➢ What is HPLC?

High pressure liquid chromatography (or high pressure liquid chromatography,

HPLC) is a form of column chromatography used frequently in biochemistry and
analytical chemistry to separate, identify, and quantify compounds based on
their idiosyncratic polarities and interactions with the column's stationary
phase.
➢ HPLC separation modes
1. Adsorption chromatography
2. Reversed phase chromatography
3. Liquid-liquid partition chromatography-The porous support material is loaded
with a liquid that acts as the stationary phase. This liquid is insoluble in mobile
phase. The molecules of the sample are distributed between liquid stationary
phase and mobile phase according to the distribution law (as extraction in
separating funnel). If a sample is more soluble in the liquid stationary phase, it
will stay more time in the stationary phase and will be eluted latter (that is it
takes long time to elute).
4. Chromatography with chemically bonded phase-The stationary phase is not
applied to porous support material in a liquids film form, instead, the stationary
phase material is covalently bonded to the support material by chemical
reaction (e.g. as in case of RP-HPLC)
5. Ion-exchange chromatography-
6. Ion-pair chromatography-Molecules of the ionic samples are masked by a
suitable counter ion. The advantages are (i) RP-HPLC system can be used, so
no ion-exchange is needed, (ii) Acids, base and neutral products can be
analyzed simultaneously.
7. Ion chromatography-is used for separating the ions of strong acids and
bases (CL-, NO3+, Na+, K+). It is an special case of ion exchange
chromatography.

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8. Size exclusion chromatography 1
9. Affinity chromatography

 Sujan Bose 
 Apparatus for HPLC: (Draw and label the schematic diagram of
HPLC machine)

Figure 1

1=solvent reservoir, 2=sintered metal frit, 3=high pressure pump, 4=pulse damper,
5=drain valve, 6=manometer, 7=pre-column, 8=injection syringe, 9=injection valve,
10=column, 11=thermostat oven, 12=detector, 13=data acquisition (recorder or
integrator), 14=fraction collector.

➢ Principle of HPLC-
The principle of HPLC is to force the sample through a column of the stationary
phase by pumping the mobile phase at high pressure. The sample to be analyzed
is introduced in small volume to the stream of mobile phase and the sample
molecules are retained by specific chemical and/or physical interactions with the
materials of the stationary phase as it travels the length of the column. The

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amount of retention depends on- (1) the nature of the sample, (2) the
composition of the stationary phase and mobile phase. The time taken by the 2
sample to travel from the point of injection to the end of the column is called the
retention time and is considered a unique identifying characteristic of a given
sample because no two compounds will have the same retention time (like no
two persons can sit in a single seat). The use of pressure increases the linear
speed giving the components less time to diffuse within the column, leading to
improved resolution in the resulting chromatogram. Common solvents used
include any miscible combinations of water or various organic liquids (the most
common are methanol and acetonitrile).

 Sujan Bose 
In HPLC separation, the compounds that tend to reside in the mobile phase, move more
quickly than those that prefer to reside in stationary phase. Phase preference can be
expressed by distribution co-efficient, K:

Cstat

Kx=-------------

Cmob
Where,

Cstat= Concentration of compound X in the stationary phase

Cmob= Concentration of X in mobile phase.

K= Partition coefficient

The stationary phase and mobile phase must be in intimate contact with each
other in order to ensure a distribution balance. The various components must
have different distribution coefficients for ease of separation.

➢ Safety in HPLC handling

There are some health risks inherent in HPLC handling. These are -
1.Toxic solvents-Short and long term risks of exposure to solvents and vapors
--Use perforated plastic lids to all feed and waste containers so that no toxic
vapor can escape into the laboratory environment and no impurities can
contaminant the highly pure solvents.
--A good ventilation system should be provided in the solvent handling areas
2. Pulmonary irritation from the stationary phase-Particles of 5 micron and less

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are used in HPLC and these may enter into lungs and may cause damage to lungs
-- As a safety measure, any operation involving possible escape of stationary
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phase dusts must be carried out in a fume cupboard
3. Dangers resulting from the high pressures- High pressure pump may be a risk
factor but liquids are less compressible in comparison to gas. A jet of liquid may
leak from a faulty fitting and may cause serious physical damage.
4. Dangers from high voltage-

➢ Causes of Band Boarding :-

 Sujan Bose 
First Cause: “Eddy Diffusion”- The column is packed with small stationary phase
particles. The mobile phase passes through and transports the sample molecules
with it (Fig 5). Some molecules are fortunate and leave the column before most
others by traveling an almost straight line path. Other sample molecules undergo
several diversions along the way.

Second Cause: Flow distribution-The mobile phase passes in a laminar flow

between the stationary phase particles (Fig 6). The flow is faster in the channel
center than it is near the particle. Eddy diffusion and floe distribution may be
reduced by packing the column with evenly sized particles (First principle).

Third Cause: Sample molecule diffusion in the mobile phase- It is also called
longitudinal diffusion effect. Sample molecules spread out in the solvent without
any external influence (Just like salt or sugar dissolves slowly in water). The
longitudinal diffusion has a harmful effect if (i) small stationary phase particles
(<10 micron) (ii) Low mobile phase velocity and (iii) large sample diffusion co-
efficient etc coincide in the HPLC system. This can be overcome by following
Second principle.

Fourth cause: Mass transfer between mobile, stagnant mobile and

stationary phase- Pore structure of a stationary phase particle show us that the
channels are both narrow and wide, some channels pass through the whole of the
particle while others are closed off (half way open and then closed). Half way
open pores are filled with mobile phase and are stagnated. The recovery process
may occur in two ways-(a) The molecules may diffuse back to the mobile flux
phase .It depends on the size of pores and viscosity of the solvents used. (b) The
molecules may interact with stationary phase and is adsorbed and then after
sometime desorbed. Hence mass transfer takes place. In both cases band
broadening decrease with increasing mobile phase flow velocity. These effects
can be overcome by Third, Fourth and Fifth principles.

Remedy:

First Principle: The packing should be composed of particles with as narrow a

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size distribution as possible (5:7.5=1:1.5)
Second principle: The mobile phase flow velocity should be selected so that
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longitudinal diffusion has no adverse effect
Third principle: Small particles or those with a thin, porous surface layer should
be used as the stationary phase
Fourth principle: Low viscosity solvents should be used
Fifth principle: High analysis speed is achieved at the expense of resolution and
vice versa.
➢ Types of HPLC
1. Normal Phase Chromatography

 Sujan Bose 
 2. Reversed phase chromatography
3. Size exclusion chromatography
4. Ion Exchange chromatography
5. Bio-affinity chromatography
6. Isocratic flow and gradient elution)
1. Normal phase HPLC (Adsorption HPL Chromatography)
Normal phase HPLC (NP-HPLC) separates samples based on polarity. This method
uses (i) a polar stationary phase and (ii) a non-polar mobile phase, and (iii) is
used when the samples is polar in nature. The polar sample dissolves in polar
stationary phase and is retained by the polar stationary phase. Adsorption
strengths increase with increase in samples polarity, and the interaction between
the polar samples and the polar stationary phase (relative to the mobile phase)
increases the elution time. NP-HPLC had fallen out of favor in the 1970's with the
development of reversed-phase HPLC because of a lack of reproducibility of
retention times as water or protic organic solvents changed the hydration state of
the silica or alumina as chromatographic media.

“Rule of thumb (Normal state of things): Polar compounds are eluted later than
non-polar compounds
Polar=Water soluble, Hydrophilic, Non-polar=Fat Soluble, Lipophilic, Protic=
Produces H+ ions (Able to donate protons”)
1.Reversed phase chromatography
In reversed phase HPLC (RP-HPLC) reverse of the following applies:
The stationary phase is very non-polar
(b) The mobile phase is relatively polar (water to dioxane)
(c) A polar solvent such as water elutes more slowly than a less polar solvent
such as acetonitrile
“Rule of Thumb: Non-polar compounds are eluted later than polar compounds”
2.Size exclusion chromatography
SEC can be subdivided into gel permeation chromatography (with organic
solvents) and gel filtration chromatography (with aqueous solutions). SEC
separates particles on the basis of molecular size, i.e. according to molecular

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mass. The largest molecules are eluted first and the smallest molecules last. It is
generally a low resolution chromatography and thus it is often reserved for the 5
final, "polishing" step of purification. It is also useful for determining the tertiary
structure and quaternary structure of purified proteins, and is the primary
technique for determining the average molecular weight of natural and synthetic
polymers (Molecular mass difference must be about 10%).
3.Ion exchange chromatography (IEC)
The stationary phase contains ionic groups (e.g. NR3+ or SO3- ) which interact
with ionic groups of the sample molecules. The method is suitable for separating
amino acids, ionic metabolic products and organic ions.

 Sujan Bose 
In IEC, retention is based on the attraction between solute ions and charged ions
bound to the stationary phase. Ions of the same charge are excluded. In general,
ion exchangers favor the binding of ions of higher charge and smaller radius. This
form of chromatography is widely used in the following applications: In purifying
water, preconcentration of trace components, Ligand-exchange chromatography,
Ion-exchange chromatography of proteins, High-pH anion-exchange
chromatography of carbohydrates and oligosaccharides, etc. Some types of Ion
Exchangers include: (1) Polystyrene resins- allows cross linkage which increases
the stability of the chain. (2) Cellulose and dextran (gels)-These possess larger
pore sizes and low charge densities making them suitable for protein separation,
(3) Controlled-pore glass or porous silica.
4.Bio-affinity chromatography
This chromatographic process relies on the property of biologically active
substances to form stable, specific, and reversible complexes. The formation of
these complexes involves the participation of common molecular forces such as
the Van der Waal's interaction, electrostatic interaction, dipole-dipole interaction,
hydrophobic interaction, and the hydrogen bond. An efficient, bio-specific bond is
formed by a simultaneous and concerted action of several of these forces in the
complementary binding sites.

Antigen Antibody
Enzyme Inhibitor
Hormone Carrier

Highly specific biochemical interaction is the basis of separation. Stationary

phase contains specific groups of molecules which can only adsorb the sample
(as in antigen-antibody reaction)

5.Isocratic flow and gradient elution

With regard to the mobile phase, a composition that remains constant throughout
the procedure is termed isocratic.
In contrast to this is the so called "gradient elution", which is a separation where

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the mobile phase changes its composition during a separation process. One
example is a gradient in 20 min starting from 10% Methanol and ending up with
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30% Methanol. Such a gradient can be increasing or decreasing. The benefit of
gradient elution is that it helps speed up elution by allowing components that
elute more quickly to come off the column under different conditions than
components which are more readily retained by the column. By changing the
composition of the solvent, components that are to be resolved can be selectively
more or less associated with the mobile phase as a result at equilibrium they
spend more time in the solvent and less in the stationary phase therefore they
elute faster.

 Sujan Bose 
➢ Pumps
Pumps for HPLC must be capable of producing high pressures. This is essential if
the mobile phase is to be forced quickly enough through the chromatographic
bed, the small particles of which offer a high resistance to flow. A high-flow rate is
essential for preparative work and column packing.
1. Gas-driven discontinuous displacement system
2. Discontinuous displacement pumps
3. Membrane piston pumps
4. Short-stroken piston pump
5. Pneumatic amplifier pumps
➢ Columns to be used as Stationary phases

Most HPLC columns are made of 316-grade stainless steel, which is chromium-
nickel-molybdenum steel, resistant to usual HPLC pressure and also relatively
inert to chemical corrosion. The inside of the column should have no rough
surfaces, grooves or micro porous structure, so the steel tubes must be either
precision drilled or polished or electro-polished after common manufacturing e.g.
by drawing. Rough surfaces can lead to up to ten times fewer plate numbers.
Glass tubes are smooth, chemically inert and the main advantage is that dirt,
cracks and the presence of air can all be easily monitored. Up to 1.5 times more
plates may be obtained than with steel tubes due to smooth interior walls. They
are also corrosion resistant and for this are strongly recommended when aqueous
buffers are the components of the mobile phase. Tantalum (Ta), Peek (plastic)
and flexible polyethylene tubes are rarely used as columns.
Parameters of Columns
1. Internal diameter and Length
The internal diameter (ID) of an HPLC column is a critical aspect that determines
quantity of sample that can be loaded onto the column and also influences
sensitivity.
Columns of i.d. 2-5mm are generally used for analytical purposes. Wider columns
of i.d. 10-25mm are used for preparative work. Analytical scale columns (4.6 mm)
have been the most common type of columns, though smaller (MICRO- AND

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CAPILLARY) columns are rapidly gaining popularity. They are used in traditional
quantitative analysis of samples and often use a UV-VIS absorbance detector. 7
Narrow i.d. columns (1-2 mm) are used for applications when more sensitivity is
desired either with special UV-vis detectors, Fluorescence detection or with other
detection methods like LC-MS. Capillary columns (under 0.3 mm) which are used
almost exclusively with alternative detection means such as Mass
spectrophotometry. They are usually made from fused silica capillaries, rather
than the stainless steel tubing that larger columns employ.

 Sujan Bose 
Length: Columns 5, 10, 15 or 25 cm long are common if micro-particulate
stationary phase of 10μm or less are used. For preparative purposes columns up
to 1m in length are used.
2. Particle size
Most traditional HPLC is performed with the stationary phase of small spherical
silica particles (very small beads). These particles come in a variety of sizes with
5μm beads being the most common. Smaller particles provide more surface area
and better separations, but the pressure required for optimum linear velocity
increases by the inverse of the particle diameter squared (Pα1/d2). This means
that changing to particles that are half as big in the same size of column will
double the performance, but increase the required pressure by a factor of four
(Pα1/(d/2)2).
3. Pore size
Many stationary phases are porous (3, 5 or 10 μm in size) to provide greater
surface area. Small pores provide greater surface area while larger pore size has
better kinetics especially for larger sample. For example a protein which is only
slightly smaller than a pore might enter the pore but not easily leave once inside.
4. Pump pressure
Pumps vary in pressure capacity, but their performance is measured on their
ability to yield a consistent and reproducible flow rate. Pressures may reach as
high 400 atmospheres. Modern HPLC systems have been improved to work at
much higher pressures, and therefore be able to use much smaller particle sizes
in the columns (< 2 μm). These "Ultra High Performance Liquid Chromatography"
systems or UHPLCs can work at up to about 1000 atmospheres.
5.2. Injection Volume -For a fixed volume auto-sampler or manual valve, this is
set by changing the loop. For a variable volume auto-sampler, it is set in the
auto-sampler method. 10 micro liter
5.3. Flow rate -Usually around 1 ml/min for an analytical column, slower if using
polymer based columns
5.4. Run time -Usually from 2-60 min. This is a little longer than it takes for the
last peak to elute. For a gradient run we have to allow for the system to return to
the starting conditions after the run is over and then re-equilibrate before the

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next injection can be made.
Column packing material, particle size, length, diameter and manufacturer are all
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important. More than one column type may work, but an HPLC method must
specify a column
Pre-columns
Pre-columns placed prior to the sample injector are used for mobile phase
conditioning. Any solvent dissolves silica to give silicic acid. A scavenger column
filled with coarse silica is advisable when silica or silica-based chemically bonded
phases are used as stationary phases. This ensures that the mobile phase is
already saturated with silicic acid on entering the column and increases its life

 Sujan Bose 
time accordingly. Moreover, even pH of 10-11 can then be tolerated for
chromatography with no problems.
On the other hand, short pre-columns (known as guard columns) are used as a
protection between the injector and the column. They are filled in exactly the
same way as the main column and prevent impurities such as water or pigments
from contaminating it. This is particularly important when biological fluids are
involved or for direct beverage analysis. Guard columns must be changed
frequently to ensure that the life time of the main column (several hundred hours
running time) is not shortened.
General information on column packing:
(1) Large no. of theoretical plates can be obtained by ensuring short diffusion
paths in the stationary phase pores, hence HPLC favors microparticles.
Shorter column length, higher flow rate
(2) The particle size distribution should be as narrow as possible , e.g., 1:1.5 or
1:2
(3) Smallest particles determines the column permeability whereas largest
particles fix the plate numbers
(4)Small particles produce a high flow resistance but large particles are
responsible for a high degree of band broadening.
(5) Particle size distribution is achieved by sieving, air separation, sedimentation,
optical methods etc.
(6) Two groups of packing materials are used (i) Fully porous particle (3,5,or 10
microm) (ii) Those with a porous layer and a non-porous core, also known
as porous layer bed (PLB, 1-3 microm thick)
Some packing materials: (See Attached sheet for details)
1. Silica
2.Chemically modified silica
3. Styrene –divinylbenzene
4.Alumina
5. Mg-silicate
7. Controlled-pore glass
8. Hydroxyalkylmethacrylate

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9. Hydroxylapatite
10. Agarose
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11. Porous graphitic carbon
12. Restricted surface access phases
6. Mobile phases- Usually a single solvent or a well-developed combination of
two or more solvents are used as mobile phase.

➢ Selection of mobile phase:The mobile phase must be chosen for its

chromatographic properties which depends on some criteria.

 Sujan Bose 
Criteria of solvents are-
1. Viscosity- Low viscosity solvents produces a lower pressure difference that
high viscosity ones. It allows faster mass transfer
2. UV transparency- If a UV detector is used the mobile phase must be
completely transparent at the required wavelength. The UV transparency of
buffers salts, ion-pair reagents and other additives must also be considered.
3.Refractive index-If a refractive index detector is used. The differenced between
the refractive indices of the mobile phase and the sample should be as great as
possible.
4. Boiling point-A lower boiling point of the mobile phase is required if the eluate
is to be recovered and further processed.
5. Purity-
-- Absence of compounds that interfere with chosen mode of operatiom
--Absence of compounds that disturb gradient elution
--Absence of non-volatile residues in case of preparative separations
--In case of UV detection hexane should not contain benzene
6. Inert with respect to sample compounds-The mobile phase must not react at
all with the sample mixtures.
7. Corrosion resistance-
8.Toxicity-
9. Price-

6.2. Preparation of Mobile Phase

Cautions to be exercised during selection and preparation of mobile phase:
(1) Only HPLC-grade products should be used
(2) Stabilizers may alter the solvent polarity completely, e.g. Chloroform +
Ethanol
(3) Additives may dramatically reduce the UV transparency or may gradually
accumulate in the column and give rise to ghost peaks
(4) Fractional distillation is used to purify large amount of eluents and for
recycling
(5) Adsorption CC is used for treating smaller amounts and removes stabilizers,

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traces of UV absorbing compounds peroxides and water. Column filled with
alumina and silica give best level of purification.
10

(6)The first few milliliters of the eluate should be

discarded as there may still be some water left.
A layer of anhydrous CuCl2 or CoCl2 at the
bottom of column provides a visual detection of
the presence of water. When the water front breaks through, the colorless salt becomes
bright blue or pink, respectively. The accumulated impurities move through the column

 Sujan Bose 
ahead of the water-hence the need for a safety zone at the very bottom of the pipe as
seen in the Figure.

(7) Cooling is the useful facility as the risk of catalytic changes becomes less and
cleaning capacity greater at lower temperature levels. The stopcock of the
column must not be greased.
(8) A volume of 150-600ml of solvent can be cleaned with a packing of 100g of
column filling, depending on polarity, purity and original Water content. A pre-
CaCl2 drying stage is an advantage. Solvents those are more polar than ethyl
acetate can not be cleaned by this Method.
(9) The pure solvent is stored in dark bottles over a desiccant.
(10) The solvent should be filtered and degassed before use in HPLC

Reasons for filtering and degassing:

Debris in solvents would damage pump valves, block the capillaries, reduce
column performance
-Air must not be allowed
-Gas bubbles-ghost peak
-Dissolved O2-absorb UV light, quenches fluorescence
(11) Degassed solvents is kept under helium, Water and lower alcohols may
dissolve large amount of air
Degassing is done by (i) reflux boiling, (ii) Helium expulsion, (iii) Ultrasonic bath
(12) The solvent level in the supply vessel must be permanently controlled to a
certain level. The pump must not be allowed to dry.

6.3. Gradient system

The composition of the mobile phase may need to be changed as the substance
migrate through the column when complex mixtures are involved. Changes must
be accompanied by an increase in mobile phase elution strength so that peaks
which would otherwise be eluted late or not at all are accelerated. Two different
gradient systems are available:
(1)Low-pressure gradient system and
(2) High-pressure gradient system
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6.4. Sample injector 11
Sample feed is one of the critical aspects of HPLC. Even a best column produces
poor separation if injection is not carried out carefully. Usually very small volume
of sample mixture is placed at the centre of the column Head and care is taken
so that air does not enter at the same time. Ways for sample injection are
(i) With syringe and septum injector
(ii) With a loop valve
(ii) With a automated injection system (autoinjector)

6.5. sample solution and sample volume

 Sujan Bose 
Sample preparation is a difficult problem. General
procedure for sample preparation are (i) filtration,
(ii) Solid phase extraction with disposable catridge, (iii) protein precipitation and
(iv) desalting.
The sample solution should pass through a preliminary filtration step to ensure
that it contains no solids. It is best to dissolve the sample in mobile phase
solvent. If the sample solvent is weaker than the mobile phase the plate number
of the column may be increased. If the sample solvent is stronger than the
mobile phase this can lead to band broadening or strange peak shapes. If the
sample solution is higher in acetonitrile, then the effect is band broadening and
then distorted peak shape. The sample volume should be kept as small as
possible. It may be preferable to dissolve the sample in a relatively large volume
to prevent mass overload at the column inlet.

7. Detectors
A detector in any instrument is compared with human brain. The detector should
be able to recognize when a substance zone is eluted from the column.
Therefore, it has to monitor the changes in mobile phase composition, convert
this into an electrical signal and then convey this signal to the monitor or display
it as a deviation from the baseline. An ideal detector should have the following
properties:
(i) It should either be equally sensitive to all eluted peaks or record only those of
interest
(ii) It should not be affected by changes in temperature or in mobile phase
composition (as in gradient elution)
(iii) It should be able to monitor small amounts of compounds (trace analysis)
(iv) It should not contribute to band broadening, hence cell volume shouldd be
small
(v) It should react quickly to pick up correctly narrow peaks which pass rapidly
through the cell
(vi) It should be easy to manipulate, robust and cheap.

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An ideal detector should have the following properties:
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Functional Non Functional

1 Sensitivity 1 Simplicity

2 Stability 2 Cost and availability

3
Versatility 3 Robustness

4 Proportionality 4 Safety

5 Reactivity

 Sujan Bose 
 6 Response Time

7 Signal Recording

Following characteristics of the detector are better considered:

Concentration or Mass sensitivity-Concentration sensitive detectors produce
a signal, ‘S’ that is proportional to the concentration, ‘c’ of the sample in the
eluate,
S α c (g/ml)
Mass sensitive detectors produce a signal that is proportional to the mass flux,
e.i. to the number , n, of sample molecules or ions per unit time , Δ t, in the
eluate,
S α n/Δt (g/s)
Selectivity-Pure mobile phase vs sample solution and UV absorption of only
those compound that absorb UV light.
Noise: High and low frequency noise (see Fig)
Sensitivity-The smallest detectable signal can not be less than double the
height of the largest noise peak. Signal-noise ratio:3:5 or 1:10
Drift-Deviation in the baseline
Linear range-Signal vs amount injected be proportional
Time Constant-0.1-0.3s
Injection/Cell Volume-8μl

➢ Types of detector
1. UV -Visual
2. Refractive Index (RI)
3. Electrochemical
4. Fluorescence
5. Other detectors
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(i) Conductivity- Anions or cations, with or without chemical suppression 13
(dual column technique).
(ii) Photoconductivity
(iii) IR
(iv) Radioactive
(v) Transport
(vi) Light scattering
(vii) Dielectric constant

 Sujan Bose 