High Performance Liquid Chromatography

(HPLC)
 HPLC is the most widely used of all the analytical
separation techniques.
 Reasons: sensitivity, suitability for separating nonvolatile
species (or thermally fragile ones), and industry, to many
fields of science and public.
 Example of such materials include:
Amino acids, proteins, nucleic acids, hydrocarbons,
carbohydrates, drugs, terpenoids, pesticides, antibiotics,
steroids, metal-organic species, and a variety of inorganic
substances.
HPLC
 HPLC Instrumentation Overview
Principle Pattern An Example

Solvent Reservoirs
Controller

Solvent Cabinet Vacuum Degasser

Binary Pump

Autosampler

Thermostatted
Column Compartment

Detector

3
HPLC
Solvent Filters

Guard
column
Injector

Precolumn Analytical
Filter Column
Solvent Inlet Filter

Precolumn Filter
Solvent Inlet Filter • Used between the injector and
• Stainless Steel or guard column.
glass with 10 micron • 2 to 0.5 micron
porosity. • Removes particulates from
sample and autosampler wear
• Removes particulates
debris.
from solvent.
• Must be well designed to
prevent dispersion.
4
 HPLC

 A liquid sample, or solid sample dissolved in a suitable

solvent, is carried through a chromatographic column by
a liquid mobile phase.
 Liquid chromatography covers a range of
chromatography systems, i.e.
(a) liquid-solid adsorption,
(b) liquid-liquid partitioning,
(c) ion exchange,
(d) size exclusion, and
(e) affinity.
HPLC
Mobile Stationary
 The principle of separation in Phase Phase
HPLC is based on the specific
interactions between sample Solvent
Bonded
Phase
molecules with both the
Separation is based on the
stationary and mobile phases. analyte’s relative solubility
between two liquid phases

 The coefficient of mass transfer in the mobile phase (CM)

in the van Deemter equation is directly related to the
square of the diameter of the particles of the column
packing. CM = (fMdp2)/DM
 The efficiency of a HPLC column should improve
dramatically as the particle size is decreased.
H = A + B/u + CSu + CMu
 HPLC

Fig.28-2 Effect of particle size of packing and flow rate upon

plate height H of liquid chromatography.
N= L/H
HPLC

WCOT, SCOT

new WCOT
HPLC

Liquid-Solid chromatography Liquid-liquid chromatography

(Adsorption chromatography) (Partition chromatography)
LSC works best for class LLC works better for the
separations or for the separation separation of homologs
of isomeric compounds
LSCs are more sensitive to steric LLCs are more sensitive to small
effects and are preferred for the molecular weight solutes and are
separation of similar compounds preferred for the separation of
having different steric members of a homologous series
configurations alkane, alkene,

LSC is less influenced by

molecular weight differences and
more by specific functional
groups compared to liquid-liquid
chromatography.
cis and trans
HPLC

Ion-exchange or ion- Exclusion (gel Affinity

pairing permeation) Chromatography
Chromatography Chromatography
(IEC/IPC) (SEC)
IEC/IPC is suitable used SEC is used when the uses immobilized
for Ionic groups and molecular weight biochemicals as the
ionizable groups exceeds approximately stationary phase to
2000 daltons for some separate one or a few
or all of the sample solutes from hundreds
components. of unretained solutes
Cation exchange resins: Separate sample The separations exploit
SO3-H+ or COO-H+ mixtures according to the "lock and key"
Anion exchange resins: the size and shape of binding that is prevalent
N(CH3)3+OH- or NH3+OH- the sample molecules in biological systems
(bioselectivity)
HPLC VERY IMPORTANT!!!!!

 Today, the most widely used type of HPLC is partition

chromatography. Basically, it is divided into
(i) Normal phase chromatography
LLC
(ii) Reverse phase chromatography
HPLC
LL

LL, LS or
bonded
phase

IEC /
IPC

SEC
Essential components of a HPLC
instrument
(a) Mobile phase reservoir – usually a mixture of
solvents Configuration of a HPLC system
Gradient
(b) Column Controller

(c) Injector •
Pump Column
(d) Detector Injector Detector

Mobile
Phases
Configuration of an HPLC system
Mobile phase
 The elution order of solutes in HPLC is governed by
polarity. buffer: protect the stationary phase

 The stability of the Si-O-Si bond involved in bonded

phases is dependent on the mobile-phase pH.
 Above pH 7, the bond is hydrolyzed and serious
degradation of the column stationary phase results.
 When acids or bases are to be separated, the solvent
pH has to be controlled by using a buffer.
The siloxane bonds are attacked only in very acidic
(pH<2) or basic (pH>9) conditions
Configuration of an HPLC system
Stationary phase
Purospher column from Merck
Suitable for acidic, basic and chelating compounds.
pH range from 1.5 to 10.5
Can use 100% H2O as MP
SiliCycle in Milan, Italy
SiliaChrom® SB C18 protect the surface from an acidic attack.
stable even at very low pH (up to 0.8)
SiliaChrom® XT Fidelity C18 resistance to extreme high pH
conditions (up to 12)
Zorbax Extend-C18 (from Agilent) pH range: 2 – 11.5

Hypercarb (Thermo Scientific) can be used for RP and NP

chromatography with a pH range from 1 to 14

XBridge BEH C18 (from Waters) can be used over an entire

range of mobile phase pH (1-12)
Configuration of an HPLC system
Mobile phase

THF: non polar, hydrocarbon

Different ratios of mobile phases

check the properties and chec the literature to start the mobile phase %
HPLC
(a) Normal-phase separation polar column
 Normal-phase liquid-liquid chromatography uses a polar
stationary phase (often hydrophilic) and a less polar
mobile phase. non polar
 To select an optimum mobile phase. Start with a pure
hydrocarbon mobile phase such as heptane.
 If the sample is strongly retained, then add small amounts
of methanol or dioxane (Relatively more polar compared
to heptane).
 In a normal-phase separation the least polar solute spends
proportionally less time in the polar stationary phase and
is the first solute to elute from the column.
Polar column, Interacts stronger with the polar solutes
HPLC stationary: polar
mobile phase: non polar
Normal-phase separation eluting too fast, add a bit of less polar phase to the system

Normal-phase LC: Effect of different combinations of

mobile phase
HPLC
Normal-phase separation
 Retention times (tR) are controlled by selecting the mobile
phase.
 For NP-LC, Less polar mobile phase leads to longer
retention times.
Because the interaction becomes stronger between the
solutes and the polar stationary phase
 For example, a separation is poor because the solutes are
eluting too quickly.
 Switch to a less polar mobile phase leads to longer
retention times and more opportunity for an acceptable
separation.
HPLC
(b) Reverse phase separation Non-polar column

 Reverse-phase chromatography uses a hydrophobic

bonded packing, usually with an octadecyl (C-18) or octyl
(C-8) functional group and a polar mobile phase.
 Hydrocarbons are retained more strongly than alcohols.
 Methanol and acetonitrile are popular solvents because
they have low viscosity and are readily available with
excellent purity.
Viscosity = the resistance of a liquid to flow.
Polar solvent = low viscosity, i.e. the flow of the mobile phase is
faster.

Q: how to improve normal phase or reverse phase. Ans : add the less or more polar solution depended on the situation
HPLC Non-polar stationary phase,
(b) Reverse phase separation and polar solvents
HPLC non-polar stationary phase,
(b) Reverse phase separation polar solvent
 In a reverse phase separation the order of elution is
reversed, with the most polar solute being the first to elute.
 Increasing the polarity of the mobile phase leads to longer
retention times.
 Shorter retention times require a mobile phase of lower
polarity.
Modification of the polarity of the mobile phase for either
technique (NP-LC or RP-LC) will affect the retention factor k
of the analytes k = (tR – tM) / tM
HPLC
Eluent Strength of Solvent

Elution power

NP-LC
RP-LC

Highest eluent strength The smaller viscosity value,

for NP-LC but lowest means the flow of the
eluent strength for RP-LC. mobile phase is faster.
HPLC
Interactions between analyte and solvent molecules
 In liquid chromatography, the sample components interact
with both the stationary phase and the mobile phase.
 The polarities of various organic solvents in increasing
order are:
Hydrocarbons < chloroform < ethers < esters < ketones <
aldehydes < amides < amines < alcohols < acetonitrile <
water.

 There are four types of interactions between analyte and

solvent molecules:
(a) Dipole interactions
(b) Dispersion interactions
(c) Dielectric interactions
(d) Molecular complex formations
HPLC
Interactions between analyte and solvent molecules
(a) Dipole interactions
 Dipole interactions occur when both the solvent and the
analyte have permanent dipole moments.
 These lead to enhanced solubility and lower values for k’.
k = (tR – tM) / tM
(b) Dispersion interactions
 Dispersion interactions between solute and solvent
molecules occur because the motions of electrons in a
molecule are random.
One part of the molecule becomes negatively charged with
respect to the other: Polarization electrostatic attraction
HPLC
Interactions between analyte and solvent molecules
(b) Dispersion interactions …
 Dispersion forces are the only attractive force between
nonpolar species.
 Strong dispersion interaction can be expected between
solvent and solute molecules having high refractive
indexes.

Refractive index is the ratio of the velocity of light in air

to the velocity of light in the solvent
(b) Dispersion interactions …
HPLC
Interactions between analyte and solvent molecules
(c) Dielectric interactions
 Dielectric interactions arise from the electrostatic
attraction between an ionized solute and a solvent with a
high permanent dipole.
 Dielectric interactions are manifested in the high
solubilities of ionic and ionizable solutes in polar solvents
such as water or methanol.
(d) Molecular complex formations
 The most common example of molecular complexing
is hydrogen bond formation between a solute proton
donor and a mobile-phase proton acceptor or the
reverse.
HPLC
Normal vs Reversed Phase HPLC Both are LLC

Normal Phase Reversed Phase

Stationary phase Polar (silica gel, alumina or Non-polar (C18)
triethylene glycol
supported by silica
particles)
Mobile phase Non-polar (organic Polar
solvents, e.g. hexane or iso- (aqueous/organic, e.g.,
propyl ether) water, methanol or
acetonitrile)
Sample movement Non-polar faster Polar faster
Separation based on Different polarities Different hydrocarbon
(functionality) content

Partition chromatography (LLC) = chromatographic separation

which involved a liquid stationary phase immobilized on an inert
support material
Normal vs Reversed Phase HPLC
Polar column, with non-polar Non-polar column, with polar
solvents solvents

If use more If use less

polar solvent polar solvent
Exercise

1. Describe the normal phase and reverse phase HPLC?

2. Draw a diagram of HPLC instrumentation set up and explain

the functions of each unit?

3. How does column type, packing material, stationary phase and

mobile phase effect the efficiency and resolution of
chromatographic peaks?