Chromatography Concept Outline

8/3/2019 Chromatography Concept Outline

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AN INTRODUCTION TO CHROMATOGRAPHIC METHODS

• GENERAL DESCRIPTION OF CHROMATOGRAPHY

Chromatography – definition

2 phases :

stationary phase Mobile phase

Classification of chromatographic Methods

o Based on nature of stationary phase

Column chromatography

Planar chromatography

o Based upon nature of mobile phase

Liquid chromatography (LC)

Gas chromatography (GC)

Supercritical fluid chromatography (SFC)

Note: In naming specific methods, the mobile phase is mentioned first

Additional terminologies:

Elution

Eluent

Partitioning

Chromatogram

Retention time

Dead time

Principle of Chromatographic Separation

o Based on differences in migration rates among the sample components

o Based on affinity of components to stationary phase and mobile phase

Information from a Chromatogram

o Retention time, tR

o Dead time, tM

o Qualitative information

Position of the peaks on the time axis can be used to identifythe components of the sample

o Quantitative information

Areas under the peaks provide a measure of the concentration

of each species

Effects of Relative Migration Rates and Band Broadening on Resolutiono With time, distance between peaks ( band separation) increases but at

the same time broadening of the bands take place

o Conditions can often be found where band broadening occurs more

slowly than band separation

o A clean separation of species is possible provided the column is

sufficiently long

o Improved separations can often be realized by the control of variables

that either 1. increase the rate of band separation

2. decrease the rate of band spreading



• MIGRATION RATES OF SOLUTES

The effectiveness of a chromatographic column as a means of separating two

solutes (A & B), depends upon the relative rates at which the two species areeluted. These rates are in turn determined by the partition ratios of the solutes

between the two phases.

Partition Ratio or Partition Coefficient, K

For the solute species A at equilibrium, Amobile Astationary

The partition ratio (or equilibrium expression) is expressed as:

Mc

Sc

K =

where cS = molar analytical concentration of solute in stationary phase

cM= molar analytical concentration of solute in mobile phase

Average Migration Rates

o Average migration rates of solutes,−

ν

R t

L ν =

−

where L = length of column packing

o Average linear velocity of mobile phase, u

MtLu =

o Relationship between migration rate and partition ratio

]

MV

SV

K 1

1u[ ν

+

=

−

o Capacity factor, k’ – used to describe the migration rates of solutes

on columns

For solute A:M

VS

V

AK '

Ak =

Since]

MV

SV

K 1

1u[ ν

+

=

−

, andMtLu = ,

]

MV

SV

K 1

1u[ ν

+

=

−

The equation rearranges to

Mt

Mt

R t

k'−

=

Interpretation of k’ value:

* If k’ < 1, elution occurs so rapidly; determination of

retention times is difficult* If k’ > 20 – 30, elution time becomes inordinately long

* Ideally, k’ lie in the range between 1 & 5

For GC, k’ can be varied by changing the temperature &

column packing

For LC, k’ can be manipulated by varying the composition of

the mobile phase and stationary phase

o Selectivity Factor, α

AK

BK

α = A

k'

Bk'

α =

where A = less strongly held soluteB = more strongly held solute

Note: α is always greater than 1.

• Band Broadening and Column Efficiency



Rate Theory of Chromatography

Quantitative Description of Column Efficiency

o Plate height, H

Also known as HETP or height equivalent to a theoretical plate

L

2σ

H=

o Number of theoretical plates, N

2

W

R t

16 N

=

o Relationship between H & N:HL N =

• Variables that Affect Column Efficiency

Refer to Table 28-2

Theory of Band Broadening

o van Deemter equation

o van Deemter diagram

o interpretation of diagram

Column Resolution

o provides a quantitative measure of its ability to separate two analytes

BW

AW

A Rt

B Rt

BW

AW

Z

S R

+

−

=+

∆=

)()[(22

o Baseline resolution is achieved when R = 1.5

o It is useful to relate the resolution to the number of plates in the

column, the selectivity factor and the retention factors of the twosolutes



o Optimization of Column Performance

• Variation in N

• Variation in H

• Variation in capacity factor, k’

• Variation in selectivity factor,α

• APPLICATIONS OF CHROMATOGRAPHY

Qualitative Analysis

Quantitative Analysis

Analyses based on peak height

Analysis based on peak areas

Calibration and standards

Internal standard method

Area normalization method

Documents

Chromatography Concept Outline