HPLC 1
HPLC 1
Chromatography
In This Section, We Will Discuss:
The chromatogram.
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You’ve Got a Problem to Solve
I need a quantitative
separation of I’ll get
carbohydrates in some on it!
of our products
as soon as possible.
3
Separation Techniques
HPLC HPLC
• No volatility requirement • Separates both polar and
non polar compounds
• Sample must be soluble
in mobile phase • PAH - inorganic ions
GC GC
• Samples are nonpolar
• Sample must be volatile
and polar
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Comparison of HPLC and GC
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Comparison of HPLC and GC
HPLC HPLC
• Analysis can take place
• No theoretical upper limit
at or below room
temperature
• In practicality, solubility is
limit.
GC GC
• Sample must be able
to survive high • Typically < 500 amu
temperature injection
port and column
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Comparison of HPLC and GC
HPLC HPLC
• Sample must be filtered
• Sample size based upon
column i.d.
• Sample should be in
same solvent as mobile
phase
GC GC
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Comparison of HPLC and GC
HPLC HPLC
• Both stationary phase • Most common UV-Vis
and mobile phase take • Wide range of non-
part destructive detectors
• 3-dimensional detectors
• Sensitivity to fg (detector
dependent)
GC GC
• Most common FID,
•Mobile phase is a universal to organic
sample carrier only compounds
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How can We Analyze the Sample?
Carbohydrates
1. fructose
2. Glucose
3. Saccharose
4. Palatinose
5. Trehalulose
6. isomaltose 5
2
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Zorbax NH2 (4.6 x 250 mm) mAU
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70/30 Acetonitrile/Water 1
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1 mL/min Detect=Refractive Index
time
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Separations
Separation in based upon differential
Injector
migration between the stationary and
mobile phases.
Mixer Stationary Phase - the phase which
remains fixed in the column, e.g. C18,
Silica
Detector
Waste
Solvents
Mixer mAU
Pumps
Column
Detector
Solvents
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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Separations
Injector Chromatogram
Mixer mAU
Pumps
Column
Detector
Solvents
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The Chromatogram
tR
mAU Area or height is proportional
to the quantity of analyte.
to
Injection time
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HPLC Analysis Parameters
Mobile Phases
Flow Rate
Composition
Injection Volume
Column
Oven Temperature
Wavelength
Time Constant
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Modes of High Performance Liquid
Chromatography
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HPLC Applications
Bioscience
Chemical proteins
peptides
polystyrenes nucleotides
dyes
phthalates
tetracyclines
Pharmaceuticals corticosteroids
antidepressants
barbiturates Consumer Products
lipids
antioxidants
sugars
Environmental
polyaromatic hydrocarbons Clinical
Inorganic ions amino acids
herbicides vitamins
homocysteine
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