Transcript PPT File

Instrumental Analysis (I) PHCM561
1. Define chromatography and its basic principles
2. Describe different development techniques of TLC
(Thin-layer chromatography) – e.g. twodimensional TLC
3. Explain how TLC can be used for quantitation of
analytes
4. Define chiral chromatographic separation systems
5. Estimate eluent strength of mobile phases and
retention of analytes on different stationary
phases
•3
Students in groups of 4-5 individuals are asked to prepare a presentation
(weight=5% of the theoretical course assessment) during the session of the last
tutorial of chromatography due from 25 to 28 November. Each group will be
asked to give a 10 min talk about the presentation and has to be prepared for
students’ and instructor’s questions and discussions. Each group will have a topic
according to the coming slide. The assessment criteria are:
Criterion
Grades (out of 10)
Choice of the topic
2
Content of the presentation
2
Comprehension of the content by members of the group
2
Quality of the presentation
2
Ability to answer students’ and instructor’s questions
2
» Some slides have been added to tutorial 5
showing structures of functional groups as
reminder. Kindly check them.
Gases (He, N2) are used in gas chromatography (called “carrier gas”), liquids are
used in any other chromatographic technique (TLC, HPLC,…)
 Pure solvents (Analytical grade):
• Generally all solvents can be used for chromatography, but they must be
immiscible with ST phase used to avoid dissolving or damaging it.
• Any chemical reactions with analytes must be avoided to keep the
integrity of the chemical nature of the sample.
• Can consist of 2,3… or more pure solvents mixed together, where
buffers or other salt solutions can be added for certain chromatographic
purposes.
•5
Isocratic elution
Gradient elution
The composition of the mobile The composition of the mobile
phase is constant during
phase changes during
chromatographic experiment
chromatographic experiment
•© Dr. Rasha Hanafi, GUC
•Lecture 8 – Chromatography, 20-11-2011
•Elution? Molecules adhering the stationary phase are washed out by the mobile
phase.
•An eluotropic series ranks solvents by their relative abilities to displace solutes from a
given adsorbent. The eluent strength 0 of a solvent is a measure of adsorption energy
per unit area of solvent.
Solvent
Polarity
Elution power on
normal phase NP
n-hexane
benzene
dichloromethane
diethyl ether
ethyl acetate
•6
acetone
ethanol
•© Dr. Rasha Hanafi, GUC
•Lecture 8 – Chromatography, 20-11-2011
Elution power on
reversed phase RP
#1
Which of the following items belongs to each chromatographic
system?
1. TLC plate
2. stationary phase
3. separation column
4. pH meter
5. light source
6. mobile phase
7. cellular phase
8. mass spectrometer
9. developing chamber
10. detector
#2
State whether right or wrong, correct if wrong:
1. Chromatography can only be used for identification of analytes, not for
quantification.
FALSE. Chromatography permits both quantification and identification of
substances.
2. Prior to any analytical investigation of analytes, separation occurs in every
chromatographic method.
TRUE.
3. Components of a mixture can only be separated by chromatography if they
have differing molecular weights.
4. Chromatography permits separation of molecules according to their chemical
functionality only: this means that it is possible to separate e.g. alcohols from
amines but impossible to separate e.g. octanol from heptanol using
chromatography.
3. & 4. FALSE. Generally all mixtures can be separated by chromatographic
methods. BUT the more different the components of the mixture are the
easier they can be separated.
#3
There are different kinds of interaction between the three
analyte – stationary phase – mobile phase that altogether
lead to chromatographic separation:
1. What are the two most important principles of separation in
chromatography?
1. Adsorption, which occurs on solution/solid interface
2. Partition, which occurs between two liquids or liquid-like phases
2. State three other principles.
ionic interaction (ion-exchange, ion-pair or ion chromatography), size
exclusion, biochemical affinity,
3. Describe how chromatography (chromatographic separation) works in
not more than one sentence.
In chromatography, different substances are moving with different speed
due to their different affinities to the stationary and the mobile phase.
Identification of analytes by comparison of their travel distances to
the travel distances of standards (reference substances).
RF value:
"ratio of fronts"
RX value:
Start to
center of
reference
spot
dsubstance
d start-spot
d
substance
hR
RFF 
*100
ddsolvent
d start-end
solvent
front
front
d substance1 d start-spot1
RX 

d substance2 d start-spot 2
Equilibria Occuring in a TLC Jar
(2)
•(1) Saturation of atmosphere
•(2) Sorptive saturation
•(3) Saturation by capillary action.
•(4) Vapor - liquid exchange
(4)
(3)
(1)
•© Dr. Rasha Hanafi, GUC
•solvent front
 most common, usual type:
• vertical, bottom to top, single development
 other techniques:
• vertical, bottom to top, multiple development
(mobile phase either changed or kept the same)
 if analytes do not move very well or equilibration (sorptiondesorption resp. partition) is restricted
• vertical, top to bottom
 to improve separation if Rf values of analytes are close (not
only capillary but also gravity action)
or horizontal (like HPTLC - for similar reasons)
• circular (wick is required for application of mobile phase)
 TLC plate rotates quickly, centrifugal force drives mobile phase
best possible reproducibility between different TLC experiments:
not more than two significant figures ! !
Rf(A) = 0.811 / 0.814 / 0.806
%sdv = 0.54%
 Rf (and RX) influenced by:
•thickness of stationary phase
 capacity of stat. phase
Rf(B) = 0.111 / 0.114 / 0.106
%sdv = 3.7%
Due to the fact that all those variables are
difficult to keep constant, a reference
compound is usually applied to the plate as
well.
•moisture content of mobile and stationary phase
 polarity, capacity of the phases
•temperature
 partition coefficient, adsorption
•degree of saturation of the developing chamber with mobile
phase vapor
•sample size
#1
If you compare methylene chloride and ethanol – which is the
stronger eluent in reversed phase chromatography? Justify.
Methylene chloride is the stronger eluent in reversed phase
chromatography."Elution" is the process where analytes are washed out
from the stat. phase, for this process we need a strong competitor with
stat. phase particles with respect to the analyte's affinity. Since in RP
chromatography the stat. phase is non-polar, the eluent strength increases
with decreasing polarity of solvents.
#2
Give 2 derivatization reactions for silanol that yield C18 RP
stationary phase.
1.
with R = (CH2)17-CH3
2.
#3
A mixture of stearic acid, palmitic acid and lauric acid is separated
using a suitable normal phase chromatographic method. Arrange
the three analytes according to the order of their elution. Justify.
stearic=C18, palmitic=C16, lauric=C12 - all are fully saturated.
normal phase: polar stationary phase = the more polar an analyte, the more it
is retained by the stat. phase = the less polar an analyte, the faster it is
eluted. polarity decreases as lipophilicity increases, consecutively the most
lipophilic of the three elutes first: stearic, second: palmitic, third (last):
lauric.
#4
Comment on the statement:
Due to its versatility, silica (silanol, SiO2) is the most used stationary
phase in chromatography.
DEFINITELY YES, versatility means:
1-chemical modification of native silanol is easily possible (make RP, different
NP or even ion-exchanger stationary phases from native silica)
2-huge variety of specifications of silica particles (shape, porosity, particle
size, pore size…)
 Spot size (diameter) correlates with amount of analyte –
 how accurate can this be measured ???
or better: 
but how can this be measured accurately ????
 quantification often is carried out in a semi-quantitave manner
only:
comparison of the analytes' spot sizes to reference spots
(from known amounts of either analyte or reference substances)
more accurate:
"scraping and dissolution technique"
•spots are scraped from the TLC sheet, analytes are separated
from stationary phase material (by extraction  solution is
obtained), and the quantities determined with any suitable
method (e.g. photometry, fluorometry…)
(Densitometer)
measuring remission
light source
detector / receiver
measuring fluorescence
light source
monochromator
detector / receiver
monochromators
TLC sheet
TLC sheet
"remission"
comprises both
absorption and
reflection
distance to origin / Rf value
emission
(fluorescence)
reduction of
remission
Resulting chromatograms:
distance to origin / Rf value
Two enantiomers of any
chiral compound exhibit all
the same physicochemical properties - so
they can only be
distinguished (thus
separated) by different
interaction with other chiral
molecules, generally by their
"differing behaviour in chiral
environment".
Approaches to chromatographic separation of
enantiomers:
1) upon reaction with any other suitable chiral compound
diastereomers are formed that can be separated due to differing
physico-chemical properties
2) a chiral stationary phase is chosen
3) a chiral reagent is added to the mobile phase
"chiral
environment"
 -cylclodextrin with a 0.78 nmdiameter opening into a chiral,
hydrophobic cavity,
it can be bonded to stationary phases for
separation of enantiomers
(the hydroxyls can be capped with groups such
as –C5H11 or –C(=O)CF3 to decrease polarity of
the faces)
enantiomers of a chiral analyte have different
affinities for the cavity; thus separation is
feasible
 pure enantiomers of chiral compounds, e.g. amino acids like
L-valine can also be bonded to stationary phases and allow
separation of enantiomers (remember: naturally occurring amino
acids all are pure “L-” enantiomers)
HO
HO
COOH
N
2
hydroxydodecyl)proline
2
2
C10H21
 (2S,4R, 2'RS)-4-hydroxy-1-(2'-
4
4
COOH
N
H
2
HO
OH
chiral analyte
HO
Cu
O
N
HO
O
H
C10H21
this is an example only ! !
H
C10H21
 this chiral selector can be used
for complexing chiral analytes
together with Cu2+
- prior to chromatographic separation a
RP (C18) stationary phase is
impregnated with this selector
(lipophilic interaction of the alkyl
chains); then Cu(II) solution is added
and the metal ion complexed by the
chiral selector
enantiomers of a chiral analyte have
different affinities for this complex;
thusly separation is feasible