April 5th, 2011

HPLC as an analytical and

preparative method
Maciej Giedyk

HPLC as an analytical and preparative method

Content:
1. Introduction and Historical Background

2. Modes of HPLC
3. Mobile Phase
4. Choice of Method

5. HPLC Equipment: pumps, injectors,

detectors, columns
6. Scale-up

7. Quantitation Analysis

Introduction and Historical Background

modern form of liquid chromatography

physical separation technique

based on differences of affinity

for the stationary and mobile phase
high performence / high pressure
advanced hardware: solvent delivery systems, injectors, detectors,
controllers

Introduction and Historical Background

from Greek: chroma colour, graphein to write

discovered by Mikhail Tswett in 1903

- separation of plant pigments on chalk

1930s: gravity-fed columns
1960s: turn into high-performance
prof. Csaba Horvath

prof. Josef Huber

Introduction and Historical Background

HPLC
Advantages
Better separation efficiency
than normal LC

Limitations
No universal detector

Fast and precise analysis

Less separation efficiency

than capillary GC

Automated operation

Relatively complicated handling

High-sensitivity detection
Quantitative sample recovery

Modes of HPLC
Normal-Phase Chromatography (NPC)
traditional separation mode
polar stationary phase, nonpolar mobile phase
useful for the separation of nonpolar
compounds and clean-up

Reversed-Phase Chromatography (RPC)

solid phase modified with nonpolar groups
(most often C18 groups)

polar mobile phase: e.g. methanol/water,

acetonitrile/water
polar or medium-polarity analytes
most popular HPLC mode

Modes of HPLC
Ion-Exchange Chromatography (IEC)
based on the exchange of ionic analytes with the counter-ions
typical cationic exchange sulfonate
typical anionic exchange quaternary ammonium
useful for the analysis of ions and biological components

Size-Exclusion Chromatography (SEC)

uses differences in molecular size
modern columns packed with cross-linked
polystyrene

typical solvents: toluene, THF

Other Separation Modes:

Affinity Chromatography, Chiral Chromatography, Electrochromatography,
Supercritical Fluid Chromatography (SFC)

Solvent

Solvent
strength [E]

n-Hexane

0.01

Toluene

0.29

Methylene chloride

0.42

Tetrahydrofuran

0.45

high solubility for the sample components

Acetonitrile

0.60

noncorrosive to HPLC system components

2-Propanol

0.82

Methanol

0.95

Water

Large

Mobile Phase

Ideal solvent:

high purity, low cost, UV transparency

low viscosity, low toxicity, nonflammability

Solvent Strength referes to the ability of a

solvent to elute solutes from a column

Mobile Phase

pH control
important when separate acidic/basic analytes on RP

weakly acidic analytes low pH

weakly basic analytes high pH
stationary phase stable with pH between 2 and 10

Another strategy for acidic/basic analytes

Ion-Pairing Additives

use detergent-like molecules:

the longer chain
the higher concentration

the higher retention

Choice of Method

Organic-Soluble Analytes
Polarity

Polar

Nonpolar

Examples of analytes

alkaloids, sugars,
nitrocompounds

steroids,
hydrocarbons

Mode

normal phase (NP)

reverse phase (RP)

hexane, chloroform

acetonitrile/water,
methanol/water,
THF/water

Mobile phase

Choice of Method

Water-Soluble Analytes
Compounds

Ions

Weak acids and basis

Mode

ion exchange (IE)

reverse phase (RP)

Mobile phase

buffer

buffers, acids, bases,

ion-pairing additives

HPLC Equipment

Hitachi

JASCO

Knauer
Shimadzu
Waters

HPLC Equipment
Solvent Delivery System
consists of one or more pump, solvent reservoirs and degassing
system

high-pressure
mixing designs

lower dwell volume

low-pressure
mixing designs

only one pump required

HPLC Equipment

Injection Systems

Manual Injectors

Autosamplers

Rheodyne 7725
Rheodyne 7125

HPLC Equipment
Detectors
monitor property

measure concentration
(or mass)
Good detector should
have high sensitivity
respond to all analytes, or have predictable specificity
be reliable and convenient to use
have a response that increases linearly with the amount of solute
be nondestructive of the solute

HPLC Equipment

UV/Vis
Absorbance

Photodiode
Array

Others

Detectors

Evaporative
Light
Scattering

Refractive
Index

HPLC Equipment

Column
heart of the HPLC system
typically: stainless steal tube filled with small
particle packing

Column Hardware
steel, PEEK, titanium etc.

Chromatographic Mode
NPC, RPC, IEC, SEC

Dimension

Support Type
silica, polymer, zirconia

HPLC Equipment

Column Length
the longer column the better resolution
but longer time
typical column: 50-250 mm
strong trend toward using shorter columns Fast LC

Particle size
affects column resolution
affects peak height and sensitivity
smaller particles chance to use shorter
columns

Scale-Up

Column Dimension

Column Type

Typical Sample
Loading

Preparative

>30 mg

Semipreparative

5 mg

Conventional

100 g

Semi-micro LC

20 g

Micro LC

1 g

determines the scale of the process

trend toward using narrower columns for analytical HPLC

Scale-Up
Principles of Scale-Up

1. establish the analysis conditions using analytical column

2. prepare the preparative column which has the same size of
packing material

3. scaling-up to preparative column

a) flow rate

b) sample amount

4. fractionate the sample

5. check peaks purity of fractionated sample
6. do necessary corrections

Quantitation Analysis

Quantitation Analysis Strategies

Peak Area % is proportional to the amount of the
component in the sample
not true ratio of the components

external standardization

internal standardization

Quantitation Analysis

external standardization

internal standardization

1. check different concentrations of

analysed compound and note peak
areas

1. add the same amount of pattern to

different concentrations of analysed
compound

2. draw a calibration graph of area

versus concentration

2. draw a calibration graph of

concentration versus ratio of peak
areas of analyte and pattern

3. analyse your sample

4. read the concentration

3. add pattern to your sample and

analyse
4. read the concentration

Literature

1. Analytical vs Preparative HPLC,Lin Yuan,

http://www.shimadzu.com/

2. A Practical Handbook of Preparative HPLC,

Donald A. Wellings, Elsevier Ltd. (2006)
3. Introduction to Modern Liquid Chromatography, Lloyd R. Snyder,
Joseph J. Kirkland, John W. Dolan, John Wiley & Sons (2010)
4. Modern HPLC For Practicing Scientists, Michael W. Dong,
John Wiley & Sons (2006)
5. Zasady dziaania oraz przykady zastosowa wysokosprawnej
chromatografii cieczowej HPLC, Dominika Pielaciska

Thank You!
Any Questions?