1

KIM3403
TEKNIK PEMISAHAN DALAM KIMIA
ANALISIS
Assoc. Prof. Dr. Marinah Mohd Ariffin
erin@umt.edu.my
09-6683437
013-9330077

Lecture Time
2

Day

Time

Venue

SUN

4-5 pm

BK1-01

MON

2-3 pm

IBH12

Synopsis:
3

Topics in this course discuss further applications of

chromatographic techniques such as
gas chromatography (GC) including GC-MS,

liquid chromatography (LC),

Supercritical-Fluid Chromatography (SFC) and

electrophoresis.

Sample preparation/pretreatment and method validation

Objectives:
4

Menerangkan prinsip-prinsip dan teori asas dalam

analisis kromatografi.
Membincangkan kaedah peralatan kromatografi dalam
menyelesaikan masalah yang behubungkait dengan
proses analisis sampel.
Membincangkan kaedah penyediaan sampel sebelum
menggunakan kaedah peralatan kromatografi.

Learning Outcomes:
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Pada akhir kursus ini, pelajar akan boleh:

 Mentakrifkan

dan memahami konsep kromatografi dalam

kimia analisis.
 Menjelaskan prinsip-prinsip dan teori asas dalam
peralatan yang melibatkan kromatografi.
 Membandingkan kegunaan berbagai alatan makmal
berdasarkan teknik kromatografi.
 Menghubungkaitkan teknik kromatografi dalam analisis
sampel.

Minggu
1-2

2-4

Tajuk Kuliah
Pengenalan kepada kaedah kromatografi

Pengkelasan kaedah kromatografi

Kelakuan kromatografi bahan terlarut

Kecekapan dan resolusi turus

Proses turus dan pelebaran jalur

Masa analisis dan resolusi

Penentuan kuantitatif
Kromatografi Gas

Prinsip asas kromatografi gas

Perkakasan dalam kromatografi gas

Turus kromatografi gas

Fasa cecair dan pemilihan turus

Pengesan untuk kromatografi gas

Pengoptimuman Ujikaji

Aplikasi kromatografi gas

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Kromatografi Cecair Prestasi Tinggi (KCPT)

Prinsip asas KCPT

Perkakasan dalam KCPT

Kromatografi partisi

Kromatografi penjerapan

Kromatografi penyisihan

Kromatografi fasa terikat dan terbalik

Aplikasi KCPT

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Kromatografi Ion (KI)

Prinsip KI

Peralatan KI

Aplikasi KI
Kromatografi Bendalir Superkritikal (KBS)

Prinsip KBS

Peralatan KBS

Aplikasi KBS

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10-11

12-14

Elektroforesis
Prinsip elektroforesis

Peralatan elektroforesis

Aplikasi elektroforesis
Penyediaan Sampel bagi Kaedah Kromatografi

Pengekstrakan pelarut

Pengekstrakan fasa pepejal

Pengekstrakan-mikro fasa pepejal

Attendance (>80%)
Individual Assignment/Quiz/Tutorial 30%
Test
30%
Final Exam
40%
Total
100%

References
1.

2.

Skoog, D.A., Holler, F.J. &

Crouch, S.R. (2007). Principles
of Instrumental Analysis. 6th
Edition. Thomson Brooks,
Canada
Rouessac, F. & Rouessac, A.
(2000). Chemical Analysis
Modern Instrumental Methods
and Techniques. John Wiley &
Sons, Chichester.
10

References
3. Christian, G.D. 1994.
Analytical Chemistry, 6th
Edition. John Wiley &
Sons, New York.

11

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INTRODUCTION TO
CHROMATOGRAPHY TECHNIQUES

KIM3403

Separation Technique In Analytical Chemistry

Quantitative Analysis
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Involves several steps and procedures ~ Analytical

Process :
Defining the problem

Obtaining a representative sample

Preparing the sample for analysis / Pretreatment

Performing the measurement

Calculating the results and presenting the data

Chromatography
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chromatography is a versatile technique for

separating mixtures

Strategy:
 flow the mixture over a material that retains some
components more than others, so different
components flow over the material at different
speeds

CHROMATOGRAPHY
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Chromatography is a separation technique in which

the components of a mixture are separated by the
distribution of substances between two phases
stationary

mobile

phase

phase.

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The Russian scientist TSWETT found that the active

components in leaves extract can be isolated by using
chromatography technique.
separating of different colored constituents of leaves
was shown when the leaves extract pass through a
column of calcium carbonate, alumina and sucrose.
So, he coined the term chromatography from Greek
where


chrom- means color

graphy means to write

Principles of chromatography

A solute equilibrates between a

mobile and a stationary phase.
The more it interacts with the
stationary phase, the slower it is
moved along a column.

Stationary phase

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Advantages of chromatography
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can separate very complex mixtures

 drugs, flavorings, foods, pesticides, tissue extracts,
fuels, air samples, water samples

very small sample sizes

separated components can be collected individually

analyses can be highly accurate and precise

Classification of Chromatography
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Chromatography can be classified according to :

(1) The type of mobile phase
(2) The physical design of stationary phase
(3) The type of equilibration process involved

1) TYPE OF MOBILE PHASE

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Liquid- Liquid chromatography (LC)

Gas-Gas chromatography (GC)

intermediate between gas & liquid



Supercritical Fluid chromatography (SFC)

2) PHYSICAL DESIGN OF STATIONARY PHASE

21

The stationary phase is in the form of a sheet/ flat

surface
TLC

PAPER CHROM

The column is filled with stationary phase and the

mobile phase solvent is allowed to pass through itexample

COLUMN CHROM eg GC and HPLC

Sheet chromatography
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Thin Layer Chromatography (TLC)



stationary phase is a thin layer of adsorbent (Al2O3

or SiO2, usually) coating a sheet of plastic or glass

some components bond to the adsorbent strongly;

others, more weakly

as with paper chromatography, components appear

as spots on the sheet

Sheet chromatography
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paper chromatography (PC)

 stationary phase is liquid soaked into a
sheet or strip of paper


mobile phase is a liquid solvent

some components spend more time in

the stationary phase than others

components appear as separate spots

spread out on the paper after drying or
"developing"

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Column chromatography
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1. Sample Injection

2. Separation

Separation Column

packing material, 3-5 m

column

different interaction
between packing material &
sample
separation can be done

Separation material/sorbent/
stationary phase
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3) TYPE OF EQUILIBRATION PROCESS INVOLVED

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The equilibration process is governed by the type of

stationary phase
bases of equilibration are
a)
b)
c)
d)
e)

Adsorption
Partition
Ion exchange
Molecular/size exclusion chromatography
Affinity

Adsorption Chromatography

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The oldest type

Utilizes a mobile liquid or
gaseous phase that is
adsorbed onto the surface of
stationary solid phase.

Partition Chromatography
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Based on a thin film formed

on the surface of a solid
support by a liquid stationary
phase

Ion Exchange Chromatography

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A resin (the stationary solid

phase) is used to covalently
attach anions or cation onto it.
Solute ions of the opposite
charge in the mobile liquid
phase are attached to the resin
by electrostatic forces.

Molecular Exclusion Chromatography

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Known as gel permeation or gel

filtration
Lacks an attractive interaction
between the stationary phase and
solute
The liquid or gaseous phase passes
through a porous gel which
separates the molecules according
to its size
The pores are normally small and
allows smaller molecules to enter the
gel
Larger molecules pass through the
column at a faster rate than smaller

Affinity Chromatography
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The most selective type

Utilizes the specific interaction
between one kind of solute
molecule and a second molecule
that is immobilized on a stationary
phase
Example :
antibody = immobilized molecule
a mixture of protein = sample
the specific protein is then eluted
by changing the ionic strength or
pH

Even though chromatographic techniques can be

classified into many categories, the main purpose of
chromatography is still the same

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to separate and quantify the target sample in mixed sample.

what will appear as the results of separation?

CHROMATOGRAM
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A plot of detector response versus elution time

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GC chromatogram

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HPLC-UV CHROMATOGRAM

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Nomenclature and Terms

H
k
N
Neff

tM
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Separation factor
Plate Height
Retention factor
Efficiency (number of
plate)
Effective theoretical
plate/Effective plate
number
Mobile-phase hold
up time

tR
t R

Retention time

wb

Bandwidth of peak

Adjusted retention
time

1) RETENTION TIME (tR)

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Parameter used to identify a sample component

Units in minute
The time shows how long the sample retained in the column.
tR depends on column types, column temperature, and flow
rate.

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Chromatogram

2) PEAK WIDTH
 wb or Wh (wb )


or peak area

parameter used to measure the quantity of the sample component

Peak height

wh
wb
tR

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tR

3) PEAK SYMMETRY
 The symmetry factor for a normal peak is 1.0 ( 0.05). If
the symmetry factor for the peak is more than 1.05 or less
than 0.95, the peak is asymmetry

a=b

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a>b

a<b

Possible cause : column overload or co-elution

Column overload
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Co-elution
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Chromatogram And Its Uses

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The chromatogram can also be used to evaluate

Column efficiency
Column performance

Column Efficiency
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Column efficiency is the ability of the column to produce

sharp peak.
The efficiency of the chromatographic column is affected
by the amount of band broadening that occurs as a
compound passes through the column
indicated by important parameters as below:
The number of Theoretical Plates (N)
2) Plate Height (H)
1)

1. THEORETICAL PLATE
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For high separation Efficincy, a large number theoretical

plates is necessary
N

separation efficiency

Each theoretical plates in chromatography can be thought

of as representing a single equilibrium step.
So, many numbers of theoretical plates, representing
many steps of equilibrium.

The number of plates/efficiency can be obtained from a

chromatogram from the expression
N= 16 tR

Intensity

wb

Wb

tR
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Time

2. PLATE HEIGHT
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The plate height (H) is the length of a column divided by the

number of theoretical plates

H= L/N

L = Column length (cm)

N= the number of TP (plate)

Thus, the more efficient the column, the bigger the N the smaller
the H
The unit of H is cm/plate.

Column Resolution
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the ability of the column to separate two peaks

from each other
Indicated by:
1)
2)
3)

Capacity factor/Retention factor (k)

Separation Factor/Selectivity ()
Resolution (Rs)

1. RETENTION FACTORS (k)

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Partitioning ratio, k where :

k=

tR-tM
tM

tR
tM

Where tM = retention time for non retained peak

tR = retention time for a solute peak
tR =adjusted retention time
k value between 1-5 is preferred

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2. SELECTIVITY ()
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Selectivity is an indication of degree of separation between

two peaks
= k2
k1

tR2-tM
tR1-tM

Two compounds cannot be separated if k is exactly the same

or =1.

tR1

tR2

tM

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3. RESOLUTION (Rs )
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The quality of a separation is measured by resolution

Rs = 2[ tR2-tR1]
Wb1 + Wb2

Rs =

-1

k2
k2 + 1

minimum resolution required for quantitation is 1.25

Separation is achieved when R = 1.5

If we increase the N and , the peak resolution also increase

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Improvement in resolution can also be achieved by adjusting

the flow rate for optimum level or adjusting temperature
(increased of temperature leads to shorter column lifetime)

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Exercise
61

Substance A and B have retention time of 16.40

and 17.63 min, respectively, on a 30.0 cm column.
An unretained species passes through the column in
1.30 min. The peak widths (at base) for A and B
are 1.11 and 1.21 min, respectively. Calculate
a.
b.
c.

The column resolution

The average number of plates in column
The plate height

Peaks/Band Broadening
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The broad peaks might consists of two peaks -overlap (poor

resolution and not efficient)
To increase the separation, we have to adjust several variables
during experiment:
i.
ii.
iii.
iv.
v.




sample size
thickness of liquid coating on stat. phase
mobile phase viscosity
temperature
linear velocity of mobile phase.

decrease (i) (iii) will increase separation

increase (iv) and (v) will decrease separation

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The Van Deemter Equation

The Van Deemter showed for GC that the broadening of a peak

is the summation of somewhat interdependent effects from
several sources.
Van Deemter equation:
H = A + B/
+ C

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A, B and C are constant and are related to the three major

factors affecting H and is the average linear velocity of the
carrier gas in cm/s.

minimum
H

C
A

optimum velocity

Average linear velocity of carrier gas velocity

A represents eddy diffusion, B represents molecular diffusion

and C represents the rate of mass transfer

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Plot of H vs linear velocity is know as the Van Deemter plot

Eddy Diffusion
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Eddy Diffusion is due to the variety of variable-length

pathways available between the particles in the column
and is independent of the gas or mobile phase velocity.
Small, uniform particles minimize eddy diffusion.

Molecular Diffusion
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Molecular diffusion of the sample components in the

mobile phase, due to concentration gradients within column

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Faster linear velocity decreases molecular diffusion.

At higher linear velocity, the population of analyte
molecules is swept through the column in a shorter period
of time

which enables the analyte band to remain more concentrated

resulting in narrower, higher efficiency peaks due to less time

for longitudinal diffusion

Rate of Mass Transfer

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Rate of mass transfer, is the finite time required for the solute
equilibrium to be establish between two phases.
The mass transfer is impacted by both linear velocity and
particle size.
The populations of analyte molecules are transported from the
mobile phase to the particle surface.
The analyte molecules then move through the mobile phase in
the pores to the bonded surface layer (e.g. C18, C8, etc.),
interact with the bonded phase and then are swept back out
of the pore into the bulk mobile phase.

Stationary phase particle

Particle pores

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However, the analyte molecules within the population travel

into and out of the pore to varying degrees.
That means as the molecules return to the bulk mobile phase,
the length of the path in which each analyte molecule has
traveled is different resulting in a widening of the analyte
band.
The amount of band spreading that occurs will depend on the
speed of the mobile phase.

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At a high linear velocity, the time lag between the molecules

interacting with the particle surface and the molecules
transferring through the mobile phase is longer.
This will lead to a broader, less concentrated analyte band,
therefore translates into a broader chromatographic peak and
lower sensitivity.
At a slow linear velocity, the time lag is shorter.
This results in a more concentrated analyte band, producing
narrower, more efficient chromatographic peaks.

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Mass transfer improves dramatically as the particle size is

decreased.
For smaller particles, it takes less time for an analyte
molecule to travel into the pores, interact with the
chromatographic surface, and be swept back into the
mobile phase.
Therefore, analyte molecules separated on smaller
particle columns diffuse much faster, resulting in a sharper,
narrower and more efficient chromatographic band.

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