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INTRODUCTION TO CHROMATOGRAPHIC SEPARATION
Lecture 9
PROBLEM SET
26-4, -12, -16, -25 27-3, -20, -25 28-6, -15 Due Thursday, May 17
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!!
Methods(of(Elimination(Interferences(in(a(Chemical(Analysis!
Method( Basis(of(Method(1.!!!!Masking! Immobilization!of!an!interferent!as!a!nonreactive!
complex!2.!!!!Mechanical!Phase!Separation! !! a.!!Precipitaion!/!Filtration! Difference!in!solubility!of!compounds!formed!! b.!!Distillation! Difference!in!volatility!of!compounds!! c.!!Extraction! Difference!in!solubility!in!two!immiscible!liquids!! d.!!Ion!Exchange! Difference!in!stability!of!reactants!in!with!ion!
exchange!resins!3.!!Chromatography! Difference!in!a!rate!of!movement!of!a!solute!
through!a!stationary!phase!4.!!Electrophoresis! Difference!in!migration!rate!in!an!electric!field!
gradient!!!
IT IS ALL ABOUT “REDUCING INTERFERENCES”
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Many determinations involve separation followed by analysis v chromatography v electrophoresis CHROMATOGRAPHY:
sample transported by mobile phase electrostatic or van der Waals' some components in sample interact more
strongly with stationary phase andare more strongly retainedsample separated into zones or bands
CHROMATOGRAPHY
The MOBILE PHASE – (the solvent moving through the column) is either a liquid or a gas. The STATIONARY PHASE – (the one that stays in place inside a column) – is a viscous liquid chemically bonded to the inside of a capillary tube unto the surface of solid particles. Fluid entering the column is the ELUENT Fluid emerging from the end of the column is ELUATE
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CHROMATOGRAPHY
COLUMN CHROMATOGRAPHY
Dilution & Peak broadening!
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CHROMATOGRAPHY: PEAK SEPARATIONS
PEAK RESOLUTION
Poor resolution
More separation
Less band spread
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DISTRIBUTION CONSTANT (recommended by IUPAC) (old term: partition coefficient)
M
Sc c
cK = stationary mobile
A mobile ↔ A stationary
K ~ constant è linear chromatography >>>K >>> Retention in the stationary phase à Retention times How to manipulate K?
CS = nS/VS, CM = nM/VM
RETENTION TIMES
tM = retention time of mobile phase (dead time) tR = retention time of analyte (solute) tS = time spent in stationary phase (adjusted retention time) L = length of the column
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CHROMATOGRAPHY: VELOCITIES LINEAR RATE OF SOLUTE MIGRATION!
Velocity = distance/time à length of column/ retention times Velocity of solute: Velocity of mobile phase: M
R
tLtLv
=
=
µ
VELOCITY/RETENTION TIME AND KC
SSMM
MM
VcVcVcv
v
v
+×=
×=
×=
µ
µ
µ
solute of moles totalphase mobile in solute of molesphase mobile in timeof fraction
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VELOCITY RELATIONSHIPS
MS
M
S
MMSS
SSMM
MM
VVKv
ccK
VcVcv
VcVcVcv
/11
Constanton Distributi
/11
+×=
=
+×=
+×=
µ
µ
µ
RETENTION FACTOR :
M
MRA
AMR
A
MSAA
MS
tttk
ktL
tL
kv
VVKkVVK
v
−=
+×=
+×=
=
+×=
11
11
Factor) (Retention /
/11
µ
µ
Adjusted retention time
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Relative retention time: RRT = tR/tRs
tRs = retention time of internal standard
SELECTIVITY FACTOR:
MAR
MBR
M
MBRB
M
MARA
A
B
A
B
tttt
tttkand
tttk
kkKK
−−
=
−=
−=
=
=
)()(
)()(
α
α
α
B retained more than A à α >1
Distribution Constant
Retention factor
Retention time
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COLUMN EFFICIENCY - THEORETICAL PLATES Plate and Rate Theories
LH
HLN
NH
2
plates ofnumber height plate
σ=
=
=
=
σ à standard deviation σ2/Là variance per unit length.
L = length of column packing
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RELATION BETWEEN COLUMN DISTANCE AND RETENTION TIMES
R
R
R
tL
tL
t
L
/
in timedeviation standard timeretention
distancein deviation standard (distance)length column
στ
τστ
σ
=
=
=
=
=
=
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RELATION BETWEEN COLUMN DISTANCE AND RETENTION TIMES
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4
4
R
R
R
R
tLW
LH
tLW
WtLtL
==
=
=
=
=
σ
σ
τ
τσ
τσ
~96% ± 2τ Tangent at
Inflection point
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DETERMINING THE NUMBER OF THEORETICAL PLATES
2
2/1
2
54.5
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pates ofnumber
⎟⎟⎠
⎞⎜⎜⎝
⎛=
⎟⎠
⎞⎜⎝
⎛=
=
WtN
WtN
N
R
R
W1/2
SUMMARY OF PLATE THEORY
§ Successfully accounts for the peak shapes and rate of movement
§ Does not account for the “mechanism” causing peak broadening
§ No indication of other parameters’ effects § No indication for adjusting experimental parameters
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RATE THEORY
§ Zone broadening is related to Mass Transfer processes
COLUMN EFFICIENCY: Kinetic variables
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ZONE BROADENING : Flow Rate of Mobile Phase
Liquid chromatography Gas chromatography Note the differences in flowrate and plates height scales
Why GC normally has high H, but also high overall efficiency?
ZONE BROADENING: Kinetic Processes
µµ )(/ MS CCBAH +++=
Van - Deemter Equation
λ and γ are constants that depend on quality of the packing. B is coefficient of longitudinal diffusion. Cs and Cm are coefficients of mass transfer in stationary and mobile phase, respectively.
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ZONE BROADENING: Kinetic Processes
µµ )(/ MS CCBAH +++=
Van Deemter Equation
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ZONE BROADENING: Multiple Pathways
Eddy Diffusion: band broadening process results from different path lengths passed by solutes.
1. Directly proportional to the
diameters of packing 2. Offset by ordinary diffusion 3. Lower mobile-phase velocity,
smaller eddy diffusion
Stagnant pools of mobile phase retained in stationary phase.
ZONE BROADENING: Longitudinal Diffusion
§ The higher the µ, the smaller the H
§ Much smaller in LC than in GC
Column
Diffusion
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ZONE BROADENING: Mass Transfer between Phases
§ Slow equilibrium of solute between mobile and stationary phases
§ Time is required for solute molecules to diffuse from the
interior of these phase to there interface where transfer occur
ZONE BROADENING: Longitude vs.Mass Transfer
§ Longitude o Parallel to the flow o Inversely proportional to the flow of the
mobile phase § Mass Transfer
o Diffusion tends to be right angles to the flow o The faster the mobile phase moves, the larger
the band broadening
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CHROMATOGRAPHY: Resolution
BA
ARBRs
BAs
BAs
WWttR
WWZR
WWZR
+
−=
+
Δ=
+
Δ=
])()[(2
22/2/
CHROMATOGRAPHIC SEPARATIONS
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CHROMATOGRAPHIC DEFINITIONS
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CHROMATOGRAPHIC RELATIONSHIPS
QUANTITATIVE ANALYSIS
o Peak areas o Peak height o Calibration and standards o Internal Standard method
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SUMMARY
v Relate to column chromatography v Retention times v Velocities of mobile and component v Height equivalent of theoretical plates v Peak or zone broadening v Resolution