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
) NH4+
(C6H5
NO
Cupferron: ionic
(C6H5
)3 Fe
NO
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
phase
solid
liqiud
Gas chromatography
GC
gas
GSC
GLC
Supercritical chromatography
Supercritical
fluid
SFC
SFC
TLC
IC
GPC,SEC
PC
Normal
phase
(HPLC-NP)
Reversed
phase
(HPLC-RP)
SFC
Liquid chromatography
LC
Liquid chromatography
LC
liquid
liquid
CE
GEL ELFO
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: V  t F
R
R
where: F, volumetric flow rate (cm3/min)
Reduced retention volume: V R   t R  F  t R  t M  F  V R  V M
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
u
L
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:
 u
moles of solute in mobile phase
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:
 u
C M VM
C M VM  C S VS
u
Since distribution constant:
KC
a A  S

a A  M

CS
CM
therefore:
  u
1
1  K C VS /VM
1
1  C S VS / C M 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:
k A  K
Vs
A
where: KA is the distribution constant for analyte A
VM
Substitution to equation earliers:
 u
1
1 KA
Rearranging:
kA 
tR  tM
tM
 
L
tR

L
tM

1
1  kA
Selectivity factor:

kB
kA

t R  2
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 quasiequilibrium takes place between stationary and mobile phase
N 
2
tR
σt
2

L
2
σL
2
Where:  standard deviation and 2
Variance
tR 
  5 , 54
w 
2
N  16 
tR

w


1/ 2 
2
w=4
Gauss equation:
HETP: Height equivivalent to the theoretical plate (H)
H 
L
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)
CdDM
u

B
u
3. Mobile phase mass transfer term (CM/u)
CMd
2
p
u
 CM u
DM
4. Stationary phase mass transfer term (CS/u)
CSMd
DM
2
p
u
 CS u
The van Deemter equation of chromatography
H  A
B
u
 CM u  CSu
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:
H  A
B
u
 CSu
Resolution
Rs 
t R 2  t R1
1
2
( w1  w 2 )
Resolution expressed with the terms of plate number,
selectivity and retention factors
RS 
1
4
N2
α 1
k 2'
α
1  k 2'
Methods to increase resolution
RS 
1
4
N2
α 1
k 2'
α
1  k 2'
Effect of increase of retention factor on resolution
RS 
1
4
How to increase retention factor:
•By decreasing eluent strength
N2
α 1
k 2'
α
1  k 2'
Effect of increase of separation factor on resolution
RS 
1
4
N2
α 1
k 2'
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
RS 
1
4
N2
α 1
k 2'
α
1  k 2'
How to increase theoretical plate number:
•Decrease of the flow rate (u)
•Increase of the column length (L)