Introduction to High Performance Liquid
Chromatography
In This Section, We Will Discuss:
 The differences between High Performance Liquid
Chromatography and Gas Chromatography.
 The components of the high performance liquid
chromatograph (HPLC).
 The separation process.
 The chromatogram.
 The most common modes of HPLC.
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You’ve Got a Problem to Solve
I need a quantitative
separation of
carbohydrates in some
of our products
as soon as possible.
I’ll get
on it!
I’ll need a separation
technique.
3
Separation Techniques
I have two separation techniques in my lab,
High Performance Liquid Chromatography
and Gas Chromatography. Which should I use?
4
Comparison of HPLC and GC
Sample Volatility
HPLC
Sample Polarity
HPLC
• No volatility requirement
• Separates both polar and
non polar compounds
• Sample must be soluble
in mobile phase
• PAH - inorganic ions
GC
• Sample must be volatile
GC
• Samples are nonpolar
and polar
5
Comparison of HPLC and GC
6
Comparison of HPLC and GC
Sample Thermal Lability
HPLC
Sample Molecular Weight
HPLC
• Analysis can take place
at or below room
temperature
GC
• Sample must be able
to survive high
temperature injection
port and column
• No theoretical upper limit
• In practicality, solubility is
limit.
GC
• Typically < 500 amu
7
Comparison of HPLC and GC
Sample Preparation
HPLC
Sample Size
HPLC
• Sample must be filtered
• Sample should be in
same solvent as mobile
phase
GC
• Solvent must be volatile
and generally lower
boiling than analytes
• Sample size based upon
column i.d.
GC
• Typically 1 - 5 L
8
Comparison of HPLC and GC
Separation Mechanism
HPLC
Detectors
HPLC
• Both stationary phase
and mobile phase take
part
GC
•Mobile phase is a
sample carrier only
• Most common UV-Vis
• Wide range of nondestructive detectors
• 3-dimensional detectors
• Sensitivity to fg (detector
dependent)
GC
• Most common FID,
universal to organic
compounds
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How can We Analyze the Sample?
Carbohydrates
1.
2.
3.
4.
5.
6.
fructose
Glucose
Saccharose
Palatinose
Trehalulose
isomaltose
5
2
Zorbax NH2 (4.6 x 250 mm)
70/30 Acetonitrile/Water
3
mAU
4
1
6
1 mL/min Detect=Refractive Index
time
10
Separations
Injector
Separation in based upon differential
migration between the stationary and
mobile phases.
Stationary Phase - the phase which
remains fixed in the column, e.g. C18,
Silica
Mixer
Pumps
Mobile Phase - carries the sample
through the stationary phase as it
moves through the column.
Column
Detector
Solvents
Waste
High Performance Liquid Chromatograph
11
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
High Performance Liquid Chromatograph
12
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
13
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
14
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
15
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
16
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
17
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
18
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
19
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
20
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
21
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
22
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
23
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
24
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
25
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
26
Separations
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
time
Column
Detector
Solvents
27
The Chromatogram
to - elution time of unretained peak
tR- retention time - determines sample identity
tR
tR
mAU
Area or height is proportional
to the quantity of analyte.
to
Injection
time
28
HPLC Analysis Parameters
Mobile Phases
Flow Rate
Composition
Injection Volume
Column
Oven Temperature
Wavelength
Time Constant
29
Modes of High Performance Liquid
Chromatography
Types of Compounds
Mode
Stationary
Phase
Mobile Phase
Neutrals
Weak Acids
Weak Bases
Reversed
Phase
C18, C8, C4
cyano, amino
Water/Organic
Modifiers
Ionics, Bases, Acids
Ion
Pair
C-18, C-8
Water/Organic
Ion-Pair Reagent
Compounds not
soluble in water
Normal
Phase
Silica, Amino,
Cyano, Diol
Organics
Ionics Inorganic Ions
Ion
Exchange
Anion or Cation
Exchange
Resin
Aqueous/Buffer
Counter Ion
High Molecular Weight
Compounds
Polymers
Size
Exclusion
Polystyrene
Silica
Gel FiltrationAqueous
Gel PermeationOrganic
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HPLC Applications
Bioscience
Chemical
polystyrenes
dyes
phthalates
tetracyclines
Pharmaceuticals corticosteroids
antidepressants
barbiturates
proteins
peptides
nucleotides
Consumer Products
lipids
antioxidants
sugars
Environmental
polyaromatic hydrocarbons
Inorganic ions
herbicides
Clinical
amino acids
vitamins
homocysteine
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