Ch' 25 High Performance Liquid Chromatography HPLC

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Ch. 25 High Performance Liquid Chromatography
(HPLC)
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HPLC

• In HPLC solvent at high pressure is forced
  through a column to obtain a separation
• The instrument is made of a solvent system, an
  injection valve, a column, and a detector

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HPLC Process

• Diffusion is much slower in a liquid than gas
• Open tube columns are generally not used in LC
  because it takes too long for solute to interact
  with stationary phase on the sides of the column
• In packed columns, the particle size used affects
  the separation results
• Particles are typically 2-10 ?m
• Smaller particles provide better resolution
  because they reduce terms A and C in the Van
  Deemter equation and

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Effects of Particle Size
The optimum flow rate is faster for smaller
particles because diffusion between the mobile
and stationary phases takes place over a shorter
distance than with big particles
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LC Column Design
Columns are steel or plastic and are
typically 20 cm in length with a diameter of a
few mm. A guard column is used to protect
the main column from impurities and dust. This
column contains the same stationary phase as the
main column. The column is usually
kept heated a few degrees above room temperature.
Decreased viscosity at high temperature reduces
the pressure needed to push solvent through the
column, so retention times are reduced.
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The Stationary Phase
The most common support is porous particles
of silica with a stationary phase bonded to the
surface. Octadecyl (C18-ODS) is the most common
stationary phase. Isobutyl groups are used to
protect Si-O bonds from hydrolysis.
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Adsorption Chromatography
Solvent molecules compete with solute molecules
for stationary phase sites in adsorption
chromatography. When a solvent displaces solute,
the solute is eluted from the column. Eluent
strength (e) is a measure of the ability of a
solvent to displace solute from a give adsorbent.
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e For Solvents on Silica
We refer to use of a polar stationary phase
(e.g. SiO) and a less polar solvent as
normal-phase LC. The more polar the solvent,
the higher the e. Reverse-phase LC is more
common. In this case the solvent is more polar
than the stationary phase.
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Elution Conditions

• Elution can be performed isocratically or with a
  gradient
• Isocratic elution is performed with a constant
  solvent (pure or constant mixture)
• In gradient elution the solvent mixture is
  altered during the run if one solvent is not
  satisfactory
• In a reverse-phase separation, the solvent is
  made gradually more polar (higher e)

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Isocratic Elution
Mixture of acetonitrile and aqueous phase buffer
solution (KH2PO4)
Separation of aromatic compounds, including
alcohols and ketones.
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Gradient Elution
Increasing ratio of acetonitrile during the
analysis behaves like temperature programming in
GC
Segmented gradient diagram
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LC Flowchart
The selection separation procedure is governed by
what the analytes are.
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Parts of the LC

• Solvent pump
• Piston pump is the most common
• Injector
• Usually a loop injector
• Column (already talked about this)
• Detectors
• Spectroscopic
• Refractive index
• Evaporative light scattering
• Electrochemical

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Piston Pump
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Loop Injector
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Spectroscopic Detector
A UV detector with a flow cell is the most
common HPLC detector because most solutes absorb
UV light. In the simplest case, absorbance at
254 nm radiation from a Hg lamp is detected.
Volume of the flow cell is 5-10 µL.
Fluorescence detection is also used for more
sensitive detection. This is not a universal
detector, but can be made very specific. Solutes
can be derivatized for better detection either
pre-column or post-column.
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LC Photodiode Array
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Refractive Index Detector
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Evaporative Light Scattering Detector
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Detector Specifications
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Criteria for Adequate Separation

• Capacity factor (k) should be between 0.5-20
• Smallersolvent distorts early peak
• Biggertoo slow a run
• Minimum resolution of 1.5 for baseline separation
  (2 is better in case of long-term effects)
• Keep pressure in operating conditions ( 15 MPa
  (150 bar 2200 psi)
• Peaks should be symmetric

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Method Development Triangle
In reverse-phase chromatography,
acetonitrile (AcN), methanol (MeOH), and
tetrahydrofuran (THF) are the most common organic
solvents. To optimize the right
mixture of solvents for a separation, a triangle
is used. The vertices are optimized separations
for each of the organic solvents/water mixtures.
Then you can optimize the separation more by
mixing equal proportions of each of the
optimized pure solvents until you get something
that meets your criteria for a good separation.
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Steps in LC Analysis

• 1) Goal of the analysis
• 2) Sample preparation
• 3) Detector
• 4) Method triangle
• A) Optimize separations with AcN/H2O, MeOH/H2O,
  and THF/H2O
• B) Mix solvents from A in 11 ratios to generate
  midpoints on triangle and center point (111)
• Use computer simulation to optimize from there!
• If by 4B you have not obtained a good separation,
  you probably need to try a different column or
  form of LC

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Specifications for LC

• Advantages
• Can analyze very complex mixtures
• Robust for multiple samples
• Can analyze polar compounds
• Disadvantages
• Potentially very complex sample preparation
• Uses many solvents
• Method development