PIS 497 Agricultural and Biological Instrumentation Chromatography

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PIS 497Agricultural and Biological
InstrumentationChromatography
The separation and possible quantification of
selected molecules. a.k.a. Analytical Separation
Techniques
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Primary References for This Lesson
Brian M. Tissue www.scimedia.com www.waters.com ww
w.grom.de www.tradeall.com
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Outline
1. Principles and types of chromatography 2.
Selecting and Developing a Procedure 3.
Calculating Performance a. theoretical plates
(N) b. types of columns and their effect on
N c. resolution (Rs) d. results
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Typical Chromatograph
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1. Chromatographic Principle
Solutes within a solution or volatiles within a
gas are placed in a mobile phase and passed over
a selected adsorbent material (the stationary
phase). The solutes or volatiles have
differential affinity for the absorbent
material and thus separate. For a given system,
each compound has a unique retention time.
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Chromatographic Principle
During separation, like molecules generally
migrate together. However, due to a combination
of natural forces (diffusion) and equipment
design (plumbing), like molecules may separate in
what is called band spreading.
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Types of Chromatography
GC HPLC TLC Paper GPC Ion
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Gas Chromatography - GC
Mobile phase is a gas. Stationary phase is liquid
on solid support or a solid only. It uses three
processes to separate compounds a. Partition
Chromatography b. Adsorption Chromatography c.
Volatility (boiling point) Differences
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Gas Chromatography - GC
Gas Supply Argon, Helium, Hydrogen
Impurities are removed Injector
Pre-heated Furnace Should be uniformly heated
Temperature gradients are possible Column
Usually glass capillary tube filled with
fused silica. Alumina or inert metals are
also used. Detector Electron capture, flame
ionization, or Mass Spectrometer
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Gas Chromatography - GC
Sample is introduced onto a column. The mixture
is eluted with a constant stream of (usually)
inert gas. Components of the mixture are
detected and analyzed.
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HPLC
High Pressure Liquid Chromatography or High
Performance Liquid Chromatography. Uses high
pressure pumps to improve efficiency of
separation. Mobile phase is one or more
solutions and stationary phase is solid. Used
for separation, identification,
purification, and
quantification.
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HPLC System
http//hplc.chem.shu.edu/BOOK/intro/int_inst.html
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HPLC Analytical Columns
1. Packed with various particles. 2. Are often
unidirectional. 3. Are often preceded by a
preparative or guard column. 4. Usually have
3-5 mm diameter but of micro-columns or capillary
columns ranges from 3 µm to 200 µm.
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TLC
Thin Layer Chromatography
1. A simple and rapid method. 2. Used to
monitor purity of organic compounds. 3.
Mobile phase is a solvent and the stationary
phase is a solid adsorbent on a flat support
(often silica gel).
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TLC
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Size Exclusion or Gel-Permeation
protein molecule
salt ions
buffer
gel particle
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2. What Factors Are Used in Selecting a
Procedure?
Cost, speed, ease, safety, accuracy, precision,
sensitivity, and freedom from interference. Howev
er, to achieve this, we must select an
appropriate mobile phase, stationary phase
(column), detector, and instrument variables
(column temperature, flow rates, etc.).
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3. Calculating Performance
When Developing a Procedure to Separate and
Quantify a Mixture of Substances, There Are
Mathematical / Theoretical Equations That Are
Used to Allow the Researcher to Select the Most
Efficient Approach
3a. Two of the Major Factors Involved in
Efficiency are1. Number of Theoretical
Plates2. Resolution
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Number of Theoretical Plates(N)
H Theoretical Plate Height L Length of the
Column. N L / H
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3b. Column Characteristics and Their Effect on N
Particle Size 3 - 10 ?m Pore Size 30 - 100 ?
10-10 m As particle size decreases, N
increases. Some columns have 40,000 to 120,000
plates per meter.
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Different Columns andTheir Diameters
Capillary columns (10 to 800 µm i.d.) Microbore
columns (1 and 2 mm i.d.) Analytical columns (3,
4 and 4.6 mm i.d.)
Preparative columns (0.8 to 10 cm i.d.)
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3c. Equation to Calculate Resolution (Rs)
? Z separation between A and B (time)
General Goal Achieve an Rs of 1.0
See next page for remaining variable definitions
Brian M. Tissue www.scimedia.com
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Typical Chromatograph
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3d. Calculating Results
To quantify the amount of a substance, the area
under the curve is calculated. If separation is
poor, the calculation is less reliable. Peak
height can be used but is not as accurate.