TUTORIAL # 6

INTRODUCTION TO
CHROMATOGRAPHY

•1 Instrumental Analysis (I)

PHCM561
 LEARNING OUTCOMES:
1. Define chromatography and its basic principles
2. Describe different development techniques of TLC
(Thin-layer chromatography) – e.g. two-
dimensional 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
•2
 CHROMATOGRAPHY : SCIENCE OF
SEPARATION
The sample mixture is transported in a mobile phase (Liquid, gas, or a
supercritical fluid*). The sample mixture is forced through an immiscible
stationary phase (fixed in a column or on a solid surface) to which it is
adsorbed or “generally attracted”.

Forcing the mobile phase occurs by a variety of mechanisms (capillarity,

gravity, high pressure or others).

mobile phase (=the eluent), while, weakly held components travel (=elute)
rapidly.

Other related terms: elution = development and eluate, what are they?
So, sample components separate into bands or zones that can be analyzed
qualitatively and quantitatively (analytical chromatography: ng or mg and
preparative chromatogrpahy: g or Kg**). •3
 MOBILE PHASES
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.
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 •4
 ELUOTROPIC SERIES
•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 Elution power

on normal on reversed
phase NP phase RP
n-hexane
benzene
dichloromethane
diethyl ether
ethyl acetate
acetone •5
ethanol
QUESTIONS ON BASICS (CONTD.)
#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 •6
10. detector
QUESTIONS ON BASICS (CONTD.)
#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
•7
methods. BUT the more different the components of the mixture are the
easier they can be separated.
QUESTIONS ON BASICS (CONTD.)
#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.
•8
QUESTIONS ON BASICS (CONTD.)
#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…)

#5
Give 2 derivatization reactions for silanol that yield C18 RP
stationary phase.

1.

with R = (CH2)17-CH3
•9
2.
 QUALITATIVE THIN-LAYER
CHROMATOGRAPHY
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

ddsubstance d start-spot d substance1 d start•10

RFF  *100
substance
hR RX  
-spot1

ddsolvent
solvent front
front d start-end d substance2 d start-spot 2
Equilibria Occuring in a TLC Jar

•(1) Saturation of atmosphere

•(2) Sorptive saturation (2)

•(3) Saturation by capillary action.

•(4) Vapor - liquid exchange solvent

(4) front

(3)
(1)

•11
DEPENDENCE OF THE RF (RX) VALUE
best possible reproducibility between different TLC experiments:
not more than two significant figures ! !

Rf(A) = 0.811 / 0.814 / 0.816 Rf(B) = 0.111 / 0.114 / 0.106

 Rf (and RX) influenced by: Due to the fact that all those variables
are difficult to keep constant, a
•thickness of stationary phase reference compound is usually applied
 capacity of stat. phase 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 •12

•sample size
VARYING TLC DEVELOPMENT METHODS
 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

• vertical, top to bottom

 to improve separation if Rf values of analytes are close (due
to 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
•13
 QUANTITATIVE THIN-LAYER
CHROMATOGRAPHY
 Spot size (diameter) correlates with amount of analyte –
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…)
•14
REAL QUANTITATIVE TLC (Densitometer)

measuring remission

"remission" comprises both absorption

and reflection

measuring fluorescence

•15
REAL QUANTITATIVE TLC (Densitometer)

measuring remission measuring fluorescence

light source detector / receiver light source detector / receiver

monochromator monochromators

TLC sheet TLC sheet

Resulting chromatograms:

(fluorescence)
reduction of
remission

emission

•16

distance to origin / Rf value distance to origin / Rf value

 EXERCISES : STATIONARY / MOBILE PHASES
#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
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.
•17
polarity decreases as lipophilicity increases, consecutively the most lipophilic of
the three elutes first: stearic, second: palmitic, third (last): lauric.
CHIRAL CHROMATOGRAPHY
Two enantiomers of any
chiral compound exhibit all
the same physico-
chemical 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
•18
2) a chiral stationary phase is chosen "chiral
environment"
3) a chiral reagent is added to the mobile phase
CHIRAL STATIONARY PHASES – LC, GC
 -cylclodextrin with a 0.78 nm-
diameter 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)

To separate chiral compounds use:

•19
1- Chiral stationary phase , or
2- Chiral Mobile Phase
CHIRAL STATIONARY PHASES – TLC
HO HO  (2S,4R, 2'RS)-4-hydroxy-1-(2'-
4 4
hydroxydodecyl)proline
2 2
N COOH N COOH this is an example
H 2
H
2

C10H21 OH HO C10H21  this chiral selector can be used

for complexing chiral analytes
together with Cu2+
- prior to chromatographic separation a
chiral analyte RP (C18) stationary phase is
impregnated with this selector
(lipophilic interaction of the alkyl
HO
Cu chains); then Cu(II) solution is added
O and the metal ion complexed by the
chiral selector
N O enantiomers of a chiral analyte have
H different affinities for this complex;
thusly separation is feasible •20

HO C10H21
GRADED PRESENTATION
1. Students in groups of 4-5 individuals are asked to prepare a
presentation (weight=5% of the theoretical course assessment)
presented in tutorial 10 in the week from 26-11-2019 till 2-12-2019.
2. Each group will be asked to give a 10 min talk about the topic and has
to be prepared for students’ and instructor’s questions and discussions.

3. The locations and timings will be sent to each group individually.

4. Each group will have a topic according to the coming slide.
The assessment criteria are:

Criterion Grades (out of 10)

Not reading from slides nor notes 2
Content of the presentation 2
Comprehension of the content by members of the 2
group
Quality of the presentation 2
Ability to answer students’ and instructor’s questions 2
TOPICS FOR PRESENTATIONS (5%)
Group Topic Group Topic
1 HPLC analysis in marine extracts 11 Biological sample treatment before
analysis by HPLC or GC
2 Chiral Chromatography 12 HPLC analysis in chocolate industry

3 UPLC (Ultra performance liquid 13 Supercritical fluid chromatography

chromatography) (1 application)
4 Derivatization reactions for non volatile 14 HPLC analysis of analgesic drugs (1
compounds prior to GC analysis EXAMPLE)
5 HPLC analysis of water contaminants 15 Analysis of dairy products by
chromatography
6 HPLC determination of 1 biomarker of 16 Analysis of Cosmetics by
your choice chromatography (1 application)
7 HPLC in peptide analysis 17 Chemometrics in analytical
Chemistry
8 HPLC in food and beverage analysis 18 Chromatographic analysis in coffee
industry
9 Refractive index detector for HPLC
10 Chromatographic analysis in Forensic
Medicine
Biotech HPLC application in the analysis of vaccines
•Lecture 6 – Chromatography