CHROMATOGRAPHY

Fundamental

Solomon Tesfalidet, Umea University, Department of Chemistry

CHROMATOGRAPHY

Mikhail Tswett 1906: Chromatography

Chroma = color
Graphein = writing
 General Principle:

Stationary Phase Mobile Phase

𝑋𝑆 𝑋𝑀

𝑋𝑆
= 𝐾𝑋
𝑋𝑀

Where 𝐾𝑋 is the distribution or partition coefficient

 Large 𝐾 = slow elution
 Small K = fast elution
Classification of chromatographic methods

Column Chromatography / Planar Chromatography

Liquid Chromatography (LC)

Liquid-Liquid (LLC)
Liquid -Solid (LSC)
Liquid-Bonded phase (LBC)
Ion exchange (IEC)
Gel-permeation (GPC)

Gas Chromatography (GC)

Gas-Liquid (GLC)
Gas-Solid (GSC)
Gas-Bonded phase (GBC)
 What is required in order to achieve separation?

 Retention

where:

t´R = tR-tM (adjusted retention time)

Capacity factor 𝑲′𝑿

t r  t M t´r n X ( S ) VS X S VS
K X   K X    KX
n X ( M ) VM X M VM
or
tM tM

where:

t´R = tR-tM (adjusted retention time)

nX = total moles of X in stationary phase (SP)
nx = total moles of X in mobile phase (MP)
VS = the volume of the stationary phase
within the column
VM = the volume of the mobile phase within
the column
Retention volume, Vr = FtR where
F = Flow rate (ml/min)
 Selectivity

The capacity of the chromatographic system to distinguish between two components

𝑡′𝑅 𝐵 𝐾′𝐵
∝= =
𝑡′𝑅 𝐴 𝐾′𝐴

∝ = selectivity factor
Column efficiency
Describes the degree of band broadening Number of theoretical
in the column (the chromatographic peak) plates
2
t 
N teor  16 r 
 w

2
 t 
N teor  5.55 r 
 w1 / 2 

Number of effective
plates

 t 
2

N eff  16 r 
 w

2
 t 
N eff  5.55 r 
 w1 / 2 
HETP: Height Equivalent to a
Theoretical Plate (H)

L
H
N

where L= the length of the

column (mm)
Resolution R:

The degree of separation

tr , B  tr , A 2tr t
R   r
wA  wB wA  wB wav
2

thus:

R is determined by the values of tr (K’ and )

and w (N)
Qualitative analysis
tR is used for qualitative analysis
tR for the sample is compared with tR for a standard sabstance:

negative verification  the substance is not present in the sample

positive verification  the substance is present in the sample 

collection of fractions for analysis, by ex. IR or MS.
Quantitative analysis

Evaluation of peak height or peak area

Calibration graph

Internal standard method can be used for

elimination of variation of peak form, ex. due
to variation in sample volume
Band-broadening (Peak broadening) in
Chromatography

Van deemter´s equation:

H  A
2k D DM

qk d 2f u


f d p2 , d c2 , u u 
u 1  k 2 DS DM

or
2 Dm  2k d 2 1  6k   11k 2 r 2 
H  A    u x
ux  3( k   1) 2
Ds 24 ( k   1) 2
Dm 

Often expressed as

B
H  A
B
 CS u  CM u or H  A  Cu x
u ux

Where:
u (u x) = linear velocity  flow rate of
mobile phase
A-term
Band broadening due to multiple flow paths
(eddy diffusion)
B-term

Peak broadening due to

diffusion in the mobile phase

B 2 kD DM

u u

Where:

kD: constant which is related to the

form and size of the packing
material

DM: Diffusion coefficient for the

sample in the mobile phase

u: linear velocity of mobile phase

 CM - term

f (d p2 , d c2 , u)
CM u  u
DM Diffusion in stagnant mobile phase

Droplets of the mobile phase containing

analyte molecules are stagnated in the pores
of the packing material

where:
Minimizes the CM term:
dp = diameter of the packing material
dc = column diameter • small particle sizes
• homogenous packing of the
stationary phase using closely
distributed packing materials
• Large diffusion coefficients
CS - term
qk d 2f u
CS u 
1  k  2 DS

df: thickness of the liquid coating on the

stationary phase

ds: diffusion coefficient of the sample in the

stationary phase

Minimizes the CS-term:

• thin and homogeneous film of stationary

phase
• low viscosity of stationary phaselow flow
rate (linear velocity) of mobile phase
The effect of Cux on the equilibration of
analyte between the mobile phas/stationary
phase

Cux = (Cs + Cm)ux

Other parameters which contribute to band-broadening:

• large injection volumes

• low injection rate

• dead volumes (Extra Column band-broadening):

- between injector and column.

- between column and detector.
- in the detector.

SUMMARY

The column efficiency can be improved by using:

small and uniform size of packing material

optimal flow rate for the mobile phase
suitable mobile phase
stationary phase of low viscosity