HPLC

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
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
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?

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April 5th, 2011

HPLC as an analytical and

HPLC as an analytical and preparative method

5. HPLC Equipment: pumps, injectors,

Introduction and Historical Background

modern form of liquid chromatography

based on differences of affinity

Introduction and Historical Background

from Greek: chroma colour, graphein to write

- separation of plant pigments on chalk

prof. Josef Huber

Introduction and Historical Background

Fast and precise analysis

Less separation efficiency

Relatively complicated handling

Reversed-Phase Chromatography (RPC)

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

Size-Exclusion Chromatography (SEC)

typical solvents: toluene, THF

Other Separation Modes:

high solubility for the sample components

noncorrosive to HPLC system components

high purity, low cost, UV transparency

Solvent Strength referes to the ability of a

weakly acidic analytes low pH

Another strategy for acidic/basic analytes

use detergent-like molecules:

the higher retention

normal phase (NP)

reverse phase (RP)

Weak acids and basis

ion exchange (IE)

reverse phase (RP)

buffers, acids, bases,

lower dwell volume

only one pump required

determines the scale of the process

trend toward using narrower columns for analytical HPLC

1. establish the analysis conditions using analytical column

3. scaling-up to preparative column

4. fractionate the sample

Quantitation Analysis Strategies

1. check different concentrations of

1. add the same amount of pattern to

2. draw a calibration graph of area

2. draw a calibration graph of

3. analyse your sample

3. add pattern to your sample and

1. Analytical vs Preparative HPLC,Lin Yuan,

2. A Practical Handbook of Preparative HPLC,

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