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Introduction and Theory of Chromatography: Business Items
Introduction and Theory of Chromatography: Business Items
Chromatography
CU- Boulder
CHEM 5181
Mass Spectrometry & Chromatography
Business Items
Chromatogram
Stationary Phase
Detector
Mobile
Phase
Laser
Stationary Phase
• Sequence of events
– At t=0 we will open the gate and let the analyte into the column
– Analyte will be carried by mobile phase
– Analyte may partition to stationary phase
– Analyte will be detected by its absorption of light at the detector
4
Schematic of Column Chromatography II
Stationary Phase
Stationary Phase
Investigate
- Effect of K
- Effect of N
- Effect of RR
• http://www.chem.uoa.gr/Applets/AppletChrom/Appl_Chrom2.html 9
Analyte A
Mobile phase
Analyte B
Key concepts:
• Enriched in
Pump component which
X=0,t=1 X=1,t=1 prefers mobile phase
• Not very good
separation in 1-step
(like thermogram). It is
repetition that makes it
great.
11
12
Factors Influencing Retention…
• are those that influence distribution (equil.)
– Stationary phase: type & properties
– Mobile phase: composition & properties
– Intermolecular forces between
• Analyte & mobile phase
• Analyte & stationary phase
– Temperature
13
Intermolecular Forces I
• Based on electrostatic forces
– “Like-attracts like” or “oil and water” (similar
electrostatic properties)
• Polar/polar & non-polar/non-polar
– Molecules with dissimilar properties are not
attracted
• Polar retention forces
– Ionic interactions (IC)
– Hydrogen bonding
(permanent dipoles)
– Dipole-Induced dipole
14
Intermolecular Forces II (Dipole)
• Polar forces (cont.):
– Energy of dipole-dipole interaction
2 A2 S2
D 6
r kT
A
dipole moment, A: analyte,
S: stationary phase
2.5
• What is the
2
resolution if this is
1.5 a mass spectrum?
Units)
1
• If it is a
chromatogram?
0.5
0
46 51 56
m/z OR t'R
19
Clicker Question
• Analysis B is more desirable than A
A. In MS & Chrom.
B. In MS but not Chrom.
C. In Chrom but not MS
D. In neither MS nor Chrom
E. I need a coffee
0.25 0.9
0.2
A 0.8 B
0.7
0.6
0.15
0.5
0.4
0.1
0.3
0.05 0.2
0.1
0 0
100 110 120 130 140 150 160 170 180 100 110 120 130 140 150 160 170 180
20
Resolution vs Peak Integration
• http://www.vias.org/simulations/simusoft_peakoverlap.html 21
1.2 Rs 1.5
22
What to do if you have too much resolution?
• If you have too much resolution, you can
– Shorten the column
– Increase temperature (GC), flow rate
=> Shorten the analysis
23
24
Separating Efficiency – Peak Asymmetry
• Tailing: some part of the
stationary phase binds analyte
molecules more strongly
• Fronting: some molecules
move ahead (inject too much
sample => saturate Stat.
Phase)
• Peak Asymmetry
b
As at 10% h
a
A. H ~ 5 m
B. H ~ 50 m
C. H ~ 500 m
D. H ~ 5 mm
E. I don’t know
27
Peak capacity of ion mobility mass spectrometry: Separation of peptides in helium buffer gas. Brandon T. Ruotolo, Kent
J. Gillig, Earle G. Stone and David H. Russell. Journal of Chromatography B 782, 1-2, 25, 2002, Pages 385-392.
http://dx.doi.org/10.1016/S1570-0232(02)00566-4 28
Diffusion: Fick’s 1st Law
Concentration (y)
• When there is a gradient in SP
Y (position in SP)
concentration of a species
that can diffuse in medium
dC A
j A DA
dy Concentration (y)
SP
Y (position in SP)
• jA: molecular flux of A (moles cm-2 s-1)
• CA: concentration of A (moles cm-3)
• DAB: diffusivity of A in B (cm2 s-1)
~ 0.1-0.01 cm2 s-1 in gases
~10-5 cm2 s-1 in liquids
29
Clicker Question
• When an analyte is diffusing in the
stationary phase, equilibrium will be
reached faster
A. When DA is small
B. When DA is large
C. When SP thickness is large
D. A and C
E. I don’t know
30
From Bird, Stewart & Lightfoot,
Transport Phenomena, 2nd Ed. 2002
• Transient concentration
• Steady state profile at long
times
WAy 0
DAB A0
S Y
– WAy: mass flux of A
– A: mass fraction of A
– DAB: diffusivity of A in B
– S: surface area; : density
d A
j Ay DAB
dy
– jAy: molecular mass flux of A 31
Stat. Phase
t=3
t=4
t=1 t=5
t=2 t=6
y (position in SP)
for diffusing species in
jout
control volume y +y 1 C(y1,t)
– Per unit area y1 jin
perpendicular to
diffusion
C A ( y1 , t )
jin jout
t
C A ( y1 , t ) C ( y , t ) C ( y y, t )
y D A 1 D A 1
t y y
33
changing in time:
C A ( y, t ) C A ( y, t )
DA
t y 2
y (position in SP)
• Start with:
C A ( y, t ) C A ( y, t )
DA
t y 2
• “Order-of-magnitude
analysis”
C A C A
DA
D Y2
• Simplifying: Y2
D
DA 35
Mobile Phase
Laser
Stationary Phase
• Non-column broadening
– Dispersion of analyte in
• Dead volume of injector
• Connection between injector & column
• Connection between column & detector
– Emphasis on minimizing dead volume
(injectors, fittings…)
• Column broadening: Van Demteer model 37
Clicker 2. B term is
A. more imp in GC
B. More imp in HPLC
C. Similar importance
D. I don’t know 41
46
Optimum Mobile Phase Velocity: GC & HPLC