Advanced Analytical Chemistry

Department of Analytical Chemistry-Faculty of Chemistry

Assoc. Prof. Nguyen Anh Mai

Assoc. Prof. Nguyen van Dong

2023
 Course introduction
 Objectives: To provide students with basic knowledge in
the field of chromatography and spectrophotometry
 Resources (for the chromatography part)
 Analytical Chemistry 2.0/2.1, David Harvey (free download)
 Quantitative chemical analysis, Daniel Harris
 Content
 Chromatography (6 sessions, 3h/session)
 Spectrophotometry (4 sessions, 3h/session)
 Evaluation: exercises, mid-term and final exams
 Introduction to
Chromatography

Assoc. Prof. Nguyen Anh Mai

Analytical Chemistry Department - HCMUS
2023
Can aniline and related compounds be
determined by acid-base titration?

Aniline Toludine

Anisidine N,N-dimethylaniline
The analysis is impossible without separation components
from a complex mixture prior to detection and quantitation!
What are benefits of separation in
analytical procedures?
 INVENTION OF (Mobile phase)
CHROMATOGRAPHY Petroleum ether

Mikhail Tsvett, 1903 xanthophills

separation of leaf pigments + chlorophylls
on a column filled with CaCO3
(Stationary phase)
 - carotene

Chromatography
 - carotene

Color writing
 Repetetive distribution of solutes
between two phases

Mobile phase

solute solute solute

solute solute solute

Stationary phase
Interactions in chromatogr. – polar forces

OH
OH
Si OH
OH Si OH
OH Si
Si Si
Si
OH OH

Si OH Si

Si
Electrostatic interaction in chromatography
Ni2+
Fe2+

Na+
Na+ Na+
SO3- Ni2+ SO3-
Fe2+ SO3-

SO3- Na+
Na+ SO3-
Na+ Na+
SO3- + SO3-
3
Na+
SO3-
Interactions in chromatogr. – van Der Waals forces
Partition coefficient (K)

AS AM
When the distribution between two
phases reaches equilibrium:

K = CS / CM

CS: concentration in SP
CM: concentration in MP
Conc. in MP
K=1

MP-SP
boundary

Conc. in SP
Conc. in MP
K=2 K=1 K = 0.5

MP-SP
boundary

Conc. in SP
How does a solute move along a column?

MP

SP
 MOBILE PHASE

CBA
CBA
C B A

DETECTOR

Column Column
INLET OUTLET
Comparison chromatograms a) and b)of same mixture
Which one would you like? And why?

0.110
AU

0.066

0.022

0.110
AU

0.066

0.022

0.0 0.1 0.2 0.3 0.4 minutes

Fundamental parameters
in chromatography
 What do peak shape & position imply?

Retention
Selectivity Efficiency
 Retention parameters
retention time (tR), adjusted retention time (t’R),
retention factor (k’)

tR

W1/2

tm

Wb
Number of theoretical plates/plate count (N)

A theoretical plate =
a hypothetical zone
in which the
distribution of a H
substance between
two phases reaches L
the equilibrium

Plate height (H)

H=L/N
Number of theoretical plates (N) -
an indicator of column separation power

N = 100
N = 1000
How to calculate number of theoretical plates

tR

W1/2

tm

Wb

b
3901

Temperature Program: isothermal at 135oC

 3
3
6
6
2
2
1
1
4
4
7
7
9
9

8
8

5
5

0 5 10
Retention time, min
Selectivity ()

 = K2 / K1
= k’2 / k’1
= t’2 / t’1
Changing pH, composition of MP to
change selectivity

Isoeluotropic

Acetonitrile:H2O
(= 40:60, v/v)

Methanol:H2O
(= 50:50, v/v)
 Problems 1
Suppose you need to separate a mixture of benzoic acid, aspartame, and
caffeine in a diet soda. The following information is available.

tR
(min) benzoic acid

pH 3.0 3.5 4.0 4.5

benzoic acid 7.4 7.0 6.9 4.4 aspartame
aspartame 5.9 6.0 7.1 8.1
caffeine 3.6 3.7 4.1 4.4

caffeine

a) Explain the change in retention time for each compound.

b) Prepare a single graph showing the retention time versus pH for each
compound.
Using your plot, identify a pH level that will yield an acceptable separation.
Resolution (Rs)
Resolution (Rs)
Approaches to improve resolution
Overlapped

a) Increase selectivity
Detector signal

DeDe b) Increase efficiency

c) Increase retention

Time
Purnell’s equation: resolution is affected
by three important parameters N, , and k’

Efficiency Selectivity Retention

 How N affect resolution ?

• Large or small N is preferred?

• Disadvantages of using long columns for
large N?
• Can we have columns with same length but
larger N? What advantages/disadvantages?
 RS as a function of k 
RS
4

Area where k has little effect

2

1
k
0 5 10 15 20
?
 RATE THEORY
(explains why peaks broaden while travelling
in chromatographic columns)
a) Multiple pathway
SP

Tightly packed + small and

uniform particles can minimized
MP
band broadening

b) Longitudinal Forward and backward

MP
diffusion diffusion in MP as band
moves along
Solutes have much higher
diffusion coefficient in
MP
gaseous MP than in liquid MP

Analyte band

c) Resistance to
mass transfer Movement
MP into SP
Low viscosity of MP and short
diffusion path length in particles SP
can enhance the mass transfer Movement
off SP
 SP van Deemter
equation
MP
A
Forward and backward
MP diffusion in MP as band
moves
+

B/u
MP

Analyte band
+

MP Movement
into SP
C.u
SP Movement
out off SP
H
Low efficiency High efficiency
van Deemter curve - combined effects

H = A + B/u + C.u
Height of theoretical plate, H

Optimum
mobile phase
velocity

Mobile phase linear velocity, U (cm/s)

Effect of particle size on column
efficiency
 General elution problem – the case of
complex mixtures
Complex mixture: consist of components with wide range of k’

Isocratic
60% ACN

C10
C11 C12 C13

C9
C14
C8

C15
Gradient vs. isocratic (in LC)

Isocratic
60% ACN

Gradient
50-100% ACN
Temperature program vs. isothermal (in GC)
Rules to design a gradient elution for better
separation and minimal analysis time?