Principles of Chromatography
Principles of
Chromatography •Introduction to Analytical Separations
Introduction to –Solvent Extraction
Analytical Separations –What is Chromatography?
–A Plumber’ s View of Chromatography
–Efficiency of Separation
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–Why Bands Spread
Ph. D.
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phases is pH-dependent [ B]
K org -2
log D
[ B ]aq pKa
•To find D, need to know
[ H ][ B]aq -4
–pH of solution Ka mainly mainly
[ BH ]aq -6 BH+ B
–Ka of acid or conjugate acid [ B ]org K Ka
D KB
–K of acid or base [ B ]aq [ BH ]aq K a [ H ] 2 4 6 8 10 12
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pH Fig.14
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What is Chromatography?
Tswett’s
•Method to separate components in a
Experiment mixture based on different distribution
coefficients between the two phases
•Same principle as solvent extraction (like
dissolves like), but one phase is “
stationary”
and one phase is “ mobile”
–no longer working with organic and aqueous
layers in a separatory funnel
–working in a column (which is just a tube)
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What is Chromatography?
Types of Chromatography
•Two phases: mobile and stationary
•Mobile phase is solvent moving through •Adsorption Chromatography
column
•Partition Chromatography
–liquid (Methanol, water, buffer)
•Ion-exchange Chromatography
–gas (He, H2, N2)
•Molecular Exclusion Chromatography
•Stationary phase fixed inside column
–viscous liquid coated on inside of column •Affinity Chromatography
–solid particles packed inside column
•Solutes have different affinities for mobile
phase and stationary phase
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Molecular Exclusion
Ion-Exchange Chromatography
Chromatography
Stationary phase Mobile phase
Anions or cations covalently Liquid Stationary phase Mobile phase
bound to solid stationary phase Porous gel Liquid
How it separates
–small molecules
How it separates
trapped in pores of
solute ions of opposite charge stationary phase
attracted to stationary phase Also called
gel filtration,
gel permeation,
or
size-exclusion
chromatography
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phase a liquid) Liquid-bonded Organic species bonded Partition between liquid and bonded
phase to solid surface surface
Stationary phase Mobile phase
Liquid-solid, or Solid Adsorption
Immobilized molecules on Liquid adsorption
liquid or solid stationary phase
Ion exchange Ion-exchange resin Ion exchange
Gas Gas-liquid Liquid adsorbed onto Partition between gas and liquid
chromatography solid surface
(GC) (Mobile
phase a gas) Gas-bonded Organic species bonded Partition between gas and bonded
phase to solid surface liquid
“
Eluent” COLUMN “Eluate”
Porous Disk
Process is called “
elution”
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t t
adjusted retention times.
time spent in stationary phase
t k' r m =
r 2 tm time spent in mobile phase
t r
1
>1 always
then separation
•Higher kindicates solute retained longer
between two components
•If k= 0, solute unretained
k’= “capacity factor” •If k= 1, solute spent same amount of time
time _ in _ stationary t r in stationary phase as in mobile phase
k
time _ in _ mobile tm
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A Plumber’
s View… Partition Coefficient Relationship
•kdescribes time spent in two phases
time _ in _ stationary moles _ in _ stationary tr
•kalso describes number of moles of solute k
time _ in _ mobile moles _ in _ mobile tm
partitioned between two phases
CV [S ] V V t
k' s s Eq’
n k
s s K s r
Cm Vm [ S ]m Vm Vm t m
•K Partition coefficient compares t k K
concentration of solute in one phase to r 2 2 2
t r
1 k1 K1
concentration of solute in the other
[ S] C V
K 2 s k' K s
What this says is that analytes with different partition
Eq’
n coefficients will have different retention times.
[S]1 Cm Vm
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A Plumber’
s View… Review
Partition coefficient describes C
•q fraction of solute in mobile phase K s
conc. of solute in SP and MP Cm
molesm Cm Vm
q
moless molesm Cm Vm Cs Vs Capacity factor describes t t V
1 1 time solute spends (or moles of k' r m K s
q solute) in SP and MP
tm Vm
CV V
1 s s 1 K s
Cm Vm Vm
Relative retention compares
1 k' retention times (or partition t ' k' K
q (1 q) r 2 2 2
1 k' 1 k ' coefficients or capacity factors) t r1' k '1 K 1
fraction in MP fraction in SP of solute 1 and 2
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Resolution = 1.7
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[S]2
•N (Theoretical plate) represents each
equilibrium between MP and SP
[S]1
•Each time a solute molecule
•Higher N corresponds to better separation
enters the SP from the mobile –indicates solute molecules enter SP a higher
phase is a theoretical plate number of times
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•Increase L to increase R
16t 2 t 2
L
H N
L N 2r r 2 Derived
N H w
•Increase to increase R or
t ' k' K
r 2 2 2 5.55t 2 t 2
t r1' k'1 K 1 N 2 r r2
–to change , must change nature of MP and SP w1
2
to change relative affinity analytes have for SP
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R N R L
N = 1.2 x 10-4
H = 1.2 mm
1 R0
k 2 0 R0
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layer of SP SP particles
coated on packed inside
inside of column column
time
Occurs only in packed columns.
Compare to solute Compare to solute Smaller particles reduce the plate height.
traveling through traveling through HA
a straw a bean bag • Open tubular columns do not provide multiple paths for solute
during tm (no A term)
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Slower-moving (more
Minority of S retained Cm
retained) S reaches Fig. 23-19
on column longer
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Peak Area
•A = ½ wbaseh
•A w½ h
-b
Speed Capacity
x= m •cut-and-weigh
concentration (M) •computer integration
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•Analyte
–Soluble in MP (more likely than being volatile) •Constructed of steel or plastic
•Mobile phase •5 –30 cm length
A
–Liquid •1 –5 mm i.d. N
•Methanol, Water, Acetonitrile, Hexane
•Easily contaminated and degraded A
–Plays more active role in separation
•Guard column L
•Stationary phase Y
–Contains same SP
–Liquid (partition) or solid (adsorption) T
–Dust, other particles, strongly adsorbed
•High Performance Liquid Chromatography(HPLC) solutes retained on guard column
I
C
–High pressure used to force solvent through column (as –Expendable A
opposed to gravity) –Extends life of analytical column L
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LC Columns LC Columns
•Almost exclusively use packed columns •Packing particle diameter (dp) crucial
–Typical particle diameters are 3 –10 m
•Solutes move much slower through liquids
–Decrease particle diameter to decrease H
than through gases
•Provide more uniform flow (low A)
–Time needed to diffuse to SP in open tubular •Less time needed for solute to get to particle (low C)
column is too long 60 10m
40
H (m)
Fig. 21-14 20 5m
S S
10
3m
0 2 4 6 8
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rate (mL/min)
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LC –Adsorption Chromatography
Microporous Silica Particles LC –Adsorption Chromatography
•Solid SP –Silica gel Aggregate of Particles Sponge-like Structure
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LC –Partition Chromatography
LC Phases
•Liquid SP coated on solid support
–Often, liquid SP covalently attached to surface •Normal-phase Chromatography
of silica gel particle –SP polar
CH3 –MP weakly polar or non polar
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Eluate in
Fig. 21-20
pathlength
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