‫کروماتوگرافی‬

:‫مرا جع‬
1-Principles of Instrumental Analysis
D.A.Skoog

2- Instrumental Analysis C.D.Chiristian and

J.G.Oeilly

3- Physical and chemical methods of separation

Berg

)‫ واحد درسی( کارشناسی ارشد‬2

‫ دکتر نژاد علی‬: ‫مدرس‬

 Introduction
 Plant Pigments
 Chlorophylls

 Xanthophylls

 Martin & synge(1952 Nobel Prize)

 A general description
chromatography
 Stationaryphase
 Mobile phase
 Classification of chromatography
 Column chromatography
 Planer chromatography
 Linear chromatography
 Partition coefficient
 K = Cs/Cm
 Cs , is molar analytical

 Cm , solute concentration in the

mobile phase
 Elution chromatography on
columns
 Elution is defined as a process
whereby a solute is washed through
a column by additions of fresh
solvent.
 Retention time (tR)

 Qualitative & Quantitative

Chromatography
 Chromatograms
 Ifa detector that responds to solute
concentration is placed at the end of
the column & its signal is plotted as
function of time (or of volume of the
added mobile phase), a series of
peaks is obtained, such a plot ,
called a chromatogram.
Theories of elution chromatography
 The effects of migration rates & zone
broadening on resolution
 Plate theory

 Kinetic theory

 L=NH
 N=L/H
 Retention time
 V=L/tR

 U=L/tM

 Where , V , u & tM are average Linear

rate of solute migration , average
Linear rate of movement of the
molecules of the mobile phase &
dead time
 The definition of plate height
 H=2/L
 N=L2/2
 Where H, is the Plate height , L , is
the column length & 2 is the
variance of measurements
Experimental Evaluation of N & H
 Definition of N

 W=4C
  =  / (L/to)
  = LW/4tR
 H = LW2/16(tR)2
 N = 16 (tR/W)2
 N = 5.54 (tR/W1/2)
 Band broadening
 The parameters that effect to the
band broadening are as follow:
 1. Eddy diffusion

 2. Longitudinal diffusion

 3. Mass transfer

 H =A + ( B/U) + CU
 Eddy diffusion
 Zone broadening in the mobile
phase arises in part from the
multitude of path ways by which a
molecule ( or ion) can find its ways
through a packed column.
 Longitudinal diffusion (B/U)
 Longitudinal diffusions results from
the tendency of molecule to migrate
from the concentrated center of a
band toward more dilute regions on
either side.
 Mass transfer
 Mass transfer to and from the
stationary phase (Cusp)
 Effect of column

 Mass transfer in the mobile phase (

CMU)
 Effect of fluent
 Relationship between retention
time and partition coefficient
A solute migrates only when it is in
the mobile phase
 V = U ( fraction of time the solute
spends in the mobile phase)

V =U x no. moles solute in mobile

 total no. moles solute
 V&U
V = U( CMVM)/(CMVM + CSVS) =
U(1/(1 + (CSVS/CMVM))

V = U ( 1/(1 + (Kvs/VM)))
 The capacity factor , K`
 K’, is related to the migration rate of solute

 K` = KVS/VM

 V = U ( 1/(1+K`))

 L/tR = L/tm (1/(1+K`))

 K` = (tR-tM)/tM = t’R/tM


 The selectivity factor, α
 The selectivity factor is the ability of
column to resolve two or more
solutes in a column
 α = KB/KA
 Where KB is the partition coefficient
for the more strongly retained
solute, B
 Or α =K’B/K’A
 Or α =((tR)B – tM)/((tR)A – tm)
 Column Resolution
 

 RS = Z/(WA/2)+ (WB/2) = 2Z/(wA +

wB) = 2[(tR)B – (tR)A ]/(wA + wB)

 Resolution more than 1.0 is good.

 Relationship between resolution
and column properties
 If wA  wB  w then :

 Rs = ((tR)B – (tR)A )/w

 Rs = ((tR)B – (tR)A )/(tR)B x (N)1/2/4

 Rs = (K’B – K’A )/(1+ K’B) x (N)1/2/4

 Rs = (N)1/2/4 ((α -1 )/α ) (K’B/(1+ K’B) )

 Relationship between , N, Rs, K`
and α
N = 16 R2s(α /(α -1))2 ((1 +K’B)/K’B)

 Rs = (N)1/2/4(α -1)(K’B/(1+K’B))

 Or N = 16 R2s(1/(α -1))2 x
((1+K’B)/K’B)
 Relationship between , Rs and
elution time
 VB = L/(tR)B

 (tR)B = (NH(1+K’B))/U

 (tR)B
= (16R2sH)/U x (α /(α -1)2 x
((1+K’B)3) / (K’B)2
 Optimization of column
performance
 Variation in α
 Variation in U
 Variation in K’
 Variation in Rs
 Variation in N
 Variation in H
 Variation in K`

 We had Rs and (tR)B so:

 Rs = (QK’B)/(1+K’B)

 And:

 (tR)B = Q ((1+K’B)3)/(K’B)2
 Optimization of the α
 1) Changing the composition of the
mobile phase
 2) Changing the pH of the mobile
phase
 3) Changing the column temperature

 4) changing the composition of the

stationary phase
 5) Using special chemical effects
The different K` in practice
 Quantitative Analysis
 Analysis based on :
 1) Peak height
 2) Peak areas & Normalization
 3) Calibration standards
 4) Peak Integration
 5) the Internal standard method
 Qualitative analysis
 X- Retention time or Retention
volume
 XX- Relative Retention time

 XXX- Retention Index or Kovats

Retention Index
Define each of the following terms:
 A- stationary phase
 B- mobile phase

 C- eluent

 D- solid support

 E- retention factor
 Calculatethe resolution of two
peaks, t1 =131sec & t2 =137sec, if
the average peak width is 8.0sec.
Would the resolution of these two
peaks be considered good ?
Gas Chromatography
 Instrumentation of GC
 Carriergas supply
 Sample injection system

 Separation column

 Oven

 Detector

 Recorder
 Sample injection system
 Overload

 Dilute samples

 Tenax-GC
 Types of Columns in GC
 Packed columns
 Capillary columns
 Packed columns
 Column dimensions:
 -Stainless steal , copper or aluminum

 -Length: 2 to 3 meters or 15cm

(Coils)
 -Inside diameters 2 to 4mm.
 Packed columns:
 -types of supports
 -specific surface area: at least 1m2/g

 -made from diatomaceous earth

(chromosorb P, wand G)
 Packed columns
 Particlesize supports
 1) Usual size : 60-80 mesh (250-
170µm)
 80 to 100 mesh ( 170-149 µm)
 Packed columns
 Adsorptionsolid supports
 Problem: present of polar sites

 Hydrolyzed silicate surface

Some craters of liquid phase in GC
 1- low volatility
 Thermal stability

 Chemical inertness

 Solvent characteristics

 These are the reasons why we use

inert gas in GC as carrier gas.
 Open Tubular Capillary Columns
 1- Wall Coated Open Tubular (WCOT)
 2- Support-Coated Open Tubular
(SCOT)
 3- Fused Silica Open Tubular (FSOT)
Characteristics of SCOT and
WCOT
 Column coating

 1- High specific area

 2- chemical inert

 3-diatoma

 4-deatomized soil
 Rohrshneider and polarity of
columns
 1- squalene, polarity=0
 2- A selected compound
polarity=100
 3- The others are between 0-100


X- Some papers about polarity of
compound in column
 Oven and column thermostating

 A- simple ( Isothermal)
thermostating
 B- programmed thempreature GC
 Detectors in GC

 Dozens of detectors (more than 100) have

been investigated and used during the
development of gas chromatography. Only
four , however , have found widespread
use:
 1- Thermal conductivity
 2- Flame ionization
 X- Thermionic
 4- Electron capture


Characteristics of the ideal detector
for GC
 1- adequate sensitivity
 2- good stability and reproducibility
 3- A linear response
 4- A temperature range
 5- A short response time
 6- high reliability
 7- similarity in response towards all
solutes
 8- nondestructive of sample
Thermal Conductivity detectors
(TCD(
 Flame Ionization Detectors (FID)
 1- for most organic compounds
 2- H2 and air or O2 is needed

 3-insensitive towards noncobustible

gases :
 H2O, CO2, SO2, and NOx


 Advantages and disadvantages of
FID and TCD
 1- FID is not sensitive to H2O
 2-TCD is nondistructive
 3- TCD is simple
 4- sensitivity of TCD ( 10-8g/ml) is less
than FID ( 10-13g/ml)
 5-FID is not general detector
 6- FID is not sensitive to rear gases and
N2 and O2
 7- the noises in FID is less than TCD

 ECD
 1- effluent from the column
 2-  emitter ( 63Ni or 3T)

 3- electron of emitter ionized the

carrier gas
 4- some compounds adsorb the
electron
 5- change in the normal current of
the detector
 Applications of ECD
 1- chlorinated insecticides
 2- peroxides
 3- halogens
 4- quinones
 5- nitrogroups

 I- AMINES
 II- ALCOHOLS
 III- HYDROCARBONS

 Reaserch Projects
 X- name some common stationary
phases in GC
 XX- what are Mcreynold constants

 XXX- how can we choice stationary

phase
 IV- what is GSC ( some papers)

 V- what is GLC ( some papers )

 GC/MS
 1- Advantages
 2- Disadvantages
 GC/FT-IR
 1- separating
 2- identifying
 ? Explain why a liquid sample
should be injected rapidly onto a
GC column
? Why is a solid support
soetimes “silanized”
? How temperature programmed GC
is able to give a faster separation
than an isothermal separation?
X- Introduce a method for the
determination of ethanol in
blood
XX- Introduce a method for the
determination of tolune in a
sample
XXX- Introduce a method for the
determination of gasoline in oil
High- Performance Liquid
Chromatography (HPLC)
 Comparison of GC and LC
 1- GC is better for the samples with
high volatilities
 2- In the GC the mobile phase is a
gas , but in the LC is liquid
 3- the permability of liquids is 105
times lower than gases
 4-the viscosity of liquids is 102 times
grater than gases
 Limiting number of theoretical
plates

 (Nlim)LC/(Nlim)GC =(GDG)/(LDL)=103

 ( G)/( L)= 102

 (DG)/(DL) = 105
 HPLC
 1- Introduction
 2- column chromatography

 3- planer chromatography
 Column liquid chromatography

 1- partion chromatography
 2- adsorption or liquid solid
chromatography
 3- Ion exchange chromatography

 4- Exclusion or gel chromatography

 Column efficiency in LC
 1- effect of particle size of packings
 2- extra column band broadening in
LC
 3- effect of sample size
 Istruments for HPLC

 1- solvent reservoirs
 2- pump
 3- injection valve
 4- column
 5- detector
 6- recorder
 Mobile phase and solvent
reservoirs
 1- Isocratic elution
 2- gradient elution
 Pumping systems
 The requierments for pums
 1- the generation of pressure of up
to 6000psi
 2- pulse free output
 3- flow rates from 0.1 – 10ml/min
 4- flow control and flow
reproducibility of 0.5% relative or
better
 5- corrosion resistant
 Some types of pumps

 1- Reciprocating
 2- Syringe or displacement

 3- Pneumatic or constant pressure

 Reciprocating pumps
 1- It is not pulse free
 2- low sample internal volume ( 35-
400ul)
 3- gradient elution

 4- high pressure ( up to 10000psi)

 Displasment pumps
 1- sample internal volume ( 250ml)
 2- pulse free

 3- syringe like chamber

 4- It dos not depend to viscosity of

solvent
 5- gradient elution
 Pneumatic pumps

 1- pulse free
 2- maximum pressure 2000psi (
disadvantage )
 3- isocratic elution
 Sample injection systems
 1- syringe injections
 2- stop flow injection

 3- sampling valves
 Analytical columns
 1- cost : 200 – 1000$
 2- inside diameter, 4 -10 mm
 3- length, 25cm
 4- N, 40000 – 60000 plates/meter
 5- particle size of packing, 5 – 10um
 Guard column
 1- removing particulate matter and
contaminates
 2- increasing the life of the analytical
column
 3- saturation the mobile phase with
the stationary phase
 4- large particle size ( than s. phase
)

X- Post column
 Detectors
 Some characteristics :
 1- sensitivity
 2- linear response
 3- free from flow rate sensitivity
 4- nondistructive of the sample
 The types of detectors

 1- general detectors
 MS
 Refractive index
 2- specific detectors
 IR
 UV-Visible
 Polarography
 Florometry
 Electrochemical detectors
 1- high sensitivity
 2- simplicity

 3- convenience

 4- applicability
X- HPLC - MS
 Mobile phases
 1- high purity
 2- ready availability
 3- A boiling point that is 20 to 50Co above
the column temperature
 4- low viscosity
 5- low reactivity
 6- immiscibility with the s. phase
 7- compatibility with the detector
 8- limited flammability and toxicity
 Types of mobile phase
 Normal phase ( polar s. phase )
 Reversed phase ( polar M. phase )
 Partition Chromatography
 Some parameters that effect on selectivity
factor and K’:
 1- solvent – solute interaction in mobile
phase
 2- dispersion interactions
 3- dipole interactions
 4- dielectic interaction
 5- molecular complex formation


 Solvent strength and K`
 P’ = log (K’g)e + log(k’g)d +log (k’g)n

 P’AB = Ap’A + Bp’B

 K’2/K’1 =10(p’1- p’2 )/2

 K’2/K’1 = 10(p’2 – P’1)/2


 Classification of solvents
 X- Snyder classification of solvents
 Packing for partition
chromatography
 Siloxane packing
 Ester formation

 Silicon/carbon

 Silicon/nitrogen bond
X- Ion pair chromatography
 LSC
 Silica - alumina s. phase

 E0 of the solvent
Ion- Exchange chromatography
Size exchange chromatography
 Vt = vg + vi + v0
 Ve = v0 + kvi
 Application of size -E-
chromatography

 Gel filtration C. ( polar solvent )

 Gelpermeation C. ( non-polar
solvent)
 Comparison, advantages,
disadvantages
 1- short time for separation
 2- non-distructive

 3- sharp peaks

 4- high sensitivity
Supercritical Fluid Chromatography (
SFC)
 Operating variables and
instrumentation
 1- effect of pressure
 2- stationary phases ( packed and
capillary)
 3- mobile phase ( in most case is
CO2)
 4- detectors ( MS, UV, R.F., FID
and…)
Planer Chromatography
 Planer chromatography

 1- paper chromatography (PC)

 2- Thin Layer Chromatography ( TLC)

 TLC
 1- glass plate
 2- s. phase

 3- m. phase

 4. origin line

 5. solvent front
Performance characteristics of TLC

 1- the retardation factor

 2- H and N

 3- the capacity factor, K’ and R

 Chromatogram development

 1- ascending flow

 2-horizontal flow

 3- one dimensional TLC

 4- two dimensional TLC

 Qualitative and Quantitative
analysis in TLC
 RF
 travel distance of analyte
 Rx =
 travel distance of standard substancea

 X- quantitative ?
 Electrophoresis and
Electrochromatography

 1-electrophoresis is defined as the

migration of particles through a
solution under the influence of an
electric filed.

 X- electrochromatography