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CHROMATOGRAPHY

Gas SFC Liquid

GSC GLC Column Planar

LSC LLC BPC IEC EC TLC PC

GPC GFC

SFC : Super Critical Fluid Chromatography PC : Paper Chromatography


GSC : Gas Solid Chromatography IEC : Ion Exchange Chromatography

GLC : Gas Liquid Chromatography EC : Exclusion Chromatography


LSC : Liquid Solid Chromatography GPC : Gel Permeation Chromatography
LLC : Liquid Liquid Chromatography GFC : Gel Filtration Chromatography
BPC : Bonded Phase Chromatography TLC : Thin Layer Chromatography
GSC : Gas Solid Chromatography
GLC : Gas Liquid Chromatography
LSC : Liquid Solid Chromatography
LLC : Liquid Liquid Chromatography
BPC : Bonded Phase Chromatography
IEC : Ion Exchange Chromatography
GPC : Gel Permeation Chromatography
GFC : Gel Filtration Chromatography
SFC : Super Critical Fluid Chromatography
Introduction to Gas
Chromatography
Introduction

 Gas chromatography is an instrumental


method for the separation and identification
of chemical compounds.
GAS LIQUID/SOLID CHROMATOGRAPHY

Principles
Partition of molecules between gas (mobile
phase) and liquid/solid (stationary phase).
The Beginning
 concept of GC announced in 1941 by
Martin and Synge (also did liquid partition
chromatography)
 10+ years later GC used experimentally
 1955, first commercial apparatus for GC
appeared on the market
Today
 estimate : 200, 000 gas chromatographs
are currently used through out the world.
 30+ instrument manufactures
 130 different models
 improvements: computers- automatic
control open tubular columns-separate a
multitude of analytes in relatively short
times
Uses of Gas Chromatography
 Determination of volatile compounds
(gases & liquids)
 Determination of partition coefficients and
absorption isotherms
 Isolating pure components from complex
mixtures
Key Information
 organic compounds separated due to
differences in their participating behavior
between the mobile gas phase and the
stationary phase in the column
 in contrast to other types of
chromatography, the mobile phase does
not interact with molecules of the analyte;
its only function is to transport the analyte
through the column
Gas Chromatography
Filters/Traps Data system
H

RESET

Regulators Syringe/Sampler

Inlets

Detectors  gas
system
Gas Carrier
Hydrogen
Air

Column  inlet
 column
 detector
 data
system
Carrier gas/ Varian 3350 Gas Computer Controls for
Regulator Chromatograph Method and Output
A sample is
 introduced into a heated injector,
 carried through a separating column by an
inert gas, and
 detected as a series of peaks on a recorder
when components leave the column.
 Chromatographic separation involves the
use of a stationary phase and a mobile
phase.
 Components of a mixture carried in the
mobile phase are differentially attracted to
the stationary phase and thus move
through the stationary phase at different
rates.
In gas chromatography

 the mobile phase is an inert carrier gas


and

 the stationary phase is a solid or a liquid


coated on a solid contained in a coiled
column.
Flow of Mobile Phase
Injector Detector
T=0

T=10’

T=20’

Most Interaction with Stationary Phase Least


MOBILE PHASE

 The mobile phase or carrier gas flows


through the instrument from a pressurized
tank.

 Flow rate is controlled by a two stage


regulator on the gas tank and additional
controls within the instrument.
Two Stage GC Flow Controller
Tank Regulator
Syarat gas pembawa: (Ar, He, N2, H2)
• inert
• kemurnian tiggi

T
h
e
V
a
The van Deemter equation:
n
D B
H  A  Cu x
e ux
e
m  H is the plate height.
t  ux is the linear velocity of the eluent
through the column.
e  A, B, and C are constant for any
r one column

P
l
o
t
Carrier Gas Velocities and Plate Height
Profil Kurva Van Demter
1.2 Gas N2, H2, dan He, pada N2
Kolom WCOT
1.0

H 0.8 He
E
T 0.6
P
H2
0.4

0.2

10 20 30 40 50 60 70 80 90

Kecepatan Alir (cm/detik)


Injector Detector

Column in Oven
Slide 10

Dilute
Solution
Pure
Sample
 When the system is ready, as indicated by
the ready light, samples are injected into
the injector port where they are vaporized
and carried into the column by the carrier
gas.
10 ml
Syringe
Injection Techniques

Split injection is used if analytes are > ~0.1% of the sample. High resolution
separations work best with the smallest amount of sample that can be detected. Split
injection also makes sure that impurities do not get onto the column in large
concentrations. Splitless injection is appropriate for trace analyses < (~0.01% of
the sample). On-column injection is for samples that thermally decompose. These
go straight onto the column rather than through an injector oven.
Types of Columns (GC)
 Packed Columns
 Less than a few meters in length
 Usually made using 1/16” (1.59mm) inner
diameter stainless steel tubing
 Packed with solid adsorbent or with solid support
with bonded stationary phase
 Used mainly for adsorption chromatography
 Have a high capacity – can handle a lot of
analyte
 Lower separation efficiencies
 Inexpensive – can make them in the lab
Types of Columns (GC)

 Packed Columns

From: www.krackeler.com/.../ Custom_Packed_Columns.jpg


Types of Columns (GC)

 Capillary columns (open tubular)

 Up to 100 meters in length


 Inner diameters are between 0.1 and 0.75 mm
(made with fused silica tubing)
 Tubing wall is coated with solid support to which
stationary phase is chemically bonded
 Used mainly for partition chromatography
 Have a low capacity – cannot handle a lot of
analyte
 Very high separation efficiencies
 Expensive – 4-8 times the cost of packed columns
Types of Columns (GC)
 Capillary columns (open tubular)

From: www.vici.com/columns/ images/crown.jpg


stationary phase
and www.cobertassoc.com/ Gccap.jpg
(0.1 – 5.0µm thick)
Open Tube Columns

Usually made of fused silica


(SiO2) coated with a polyimide
that can withstand 350°C.
Columns are 15-100 m in length.
Open tube columns give
higher resolution, greater
sensitivity, and shorter analysis
times compared to packed
columns.
Types of Stationary Phase (GC)

 For partition chromatography (the most


common), the stationary phase is selected
according to polarity.
 The stationary phase itself is a long chain
polymer, bonded to a support (usually silica
based).
 Non-polar phases are better for separating non-
polar compounds (compounds separate according
to molecular weight).
 Polar phases are better for separating polar
compounds (compounds separate according to
polarity).
Most Common Stationary Phases

1. Separation of mixture of polar compounds


Carbowax 20M (polyethylene glycol)

2. Separation of mixtures of non-polar compounds


OV101 or SE-30 (polymer of methylsilicone)

3. Methylester of fatty acids


DEGS (diethylene glycol succinate)
Gas Chromatography
A) m.p.(gas) - s.p.
1) s.p.: solid(using adsorption)ex: SiO2
column ages: Si-O-H cause tailing peak.
2) s.p.: liquid(GLC, using partition)
a range of polarities , “like dissolves like”
Decrease thickness of stationary phase leads to
a) Resolution (H)
b) tr 
c) Sample capacity
22.1 Gas Chromatography -4
ex

-ol: -OH
-oate: -COOR
-al :-HC=O b.p.
-one:-C=O
N Massa Molekul
Nama Senyawa Rumus Molekul Titik Didih (˚C) Kepolaran
o Relatif

1 Asetaldehid C2H4O 20,2 Polar 44,05 g/mol

2 Methanol CH3OH 64,5 Polar (0,762) 32,04 g/mol

3 Ethanol C2H5OH 78,37 Polar 46 g/mol

4 Etil Asetat C4H8O2 77,1 Semipolar 88,105 g/mol

5 2 Methyl-2 Propanol C4H10O 83 Polar 74,12 g/mol

6 1.Buthanol C4H10O 117,7 Polar (0,586) 74,12 g/mol

7 2 Methyl-1 Buthanol C5H12O 128,7 Polar (0,552) 88,19 g/mol

8 3 Methyl-1 Buthanol C5H12O 132 Polar 88,15 g/mol

9 Isoamil Asetat C7H14O2 142 Non-polar 130 g/mol

10 Ethyl Carpoate C8H16O2 168 Non-polar 144,214 g/mol

11 2-Phenylethanol C8H10O 225 Non-polar 122,167 g/mol


22.1 Gas Chromatography -5
B) The effects of column polarity on separation
Like dissolves like (a) S.P: nonpolar, b.p. dependent (b) S.P: polar
22.1 Gas Chromatography -6
C) Common solid s.p. :
a) Porous carbon :
larger molecules bind more tightly
than small ones, flexible molecules
bind more than rigid ones
b) Molecular sieves :
retain & separate small molecules :
H2, O2, N2, CO2, CH4. (Fig. 22-5)
22.1 Gas Chromatography -7
packed column vs. open tubular column

higher resolution
lower sample capacity
22.1 Gas Chromatography -9
22.1 Gas Chromatography -10

4. Carrier Gas
22.1 Gas Chromatography -11
5. Sample Injection-1
1) gasses, liquids, or solids
 vaporized, not decomposition
2) injection time   bands broader
3) injected by syringe
(manual or automatic injection)
 The column is contained in a heated oven
that is preceded by a heated injector port
and followed by a heated detector unit
which produces the output.

 A set of preprogrammed parameters


regulate the operation of the system.
THERMOSTAT OVEN

Isothermal

Mengatur suhu
kolom
Temperatur
terprogram

a T : suhu kolom (K)


log t   b
'
r
T a, b: konstanta
Temperature Programming
 During most GC analyses, the temperature of the
column is raised during the analysis
 This is called temperature programming
 It decreases retention time and sharpens the
peaks
Temp Prog
Isothermal
(50-250°C)
(150°C)
8°/min
Suatu senyawa terelusi dari kolom kromatograf,
dengan data sebagai berikut:
Pada saat suhu kolom 363K, tr’=20,0menit. Bila
suhu kolom dinaikkan menjadi 373K,
tr’=15,0menit. Berapa waktu retensi terkoreksi bila
suhu kolom dinaikkan menjadi 383K?
a = 1,69 x 103K ; b = - 3,36
 Separation of the components of the
mixture occurs in the column.

 Compounds differentially retained in the


stationary phase reach the detector at
different times to produce a set of peaks
along the time line.
Enter from Exit to
Slide 12
Injector Detector

Packed Column
installed in Oven
Compartment.
 The detector response is sent to a
computer system where the progress of
the sample is monitored on the computer
monitor in graphical form that displays
detector response as a function of run
time.
The Ideal Detector

Adequate sensitivity - range 10–18 to 10–15 g analyte/s


Good stability and reproducibility
A linear response to analyte that extends over several orders of
magnitude
A temperature range from room temperature to at least 400o C
A short response time that is independent of flow rate
High reliability and ease of use
Similarity in response toward all analytes of alternatively a higher
Predictable and selective response toward + classes of analytes
Nondestructive of sample

http://www.people.virginia.edu/~roa2s/chem_551/8/tsld002.htm
Detector Requirements
1) High Sensitivity:
Sensitivity refers to the change in detector response as a function
of the change in the amount or concentration of the analyte.

S = dR / dC
or S = dR / dQ

where S is sensitivity, R is detector response, C is concentration of


the analyte in the detector, and Q is the total quantity of the analyte
in the detector.
Detector sensitivity is best measured as the slope of the calibration
graph, a plot of detector response vs. analyte concentration or
quantity.
The range over which the detector sensitivity is constant is called the
linear dynamic range, and the entire range over which response
varies with concentration or quantity is called the dynamic range of
the detector.
The lower limit of detection is a function not only of the detector
sensitivity but of detector noise. Detector noise is defined as the
standard deviation of the detector response when no sample is
present and is referred to as the root-mean-square noise (Nrms).
The detection limit (DL) is defined as the quantity or concentration
required to produce a response which is three times the detector
noise).

DL = 3 Nrms / S

H.H. Hill and D.G. McMinn (Ed.), Detectors for Capillary


Chromatography, Wiley, 1992. pp 2-3.
2) High Selectivity
The selectivity of a given compound over a potentially interfering
compound can be measured by the ratio of the detector sensitivities.
Selectivity (SEL) is reported in terms of relative molar response or as
relative weight response.

SEL = S1 / S2

Where S1 is the detector sensitivity of the compound of interest and S2


is the detector sensitivity of the potentially interfering compound.
When selectivity is greater than three orders of magnitude for most
potentially interfering compounds, it is sometimes referred to as
specificity and the detector is said to be specific for that compound
or class of the compounds.
Detector types are:

1) FID ( flame ionization detector )


2) TCD ( thermal conductivity detector )
3) ECD ( electron capture detector )
4) FPD ( flame photometric detector )
5) HID ( helium ionization detector )
6) NPD ( nitrogen-phosphorus detector )
7) PID ( photo ionization detector )
8) TID ( thermionic ionization detector )
9) CCD ( catalytic combustion detector )
10) NPD/DELCD ( combination NPD and dry electrolytic conductivity
detector )
11) FID/DELCD ( combination FID and dry electrolytic conductivity
detector )
12) FID/FPD ( combination FID and FPD )
13) Dual FPD ( dual wavelength FPD for both sulfur and phosphorus )
14) FID dual FPD ( dual FPD plus FID combination )
GC Detector Specifications

Detector Minimum detectable Linear dynamic


amount range
Thermal Conductivity Detector typically 600pg ethane/mL 106
(TCD) helium carrier
Flame Ionization Detector (FID) (with makeup gas) : >106
2×10-12g C /sec
Electron Capture Detector (ECD) <10fg of lindane >104
Nitrogen/Phosphorus Detector 5 × 10-14g N /s >104
(NPD) and 2 × 10-14g S /s
Flame Photometric Detector (FPD) 1 × 10-13g P /s >104 (P)
and 5 × 10-12g S /s
Photoionization Detector (PID) 1 × 10-12g benzene and 1 × >105
10-12g toluene, using 10.6
eV lamp
Electrolytic Conductivity Detector 5pg heptachlor 106
(ELCD)
Flame Ionization Detector (Nanogram - ng)

High temperature of hydrogen flame (H2 +O2 + N2)


ionizes compounds eluted from column into flame.
The ions collected on collector or electrode and were
recorded on recorder due to electric current.
FID
The FID is now the most common detector for GC
because it is nearly universal but has greater
sensitivity than a TCD.
In an FID, eluate is burned in a mixture of H2 and
air. Most carbon atoms (except C=O) produce
radicals that produce CHO+ in the flame:

CH  O  CHO   e 

Electrons are used to neutralize the CHO+ atoms


and the electron current can be measured. This
current is proportional to the number of molecules
present, and exhibits a linear response over ~7
orders of magnitude. The detection limits are lower
than the TCD detector by a couple orders of
magnitude.
Schematic Diagram of Flame Ionization Detector

Exhaust

Chimney

Igniter Collector Electrode


Polarizing Electrode

Hydrogen Column
Inlet Effluent
Schematic Diagram of Flame Ionization Detector

Collector
Detector electronics

 - 220 volts

Flame
Chassis ground

Jet

Column Signal output


Thermal Conductivity Detector

Measures the changes of thermal conductivity due


to the sample (mg). Sample can be recovered.
Thermal Conductivity Detector
Principal: The thermal balance of a heated filament
Electrical power is converted to heat in a resistant
filament and the temperature will climb until heat
power loss form the filament equals the electrical
power input.
The filament may loose heat by radiation to a cooler
surface and by conduction to the molecules coming
into contact with it.
TCD The TCD was the most common detector for a
long time in GC because of its universal detection.
The TCD measures how much a substance can
transport heat from a hot to cold region. Helium has
a high thermal conductivity (2nd only to H2), so if it is
used as the carrier gas, anytime an analyte emerges
from the column with it, conductivity will decrease.

Filament gets hotter in presence of analyte,


changing voltage
Thermal Conductivity Detector
Thermal Conductivity Detector

The detector contains two filaments: one exposed only


to carrier gas, while the other is exposed to the carrier
gas for sample analysis.
When the gas for the sample analysis is only carrier
gas , the two filaments can be balanced.
Instead of a direct measurement of filament
temperature, the filament resistant, which is a function
of temperature, is measured.
Thermal Conductivity Detector

When a compound elutes, the thermal


conductivity of the gaseous mixture of carrier gas
and compound gas is lowered, and the filament in
the sample column becomes hotter than the other
control column.
Its resistance increased, and this imbalance
between control and sample filament resistances is
measured by a simple gadget and a signal is
recorded
Thermal Conductivity Detector

The TCD will respond to any substance


different from the carrier gas as long as its
concentration is sufficiently high enough.
Thermal Conductivity Basics

The TCD is a nondestructive, When the carrier gas is contaminated by


concentration sensing detector. A sample , the cooling effect of the
heated filament is cooled by the flow of gas changes. The difference in cooling
carrier gas . is used to generate the detector signal.
Flow

Flow
Thermal Conductivity Detector

The ability of a colliding molecule to carry off heat


depending on its thermal conductivity. Hydrogen
and helium have high thermal conductivity and
therefore will be more efficient at “cooling” a
heated filament than other gases will
Relative Thermal Conductivity

Relative Thermal
Compound
Conductivity
Carbon Tetrachloride 0.05
Benzene 0.11
Hexane 0.12
Argon 0.12
Methanol 0.13
Nitrogen 0.17
Helium 1.00
Hydrogen 1.28
Thermal Conductivity Detector

• Responds to all compounds


• Adequate sensitivity for many compounds
• Good linear range of signal
• Simple construction
• Signal quite stable provided carrier gas glow rate,
block temperature, and filament power are controlled
• Nondestructive detection
Electron Capture Detector

For pesticide analysis (picogram).

Accept electrons of carrier gas.


Electron Capture Detector
ECD detects ions in the exiting from the gas chromatographic
column by the anode electrode.
3H or 63Ni which emits  particles.
Ionization : N2 (Nitrogen carrier gas) +  (e) = N2+ + 2e
These N2+ establish a “base line”

X (F, Cl and Br) containing sample +  (e)  X-


Ion recombination : X- + N2+ = X + N2

The “base line” will decrease and this decrease constitutes the signal.

Insecticides, pesticides, vinyl chloride, and fluorocarbons


Electron Capture Detector

Electron capture compound, X (highly electonegative element), tends


to capture free electrons and increase the amount to ion recombination

X (F, Cl and Br) + e  X-


Ion recombination : X- + N2+ = X + N2
The current will decrease and this decrease constitutes the signal.

Halogens, lead, phosphorous, nitro groups, silicone and polynuclear aromatics.


Insecticides, pesticides, vinyl chloride, and fluorocarbons
ECD

The ECD is sensitive to compounds with


high electron affinities (halogen-containing,
conjugated carbonyls, nitriles, nitro-
compounds). Gas entering the detector is
ionized by a high energy radioactive source that
gives off electrons (often 63Ni). When analytes
of high e- affinity enter the detector, they
capture some of the electrons.

A carrier or makeup gas of N2, H2, or CH4/Ar


must be used. The ECD is an extremely
sensitive detector for those compounds that it
detects.
Electron Capture Detector
Electron Capture Detector
Electron Capture Detector
Gas Chromatography
Application
SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS

Detector Response Peak Area (cm2 )


C18 10

8
C 16
6

4
C14
2

0.5 1.0 1.5 2.0 2.5 3.0


Sample Concentration (mg/ml)

Retention Time
C
The content % of C14 fatty acids =  
C + C+ C 


= the content % of C14 fatty acids


TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS

Response

Mixture of known compounds

Octane
Decane
1.6 min = RT

Hexane

GC Retention Time on Carbowax-20 (min)

Response

Unknown compound may be Hexane

1.6 min = RT

Retention Time on Carbowax-20 (min)


Retention Times
Response

RT= 4.0 min on SE-30


Hexane

GC Retention Time on SE-30

Response

RT= 4 min on SE-30

Unknown compound

GC Retention Time on SE-30


Gas Chromatogram of Methyl Esters of Fatty Acids
Slide 13
 Each component of the mixture reaches
the detector at a different time and
produces a signal at a characteristic time
called a retention time.

 The area under a peak is related to the


amount of that component present in the
mixture.
 The detector information can also sent to a
printer that produces hard copy of the
chromatographic run.
In the printout of the chromatographic analysis:
the number of peaks correlates with the number
of components in the sample,
the area under each peak correlates with the
relative amount of that component in the
sample,
and if standard information is available, the
retention time under defined conditions can be
used to identify each component.
GC Analysis

Qualitative --- determine what is present

1) Chromatographic
a) tR or Retention Index
b) Spiking

2) Spectroscopic

a) Sample collection --- MS, IR


b) Dynamic GC/MS
c) IR, GC/FTIR spectromete
d) NMR

Quantitative -- determine how much is present

use peak height h or area A


Qualitative analysis : tR
Standard ---- methanol, MEK(tR ), toluene
Unknown ----- same tR (X)
Conclude (X) = MEK

Retention time
methan MEK limitations
ol
toluene tR changes with flow rate,
column temperature,
liquid phase, column
history,
X sample size

*** WARNING
tR identical retention times do
not confirm peak identity
Spiking

Step 1 Peak X --- toluene ?


Step 2 Toluene added to sample
Step 3 Peak X identified as toluene

methan MEK
ol
toluene

Toluene added to sample

tR
Qualitative Analysis –
Retention Index (I)
 I for an analyte is a measure of the rate at which it is
carried through a column compared with the rate of
movement of two normal alkanes one that moves faster
than the analyte and the other that moves more slowly.
 I of alkanes, by definition, is 100 times the number of
carbon atom they contain regardless of the column
packing, temperature or other conditions
e.g. butane, I = 400
pentane, I = 500
The Kovats Index

 The Kovats retention index (I) is defined for


alkanes where I=#C atoms*100
 So, for butane, I=400, but for octane I=800
 Every other compound has a retention index I
based on what alkanes it elutes between

 log tr' (unknown )  log tr' (n) 


I  100n  ( N  n) 
 log t '
r ( N )  log t '
r ( n ) 

N:# carbon atoms in larger alkane


n:# carbon atoms in smaller alkane
Sample Preparation
 In the simplest case, a sample must be prepared for
analysis by GC
 This involves converting it to a volatile form if
necessary
 Steps in preparation
 Extraction
 Preconcentration
 Removing or masking interference(s)
 Derivatization
 For GC
 Solid phase microextraction (SPME)
 Purge and trap
SPME
SPME is used to extract from liquid, air, or
sludge without using any solvents. A silica
fiber coated with a stationary phase for a GC
is attached to a syringe. The fiber is exposed
to sample for a certain time to allow the phase
to become saturated with analyte.
After sampling, the fiber is retracted into
the syringe and the syringe gets injected into
the inlet of the GC. SPME does not remove
all of the analyte because it is an equilibrium
reaction. It usually can obtain 30-50% of the
molecules.
Because the binding to the stationary phase
of the fiber is a partitioning reaction, there is
an equilibrium involved. Equilibration time
for analytes must be obtained using
calibration experiments.
GLC ADVANTAGES

1. Very good separation


2. Time (analysis is short)
3. Small sample is needed - ml
4. Good detection system
5. Quantitatively analyzed
DISADVANTAGES OF GAS CHROMATOGRAPHY

Material has to be volatilized at 250C without decomposition.

Fatty Acids Methylester

O O
R C OH + CH 3 OH + H 2 SO4 R C O CH3
Reflux
Volatile in Gas
Chromatography
O
CH 2 O C R

O CH 3 ONa O
CH O C R + CH 3 OH 3 R C O CH3
Volatile in Gas
O Chromatography
CH 2 O C R
The Effects of OH groups of Carbohydrates

6
CH2 OH
O
5 6
4 CH2 OH
OH 1
HO O
5
3 2 OH 4
OH OH 1
HO
3 2 OH
OH
Derivation of Glucose with Trimethylchlorosilane

6
CH2 OH
O CH 3
5
4
OH 1 + 5Cl Si CH 3
HO
3 2 OH CH 3
OH
Glucose Trimethylchlorosilane
6
CH2 O-Si(CH3)3
O
5
4 + 5HCl
O-Si(CH ) 1
3 3
(CH3)3-Si-O
3 2 O-Si(CH3)3
O-Si(CH3)3
Effects of Derivation

1. Time consumption
2. Side reaction
3. Loss of sample

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