is the diffusion coefficient of solute in the mobile phase. It may be

considered as the ratio of the time required to displace solute molecules a distance
equal to one particle diameter to the time needed for the same displacement by
molecular diffusion. It expresses the balance between mass transport by diffusion or
molecular motion across a single particle. Substituting the value of u (= L/tm), reduced
velocity may be expressed as

49

v = L.dp/tm.DM

(8.3)

Thus, the complete equation for the dependence of the reduced plate height may be
represented as modified van Deemter equation
h = B/v + A.v0..33 + C.v

(8.4)

where, B = 1.2 for solid core (pellicular) packing and 2.0 for completely porous
column packing. Also, A = 0 for well packed column and C = 0.05 for porous particles
decreasing to 0.003 for pellicular particles. No theory accurately describes the
dispersion from flow in homogeneity in the mobile phase. A logarithmic plot of
Eq. (8.4) is shown in Fig. 8.2. The reduced plate height has a minimum value in the
range 2-3 for intermediate region of velocities where reduced velocity is 3 -5.
It may be observed from Fig. 8.2 that A term dominates all along whereas B term
arising from axial and longitudinal diffusion, dominates at law reduced velocities. This
region of h/v curve is usually avoided. At high velocities, however, C term responsible
for increase in reduced plate height, dominates. As explained earlier, C term contains
the contributions from mass transfer kinetics and stagnant pockets of mobile phase.
You can see that Eq. 8.4 representing the reduced plate height is independent of
particle diameter of the column packing. The constants A, B and C are dependent on
the packing of column. The number of plates in a reasonable time may be optimized
while operating the

Fig. 8.2: Logarithmic plot of reduced plate height, h against reduced velocity, with a
set of values of constants, A = 1, B = 2 and C = 0.1

column at the minimum in the h/v plot of Fig. 8.2. The column length and particle size
of the tm and N are chosen under the experimental conditions of eluent viscosity as
illustrated by the following example.
Assuming desired plate counts, N = 5000, reduced plate height, h = 5 and a column
length, L = 250 mm, required plate diameter, dp may be calculated using Eq. (8.1).
dp = L/N.h = 250/5000 5 = 1/100 mm = 10 m
Similarly, using viscosity parameter () and specific column resistance () for a fully
porous packing, pressure drop (P) may be calculated using the expression.

P =

50

LvDM
d 3p

N 2 h 2
tM

(8.5)

Combinations of column lengths and particle sizes including operating pressures for
different plate counts and retention times are available in literature.

SAQ 1
What are the various synonyms used for HPLC. Write each one of them.
...
...

8.3

INSTRUMENTATION

General instrumentation for HPLC has following components.

i)

One or more solvent reservoirs for the mobile phase.

ii)

A pump to deliver the mobile phase with varying range of pressures up to

several hundred atmospheres to achieve reasonable flow rates.

iii)

Sampling valves or loops where the sample may be injected into the flowing
mobile phase. Sample may be dissolved in mobile phase.

iv)

A guard column or an on-line filter to prevent contamination of the main

column.

v)

A pressure gauge, inserted in front of the separation column, to measure column

inlet pressure.

vi)

Separation column containing packing to accomplish desired separation. These

may be modified silica gel, ion-exchange resin, gel or some other unique
packing.

vii)

A detector capable enough of measuring the solute concentrations.

viii) Display and recording device for plotting time vs peak intensity.
Besides, other electronic accessories for data manipulations are also required. These
are schematically shown in Fig. 8.3.

Fig. 8.3: Schematic illustration of various components of HPLC instrument

51

The individual components are described below:

8.3.1 Sample Injection System

It is a limiting factor in the precision of HPLC measurement because of reproducibility
with which samples may be introduced onto the column packing. Insertion of the
sample into the column must be through a narrow plug so that peak broadening is
minimized and the system should have no dead volume by itself. Generally, samples
are dissolved in a mobile phase solvent to avoid solvent peak and 10 to 50 L is
introduced through micro sampling valves. These devices form an integral part of
liquid chromatography equipment having interchangeable loops with a choice of
sample size from 5 to 500 L. The most widely used method of introduction is based
on sampling loop as shown in Fig. 8.4. It is filled by thoroughly flushing it using a

Fig. 8.4: Schematic of injector valve with external sample loop in a microvolume sampler

sample solution by means of a microsyringe at pressures up to 7000 psi. A rotation of

the valve rotor places the sample filled loop into the high pressure mobile phase
stream whereby the sample is sent to the column. The system can be located within a
temperature controlled oven if handling at elevated temperatures is required. Many
HPLC instruments incorporate an auto sampler with an automatic injector that can
inject variable volumes as per requirement. In stopped flow injection method, pump is
turned off till atmospheric pressure is attained, syringe is inserted and the sample
injected. The flow of sample can be brought to zero and rapidly resumed by diverting
the mobile phase using a three way valve placed before the injector. This method is
especially very useful for very high pressures. For best results, a two to fivefold excess
of sample should be passed through the loop to ensure that previous sample has been
purged thoroughly.

8.3.2 Column
It is the heart of the HPLC instrument where actual separation occurs. Separation
column in HPLC is usually made of heavy wall, glass lined metal or 316 grade
stainless steel tubing, that can withstand high pressure and which is inert to the
chemical corrosion due to mobile phase. The interior of the tubing must be smooth
with a uniform bore diameter. Straight columns that can be operated in vertical
position are preferred. Some typical tubing materials used in HPLC column are listed
in Table 8.1.

52

Table 8.1: Column Tubing Materials and its Uses

Material

Use

316 Stainless steel (SS)

General utility material, good for high

pressure system

Poly (ether-ether) ketone

Inert to most organic solvents except

methylene chloride, THF, DMSO and conc.
Sulphuric and nitric acids. Holds pressures
up to 5000 psi (34MPa).

(PEEK)

Good for metal-free biological systems.

Tefzel

Inert. Common for metal-free applications.

Titanium

Withstands pressures up to 5000 psi,

corrosion resistant; expensive

Fused silica Glass

Used for capillary LC.

Glass

Limited pressure range.

Glass-lined SS

Inert, withstands pressures but difficult to

know when the glass is broken.

Column fittings and connectors must be so designed that void volume is zero avoiding
unswept corners. Column length ranges10 to 30 cm with inner diameter of 2 to 5 mm
providing 40,000 to 60,000 plates per inch. However, shorter columns of 3 to 8 cm are
also used for fast separations but in such cases, sample size will become limited. The
length of the column may not only affect the resolution of a given separation the
longer the column the larger number of plates but also the speed of separation.
Standard lengths vary with the manufacturer but most common values are 30, 25, 15,
12.5, 10 and 7.5 cm. It may be noted that shorter columns are described as high speed
columns. The columns packed with the finer particles are more expensive than the
standard 5 m packing.
Guard column: In order to increase the life of analytical column, a short guard
column, also called precolumn, is placed before the main column as shown in Fig. 8.3.
It removes contamination from the solvent. Guard column serves to saturate the
mobile phase with the stationary phase so that losses of stationary phase in the column
are minimized. However, it is essential that the composition of the guard column
should be similar to that of the analytical column but its particle size may be larger to
minimize the pressure drop.

8.3.3 Packing Material or Stationary Phase

The packing used in modern HPLC consists of small, rigid particles having a narrow
particle size distribution. The most common packing material used for LC is prepared
from silica (acidic) and alumina (basic) particles which are synthesized by
agglomerating submicron size particles under conditions that lead to larger particle
diameter. These are often coated with thin organic films which are physically or
chemically bonded to the surface. For nearly all HPLC applications, chemically
modified or unmodified micro particulate silicas of 3, 5 or 10 m diameter are
preferred. This form of LLC, in which both monomeric and polymeric phases have
been bonded to a wide range of support materials, is called bonded phase
chromatography (BPC). Characteristics of typical packing materials used in HPLC are
listed in Table 8.2. The particles used in HPLC, which are totally porous
(macroporous) or superficially porous (pellicular) support, may be spherical or
irregular in shape but it is essential that the size range is as narrow as possible to

53

Table 8.2: Characteristics of Some Commercial HPLC Column Packing

Materials
Type
Silica

Pellicular

Microprous

Corasil (37-50m)

Lichrosorb (5,10 & 20 m)

Vydac (30-40m)

Micropak (5&10 m)
Porasil (15 & 20 m)
Spherisorb (5 m)

Alumina

Perisorb PA

Micropak Al (5 & 10 m)
Spherisorb Al

ensure high column efficiency and permeability. These adsorbent packings retain
solute molecules almost exclusively on the internal surface of the pores, thus,
separating these from others. Various types of bonded phases used in HPLC are
schematically shown in Fig. 8.5.

Fig. 8.5: Various shapes of stationary phase packings used in HPLC:

(a) Bonded (spherical) phase; (b) Irregular large porous phase;
(c) Pellicular particle beads and (d) Porous microparticle

The characteristics of various types of bonded phases are described below:

54

A.

Spherical bonded phase: These spherical packings consist of a solid, spherical

nonporous core (usually a glass bead) with a layer of attached functional groups
forming an outer shell containing unmodified or modified silica gel, resin,
polyamide, etc. Various functional groups are used depending on the nature of
solutes to be separated.

B.

Porous layer beads: A porous or pellicular layer bead type packing material
consists of a solid, spherical with an average particle diameter 30-40 m coated
with a thin porous outer shell, typically of 1-3 m thick. It may be a silica gel
layer, a network of small spherical particles bonded to the solid core. It may also
be monomeric or polymeric organic phase. Surface areas of the porous layer
beads range from 5 to 15 m2/g . These materials are easy to be packed because
of its dense core but suffer from limited sample capacity due to small surface
areas. Porous layer packings exhibit good efficiency because of improved mass
transfer within the stationary phase. Longer columns are possible because the
pressure drop is lower due to larger particle size of porous layer supports.
Thicker coatings give rise to slower mass transfer but have increased sample
capacity.

C.

Porous particles: Totally porous particles have a large surface area in the range
100 to 860 m2/g with average being 400 m2/g. The mean pore diameter is
inversely related to the specific surface area where small molecules enter the
pores. The particles can be packed into the HPLC column of efficiencies up to
800 theoretical plates per centimeter if 5 m particle sizes are used. However,

larger particles give proportionately small number of theoretical plates whence

the efficiency of separation goes down.
D.

Macroporous particles: These are recently introduced graphitized carbon and

styrene-divinylbenzene polymers having large channels besides micropores. The
rigid, porous polymeric macroporous beads do not swell or shrink with changes
in the ionic strength of the mobile phase (can be used over an extended pH range
of 1 and 13) or deform at high velocities and are most suited for separations in
nonaqeous media. These materials have increased the choice of stationary
phases and the scope of HPLC, particularly for highly polar and basic
substances.

An illustration of various types of bonded phases used in HPLC is shown in Fig. 8.6
where different topographies are obtained depending on the nature of the ligand . It
may be noted that different packing materials are used in different type of techniques
of adsorption, partition, ion-exchange, size exclusion chromatography.

(a)

(b)

(c)

Fig. 8.6: Various shapes of bonded HPLC column packing materials

(a) Types of bonded phases, (b) Topography of ligands and (c) Size of ligands

SAQ 2
Explain why small particle size is required in HPLC? How is it important in attaining
higher efficiency?
...
...
...

SAQ 3
Choose the correct answer from the choices given.
i)

Which one of the following is the most appropriate particle size (in m) for
packing material in HPLC?
a) 1-5

ii)

b) 3-5

c) 10-20

d) 20-50

Which one of the following column length (in cm) should be used for faster
HPLC separation?
a) 2-5

b) 5-10

c) 10-15

d) 20-30

55

iii)

Which of the following materials meet the requirements to fabricate HPLC

column?
a) Glass lined metal

iv)

c) Stainless steel

d) Steel

What is the average surface area ( in m2/g) of porous particles in HPLC column?
a) 100

v)

b) Quartz

b) 300

c) 800 d) 400

Which one of the following ranges of flow rates (in mL/min) should be adequate
for analytical HPLC?
a) 0.02 1.0 b) 0.05 - 2.0

c)

1.0-2.0

d) 0.5 2.0

Let us now study about the stationary phases used in various chromatographic modes.
i)

Adsorption Chromatography
In majority of the cases of adsorption chromatography, silica column packings
are used where main mechanism is the interaction of its OH groups with the
polar or unsaturated functional groups of a solute/solvent molecule by hydrogen
bonding or dipole interaction. The slightly acidic silanol (Si-OH) groups in
silica gel are at the surface and extend out from the surface in the internal
channels of the pore structure. The number and topographical arrangement of
the several types of OH groups, as shown in Fig. 8.7, determine the activity of
the adsorbent and thereby the retention of the solutes. These OH groups can be
divided into three types:

silanol (free OH),

siloxane bond (Si-O-Si) and

hydrogen bond (Si-OHO).

Fig. 8.7: Structure of silica gel depicting the various types of hydroxyl groups that
interact with the functional groups of solute/solvent molecules

Each of these groups has different activity that increases in the following order:
Bound < free < H-bond.
According to current models of adsorption process, it is assumed that adsorption
sites are completely covered by either of solute or solvent molecules that are
adsorbed depending on their relative strength in this competitive interaction. The
56

competition between the solute and the mobile phase molecules for an active
site provides the driving force and selectivity in separations. Interaction between
a solute molecule and the adsorbent surface is best when functional groups
overlap adsorption sites. Adsorption chromatography is less influenced by
difference in molecular weight but certainly more by functional groups. For
compounds of low to moderate polarity, adsorption chromatography often
makes possible the separation of complex mixtures into classes of compounds
with similar chemical functionality. Typical examples of group separations are
polynuclear aromatics from a petroleum sample and the triglycerides from a
liquid extract.
ii)

Partition Chromatography
It can be subdivided into liquid-liquid chromatography (LLC) and bonded phase
chromatography (BPC), the difference being in the method by which stationary
phase is held on the support particles of the packing. In case of LLC, a liquid
stationary phase is retained on the surface of the packing by physical adsorption.
With bonded phase, the stationary phase is bonded chemically to the surface of
inert support. Of late bonded phase has become predominant over liquid phase
because of certain disadvantages. The packings for bonded phase are prepared
from rigid silica or silica based compositions. These are formed as uniform,
porous, mechanically sturdy particles commonly having diameters 3, 5 or 10
m. The surface of fully hydrolysed silica is made up of chemically silanol
groups. The most useful bonded phase coatings are siloxanes formed by the
reaction of hydrolysed surface with an organochlorosilane as shown below:
CH3
Si

OH + Cl

Si

CH3
R

Si

Si

R + HCl

CH3

CH3

CH3

CH3
Si
Si

OH + Cl

Si

Cl

H2O

CH3
Si

OH

Si

Si

Si

CH3

CH3

CH3

CH3

Si
CH3

Si

Cl

Cl

CH3

Surface coverage by silanization is limited to 4 mol/m2 or less because of steric

effects. The unreacted SiOH groups impart an undesirable polarity to the
surface, which may lead to chromatographic tailing of the peaks. In order to
avoid this effect, siloxane packings are often capped by further reaction with
chloromethylsilane that can react with many of the unreacted silanol groups.
Two types of partition chromatography have been recognized based on relative
polarities of stationary phase and mobile phase. In normal phase LC or HPLC,
stationary phase consists of highly polar water or triethyleneglycol supported on
silica or alumina particles and a nonpolar mobile phase solvent such as hexane is
used. In contrast, in the reversed phase chromatography, the stationary phase is
nonpolar, often a hydrocarbon and the mobile phase is polar such as water,
methanol or acetonitrile where most the polar component appears first. Perhaps
three quarters of all the HPLC is currently being carried out in columns with
reversed phase.

57

Most commonly, the R group of the siloxane in these coatings is a n-octyl (C-8
chain) or n-octadecyl (C-18 chain). With such preparations, the long chain
hydrocarbon groups are aligned parallel to one another and perpendicular to the
particle surface, giving a brush or bristle-like structure as illustrated in Fig. 8.6.
The relationship the between polarity of the sample with that of the column
packing material and mobile phase is illustrated in Fig. 8.8. Retention increases
with the hydrophobic character of the solute samples. Generally, the lower the
polarity of the mobile phase, the higher is its eluent strength. The effect of chain
length of the alkyl group upon column performance is illustrated in Fig. 8.8
where it is observed that longer chains produce packings that are more retentive.
For example, maximum sample size for a C18 packing is roughly double that for
a C4 preparation under similar experimental conditions.

Fig. 8.8: Relationship between the polarity of the sample with that of the packing
material and the mobile phase in reverse phase HPLC

In commercial normal-phase bonded packings, the R in the siloxane structure is

a polar functional group such as cyano (C2H4CN), diol
(C3H6OCH2CHOHCH2OH), amino (C3H6NH2), and dimethylamino
(C3H6N(CH3)2). The polarities of these packing materials vary over a
considerable range with the cyano type being the last polar and the amino types
the most. Diol packings are intermediate in polarity. With normal phase
packings, elution is carried with relatively non-polar solvents such as ethyl
ether, chloroform and n-hexane.
iii)

Ion-exchange Chromatography
In this case, column packings have charge bearing functional groups attached to
a polymer matrix. The functional groups are permanently bonded ionic groups
associated with counterions of the opposite charge. Some ion-exchange
packings bear negatively charged groups and are used for exchanging cationic
species whereas others are designed for exchanging anionic species. Similarly,
some functional groups such as COOH or -PO32 have weak acidic or basic
properties whereas some others have considerable affinity for heavy metal
cations. Several structural types of packings, as shown in Fig. 8.9, have been
used in ion-exchange HPLC.
Of these, the pellicular type consists of a resin coating, about 1-2 m thick, on a
glass bead of 30-40 m diameter. Superficially porous resins are obtained by
coating glass beads with a thin layer of silica microspheres on which ion
exchanger is bonded. This increases the interface between the resin and mobile
phase. Either type of these packings have low exchange capacity, 0.01 0.1
meq/g. The exchanger may also be bonded to silica microparticles by means of
silylation reactions or polymerized into pores of a superficially porous silica gel.

58

(a)

(b)

(c)

(d)

Fig. 8.9: Various structural types of ion-exchange packings: (a) pellicular with
ion-exchange film; (b) exchanger beads coated superficially with porous resin;
(c) macroreticular resin bead and (d) anion exchanger surface sulfonated and
bonded electrostatically

During preparation of ion exchanger by silylation, a vinyl group is chosen for R3

in -SiOSiR1R2R3 leading to a vinylated silica which is then polymerized with
styrene.
CH = CH2 + CH = CH2

CH

CH2

C 6H 5

CH

CH2

C6H5

Afterwards, the bonded phase is treated with chloromethyl ether and

subsequently trimethylamine or hydroxyethyldimethylamine to prepare the
quaternary amine exchanger as is shown below:
CH2

CH2 CH

CH2 + ClCH2OCH3

C6H5

CH2 CH2

CH

CH2 + CH3OH

C6H5CH2Cl

RNH2

N(CH3)2CH2CH2OH
CH2

CH2 CH

CH2

+
C6H5CH2NH2-R
Weak anion exchanger

CH2

CH2

CH

CH2

+
C6H5CH2N(CH3)3
Strong anion exchanger

Hydrophilic polymers allow the separation of proteins, nucleic acids and other
large ionic molecules. The microporosity of these ion exchangers minimizes
possible exclusion effects.
iv)

Size Exclusion Chromatography

In this case, column packings are either semi-rigid, cross-linked macromolecular
polymers or rigid, controlled pore size glasses or silicas. The semi-rigid
materials swell and care must be taken to their use limited to a maximum
pressure of 300 psi due to bed compressibility. The styrene-divinyl benzene
polymers allow fractionation within a molecular weight range of 100 to 5000
million. Partially sulphonated polystyrene beads are compatible with aqueous
systems and non-sulphonated ones with non-aqueous systems with bead
diameters ~5 m.
Another class of hydrophilic porous packing is prepared by suspension
polymerization of 2-hydroxyethyl methacrylate with ethylene dimethacrylate.
These packings can withstand pressures up to 3000 psi and are usable with
aqueous systems and with a variety of polar organic solvents. Porous glasses and
silicas cover a wide range of pore size diameters. For example, a series of
particle size diameters and operating ranges of molecular weights are listed in
Table 8.3.

59

Table 8.3: Correlation of Pore Size Diameter and Operating Range of Mol. Wt
Pore-size diameter
(m)

Operating range

1000-8000

10

1000-30000

25

2500-125000

55

11000-350000

150

100000-1000000

250

200000-1500000

(Daltons)

These packings are chemically resistant at pH <10 and can be used with aqueous
and polar organic solvents. With nonpolar solvents, it is desirable to deactivate
the surface by silylation. Porous inorganic packings have distinct advantages
over organic exclusion packings. The surface of a typical hydrophilic packing
has the following structure;
Si

CH2

CH2 CH2

CH2 CH2

C
H

CH2OH

Columns can be used routinely and indefinitely after calibration, without any
possibility of sample contamination or biodegradation. Properties of some
commercial size exclusion packings are listed in Table 8.4.
Table 8.4: Properties of Typical Commercial Packings for Size-Exclusion
Chromatography
Type

Polystyrenedivinylbenzene

Silica

v)

Particle
Size, m

Average Pore

Molecular eight

Size,

Exclusion Limit

10

102

700

103

(0.1 20) 104

104

(1 20) 104

105

(1 20) 105

106

(5 >10) 106

125

(0.2 5) 104

300

(0.03 1) 105

500

(0.05 5) 105

1000

(5 20) 105

10

Ion Chromatography
It differs from ion-exchange chromatography in the nature of exchange resins.
The technique involves an ion-exchange column and a means of suppressing
(removing) ionic species other than the sample ions in the eluting mobile phase
to facilitate detection of the sample by a conductivity monitor as schematically
illustrated in Fig. 8.10.

60

Electric
integrator

Fig. 8.10: Schematic diagram of ion chromatograph with separation column

The column packing consists of a neutral polymer core of ~ 10 m diameter

depending on whether the packing will be used for the separation of cations or
anions. Contrary to the conventional ion-exchange chromatography where core
is sulphonated or aminated leading to the formation of sulfonic acid or
quaternary amine groups, in ion chromatography, a monolayer of aminated or
sulphonated polymeric anion exchange beads is used.
Similarly, for a cation exchanger, there would be an intermediate layer of
aminated groups covered by a thin layer of sulphonated resin beads. Due to the
proximity of all the active sites to the eluent-resin interface, this type of
exchanger has favorable mass transfer characteristics. It has low exchange
capacity, about 0.020 meq/g of copolymer. In most applications, silica based
materials are inappropriate due to their degradation in the presence of aqueous
eluents and their poor selectivity for some ionic species. The eluent passes
through a suppressor column where the eluting or background electrolyte is
effectively removed by converting it into water or, water and carbon dioxide i.e.,
sodium ions are replaced by hydronium ions or methylsulfonate ions with
hydroxyl ions.
A miniaturized self-regenerating suppressor cartridge incorporating an
electrolysis cell is also available where H3O+ and O2 are continually formed by
the electrolysis of a stream of deionized water passing through an anode
compartment and similarly, OH and H2 are formed in a cathode compartment.
Both compartments are separated from the eluent either by cation or anionexchange membranes depending on whether anionic or cationic analytes are to
be separated.
vi)

Chiral Chromatography
Quite often only one enantiomer possesses the desired therapeutic activity
whereas the other may be inactive or even harmful. The separation of
enantiomers by HPLC using chiral stationary phase (CSP) is based on the
formation of transient diastereoisomeric compounds between the

61

enantiomorphs of the solute and the chiral selector which is an integral part of
the stationary phase. The difference in stability between these complexes results
in difference in their retention times, the enantiomer forming the less stable
complex being eluted first.
A large number of chiral phases are commercially available. All of these are
coated on silica gel support. The coating itself is a polymeric material to which
an optically active isomer is bonded. For example, the l form of the amino acid,
proline has been bonded to polystyrene-p-divinylbenzene, a cross linked
copolymer to give an optically active stationary phase for the separation of
racemic mixtures of amino acids. In this case, Cu2+ ions are introduced into the
solution of the analyte enantiomers to be separated whereby a ternary complex,
as shown in Fig. 8.11. is formed between the stationary phase, amino acid anion
and Cu2+. The formation constant for this complex differs for d and l forms of
the analyte amino acid; thus, making their separation possible.

Fig. 8.11: Illustration of a ternary complex formed between an L-proline bonded phase,
an analyte amino acid and a Cu2+ ion

Cyclodextrin-bonded stationary phases have been demonstrated to be

particularly efficient in resolving structural isomers. Some examples areprostaglandin A1, A2 and B1B2, - and -naphthols, o,o and p, p-biphenyls and
the ortho-, meta- and para- isomers of nitrophenol, nitroaniline, xylene, cresol
and aminobenzoic acid.
Recently introduced graphitized carbon and new generation of rigid porous
polymeric microbeads based on styrene/divinyl benzene as alternatives to silica
can be used over a wide range of pH between 1 to 13. Some examples of column
packings used in HPLC and their applications are listed in Table 8.5.
Table 8.5: Some Typical Column Packings Used in HPLC
Packing

Mode of HPLC

Applications

Microparticulate silicas;
spherical or irregular
particles; mean particle size
3, 5 and 10m
chemically modified
versions of the above
(bonded-phase packings)

LSC (adsorption)

Non-polar to moderately polar

mixtures, e.g., polyaromatics, fats, oils,
mixtures of isomers

Octadecyl (ODS or C18)

BP (bonded phase)
and Ion Pair
Chromatography
(IPC)

Wide range of moderately polar

mixtures, e.g., pharmaceuticals and
drugs, amino acids

Octyl (C8)

BPC, IPC

More polar mixtures, e.g., pesticides,

herbicides, peptides, metabolites in
body fluids
Table continued on next page

62

Short chain (C3 or less)

BPC, IPC

IPC applications of above three

packings include bases, dyestuffs and
other multiply charged species; used
instead of IEC

Diol

BPC

Very polar and water-soluble

compounds, e.g., food and drink
additives

Nitrile

Normal phase and

BPC

Alternative to silica,
can give better results

Aminoakyl

BPC

Carbohydrates including sugars

Anion and cation

exchangers
(tertiary amine or sulphonic
acid)

IEC (Ion-exchange
chromatography)

Ionic and ionizable compounds, e.g.,

vitamins, water-soluble drugs, amino
acids, food and drink additives

Controlled porosity silicas

(chemically modified to
reduce adsorption effects)

Size exclusion
Chromatography

Polymer mixtures, screening of

unknown samples. Increasing use for
separating mixtures of smaller
molecules before other modes of HPLC

Chiral amino acids bound to

aminopropyl

Chiral
Chromatography
(CC)

Chiral peptides

CC

Cyclodextrins

CC

Mixtures of enantiomers especially of

drugs

It may be mentioned that besides various modes of HPLC discussed above, thin layer
chromatography is another mode which is already discussed in Unit 6. Hence, it is not
included in the discussion here.

SAQ 4
Complete the following sentences with suitable words.
i)

Silylation is a process where...................................................................................

ii)

While using normal phase packing, elution is carried out using.............................

.

iii)

Functional groups such as ......................................have weakly acidic properties.

iv)

Styrene-divinyl benzene polymers allow fractionation of substances having

molecular weight in the range of.............................................................................

v)

Background electrolyte is effectively removed in.....................................where it

is converted into .....................................................................................................

vi)

Chiral stationary phase is used for the separation of .............................................

and is based on the formation of ............................................................................

8.3.4 Solvent Supply System

Nature and composition of mobile phase in LC plays an important role as it provides a
dimension in terms of retention time for experimental manipulation. In case of HPLC,
high purity solvents without any dissolved gases should be used because any impurity
may affect the retention time and hence separation of the constituents. The eluent
system consists of reservoirs from which one or more solvents can be selected.
63

Essential features of a modern HPLC system includes flow control and inlet filter
through a Millipore filter under vacuum. Also degassing facility such as a supply of an
inert gas is a must. It helps in removing dissolved gases that may have adverse effect
on the column performance. The general criteria for the selection of a mobile phase
are:

It should dissolve the sample.

It should keep the column stable.

It should be compatible with the detector.

It should be immiscible with the stationary phase.

Its viscosity should not be high.

Active fluorides should be avoided when using glass column.

The eluting power of a solvent is determined by its overall polarity, the polarity of the
stationary phase, and the nature of sample components. The capacity factor, k, is
controlled by the strength of solvent which can be easily predicted in adsorption
chromatography. Snyder has defined solvent strength parameter, o, as the adsorption
energy per unit area of adsorbent.
Some common solvents used in adsorption chromatography are listed in Table 8.6 in
the order of increasing solvent strength. It also includes adsorption strength of the
various functional groups of solute molecules. Such a list is also called eluotropic
series of solvents. It has been observed that log k for a given solute varies linearly
with o. Other properties of solvents which must be taken into account include boiling
point and viscosity, detector compatibility, flammability and toxicity. Generally, the
lower boiling and hence, the low viscosity solvents give higher chromatographic
efficiency and lower back pressure.
Table 8.6: Solvent Strength Parameter, o and the Physical Properties of Selected
Solvents Used in HPLC
Solvent

Pentane
Hexane
Cyclohexane
Carbon disulphide
Carbon tetrachloride
1-Chlorobutane
Di-isopropyl ether
2-Chloropropane
Benzene
Diethyl ether
Chloroform
Methylene dichloride
Tetrahydrofuran
Acetone
1,4-Dioxane
Ethyl acetate
1-Pentanol
Acetonitrile
Methanol
Water

64

o(SiO2)

o (Al2O3)

0.00

0.00
0.00
0.04
0.15
0.18
0.26
0.28
0.29
0.32
0.38
0.40
0.42
0.45
0.56
0.56
0.58
0.61
0.65
0.95
Large

0.05
0.14
0.14

0.25
0.38
0.26

0.47
0.49
0.38
0.50

Viscosity,
20C (mN
sec m2)
0.23
0.313
0.980
0.363
0.965
0.47
0.379
0.335
0.65
0.23
0.57
0.44
0.55
0.32
1.54
0.45
4.1
0.375
0.60
1.00

Refractive
index, 20C
1.358
1.375
1.426
1.628
1.460
1.402
1.368
1.378
1.501
1.353
1.443
1.425
1.407
1.359
1.422
1.370
1.410
1.344
1.329
1.333

As the column flow rate is proportional to the product of the linear velocity and the
cross sectional area of the column, the solvent consumption is considerably reduced as
illustrated in Table 8.7.
Table 8.7: Solvent Consumption for Different Diameter HPLC Columns
ID
(mm)

Flow rate for linear velocity of 0.14

cm/sec (mL/min)

Volume in a
8 hr day (mL)

0.51

0.02

6.9

0.71

0.027

13

1.02

0.044

24

1.29

0.079

38

1.59

0.12

57

4.6

1.00

480

When two more solvents with a fixed composition are used, it is called isocratic
elution. This, however, is a very cumbersome process and instead gradient elution is
used.
Gradient Elution: It involves continuous change in the composition of the mobile
phase by allowing a more polar solvent to flow into the reservoir containing a less
polar one, whence the mixture flows to the column as illustrated in Fig. 8.12. Thus, a
complex mixture of solutes that cannot be separated by isocratic separation can be
separated by gradient elution. It is especially useful for separating components that
differ widely in polarity. For gradient elution using a low pressure mixing system, the
solvents from different reservoirs are fed to a mixing chamber and then mixed solvent
is pumped into the column.

Fig. 8.12: Schematic illustration of low pressure gradient using three solvents
of different polarity

Time proportionating electrovalves used in modern instruments are regulated by a

microprocessor; thus, the resolution for each chromatogram. It can reduce the run
time and increase the sensitivity. As the gradient develops, tailings are made to elute
quicker.
The commercial liquid chromatographs are designed to mix two or more solvents in a
progressive manner from 0 to 100% of one component. If one of the solvents gives an
appreciable response at the detector, then the generation of a solvent gradient will also
introduce a baseline drift in response. In such a case, column will also need time to
65

regenerate the starting solvent composition each time a fresh gradient run is started
and ideally, a blank gradient is run between samples to prevent the occurrence of
artifact peaks which can be observed. This can make gradient elution seem slower than
literature values. It may be noted that gradient elution produces effects similar to
temperature programming in gas chromatography.

8.3.5 Pumps
A variety of pumps are used to maintain flow rate and pressure of the mobile phase.
Also a degasser is needed to remove dissolved air and other gases from the solvent. A
desirable feature of the delivery system is the capability of generating solvent gradient.
A pump should be able to operate up to a pressure of 100 atm (1500 psi) though in
some cases 400 atm (6000 psi) is desired. For most analytical columns, only moderate
flow rates of 0.5 2 mL/min may be required. However, for microbore columns, low
flow rates of only a few microlitres/min may be sufficient. Also, a pump should have a
small hold up volume. Some typical pumps are described below:
i)

Reciprocating piston pump: It is the most popular type of pump as it is

inexpensive and can permit a wide range of flow rates. It consists of a small
motor driven piston moving rapidly back and forth in a hydraulic chamber that
may vary from 35 to 400 L. The piston sucks in solvent from the mobile phase
reservoir by means of check valves. Usually, a hydraulic fluid transmits the
pumping action to the solvent via a flexible diaphragm; thus, minimizing solvent
contamination and corrosion problems with pump parts.
A wide range of flow rates may be obtained by varying either the stroke volume
during each cycle of the pump or the stroke frequency. Delivery of solvent
through reciprocating pump is continuous without any restrictions on the
reservoir or operating time. These have very small initial volume and accurate
elution gradient. Its advantages include small internal volume (35 to 400 L),
their high output pressures (up to 10,000 psi), their ready adaptability to gradient
elution and their constant flow rates which are largely independent of column
back pressure and solvent viscosity.

66

ii)

Syringe type displacement pump: These pumps work through positive solvent
displacement by a mechanically driven piston at a constant flow rate. The piston
is actuated by a screw feed drive through a gear box usually run by a digital
stepping motor. The rate of solvent delivery is controlled by changing the
voltage of the motor. The solvent chamber has finite capacity of 250-500 mL
which may be refilled if need be. Pulse less flow is achieved along with high
pressure capability of 200-475 atm.

iii)

Constant pressure pump: In this type of pump, pressure delivered through a

large piston drives the mobile phase. Since the pressure on the solvent is
proportional to the ratio of the area of the two pistons, usually between 30:1 and
50:1, a low pressure gas source of 1-10 atm can be used to generate high liquid
pressures (1-400 atm). A valving arrangement permits the rapid refill of the
solvent chamber whose capacity is about 70 mL. This system provides pulse less
and continuous pumping, including high flow rates for preparative applications.
This type of pump is useful for pumping columns but inconvenient for solvent
gradient columns.

iv)

Pneumatic pump: These types of pumps are simple, inexpensive and pulse free
but suffer from limited capacity and pressure output. In this case, mobile phase
is contained in a collapsible container housed in a vessel that can be pressurized
by a compressed gas. These are not amenable to gradient elution and are limited
to pressures less than 2000 psi.

Most commercial instruments are equipped with computer controlled devices for
measuring the flow rate by determining the pressure drop across a restrictor
located at the pump outlet. Any difference in signal from a preset value is then
used to decrease or increase the speed of the pump motor. Composition of
solvents may be continuously varied or in a stepwise fashion.

8.3.6 Detectors
A detector is an important part of the HPLC instrument and should be chosen very
carefully for selective separation and accurate determination. The single most crucial
factor is continuous detection based on the progress of separation of a component
which may be immediately displayed and then recorded. However, a good detector
must have following characteristics:

It should have linear response to solute concentration in the range 0.1 g/mL to
1 ng/mL.

It should respond to solute only and not to the solvent or change in solute to
solvent ratio.

It should be insensitive to change in temperature, pressure and flow rate.

Though highly sensitive detectors have been developed for HPLC but there is no
universal detector which could be used for all kinds of samples and for all
concentration ranges. The choice of a detector depends on the problem at hand though
sometimes more than one detector may be used. These are of two basic types.
i)

Bulk Property Detectors

These types of detectors measure on overall change in a physical property of the
mobile phase with and without solute e.g. refractive index, dielectric constant,
density, electrical and thermal conductivity, vapour pressure etc.. These types of
detectors are somewhat insensitive and require good temperature control.

ii)

Solute Property Detectors

These respond to a physical property of the solute that is not exhibited by the
pure mobile phase. These are highly sensitive with a detection signal for a few
ng or even lesser amount of sample. For example absorbance, fluorescence,
diffusion current and electrochemical detectors are considered in this category.

Besides low detection limit, a HPLC detector must meet following requirements:

Selective response towards one or more classes of solutes.

It must be small and compatible with liquid flow.

It should have good stability and reproducibility.

It must have low dead volume to minimize extra-column band broadening.

It must have high reliability and ease of use.

It must have small response time, at least 10 times less than the peak width of a
solute.

It should have a linear response to solute that extends over several orders of
magnitude.

Some characteristics of commonly used detectors in HPLC systems are listed in

Table 8.8.

67

Table 8.8: Performance of some HPLC Detectors

Detector property
Absorbance
Fluorescence
Electrochemical
Conductivity
Refractive Index
Mass spectrometry
FTIR
Light scattering
Optical activity
Photo ionization
Element selective

Typical LOD*
(mass)
10 pg
10 fg
100 pg
100 pg-1 ng
1 ng
<1pg
1g
1g
1 ng
<1pg
1 ng

Linear range
3-4
5
4-5
5
3
5
3
5
4
4
4-5

Commercial
availability
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No

*Actual LOD will depend on the compound.

It is essential to know the band spreading for estimating detection limit of a particular
detection system. Connecting tubing must be minimum (not longer than 20 cm).
Tubing diameter (< 0.25 mm) is most critical as this would create a 10L volume and
so also dilution factor is important for detection limit. A detector must have a linear
dynamic range so that major and trace components can be determined in a single
analysis.
A.

Optical Detectors

These include uv-visible spectrophotometers and are the most widely used ones in
HPLC instruments. Three types of absorbance detectors are available;

fixed wavelength (UV) detector,

variable wavelength detector using deuterium lamp, and

scanning wavelength detector.

These have noise limitations due to thermal instability in the flow cell and in the
optical and electronic components. Therefore, thermosetting to 0.01 oC is required as it
may put noise limitation of 106 absorbance units. A quartz collimating lens focuses
the radiation on the sample and reference cell with detector cell volume of 8L/cm
optical path length. A schematic diagram of a typical flow through cell for absorbance
measurements in eluents is shown in Fig. 8.13. A rise time of 0.1 sec is needed for fast
HPLC measurements. It is essential that the mobile

Fig. 8. 13: Schematic of an ultraviolet detector for HPLC system

68

phase solvent must not absorb or may absorb only weakly. Water, methanol, hexane
and acetonitrile all permit operation in the for uv to at least 210 nm. Many absorbance
detectors are double beam devices where one beam passes through the element cell
and the other through a filter to reduce its intensity. Alternatively a chopped beam
system in conjunction with a single phototube is used. Detection limit can be
estimated if the noise level and the approximate molar absorptivity are known at the
operating wavelength. Assuming 1 cm path length and a noise level of 0.00004
absorbance unit, detection limit is
2 (noise) / b = 0.00008 / mol cm1litre1
If =10000, the minimum detectable concentration is 8 nM/L or 4 ng/mL for a
compound having mol wt = 500. If it is required in terms of sample weight instead of
concentration, the sample volume and system dilution factor must be considered. For
5L sample and a dilution factor of 20 (Mol wt 500), detection limit is

i)

ii)

2(0.00004) 20 (5 106 L) 500

= 0.4 ng
(10,000 L mol-1 cm -1)1cm
Fixed wavelength detector: It uses a light source that emits maximum light
intensity at one or more discrete wavelengths that are isolated by appropriate
filters. These have minimum noise but no free choice of wavelength. A medium
pressure mercury lamp can be selected for wavelengths of 254, 280, 313, 334
and 365 nm by the use of narrow band pass interference filters. Visible region
wavelengths can be accomplished by using a quartz iodine lamp and appropriate
filters. These have short terms noise levels usually <0.0001 absorbance unit.

Variable wavelength detector: It is usually a wide band pass uv-visible

spectrophotometer coupled to a chromatographic system. It offers a wide
selection of uv and visible wavelength range. A versatile detection system is
based on a spectrophotometer fitted with a grating monochromator and
continuum source e.g. deuterium lamp for the uv region and and a tungstenhalogen lamp for the visible region as shown in Fig. 8.14. These have double
beam optics, stable low noise electronics and are microprocessor controlled.
Some can be programmed to select a sequence of optimum monitoring
wavelengths during chromatographic runs and recording of a complete uv
spectrum.

Fig. 8.14: A schematic diagram of a variable wavelength detector

iii)

Scanning wavelength detector: Solid state diode arrays are used to record
spectrum for each solute simultaneously monitoring all wavelengths. Thus, a
complete spectrum for 190 to 600 nm can be obtained in just 0.01 sec. Some
instruments can be configured for scanning complete spectrum for monitoring of

69

independent signals. All the signals can be integrated between two preset
wavelengths and thus, multi-component complex samples can be detected.
iv)

Infrared absorbance detector: Infrared detector cells are similar in

construction to those used with uv radiation except that windows are made of
sodium chloride or calcium fluoride and the cell lengths range from 0.2 to 1.0
mm with volumes 1.5 to 10 L. The simpler ir instruments can be operated at
one or more single wavelength settings. Alternatively, the spectra for peaks can
be scanned by stopping the flow at the time of elution. Two types of ir detectors
are used. The first is with wavelength scanning being provided by three semi
circular wedges with a range of 2.5 to 14.5 m or 4000 to 690 cm1. The second
type of ir detector is based upon Fourier transform (FT) instruments which can
be attached with HPLC detectors.

v)

Fluorescence detector: This is similar to the design of fluorometers and

spectrofluorometers where a photoelectric detector is located at 90o to the
excitation beam. The simplest detectors use a mercury excitation source and one
or more filters to isolate a band of emitted radiation. More sophisticated
instruments are based upon a xenon source and employ a grating
monochromator to isolate the fluorescent radiation. These have the advantage of
being highly sensitive, typically greater by more than an order of magnitude
than most spectrophotometers and selective (specific) in picking out a specific
component of a mixture which fluoresces from a host of other components.
These are typically in the range of 109 to 1012 g/mL. Quite often the number of
fluorescent species can be enlarged by preliminary treatment of samples with
fluorescent derivative forming reagents. Further developments are taking place
based on tunable laser sources leading to enhanced sensitivity and selectivity.

B.

Differential Refractometer

It monitors the difference in refractive index between the references (mobile phase)
and the column eluent. It responds to any solute where refractive index is significantly
different from that of the mobile phase. A schematic illustration of a differential
refractive index detector is shown in Fig. 8.15 where solvent passes through one half
of the cell and then it passes through another chamber where eluent flows.

Fig. 8. 15: A schematic illustration of a differential refractive index detector

Two compartments are separated by a glass plate mounted at an angle such that
bending of the incident beam occurs if the two solutions differ in refractive index. The
resulting displacement in beam with respect to the photosensitive surface of a detector
causes variation in the output signal.
These detectors based on Freshnels law of reflection have the advantage of
responding to most solutes and have a wide range of linearity where one cell covers
the entire refractive index range. The cell volume is 15-25 L. In addition, these are
highly reliable and remain unaffected by flow rate. However, these are temperature
sensitive and do not yield sensitive results. Reflection type refractometer measures the
change in percentage of reflected light at a glass-liquid interface as the refractive index
70

of the liquid changes. Two collimated beams from the projector light illuminate the
reference and the sample cells made of Teflon gasket clamped between the cell prism
and a stainless reflecting back plate. As the light beam is transmitted through the
interface, it passes through the flowing liquid film and impinges on the surface of the
reflecting back plate. This diffuse reflected light appears as two spots of light that are
imaged by lenses onto dual photo detectors. Since the ratio of reflected light to
transmitted light is a function of the refractive index of the liquids, the illumination of
the cell back plate is a direct measure of the refractive index in each chamber. With
mobile phase flowing through both compartments, coarse zero is adjusted by rotating
the entire projector assembly. Fine adjustment is done with the optical plate, a glass
plate which can be rotated 30o from normal. Two different prisms must be used to
cover the useful range of refractive index.

C.

Electrochemical Detectors

These can be used only if solute molecules in aqueous and aqueous-organic phase
have voltammeteric characteristics. These are of several types depending on
amperometry, polarography, coulometry and conductometry. Electrochemical
detectors have not been exploited to the extent of optical detectors though these have
advantage of being simple, highly sensitive, convenient and wide spread applicability.
A variety of HPLC/ electrochemical detectors are available commercially. These are
potential universal detectors for fulfilling long time need. A typical thin layer
amperometer detector is shown in Fig. 8. 16. It can measure nanoampere level current
at a controlled potential as a function of time and consists of a flow cell in a 50 m

Fig. 8.16: A Schematic diagram of an amperometric thin layer detector for HPLC

thick polyfluorocarbon gasket sandwiched between two blocks, one plastic and the
other stainless steel. A working electrode of Pt, Au, glassy carbon or a carbon paste is
placed on one side of the channel and a reference electrode (usually Ag/AgCl) is
connected to the working region by tubing. Its cell volume is 1-5L.
A polarographic detector consists of a mechanically controlled dropping mercury
electrode where eluent flows around the mercury droplet. The potential of the
electrode is then maintained at a suitable level during elution. Plot of current vs time
provides elution pattern for species that are reduced at the chosen potential.
A dual electrode detector offers additional specificity. In one configuration, two
working electrodes are placed parallel with the flowing stream where each electrode is
held at a different potential, thus, generating two simultaneous chromatograms. The
current ratio at the two potential settings is calculated and used for peak confirmation.
The second configuration has two electrodes arranged in series. The upstream working
electrode generates an electroactive product from the analyte which is subsequently
detected at the downstream working electrode. The series of arrangement limits the

71

number of electroactive compounds that are detected. Only the compounds that
generate stable electroactive product and reach the second electrode are sensed.

D.

Mass Spectrometric Detector

These are considered as the most sensitive detectors but there is a fundamental
problem in coupling a liquid chromatograph with a mass spectrometer due to
mismatch between the relatively large solvent volume and the vacuum requirements.
Several interfaces have been developed for solving this problem as discussed later in
Sec. 8.8 of this Unit.
An LC-MS instrument has three basics components: a liquid chromatograph, an
interface and a mass spectrometer. In a commercial instrument, column is split with a
tiny fraction introduced directly into the mass spectrometer. Direct liquid introduction
systems are used in conjunction with the microbore column having typical flow rate in
the range, 10 to 50 L/min. In another type of interface, the eluent is deposited on
continuously moving belt/wire that transports the solvent and analyte to heated
chamber for removal by volatilization. After evaporation of the solvent, analyte
residue on the belt/wire passes into the ion source area where desorption-ionization
occurs.
A new and promising interface called thermo spray has become available
commercially and is useful in biochemical field. It permits direct introduction of the
total effluent from a column at high flow rates of 2L/min. In this case, the liquid is
vaporized as it passes through a heated capillary tube of stainless steel to form an
aerosol jet of solvent and analyte molecules. The analyte in the spray is ionized
through a charge exchange mechanism with a salt such as ammonium acetate
incorporated in the eluent. Thus, the thermospray is not only an interface but also an
ionization source. However, this has the disadvantage of being applicable to polar
analyte molecules and polar mobile phases that may dissolve ammonium acetate.
Fourier transform mass spectrometers based on ion cyclotron resonance also hold
immense potential for the analysis of thermospray produced ions. Thermospray
interface provides spectra for a wide range of non-volatile and thermally stable
compounds such as peptides and nucleotides with detection limits down to 1 to10 pg.
Mass spectrometric detectors use computer control and data storage in real-time and
computer reconstructed chromatograms. To achieve full benefit of an LC-MS
combination, besides being low cost a mass spectrometer should have high sensitivity,
high scan speed, adequate mass range and reasonable mass resolution. Time of flight
mass spectrometers also have useful features such as unlimited mass range, high
sensitivity, very high spectrum acquisition rate, multiplex detection capability.
Besides there are some detectors based on density, vapor pressure, heat of absorption,
thermal and electrical conductivity measurements. Also, if the analyte sample has
radioactive species formed as result of bombardment in a nuclear reactor then its
radioactivity can be measured using nuclear detectors.
There are also special types of detectors based on spray impact, electron capture or
transport which may measure electric charge in aerosol, absorption of electrons or
isolating the sample followed by vaporization, respectively.

Comparison of various HPLC detectors

In general, detectors based on absorbance measurements are insensitive to temperature
variations of the sample. The main advantage of these detectors is their low cost and
high sensitivity for many chemical and biological compounds of interest.
Comparatively speaking, a variable wavelength detector offers a range of
wavelengths, 190 to 600 nm which permits choice of wavelength depending on the
72

nature of solute. It is also possible to select a wavelength that can suppress the
absorption of an interfering solute or the mobile phase. However, their noise levels are
greater and hence they are less sensitive.
Simultaneous monitoring of radiation at many wavelengths and the acquisition of data
may present three dimensional chromatograms which may be stored. In case of single
fixed wavelength detector, it is not possible to go back and look for other information
at other wavelengths. However, with diode array it is possible to extract data at other
wavelengths from the memory. Comparison of absorption spectra with spectra in a
user generated library often gives positive identification of sample components. It is
now possible to evaluate a peak for purity by software data manipulation rather than
by iteration and refinement of the chromatographic separation.
Fluorescence is a means of increasing selectivity and sensitivity of HPLC analysis.
Certainly, selectivity is enhanced because all compounds do not fluoresce at the
absorbing radiation. Although many fluorescent derivatives can be prepared but that
puts limitation on their use. Typical fluorescing compounds are polynuclear aromatics,
steroids, plant pigments, vitamins, alkaloids, aflatoxins and porphyrins. Sensitivity is
improved because the fluorescing signal is measured against a low background
assuming that the mobile phase does not fluoresce. In general, a fluorescence detector
is 100 to 1000 times more sensitive than absorbance detector and is approximately
1ng/mL for strongly fluorescing compounds. Though it is a powerful tool for specific
applications of selective detection of trace components but it is not meant for general
use.
Electrochemical detectors have been found to be especially useful for polar mobile
phase. These detectors provide considerable selectivity because only a few
components in a complex mixture are likely to be electoractive. Sensitivisity is very
high, in many cases a picomole or less can be detected. Phenols and aromatic amines
of biochemical interest are the most important class of compounds where
electrochemical detectors can be used.
Differential refractometers are the universal type of detectors except when refractive
index of data sample component is same as that of the mobile phase. However, these
have limitations of having poor detection sensitivity, lack of selectivity and extreme
sensitivity to temperature and flow changes. It is essential to maintain cell temperature
within 0.001C. Response time is also somewhat large, about 2 sec. Photograph of an
Agilent 1100 (USA) HPLC set up is shown in Fig. 8.17. Besides, there are several
other manufacturers such as Perkin-Elmer Corporation (USA), Beckman Instruments
(USA), Shimadzu Scientific Instruments (Japan), Bio-Rad Laboratories (USA),
Hewlett-Packard Co (USA) wherefrom or through their representatives the instrument
or its accessories can be procured.

Fig. 8.17: Photographic representation of Agilent 1100 (USA) HPLC set up. On the top
are shown solvent reservoirs

73

SAQ 5
Complete the following statements:
i)

Solvent is delivered to the column through.....

.

ii)

The composition of mobile phase may be estimated from..

.

iii)

The most sensitive detector works on the principle of

.

iv)

Electrochemical detectors are specially useful for..

.

v)

Elution pattern from a polarographic detector is obtained in terms of

.

vi)

The purity of a compound may be evaluated from a peak by.

.

SAQ 6
Write the options available for the following:
i)

Essential requirements for choosing a detector

.

ii)

Material used as windows of infrared spectrophotometer

.

iii)

Features of time of flight mass

.
.

8.4

OPTIMIZATION OF SEPARATION

The primary aim of the separation of components of a complex mixture is the adequate
resolution with highest purity in the shortest possible time. Though it is a common
practice to follow trial and error method for the optimization of chromatographic
separation conditions but it is not always possible to do so. First the proper HPLC
system must be selected and all the parameters of stationary and mobile phase are
selected. The compound of interest should take five to ten times longer time to transit
through than unretained peak, tM. The type and characteristics of the column packing
(porosity, particle size range, good packing procedure, and high quality packing
material) also influence column length and the particle size.
The development of computer controlled HPLC systems has enabled systematic
automatic optimization techniques based on statistical experimental design and
mathematical resolution function. First of all column (stationary phase) and detector
are carefully chosen followed by the mobile phase composition and other parameters
such as flow rate, temperature etc. Though this can be done manually but computer
controlled optimization has several advantages. Sometimes gradient elution is used as
a preliminary step for unknown samples so as to indicate mobile phase composition
conditions. A typical example of parathion of a six component mixture using five
different proportions of methanol, tetrahydrofuran (THF) and water is illustrated in

74

Fig. 8.18. It is observed that as the water content is increased and the composition of
methanol and tetrahydrofuran is adjusted, six peaks corresponding to benzyl alcohol,
phenol, 3-phenylpropanol, 2,4-dimethylphenol, benzene and diethyl o-phthalate are
better resolved.

Fig. 8.18: Illustration of optimization of HPLC separation conditions using five ternary
phases. Peaks; 1. Benzyl alcohol, 2. Phenol, 3. 3-Phenylpropanol,
4. 2,4-dimethylphenol, 5. Benzene, 6. Diethyl o-phthalate

If complete separation is required in minimum possible time then first chromatogram

is the method of choice. If internal standard is to be added within a space of
chromatogram then either of next two chromatograms may be used. However, if
reaction products need to be separated or impurities are expected then any one of the
last two methods best meets the requirement.
In general, mobile phases with no or little polarity are used with polar bonded phases.
For non-polar bonded phases, however, mobile phases are selected from solvents with
high polarities. Thus, the mobile phase composition is adjusted to change the overall
retention time and/or to pull specific peak pairs apart as illustrated above. Solvent
optimization involving four solvents have also been described. Thus, the separation of
solutes with different functional groups may be improved by the use of ternary mobile
75

phases for the precise control of mobile phase-eluent strength in conjunction with
solvent programming. More sophisticated automated methods of mobile phase
optimization are commercially available. Most software packages are designed to use
one of the two alternative approaches.
A linear hydrocarbon chain is a popular bonded phase where alkyl group may have a
variety of chain length, usually a methyl (C-1), ethyl (C-2), octyl (C-8) or octadecyl
(C-18). The effect of chain length of the alkyl group upon performance of siloxane
column in reverse phase chromatography resulting in better resolution is illustrated in
Fig. 8.19. It is observed that poor resolution is observed with methyl group but it
becomes better with octyl and still better with octadecyl group. Thus, longer chains

Fig. 8.19: Effect of chain length on performance of reverse phase siloxane column packed
with 5 m particles. Peaks;1. Uracil, 2. Phenol, 3. Acetophenone,
4. Nitrobenzene, 5. Methyl benzoate, 6. Toluene

produce more retentive packings. On the basis of this observation, it may be

concluded that octadecyl packing can be used for application where maximum
retention is required. In order to emphasize this point further, a comparison of
chromatograms obtained on columns of octadecyl and octyl packings under the same
mobile phase conditions of methanol/water (50:50) is presented in Fig. 8.20. Mixture
of sample represents a variety of functional groups. Thus, bonded octyl packings
represent a good compromise for the separation of compounds with low to high
polarity and samples with wide ranging polarities. It seems that bonded alkyl phases
permit rapid analyses and rapid reequilibration when the mobile phase is altered as in
solvent programming.

(a)

(b)

Fig. 8.20: Comparison of chromatograms for nine compounds of different polarity as

obtained on (a) octadecyl and (b) octyl packings under the same mobile
phase conditions, methanol/water (50:50). Peaks; 1. Phenol, 2. Benzaldehyde,
3. Acetophenone, 4. Nitrobenzene, 5. Methyl benzoate, 6. Methoxybenzene,
7. Fluorobenzene, 8. Benzene, 9. Toluene

76

In many cases when the bands overlap, the selectivity factor is made larger by
adjusting to a suitable level. Such a change is conveniently made by changing the
chemical nature of the mobile phase as typically shown for the separation of six
steroids in Fig. 8.21 by reverse-phase chromatography following a four solvent
optimization procedure consisting of methanol, acetonitrile, tetrahydrofuran and water.
It was developed for finding a suitable solvent system to resolve a given mixture in a
minimum possible time. Three compatible solvents were used to adjust the strength of
the mixture to yield a suitable value of . The first two chromatograms in (a) and (b)

Fig. 8.21: Choice of mobile phase on the selective separation of six steroids using 5 m C8
bonded reversed phase particles. Peak ; 1. Prednisone, 2. Cortisone, 3.
Hydrocortisone, 4. Dexamethasone, 5. Corticosterone, 6. Cortoexolone. Effect
of % water to adjust in (a) and (b). Further separation factor is varied at
constant in (b), (c), (d) and (e)

show the results from initial experiments to determine minimum value of which is
estimated to be 10. However, it is observed that values for components 1 & 3 and
that of 5 & 6 do not yield satisfactory resolution. In further experimentation for
finding better values, water was added to get = 10. It is observed that results of
methanol/water and tetrahydrofuran/water in (c) and (d) show better resolution. A
77

mixture of acetonitrile, THF and water was also attempted as shown in (e) where it is
found to be the best mobile phase for the separation of six steroids in a mixture.

8.5

ADVANTAGES

HPLC is a versatile technique offering a number of selective variants to resolve

complex mixtures of biological, environmental and pharmaceutical samples. It is more
a concept which has been employed to a variety of chromatographic techniques based
on adsorption, partition using normal bonded and reversed phase, ion-exchange, size
exclusion, etc. It has the advantage of the possibility of controlling solute retention and
better selectivity by manipulating the stationary phase, mobile phase and other
experimental conditions. Some of the advantages may be summarized as follows:
i)

It is a very fast separation method with analysis time as small as less than a
minute in some cases.

ii)

HPLC is a useful separation method with high resolving power and a

quantitative analysis method with low detection limits and high accuracy.

iii)

It is especially useful for resolving optically active compounds though different

types of columns with chiral stationary phase are used.

iv)

The technique is applicable to small amount of samples where even trace

amounts of solutes may be determined.

v)

All kinds of solids soluble in suitable organic solvent and liquid samples can be
analyzed though gases can not be anayzed.

vi)

HPLC has much wider applicability in pharmaceutical and food processing

industry including forensic and environmental samples compared to GC which
has been found to be more useful in petroleum industry.

vii)

HPLC is especially useful for continuous monitoring of the column effluent and
thus it can be used for any on-line process where analytical procedures may be
automated and data need not be handled manually.

viii) HPLC provides repetitive and reproducible analysis using the same column.
ix)

HPLC is being widely used for the speciation of ionic and non-ionic species
such as various organic and inorganic forms of arsenic.

x)

It is more versatile than gas chromatography since it is not limited to volatile

and thermally stable samples with wider choice of stationary and mobile phases.

xi)

It is a non-destructive method which can be used for preparative and process

scale separations.

xii)

It can be used for the separation of closely related compounds as well as for the
purification of compounds.

Besides so many advantages, the method also suffers from many disadvantages as
given below:

78

i)

It cannot be used for the analysis of volatile compounds such as hydrocarbons.

ii)

It cannot be used for the analysis of industrial products such as alloys, polymers
etc.

iii)

High purity solvents are required because any impurity may affect the separation
and resolution.

iv)

It requires extensive training in order to operate the instrument and optimize the
conditions.

v)

Sample preparation is often required e.g. dissolution, dilution, etc.

8.6

COMPARISON WITH GAS CHROMATOGRAPHY

It has been observed in our discussion on GC in Unit 7 and preceding discussion on

HPLC that both the techniques have many similarities and dissimilarities. A common
parameter in two cases is the retention data (retention time tr, relative retention and
separation factor s) that is most useful means of qualitative identification of
components in a complex mixture. For quantitative analysis, however, peak height or
peak area is measured, the former being suitable for sharp, early eluting peaks where
peaks are fully resolved. In both the cases, the stationary phase may be similar but the
nature of solute analyte and mobile phases are quite different. Both the techniques are
highly sensitive with low detection limits, of the order of 1012 to 1015 moles which,
however, depend on the type of detector used. For example, the use of mass
spectrometer (MS) interface may further enhance the sensitivity in both cases. Also,
both the techniques are highly reproducible, of the order of 1% and have comparable
analysis time (2 to 10 min). Similarly, there are many points of similarity and
dissimilarities as summarized below:
i)

GC can be used to separate gaseous or low boiling. pt. liquid solutes can be
analyzed whereas in HPLC can be used to separate volatile and nonvolatile,
including solids soluble in organic solvents.

ii)

The amount of sample required in GC is of the order of a few nanograms per

mL whereas in HPLC even a fraction of microlitre may be sufficient. However,
in both the cases, the sample is introduced using a microsyringe.

iii)

The column tubing in GC can be circular, in a loop or bent so as to have long

column but in HPLC it should always be a straight column or else mobile phase
will not flow smoothly. No bending is allowed or else pressure will not be
uniform.

iv)

The instrumental set up in two cases is widely different. In case of GC, it is

essential to have the sample injection chamber, column and detector, all housed
in a thermo stated oven and an inert gas carrier is used. On the other hand, in
HPLC, quite often a mixture of high purity solvents with low pressure gradient
is used and then it is allowed to pass through stationary phase column under
high pressure.

v)

Though some detectors are common for GC and HPLC but not all the detectors
used in GC or HPLC can be used by another. Flame ionization (FID) or electron
capture (ECD) detectors commonly used in GC cannot be used in HPLC.
Similarly, a fluorescence or refractive index detector used in HPLC cannot be
used in GC. In principle, GC can be coupled to an UV detector but it is rarely
done. On the other hand, GC is often coupled to infrared detector though a fast
scanning and sensitive detector is required. However, HPLC frequently uses
fixed wavelength UV detector though variable wavelength detectors can also be
used. Infrared detection of eluting compounds can also be carried out.

vi)

The optimization of experimental procedure in GC is easier whereas in case of

HPLC, it is difficult.

vii)

The basic mechanism of separation in GC is adsorption or partition whereas in

HPLC different mechanisms of adsorption, partition, ion-exchange, etc. are
operative.
79

viii) GC has limited applicability for gaseous solutes only though it is useful for the
identification of hydrocarbons of a homologous series. On the other hand, HPLC
has much wider applicability to a variety of organic and inorganic compounds.
ix)

GC cannot be used for the separation of ionic species whereas such species can
be easily separated by HPLC.

SAQ 7
Write brief answers for the following:
i)

Explain the role of computer controlled HPLC systems.

.
.....
.....
.....

ii)

What is the effect of chain length of the alkyl group on the performance of
siloxane column in HPLC?
.
.....
.....
.....
.....

iii)

Explain the role of k in improving the resolution of HPLC chromatograms.

.
.....
.....
.....

iv)

What is the most important common factor between GC and HPLC?

.
.....
.....
.....

v)

Explain how HPLC is a non-destructive method of analysis?

.
.....
.....
.....
.....

80

8.7

APPLICATIONS

The applications of HPLC in all its different forms have been steadily increasing day
by day. This is primarily because the technique is well suited to a wide variety of
compounds including organic, inorganic and biological compounds, small ions to
macromolecules, polymers, chiral compounds and labile materials. The most
appropriate choice of mode of HPLC for a given separation problem is based on the
relative molecular mass, solubility characteristics and polarity of the compounds to be
separated, as is illustrated in Fig. 8.22.

Fig. 8.22: Selection of suitable liquid chromatographic method for the separation
analysis depending on structure and properties of solute

The HPLC is the most successful technique in separating compounds as diverse as

aminoacids, nucleic acids, and proteins in physiological samples, active drugs,
steroids, carbohydrates, vitamins, dyestuffs, pesticides, polymers etc. It is used
routinely for the assay of pharmaceutical products, the monitoring of drugs and
metabolites in body fluids and for other biomedical, biochemical and forensic
applications such as the detection of drugs of abuse. The determination of additives in

81

foodstuffs and beverages including sugars, vitamins, flavorings and colorings and the
quality control of polymers, plastics and resins are further examples of the wide and
growing scope of HPLC. A summary of typical applications in various fields are listed
in Table 8.9.

Table 8.9: Typical Applications of HPLC Methods to Various Fields

Sl.
No.

Field

Typical mixtures

1.

Pharmaceuticals

Antibiotics, sedatives, steroids, analgesics

2.

Biochemicals

Aminoacids, proteins, carbohydrates, lipids

3.

Food products

Artificial sweetners, antioxidants, aflatoxins,

additives

4.

Industrial chemicals

Condensed aromatics, dyes, surfactants, propellants.

5.

Clinical medicines

Bile acids, drug metabolites, urine extracts, estrogens

6.

Polymers

Molecular weight determination and distribution.

7.

Forensic chemistry

Drugs, poisons, blood alcohol, narcotics

8.

Pollutants

Pesticides, herbicides, PCBs, phenols in

environmental samples

9.

Quality control

Purification from mixrure

8.7.1 Polyaromatic Hydrocarbons

Silica is well suited for the separation and analysis of non-ionizing, water insoluble,
and relatively simple molecules which are very closely related such as polyaromatic
hydrocarbons and fats and oils with different functional groups The order of
adsorption follows the general polarity scale for various classes of compounds. It is
less influenced by molecular weight differences and more by specific functional
groups. Therefore, the separation of compounds differing in the degree or type of alkyl
substitution, such as members of a homologous series is usually by adsorption.
However, adsorption chromatography has been used to isolate a number of
polynuclear aromatics from a petroleum sample, as illustrated in Fig. 8.23, from a
totally porous silica column having dimensions of 25 0.46 cm and acetonitrile/water
(70:30) as mobile phase.

Fig. 8.23: Separation of polynuclear hydrocarbons on porous and spherical silica.

Peaks; 1. Naphthalene, 2. Fluorene, 3. Phenanthrene, 4. Anthracene, 5. Pyrene

8.7.2 Isomeric Compounds

The adsorption chromatography has a particular strength, not shared by other methods,
in its ability to differentiate among the isomeric compounds in a mixture. Generally,
the most polar group of a polyfunctional compound governs its adsorption
characteristics. Often, only one functional group is geometrically positioned with
respect to the adsorption site. However, some polyfunctional solutes are better
matched to the adsorbent surface than other isomeric counterpart as typically
82

illustrated in Fig. 8.24 showing the separation of positional isomers of syn- and antipyrazolines also called cis- and trans- forms. A 100 0.3 cm pellicular silica along
with methylene chloride/isooctane (50:50) mobile phase was used at a flow rate of
0.225 mL/min. A fixed wavelength of 254 nm UV detector was used.

Fig. 8.24: Separation of syn- and anti-pyrazoline isomers

8.7.3 Sugars in Popular Drinks

A typical application of HPLC may be illustrated by the identification of glucose,
fructose and sucrose in a popular drink such as Pepsi using a 4 mm 30 cm column of
Macropak-NH4 and acetonitrile-water system at a flow rate of 2.0 mL/min as shown in
Fig. 8.25. It is observed that fructose is eluted first followed by glucose and fructose
and the whole separation takes about 15 min.

Fig. 8.25: The HPLC separation of glucose, fructose and sucrose in Pepsi shown as three
distinct peaks

83

However, size exclusion chromatography has been used for qualitative identification
and quantitative determination of these sugars in four types of canned fruit juices of
apple, orange, pineapple and cranberry, as shown in Fig. 8.26. Also shown is
chromatogram for the standards which can be used for quantitative determination of
the constituent sugars. The packing which had an exclusion limit of 1000, was a cross
linked-polystyrene polymer made hydrophilic by sulphonation. A 25 cm long column
with this packing contained 7600 plates at 80 oC.

Fig. 8.26: Size exclusion HPLC method for the determination of glucose (G), fructose
(F) and sucrose (S) in canned fruit juices. Also shown is the standard on RHS
for quantitative determination of sugars

The size exclusion chromatography or gel permeation chromatography is the most

suited technique for the solutes with molecular weights 2000 or more and is also
useful for preliminary investigation of unknown samples. It can be used for the
determination of relative mass distribution for components of biochemical and
polymer systems with an accuracy of 10%. Desalting is commonly employed to isolate
the macromolecules from biochemical materials, where both simple and
macromolecules may be present in an electrolyte solution. Dilute solutions of macromolecules can be concentrated and isolated by adding dry gel beads to absorb the
solvent relative molecular mass solutes.

8.7.4 Drug Abuse

The HPLC is being widely used for the detection of drug abuse especially for the
steroids by the athletes at the International games. A typical case of drug abuse is
illustrated in Fig. 8.27 where oxphenbutazone, phenylbutazone and furosemide were
detected in horse plasma.
84

Fig. 8.27: Reversed phase HPLC separation of drug abuse in horse plasma

The sample is filtered through 2 m membrane prior to direct injection into a

15 cm 4.6 mm with 5 micron ISRP packing. A ternary mobile phase of isopropanol,
tetrahydrofuran along with potassium hydrogen phosphate buffer was used at a flow
rate of 1 mL/min.

8.7.5 Separation of Nucleic Acids

The HPLC is a rapid separation method as illustrated by a comparison of separation of
nucleic acids by HPLC and conventional ion-exchange chromatography in Fig. 8.28.

(a)

(b)

Fig. 8.28: HPLC separation of nucleic acids: (a) on Zipax cation exchange packing using
HPLC; and (b) using conventional ion-exchange chromatography

85

Whereas conventional ion-exchange method takes more than 2 hours for the
separation of cytosine, uracil, guanine and adenine, it takes only 5 minutes by HPLC
method requiring only microgram quantities.

8.7.6 Analysis of Amino Acids

Commercial amino acid analyzers have been available for many years. They work on
the principle of HPLC and are widely used for the separation analysis of a large
number of amino acids from a mixture as they form cations by adding protons in the
pH range below their isoelectric point. They are separated on a cation exchange
column where more acidic component emerges first and the most basic in the last. A
typical chromatogram of 17 amino acids containing 10 nM of each is shown in
Fig. 8.29. The complex of amino acid-ninhydrin is photometrically measured at
570 nm.

Fig. 8.29: Analysis of amino acids from a complex mixture

8.7.7 Partition Chromatography

As already pointed out it is being used in two forms of bonded-phase (BPC) and
reversed phase chromatography (RPC). Both of these forms are suitable for most
HPLC separations ranging from mixtures of weakly polar to highly polar and ionic
compounds. Reverse phase chromatography using octadecyl (C18) columns and
methanol/aqueous buffers or acetonitrile/water mobile phases is by far the most widely
used. Ion pair chromatography offers the advantages of greater efficiency and column
stability and more selectivity in the separation of ionic compounds compared to
bonded phase or conventional ion exchangers.
The reverse phase technique comprises nearly half of all the LC methods described in
literature. It provides optimum retention and selectivity when compounds have no
hydrogen bonding groups or have a predominant aliphatic or aromatic character as
typically illustrated in Fig. 8.30 showing separation of solutes based on the size and
structure of alkyl groups in a series of phthalate esters.
86

Fig. 8.30: Chromatogram of o-phthalate esters run on a column with octadecyl packing
and methanol/water (90:10) as eluent. Peaks; 1. Dimethyl, 2. Diethyl, 3. Dipropyl, 4.
Dibutyl, 5. Dipentyl, 6. Dihexyl, 7. Diheptyl, 8. Dioctyl.

Applications of bonded phase partition chromatography have been explored in a

variety of fields. Of several thousands, two have been illustrated in Fig. 8.31 with the
examples of analysis of consumer and industrial products such as additives in soft
drinks and phosphate insecticides.

(a)

(b)

Peaks; 1. Methyl parathion,

2. Ciodrin, 3. Parathion,
4. Dyfonate, 5. Diazinon, 6. EPN,
7. Ronnel, 8. Trithion
Fig. 8.31: Typical applications of bonded phase chromatography: (a) Additives in soft
drinks; and (b) Organophosphate insecticides
Peaks; 1. Vitamin C, 2. Saccharin,
3. Caffeine, 4. Sodium benzoate

87

8.7.8 Ion Chromatography

It is being routinely used for the separation of inorganic anions such as Cl, Br, F,
SO 24 , NO 3 , NO 2 , and PO 34 at ppm levels in surface waters, industrial effluents,
food products, pharmaceuticals and clinical samples. Typical ion chromatograms in
Fig. 8.32 shows the analysis of various anions and cations using two column flow
method. Thus, inorganic cations such as Na+, K+ and NH4+ can be monitored in foods
such as dietetic foods low in sodium, and urine samples where the efficiency of the
separation is markedly influenced by the use of complexing agent in the eluent.
However, separation of organic acids and bases, alkali, alkaline earth and transition
metal cations are also being achieved. Ion-exchange resins having a proportion of the
ionic sites replaced with hydrophobic reversed phase groups commonly used in HPLC
columns, typically octadecylsilane, enable separation of both non-ionic and ionic
species in a mixture. It has become a widely used technique in a pathological and
environmental analysis laboratory where commercial instruments are used.

Fig. 8.32: Applications of Ion chromatography for the separation of anions (a) and
cations (b) using two column flow method.

Another version of reverse-phase chromatography is ion-pair chromatography which

deals with separation of ionized or ionizable species on a reverse phase column. The
method can handle samples that are very polar, multiply ionized and/or strongly basic.
In ordinary reverse phase HPLC, organic ions show poor peak shapes and inadequate
retention. Ion-suppression method is limited to the pH range 2.0 to 7.5 by the
instability of stationary bonded phases outside this pH range. In case of ion-pair
88

chromatography, an ion pair reagent, a large organic counter-ion which is ionized, is

added at low concentration to the mobile phase. One ion of the reagent is retained and
separate organic solute ions of opposite charge by forming a reversible ion-pair
complex with the ionized sample as represented by the equilibrium
RCOO

R4N+

[R4N+,

OOCR]o ion-pair

Thus, electrically neutral compounds are partitioned between the mobile and non-polar
stationary phases. Unlike conventional ion-exchange, ion-pair partition can separate
non-ionic and ionic compounds in the same sample. First of all separation of the
nonionic solutes is optimized and then counterion is added to the mobile phase
whereby ionic solutes are retained.
Let us consider the separation of water soluble vitamins, strongly ionic thiamine, nonionic riboflavin and less ionic pyridoxine and niacinamide by a two step process. In
the first step, water/methanol ratio is adjusted to obtain good retention of non-ionic
riboflavin and then the organic counterion is added to the eluent to separate three ionic
compounds which is affected by the alkyl chain length of the counterion. Thus,
thiamine, a quaternary amine shows greatest sensitivity to change in counterion. The
optimum separation is achieved with a 50/50 mixture of C-5/C-7 alkyl sulphonic acids
as shown in Fig. 8.33 where a mixture of counterions is added to the mobile phase
producing a retention proportional to the concentration of each counterion.

Fig. 8.33: Separation of ionic and nonionic compounds in a mixture by ion-pair

chromatography

8.7.9 Chiral Separation of Enantiomers

A typical example of the separation of enantiomers of benzodiazepine Temazepam
drug is illustrated in Fig. 8.34 where immobilized human serum albumin (HSA)
bonded to silica was used as a reverse phase material and phosphate buffer-acetonitrile
as mobile phase. A number of manufacturers now supply these columns specifically
tailored for particular application.

89

Fig. 8.34: Chiral separation of enantiomers of the benzodisazepine Temazepam drug

showing separate peaks corresponding to d and l forms

8.7.10 Ion-Exclusion Chromatography

Similar to ion chromatography, it also employs ion-exchange columns to achieve
separations. However, it differs from ion chromatography in that it is used for the
separation of neutral species rather than ions as typically illustrated by the separation
of simple carboxylic acids in Fig. 8.35. A cation exchange resin in acidic form was
used and elution was accomplished with dil HCl. The analytical column was followed
by a suppressor column packed with a cation exchange resin in silver form where H+
were exchanged for Ag+ which then precipitated Cl thus removing the ions
contributed by the eluent. The undissociated analyte acids were

Fig. 8.35: An ion-exclusion chromatogram for a mixture of six weak acids

90

distributed between the mobile phase in the column and the immobilized liquid held in
the ores of the ion packing. Ion-exclusion chromatography has found numerous
applications for the identification and determination of acidic species in milk, coffee,
wine and other commercial products. Similarly, weak bases and their salts can also be
separated by using anion exchange column in OH form.

8.7.11 Speciation Studies

It has been well emphasized in the preceding discussion that HPLC can be
successfully used for the separation of a wide variety of ionic, non-ionic and polar
compounds. In many cases such as that arsenic, selenium, mercury ionic and organic
forms are formed. Their detection is very difficult because of occurrence in very low
concentrations in the environmental samples. In a typical case, four species of As (III),
As (V) and organic species such as monomethylarsine (MMA) and dimethylarsine
(DMA) and arsenocholine (As Choline) have been identified and quantified by HPLC
as shown in Fig. 8.36 using a 15 cm 4.6 mm column packed with Supelcosil reverse
phase material.

Fig. 8.36: HPLC separation of various arsenic species along with a peak due to carbonic
acid as impurity

The mobile phase was sodium phosphate buffer at pH 6.0 and 5 mM

tetrabutylammonium hydroxide as ion interaction reagent at a flow rate of 2 mL/min.
Quantitative HPLC analysis ideally requires a linear relationship between the
magnitude of the signal and the concentration of any particular solute in the sample.
Most HPLC systems have in built computerized data handling system and prepare
calibration curves using standard concentrations. It is possible to obtain an automatic
set of results for the concentrations of individual components in samples matched with
their corresponding retention times. It may be remembered that detector response may
be different for various components of a mixture introducing a factor of uncertainty in
results.

8.8

INTERFACING HPLC WITH MASS

SPECTROMETRY

As it has been emphasized earlier, mass spectrometer is the most sensitive and ideal
detector for liquid chromatography. It can provide both structural information and
quantitative analysis for the separated compounds. When it is coupled with HPLC, it
becomes a hyphenated technique similar to GC-MS. Unlike in GC where separated
compounds are more or less in pure state, in case of HPLC inherent difficulty was in
91

removing the liquid mobile phase while allowing the analytes to pass into the mass
spectrometer. Therefore, the main problem encountered is the mismatch between the
mass flows involved in HPLC (about 1g/min) which is two or three order of
magnitude larger than can be accommodated by conventional mass spectrometer
vacuum systems. Another problem is the difficulty in vaporizing nonvolatile and
thermally labile molecules without degrading them. Several designs of interface have
been developed, the main difference between them being the means of separating
analytes from the mobile phase and the method of ionization employed. Many
compromises have been suggested in the operating conditions of either the
chromatograph or the mass spectrometer. Some approaches followed are described
below and schematically shown in Fig. 8.37.

8.8.1 Thermospray Method

It depends on the thermal generation of a spray and separated heat treatment of that
spray to yield desolvated ions. The HPLC effluent is fed into a micro furnace, a
capillary tube maintained at up to 400 oC that protrudes into a region of reduced
pressure. The heat creates a supersonic expanding aerosol jet containing a mist of fine
droplets of solvent vapor and sample molecules. The droplets vaporize on their way
downstream. The excess vapors are pumped away by an added mechanical pump
directly coupled to the ion source. No external ionizing source is required to achieve
molecular ions from many nonvolatile solutes at the sub-nanogram level. The ions of
sample molecule are formed in the spray either by direct desorption or by chemical
ionization when used with polar mobile phase containing appropriate buffer such as
ammonium ethanoate. With weakly ionized mobile phase, a conventional electron
beam is used to provide gas phase reagent ions for the chemical ionization of solute
molecules. Chemical ionization (CI) spectra are typically accompanied by electronimpact spectra. The ions formed are led into a quadrupole or magnetic sector mass
spectrometer as shown in Fig. 8.37 (a). An electron beam is used to enhance the
production of ions by CI.

8.8.2 Particle Beam Interface

It employs helium gas to nebulize the mobile phase, producing an aerosol from which
the sample is evaporated at near ambient temperature and pressure. Thus, it consists of
three sections; aerosol generator, desolvation chamber and two stages aerosol-beam
pressure reducer. The mixture of He, solvent vapor and analyte molecules is
accelerated into a low pressure two stage momentum separator where it expands
supersonically. The two stage aerosol-beam separator consists of two nozzle and
skimmer devices which reduce the pressure from an initial value close to atmospheric
pressure in the desolvation chamber to a final value close to the pressure in the ion
source. In the desolvation chamber maintained at room temperature, solvent
evaporates. The separator allows solute particles from the initial aerosol to be
preferentially transferred through the system while dispersion gas and solvent vapor
are pumped away. After He and solvent are pumped off, the heavier analyte molecules
pass directly through two skimmer plates and along a narrow probe into a heated
ionization chamber where electron impact (EI) ionization occurs as shown
in Fig. 8.37 (b).

8.8.3 Atmospheric Pressure Chemical Ionization (APCI)

This interface uses nitrogen as a nebulizing gas and a heated nebulizer probe. The
mobile droplets and the gas are heated to `120 oC in a desolvation chamber where a
corona discharge generates electrons that ionize the mobile molecules to give reactant
ions as shown in Fig. 8.37 (c). These ions then collide with analyte molecules to yield
pseudomolecular ions by chemical ionization. The analyte ions are accelerated through
skimmer-cones into the spectrometer whereas the uncharged solvent molecules are
removed by differential pumping.
92

(a) Schematic of thermospray where particle enters in at a and transfer line is suddenly
heated at b resulting in spray formation at c

(c) Schematic of atmospheric pressure chemical ionisation (APCI) source

Fig. 8.37: Various interfaces of HPLC-MS depicting Thermospray, Particle beam, APCl
and electrospray source.

93

8.8.4 Electrospray Interface

It also operates at atmospheric pressure and consists of a metal capillary tube through
which column effluent is passed at a relatively low flow rate of 1-20 L/min. An
electric field is generated at the capillary exit by applying a 3-6 kV potential between
the tube and a counter electrode placed at a distance away as shown in Fig. 8.37 (d).
The field induces an accumulation of charge on the surface of the liquid emerging
from the capillary resulting in the production of highly charged droplets. As the
solvent evaporates, droplets shrink from their surface, which increases the charge
density and leads to their explosive rupture and the creation of smaller charged
droplets.
This process is repeated many times and finally multiply charged analyte species are
formed. These are then passed through skimmers into the mass spectrometer where
uncharged solvent molecules are pumped away. A variant, known as ion spray,
involves pneumatic nebulization to increase the flow rate and an earthed screen to
inhibit droplet condensation that would otherwise destabilize the spray.

8.8.5 Moving Belt Interface

In this case, effluent is placed by spray deposition onto a continuous moving belt that
is woven from ultrafine quartz fibre. As the spray deposition is essentially a dry
process, solvent need not be removed. The belt passes through two successive vacuum
locks where the pressure is reduced to 0.1 Torr before moving into the ion source
housing of the fast ion bombardment (FAB) mass spectrometer. After the belt leaves
the FAB unit, it exits through the two vacuum chambers. Any residual sample or
solvent is removed by a wash bath. Though moving belt interface is cumbersome but it
can be used with magnetic sector or quadruple instruments and in either of the
election-impact or chemical ionization mode.
The technology and value of the HPLC-mass spectrometry has increased in parallel
with the developments in mass spectrometry. As of now very accurate molecular mass
measurements can be made using new generation of compact time of flight
spectrometers whose performance is comparable to much larger and more expensive
magnetic sector instruments.

8.9 SUMMARY
High performance (or pressure) liquid chromatography (HPLC) is a type of liquidliquid chromatography (LLC) where a narrow width (2-5 mm diameter) and about
50 cm long column is packed with ultrafine material (5-10 m) so as to increase its
surface area. It is used for the separation/analysis of a variety of solutes from a
complex mixture in small amounts. Its various modes such as adsorption, partition (or
bonded phase), reverse phase, exclusion, ion-exchange and ion chromatography are
described with respect to stationary phase packing materials.
In addition, solvent delivery system including reservoir, pumps and characteristics of
mobile phase and various types of detectors are briefly described. Optimization
procedure is described with typical examples. Various interfaces of HPLC with mass
spectrometry are also described. A comparison is made between gas chromatography
(GC) and HPLC especially with regard to the types of solutes analyzed and
instrumentation. Further, its advantages and some typical applications in various
modes including typical cases of drug abuse, additives in canned fruit juice, chiral
separation, and separation of amino acids, nucleic acids, cations and anions and
isomers are explained.

94

8.10 TERMINAL QUESTIONS

1.

Explain the following terms briefly:

i)

Sample injection

ii)

Bonded phase

iii)

Pellicular packing

iv)

Bulk property detector

v)

Normal phase packing

vi)

Characteristics of a detector

2.

Explain the acidic character of silica and basic character of alumina and their
usefuness as inert core material.

3.

Compare conventional liquid chromatography with reverse phase high

performance chromatography with suitable examples and applications.

4.

Explain the linear response range of a detector. What will happen if you work
beyond this range?

5.

What are guard and suppressor columns. In what respects they differ from each
other?

6.

In a normal phase column of 15 cm length, a solute showed a retention time of

17.8 min whereas an unretained sample had a retention time of 0.73 min when
the mobile phase was chloroform/benzene(1:1). Calculate capacity factor k of
the solute. How can it be altered. If the number of plates were 10200, calculate
the plate height.

7.

A two component pharmaceutical product was separated by using a 15.0 cm

long HPLC column yielding following retention and peak width data:
Component
time

Retention
width

Peak width

Half peak

Component A

7.38 min

0.65 min

0.31 min

Component B

8.63 min

0. 73 min

0. 34 min

If the solvent showed up a peak at 1.37 min then calculate (a) capacity factors
for each of the two components (b) number of plates using peak width and half
peak width and (c) the resolution of the two compounds using full peak width
and half peak width.
8.

Explain the role of solvent mixture in the separation of components in a mixture.

8.11 ANSWERS
Self Assessment Questions
1.

High performance liquid chromatography

High pressure liquid chromatography
High speed liquid chromatography
High resolution chromatography
High efficiency liquid chromatography

95

2.

Smaller the particle size of stationary phase, larger is the surface area for solute
particles to interact with. It increases the efficiency of separation.

3.

i)

[B]

ii)

[B]

iii)

[C] and [D]

iv)

[D]

v)

[D]

i)

Silanol group is hydrolysed with organochlorosilane

ii)

nonpolar solvents such as n-hexane, chloroform

iii)

COOH, PO 34

iv)

100 to 500 millions

v)

ion chromatography, water and CO2

vi)

enantiomers, transient diastereoisomeric compounds

i)

Reciprocating pump

ii)

Gradient elution

iii)

Fluorescence

iv)

Polar mobile phase

v)

Current vs time

vi)

Software data evaluation

i)

Selective separation, accurate determination and continuous detection

ii)

Sodium chloride, calcium fluoride

iii)

Unlimited mass range, high sensitivity, multiplex detection capability

i)

It enables systematic automatic optimization of experimental conditions.

ii)

The retention time increases with the increasing chain length of the alkyl
group and the resolution of separated components becomes better.

iii)

The capacity factor k is defined as equal to K.Vs /Vm where K is

distribution ratio and Vs and Vm are the volumes of solvent in the
stationary and the mobile phases, respectively. It varies with retention
time tr and hence, the resolution of separation.

iv)

The most important common factor between GC and HPLC is the

retention time.

v)

As the sample for HPLC should be liquid or solid dissolved in a solvent

from where the solute can be recovered, this is considered as a nondestructive method.

4.

5.

6.

7.

Terminal Questions

96

1.

The answers to Questions 1 to 3 are descriptive and you can refer to the relevant
sections of the Unit.

2.

It is essential that detector response should vary linearly with the concentration
of analyte in a solute mixture. However, it is not always true especially in higher

concentration ranges where convex or concave behaviour is observed depending

on the nature of solute. In such a case, one should use the detector within the
linear range only.
See Sec. 8.3.2

4.

You have already learnt that tR = tM (1+ k ) wherefrom after substitution of

retention time values, k = 22.7. Similarly, plate height, h = 14.7 m

5.

a)

Capacity factors using the formula,

b)

Number of plates using full width formula, n = 16 (tR / w)2; 2063 and 2236

3.

= (tR tM) / tM; 4.39 and 5.29

using half width formula, n = 5.54 (tR / w)2 ; 3140 and 3569
c)
6.

Resolution using the full width formula, R = 2(t2 t1)/ (w1 + w2); 1.81
half width formula, R = 2(t2 t1)/ 1.69 (w+ w); 2.28

Choice of solvent or mixture of solvent plays an important role in the separation

of various components in a mixture. By varying the composition of solvents, its
polarity and capacity factor can be adjusted for improving the separation.

Further Readings
1.

Vogels Textbook of Quantitative Chemical Analysis, By J. Menham, R.C.

Denney, J.D. Barnes and M.J.K. Thomas, 6th Edn, Low Price Edition, Pearson
Education Ltd, New Delhi (2000).

2.

Quantitative Analysis, By R. A. Day and A. L. Underwood, 6th Edn, Prentice

Hall of India, New Delhi (2001)

3.

Instrumental Analysis, Editors, H. H. Bauer, G. D. Christian and J. E. OReilly,

2nd Edn, Allyn and Bacon, Inc., Boston (1991)

4.

Principles of Instrumental Analysis By D. A. Skoog, F. J. Holler and T. A.

Nieman, 5th Edn, Thomson Brooks/Cole, Bangalore (2004)

5.

Fundamentals of Analytical Chemistry By D. A. Skoog, D. M. West, F. J. Holler

and S. L. Crouch, 8th Edn, Thomson Brooks/Cole, Bangalore (2004).

6.

Analytical Chemistry By G. D. Christian, 6th Edn, John Wiley & Sons Inc,
Singapore (2003)

7.

Principles and Practice of Analytical Chemistry By F.W. Fifield and D. Kealey,

5th Edn, Blackwell Science Ltd, New Delhi (2004)

8.

Instrumental Methods of Analysis, 7th Edition, By H. H. Willard, L.L. Merritt,

J. A. Dean and F. A. Settle, CBS Publishers & Distributors, New Delhi (1986)

9.

Handbook of Instrumental Techniques for Analytical Chemistry, Editor, F.

Settle, Low Price Edition, Pearson Education Inc, New Delhi (2004)

10.

Instrumental Methods of Chemical Analysis By G. W. Ewing, 5th Edn,

Mc-Graw Hill, Singapore (1985)

11.

High Performance Liquid Chromatography By S. A. Lindsay, Wiley, New York

(1992)

97

12.

98

Separation Methods By M. N. Sastri, 3rd Edn. Himalaya Publishing House,

Mumbai (2005)

INDEX
Adjusted retention time 7
Adsorption energy per unit area of adsorbent 64
Advantages 78
Air peak 7
Alkali flame detector 31, 35
Applications of gas chromatography 40
Examples 41
Identification of compounds 40
Quantitative analysis 40
Area normalization method 40
Internal standardization method 40
Comparison method 41

Applications 81
Analysis of amino acids 86
Chiral separation of enantiomers 89
Drug abuse 84
Ion chromatography 88
Ion-exclusion chromatography 90
Isomeric compounds 82
Partition chromatography 86
Polyaromatic hydrocarbons 82
Separation of nucleic acids 85
Speciation studies 91
Sugars in popular drinks 83

Bacterial identifications 41
Bonded phase chromatography (bpc) 57
Bulk property detectors 67
Capacity factor (k) 49
Carrier gas 12, 15
Cell voltage 34
Chromatogram 6
Chromatogram 6, 8
Chromosorb A 20
Chromosorb G 20
Chromosorb P 20
Chromosorb W 21
Column 52
Guard column 53
Precolumn 53

Columns 18
of a Gas chromatograph 18
Open tubular column 19
Capillary column 19
Packed columns 19

Column and solvent efficiency 6

Column diameter 11
Column efficiency 8
Liquid stationary phase 8
Flow rate of carrier gas 8
Sample size 8
Plate number 9
Plate height 9
Height equivalent to one theoretical plate (hetp) 9
Resolution, RS 9

Column temperature 26
Columns 18
Comparison of various HPLC detectors 72
Agilent 1100 (usa) hplc set up 73

99

Dead volume 7
Detectability 30
Detector response 30
Detector sensitivity 30
Detectability 30
Sensitivity 30

Detectors 27
Differential chromatogram 29
Differentiating detector 28
Electron capture detector (ECD) 31,33
Flame emission detector (FED) 31,35
Flame ionization detector (FID) 31, 32
Helium ionization detector (HID) 31, 34
Integral chromatogram 28
Integrating detector 28
Performance of some HPLC detectors 68
Plug flow 29
Solute property detectors 67
Solvent efficiency detectors 67
Thermal conductivity detector (TCD) 31

Differential refractometer 70
Diffusion coefficient 49
Eddy diffusion 10
Electrochemical detectors 71
Amperometric thin layer detector 71

Electron capture detector (ECD) 31, 33

Cell voltage 34
Analysis of lindane 34

Eluotropic series of solvents 64

Environmental analysis 41
Air analysis 41
Clinical and toxicological analysis 42
Forensic toxicology 42
Water analysis 41

Flame emission detector (alkali flame detector) 31, 35

Flame ionization detector (FID) 31, 32
Flow rate 11
Gas chromatograph 6
Gas chromatography 5
Applications 40
Instrumentation 15

Gas- liquid chromatography (GLC) 5

Gas sampling valve 39
Gas- solid chromatography (GSC) 5
Gradient elution 65
HETP 9
Helium ionization detector (HID) 31,35
High pressure liquid chromatograph (HPLC) 47
Advantages 78
Applications 81
Comparison with gas chromatography 79
High efficiency chromatography 48
High performance liquid 47
High resolution chromatography 48
High speed chromatography 48

Hold-up volume 7
Instrumentation 15
Rotometer 18

Instrumentation 15

100

Interfacing HPLC with mass spectrometry 91

Atmospheric pressure chemical ionization (APCI) 92
Electrospray interface 94
Moving belt interface 94
Particle beam interface 92
Thermospray method 92

Lindane
Analysis of 34

Linear detector range 31

Linear range 31

Liquid phase percentage 25

Liquid phases 21
Mass spectrometric detector 72
Mobile phase 15
Molecular diffusion 10
Noise and minimum detectable quantity 30
Minimum detectable quantity 31

Number of plates 13
Operation 6
Optical detectors 68
Fixed wavelength detector 69
Fluorescence detector 70
Infrared absorbance detector 70
Scanning wavelength detector 69
Ultraviolet detector 68
Variable wavelength detector 69

Optimization of separation 74
Packing material 53
Stationary phase 53
Macroporous particles 55
Particle diameter 11
Porous layer beads 54
Porous particles 54
Spherical bonded phase 54

Particle diameter 11
Plate count (n) 49
Plate number 9
Plate height 9
Plug flow 29
Porapak 26
Pressure drop ( p) 50
Principle 48
Pumps 66
Constant pressure pump 66
Pneumatic pump 66
Reciprocating piston pump 66
Syringe type displacement pump 66

Rate theory10
Multiple path effect 10
Eddy diffusion (a term) 10
Molecular diffusion 10
van Deemter equation 11
Column efficiency 11
Particle diameter 11
Flow rate 11
Carrier gas 11, 15
Column diameter 11

Relative retention () 49
Resolution, RS 9
Retention time 6

101

Retention time of air peak 7

Retention volume 7
Rotometer 18
Sample injection system 52
Sampling
Steps 37
Hazards 37
Introduction of the sample 37
Injection systems 37, 38
Direct injection 38
On- column injection 38
Purge and trap injection 38
Split injection 38
Solid injection 38
Thermal desorption 38
Valve injection 38

Sample size 37
Sample injection port 37, 38
Sampling syringe 38
Silicon septum 39
On-column operation 39

Sampling syringe 38
Separation factor 8
Sensitivity 30
Solvent efficiency 6, 11, 13
Debye forces 12
Dispersion forces 12
Induced dipole 12
Keesom forces 12
London forces 12
Nonpolar forces 12
Orientation 12
Specific interaction forces 12
Temperature 13

Solvent strength parameter, o 64

Some examples of applications 40
Specific column resistance () 50
Stationary phase support 20
and Liquid phases 20
Diatomite 20
Diatomaceous silica 20
Diatomaceous earth 20
Kieselguhr 20

Stationary phases 56
Adsorption chromatography 56
Chiral chromatography 61
Column packing 58
Column packings used in hplc 62
Hydrophilic porous packing 59
Ion chromatography 60
Ion-exchange chromatography 58
Liquid-liquid (LLC) chromatography 57
Partition chromatography 57
Schematic diagram of ion chromatograph 61
Size exclusion chromatography 59
Strong anion exchanger 59
Structure of silica gel 56
Weak anion exchanger 59

Super selective liquid phases 24

TCEPE 24
Liquid crystals 24

Thermal conductivity detector (katharometer) TCD 31

T.c. Wheatstone bridge circuit 32

102

van Deemter equation 11

Viscosity parameter () 50
Void volume 7

103