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Components in a mixture
Gas (FG)

Fase gerak : gas inert (N2, He, Ar)

Fase diam : Kolom berisi bahan


Padat : partikel
Cair : cairan yang diembankan
pada padatan pendukung
Ketika fase gerak/gas dialirkan akan membawa
sampel bergerak komponen-komponen
sampel terdistrisbusi pada FG dan FD fraksi
berbeda Ditunjukkan oleh K (Koefisien






Setiap komponen terdistribusi dalam fase diam

dan fase gerak dengan kecepatan yang berbeda

Dengan adanya kesesuaian solven dan fase diam dengan

komponen , komponen-komponen sampel akan bergerak
dengan kecepatan yang berbeda terjadi pemisahan


Dalam kromatografi : 2 komponen dapat terpisah karena adanya

perbedaan Koefisien distribusi (k) antara komponen dalam

sampel dalam fase diam dan fase gerak
Koefisien distribusi (k)


Konsentrasi komponen A dalam fase diam

Konsentrasi komponen A dalam fase gerak
CS : Konsentrasi komponen A dalam fase diam
CM : Konsentrasi komponen A dalam fase gerak



Deteksi terjadinya pemisahan mis.4 komponen : A, B, C and D

Res pons e

4 signals/peaks

4 spots




Retention Time

Kromatografi planar
(PC or TLC)

Kromatografi kolom
terukur secara kontinyu


Instrumen kromatografi gas (GC)

Instrument of GC
Injection port
Syringe needle


N2, He, Ar

Injection port :
tempat dimana sampel
diinjeksikan dan masuk ke
dalam kolom
Dibuat dari karet silikon
tebal 1mm dan diamtere 6-8

Syringe needle :
Jarum suntik untuk
memasukkan sampel melalui
Injection Port
Volume : 1-10L (liquid) and
0.1-5 mL (gas )

Jenis kolom dalam GC

Bagian yang sangat penting dalam alat GC
Kapiler (open tubular)
Dinding kolom bagian dalam dilapisi dengan lapis tipis (1
m) dari cairan film dengan diameter dalam 0.3 - 0.5 mm
dan panjang 10 - 100 m
Isi (Packed )
Padatan partikel yang berpori ataupun tidak yang dilapisi
dengan cairan film yang tipis (1 m) dengan diameter
dalam 1- 8 mm dan panjang 1 - 10 m

Fase diam (cairan) dalam kolom GLC

Volatilitas rendah (Titik didih tinggi)
high thermal stability
Tidak rekatif secara kimia
Contoh :
Carbowax (polyethylene glycol)

Process in the column

Samepel diinjeksikan ke dalam Injection port A yang mempunyai
suhu tinggi sampel cair akan menguap membentuk gas
Sampel gas dibawa oleh gas Pembawa (FG) di sepanjang kolom, di
mana komponen-komponen terdistribusi di dalam FG dan FD
dengan afinitas/retardasi yang berbeda melaju dengan kecepatan
yang berbeda

The types of column in GC :

Isothermal (GC)
Programmed temperature (GC)
Raising column temperature (GC)
Decreases retention time
Sharpens peaks

Detector : for detecting the component eluted from the

sensitivity :
Response/ Concn
Universal or selective
selectivity - ability to
distinguish between
Rapid response
Linearity - concentration
range over which signal
proportional to
Stability with respect to
noise (baseline noise) and
time (drift)

Types :
Electron capture (ECD)
good for X-, NO2- and conjugated
Thermal conductivity (TCD)
change in resistance of heated wire
Flame ionization (FID)
destruction of combustible sample in
flame produces measurable current
Fourier transform infrared (FTIR)
Mass spectrometry (MS)

Ion formation of the sample when passing through the flame (O2/H2)

at 2100oC
The flame produce + and ions the ions and the electrons
(from sample burned) will be collected by a positively charge wire
ring surrounding the flame current electrical signal
Component is burned to form ion the number of ion proportional to
the carbon content of the sample
CO2 and CO (resulted form organic combution) are undetected
For organic compounds
The detector Temperature = 400oC
Is insensitive in temperature change
As sensitive as nose

For all compounds
Less sensitive
A thin filament of wire in the end of column
Heated by passing a constant current through it to a

trempetarutre T1, greater than Tdetector (T2).

A molecule gas strike the hot wire take a bit of heat away
the wire getting cooler
As the wire is cooled its resistance changes, reaching
equilibrium and produce baseline voltage
When a sample passes over the hot wire because each
component has different velocity and mass will cool the wire
in different amount than the carrier gas does the resistance
is cahnged the current is constant the volatge change a
signal is produced

Some terms in Chromatography GC

Distribution coefficient (k)
Retention factor/ Retardation factor (Rf) =factor

Retention time (tr) and Retention volume (Vr)
Resolution (R)
Distribution coefficient (k)



Principle : each component has different k

in the mixture can be separated

Retardation factor (Rf)

tr Rf =



Chromatogram :
Intensity/concentration/response/detector signal versus time or

volume required for mobile phase to bring the component out

from the column
Res pons e



Retention Time



Retention Time

(tR ) and dead time (tm):

Retention Time (tR )

The time between sample
injection and an analyte peak
reaching a detector at the end of
the column
Each analyte in a sample will
have a different retention time.

Dead time (tM) :

The time taken for the mobile phase to
pass through the column

Other terms
The baseline is any part of the chromatogram where only mobile phase
is emerging from the column.
The peak maximum is the highest point of the peak.
The injection point is that point in time/position time when/where the
sample is placed on the column.
The dead point is the position of the peak-maximum of an unretained
The dead time (tm) is the time elapsed between the injection point and
the dead point.
The retention volume (Vr) is the volume of mobile phase passed
through the column between the injection point and the peak maximum.

The dead volume (Vm) is the volume of mobile phase passed through the
column between the injection point and the dead point.


The retention time (tr) is the time elapsed between the injection point and the
peak maximum. Each solute has a characteristic retention time.

The retention volume (Vr) is the volume of mobile phase passed through the
column between the injection point and the peak maximum.


The corrected retention time (t'r) is the time elapsed between the dead point and
the peak maximum.

The corrected retention volume (V'r) is the volume of mobile phase passed
through the column between the dead point and the peak maximum. It will also
be the retention volume minus the dead volume.


The peak height (h) is the distance between the peak maximum and the base
line geometrically produced beneath the peak.

V m = Q tm

where Q is the flow rate in ml/min.

Vr = Q tr where Q is the flow rate in ml/min.

V'r = Vr - Vm = Q(tr - tm) where Q is the flow rate in ml/min.

The peak width (w) is the distance between each side of a peak measure
at 0.6065 of the peak height (ca 0.607h).

The peak width measured at this height is equivalent to two standard deviations
(2s) of the Gaussian
curve and thus has significance when dealing with chromatography theory.

The peak width at half height (w0.5) is the distance between each side of a peak
measured at half the peak height.

The peak width measured at half height has no significance with respect to
chromatography theory.

The peak width at the base (wB) is the distance between the intersections of the
tangents drawn to the sides of the peak and the peak base geometrically

The peak width at the base is equivalent to four standard deviations (4s) of the
Gaussian curve and thus also has significance when dealing with
chromatography theory.

to describe the migration

rate of an analyte on a column.
Or called as Capacity factor (k)
Retardation/retention factor (Rf)

k =

tr - tm


tr and tm : determined from chromatogram

k < 1 elution is so fast that accurate determination
of the retention time is very difficult
k > 20 elution takes a very long time inefficient
1> k > 5 ideal

Selectivity factor () for 2 components (A and B)

Describes the separation of two species (A and B) on the column



KA and KB : partition coefficient of A or B

kA and kB : capacity factor of A and B

> 1 B is retarded longer/elution is slower

than A separation

Determination of based on chromatogram and tr


tr (B) - tm
tr (A) - tm

Resolution (R) : quality of the separation

Efficiency : high resolution (R)
Effectiveness : there is a difference of k between 2 components

3 types of resolution
Less effective separation



t r1




t r2

t r1


t r2


Band broadening and column efficiency

To obtain optimal separations sharp and symmetrical chromatographic

peaks must be obtained.

This means that band broadening must be limited.
It is also beneficial to measure.

Column efficiency
The number of theoretical plates
The height/length of the column

Why Broadening peak?

The mobile phase moves through the column which is packed
with stationary phase. Solute molecules will take different
paths through the stationary phase at random. This will cause
broadening of the solute band, because different paths are of
different lengths.

Column efficiency
The Theoretical Plate Model of

The plate model supposes that the chromatographic column is

contains a large number of separate layers, called theoretical

Separate equilibrations of the sample between the stationary and
mobile phase occur in these "plates".
The analyte moves down the column by transfer of equilibrated
mobile phase from one plate to the next.

It is important to remember that the plates do not

really exist; they are a figment of the imagination that

helps us understand the processes at work in the
They also serve as a way of measuring column
efficiency, either by stating the number of theoretical
plates in a column, N (the more plates the better), or
by stating the plate height; the Height Equivalent to a
Theoretical Plate (HETP) (the smaller the better).

HETP : Height Equivalent to a Theoretical Plate

HETP : in cm
L : the length of the column (cm)
N : the number of the TP

HETP = L / N

: length factor (cm)

HETP = 2/L
= L W/4 tr

HETP= 2/L = (L W/4 tr)2/L

= L W2/16 tr2

N = L/ HETP= L/ L W2/ 16 tr2 = 16 tr2/ W2


Resolution (Rs) :
Rs =

2(trA trB)

(WA +WB)

(trA trB)
(W A +W B)

Ideal resolution (efficient resolution) Rs = 1.5

Practically Rs =1 98% resolution

a. Rs = 0.75, b. Rs =1.0 and c. Rs = 1.5

The influences of capacity factor and selectivity on

Resolution :

Rs =

N -1

1 +ki

1 +ki
N = 16 Rs2 ( )2 (
To obtain high resolution, the three (N, , and ki ) terms must be maximized.
An increase in N ( the number of theoretical plates) , by lengthening the column, leads to an
increase in retention time and increased band broadening - which may not be desirable.
Instead, to increase the number of plates, the height equivalent to a theoretical plate
(HETP) can be reduced by reducing the size of the stationary phase particles.

Factors influencing HETP :average flow rate of the gas (MF)

Low f(slow) : takes longer time greater extent diffusion peak

broadening and inefficient resolution high HETP

Increasing flow rate decrease of time for diffusion decreasing
Very high flow rate (fast) small elution increasing HETP

The influence of flow rate on HETP

HETP = A + B / u + C u
u is the average velocity of the mobile
phase. A, B, and C are factors which
contribute to band broadening.

Van Demteer plot in detail

HETP = A + B / u + C u

The equation consists of three basic terms.

A : Packing related term
B : Gas (mobile phase) term
C : Liquid (stationary phase) term

A term : Eddy diffusion

A = 2 dp

dp : the particle diameter of the packing,

: a constant that depended on the quality of the packing

Once the column is packed, nothing can be done to reduce the

A term. A term has no influence on HETP with various flow
Its effect can be reduced by using
Regular sized packing
small diameter packing
not allowing any loose packing or dead space in the

Influence of particle size to van Demteer plot

B -Longitudinal diffusion

B = 2 Dm
The effect of the B term is flow dependent.
Increasing the flow (faster), the time for diffusion is reduced HETP
decreases better resolution.
The flow rate is adjusted as slow as possible, untill impossed by C term
The longer the solute band remains in the column (slow flow rate) , the greater will
be the extent of diffusion. The time the solute remains in the column is inversely
proportional to the mobile phase velocity, so, the dispersion will also be
inversely proportional to the mobile phase velocity.

The concentration of analyte is less at the edges of the band than at the center.
Analyte diffuses out from the center to the edges. This causes band
broadening. If the velocity of the mobile phase is high then the analyte spends
less time on the column, which decreases the effects of longitudinal diffusion.

C term : resistance to mass transfer

C = fk dp2/ Dm
The analyte takes a certain amount of time to equilibrate between the stationary
and mobile phase. If the velocity of the mobile phase is high, and the analyte has
a strong affinity for the stationary phase, then the analyte in the mobile phase
will move ahead of the analyte in the stationary phase. The band of analyte is
broadened. The higher the velocity of mobile phase, the worse the broadening
becomes. HETP increases

The effect of the C term can be minimized by:

Using thin coatings of the stationary phase on a solid support.
Use less viscous phases.
Keep the flow as low as possible - limited by the effect of the B term.

Terms A and C : are functions of the particle diameter. Thus for smaller
particles the plate height will be lower.

Sample :
Small volume quick vaporization enter into

column in gas phase all gas molecules is

travelled by MF at the same time
Large volume more time vaporization some
molecules travel faster than others broadening

Fronting and Tailing peak

Fronting peak : when some
molecules move ahead due to
the less retention on the SF (too
large sample being introduced)

Fronting peak

Ideal chromatographic peak

symmetrical (Gaussian)
shape : normal dispersion

Tailing peak : when some

molecules are retained strongly
on the SF (high intermolecular
force (hydrogen bonding)

Tailing peak

For a sample containing many compounds :

If the column is maintained at a low temperature for the duration of a sample
run, the first peaks to elute will likely be well-spaced, but the components
staying on the column longer will find themselves bound to the stationary
phase for longer periods of time; this results in large band-broadening, and
long run times.
At higher temperatures, these components spend more time in the mobile
(gas) phase, helping them elute faster and minimizing band-broadening; the
faster peaks also elute faster however, pressing the peaks so close together
that they may not be resolved.

Solution : by temperature programmed technique

In temperature programming, this effect is overcome by maintaining a low temperature for

a short period of time, and increasing the temperature to help force out the longersticking compounds.

Example of Temperature programmed GC

Determine the k, , N , HETP, and Rs for toluene in the
following analysis :


tr (min)

Column length = 10 meters

Flow rate = 30 ml/min
isothermal conditions

W1/2, (min)

Types of the resolution


k = k (Vs/Vm)
tR = tm (1 +k) = tm (1+ k (Vs/Vm))

General Factors Influencing retention :

Composition and properties of the mobile phase

Type and properties of the stationary phase GC;
Intermolecular force between the components and
the SP and MP (adsorption, ion exchange C)
Temperature GC

Intermolecular Forces influencing retardation of components

Coulombs law : like attracts like

attraction occurs between molecules with similar electrostatic

properties, but molecules with dissimilar properties are repelled

Electrostatic interactions between molecules : 2 main types
Polar van der Waals retention forces : between molecules having

surface charge
Non-polar dispersion forces : between neutral molecules or functional

Polar van der Waals retention forces :

A consequence of dipole-dipole interaction and
hydrogen bonding






hydrogen bonding

dipole-dipole interaction

Components (solut) with

dipole similar to the SP
(solvent) will disperses
compo-SP pair