Introduction to Chromatographic Separations

• Due to lack of analytical specificity, separations are often necessary

• Chromatography is about separations– need minimum of two phases

– stationary phase

– mobile phase

– analyte and matrix must have differing affinities for one or more phases

Example of Chromatographic Experiment

In this example, compound B is more attracted tothe stationary phase than is compound A

The compounds A and B which are attracted to thestationary phase form bands, or zones,along the length of the stationary phase

Intro to Band Broadening, I

• As analytes migrate farther through the stationary phase, band broadening occurs

Intro to Band Broadening, II

• Band broadening decreases chromatographic resolution

we need to optimize the chromatographic method

Chromatographic 'Figures of Merit'

• Table 26-5 in text, 'Calculation of Derived Quantities'– a series of formulas that describe, in various ways,

aspects of chromatographic performance

– we will not examine the derivation of these here, but you may find it helpful to follow the derivation in the text as an aid to understanding Table 26-5

– many of the derived quantities in Table 26-5 are themselves functions of derived quantities

– the question becomes, what can we measure? (and how does that relate to the derived quantities)

What we can measureExperimental Quantities (see Table 26-4)

• migration time of unretained species– (dead time) tM

• retention time (species A and B) (tR)A, (tR)B

• adjusted retention time (species A) (tR)A- tM

• peak width (species A) WA

• length of column packing L

• flow rate of mobile phase F

• volume of stationary phase VS• concentration of analyte in mobile

and stationary phases cM, cS– actually, cM, cS aren't practical to measure

Chromatographic figures of merit• resolution

• capacity factor

• selectivity

Resolution

• From experimental data

• As a derived quantity (rearranged from last equation in Table 26-5)

– u is the linear velocity of the mobile phase, also related to the Height Equivalent of a Theoretical Plate (HETP) or H

is the selectivity factor

– k' is the capacity or retention factor

BA

ARBRs WW

ttR

2

3'

2'2

1

1

16B

BBRs

k

k

H

tuR

The Capacity or Retention Factor• Experimentally

• gives relative value for attraction of analyte to the stationary phase

M

MR

t

ttk

'

• Experimentally

• Derived

• Info on the preferential attraction of A for the stationary phase relative to B (1 )

A

B

K

K

.,etcc

cK

BM

BSB

MAR

MBR

tt

tt

The Selectivity Factor

Plates, u and the van Deempter Equation

• plate – conceptually separations unit

• the more plates, N, the better the separation

• the shorter the plate, the more that can be stuffed onto fixed length, L, column

– experimentally

– empirically (the van Deempter equation)

uCCu

BAH MS

2

2

4 Rt

LW

N

LH

van Deempter Plots

Gas Chromatography

Liquid Chromatography

Laundry List of Experimental Parameters of Concern

• number of plates, HETP L

• stationary phase column type, head pressure

• mobile phase F, head pressure, mixture, gradient

• temperature (programmable) T

• quality of column packing column type

The 'General Elution Problem'