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Introduction and Theory of Chromatography
CU- Boulder CHEM 5181
Mass Spectrometry & Chromatography
Prof. Jose L. Jimenez
Reading: Braithwaite & Smith Chapters 1 & 2Last Updated: Nov. 2013
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Business Items
Next Tue: invited lecture from Lucas Smith, LECO HW due Tue (could extend some of it, but overlap with
1st LV HW) FCQs online + our own feedback form, response rate
counts towards class participation grade for everyone
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Chromatogram
Why is Chromatography So Successful?
Single-Step Thermogram
Much higher selectivity, by doing single step over and over
Ziemann et al.UC-Riverside(now CU-Boulder)
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Schematic of Column Chromatography
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
Stationary Phase
Stationary Phase
MobilePhase
Detector
Data to Computer
Lase
r
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Schematic of Column Chromatography II
For simplicity, we will assume that the mobile phase moves in steps rather than continuously
If analyte had no affinity to the stationary phase, it would just follow the mobile phase Emerge at the detector after tM (mobile phase time)
Stationary Phase
Stationary Phase
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Schematic of Column Chromatography III
If analyte has some affinity to the stationary phase, it will be retarded Equilibrium Kinetics
Molecular mass transfer: diffusion Emerge at the detector after retention time tR
SP
MCCK s Distribution Ratio
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Mechanisms of Partitioning to the Stationary Phase
Stick to the surfaceDissolve into the bulk
Q: (A) Left is Absorption, Right is Adsorption(B) Left is Adsorption, Right is Absorption(C) I dont know
Fig. from McNair & Miller, Basic Gas Chromatography, Wiley, 1998
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Retention and Mobile Phase Time
Simplest chromatogram w/ 2 components Unretained peak (tM) Analyte peak (tR) Corrected retention time (tR)
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Chromatography Simulator
http://www.chem.uoa.gr/Applets/AppletChrom/Appl_Chrom2.html
Investigate- Effect of K- Effect of N- Effect of RR
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Using the Distribution Ratio: Single Step (Plate)
Mobile phase
Stationary Phase
Analyte A
Analyte B
Q: If each symbol represents a mol, given 10 cm2 of surface to which analytes adsorb, and 100 cm3of liquid in which analytes are dissolved, what are KA & KB? Large K has more affinity for stationary phase
Small K has more affinitiy for the mobile phase
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The first step in a separationX = 0 , t = 0
X = 1 , t = 1X = 0 , t = 1Pump
Q: does molecular-level kinetics matter?
Key concepts: Enriched in component which prefers mobile phase Not very good separation in 1-step (like thermogram). It is repetition that makes it great.
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Partitioning in Real Chromatography
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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
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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
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Intermolecular Forces II (Dipole) Polar forces (cont.):
Energy of dipole-dipole interaction
Factor of 10 variation on permanent dipole moment Factor of 104 variation on interaction energies
As r6 => mainly at the surfaces
kTrSA
D 6
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A dipole moment, A: analyte, S: stationary phase
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Intermolecular Forces III (London) Londons Dispersion Forces
Most universal interaction between molecules Only one for non-polar species
Due to induced dipoles Relatively weak
Energy of interaction:
is the polarizability, I: ionization potential, A: analyte, S: stationary phase
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6SA
SAPAL IIr
II
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Clicker QuestionA. The chemical nature of the mobile phase
affects distribution in gas-chromatography (GC) & liquid chromatography (LC)
B. The mobile phase affects distribution in GC but not LC
C. The mobile phase affects distribution in LC but not GC
D. The mobile phase affects doesnt affect distribution in either GC or LC
E. I dont know
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Separating Efficiency Peak WidthAssume Gaussian Peaks FWHM = wh = 2.36HWHM = 1.18
Base Width of Peak (wb) Width at intersection of
tangents at inflexion points (+/- ) and the baseline
wb = 4 95.5% of molecules are
within wb Assuming that the
Gaussian model holds!
Figure 2.3, Braithwaite & Smith
When in doubt, use NORMDIST in Excel or GAUSS in Igor
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Resolution in Chromatography Objective: accurate measurement
of individual peak areas (spectra) FWHMtRs *2
What is the resolution if this is a mass spectrum? If it is a chromatogram?
0
0.5
1
1.5
2
2.5
46 51 56
m/z OR t'R
Sign
al In
tens
ity (A
rbitr
ary
Uni
ts)
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Clicker Question
Analysis B is more desirable than AA. In MS & Chrom.B. In MS but not Chrom.C. In Chrom but not MSD. In neither MS nor ChromE. I need a coffee
0
0.05
0.1
0.15
0.2
0.25
100 110 120 130 140 150 160 170 1800
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
100 110 120 130 140 150 160 170 180
A B
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Resolution vs Peak Integration
http://www.vias.org/simulations/simusoft_peakoverlap.html
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Can One Have Too Much Resolution?
5.12.1 sR
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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
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Separating Efficiency Peak Asymmetry
Q: what can cause peak asymmetry?
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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
0.9 < As < 1.2 for acceptable chromatography
h 10%at s abA
Equations for Calculation of Chromatographic Figures of Merit for Ideal and Skewed Peaks, J.P. Foley and J.G. Dorsey, Anal. Chem., 55: 730-737, 1983
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Separating Efficiency Peak Width II Described by variance 2 (units: s2)
determined from Gaussian fit to peak Classical chromatography theory
Separation in N discrete steps (plates)
or
Also broadening increases as tR increases:
and in practice:
N12
N1
NtR
1 2
RtN
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Height Equivalent of a Plate
Height Equivalent to One Theoretical Plate (HETP)N
LH
Application: Calculate H and N for peak 18, assuming a 60 m column
A. H ~ 5 mB. H ~ 50 mC. H ~ 500 mD. H ~ 5 mmE. I dont know
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Concept of Peak Capacity ()
Fig. 4. Mobilitymass plot of a complex mixture containing multiple classes of ions. In this case peptide, DNA, and carbon cluster ions are observed. Lines are superimposed onto the plot to indicate the mobilitymass trends for each class of molecule.
of IMS-MS for peptides ~ 2600 for LC-FTICR ~ 6 x 107
PeakIndividualan ofAreaor TimeSeparationfor Available Areaor Time
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
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Diffusion: Ficks 1st Law
dydCDj AAA
When there is a gradient in concentration of a species that can diffuse in medium
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
Concentration (y)
Y (position in SP)
SP
Concentration (y)
Y (position in SP)
SP
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Clicker Question When an analyte is diffusing in the
stationary phase, equilibrium will be reached faster
A. When DA is smallB. When DA is largeC. When SP thickness is largeD. A and CE. I dont know
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Mass Transfer Kinetics: Ficks Law
Stat. Phase
From Bird, Stewart & Lightfoot, Transport Phenomena, 2nd Ed. 2002
YD
SW A
ABAy 00
dydDj AABAy
Initially no substance A Pulse at the surface Transient concentration Steady state profile at long
times
WAy: mass flux of A A: mass fraction of A DAB: diffusivity of A in B S: surface area; : density
jAy: molecular mass flux of A
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Situation in Column Chromatography
Stat. Phase
t = 1
t = 2
Mass transfer takes time => separation + limit on resolution
t = 3
t = 4
t = 5
t = 6
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Diffusion: Ficks 2nd Law
Conservation of mass for diffusing species in control volume Per unit area
perpendicular to diffusion
Concentration (y)
y (position in SP)
SP
outinA jj
ttyC
),( 1
jin
joutC(y1,t)
ytyyCD
ytyCDy
ttyC AAA
),(),(),( 111
y1y1+y
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Diffusion: Ficks 2nd Law II
When things are changing in time:
Once CA is the same everywhere, we have reached equilibrium in the SP
Concentration (y)
y (position in SP)
SP
2
),(),(y
tyCDt
tyC AA
A
Q: can we estimate order-of-magnitude of time needed?
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Time Scale of Mass Transfer For transfer across SP
thickness Y Start with:
Order-of-magnitude analysis
Simplifying:
2
),(),(y
tyCDt
tyC AA
A
2YCDC AA
D
A
AD D
Y 2
Concentration (y)
y (position in SP)
SP
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Numerical Example of Mass Transfer Assume
Column diameter = 100 m Film thickness: Y = 1 m Diffusivity of analyte in stationary phase
DAB= 10-6 cm2 s-1 Solubility of A in B is 1% of volume MWA = 100 g mol-1
Questions What is the time scale of mass transfer? What should be the time scale of flow along 1 mm of the
column? What is the max. amount of analyte that can be in the
stationary phase per 1 mm of column length?
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Band Broadening Processes
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
Stationary Phase
Stationary Phase
Mobile Phase
Detector
Lase
r
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Schematic of Column Chromatography III
If analyte has some affinity to the stationary phase, it will be retarded Equilibrium Kinetics
Molecular mass transfer: diffusion Emerge at the detector after retention time tR
SP
MCCK s
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GC vs. HPLC ColumnsGC HPLC
Most GC columns do NOT have particles Most HPLC columns do have particles Why?
A. Particles are needed to prevent liquid flow for being too fastB. Particles are needed in HPLC to shorten diffusion distance in MPC. Particles are not needed in GC because diffusion is very fastD. B & CE. I dont know
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Effect of Mobile Phase velocity on H
Experiment Repeat the same
separation, same column and mobile phase
Determine H vs. flow rate
Observe an optimum, H increases to both sides
Goethe: there is nothing more practical than a good theory
Skoog & Leary, 4th Ed
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Van Deemter Model: B Term Longitudinal Diffusion Basically molecular diff., as ifmobile phase was not moving
Clicker 1A. B term as u B. B term as u C. B term D. Dont know
)(uf
Clicker 2. B term isA. more imp in GCB. More imp in HPLCC. Similar importanceD. I dont know
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Van Deemter Model: A Term Broadening
Eddy diffusion &unequal pathways Molecules may travel unequal distances Particles (if present) cause eddies & turbulence A depends on size of stationary particles (want small) and
their packing (want uniform) (or coating in TLC plate) GC: 150 m, HPLC: 5-10 m
PdA ClickerA. A term as u B. A term as u C. A term D. Dont know
)(uf
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Van Deemter Model: C Terms I Accounts for finite time for mass transfer (equil. btwanalyte in stationary and mobile phase not instantaneous) Most important effect in GC & HPLC CS accounts for stationary phase mass transfer
df: stationary phase film thickness DS: diffusion coeff. of analyte in SP Thinner films reduce mass transfer time& broadening
But also reduce capacity of the column
s
fs D
dC
2
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Van Deemter Model: C Terms II CM accounts for mass transfer on themobile phase interface with the SP In packed columns:
dp is particle diameter In open columns
dc is column diameter
M
PM D
dC2
M
CM D
dC2
Clicker 1A. C term as u B. C term as u C. C term D. Dont know
)(uf
Clicker 2. CM term isA. More important in GCB. More important in HPLCC. Similar importanceD. I dont know
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Van Deemter Model of Band Broadening
Tries to explain previous experiment H: plate height u: average linear velocity
H: as small as possible (calculate Hmin?) Some terms decrease, other increase with u
There should be optimum u There are alternative models (see reading)
uCuCuBAH MS
MtLu
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Optimum Mobile Phase Velocity We want N highest, H lowest Do we want something else?
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Optimum Mobile Phase Velocity: GC & HPLC
Q: differences in A, B, C between Supercritical Fluid Chrom. & HPLC?