CHAPTER 4CHAPTER 4(AN INTRODUCTION TO (AN INTRODUCTION TO CHROMATOGRAPHY SEPARATION)CHROMATOGRAPHY SEPARATION) 1

4.1 GENERAL DISCRIPTION OF CHROMOTOGRAPHY

Method Basis of Method

Mechanical phase separation• Precipitation and filtration• Distillation• Extraction

• Ion exchange

Difference in solubility of compounds formedDifference in volatility of compoundsDifference in solubility in two immiscible liquids

Difference in interaction of reactants with ion-

exchange resin

Chromatography Difference in rate of movement of a solute through a stationary phase

Electrophoresis Difference in migration rate of charged species in an electric field

Field-flow fractionation Difference in interaction with a field or gradient applied perpendicular to transport direction

2

4.1.1 Definition of Chromatographic Terms

a. Chromatography

» A technique

in which the components of a mixture are separated

based on differences in the rates

at which they are carried

through a fixed or stationary phase

by a gaseous or liquid mobile

phase.

b. Stationary Phase

» A solid or an immobilized liquid

on which analyte

species are

partitioned

during passage of a mobile phase.

c. Mobile Phase

» A liquid or a gas

that carries analytes

through a liquid or solid

stationary phase. 3

4.1.2

Classification of Chromatography• Chromatography methods are of two basic types :

Column Chromatography

The stationary phase

is held in

a narrow tube called column,

and the mobile phase

is forced

through the tube under pressure

or by gravity.

4

Planar Chromatography

The stationary phase

is supported on a flat plate or in the pores

of a paper. The mobile phase

moves through the stationary

phase by capillary action or under the influence of gravity.

5

• Classification of chromatography column

6

V

VV

VV

V

VV

V V

V

V

Elution

V

VV

V

V

V

XX

XA

A

A

A

A

A

X

XX

X

X

X A

AA

A

A A

X

X

XX

XX

A

A

AA

A

A

Injection

Interaction

7

4.1.3 Elution of Chromatography4.1.3 (a) Terms • Elution :

A process in which solutes are washed through a stationary

phase

by the movement of a mobile phase.

• Eluate :

The mobile phase

that exits the column.

• Eluent :

A fresh solvent

used to carry the components of a mixture

through a stationary phase.

4.1.3 (b) Mechanism• Initially, a solution of a mixture of A and B in the mobile phase is

introduced at the head of the column at time to

.

• The two components separate themselves between the mobile

phase and stationary phase.8

• Elution then occurs

by forcing the sample components through the

column by continuously adding fresh mobile phase.

• With the first introduction of fresh mobile fresh, the eluent

moves

down the column, where further partitioning between the mobile

phase and the stationary phase takes occurs (time t1

).

• Further additions of solvent carry solute molecules down

the

column in a continuous series of transfers between the two phases.

• Because solute movement can occur only in the mobile phase, the

average rate at which a solute migrates depends on the fraction of

time it spends in that phase.

9

• This fraction is small

for solutes that are strongly retained

by the

stationary phase (component B), and large

when retention in the

mobile phase is more likely

(component A).

• The resulting differences in rates

cause the components in a

mixture into bands or zones

along the length of the column.

• Isolation of the separated species

is then accomplished by passing a

sufficient quantity of mobile phase

through the column to cause the

individual bands to pass out the end, where they can be collected or

detected

(times t3

and t4

).

10

a)

Diagram showing the separation of a mixture of components A and B by elution chromatography

b)

The detector signal at the various stages of elution shown in a

11

12

4.1.3 (c) Chromatograms• If a detector that responds to solute concentration is placed at the

end of the column during elution and its signal is plotted as a

function of time

(or of volume of added mobile phase), a series of

peaks is obtained.

• Such a plot is called a chromatograms, which is useful for both

qualitative and quantitative analysis.

• The positions of the peaks on the time axis

can be used to identify

the components of the sample.

13

14S

S

• On a chromatogram, the perfect elution

would have the same as the

graphical representation

of the law of Normal distribution of

random errors a (Gaussian curve).

• Gaussian Distribution

: a theoretical bell-shaped distribution

of

results obtained for replicate measurements that are affected by

random errors.

• In keeping the classical notation, μ, would correspond to the

retention time of the eluting peak while σ

to the standard deviation of

the peak ( represents the variance

σ2). y

represents the signal as a

function of time, x, from the detector located at the outlet of the

column.

15

4.1.4 Methods of Improving Column Performance•

By changing experimental conditions, non-separated bands can be separated : (a) Adjust migration rates for A and B

(increase band separation)(b) Adjust zone broadening

(decrease band spread)

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4.2 MIGRATION RATES OF SOLUTE•

The effectiveness of a chromatographic column in separating two

solutes

depends on the relative rates at which the two species are

eluted.

•

These rates in turned are determined by the ratios of the solute

concentrations in each of the two phases.

•

Other factors:

Distribution constant

Retention times

Retention factor

Selectivity factor

17

4.2.1 Distribution Constant• Distribution constant for a solute in chromatography is equal to the

ratio of its molar concentration in the stationary phase (cs) to its

molar concentration in the mobile phase (cm).

• Analyte

A in equilibrium with two phases :

where K

is distribution constant

• K

is constant over a wide range

of solute concentration; that is Cs

is

directly proportional to Cm. 18

mobile stationary

stationary

mobile

A A

CK

C

↔

=

4.2.2 Retention Time• In chromatograms, the small peak

on the left is for a species that is

not retained by the stationary phase.

• Time tM

between sample injection and the appearance

of this peak is

called dead time or void time.

• Dead time/ void time

: time it takes for an unretained

species to pass

through a chromatographic column.

19

• The larger peak

on the right is that of an analyte

species.

• The time required for this zone to reach the detector after sample

injection is called the retention time, tR

.

• The analyte

has been retained because it spends a time tS

in the

stationary phase.

The retention time is then :

• Retention time :

is the time between injection of a sample and the

appearance

of a solute peak at the detector of a chromatographic

column In chromatograms.

20

R M St t t= +

• Average linear rate of solute migration :

where L

= length of column packing

• Average linear rate of mobile-phase migration :

R

Lt

υ =

M

Lut

=

21

4.2.3 Retention Factor• The retention factor is an important experimental parameter that is

widely used to compare the migration rates of solutes on columns.

• Retention factor for a solute A:

• Retention factor

: Retention factor kA

for a solute A is related to the

rate at which A migrates through a column. It is the amount of time a

solute spends in the stationary phase relative to the time it spends

in the mobile phase.

M

S

M

MRA t

ttttk =

−=

22

•

Value of

kA

:

when kA is ≤ 1.0 » separation is poor

when kA is > 30 » separation is slow

when kA is = 2 – 10 » separation is optimum

4.2.4 Selectivity Factor•

Selectivity factor :

the ratio of the distribution constant of the more

strongly retained solute

(B) to the distribution constant for the

less strongly held solute

(A).

•

Selectivity factor of a column for the two solutes is defined as

:

A

B

KK

=α23

where KB

is the distribution constant for the more strongly retained

species B.

KA

is the constant for the less strongly held or more rapidly

eluted species A.

•

α

is always greater than unity

and larger α

»

better separation

•

Selectivity factor can also be described with retention factor.

:

where kB

and kA

are the retention factors for B and A.

A

B

kk

=α

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4.3 THE EFFICIENCY OF CHROMATOGRAPHIC COLUMN

•

The efficiency of a chromatographic column

is affected by the amount of band broadening

that occurs as a compound passes through the column.

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4.3.1 Quantitative Description of Column Efficiency•

Two related terms are widely used as quantitative measures of chromatographic column efficiency :

Plate height HPlate count or number of theoretical plates N

•

The two are related by the equation :

where L

is length (in cm) of column packing.

•

The efficiency of chromatographic columns

increases

as the plate

count N becomes greater

and as the plate H becomes smaller.

HLN =

26

•

Thus, H is needed to be as small as possible »

more plates can be fitted into a column.

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HLN =

5 Plates

10 Plates(Larger H) (Smaller H)“Thicker”

“Thinner”

L, columnlength

Plate

↓

H ↑ No of Plate ↑ Efficiency

•

Because chromatographic bands are usually Gaussian

and because

the efficiency of a column is reflected in the breadth of

chromatographic peaks, the variance per unit length of column is

used by chromatographers as a measure of column efficiency.

•

That is the column efficiency

is defined by :

•

Efficient column has small plate height

»

less zone broadening.

•

Thus, unit H is in cm.

LH

2σ=

28

(a)

Gaussian distribution of sample molecules

(b)

Column length as the distance from the sample entrance point to the detector 29

•

In simple relationship, N can be approximated from the width, W (unit in time) of the base of the chromatographic peak :

30

2

16 R

B

tNW

⎛ ⎞= ⎜ ⎟

⎝ ⎠

4.3.2 Other Variables That Affect Columns Efficiency•

Band broadening reflects a loss of column efficiency.

•

The slower the rate of mass transfer processes occurring while a

solute migrates through a column, the broader the band at the column exit.

•

Some of the variables that affect mass-transfer rates are controllable can improve the separation.

4.3.2 (a)

The Effect of Mobile-Phase Flow Rate

•

Higher mobile phase velocity, less time in column »

less zone broadening.

•

However, plate height, H, can changes with flow rate. The plot of H versus u

»

Van Deemter

plot. 31

where

H is the plate height, u

is the average linear velocity of the

mobile phase, and A, B, and C

are (positive) constants determined

by various physical properties of the mobile and stationary phases.

CuuBAH ++= CuuBAH ++=

CuuBAH ++=

32

33

34

i. Multipath

Term/ Eddy Diffusion (A)•

Eddy diffusion, which is caused by non-uniform packing of chromatographic columns.

35

•

Molecules moves through different paths.

•

Larger difference in pathlengths

for a larger particles.

•

At low flow rates, diffusion allows particles to switch between paths

quickly and reduces variation in transit time.

36

ii. Longitudinal Diffusion Term (B)

37

•

Longitudinal diffusion, which is caused by the tendency of a solute species to diffuse from regions of high concentration

(such as the center of a chromatographic band) to regions of low concentration(such as the leading or trailing edge of a chromatographic band).

•

Proportional to mobile phase diffusion coefficient.•

Inversely proportional to flow rate

»

high flow rate, less time for diffusion.

38

iii. Mass Transfer Coefficients (C)

39

•

Mass transfer in the form of partitioning

of the solute into the stationary phase, which does not occur instantaneously and depends on the solute’s partition and diffusion coefficients.

•

All species need a certain amount of time to equilibrate between

the stationary and mobile phases.

•

High flow rates » species with strong affinity for stationary phase will be slowed

and the molecules with no affinity to the stationary phase move ahead which causes band broadening.

• 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 flowrate

» less time to approach equilibrium.

( )uCCuBAH MS +++=

( )uCCuBAH MS +++=

4.4 COLUMN RESOLUTION•

The resolution Rs

of a column »

how far apart two bands are relative to their widths.

•

The resolution provides a quantitative measure of the ability of the

column to separate two analytes.

•

The chromatogram consists species A and B on three columns with

different resolving powers.

•

Resolution :

0.75 = no separation occur

1.0 = zone A contain ~ 4% B and zone B contain ~ 4% A

1.5 = complete separation of A and B (overlap 0.3%)

•

The resolution for a given stationary can be improved by lengthening column »increasing the no of plates.

41

•

42

( )( ) ( )

2

2

SA B

R B R A

A B

ZRW W

t tW W

Δ=

+

−=

+

4.4.1 Effect of Retention and Selectivity Factors on Resolution

•

Resolution equation

of a column that relates to the

no of plates with the retention time and selectivity

factors of a pair of solutes

on the column :

where kB

= retention factor of the slower moving

species and α

= the selectivity factor

⎟⎟⎠

⎞⎜⎜⎝

⎛+

⎟⎠⎞

⎜⎝⎛ −

=B

Bs k

kNR1

14 α

α

43

4.4.2 Effect of Resolution on Retention Time•

The goal of chromatography

is the highest possible resolution in the shortest possible elapsed time.

•

Unfortunately, these goals tend to be incompatible, and a

compromise between the two usually necessary.

•

The time (tR

)B

required to elute the two species with a resolution Rs

is given by:

where u

is the linear velocity of the mobile phase

•

The resolution will increase

if the u

is low.

( ) ( )( )2

322 11

16

B

BSBR k

kuHRt +

⎟⎠⎞

⎜⎝⎛

−=

αα

44

4.5 APPLICATION OF CHROMATGOGRAPHY•

Gas Chromatography (GC)•

High Liquid Performance Chromatography (HPLC)

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