Unit 8 High Performance Liquid Chromatography HPLC

57
47 UNIT 8 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) Structure 8.1 Introduction Objectives 8.2 Principle 8.3 Instrumentation Sample Injection System Column Packing Material or Stationary Phase Solvent Supply System Pumps Detectors 8.4 Optimization of Separation 8.5 Advantages 8.6 Comparison with Gas Chromatography 8.7 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 μm 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 μm 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.

description

High Performance Liquid Chromatography HPLC

Transcript of Unit 8 High Performance Liquid Chromatography HPLC

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47

UNIT 8 HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC)

Structure

8.1 Introduction Objectives

8.2 Principle

8.3 Instrumentation Sample Injection System

Column

Packing Material or Stationary Phase

Solvent Supply System

Pumps

Detectors

8.4 Optimization of Separation

8.5 Advantages

8.6 Comparison with Gas Chromatography

8.7 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 µm 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 µm 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.

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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.

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(a) (b) (c)

Particle size: >150 µm 40-70 µm 5-10 µm

Column diameter: 10-50 mm 1 – 3 mm 2 – 6 mm

Column length: 50-200 cm 50-100 cm 10-50 cm

Pressure: < 1 atm 30-50 atm 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 (α), 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

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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, ν 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 µm

Similarly, using viscosity parameter (η) and specific column resistance (ф) for a fully

porous packing, pressure drop (∆P) may be calculated using the expression.

2 2

3

M

p M

LvD N hP

d t

φη φη∆ = = … (8.5)

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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

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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 µL 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 µL. 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.

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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

(PEEK)

Inert to most organic solvents except

methylene chloride, THF, DMSO and conc.

Sulphuric and nitric acids. Holds pressures

up to 5000 psi (34MPa).

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

Glass

Used for capillary LC.

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 µm 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 µm 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

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Table 8.2: Characteristics of Some Commercial HPLC Column Packing

Materials

Type

Pellicular Microprous

Silica Corasil (37-50µm) Lichrosorb (5,10 & 20 µm)

Vydac (30-40µm) Micropak (5&10 µm)

Porasil (15 & 20 µm)

Spherisorb (5 µm)

Alumina Perisorb PA Micropak Al (5 & 10 µm)

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:

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 µm coated with a thin porous outer shell, typically of 1-3 µm 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 m

2/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 µm particle sizes are used. However,

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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 µm) for

packing material in HPLC?

a) 1-5 b) 3-5 c) 10-20 d) 20-50

ii) 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

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iii) Which of the following materials meet the requirements to fabricate HPLC

column?

a) Glass lined metal b) Quartz c) Stainless steel d) Steel

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

a) 100 b) 300 c) 800 d) 400

v) 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-OH…O).

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

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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

µm. 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:

Si OH Cl Si R

CH3

CH3

Si O Si R

CH3

CH3

+ + HCl

Si OH Cl Si Cl

Si OH

Si O Si O Si Cl

CH3

CH3CH

3

CH3

Si O Si O Si Cl

CH3

CH3

CH3

CH3

CH3

+ H2O

Surface coverage by silanization is limited to 4 µmol/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.

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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 -PO3

2– 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 µm thick, on a

glass bead of 30-40 µm 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.

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(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

CH CH2

C6H

5 C6H

5

= + =

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 CH

2ClCH

2OCH

3CH

2CH

2CH CH

2CH

3OH

C6H

5CH

2ClC

6H

5

CH2

CH2 CH CH

2

C6H

5CH

2NH

2-R

CH2

CH2 CH CH

2

C6H

5CH

2N(CH

3)

3

N(CH3)

2CH

2CH

2OH

RNH2

++

+

Weak anion exchanger 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 µm.

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.

Page 14: Unit 8 High Performance Liquid Chromatography HPLC

60

Table 8.3: Correlation of Pore Size Diameter and Operating Range of Mol. Wt

Pore-size diameter

(µm)

Operating range

(Daltons)

4 1000-8000

10 1000-30000

25 2500-125000

55 11000-350000

150 100000-1000000

250 200000-1500000

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 CH

2CH

2O CH

2CH

2 C CH2OH

H

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 Particle

Size, µm

Average Pore

Size, Ǻ

Molecular eight

Exclusion Limit

Polystyrene-

divinylbenzene

10 102 700

103 (0.1 – 20) × 10

4

104 (1 – 20) × 10

4

105 (1 – 20) × 10

5

106 (5 – >10) × 106

Silica 10 125 (0.2 – 5) × 104

300 (0.03 – 1) × 105

500 (0.05 – 5) × 105

1000 (5 – 20) × 105

v) 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.

Page 15: Unit 8 High Performance Liquid Chromatography HPLC

61

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

The column packing consists of a neutral polymer core of ~ 10 µm 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 anion-

exchange 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

Electric

integrator

Page 16: Unit 8 High Performance Liquid Chromatography HPLC

62

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 are-

prostaglandin A1, A2 and B1B2, α- and β-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 10µm

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

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63

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.

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64

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, εo, 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 εo. 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, εo and the Physical Properties of Selected

Solvents Used in HPLC

Solvent εo(SiO2) ε

o (Al2O3) Viscosity,

20ºC (mN

sec m─2

)

Refractive

index, 20ºC

Pentane 0.00 0.00 0.23 1.358

Hexane 0.00 0.313 1.375

Cyclohexane —0.05 0.04 0.980 1.426

Carbon disulphide 0.14 0.15 0.363 1.628

Carbon tetrachloride 0.14 0.18 0.965 1.460

1-Chlorobutane 0.26 0.47 1.402

Di-isopropyl ether 0.28 0.379 1.368

2-Chloropropane 0.29 0.335 1.378

Benzene 0.25 0.32 0.65 1.501

Diethyl ether 0.38 0.38 0.23 1.353

Chloroform 0.26 0.40 0.57 1.443

Methylene dichloride 0.42 0.44 1.425

Tetrahydrofuran 0.45 0.55 1.407

Acetone 0.47 0.56 0.32 1.359

1,4-Dioxane 0.49 0.56 1.54 1.422

Ethyl acetate 0.38 0.58 0.45 1.370

1-Pentanol 0.61 4.1 1.410

Acetonitrile 0.50 0.65 0.375 1.344

Methanol 0.95 0.60 1.329

Water Large 1.00 1.333

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65

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

Page 20: Unit 8 High Performance Liquid Chromatography HPLC

66

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 µL. 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 µL),

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.

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.

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67

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 µg/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.

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68

Table 8.8: Performance of some HPLC Detectors

Detector property Typical LOD*

(mass)

Linear range Commercial

availability

Absorbance 10 pg 3-4 Yes

Fluorescence 10 fg 5 Yes

Electrochemical 100 pg 4-5 Yes

Conductivity 100 pg-1 ng 5 Yes

Refractive Index 1 ng 3 Yes

Mass spectrometry <1pg 5 Yes

FTIR 1µg 3 Yes

Light scattering 1µg 5 Yes

Optical activity 1 ng 4 No

Photo ionization <1pg 4 No

Element selective 1 ng 4-5 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 10µL 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 10–6 absorbance units. A quartz collimating lens focuses

the radiation on the sample and reference cell with detector cell volume of 8µL/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

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69

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) / bε = 0.00008 / ε mol cm–1

litre–1

If ε =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

5µL sample and a dilution factor of 20 (Mol wt 500), detection limit is

ng0.4cm1)cmmolL(10,000

106 L) 500 (5 202(0.00004)-1-1

=××

i) 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.

ii) 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 tungsten-

halogen 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

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70

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 µL. 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 µm or 4000 to 690 cm–1. 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 10–9 to 10–12 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 Freshnel’s 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 µL. 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

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71

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 µm

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 1-5µL.

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

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72

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 µL/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 2µL/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

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73

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 0.001ºC. 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

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74

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

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75

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

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76

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 µm 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

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77

In many cases when the bands overlap, the selectivity factor is made larger by

adjustingk ′ 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 ofk ′ . The first two chromatograms in (a) and (b)

Fig. 8.21: Choice of mobile phase on the selective separation of six steroids using 5 µm C8

bonded reversed phase particles. Peak ; 1. Prednisone, 2. Cortisone, 3.

Hydrocortisone, 4. Dexamethasone, 5. Corticosterone, 6. Cortoexolone. Effect

of % water to adjust k ′ in (a) and (b). Further separation factor α is varied at

constant k ′ in (b), (c), (d) and (e)

show the results from initial experiments to determine minimum value of k ′ which is

estimated to be 10. However, it is observed that α values for components 1 & 3 and

that of 5 & 6 do not yield satisfactory resolution. In further experimentation for

finding better α values, water was added to get k ′ = 10. It is observed that results of

methanol/water and tetrahydrofuran/water in (c) and (d) show better resolution. A

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78

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:

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.

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79

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 α 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 10–12 to 10–15 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.

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80

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?

………………………………………………………………………………….…

……...……………………………………………………………..………………

…………...………………………………………………………..………………

…………...………………………………………………………..………………

…………………...………………………………………………..………………

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81

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

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82

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

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83

illustrated in Fig. 8.24 showing the separation of positional isomers of syn- and anti-

pyrazolines 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

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84

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 macro-

molecules 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.

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85

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

The sample is filtered through 2 µm 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

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86

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.

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87

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)

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

Peaks; 1. Methyl parathion,

2. Ciodrin, 3. Parathion,

4. Dyfonate, 5. Diazinon, 6. EPN,

7. Ronnel, 8. Trithion

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88

8.7.8 Ion Chromatography

It is being routinely used for the separation of inorganic anions such as Cl–, Br–, F–,

,SO 2

4

− −

3NO , −

2NO , and −3

4PO 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

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89

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, non-

ionic 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.

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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

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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

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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 electron-

impact 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.

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(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.

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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 µL/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 liquid-

liquid chromatography (LLC) where a narrow width (2-5 mm diameter) and about

50 cm long column is packed with ultrafine material (5-10 µm) 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.

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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

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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]

4. i) Silanol group is hydrolysed with organochlorosilane

ii) nonpolar solvents such as n-hexane, chloroform

iii) COOH, −34PO

iv) 100 to 500 millions

v) ion chromatography, water and CO2

vi) enantiomers, transient diastereoisomeric compounds

5. i) Reciprocating pump

ii) Gradient elution

iii) Fluorescence

iv) Polar mobile phase

v) Current vs time

vi) Software data evaluation

6. i) Selective separation, accurate determination and continuous detection

ii) Sodium chloride, calcium fluoride

iii) Unlimited mass range, high sensitivity, multiplex detection capability

7. 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 non-

destructive method.

Terminal Questions

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

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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.

3. 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 µm

5. a) Capacity factors using the formula, k ′ = (tR – tM) / tM; 4.39 and 5.29

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

using half width formula, n = 5.54 (tR / w½)2 ; 3140 and 3569

c) Resolution using the full width formula, R = 2(t2 – t1)/ (w1 + w2); 1.81

half width formula, R = 2(t2 – t1)/ 1.69 (w½′+ w½′′); 2.28

6. 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. Vogel’s 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. O’Reilly,

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)

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

Mumbai (2005)

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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

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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

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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 (α) 49

Resolution, RS 9

Retention time 6

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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

Non–polar forces 12 Orientation 12

Specific interaction forces 12

Temperature 13

Solvent strength parameter, εo 64

Some examples of applications 40

Specific column resistance (ф) 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

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van Deemter equation 11

Viscosity parameter (η) 50

Void volume 7