Analytical Techniques 4

HPLC

Outline
What is HPLC?

An Overview

Types of HPLC

Ion Chromatography
Size-Exclusion Chromatography
Adsorption Chromatography
Etc.,

What is HPLC?
The most widely used analytical separations
technique
Utilizes a liquid mobile phase to separate
components of mixture
uses high pressure to push solvent through
the column
Popularity:

sensitivity
ready adaptability to accurate quantitative
determination
suitability for separating nonvolatile species or
thermally fragile ones

HPLC is.
Popularity:

widespread applicability to substances that are of

prime interest to industry, to many fields of science,
and to the public

Ideally suited for separation and identification

of amino acids, proteins, nucleic acids,
hydrocarbons, carbohydrates, pesticides,
pigments, antibiotics, steroids, and a variety of
other inorganic substances

History lesson
Early LC carried out in glass columns
diameters: 1-5 cm
lengths: 50-500 cm
Size of solid stationary phase
diameters: 150-200 m
Flow rates still low! Separation times long!
HPLC - Decrease particle size of packing causes increase in
column efficiency!
diameters 3-10 m
This technology required sophisticated instruments
new method called HPLC

Components
Instruments required:

Mobile phase reservoir

Pump
Injector
Column
Detector
Data system

Elution methods

Isocratic elution

single solvent of constant composition

Gradient elution

2 or more solvents

Pumping System I
Provide a continuous constant flow of the solvent
through the injector
Requirements
pressure outputs up to 6000 psi
pulse-free output
flow rates ranging from .1-10 mL/min
flow control and flow reproducibility of 0.5% or
better

Pumping System II
Two types:
constant-pressure
constant-flow
Reciprocating pumps
motor-driven piston
disadvantage: pulsed flow creates noise
advantages: small internal volume (35-400 L),
high output pressures (up to 10,000 psi), ready
adaptability to gradient elution, constant flow
rates

Pumping System III

Displacement pumps(pulse free pumps)
syringe-like chambers activated by screw-driven
mechanism powered by a stepper motor
advantages: output is pulse free
disadvantage: limited solvent capacity (~20 mL)
and inconvenience when solvents need to be
changed
Flow control and programming system
computer-controlled devices
measure flow rate
increase/decrease speed of pump motor

Several column types

(can be classified as )
Normal phase

Reverse phase
Size exclusion
Ion exchange

Normal phase

In this column type, the retention is

governed by the interaction of the polar
parts of the stationary phase and solute. For
retention to occur in normal phase, the
packing must be more polar than the mobile
phase with respect to the sample

Reverse phase

In this column the packing material is

relatively nonpolar and the solvent is polar
with respect to the sample. Retention is the
result of the interaction of the nonpolar
components of the solutes and the nonpolar
stationary phase. Typical stationary phases
are nonpolar hydrocarbons, waxy liquids, or
bonded hydrocarbons (such as C18, C8, etc.)
and the solvents are polar aqueous-organic
mixtures such as methanol-water or
acetonitrile-water.

Size exclusion

In size exclusion the HPLC column is

consisted of substances which have
controlled pore sizes and is able to be
filtered in an ordinarily phase according to
its molecular size. Small molecules
penetrate into the pores within the packing
while larger molecules only partially
penetrate the pores. The large molecules
elute before the smaller molecules.

Ion exchange

In this column type the sample components

are separated based upon attractive ionic
forces between molecules carrying charged
groups of opposite charge to those charges
on the stationary phase. Separations are
made between a polar mobile liquid, usually
water containing salts or small amounts of
alcohols, and a stationary phase containing
either acidic or basic fixed sites.

Sample Injection Systems

For injecting the solvent through the column
Minimize possible flow disturbances
Limiting factor in precision of liquid chromatographic
measurement
Volumes must be small
0.1-500 L
Sampling loops
interchangeable loops (5-500 L at pressures up to 7000
psi)

Sample Injection System

The valve handle as shown on the left, the loop is filled from the syringe,
and the mobile phase flows from the pump to column,
Valve is placed in position on the right, the loop is inserted
between the pump and the column so that the mobile phase
sweeps the sample onto the column

Liquid Chromatographic Column

Smooth-bore stainless steel or heavy-walled glass
tubing
Hundreds of packed columns differing in size and
packing are available from manufacturers ($200$500)
Add columns together to increase length

Liquid Chromatographic Columns II

Column thermostats
maintaining column
temperatures constant to a
few tenths degree
centigrade
column heaters control
column temperatures (from
ambient to 150oC)
columns fitted with water
jackets fed from a constant
temperature bath

Detector
Mostly optical
Equipped with a flow cell
Focus light beam at the center for
maximum energy transmission
Cell ensures that the separated
bands do not widen
UV-Vis
PDA

Some Properties of Detector

Adequate sensitivity
Stability and reproducibility
Wide linear dynamic range
Short response time
Minimum volume for reducing zone broadening
High reliability and ease of use
Similarity in response toward all analytes
Selective response toward one or more classes of analytes

Types of Detector

Refractive index
UV/Visible
Fluorescence
Conductivity
Evaporative light scattering
Electrochemical

Refractive Index I
Measure displacement of beam with respect to
photosensitive surface of dectector

Refractive Index II
Advantages

universal respond to nearly all solutes

reliable
unaffected by flow rate
low sensitive to dirt and air bubbles in the flow
cell

Disadvantages

expensive
highly temperature sensitive
moderate sensitivity
cannot be used with gradient elution

UV/Visible I

Mercury lamp
= 254nm
= 250, 313, 334 and 365nm with filters
Photocell measures absorbance
Modern UV detector has filter wheels for rapidly
switching filters; used for repetitive and
quantitative analysis

Advantages

high sensitivity
small sample volume required
linearity over wide concentration ranges
can be used with gradient elution

Disadvantage

does not work with compounds that do not absorb light at

this wavelength region

Fluorescence I
For compounds having natural
fluorescing capability
Fluorescence observed by
photoelectric detector
Mercury or Xenon source with
grating monochromator to
isolate fluorescent radiation

Fluorescence II
Advantages

extremely high sensitivity

high selectivity

Disadvantage

may not yield linear response over wide range of

concentrations

Conductivity

Measure conductivity of
column effluent
Sample indicated by
change in conductivity
Best in ion-exchange
chromatography
Cell instability

Evaporative Light Scattering I

Nebulizer converts eluent into mist
Evaporation of mobile phase leads to
formation of fine analyte particles
Particles passed through laser beam;
scattered radiation detected at right angles
by silicon photodiode
Similar response for all nonvolatile solutes
Good sensitivity

Evaporative Light Scattering II

Electrochemical I

Based on reduction
or oxidation of the
eluting compound
at a suitable
electrode and
measurement of
resulting current

Electrochemical II
Advantages

high sensitivity
ease of use

Disadvantages

mobile phase must be made conductive

mobile phase must be purified from oxygen,
metal contamination, halides

Data System
For better accuracy and precision
Routine analysis

pre-programmed computing integrator

Data station/computer needed for higher

control levels

add automation options

complex data becomes more feasible
software safeguard prevents misuse of data
system

Advantages to HPLC

Quantification
Separation
Higher resolution and speed of analysis
HPLC columns can be reused without
repacking
Greater reproducibility due to close control
of the parameters affecting the efficiency of
separation
Easy automation of instrument operation and
data analysis
Adaptability to large-scale, preparative
procedures