HPLC

2. BP_701_T – Instrumental Methods of Analysis
4.2 High Performance Liquid
Chromatography-Part 1
Archana S Gurjar
Assistant Professor, Pharm Chem dept.
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For Educational Purpose only

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4.2 High performance liquid chromatography (HPLC)-Introduction,
theory, instrumentation, advantages and applications. 4hrs

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• HPLC is a chromatographic technique in which Mb phase is passed under
pressure so k/n as High Pressure Liquid Chromatography.
• Provides higher efficiency for sepn of compds : High Performance Liquid
Chromatography
• Technique needs sophisticated instrumentation as compared to Classical
Liq Chg, which increases cost of instrumentation: High Priced Liquid
Chromatography
• Column chromatography.
• Liquid Chromatography.
• Key pts: Reduced time of Analysis with reasonable Mobile phase flow rate
(achieved due to high pressure) and reduced particle size of stationary
phase, dense packing & Increased no. of plates (good Column efficiency)
HPLC

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High Performance Liq Chg (HPLC)
• Common two types: 1. Normal phase HPLC 2. Reversed Phase HPLC.
• Has high resolution and separation capacity.
• Used as qualitative as well as quantitative analysis.
• Used to separate a mixture of compounds in analytical chemistry and
biochemistry with the purpose of identifying, quantifying or purifying the
individual components of the mixture.

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Normal Phase: Separation of polar analytes by partitioning onto a polar,
bonded stationary phase.
Reversed Phase: Separation of non-polar analytes by partitioning onto a non-
polar, bonded stationary phase.
Adsorption: In Between Normal and Reversed. Separation of moderately
polar analytes using adsorption onto a pure stationary phase (e.g. alumina or
silica)
Ion Chromatography: Separation of organic and inorganic ions by their
partitioning onto ionic stationary phases bonded to a solid support.
Size Exclusion Chromatography: Separation of large molecules based in the
paths they take through a “maze” of tunnels in the stationary phase.11
Diff Types Of HPLC Separations

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From Waters Corporation

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Schematic dig of HPLC

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Instrumentation in HPLC
Components:
1. Solvent reservoir & degassing system
2. Pumps
3. Sample injection system
4. Columns
5. Temperature controller (Thermostat)
6. Detectors

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Instrumentation in HPLC: 1. Solvent reservoir & degassing system
• Has Mb phase - Single solvent/ Mixt of solvents for elution of solutes from
mixture
• Isocratic elution: single Mb phase
• Gradient elution: Elution of solutes of extreme polarities from mixt carried
out using two or more Mb phases with significantly difference in polarity.
• Modern HPLC equip: two or more reservoirs from which diff solvents can
be introduced into a chamber at varied rates to adjust Mb phase polarity.
• Glass or stainless-steel 500ml -1 lt capacity
• Inert to a variety of aqueous and non aqueous mobile phases (s.a. Water,
MeOH, Acetonitrile, pure organic solvents or aq solns of salts and buffers)
• Stainless steel should be avoided for use with solvents containing halide
ions.

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Instrumentation in HPLC: 1. Solvent reservoir & degassing system
• HPLC column very sensitive to impurities so use HPLC grade solvents
• Degassing of solvents to remove dissolved air/ oxygen- Can rupture column
packing/ produce unwanted peaks in Chgm.
Degassing of Mb phase done by four methods:
• Filtration under vacuum: Removes all dissolved air / O2. Millipore filters are
used commonly
• Distillation of Mb phase: under vacuum to remove all dissolved air / O2
• Ultrasonication: Sonicatn with sound energy (Freq > 20 KHz) in ultrasonic
bath or probe k/n as Sonicator. Alternative to
• Sparging inert gas of low solubility: Also k/n as Gas flushing. Involves
bubbling chemically inert gas of low solubility s.a Ar/ He thru Mb phase

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Instrumentation in HPLC: 2. Pumps
• Mobile phase passed through a column at high pressures: pump output at
least 1000 to 6000 pounds per sq inch (psi, lb/in²) or 414 bar with flow
delivery rate of at least 3ml/ min (range 0.1 to 10ml/min)
• As particle size of stationary phase is smaller (5 to 10μ) so resistance to flow
of solvent is high. Hence high pressure is recommended.
• Flow control & flow reproducibility of 0.5 % or better.
• Corrosion resistant components: Stainless steel/ Teflon/ Sapphire
• Pumps should be capable of taking solvent from a single reservoir or more
than one reservoir containing different solvents simultaneously.

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Pumps
Reciprocating
Displacement /
Syringe type
Pneumatic/
Const. pressure

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Reciprocating Pumps • Most commonly used &
commercially available
• Piston in syringe type chamber
• Connected to two ball check
valves on two ends which open
and close alternately.
• Solvent is in direct contact with
piston
• Piston drawn back: Ball position A & B so Mb phase fills in Chamber 1 , no Mb phase goes to Column
• Piston direction reversed: Ball position A’ & B’ so Mb phase enters in Chamber 2
• Vol of Mb phase discharged from pump/ unit time can be changed by altering dist that piston travels
• Mb phase enters Column, Chamber 1 is empty. This is reason for pulsed flow of Mb phase in these
pumps.

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Reciprocating Pumps
Drawback: Pulsed flow of Mb phase overcomed in two ways:
• Reduce time of filling- reduces dead time between cycles
• Provide dual piston pump with rapid filling phase- reduces pulsation in flow as
one fills other discharges
Adv:
• Internal vol. of pump is small (35- 400 µL). Imp for Gradient elution- needs
removal of one Mb phase before another is introduced. This changeover
achieved fast due to small internal volume
• High output pressure upto 10,000 psi
• Const flow rate of Mb phase- independent of Col. Back pressure & solvent
viscosity.
Disadv: Not major one- Pulsed flow as can be overcomed

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Displacement/ Syringe
type Pumps
• Piston/ plunger inside
chamber can move to & fro
with help of screw-feed drive
connected to stepping motor.
• Mb phase is present in
chamber of pump. Electric
supply to motor allows
continuous & uniform
movement of piston inside
chamber so definite vol
delivered/ unit time
depending on voltage applied

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Displacement/ Syringe type Pumps
Adv:
• Produces pulse free flow of Mb phase
• Flow rate is independent of back pressure in column & viscosity of Mb
phase
Disadv:
• Internal vol of pump is very high so not suitable for Gradient elution

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Pneumatic/ Constant pressure Pumps
• Compressed gas squeezes collapsible bag
so mb phase comes out from outlet
Adv:
• Simple in construction, cheap
• Provide pulse free flow of Mb phase
Disadv:
• Mb phase depends on its viscosity and back
pressure in the column
• Pressure provided is <2000 psi
• Not useful for gradient elution

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Instrumentation in HPLC: 3. Sample injection system
Several injector devices are available either for manual or auto
injection of sample.
• Septum Injector
• Stop Flow Injector
• Rheodyne Injector
• Rotary sampling Valve
• Slider valve
• HPLC auto injectors
Used to introduce fixed volume of spl soln into Mb. Phase as sensitivity of
HPLC is very high so accurate qty of spl should be introduced in column for
reproducibility of results

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• SEPTUM INJECTOR
Used for injecting sample through rubber septum.
Cannot be commonly used , since septum has to withstand high pressures.
• Stop Flow
Flow of mobile phase is stopped for a while & the sample is injected through a
valve.
• RHEODYNE INJECTOR
Most popular injector and widely used.
Has a fixed volume of loop, for holding sample until its injected into column, like
20μL, 50μL or more.
Through injector sample is introduced into column.
Injector is positioned just before inlet of column.

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Handle on left Handle on Right

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HPLC Auto injector

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Instrumentation in HPLC: 4. Columns
Types: Precolumn (Guard), Analytical column
Success or failure of analysis depends upon
choice of analytical column.
Actual separation is carried out here.
Stainless –steel tube
Size – length - 15-150 cm
Internal diameter – 2 to 3 mm
Analytical Column is filled with finely divided particles of st. phase: 5 – 10 µm.
Separation is result of different components adhering to or diffusion into
packing particles when mobile phase is forced through column.

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Instrumentation in HPLC: 4. Columns
• Precolumn: Also c/d Guard Column
• Introduced before analytical column to increase its shelf life or Placed
between pump & spl injection system
• Short and Broader than analytical
• Has identical st. phase as analytical but bigger particle size, small in
length
• Can be sacrificed to protect expensive Analytical column
• Mainly used :
-To remove impurities present in Mb. Phase so they don’t
contaminate analytical column or interfere with spl separation
- Saturates Mb. Phase so when passing through analytical col. It
doesn’t strip off its st. phase. Col. Remains intact

27. HPLC Columns & Packings
• Made of glass / special quality stainless steel
• Glass columns can bear low pressures, maxm upto 1000 psi
• Stainless steel can work at higher pressures ranging between 2000 & 6000 psi
• Longer columns are prepd by joining small portions together.
• Two basic types: Porous Particle and Pellicular
Another is Monolithic
• Porous Particle: Porous finely divided micro particles of st. phase: 3 – 10 µm.
Composed of commonly silica particles. Prepd by agglomerating submicron
silica particles under condns to form large particles with highly uniform
diameters. These are coated with thin organic films chemically or physically
bonded to the surface.
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28. HPLC Columns & Packings
• Others: alumina, celite (diatomaceous earth), synthetic resin (polystyrene-
divnyl benzene) or ion-exchange resin
• Pellicular packings: spherical, nonporous, glass or polymer beads with
diameters of 30- 40µm, surface coated with 1-3 µm layer of porous material
s.a. silica gel, alumina, synthetic resin (polystyrene-divnyl benzene) or ion
exchange resin.
• For some applications an additional coating is applied consist of liq st.
phase, held in place by adsorption
• Alternatively beads may be ttd chemically to give an organic surface layer.
• Currently Pellicular packings are used largely for Guard columns and not
Analytical columns
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29. HPLC Columns & Packings
Pellicular packings
Adv : Higher rate of mass transfer from one phase to other due to thin st. phase
layer resulting in higher col. Efficiency
Drawback: Occupy lot of space hence reduce loading capacity of column
(1/10th of porous packing column)
• Monolithic packing: internal structure of monolithic column is created in such
a way that many channels form inside the column.
• Material inside column which separates the channels can be porous and
functionalized.
• Most HPLC configurations use particulate packed columns; in these
configurations, tiny beads of an inert substance, typically modified silica, are
used inside the column.
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30. HPLC Columns & Packings
• Monolithic packing: Monolithic columns can be broken down into two
categories, silica-based and polymer-based monoliths.
• Silica-based monoliths are known for their efficiency in separating smaller
molecules while, polymer-based are known for separating large protein
molecules.
• Normal phase HPLC : polar st. phase & less polar/ nono-polar Mb. Phase
• Reverse phase: st. phase less polar/ non-polar compared to mobile phase
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• Reverse phase: Surface of silica gel is modified by forming covalent bond
between silica gel & chlorooctadecylsilane reagent. This reverses polarity.
Such columns are c/d Bondapack columns.
HPLC Columns & Packings

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• C8 and C18 columns are examples of reversed phase liquid
chromatography (RP).
• Difference between two columns will be in the length of the C-chain
attached to the silica surface.
• C8 HPLC columns have packing material composed of silica particles
attached to C8 carbon units.
• C18 will have packing materials coated with octadecylsilane.
• Categorically both are reversed phase but C18 columns more
"hydrophobic rather than the C8 columns.
HPLC Columns & Packings

33. Instrumentation in HPLC: Thermostats
• Normally HPLC is performed at RT
• If solute takes long time for elution then done at High temps.
• Water jacketed columns are available commercially for precise temp
control
• Increase in temp of col. decreases retention time of compd in the
column.
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