Introduction to High Performance Liquid Chromatography

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Introduction to High Performance Liquid
Chromatography
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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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Youve Got a Problem to Solve
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Separation Techniques
I have two separation techniques in my lab, High
Performance Liquid Chromatography and Gas
Chromatography. Which should I use?
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Comparison of HPLC and GC
Sample Volatility
Sample Polarity
HPLC
HPLC

• Separates both polar andnon polar compounds
• PAH - inorganic ions

• No volatility requirement
• Sample must be solublein mobile phase

GC
GC

• Samples are nonpolarand polar

• Sample must be volatile

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Comparison of HPLC and GC
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Comparison of HPLC and GC
Sample Thermal Lability
Sample Molecular Weight
HPLC
HPLC

• Analysis can take placeat or below
  roomtemperature

• No theoretical upper limit
• In practicality, solubility islimit.

GC
GC

• Sample must be able to survive high
  temperature injection port and column

• Typically lt 500 amu

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Comparison of HPLC and GC
Sample Preparation
Sample Size
HPLC
HPLC

• Sample must be filtered
• Sample should be insame solvent as mobilephase

• Sample size based uponcolumn i.d.

GC
GC

• Solvent must be volatileand generally lower
  boiling than analytes

• Typically 1 - 5 ?L

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Comparison of HPLC and GC
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How can We Analyze the Sample?
Carbohydrates 1. fructose 2. Glucose 3.
Saccharose 4. Palatinose 5. Trehalulose 6.
isomaltose
Zorbax NH2 (4.6 x 250 mm) 70/30
Acetonitrile/Water 1 mL/min DetectRefractive
Index
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Separations
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
Mobile Phase - carries the sample through the
stationary phase as it moves through the column.
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Separations
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Separations
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Separations
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Separations
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Separations
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Separations
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Separations
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Separations
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Separations
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Separations
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Separations
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Separations
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Separations
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Separations
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Separations
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Separations
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The Chromatogram
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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