Introduction to HPLC
Introduction to LC and the HPLC ChemStation
Course H5929A
This 4 day laboratory and lecture course is designed to introduce users
to the practical aspects of performing routine analyses.
What is HPLC
From Wikipedia, the free encyclopedia
High-performance liquid chromatography (HPLC; formerly referred to as
high-pressure liquid chromatography), is a technique in analytical
chemistry used to separate the components in a mixture, to identify each
component, and to quantify each component.
It relies on pumps to pass a pressurized liquid solvent containing the sample
mixture through a column filled with a solid adsorbent material.
Each component in the sample interacts slightly differently with the
adsorbent material, causing different flow rates for the different components
and leading to the separation of the components as they flow out the
column.
Comparison of HPLC and GC
Mass Analyzer
The spectra are used to determine the elemental or isotopic signature of
a sample, the masses of particles and of molecules, and to elucidate the
chemical structures of molecules, such as peptides and other chemical
compounds.
Octopole
Ion Pulser
DC Quad
Rough
Pump
Turbo 1
Turbo 1
Turbo 1
Turbo 2
Reversed-Phase Chromatography
Why RPC?
• Mostly Water as mobile phase
• Gradients
• pH selective
• Hydrophobic stationary phases work for most organic
analytes.
29
Sample Preparation
A typical requirement of the pharmaceutical industry is the
quality control of active ingredients ( ) in drugs.
It is very important to identify drug impurities (
) that
may occur during synthesis or by decomposition of the
active ingredient.
Quality control
ensures patient
safety.
Page 35
Step 1: Sample made soluble
Page 36
Step 2: Separation
Column
Stationary Phase
Compound separation
occurs in a separation
column.
The column consists of a
metal tube filled with silica
gel.
The silica gel is considered
the stationary phase.
Page 37
Step 2: Separation
Column
The dissolved compounds
are injected onto the
separation column.
Stationary Phase
Page 38
Useful Solvents for Reversed-Phase
Chromatography
Water
Methanol
Acetonitrile
Isopropanol
Dioxane
Tetrahydrofuran
Elution
Strength
Solvent Miscibility
Name
Acetic Acid
Acetone
Acetonitrile
Benzene
Butyl Alcohol
Carbon Tetrachloride
Immiscible
Miscible
Chloroform
Cyclohexane
Cyclopentane
Dichloroethane
Dichloromethane
Dimethylformamide
Dimethyl Sulfoxide
Dioxan
Ethylacetate
Ethyl Alcohol
Di-Ethylether
Heptane
Hexane
Methyl Alcohol
Methylethyl Ketone
I-Octane
Pentane
I-Propyl Alcohol
Di-Propylether
Tetrachloroethane
Tetrahydrofuran
Toluene
Trichloroethane
Water
Xylene
2-Propanol is an
excellent intermediate
solvent
Structures and pKa values
Benzoic Acid pKa 4.19
Phenylacetic Acid pKa 4.3
Vanillin pKa 7.38
Phenol pKa 10
Sorbic Acid pKa 4.8
Structure graphics: Wikipedia
pH Blending C and D (C+D=10%)
meter pH
9.00
8.00
7.81
7.00
Measured pH
6.00
5.00
4.00
3.00
3.07
3.66
5.34
5.06
4.85
Acetate pK 4.7
4.42 4.63
4.24
4.00
5.78
meter pH
2.00
1.00
0.00
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Fraction of 7.8 buffer vs. 3.07
Where C = 1M HOAc, D=~1M HOAc pH ~7.8 (NH4OH) and the final molarity is 0.1M
The pKa of the acetate ion is approximately 4.7, while the formate pKa is 3.7
Selectivity vs. pH AmmOAc 0.1M
1
Initial test
pH 3.07
10% C
1 Vanillin
2 Phenol
3 Benzoic
4 Sorbic
5 Phenylacetic
3,4
2
5
Selectivity vs. pH AmmOAc 0.1M
3
1
5
4
2
Mid-pH test
pH 4.63
5% C, 5% D
1 Vanillin
2 Phenol
3 Benzoic
4 Sorbic
5 Phenylacetic
Selectivity vs. pH AmmOAc 0.1M
3
4
5
1
Optimized
pH 4.85
4% C, 6% D
2
1 Vanillin
2 Phenol
3 Benzoic
4 Sorbic
5 Phenylacetic
Quick Evaluation of Different pH on Selectivity
7
pH 2.7 0.1% formic acid/acn
4 6
1
3
2
5
0
2
4
6
pH 4.8 NH4AC/acn 1
0
2
pH 7 NaPO4/acn
4
5
4
2
3
8
6
7
min
2
1
3
4
0
min
6
2
7
8
4
6
6
5
8
Group/Presentation Title
Agilent Restricted
Month ##, 200X