Separation Science
Lecturer: Dr. S. D. M. Chinthaka
Venue : C 1 lecture theater
Time
: Saturday 9.00 – 12.00 am
References
: Skoog and Leary: Principals of instrumental
analysis, 6t dtion
C.F. Poole, Essence of chromatography,
2nd edtion
Separation Science
Chromatography
Most widely used analytical separation method
Applications are grown to every branches of science
First introduced by Russian botanist M.S. Tswatt (1903)
Not an established method until 1930
Before 1930, Chemical separations were limited to crystallization, liquidliquid partitioning, and distillation
Chromatography is essentially a physical method of separation in which the
components to be separated are distributed between two phases
One of these is stationary : (stationary phase)
The other is moving in a definite direction (Mobile phase)
Separation Science
Important considerations
 Physical method of separation
 Two distinct phases are involved
 Separation results from differences in the distribution constants of
the individual sample components between two phases
Separations depend on a favorable contributions from
thermodynamic and kinetic properties of the compounds to be
separated
Family Tree of chromatography
Chromatography
Supercritical
fluid
Gas
Solid
(GSC)
Adsorption
(LSC)
Liquid
(GLC)
Size
exclusion
(SEC)
Liquid
Solid
(SFC)
Solid
Ion
exchange
(IEC)
Affinity
(AC)
Liquid
(LLC)
Micells
(MEKC)
Sorption
(RPC)
Sorption
(CEC)
Selective zone migration
 Transportation of solutes zones in column chromatography occurs
entirely in the mobile phase
Elution chromatography
 The mobile phase and stationary phase are normally at
equilibrium
 Sample is applied to the column as a discrete band
Mobile phase must compete with the stationary phase for
the sample components
 The distribution constants for the sample components
resulting the competition must be different
The average rate that species migrate depends on the
fraction of time it spend in mobile phase
 Most convenient method
Zone migration
Chromatogram
 The information obtained from a chromatographic experiments is
contained in the chromatogram
 Information readily extracted from chromatograph
Sample complexity (no of peaks)
Qualitative identification of sample components (peak position)
Quantitative assessment of the relative concentrations
Physical properties of a solute in chromatographic systems
(peak positions and profiles)
Retention
Partition coefficients
Amobile
Astationary
cS
K
cM
Retention time
Detector signal
tR
tM
Time
Relationship between retention time and partition coefficient
Average rate of analyte migration
Average rate of mobile phase migration
 u
1
1  K Vs
VM
L

tR
u
1
1
1

t R t M 1  K Vs
Vs = volume of stationary phase, VM = Volume of mobile
phase
L
tM
VM
The rate of solute migration: capacity/retention factor ( kA’)
k A 
K AVS
VM
tR  tM
k A 
tM
If the capacity factor is
 less than unity, elution is so rapid and determination of retention
time is difficult
 When the capacity factor Is larger, elution time becomes longer
 Separations are performed under conditions in which capacity
factor for a given analyte in the mixture lie in the range 1 to 5,10
Selectivity factor ()
For species A and B
KB

KA
k B

k A
t R B  t M

t R A  t M
Plate theory column efficiency
 Chromatographic peaks are generally Gaussian in shape
 During migration analyte particles undergo thousands of transfers
between the stationary phase and mobile phase
 Sometimes residence time is transitory or longer period
 Particles are eluted during residence in the mobile phase
Methods describing column efficiency- Plate theory
Plate height (H) and number of theoretical plates (N)
 The smaller the plate height, the greater the column efficiency
 The higher the number of theoretical plates, the greater the column
efficiency
2
LW
H
16t R2
 tR 
N  16 
W 
2
 t 
N  5.54 R 
 W1 
 2
2
van Deemter plot
A
Column resolution
Provides a quantitative measure of its ability to separate two analytes
2t R B  t R A 
Rs 
W A  WB
Column resolution
4% overlap
0.3% overlap
The resolution of given stationary phase can be improved by
lengthening the column : increasing number of plates
Increase the time required for separation
Effect of capacity factor on resolution and elution time
General elution problem
Solution for the general elution problem
 Change of the conditions that determine the values of k’ as
separations proceeds
 For LC: variations in the composition of mobile phase
(gradient elution)
 For GC: Temperature programming
General elution problem
General elution problem
The function of GC
To provide
conditions required by column for achieving a
separation without adversely effecting its performance
 Operation of the column requires
 A regulated flow of carrier gas
 An Inlet system to vaporize and mix the sample with the
carrier gas
 A Thermostated oven to optimize the temperature for
separation
 An online detector to monitor the separation
 Associated electronic components to control and to monitor
instrument conditions and to record, manipulate, and format the
chromatographic data
Instrumentation
Gas chromatography
Pneumatic system of GC
Sample inlet system
Sample inlet
Vaporize the sample and mixed with carrier gas prior to the start
of the separation
This is achieved
Without reduction of the separation potential of the column
In the absence of thermal degradation, adsorption or rearrangement
of sample components
Without discriminating of sample components by boiling point,
polarity, or molecular weight
Changes in column operating conditions should not affect the
sampling processes
Types of injectors used in GC
 Flash vaporization injectors
 Hot split/splitless injectors
 On column injections
Flash vaporization injectors
 Injector must have sufficient thermal mass
 Carrier gas must be pre heated to avoid
condensation
 Temperature range 25- 400 °C
 Generally sets 50 °C higher than maximum
column temperature
 Because of high temperature, septum bleed must
be avoided
Use of low bleed septa
Use of septum purge devices
Flash vaporization injector
Hot split/splitless injector
Hot split/splitless injectors
 Allows injection of mixtures virtually independent of the choice of
solvent
 Reduce band broadening
 Sample is isothermally evaporated and mixed with carrier gas
 Divide the sample into two streams of different flow
 Chamber is constructed from stainless steal tube lined with
removable glass liner
 Injection volume is 0.2 to 2 μL with split ration 1:10 to 1: 1000
 Split ratio depends on
Sample volatility range, solvent, injected volume,
injector temperature
Splitless injection
 More suitable for quantitative analyses of trace
compounds in dirty sample
 Volume of the chamber must be large enough to hold
the entire sample
 Cold trapping is needed to refocus the sample
 Method allows the analysis of dilute samples without
preconcentration
 Reduces solute discrimination
Problems with hot vaporizing injectors
 Contamination by nonvolatile residues
 Nonvolatile droplets slowly release certain solute components to clean
samples
 Degradation of sample components
Detectors in GC
Ideal detector
 Adequate sensitivity
 10-8 10-15 g analyte/s
 Good stability and reproducibility
 Wide range of linear response (dynamic range)
 Temperature range RT- 400 °C
 Short response time that is independent of flow rate
 High reliability and easy use
 Universal response
 Nondestructive
Detectors in GC
 Online detection of organic vapors
 Ionization, bulk physical property, optical and electrochemical
detectors
Based on the detector response
 Universal (flame ionization, thermal conductivity)
 Element selective (flame photometric, thermoionic ionization, and
atomic emission detectors
 Structure selective (Electron capture, photoionization)
Ionization detectors
 Flame ionization detector (FID)
 Thermoionic ionization detector (TID)
 Photoionization detector (PID)
 Electron capture detector (ECD)
 Helium ionization detector (HID)
Each detector employs different method of ion production
Detector operation is based on the fluctuation of an ion
current in the presence of organic vapors
Flame Ionization detector
 Background current 10-14A
 Increase to 10-12-10-5 A
 No response to He, Xe, H2 N2, N2O, NO, CO2, CS2, COS, NH3 , SO2 , and
H2O and formic acid
Flame Ionization detectors
 Produces nearly universal response to organic compounds
 Minimum sample detection limit is 10-13 g carbon/s: very low
detection limit
 Long-term stability
 Simplicity of operation
 Fast signal response
 Exceptional liner response range: dynamic range is 106-107
Electron capture detector
Electron capture detector
 The second most widely used ionization detector
 Good for the compounds with high electron affinity
(pesticides, industrial chemicals, assessment of ozone
depleting chemicals)
 Determination of drugs and hormones in bio fluids
63N
 β + M  e + M+ (plasma)
E + e  EE- + M+  EM
LC instrumentation
LC instrumentation
 Favorable kinetic properties yield higher efficiency and
shorter separation time in GC
 LC operates with modest number of theoretical plates at
optimized selectivity achieved by appropriate selection of
separation mode , stationary phase , mobile phase composition
LSC : interfacial adsorption
SEC : restricted permeability of porous solids
LLC/BPC : Partition
IEC : Electrostatic interactions
AC : Structure specific binding
Instrumental aspect of LC
Operation conditions
 Must be capable of operating at very high pressure
 Accurate preparation of mobile phase composition
 Requires stable mobile phase flow
 Online detection with small operation volume
 Requires desired mobile phase composition to the head of the
column as continuous and pulse free stream at known pressure
and volumetric flow rates
 Sample injection requires insertion of known volume of sample
into fully pressurized mobile phase flow
Solvent reservoir
 Container resistant to chemical attack by mobile phase
 2 μm filter is required for particle separation
 Degassing is required to prevent gas bubble formation when
two solvents are mixed
 O2 must be removed to increase sensitivity and increase
baseline separation
Applying vacuum
Ultrasonic treatments
Helium spraying
LC pumps
Constant pressure pumps : pneumatic amplifier pumps
Constant volume pumps : Syringe pumps
Reciprocating piston pumps
Syringe pump

Pulse free output

Very high pressure can be obtained

Gradient and flow programming are possible

Finite time is required for achieving constant volumetric flow rate

Limited solvent reservoir capacity
Pumps
Reciprocating piston pumps
 Displaces a small volume (10- 400 μL)
 Made with material that can withstand high pressure and resistant to
chemical attack
 Suitable for use at 5000 p.s.i
 Output flow is unstable
Uses single piston with asymmetric cam
Dual head reciprocating pump
Composition gradients
 Achieved by mixing two or more solvents either
incrementally or continuously
 Low pressure mixing
 Little influence by
compressibility effects
 Eliminates thermodynamic
volume change error
 Air bubble formation is
avoided
High pressure mixing
 Solvent compressibility and
thermodynamic volume change
influence the composition
 Needs separate pumps for each
solvents
LC Sample inlet system
LC Sample inlet system
 Introduction of sample into highly pressurized mobile phase as
a sharp plug
 Use external sample loop method
 Materials must be inert and non sorptive to avoid memory effect
 Leak tight to 7-10,000 p.s.i at RT
 Sample loop size is 5 μl- 5 mL
 Loop must filled completely/partially
 Sample must be prepared in same solvent as mobile phase or in
weaker solvent but miscible with mobile phase
Sample inlet system
Sample inlet system
Sample inlet system
Detectors in LC
 No universal detectors for LC
 Should have minimal internal volume
Two types of detectors are available

Bulk property detectors: response to mobile phase bulk property
e.g., refractive index, dielectric constant, density
 Solute property detectors: response to some property of solute
e.g., UV absorbance, fluorescence, diffusion current
Z shaped flow cell
1-10 μL volume
2- 10 mm in length
Minimizes stagnant flow
regions
Reduce peak tailing
Low volume fiber optic cell
Diode array detectors
Fluorescence detectors
Columns in Chromatography
Selection of mobile phases
 Non solvating gases are ideal for GC
H2, He, N2
 Behave almost ideally at low pressures and typically high GC
temperatures
 Do not influence the selectivity
 No competition with solute molecules for stationary phase
 Flow rate influence the diffusion rate of the solute
 Cost, purity, safety, reactivity, and detector compatibility must
be considered
Choice of carrier gas vs plate height
Columns in Chromatography
Types of columns in GC
Classical packed columns ( 2 mm id, 100-200m particles)
Micropacked columns ( 1 mm id, 100-200m particles)
Support coated open tubular columns (SCOT)
Porous Layer open tubular columns (PLOT)
Wall-Coated open tabular columns (WCOT)
Stationary phases in GC
 Liquid must be unreactive
 Low vapor pressure
 Good coating characteristics
 Reasonable solubility in common organic solvents
 Must have wide temperature operating range (-60 - 400°C)
Hydrocarbon, ether and ester stationary phases
T/ °C
Hexadecane
20- 50
Squlane
20-120
PPE-5 (polyphenyl ether)
20-200
EGS (polyethelene glycol succinate)
100-220
Carbowax 20M (poltethelene glycol)
60-225
Tetrabutylammonium tetrafluoroborate
162- 290
Poly siloxane stationary phases
Most widely used in stationary phases in GC
Wide temperature operating ranges
Low vapor pressure
Low temperature glass transition point
Chemical inertness
Good film forming property
Easy of synthesis with wide range of chromatographic selectivity
Easy of immobilization
Some common polysiloxane stationary phases
T/C°
Dimethyl siloxane (OV-1)
100-350
Phenylmethyldimethylsoloxane (OV-7)
20-350
Tryfluoropropylmethylsiloxane (OV-210)
20-275
Cyanopropylphenylsiloxane (Silar 7CP)
50-250
Columns in LC
 Inorganic oxides and porous polymers with various surface functional
groups used in early LC
 Chemically bonded phases almost completely replaces all the other
materials
 Development of hydrophobic surfaces
 Allows use of polar mobile phase (water) : reverse phase liquid
chromatography (RPC)
 RPC is the most widely used separation technique in LC because of life
science applications
 Provides the separation of neutral, polar, and ionic samples of wide
molecular weight ranges
Column packing materials
 Porous inorganic materials
 Porous polymer materials 30-200 μm
 Porous/nonporous silica microparticals 0.1-10 μm
Silica: granular and irregular
Alumina, Titania and Zirconia
Mechanically strong and hydrolytically stable
Surface chemistry is more complex
Chemically bonded inorganic oxides
Organosiloxane-bonded phases
Porous silica substrate modified by reaction with an organosilane reagents
Monomeric synthesis
Solution polymerization
Deactivation of silanol groups