1.1 General description - Sample dissolved in and transported by a mobile phase - Some components in sample interact more strongly with stationary phase.

1.1 General description- Sample dissolved in and transported by a mobile phase- Some components in sample interact more strongly with stationary phase and

are more strongly retained- Sample separated into zones or bands

1.2 Classification(based one the types of mobile and stationary)

- Gas chromatography (GC)- Liquid chromatography (LC)- Supercritical fluid chromatography (SFC)

Classification based on the types of mobile and stationary phases

1.3 Elution chromatography:Flushing of sample through column by continual mobile phase addition

- only eluent (portion of sample in

mobile phase) moves down

- migration rate fraction of time spent in mobile phase

Fig. 26-1 (p.764) (a) The separation of a mixture of components A and B by column elution chromatography, (b) the output of the signal detector

Fig. 26-2 (p.765) Concentration profiles of solute bands A and B at two different times in their migration down the column.

- Chromatogram (concentration versus elution time)

- More strongly retained species elutes last (elution order)

- Analyte is diluted during elution (dispersion)

- Zone broadening proportional to elution time

- Adjust migration rates for A and B (increase band separation)- Adjust zone broadening (decrease band spread)

Fig. 26-3 (p.765) Two-component chromatogram illustrating two methods for improving overlapping peaks.

2.1 Distribution constant

Analyte A in equilibrium with two phases

constanton distributi mobile

stationary

stationarymobile

c

cK

AA

tM time for unretained species (dead time),same rate as mobile phase moleculesaverage migration rate

tR retention time for retained speciesaverage migration rate

Ideally: tR independent of volume injected, produces a Gaussian peak

Fig. 26-4 (p.767) A typical chromatography for a two component mixture.

lengthcolumn :L Rt

Lv

Mt

Lu

2.2 Retention time

2.3 Relationship between tR and K

A

MSA

MS

MS

MMS

SSMM

M

A

ku

VVK

estimeatedVV

VV

VcV

VcVc

Vtotal

u

fractionuv

1

1

kfactor retention :/

packingcolumn from :/

)/(K1

1u

/c1

1u

cu

A of mols

phase mobilein A of moles

phase mobilein spendsA timeof

A

A

S

M

2.4 retention factor k

kA is 1.0, separation is poor

kA is >20-30, separation is slow

kA is between 1-10, separation is optimum

M

MRA

AMR

t

ttk

kt

L

t

L

1

1

2.5 Relative migration rate: Selectivity factor ()

Selectivity factor

Larger = better separation

timesretention )(

)(t

factorsretention

constantson distributi

R

MAR

MB

A

B

A

B

tt

t

k

k

K

Ka

3.1 Rate theory of chromatography

- Individual molecule undergoes “random walk”, and many thousands of adsorption/desorption processes.

- Some travel rapidly while other lag add up to give Gaussian peak (like random errors)

- Breadth of band increases down column because of more time

- Zone broadening is affecting separation efficiency – high efficiency requires less broadening

3.2 Column efficiency

N: number of plates

L: length of column

H: height of 1 theoretical plate

Plates are only theoretical –

column efficiency increase with N

H

LN

Fig. 26-6 (p.770) Definition of H. Efficient column has small plate height

– less zone broadening

Experimentally, H and N can be approximated from the width of the base of chromatographic peak.

Fig. 26-7 (p.770) Determination of N.

2)(16W

tN R

3.3 Kinetic variables affecting column efficiency (H)

3.3.1 Mobile phase velocity

- Higher mobile phase velocity, less time on column, less zone broadening

- However, plate height H also changes with flow rate

Fig. 26-7 (p.770) Effect of mobile-phase flow on plate height for GC.

3.3.2 van Deemter Equation

A: multipath term

- Molecules move through different paths

- Larger difference in path length for larger particles

- At low flow rates, diffusion allows particles to switch between paths quickly and reduces variation in transit time

Fig. 26-9 (p.773) Typical pathways of two molecules during elution.

uCuCu

BAH Ms

B/: Longitudinal diffusion term- Diffusion from central zone to front and tail- Proportional to analyte diffusion coefficient- Inversely proportional to flow rate - high flow, less time for diffusion

C:Mass transfer coefficients (CS and CM)

- CS is rate for adsorption onto stationary phase

- CM is rate for analyte to desorb from stationary phase

- Effect proportional to flow rate – at high flow rates less time to approach equilibrium

Fig. 26-10 (p.774) van Deemter plot.

Column resolution

Fig. 26-12 (p.776) Separation at three resolution values

BA

ARBR

BAs WW

tt

WW

ZR

])()[(22

- u (linear flow rate): low flow rate favors increased resolution (van Deemter plot)

-H (plate height) (or N number of plates): use smaller particles, lengthen column, reduce viscosity of mobile phase (diffusion)

- (selectivity factor): vary temperature, composition of column/mobile phase

- kA (retention factor): vary temperature, composition of column/mobile phase

Effect of k and on R

When A and B have similar retention

41

][

4)(

)()(

]))(

(16[

)()(

2

N

k

kkR

t

ttk

N

t

ttR

W

tN

W

ttR

WWW

B

ABs

M

MR

B

ARBRs

BR

ARBRs

BA

R

222222

A

)1

()1

(16)1

()1

(16

2

kk )

1)(

1(

4)

1)(

1(

4

][

k

k

a

aR

k

k

a

aRN

k

k

k

a

aN

k

k

a

aNR

kka

sB

Bs

B

B

Bs

A

B

Effect of R on t

2

32

2 )1()

1(

16

)1()1()(

]1

1 ,

)([

)(

B

Bs

BBBR

BB

B

M

MBRB

B

BR

k

k

u

HR

u

kNH

u

kLt

kuv

uLuL

v

L

t

ttk

v

Lt

Fig. 26-15 (p.780) The general elution problem in chromatography

General elution problem: for multiple components, conditions rarely optimum for all components.

1. Change liquid mobile phase composition – gradient elution or solvent programming

2. Change temperature for gas chromatography – temperature programming

Section 26E

concepts and formulas in

Table 26-4 and 26-5 (p781, Reading assignment)