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

12