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Unit 8
Unit 8
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.
48
(a) (b) (c)
The plate height (H = L/N where, L is the column length and N the number of plates) is
reduced by the particle diameter (dp) and may be represented as
h = H/dp = L/N.dp ... (8.1)
It actually states the number of particle diameter (dp) that constitutes one plate height.
Thus, reduced velocity may be represented as
v = u.dp/ DM … (8.2)
where, DM is the diffusion coefficient of solute in the mobile phase. It may be
considered as the ratio of the time required to displace solute molecules a distance
equal to one particle diameter to the time needed for the same displacement by
molecular diffusion. It expresses the balance between mass transport by diffusion or
molecular motion across a single particle. Substituting the value of u (= L/tm), reduced
velocity may be expressed as
49
v = L.dp/tm.DM … (8.3)
Thus, the complete equation for the dependence of the reduced plate height may be
represented as modified van Deemter equation
h = B/v + A.v0..33 + C.v … (8.4)
where, B = 1.2 for solid core (pellicular) packing and 2.0 for completely porous
column packing. Also, A = 0 for well packed column and C = 0.05 for porous particles
decreasing to 0.003 for pellicular particles. No theory accurately describes the
dispersion from flow in homogeneity in the mobile phase. A logarithmic plot of
Eq. (8.4) is shown in Fig. 8.2. The reduced plate height has a minimum value in the
range 2-3 for intermediate region of velocities where reduced velocity is 3 -5.
It may be observed from Fig. 8.2 that A term dominates all along whereas B term
arising from axial and longitudinal diffusion, dominates at law reduced velocities. This
region of h/v curve is usually avoided. At high velocities, however, C term responsible
for increase in reduced plate height, dominates. As explained earlier, C term contains
the contributions from mass transfer kinetics and stagnant pockets of mobile phase.
You can see that Eq. 8.4 representing the reduced plate height is independent of
particle diameter of the column packing. The constants A, B and C are dependent on
the packing of column. The number of plates in a reasonable time may be optimized
while operating the
Fig. 8.2: Logarithmic plot of reduced plate height, h against reduced velocity, 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.
φη LvDM N 2 h 2φη
∆P = = … (8.5)
d 3p tM
50
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.
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The individual components are described below:
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 Inert to most organic solvents except
(PEEK) 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 Used for capillary LC.
Glass Limited pressure range.
Glass-lined SS Inert, withstands pressures but difficult to
know when the glass is broken.
Column fittings and connectors must be so designed that void volume is zero avoiding
unswept corners. Column length ranges10 to 30 cm with inner diameter of 2 to 5 mm
providing 40,000 to 60,000 plates per inch. However, shorter columns of 3 to 8 cm are
also used for fast separations but in such cases, sample size will become limited. The
length of the column may not only affect the resolution of a given separation –the
longer the column the larger number of plates but also the speed of separation.
Standard lengths vary with the manufacturer but most common values are 30, 25, 15,
12.5, 10 and 7.5 cm. It may be noted that shorter columns are described as high speed
columns. The columns packed with the finer particles are more expensive than the
standard 5 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.
53
Table 8.2: Characteristics of Some Commercial HPLC Column Packing
Materials
Type Pellicular Microprous
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.
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 m2/g. The mean pore diameter is
inversely related to the specific surface area where small molecules enter the
pores. The particles can be packed into the HPLC column of efficiencies up to
800 theoretical plates per centimeter if 5 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.
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.
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
55
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
56
competition between the solute and the mobile phase molecules for an active
site provides the driving force and selectivity in separations. Interaction between
a solute molecule and the adsorbent surface is best when functional groups
overlap adsorption sites. Adsorption chromatography is less influenced by
difference in molecular weight but certainly more by functional groups. For
compounds of low to moderate polarity, adsorption chromatography often
makes possible the separation of complex mixtures into classes of compounds
with similar chemical functionality. Typical examples of group separations are
polynuclear aromatics from a petroleum sample and the triglycerides from a
liquid extract.
Si OH + Cl Si R Si O Si R + HCl
CH3 CH3
CH3 CH3
Si O Si O Si Cl
CH3 CH3
57
Most commonly, the R group of the siloxane in these coatings is a n-octyl (C-8
chain) or n-octadecyl (C-18 chain). With such preparations, the long chain
hydrocarbon groups are aligned parallel to one another and perpendicular to the
particle surface, giving a brush or bristle-like structure as illustrated in Fig. 8.6.
The relationship the between polarity of the sample with that of the column
packing material and mobile phase is illustrated in Fig. 8.8. Retention increases
with the hydrophobic character of the solute samples. Generally, the lower the
polarity of the mobile phase, the higher is its eluent strength. The effect of chain
length of the alkyl group upon column performance is illustrated in Fig. 8.8
where it is observed that longer chains produce packings that are more retentive.
For example, maximum sample size for a C18 packing is roughly double that for
a C4 preparation under similar experimental conditions.
Fig. 8.8: Relationship between the polarity of the sample with that of the packing
material and the mobile phase in reverse phase HPLC
In commercial normal-phase bonded packings, the R in the siloxane structure is
a polar functional group such as cyano (−C2H4CN), diol
(–C3H6OCH2CHOHCH2OH), amino (−C3H6NH2), and dimethylamino
(C3H6N(CH3)2). The polarities of these packing materials vary over a
considerable range with the cyano type being the last polar and the amino types
the most. Diol packings are intermediate in polarity. With normal phase
packings, elution is carried with relatively non-polar solvents such as ethyl
ether, chloroform and n-hexane.
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.
58
(a) (b) (c) (d)
Fig. 8.9: Various structural types of ion-exchange packings: (a) pellicular with
ion-exchange film; (b) exchanger beads coated superficially with porous resin;
(c) macroreticular resin bead and (d) anion exchanger surface sulfonated and
bonded electrostatically
During preparation of ion exchanger by silylation, a vinyl group is chosen for R3
in -SiOSiR1R2R3 leading to a vinylated silica which is then polymerized with
styrene.
CH = CH2 + CH = CH2 CH CH2 CH CH2
C 6H 5 C6H5
C6H5 C6H5CH2Cl
RNH2
N(CH3)2CH2CH2OH
Hydrophilic polymers allow the separation of proteins, nucleic acids and other
large ionic molecules. The microporosity of these ion exchangers minimizes
possible exclusion effects.
59
Table 8.3: Correlation of Pore Size Diameter and Operating Range of Mol. Wt
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;
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.
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.
60
Electric
integrator
61
enantiomorphs of the solute and the chiral selector which is an integral part of
the stationary phase. The difference in stability between these complexes results
in difference in their retention times, the enantiomer forming the less stable
complex being eluted first.
A large number of chiral phases are commercially available. All of these are
coated on silica gel support. The coating itself is a polymeric material to which
an optically active isomer is bonded. For example, the l form of the amino acid,
proline has been bonded to polystyrene-p-divinylbenzene, a cross linked
copolymer to give an optically active stationary phase for the separation of
racemic mixtures of amino acids. In this case, Cu2+ ions are introduced into the
solution of the analyte enantiomers to be separated whereby a ternary complex,
as shown in Fig. 8.11. is formed between the stationary phase, amino acid anion
and Cu2+. The formation constant for this complex differs for d and l forms of
the analyte amino acid; thus, making their separation possible.
Fig. 8.11: Illustration of a ternary complex formed between an L-proline bonded phase,
an analyte amino acid and a Cu2+ ion
Cyclodextrin-bonded stationary phases have been demonstrated to be
particularly efficient in resolving structural isomers. Some examples 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.
62
Short chain (C3 or less) BPC, IPC IPC applications of above three
packings include bases, dyestuffs and
other multiply charged species; used
instead of IEC
Diol BPC Very polar and water-soluble
compounds, e.g., food and drink
additives
Anion and cation IEC (Ion-exchange Ionic and ionizable compounds, e.g.,
exchangers chromatography) vitamins, water-soluble drugs, amino
(tertiary amine or sulphonic acids, food and drink additives
acid)
Controlled porosity silicas Size exclusion Polymer mixtures, screening of
(chemically modified to Chromatography unknown samples. Increasing use for
reduce adsorption effects) separating mixtures of smaller
molecules before other modes of HPLC
Chiral amino acids bound to Chiral
aminopropyl Chromatography
(CC) Mixtures of enantiomers especially of
drugs
Chiral peptides CC
Cyclodextrins CC
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 ............................................................................
63
Essential features of a modern HPLC system includes flow control and inlet filter
through a Millipore filter under vacuum. Also degassing facility such as a supply of an
inert gas is a must. It helps in removing dissolved gases that may have adverse effect
on the column performance. The general criteria for the selection of a mobile phase
are:
• It should dissolve the sample.
• It should keep the column stable.
• It should be compatible with the detector.
• It should be immiscible with the stationary phase.
• Its viscosity should not be high.
• Active fluorides should be avoided when using glass column.
The eluting power of a solvent is determined by its overall polarity, the polarity of the
stationary phase, and the nature of sample components. The capacity factor, k′, is
controlled by the strength of solvent which can be easily predicted in adsorption
chromatography. Snyder has defined solvent strength parameter, 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) εo (Al2O3) Viscosity, Refractive
20ºC (mN index, 20ºC
sec m─2)
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
64
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.
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.
Fig. 8.12: Schematic illustration of low pressure gradient using three solvents
of different polarity
Time proportionating electrovalves used in modern instruments are regulated by a
microprocessor; thus, the resolution for each chromatogram. It can reduce the run
time and increase the sensitivity. As the gradient develops, tailings are made to elute
quicker.
The commercial liquid chromatographs are designed to mix two or more solvents in a
progressive manner from 0 to 100% of one component. If one of the solvents gives an
appreciable response at the detector, then the generation of a solvent gradient will also
introduce a baseline drift in response. In such a case, column will also need time to
65
regenerate the starting solvent composition each time a fresh gradient run is started
and ideally, a blank gradient is run between samples to prevent the occurrence of
artifact peaks which can be observed. This can make gradient elution seem slower than
literature values. It may be noted that gradient elution produces effects similar to
temperature programming in gas chromatography.
8.3.5 Pumps
A variety of pumps are used to maintain flow rate and pressure of the mobile phase.
Also a degasser is needed to remove dissolved air and other gases from the solvent. A
desirable feature of the delivery system is the capability of generating solvent gradient.
A pump should be able to operate up to a pressure of 100 atm (1500 psi) though in
some cases 400 atm (6000 psi) is desired. For most analytical columns, only moderate
flow rates of 0.5 – 2 mL/min may be required. However, for microbore columns, low
flow rates of only a few microlitres/min may be sufficient. Also, a pump should have a
small hold up volume. Some typical pumps are described below:
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.
66
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.
Besides low detection limit, a HPLC detector must meet following requirements:
• Selective response towards one or more classes of solutes.
• It must have small response time, at least 10 times less than the peak width of a
solute.
• It should have a linear response to solute that extends over several orders of
magnitude.
Some characteristics of commonly used detectors in HPLC systems are listed in
Table 8.8.
67
Table 8.8: Performance of some HPLC Detectors
Detector property Typical LOD* Linear range Commercial
(mass) 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
68
phase solvent must not absorb or may absorb only weakly. Water, methanol, hexane
and acetonitrile all permit operation in the for uv to at least 210 nm. Many absorbance
detectors are double beam devices where one beam passes through the element cell
and the other through a filter to reduce its intensity. Alternatively a chopped beam
system in conjunction with a single phototube is used. Detection limit can be
estimated if the noise level and the approximate molar absorptivity are known at the
operating wavelength. Assuming 1 cm path length and a noise level of 0.00004
absorbance unit, detection limit is
2 (noise) / bε = 0.00008 / ε mol cm–1litre–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
2(0.00004) 20 × (5 × 106 L) 500
= 0.4 ng
(10,000 L mol-1 cm -1)1cm
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.
69
independent signals. All the signals can be integrated between two preset
wavelengths and thus, multi-component complex samples can be detected.
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.
70
of the liquid changes. Two collimated beams from the projector light illuminate the
reference and the sample cells made of Teflon gasket clamped between the cell prism
and a stainless reflecting back plate. As the light beam is transmitted through the
interface, it passes through the flowing liquid film and impinges on the surface of the
reflecting back plate. This diffuse reflected light appears as two spots of light that are
imaged by lenses onto dual photo detectors. Since the ratio of reflected light to
transmitted light is a function of the refractive index of the liquids, the illumination of
the cell back plate is a direct measure of the refractive index in each chamber. With
mobile phase flowing through both compartments, coarse zero is adjusted by rotating
the entire projector assembly. Fine adjustment is done with the optical plate, a glass
plate which can be rotated ±30o from normal. Two different prisms must be used to
cover the useful range of refractive index.
C. Electrochemical Detectors
These can be used only if solute molecules in aqueous and aqueous-organic phase
have voltammeteric characteristics. These are of several types depending on
amperometry, polarography, coulometry and conductometry. Electrochemical
detectors have not been exploited to the extent of optical detectors though these have
advantage of being simple, highly sensitive, convenient and wide spread applicability.
A variety of HPLC/ electrochemical detectors are available commercially. These are
potential universal detectors for fulfilling long time need. A typical thin layer
amperometer detector is shown in Fig. 8. 16. It can measure nanoampere level current
at a controlled potential as a function of time and consists of a flow cell in a 50 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.
71
number of electroactive compounds that are detected. Only the compounds that
generate stable electroactive product and reach the second electrode are sensed.
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.
72
nature of solute. It is also possible to select a wavelength that can suppress the
absorption of an interfering solute or the mobile phase. However, their noise levels are
greater and hence they are less sensitive.
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
73
SAQ 5
Complete the following statements:
i) Solvent is delivered to the column through…………………………………….....
…………………………………………………………………………………….
ii) The composition of mobile phase may be estimated from………………………..
…………………………………………………………………………………….
iii) The most sensitive detector works on the principle of……………………………
…………………………………………………………………………………….
iv) Electrochemical detectors are specially useful for………………………………..
…………………………………………………………………………………….
v) Elution pattern from a polarographic detector is obtained in terms of……………
…………………………………………………………………………………….
vi) The purity of a compound may be evaluated from a peak by…………………….
…………………………………………………………………………………….
SAQ 6
Write the options available for the following:
i) Essential requirements for choosing a detector
…………………………………………………………………………………….
ii) Material used as windows of infrared spectrophotometer
…………………………………………………………………………………….
iii) Features of time of flight mass
…………………………………………………………………………………….
…………………………………………………………………………………….
74
Fig. 8.18. It is observed that as the water content is increased and the composition of
methanol and tetrahydrofuran is adjusted, six peaks corresponding to benzyl alcohol,
phenol, 3-phenylpropanol, 2,4-dimethylphenol, benzene and diethyl o-phthalate are
better resolved.
Fig. 8.18: Illustration of optimization of HPLC separation conditions using five ternary
phases. Peaks; 1. Benzyl alcohol, 2. Phenol, 3. 3-Phenylpropanol,
4. 2,4-dimethylphenol, 5. Benzene, 6. Diethyl o-phthalate
If complete separation is required in minimum possible time then first chromatogram
is the method of choice. If internal standard is to be added within a space of
chromatogram then either of next two chromatograms may be used. However, if
reaction products need to be separated or impurities are expected then any one of the
last two methods best meets the requirement.
In general, mobile phases with no or little polarity are used with polar bonded phases.
For non-polar bonded phases, however, mobile phases are selected from solvents with
high polarities. Thus, the mobile phase composition is adjusted to change the overall
retention time and/or to pull specific peak pairs apart as illustrated above. Solvent
optimization involving four solvents have also been described. Thus, the separation of
solutes with different functional groups may be improved by the use of ternary mobile
75
phases for the precise control of mobile phase-eluent strength in conjunction with
solvent programming. More sophisticated automated methods of mobile phase
optimization are commercially available. Most software packages are designed to use
one of the two alternative approaches.
A linear hydrocarbon chain is a popular bonded phase where alkyl group may have a
variety of chain length, usually a methyl (C-1), ethyl (C-2), octyl (C-8) or octadecyl
(C-18). The effect of chain length of the alkyl group upon performance of siloxane
column in reverse phase chromatography resulting in better resolution is illustrated in
Fig. 8.19. It is observed that poor resolution is observed with methyl group but it
becomes better with octyl and still better with octadecyl group. Thus, longer chains
Fig. 8.19: Effect of chain length on performance of reverse phase siloxane column packed
with 5 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
76
In many cases when the bands overlap, the selectivity factor is made larger by
k
adjusting ′ to a suitable level. Such a change is conveniently made by changing the
chemical nature of the mobile phase as typically shown for the separation of six
steroids in Fig. 8.21 by reverse-phase chromatography following a four solvent
optimization procedure consisting of methanol, acetonitrile, tetrahydrofuran and water.
It was developed for finding a suitable solvent system to resolve a given mixture in a
minimum possible time. Three compatible solvents were used to adjust the strength of
k
the mixture to yield a suitable value of ′ . The first two chromatograms in (a) and (b)
Fig. 8.21: Choice of mobile phase on the selective separation of six steroids using 5 µm C8
bonded reversed phase particles. Peak ; 1. Prednisone, 2. Cortisone, 3.
Hydrocortisone, 4. Dexamethasone, 5. Corticosterone, 6. Cortoexolone. Effect
k
show the results from initial experiments to determine minimum value of ′ 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
k
finding better α values, water was added to get ′ = 10. It is observed that results of
methanol/water and tetrahydrofuran/water in (c) and (d) show better resolution. A
77
mixture of acetonitrile, THF and water was also attempted as shown in (e) where it is
found to be the best mobile phase for the separation of six steroids in a mixture.
8.5 ADVANTAGES
HPLC is a versatile technique offering a number of selective variants to resolve
complex mixtures of biological, environmental and pharmaceutical samples. It is more
a concept which has been employed to a variety of chromatographic techniques based
on adsorption, partition using normal bonded and reversed phase, ion-exchange, size
exclusion, etc. It has the advantage of the possibility of controlling solute retention and
better selectivity by manipulating the stationary phase, mobile phase and other
experimental conditions. Some of the advantages may be summarized as follows:
i) It is a very fast separation method with analysis time as small as less than a
minute in some cases.
ii) HPLC is a useful separation method with high resolving power and a
quantitative analysis method with low detection limits and high accuracy.
iii) It is especially useful for resolving optically active compounds though different
types of columns with chiral stationary phase are used.
iv) The technique is applicable to small amount of samples where even trace
amounts of solutes may be determined.
v) All kinds of solids soluble in suitable organic solvent and liquid samples can be
analyzed though gases can not be anayzed.
vi) HPLC has much wider applicability in pharmaceutical and food processing
industry including forensic and environmental samples compared to GC which
has been found to be more useful in petroleum industry.
vii) HPLC is especially useful for continuous monitoring of the column effluent and
thus it can be used for any on-line process where analytical procedures may be
automated and data need not be handled manually.
viii) HPLC provides repetitive and reproducible analysis using the same column.
ix) HPLC is being widely used for the speciation of ionic and non-ionic species
such as various organic and inorganic forms of arsenic.
x) It is more versatile than gas chromatography since it is not limited to volatile
and thermally stable samples with wider choice of stationary and mobile phases.
xi) It is a non-destructive method which can be used for preparative and process
scale separations.
xii) It can be used for the separation of closely related compounds as well as for the
purification of compounds.
Besides so many advantages, the method also suffers from many disadvantages as
given below:
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.
78
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.
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.
79
viii) GC has limited applicability for gaseous solutes only though it is useful for the
identification of hydrocarbons of a homologous series. On the other hand, HPLC
has much wider applicability to a variety of organic and inorganic compounds.
ix) GC cannot be used for the separation of ionic species whereas such species can
be easily separated by HPLC.
SAQ 7
Write brief answers for the following:
i) Explain the role of computer controlled HPLC systems.
………………………………………………………………………………….…
……...……………………………………………………………………………..
……...……………………………………………………………………………..
……………...……………………………………………………………………..
ii) What is the effect of chain length of the alkyl group on the performance of
siloxane column in HPLC?
………………………………………………………………………………….…
…...……………………………………………………………..…………………
………...………………………………………………………..…………………
…………...………………………………………………………..………………
…………………...………………………………………………..………………
iii) Explain the role of k ′ in improving the resolution of HPLC chromatograms.
………………………………………………………………………………….…
……...……………………………………………………………..………………
…………...………………………………………………………..………………
…………………...………………………………………………..………………
iv) What is the most important common factor between GC and HPLC?
………………………………………………………………………………….…
……...……………………………………………………………..………………
…………...………………………………………………………..………………
…………………...………………………………………………..………………
v) Explain how HPLC is a non-destructive method of analysis?
………………………………………………………………………………….…
……...……………………………………………………………..………………
…………...………………………………………………………..………………
…………...………………………………………………………..………………
…………………...………………………………………………..………………
80
8.7 APPLICATIONS
The applications of HPLC in all its different forms have been steadily increasing day
by day. This is primarily because the technique is well suited to a wide variety of
compounds including organic, inorganic and biological compounds, small ions to
macromolecules, polymers, chiral compounds and labile materials. The most
appropriate choice of mode of HPLC for a given separation problem is based on the
relative molecular mass, solubility characteristics and polarity of the compounds to be
separated, as is illustrated in Fig. 8.22.
Fig. 8.22: Selection of suitable liquid chromatographic method for the separation
analysis depending on structure and properties of solute
The HPLC is the most successful technique in separating compounds as diverse as
aminoacids, nucleic acids, and proteins in physiological samples, active drugs,
steroids, carbohydrates, vitamins, dyestuffs, pesticides, polymers etc. It is used
routinely for the assay of pharmaceutical products, the monitoring of drugs and
metabolites in body fluids and for other biomedical, biochemical and forensic
applications such as the detection of drugs of abuse. The determination of additives in
81
foodstuffs and beverages including sugars, vitamins, flavorings and colorings and the
quality control of polymers, plastics and resins are further examples of the wide and
growing scope of HPLC. A summary of typical applications in various fields are listed
in Table 8.9.
82
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. 8.25: The HPLC separation of glucose, fructose and sucrose in Pepsi shown as three
distinct peaks
83
However, size exclusion chromatography has been used for qualitative identification
and quantitative determination of these sugars in four types of canned fruit juices of
apple, orange, pineapple and cranberry, as shown in Fig. 8.26. Also shown is
chromatogram for the standards which can be used for quantitative determination of
the constituent sugars. The packing which had an exclusion limit of 1000, was a cross
linked-polystyrene polymer made hydrophilic by sulphonation. A 25 cm long column
with this packing contained 7600 plates at 80 oC.
Fig. 8.26: Size exclusion HPLC method for the determination of glucose (G), fructose
(F) and sucrose (S) in canned fruit juices. Also shown is the standard on RHS
for quantitative determination of sugars
The size exclusion chromatography or gel permeation chromatography is the most
suited technique for the solutes with molecular weights 2000 or more and is also
useful for preliminary investigation of unknown samples. It can be used for the
determination of relative mass distribution for components of biochemical and
polymer systems with an accuracy of 10%. Desalting is commonly employed to isolate
the macromolecules from biochemical materials, where both simple and
macromolecules may be present in an electrolyte solution. Dilute solutions of macro-
molecules can be concentrated and isolated by adding dry gel beads to absorb the
solvent relative molecular mass solutes.
84
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.
(a) (b)
Fig. 8.28: HPLC separation of nucleic acids: (a) on Zipax cation exchange packing using
HPLC; and (b) using conventional ion-exchange chromatography
85
Whereas conventional ion-exchange method takes more than 2 hours for the
separation of cytosine, uracil, guanine and adenine, it takes only 5 minutes by HPLC
method requiring only microgram quantities.
The reverse phase technique comprises nearly half of all the LC methods described in
literature. It provides optimum retention and selectivity when compounds have no
hydrogen bonding groups or have a predominant aliphatic or aromatic character as
typically illustrated in Fig. 8.30 showing separation of solutes based on the size and
structure of alkyl groups in a series of phthalate esters.
86
Fig. 8.30: Chromatogram of o-phthalate esters run on a column with octadecyl packing
and methanol/water (90:10) as eluent. Peaks; 1. Dimethyl, 2. Diethyl, 3. Dipropyl, 4.
Dibutyl, 5. Dipentyl, 6. Dihexyl, 7. Diheptyl, 8. Dioctyl.
Applications of bonded phase partition chromatography have been explored in a
variety of fields. Of several thousands, two have been illustrated in Fig. 8.31 with the
examples of analysis of consumer and industrial products such as additives in soft
drinks and phosphate insecticides.
(a) (b)
87
8.7.8 Ion Chromatography
It is being routinely used for the separation of inorganic anions such as Cl–, Br–, F–,
SO 24 − , NO 3− , NO −2 , and PO 34− at ppm levels in surface waters, industrial effluents,
food products, pharmaceuticals and clinical samples. Typical ion chromatograms in
Fig. 8.32 shows the analysis of various anions and cations using two column flow
method. Thus, inorganic cations such as Na+, K+ and NH4+ can be monitored in foods
such as dietetic foods low in sodium, and urine samples where the efficiency of the
separation is markedly influenced by the use of complexing agent in the eluent.
However, separation of organic acids and bases, alkali, alkaline earth and transition
metal cations are also being achieved. Ion-exchange resins having a proportion of the
ionic sites replaced with hydrophobic reversed phase groups commonly used in HPLC
columns, typically octadecylsilane, enable separation of both non-ionic and ionic
species in a mixture. It has become a widely used technique in a pathological and
environmental analysis laboratory where commercial instruments are used.
Fig. 8.32: Applications of Ion chromatography for the separation of anions (a) and
cations (b) using two column flow method.
Another version of reverse-phase chromatography is ion-pair chromatography which
deals with separation of ionized or ionizable species on a reverse phase column. The
method can handle samples that are very polar, multiply ionized and/or strongly basic.
In ordinary reverse phase HPLC, organic ions show poor peak shapes and inadequate
retention. Ion-suppression method is limited to the pH range 2.0 to 7.5 by the
instability of stationary bonded phases outside this pH range. In case of ion-pair
88
chromatography, an ion pair reagent, a large organic counter-ion which is ionized, is
added at low concentration to the mobile phase. One ion of the reagent is retained and
separate organic solute ions of opposite charge by forming a reversible ion-pair
complex with the ionized sample as represented by the equilibrium
RCOO– + R4N+ ↔ [R4N+, –
OOCR]o ion-pair
Thus, electrically neutral compounds are partitioned between the mobile and non-polar
stationary phases. Unlike conventional ion-exchange, ion-pair partition can separate
non-ionic and ionic compounds in the same sample. First of all separation of the
nonionic solutes is optimized and then counterion is added to the mobile phase
whereby ionic solutes are retained.
Let us consider the separation of water soluble vitamins, strongly ionic thiamine, 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.
89
Fig. 8.34: Chiral separation of enantiomers of the benzodisazepine Temazepam drug
showing separate peaks corresponding to d and l forms
90
distributed between the mobile phase in the column and the immobilized liquid held in
the ores of the ion packing. Ion-exclusion chromatography has found numerous
applications for the identification and determination of acidic species in milk, coffee,
wine and other commercial products. Similarly, weak bases and their salts can also be
separated by using anion exchange column in OH– form.
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.
91
removing the liquid mobile phase while allowing the analytes to pass into the mass
spectrometer. Therefore, the main problem encountered is the mismatch between the
mass flows involved in HPLC (about 1g/min) which is two or three order of
magnitude larger than can be accommodated by conventional mass spectrometer
vacuum systems. Another problem is the difficulty in vaporizing nonvolatile and
thermally labile molecules without degrading them. Several designs of interface have
been developed, the main difference between them being the means of separating
analytes from the mobile phase and the method of ionization employed. Many
compromises have been suggested in the operating conditions of either the
chromatograph or the mass spectrometer. Some approaches followed are described
below and schematically shown in Fig. 8.37.
92
(a) Schematic of thermospray where particle enters in at a and transfer line is suddenly
heated at b resulting in spray formation at c
Fig. 8.37: Various interfaces of HPLC-MS depicting Thermospray, Particle beam, APCl
and electrospray source.
93
8.8.4 Electrospray Interface
It also operates at atmospheric pressure and consists of a metal capillary tube through
which column effluent is passed at a relatively low flow rate of 1-20 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.
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.
94
8.10 TERMINAL QUESTIONS
1. Explain the following terms briefly:
i) Sample injection
ii) Bonded phase
iii) Pellicular packing
iv) Bulk property detector
v) Normal phase packing
vi) Characteristics of a detector
2. Explain the acidic character of silica and basic character of alumina and their
usefuness as inert core material.
3. Compare conventional liquid chromatography with reverse phase high
performance chromatography with suitable examples and applications.
4. Explain the linear response range of a detector. What will happen if you work
beyond this range?
5. What are guard and suppressor columns. In what respects they differ from each
other?
6. In a normal phase column of 15 cm length, a solute showed a retention time of
17.8 min whereas an unretained sample had a retention time of 0.73 min when
the mobile phase was chloroform/benzene(1:1). Calculate capacity factor k ′ of
the solute. How can it be altered. If the number of plates were 10200, calculate
the plate height.
7. A two component pharmaceutical product was separated by using a 15.0 cm
long HPLC column yielding following retention and peak width data:
If the solvent showed up a peak at 1.37 min then calculate (a) capacity factors
for each of the two components (b) number of plates using peak width and half
peak width and (c) the resolution of the two compounds using full peak width
and half peak width.
8. Explain the role of solvent mixture in the separation of components in a mixture.
8.11 ANSWERS
Self Assessment Questions
1. High performance liquid chromatography
High pressure liquid chromatography
High speed liquid chromatography
High resolution chromatography
High efficiency liquid chromatography
95
2. Smaller the particle size of stationary phase, larger is the surface area for solute
particles to interact with. It increases the efficiency of separation.
3. i) [B]
ii) [B]
iii) [C] and [D]
iv) [D]
v) [D]
5. i) Reciprocating pump
ii) Gradient elution
iii) Fluorescence
iv) Polar mobile phase
v) Current vs time
vi) Software data evaluation
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
96
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.
k
5. a) Capacity factors using the formula, ′ = (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
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).
6. Analytical Chemistry By G. D. Christian, 6th Edn, John Wiley & Sons Inc,
Singapore (2003)
97
12. Separation Methods By M. N. Sastri, 3rd Edn. Himalaya Publishing House,
Mumbai (2005)
98
INDEX
Adjusted retention time 7
Adsorption energy per unit area of adsorbent 64
Advantages 78
Air peak 7
Alkali flame detector 31, 35
Applications of gas chromatography 40
Examples 41
Identification of compounds 40
Quantitative analysis 40
Area normalization method 40
Internal standardization method 40
Comparison method 41
Applications 81
Analysis of amino acids 86
Chiral separation of enantiomers 89
Drug abuse 84
Ion chromatography 88
Ion-exclusion chromatography 90
Isomeric compounds 82
Partition chromatography 86
Polyaromatic hydrocarbons 82
Separation of nucleic acids 85
Speciation studies 91
Sugars in popular drinks 83
Bacterial identifications 41
Bonded phase chromatography (bpc) 57
Bulk property detectors 67
Capacity factor (k’) 49
Carrier gas 12, 15
Cell voltage 34
Chromatogram 6
Chromatogram 6, 8
Chromosorb A 20
Chromosorb G 20
Chromosorb P 20
Chromosorb W 21
Column 52
Guard column 53
Precolumn 53
Columns 18
of a Gas chromatograph 18
Open tubular column 19
Capillary column 19
Packed columns 19
Column and solvent efficiency 6
Column diameter 11
Column efficiency 8
Liquid stationary phase 8
Flow rate of carrier gas 8
Sample size 8
Plate number 9
Plate height 9
Height equivalent to one theoretical plate (hetp) 9
Resolution, RS 9
Column temperature 26
Columns 18
Comparison of various HPLC detectors 72
Agilent 1100 (usa) hplc set up 73
99
Dead volume 7
Detectability 30
Detector response 30
Detector sensitivity 30
Detectability 30
Sensitivity 30
Detectors 27
Differential chromatogram 29
Differentiating detector 28
Electron capture detector (ECD) 31,33
Flame emission detector (FED) 31,35
Flame ionization detector (FID) 31, 32
Helium ionization detector (HID) 31, 34
Integral chromatogram 28
Integrating detector 28
Performance of some HPLC detectors 68
Plug flow 29
Solute property detectors 67
Solvent efficiency detectors 67
Thermal conductivity detector (TCD) 31
Differential refractometer 70
Diffusion coefficient 49
Eddy diffusion 10
Electrochemical detectors 71
Amperometric thin layer detector 71
Electron capture detector (ECD) 31, 33
Cell voltage 34
Analysis of lindane 34
Eluotropic series of solvents 64
Environmental analysis 41
Air analysis 41
Clinical and toxicological analysis 42
Forensic toxicology 42
Water analysis 41
Flame emission detector (alkali flame detector) 31, 35
Flame ionization detector (FID) 31, 32
Flow rate 11
Gas chromatograph 6
Gas chromatography 5
Applications 40
Instrumentation 15
Gas- liquid chromatography (GLC) 5
Gas sampling valve 39
Gas- solid chromatography (GSC) 5
Gradient elution 65
HETP 9
Helium ionization detector (HID) 31,35
High pressure liquid chromatograph (HPLC) 47
Advantages 78
Applications 81
Comparison with gas chromatography 79
High efficiency chromatography 48
High performance liquid 47
High resolution chromatography 48
High speed chromatography 48
Hold-up volume 7
Instrumentation 15
Rotometer 18
Instrumentation 15
100
Interfacing HPLC with mass spectrometry 91
Atmospheric pressure chemical ionization (APCI) 92
Electrospray interface 94
Moving belt interface 94
Particle beam interface 92
Thermospray method 92
Lindane
Analysis of 34
Linear detector range 31
Linear range 31
Liquid phase percentage 25
Liquid phases 21
Mass spectrometric detector 72
Mobile phase 15
Molecular diffusion 10
Noise and minimum detectable quantity 30
Minimum detectable quantity 31
Number of plates 13
Operation 6
Optical detectors 68
Fixed wavelength detector 69
Fluorescence detector 70
Infrared absorbance detector 70
Scanning wavelength detector 69
Ultraviolet detector 68
Variable wavelength detector 69
Optimization of separation 74
Packing material 53
Stationary phase 53
Macroporous particles 55
Particle diameter 11
Porous layer beads 54
Porous particles 54
Spherical bonded phase 54
Particle diameter 11
Plate count (n) 49
Plate number 9
Plate height 9
Plug flow 29
Porapak 26
Pressure drop ( ∆ p) 50
Principle 48
Pumps 66
Constant pressure pump 66
Pneumatic pump 66
Reciprocating piston pump 66
Syringe type displacement pump 66
Rate theory10
Multiple path effect 10
Eddy diffusion (a term) 10
Molecular diffusion 10
van Deemter equation 11
Column efficiency 11
Particle diameter 11
Flow rate 11
Carrier gas 11, 15
Column diameter 11
Relative retention (α) 49
Resolution, RS 9
Retention time 6
101
Retention time of air peak 7
Retention volume 7
Rotometer 18
Sample injection system 52
Sampling
Steps 37
Hazards 37
Introduction of the sample 37
Injection systems 37, 38
Direct injection 38
On- column injection 38
Purge and trap injection 38
Split injection 38
Solid injection 38
Thermal desorption 38
Valve injection 38
Sample size 37
Sample injection port 37, 38
Sampling syringe 38
Silicon septum 39
On-column operation 39
Sampling syringe 38
Separation factor 8
Sensitivity 30
Solvent efficiency 6, 11, 13
Debye forces 12
Dispersion forces 12
Induced dipole 12
Keesom forces 12
London forces 12
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
102
van Deemter equation 11
Viscosity parameter (η) 50
Void volume 7
103