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THEORY OF CHROMATOGRAPHY

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The Chromatogram

A chromatogram is a graph showing the

detector response as a function of elution

time.

The retention time, tR, for each component

is the time needed after injection of the

mixture until that component reaches the

detector.

Retention volume, VR, is the volume of

mobile phase required to elute a particular

solute from the column:

VR = tRF

where F is the mobile phase flow rate

The dead time, tm, is the time of travel of

unretained mobile phase through the

column.

The adjusted retention time, tR, for a solute

is the additional time required for solute to

travel the length of the column beyond the

time required by unretained solvent:

tR = tR tm

In GC, tm is usually taken as the time

needed for CH4 to travel through the

column.

t R' 2

= '

t R1

For any two components 1 and 2, the

relative retention, , is the ratio of their

adjusted retention times:

t

=

t

'

'

t

>

t

where R 2 R1,

'

R2

'

R1

so > 1.

For each peak in the chromatogram, the

capacity factor, k, is defined as:

tR tm

k'=

tm

time solute spends in stationary phase

k'=

time solute spends in mobile phase

k'=

=

time solute spends in mobile phase

moles of solute in mobile phase

C sV s

k =

C mVm

'

Cs

=K

Cm

'

V

t

t

t

'

s

R

m

R

k =K

=

=

Vm

tm

tm

Expressions

t

=

t

'

R2

'

R1

'

2

'

1

k

K2

=

=

k

K1

Efficiency of Separation

Resolution

Solute moving through a column spreads

into a Gaussian shape with standard

deviation . Common measures of breadth

are:

The width w measured at half-height

The width w at the baseline between

tangents drawn to the steepest parts of the

peak (inflection points).

Resolution (cont.)

It can be shown that:

w = 2.35

and

w = 4

Resolution (cont.)

In chromatography, the resolution of two peaks

from each other is defined as

t R VR 0.589t R

Rs =

=

=

wav

wav

w 1 av

2

and wav is the average width of the two peaks.

Resolution

So, separation of mixtures depends on:

width of solute peaks (want narrow)

efficiency

spacing between peaks (want large spacing)

selectivity

Example

What is the resolution of two Gaussian

peaks of identical width (3.27 s) and height

eluting at 67.3 s and 74.9 s, respectively?

ANS: Resolution = 2.32

Diffusion

A band of solute broadens as it moves

through a column. Ideally, an infinitely

narrow band applied to the inlet of the

column emerges with a Gaussian shape at

the outlet.

Diffusion (cont.)

One main cause of band spreading is

diffusion. The diffusion coefficient

measures the rate at which a substance

moves randomly from a region of high

concentration to a region of lower

concentration.

Diffusion (cont.)

The number of moles crossing each square meter

per second, called the flux, is proportional to the

concentration gradient:

dc

mol

flux 2 J = D

dx

m s

Broadening of Chromatographic

Band by Diffusion

If solute begins to move through a column in an

infinitely sharp layer with m moles per unit crosssectional area of the column and spreads by

diffusion alone, then the Gaussian profile of the

band is described by

m

c=

e

4Dt

x2

( 4 Dt )

= 2 Dt

Column Efficiency

Plate theory - older; developed by Martin &

Synge

Rate theory - currently in use

View column as divided into a number (N)

of adjacent imaginary segments called

theoretical plates

within each theoretical plate complete

equilibration of analytes between stationary

and mobile phase occurs

Significance?

Greater separation occurs with:

greater number of theoretical plates (N)

as plate height (H or HETP) becomes smaller

L = NH or H = L / N

where L is the length of column, N is the

number of plates, and H is the plate height

Plate Theory

Band spreading - the width of bands increases

as their retention time (volume) increases:

Plate height is the constant of proportionality

between the variance of the band and the

distance it has traveled:

x 2D

x = Hx

= 2 Dt = 2 D =

ux ux

2

Plate Theory

The smaller HETP, the narrower the

eluted peak

Considerations

Not unusual for a chromatography column

to have millions of theoretical plates

Columns often behave as if they have

different numbers of plates for different

solutes present in same mixture

VR

N = 16

wb

This equation is a measure of the efficiency of

a column.

at the bandwidth at half-height w1/2:

VR

N = 5.54

w 1

2

Asymmetric Peaks

The Dorsey-Foley equation:

41.7 R

w

0.1

N=

A + 1.25

B

from a Chromatogram

Knowing the number of theoretical plates

and the length of the column,

we can determine the HETP,

height equivalent to a theoretical plate:

L

L wb

=

H = HETP =

N 16 V R

L wb

=

16 t R

Plates

Introduced to characterize open tubular columns

uses adjusted retention volume VR in lieu of total

retention volume VR:

N eff

t

V

= 16 = 16

wb

wb

'

R

'

R

Plates (cont.)

The Neff value is useful for comparing a packed and

an open tubular column when both are used for the

same separation.

N and Neff are related by the expression

N eff

k'

= N

k '+1

Rate Theory

Based on a random walk mechanism for the

migration of molecules through a column

Takes into account:

mechanism of band broadening

effect of rate of elution on band shape

availability of different paths for different

solute molecules to follow

diffusion of solute along length

Height

B

H = A+

+ Cu x

ux

Multiple paths

Longitudinal

diffusion

Equilibration

time

contribute to band broadening

In open tubular columns, A is zero

In capillary electrophoresis, both A and C

go to zero

H min = A +

B

C

H min = A + BC

u opt =

B

C

Longitudinal Diffusion

Gives rise to B/ux term

Solute continuously diffuses away from the

concentrated center of its zone

The greater the flow rate, the less time is

spent in the column and the less

longitudinal diffusion occurs

The variance resulting from diffusion is

2 Dm L

= 2 Dm t =

ux

2

2 Dm

B

HD =

=

L

ux

ux

In packed columns, the tortuosity coefficient is

used to account for irregular diffusion patterns and

is usually less than unity ( ~ 0.6), because

molecular diffusivity is smaller in packed columns

than in open tubes ( = 1):

2Dm

HD =

ux

Because longitudinal diffusion in a gas is

much faster than in a liquid, the optimum

flow rate in gas chromatography is higher

than in liquid chromatography

Transfer Term)

This term comes from the finite time required for

the solute to equilibrate between the mobile and

stationary phases

Some solute is stuck in the stationary phase, but

the remainder in the mobile phase moves forward

resulting in spreading of the zone

The slower the flow rate, the more complete

equilibration is and the less band broadening

occurs

Plate height due to finite equilibration time:

where Cs describes the rate of mass transfer

through the stationary phase and Cm describes the

rate of mass transfer through the mobile phase.

Specific equations for Cs and Cm depend on the

type of chromatography.

For gas chromatography in an open tubular

column, the terms are:

Mass transfer in stationary phase:

Cs =

2k '

d 2f

2

3(k '+1) D s

1 + 6k '+11k ' 2 r 2

Cm =

2

Dm

24(k '+1)

k the capacity factor

df the thickness of stationary phase film

Ds the diffusion coefficient of solute in the

stationary phase

r the column radius

Dm the diffusion coefficient of solute in

the mobile phase

Efficiency is increased by:

Decreasing stationary phase thickness

Reducing column radius

Increasing temperature

(Eddy Diffusion)

The term A accounts for the multitude of pathways

that could be followed through the column by one

molecule:

A = 2d p

dimensionless constant characteristic of

packing

dp the particle diameter

(Eddy Diffusion) (cont.)

The A term can be minimized by:

Eliminating column packing (open tubular

columns)

Reducing the particle size of the packing

Packing the column more uniformly

(spherical particles of similar size)

The Knox Equation

Used to compare column efficiencies

Makes use of so-called reduced parameters

(dimensionless quantities):

Reduced plate height: h = H/dp

Reduced velocity: v = udp/Dm

h = a

+ c

For well-packed columns of varying particle

size and differing conditions, the

coefficients a, b and c will be roughly

constant: e.g. a =1, b = 2, and c = 0.05 for

porous particles.

Equation: the Giddings Equation

Giddings realized that the eddy diffusion and

resistance to mass transfer in the mobile phase

must be treated dependently:

5

i =1

1 + 1

A

C1u

H =

B

+ + C s u + Cm u + H e

u

He extracolumn band broadening

cb d p2

C1 =

D

m

cb a proportionality constant; cb 1

broadening are:

Through channels between particles

Through particles

Resulting from uneven flow channels

Between inhomogeneous regions

Throughout the entire column length

A more usable expression for resolution is

1

Rs =

4

1 k '

k '+1

N req

= 16 R

1

2

s

k 2' + 1

'

k2

If k2 and are known, the required number of

plates can be calculated:

N req

= 16 R

1

2

s

k + 1

'

k2

'

2

The Nreq parameter can be used to determine the

length of column necessary for a separation. We

know that N = L/H; thus

Lreq

= 16 R H

1

2

s

k +1

'

k2

'

2

The minimum analysis time tmin is

t min

H

= 16 R

u

2

s

k +1

' 2

k2

'

2

( )

Chromatography

The goal in chromatography is the highest

possible resolution in the shortest possible

time. Optimization techniques aim at

choosing conditions that lead to a desired

degree of resolution with a minimum

expenditure of time

Optimization Techniques

Minimizing plate height:

Reducing dp

Reducing column diameter

Changing column temperature

Reducing the thickness of the liquid film

Optimizing the flow rate of the mobile phase

Resolution also improves with L, but it

expensive in terms of time of analysis

Variation in the selectivity factor:

Changing the composition of the mobile phase

(in HPLC)

Changing the column temperature

Changing the stationary phase

Using special chemical effects

Resolution and Elution Time

Increases in k enhance resolution but at the

expense of elution time

The optimum k values are from 1 to 5

The easiest way to improve Rs is by

optimizing k

In GC, k can be improved by temperature

programming

In LC, k is improved by gradient elution

Columns

tubular columns can provide:

Higher resolution

Shorter analysis time

Increased sensitivity

Lower sample capacity

Columns (cont.)

Compared with packed columns, open

tubular columns allow

Increased linear flow rate or a longer

column or both

Decreased plate height, which means higher

resolution

Bandshapes

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