Sac ki anh mai - anh mai
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Nguyễn Ánh MaiAnalytical Chemistry – University of Science HCMC
2011
What is chromatography?
xanthophills+ chlorophylls
β - carotene
α - carotene
Tswett, 1903
separation of leaf pigments
on carbonate calcium
Chromatography
Color writing
Petroleum ether
Stationary phase Mobile phaseAnalyteSorption Desorption
Mobile phase
Stationary phase
Mobile phase
ChromatographyChromatography-- a a separationseparation techniquetechnique
The classification of
chromatographic techniques is based on …..
Select an appropriate chromatographic technique
Gas-Liquid Gas-Solid(GLC) (GSC)
Gas Chromatogr. (GC) Liquid Chromatogr. (LC)
Reversed Phase(RPC)
Normal Phase(NPC)
Ion Exchange(IEC)
Affinity(AC)
Size Exclusion(SEC)
…..
How an analyte interact with the phases?
(types of interaction force)
Interactions in chromatography
♦ Dispersion force (non-polar compounds, e.g. hydrocarbons)
♦ Polar force (dipole-dipole/dipole-induced dipole)♦ Ionic force (ion-ion)
Electrostatic nature!
♦ Dispersion force2 molecules interacting
and held togetherby dispersion force
Interacting plane
Charge fluctuation
Hydrocarbonsaliphatic, aromatic
♦ Polar forces Two molecules interacting and held together by dispersive forces and polar forces from
permanent / induced dipoles
Permanent dipole e.g.alcohols, esters, amines, nitrile
Hydrogen bonding
Induced dipole e.g. benzene
+
+
♦ Ionic force
Two molecules interacting and held together by dispersive forces and ionic forces between net ionic charges
+
Sodium dodecyl sulfonate
Cetyl trimethyl ammonium bromide
Mixed-mode interaction in chromatography!!
Weak electrostaticinteraction
Weak electrostaticinteraction
Hydrophylicpartitioning
ANALYTE
ANALYTE
ANALYTE
ZIC-HILIC (Merck-Sequant)
* octanol-water partition coefficient
Acclaim Trinity P1 (Dionex)
Simultaneous separation of pharmaceutical counter ions
The time an analyte take to pass a chromatographic column (retention time)is a function of…
Factors governing the retention
Partition Coefficient, K
The difference in partitioning of an analyte between the mobile and stationary phases is governed by the partition coefficient K
CS: concentration in stationary phaseCM: concentration in mobile phase
K = CS / CM
Theoretical plate ?
Plate theory
A chromatographic column - is envisioned as repetitive liquid-liquid extraction process
or a distillation column - composed of a series of discrete, contiguous horizontal layers
Question: draw the concentration profiles of A and B in the mobile phase after they pass through 5 theoretical plates?
A → KA = 1B → KB = 2
Concentration profiles of A, B, C (KA = 1/9, KB = 1, KC = 2) after passing a) 10 and b) 20 theoretical plates
Which is A, B or C?
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0 2 4 6 8 10
-0.2
-0.1
0
0.1
0.2
0.3
0 5 10 15 20
a) b)
The column efficiency increases with the number of successive equilibrations, and the number of theoretical plates,N, has become a way of determining the column separation efficiency.
Solute transferring from the MP to the SP at the front of the peak profile
Solute transferring from the SP to the MP at the back of the peak profile
How does a solute migrate along a column?
Migration of a solute mixture in a column
Is K (partition coefficient) always constant
regardless the concentration of analytes?
Linear and non-linear chromatographyIsotherm effect on peak shape
(1)
(2)
(3)
Cs
CM
(1): linear Gaussian peak(2): concave fronting (due to e.g. solute-solute interaction in the case of overloading)(3): convex tailing (heterogeneous surface of stationary phase with strong active sites)
Peak asymmetry
What else affect the retention of a compound in the column?
n A (S) , n A (M) : number of moles of A in the SP and MP, respectivelyVS , VM : volume of the SP and MP, respectivelyβ : phase ratio
k’ = nA(S) / nA(M) = K (VS/VM) = K / β
k’ = t’R / t’0
Factors governing the retention
Capacity/retention factor, k’ (k)
The role of phase ratio !
van Deemter curve
Mobile phase velocity, mm/sec
Pla
te h
eigh
t, μm
H
U
van Deemter equation
CCSS, C, CMM: mass transfer coefficients related to the : mass transfer coefficients related to the properties of the phases and the soluteproperties of the phases and the solute
• A term: eddy diffusion• B term: longitudinal diffusion• C term: mass transfer resistance
in stationary and mobile phases
H: plate heightu: linear velocity (cm/s)H = A + B/u + Cu
C = CS + CM
H
U (cm/s)Uopt
van Deemter curve
Factors contributing to band broadening
• Eddy Diffusion (A)Band broadening arises in part from a multitude of pathways thata solute molecule can find through a packed column.
Rate theory
Well-packing particles with narrow size distribution is preferred !
dp: particle diameterλ: packing properties
narrower size distribution of particles → smaller λ(0.5 – 1.5)
He = 2λdp
He = 0 in open tubular column (GC)
• Longitudinal Diffusion (B/u)Results from the tendency of molecules to diffuse from regions of high concentration to regions of low concentration
Rate theory
Longitudinal diffusion occurs both in the mobile and the stationary phase,but it is significant only in the mobile phase, and only when this is a gas.
t1 < t2 < t3
Ψ: obstruction factor~ 0.6 for a packed bed and 1 for an open tube
DM: diffusion coefficient of solute in the mobile phase
B /u = 2ΨDM / u
The larger molecules → the slower diffusion
• Mass transfer TO and FROM stationary phase (CSu)Influenced by the rate at which analyte molecules can be transferred to and from the stationary phase.
Rate theory
THIN stationary liquid films in open tubular column are advantageous !
df : stationary film thicknessq : shape factor, ~2/3 for uniform filmDS: diffusion coefficient of solute in stationary phase
for GC!!!CSu = u [qk’df2] / [(1 + k’)2.DS]
“CSu” term in LC is more complex, dependent on the surface (pore) morphologyand stationary phase film thickness.
Remember that the surfaces are usually porous!
Solutes get into stagnant mobile phase “pool” to interact with functional groups by diffusion
Stagnant mobile phase
Stationary phasefilm
• Mass transfer To and From Stationary Phase (CSu)
Thick film of stationary phase, too small, deep and tortuous pores on the surface increase CS
Rate theory
The contribution of CM u to plate height is not linear,
but bears a complex dependency on mobile phase velocity.
Mass transfer in mobile phase
CM u = u × f (dp2, u) / DM
Comparison of different efficiencies of carrier gases in OT column GC
Which carrier gas do you prefer regarding efficiency and analysis time?
Band broadening in open tube and porous media
Mobile phase flow profile for an open tube and a packed column with pressure-driven and electroosmotic flow
Flow profile in inter-particle space
particle particle
Number of theoretical plates –an indicator of column quality
• HETP (Height Equivalent to a Theoretical Plate), HThe column length corresponding to one theoretical plate
• Number of theoretical plates, N
N = (tR / σ)2
N = 5.54 (tR / W1/2)2
N = 16 (tR / Wb)2
• Assuming a Gaussian peak shape
1.000
0.882
0.607
0.500
0.324
0.134
0.044
σ
2σ
W1/2=2.354 σW1/2
3σ
4σ5σ
Wb=4σ
Gaussian peak
L: column length
WHY ?
Classical chromatographic theory considers that a separation process take place by a succession of equilibrium steps, the more equilibrium steps in the column the greater column efficiency with less band broadening (σ), therefore
In practice the proportionality constant is 1, therefore
Where , the standard deviation of the Gaussian peak, describes the spread of the molecules in the band. Band broadening is also a function of time, the longer the band takes to elute the more time the molecules have to spread out, therefore
Selectivity factor, α
α = K2 / K1= k’2 / k’1 = t’2 / t’1
Shows how much difference in (relative) interaction strength of two solute 1 and 2 with the stationary phase
Peak Resolution
(for 2 closely spaced peak)
RS = 2Δt / (Wb1 + Wb2) ≈ Δt / Wb2
In general, what factors affect the resolution of a column?
Resolution -Relationship to column properties
Purnell’s equation for resolution factor of two closely spaced peaks
How to vary α , k’, N (in practice) to improve resolution?
RS = [N1/2 /4] [(α -1) / α] [k’2 / (1+k’2)]
How to optimize a separation regarding resolution and analysis time?
k’2k’ 2/(
1+k’ 2)
α
Effect of k’, α, Non resolution
k’ = 3.0; α = 1.10; N = 3500; Rs = 1.00
k’ = 3.0; α = 1.20; N = 3500; Rs = 1.83
k’ = 3.0; α = 1.10; N = 7000; Rs = 1.42
k’ = 6.0; α = 1.10; N = 3500; Rs = 1.16
(B)(D)(C)(A)
Resolution and relative peak area(*)
totR, VR
t’R, V’R
Retention time/volume
Vo
to: dead time (time an un-retained solute spends in the column)tR: retention time (total time a retained solute spends in the column)t’R: corrected retention time (time a solute spends in the stationary phase)
F: flow rate (mL/min)t: time (min)V = F.t
QUANTITATION IN CHROMATOGRAPHY
Peak detection bya) slope and b) area sensitivity
CORRECTincorrectincorrect
Analysis of merged peak
PEAK HEIGHT OR AREA IS BETTER FOR QUANTITATION?
Quantitation
• CalibrationInternal calibration, internal standard (IS)External calibrationAddition calibration
• Limit of detection/quantitation (LOD, LOQ)
Chlorophyll C1
Chlorophyll A/B/D/C2with different side chains of chlorin ring
β-carotene
α-carotenewith double bond 1 → 2
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