ANALYTICAL SEPARATIONS

Precipitation

Gravimetry

•Precipitation
•Filtration
•Washing
•Drying or ignition
•Measuring
•Calculation
Separating species by distillation

Determination of ammonia

Determination of carbon dioxide

Extraction

Distribution between organic and water phase

Separation of metal ions as chelates

Ions are: soluble in water

insoluble in non polar-organic phase

Separation of Fe3+ ion

O O
(C6H5 ) NH4+ (C6H5 )3 Fe
NO NO

Cupferron: ionic ferric cupferrate: neutral

Separating ions by ion exchange
Cation exchange:
xRSO3-H+ + Mx+  (RSO3-)xMx+ + xH+
solid soln solid soln

where: Mx represents a cation and R a part of resin containing sulfonic

acid group

Anion exchange:
xRN(CH3)3+OH- + Ax-  [RN(CH3)3]xAx- + xOH-
solid soln solid soln
where: Ax- represents an anion and R a part of resin containing
trimethyl ammonium group

After ion exchange cations or anions are on the resin, it

should remove them
Chromatography
 Classification of chromatographic methods
Stationary phase
Mobile
solid liqiud
phase
Gas chromatography gas GSC GLC
GC
Supercritical chromatography Supercritical SFC SFC
SFC fluid

TLC PC
IC
Liquid chromatography GPC,SEC
LC
liquid Normal Reversed
phase phase
(HPLC-NP) (HPLC-RP)
CE
Liquid chromatography liquid GEL ELFO
LC
 Modes of chromatographic separation

Frontal chromatography
Displacement chromatography
Elution chromatography
 Interactions in chromatography
1. Physical interactions
-sorption: adsorption
absorption (solvation, distribution)
chemisorption
-hydrofil-interactions
-hydrofob-interactions
-interactions based on size exclusion
2. Chemical interactions
-acid-base interactions
-complex formation
-H-bond interactions
3. Biochemical interactions
-biochemical affinity
 The chromatographic process

Consequences:
•Analytes are moving with different rates (differential migration)
•In the course of chromatographic process band are wider and
wider (band broadening)
 Retention data

Retention time: tR

Dead time: tM (t0)

Reduced retention time:

tR’ = t R - t M

Retention volume: VR  tR F
where: F, volumetric flow rate (cm3/min)

Reduced retention volume: VR  t R F  tR  tM  F  VR  VM

 The average linear rate of solute migration,  (usually cm/s)

L
 
tR
where L is the length of the column, tR retention time

The average linear velocity of the mobile phase molecules, u

L
u
tR
The relationship between migration rate and distribution constant

The rate as a fraction of the velocity of mobile phase:

  u  fraction of time of solute spend in mobile phase

This fraction equals the average numbers of moles of solute

in the mobile phase at any instant divided by the total
number of moles of solute in the column:
moles of solute in mobile phase
 u
total moles of solutes

The total number of moles of solute in the mobile phase is :

nM = CM x VM
in stationary phase:
nS = CS x VS
Therefore:

C M VM 1
 u u
C M VM  C S VS 1  C S VS / C M VM

Since distribution constant:

a A S CS
KC  
a A M CM

therefore:

1
  u
1  K C VS / VM
 The retention factor: k’
•Time spended of analyte in the stationary phase relating to
the mobile phase

k’: relative number of

moles of analytes in
the stationary and
mobile phase

k’ = nS/nM
Other definition of retention factor for analyte A:

Vs
k A  K A where: KA is the distribution constant for analyte A
VM

Substitution to equation earliers:

1 L L 1
 u   
1 KA tR tM 1  k A
Rearranging:

tR  tM
kA 
tM
Selectivity factor:

k B t R 2
 
k A t R 1

Always greater than 1.0

 Column efficiency and band broadening
The plate theory of chromatography
One theoretical plate (N): the part of the column, where quasi-
equilibrium takes place between stationary and mobile phase

2
tR L2
N 2  2
σt σL
Where:  standard deviation and 2
Variance
w=4
 tR   tR 
2 2

N  16    5,54  
w   w 1/ 2 
Gauss equation:

HETP: Height equivivalent to the theoretical plate (H)

L
H
N
 The rate theory of chromatography (van Deemter)

Porous silica particle

particle size (diameter): dP

Theory of band broadening

1. Eddy diffusion term (A)
multiple path effects

C ed p  A
2. The longitudal diffusion term (B/u)

C d DM B

u u
3. Mobile phase mass transfer term (CM/u)

2
C Md p u
 CM u
DM

4. Stationary phase mass transfer term (CS/u)

C S Md p 2 u
 CS u
DM
 The van Deemter equation of chromatography

B
H  A  C M u  CS u
u
H

u
The equation has an optimum (Hopt) where the column efficiency is highest.
This optimum has been found at a linear velocity:
for gas chromatography at. 0.1 – 0.5 cm/s
for liquid chromatography at: 1.0 – 5.0 cm/s
At high linear velocities equation can be estimated as:

B
H  A  CS u
u
 Resolution

t R 2  t R1
Rs 
1
( w1  w2 )
2

Resolution expressed with the terms of plate number,

selectivity and retention factors
1 α 1 k 2'
RS  N2
4 α 1  k 2'
Methods to increase resolution

1 α 1 k 2'
RS  N2
4 α 1  k 2'
 Effect of increase of retention factor on resolution

1 α 1 k 2'
RS  N2
4 α 1  k 2'

How to increase retention factor:

•By decreasing eluent strength
Effect of increase of separation factor on resolution

1 α 1 k 2'
RS  N2
4 α 1  k 2'

How to increase separation factor:

•By change chemical quality of the mobile phase
•By change quality of column
Effect of increase of plate number on resolution

1 α 1 k 2'
RS  N2
4 α 1  k 2'

How to increase theoretical plate number:

•Decrease of the flow rate (u)
•Increase of the column length (L)