Chapter 6 (HPLC) rev a

Y
CHAPTER 6
(HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY)

6.1 HPLC INTRODUCTION
6.1.1 Founder of Liquid Chromatography
Mikhail Tsvet
Born on May 14, 1872
Asti, Italy
Invented chromatography in 1901 while
performing research on plant pigments
Column adsorption liquid
chromatography

6.1.2 HPLC Analysis
Qualitative Analysis
Identification of compound identity
Require a known standard
Identified by comparing retention time
Quantitative Analysis
Identify the amount/concentration of the compound
Require a standard with known amount of concentrations
Identified by interpolating the area of unknown into a set of
standards with known concentration

Qualitative Analysis » Comparing Retention Time
Quantitative Analysis » Comparing Peak Area/Height

6.1.3 Basic Concept of HPLC
Sample are injected into a HPLC system.
Interactions happen between the samples with the mobile phase
and stationary phase (column) which results in separation of
samples which is detected through the detector and converted into
a chromatogram.

V
V
V
V
V
V
V
V
V
V
V
V
Elution
V
V
V
V
V
V
X
X
X
A
A
A
A
A
A
X
X
X
X
X
X
A
A
A
A
A A
X
X
X
X
X
X
A
A
A
A
A
A
Injection
Interaction

6.1.4 HPLC Peaks
A separation by changing the relative speed of each analyte band
(Competition between the mobile phase and stationary phase).

As analyte “Bands” pass
through Detector Flow Cell, an
electrical signal is sent to the
Computer Data Station
(recorder) to draw the “Peak”.
Blue band is now the broadest as it
exits the column since it was
moving so slowly in the column, it
takes a lot of mobile phase to
finally sweep it all out – this is why
the latest eluting peaks are the
broadest.
How Peaks Are Created?
ISOCRATIC
CONDITIONS

Few, Earliest
Arriving
Analyte
Molecules
Front
Apex
Back
Chromatogram
Analyte “Band” flowing
Into Detector Cell
Most Analyte
Molecules
Arrive
(Highest
Concentration)
Few, Latest
Arriving
Analyte
Molecules
Computer
Data Station
Signal from the
Analyte “Band” in the
Detector
Flow Cell
is Translated into a
“Peak”
The higher the
analyte
concentration, the
higher the peak
height –
better Sensitivity
Most Concentrated
Point in the
Analyte “Band”
Time
D
E
T
E
C
T
O
R
Mobile
Phase
Mobile
Phase
Mobile Phase
Base Line
Mobile Phase
Base Line0
How an Analyte Becomes a Peak?

6.1.5 Band Spreading
Narrow Band –
Narrow Peak
More Concentrated –
Increased Peak
Height/Sensitivity
More Resolution
Capability
Broad Band – Broad Peak
Less Concentration –
Less Sensitivity
Less Resolution Capability
Broader “Band”
More “Band Spreading”
Broader Peak
Narrower “Band”
Less “Band Spreading”
Narrower, Taller Peak
Mobile
Phase
Mobile
Phase
Mobile
Phase
Less
Concentrated
0 0
Mobile
Phase

Better separation
More concentrated “Bands”
Higher Sensitivity
In this region, both analytes (blue and red)
are not separated [a partial co-elution –
shown as a “purple” band]
System with
MORE
Band Spreading
System with
LESS
Band Spreading
Narrower, Sharper Bands create Narrower Sharper Peaks, which
provide a better separation.
This results in better Resolution between the 2 peaks and
greater peak heights for better Sensitivity

6.2 TYPES OF HPLC
HPLC : is a type of chromatography that employs a liquid mobile
phase and a very finely divided stationary phase.
The types of HPLC are often classified by separation mechanism or
by the type of stationary phase :
Partition / Liquid-liquid chromatography
Adsorption / liquid solid chromatography
Ion exchange
Size-exclusion
Affinity chromatography
Chiral chromatography

The selection separation procedure is governed by what the
analytes are.

6.3 APPARATUS
In HPLC solvent at high pressure is forced through a column to
obtain a separation
The instrument is made of a solvent system, an injection valve, a
column, and a detector.

HPLC system have :
1. Solvent reservoir 7. Sample Loop
2. Degasser 8. Guard Column
3. Gradient Valve 9. Column
4. Mixer 10. Detector
5. High Pressure Pump 11. Acquisition Computer
6. Switching Valve 12. Waste Reservoir

6.3.1 (a) Mobile Phase Reservoir and Solvent Treatment
Systems
Solvent reservoir is made from glass and it contains 500 mL or
more of a solvent volume.
However, dissolved gas and dust from liquid can form bubbles in
column » band spreading. Thus, Degassers is needed for a
sparging process.
Sparging : the removal of an unwanted dissolved gas by aeration
with an inert gas.
Elution can be performed isocratically or with a gradient :
Isocratic elution is performed with a constant solvent (pure or
constant mixture).
Gradient elution is the solvent mixture is altered during the run
if one solvent is not satisfactory.

Isocratic elution
Mixture of acetonitrile and aqueous
phase buffer solution (KH2PO4)
Separation of aromatic compounds,
including alcohols and ketones.

Gradient elution
Increasing ratio of acetonitrile during
the analysis behaves like
temperature programming in GC

6.3.1 (b) Pumping System
The requirement of pump :
Ability to generate pressures up to 6000 psi
Pulse-free output
From rate ranging 0.1 to 10 mL/min
Flow reproducibilities of 0.5% relative or better
Resistance to corrosion by a
variety of solvent
There are 3 types of pumps
Syringe-type pumps
Reciprocating pump
Pnuematic or constant pressure pump

A reciprocating pump for HPLC
6.3.1 (c) Sample Injection System
The most widely used method of sample introduction in liquid
chromatography is based on a sampling loop.
These devices are an integral part of some liquid chromatography
equipment.

Interchangeable loops are available to provide
a choice of sample sizes ranging from 5 to 500 μL.
Many HPLC instruments have autosampler
with an automatic injector.

6.3.1 (d) Analytical Columns
HPLC column :
made from stainless steel
10 – 30 cm long
2 – 5 mm internal diameter
Particles size for column packing is 3 – 10 μm and provide
40000 – 60000 plates/m
Microcolumn :
3 – 7.5 cm long
1 – 4.6 mm internal diameter
Particles size for column packing is 3 – 5 μm and provide
100000 plates/m
Have disadvantage of speed and minimal solvent consumption.

The most packing for liquid chromatography is prepared from silica
particles which are synthesized by agglomerating submicron silica
particles under conditions that lead to larger particles with highly
uniform diameters.
The resulting particles are coated with thin organic films, which are
chemically or physically bonded to the surface.

Guard Column
Positioned ahead of the analytical column to increase the life of
the analytical column by removing particulate matter and
contaminants from solvents.
It also serves to saturate the mobile phase with the stationary
phase so that losses of stationary phase from the analytical
column are minimized.
Composition is similar to analytical column
except particle size
is larger to minimize pressure drop.

Column Thermostats
Better chromatograms are obtained by maintaining column
temperatures constant to a few tenths of a degree celcius.
Equipped with heaters to control column temperatures.
Columns may be fitted with water jackets from a constant-
temperature bath to give precise temperature control.

6.3.1 (e) Detectors
Detectors for HPLC must have low dead volume to minimize extra-
column band broadening.
The detector :
small and compatible with liquid flow
not highly sensitive
universal detector system
There are 2 types of detector :
Bulk property detector : measure property of mobile phase
(refractive index, dielectric constant, density).

Solute property detector : measure property of solute
present in mobile phase (UV absorbance, fluorescence, IR
absorbance).
The most widely used detectors for liquid chromatography are
based on absorption of ultraviolet or visible radiation.
Both photometer and spectrophotometer specifically designed for
use with chromatographic column are commercially available.
Source :
single line (arc or hollow cathode lamp)
continuum (Xe, D2 lamp)
Detector :
photodiode/ photomultiplier tube
photodiode array

6.4 HIGH PERFORMANCE PARTITION
CHROMATOGRAPHY
The most widely used and popular type of HPLC → the stationary
phase is a second liquid that is immiscible with the liquid mobile
phase.
Sample molecules equilibrate (PARTITION) between liquid
stationary phase and mobile phase.
Retention depends on a sample molecule's escaping tendency into
the mobile phase versus its solubility in the stationary phase.

It can be divided by 2 types, liquid-liquid and liquid-phase-bounded
chromatography.
i. Liquid-liquid partition chromatography » the stationary phase is a
solvent that is held in place by absorption on the surface of
packing material.
ii. Liquid-bounded-phase chromatography » the stationary phase is
an organic species that is attached to the surface of the packing
particles by chemical bonds.

6.4.1 Bonded-Phase Packings
Most of the bonded-phase packings are prepared by rxn of an
organochlororsilane with the ―OH groups formed on the surface of
silica particles (3, 5 or 10 μm) by hydrolysis in hot, dilute HCL to
produce organosiloxane.
Advantage : not required periodic recoating of stationary phase
due to thee phase will be dissolved away by mobile phase and very
practical for gradient elution.

6.4.2 Normal and Reversed-Phase Packings
Base on the relative polarities of the mobile and stationary phase :
Normal-Phase HPLC → nonpolar (solvent)/ polar (column)
Reversed-Phase HPLC → polar (solvent)/ nonpolar (column)

In Normal-Phase Chromatography → the least polar component is
eluted first » ↑ the polarity of the mobile phase, ↓ the elution time.
In Reversed-Phase Chromatography → the most polar components
elutes first, ↑ the polarity of the mobile phase, ↑ the elution time.
6.4.3 Choice of Mobile and Stationary Phases
The partition chromatography requires a proper balance of
intermolecular force among analyte, mobile phase and stationary
phase.
This intermolecular force are describes qualitatively in terms of the
relative polarity posses by each components.
The order of polarities of common mobile phase solvents » water>
acetonitrile> methanol> ethanol > tetrahydrofuron > propanol >
cyclohexane > hexane.

For stationary phase choice : choose column with similar polarity to
analyte for maximum interaction.
Reversed-Phase » column (nonpolar)
Normal-Phase » column (polar)
For mobile phase choice : Polar strong solvent interacts most with
polar analyte (solute) – elute faster but less resolution.

6.4.4 Applications
Field Typical Mixture Separated
Pharmaceuticals Antibiotics, sedatives, steroids
Biochemicals Amino acids, proteins, carbohydrates,
lipids
Food Products Antioxidants, additives
Industrial Chemicals Dyes, propellants, surfactants
Pollutants Pesticides, herbicides, phenols
Forensic Chemistry Drugs, poisons, blood alcohols

6.5 HIGH PERFORMANCE ION-EXCHANGE
CHROMATOGRAPHY
The interactions between the chromatographic medium and the
proteins in the mixture are based primarily on ionic charge.
Ion exchangers are resins often coupled on cross-linked
polysaccharides that can exchange ions with water solutions.

Most common types of ion-exchangers:
DEAE (diethylaminoethyl)-cellulose; an anion exchange resin
used primarily for neutral and acidic proteins
CM (carboxymethyl)-cellulose; a cation exchanger used
primarily for the separation of neutral and basic proteins.
Elution from the column:
by altering the pH of the elution buffer
by increasing the ionic strength of the elution buffer
Two types of ion chromatography in use :
Suppressor based
Single Column

6.5.1 Ion Chromatography Based on Suppression
In suppression-based ion chromatography, the ion exchange
column is followed by a suppressor column or by a suppressor
membranes that converts an ionic eluent into a non-ionic species
that does not interfere with the conductometric detection of analyte
ions.
6.5.2 Single Column Ion Chromatography
In single-column ion-exchange chromatography, analyte ions are
separated on a low-capacity ion exchanger by means of low-ionic
strength eluent that does not interfere with the conductometric
detection of analyte ions

6.6 HIGH PERFORMANCE SIZE-EXCLUSION
CHROMATOGRAPHY
It is based on molecular size.
Used for large MW compounds protein and polymers.
Separation mechanism is sieving not portioning.
Stationary phase » porous silica or polymer particles (Polystyrene,
polyacrylamide)(5 – 10 μm)

6.6.1 Column Packing
Well-defined pore sizes (40 – 2500 Å)
Large molecules excluded from pores – not retained, first
eluted (exclusion limit – term of MW).
Intermediate molecules – retained, intermediate elution times.
Small molecules permeate into pores – strongly retained last
eluted (permeation limit – term of MW).
6.6.2 Applications
Separation of sugars in cane
Separation of proteins/ peptide
Determination of molecular mass of large polymers or natural
products

Chapter 6 (HPLC) rev a

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