Transcript
Introduction to and Theory of
Chromatographic Separations
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What is Chromatography? Chromatography is the separation of an
analyte from a complicated mixture of similar constituents for qualitative or quantitative identificationApplies to both organic and inorganic
compounds
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Basic Separation Principles and Terms Compounds (analytes) are separated from a
mixture by passing them through a stationary phase using a mobile phase carriermobile phase = solventstationary phase = column packing material
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Basic Separation Principles and Terms Separations occur because analytes in the
mobile phase interact with the stationary phase at different ratesProduces varying migration rates for analytes
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Basic Separation Principles
Example: Column elution chromatography
Introduce two solutes (A & B) onto a packed column through which a mobil phase (i.e. solvent) or eluent is continuously pumped
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Basic Separation Principles Separations depend on the extent to which
solutes partition between the mobile and stationary phases
We define this interaction with a partition coefficient:
K = cs/cmWhere:cs = stationary phase concentration (molar)cm = mobile phase concentration (molar)
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Types of Chromatography
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ChromatographyPlanar Column
Liquid Gas Supercritical FluidPartition
Bonded PhaseAdsorption
Ion ExchangeSize Exclusion
Gas - LiquidGas – Bonded Phase
Gas - Solid
Classification of Column Chromatography Methods
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The Chromatogram
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Sample Mixture
Chromatogram
0 5 10 15 20Time (minutes)
Abun
danc
e AB
C
D
E
ChromatographThe time that a peak appears (it’s retention time) is diagnostic for a given compound
The relative size of a peak (area or height) is proportional to the relative abundance of the compound in the mixture
Chromatography Terms A number of terms can be defined from
the following chromatogram
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Chromatography Terms Retention Time (tR) = the time it takes after
injection for a solute to reach the detector Dead time (tM) = the time for an unretained
species to reach the detector Mobile phase velocity (u) = L/tM; the average
linear rate of movement of molecules in the mobile phaseL = length of chromatographic column
Linear Rate of solute migration ( ) = L/tR
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v
Chromatography Terms Retention Factor or Capacity Factor (k’A) = a
term used to describe the migration rate of solutes (analytes) on columns
Where:KA = is the distribution constant for species AVS = volume of stationary phaseVM = volume of the mobile phase
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M
SAA V
VKk '
Chromatography Terms The retention factor can be determined
directly from a chromatogram using:
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M
MRA t
ttk '
Chromatography Terms Interpreting the retention factor
If k’A < 1; the elution is too rapid for accurate determination of tR.
If k’A > approx. 10; the elution is too slow to be practical
The preferred range for k’A is between 1 and 5
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Chromatography Terms Selectivity Factor () = KB /KA
Where KB is the distribution constant of the more strongly retained species (so that >1
The selectivity factor can also be defined in terms of retention factors and retention times:
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MAR
MBR
A
B
tttt
kk
)()(
''
Chromatography Terms Zone Broadening or Band Broadening
Refers to the shape of a peak as a solute migrates through a chromatographic column; it will “spread out” and “shorten in height” with distance migrated
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Chromatography Terms Plate Height (H) and Theoretical Plates (N)
Terms used to quantitatively describe chromatographic column efficiency
Column “efficiency” increases as N increases
N = L/HWhere:
N = the number of interactions (i.e. transitions between mobile and stationary phases) that a solute has during its residence in the column
H= the distance through the column a solute travels between interactions (typically given in centimeters)
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Chromatography Terms Plate Theory – used to describe solute migration
through a column and the Gaussian shape of a peakIf the shape of a chromatographic peak is assumed to be
Gaussian, then the plate height can be defined in statistical terms involving standard deviation (σ)
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LH
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Chromatography Terms Plate Theory (cont.)
H is defined as variance per unit length of column
H is the length of column that contains the fraction of analyte between L and L – σ
This is 34% of the analyte (1/2 of 1 std. dev.)
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Calculation of N From a Chromatogram
Where:W = magnitude of the
base of the triangle (in units of time)
tM = retention time of an unretained solute
tR = retention time of the solute
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2
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WtN R
Chromatography Terms Rate Theory (or Kinetic Theory) – used to explain (in
quantitative terms) the shapes of chromatographic peaks and the effects of chromatographic variables on peaks as a solute migrates through a columnThe Gaussian shape of an peak can be attributed to the
additive combination of the random motions of individual solute molecules
The migration of an individual molecule through a column is irregular (based on “random walk” mechanism)
The time an individual molecule spends in a phase depends upon (accidentally) gaining sufficient thermal energy to change phases
Results in a symmetric spread of velocities around the mean
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Chromatography Terms Column Resolution (RS)
is a quantitative measure of the ability of a column to separate two analytes:
To increase resolution increase column length (L) Limitation: longer time and
broader bands Usually compromise
between resolution and speed of analysis
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BA
ARBRS WW
ttR
])()[(2
Summary of Important Terms
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Summary of Important Terms
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Variables Effecting Separation Efficiency in Column Chromatography
1. Particle Size of Packing (as size , Nand H)2. Immobilized Film Thickness (as film thickness , Nand H) -
due to faster diffusion rates in film3. Viscosity of Mobil Phase (as viscosity , Nand H)4. Temperature (as T , k' , but remains approximately
constant)5. Linear Velocity of Mobil Phase; u = L/tM (u is proportional to
1/tM because L = constant)6. Column Length (as L , N but H = constant, and separation
efficiency )
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In general, Separation (or Column) Efficiency In general, Separation (or Column) Efficiency , as N , as N and H and H
Variables Effecting Column Efficiency Mobile Phase Flow Rate
Optimum flow rate corresponds to minimum H
H is much smaller for HPLC than for GC (more efficient), but in practice GC columns are much longer than for HPLC - hence greater N for GC.
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Liquid Chromatography
Gas Chromatography
Variables Effecting Column Efficiency Particle Size of Column Packing
The smaller the packing particles, the greater the column efficiency.
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The General Elution Problem
It is often difficult to find a set of conditions which will resolve all peaks to the same degree and also permit reliable quantification
SolutionsMultiple runs at
different conditionsProgrammed elution
(i.e. change conditions during the run)
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