Gas Chromatography

Invention of Chromatography
Mikhail Tswett invented
chromatography in 1901
during his research on
plant pigments.
He used the technique to
separate various plant
pigments such as
chlorophylls, xanthophylls
and carotenoids. Mikhail Tswett
Russian Botanist
(1872-1919)
Original Chromatography Experiment
tart: A glass End: A
olumn is filled series of
ith powdered colored
imestone bands is
CaCO3). seen to
n EtOH extract Later form,
f leaf pigments correspondin
s applied to the g to
op of the column. the
different
tOH is used to
pigments in
lush the pigments
the original
own the column.
plant
Chromatography: (Greek = chroma “color” and
graphein “writing” ) Tswett named this new technique
chromatography based on the fact that it separated the
components of a solution by color.
Common Types of Chromatography
Tswett’s technique is based on Liquid Chromatography.
There are now several common chromatographic
methods. These include:
Paper Chromatography
Thin Layer Chromatography (TLC)
Liquid Chromatography (LC)
High Pressure Liquid Chromatography (HPLC)
Ion Chromatography
Gas Chromatography (GC)
Paper and Thin Layer Chromatography
e solvent moves up paper by capillary actio
rrying mixture components at different rate

solvent
front

Later

solvent
 How Does Chromatography Work?
In all chromatographic separations, the sample is transported
in a mobile phase. The mobile phase can be a gas, a liquid,
or a supercritical fluid.
The mobile phase is then forced through a stationary phase
held in a column or on a solid surface. The stationary phase
needs to be something that does not react with the mobile
phase or the sample.
The sample then has the opportunity to interact with the
stationary phase as it moves past it. Samples that interact
greatly, then appear to move more slowly. Samples that
interact weakly, then appear to move more quickly. Because
of this difference in rates, the samples can then be separated
into their components.
Chromatography is based on a physical
equilibrium
that results when a solute is
transferred between the K
mobile
= and a
stationary
A phase. distribution
A A
A A coefficient or
C S
A A K 
A C M ratio
partition
A Where CS is the molar
A
A A concentration of the
solute in the stationary
Cross Section of Equilibrium in a column. phase and CM is the
“A” are adsorbed to the stationary phase. molar concentration in
“A” are traveling in the mobile phase.
the mobile phase.
 In a chromatography column,
flowing gas or liquid
continuously replaces saturated mobile
phase and results in movement of A
through the column. Flow

Column is packed
with particulate
stationary phase.

As a material travels through the

column, it assumes a Gaussian
concentration profile as it distributes
between the stationary packing phase and
In a mixture, each component has a different
distribution coefficient, and thus spends a
different amount of time absorbed on the solid
packing phase vs being carried along with the
Flowgas
flowing

Flow

Flow

Flow

More volatile materials are carried through

the column more rapidly than less volatile
materials, which results in a separation.
If a detector is used to determine when
the components elute from the column, a
series of Gaussian peaks are obtained,
one for each component in the mixture
that was separated by the column.

The first two components were not completely sep

aks in general tend to become shorter and wider wit
 The Theoretical Plate
Theoretical plate is a term coined
by Martin & Synge. It is based on a
study in which they imagined that
chromatographic columns were analogous
to distillation columns and made up or
numerous discrete but connected narrow
layers or plates. Movement of the
solute down the column then could be
treated as a stepwise transfer.

Theoretical plates (N) measure how

efficiently a column can separate a
mixture into its components. This
umber of theoretical plates (a measure of effic
tR 2
N  16( )
wb
tR

wb

tR is the retention time; it is measured

from the injection peak
(or zero) to the intersection of the
tangents.
 tR 2
N  16( )
wb
tR
tR

wb wb
Larger N Smaller N

When the retention time, tR, is held constant,

the column that produces peaks with narrower
bases, wb, will be more efficient – have a
greater N value.
Likewise a column that produces wider peaks
will be less efficient – have a smaller N
value.
 Gas Chromatography
 Good for volatile samples (up to about 250 oC)
 0.1-1.0 microliter of liquid or 1-10 ml vapor
 Can detect <1 ppm with certain detectors
 Can be easily automated for injection and data analysis
Components of a Gas Chromatograph
Gas Supply: (usually N2 or He)
Sample Injector: (syringe / septum)
Column: 1/8” or 1/4” x 6-50’ tubing packed with
small uniform size, inert support coated with
thin film of nonvolatile liquid
Detector: TC - thermal conductivity
FID - flame ionization detector
Schematic of a Commercial Gas Chromatograph
Gas chromatography is a technique used for
separation of volatile substances, or
substances that can be made volatile, from
one another in a gaseous mixture at high
temperatures. A sample containing the
materials to be separated is injected into
the gas chromatograph. A mobile phase
(carrier gas) moves through a column that
contains a wall coated or granular solid
coated stationary phase. As the carrier gas
flows through the column, the components of
the sample come in contact with the
stationary phase. The different components
of the sample have different affinities for
the stationary phase, which results in
differential migration of solutes, thus
leading to separation
 Gas chromatography can be used for
both qualitative and quantitative
analysis. Comparison of retention times
can be used to identify materials in the
sample by comparing retention times of
peaks in a sample to retention times for
standards. The same limitations for
qualitative analysis
There are two types of Gas chromatography
Gas - Solid Chromatography (GSC)
Gas - Liquid Chromatography (GLC)
 Gas - Solid
Chromatography (GSC)
The stationary phase, in this case, is
a solid like silica or alumina. It
is the affinity of solutes towards
adsorption onto the stationary phase
which determines, in part, the
retention time. The mobile phase
is, of course, a suitable carrier
gas. This gas chromatographic
technique is most useful for the
separation and analysis of gases
like CH4, CO2, CO, ... etc.
 Gas - Liquid
Chromatography (GLC)
The stationary phase is a liquid with
very low volatility while the mobile
phase is a suitable carrier gas.
GLC is the most widely used
technique for separation of volatile
species. The presence of a wide
variety of stationary phases with
contrasting selectivities and easy
column preparation add to the assets
of GLC or simply GC.
 Gas Chromatography (GC)
*Gas chromatography is a chromatographic technique that
can be used to separate volatile organic compounds.
*It consists of
a flowing mobile phase
an injection port
a separation column (the stationary phase)
an oven
a detector.
 Principle

The organic compounds are separated due to

differences in their partitioning behavior between
the mobile gas phase and the stationary phase in
the column.
Mobile phases are generally inert gases such as
helium, argon, or nitrogen.
The injection port consists of a rubber septum
through which a syringe needle is inserted to inject
the sample.
The injection port is maintained at a higher
temperature than the boiling point of the least
volatile component in the sample mixture.
Since the partitioning behavior is dependent on
temperature, the separation column is usually
contained in a thermostat-controlled oven.
Separating components with a wide range of boiling
points is accomplished by starting at a low oven
temperature and increasing the temperature over time
to elute the high-boiling point components.
A gas chromatography oven, open
to show a capillary column
 Gas Supply Pressure
regulator
Flow
controller

Gas
supply

The mobile phase (carrier gas) should be chemically inert,

dry and free from O2 (helium, argon, nitrogen and hydrogen).
The carrier gas should be of high purity; impurities in the
carrier gas such as water vapour, air and trace gaseous
hydrocarbons can cause reactions with sample and cause
column deterioration and affect detector performance.
The gas supply is associated with pressure regulator and
flow controller.
 Flow meter
Flow
Sample Injection System controller Injector Septum
Pressure
regulator

Detector
Recorder
GC
chart

Preparation of the Sample : Oven

Column Column
Samples GC must be volatile. Gas
supply

Samples which are non volatile are converted into a volatile derivative.
GC Column
Most GC columns are made from high-purity fused silica capillary, the inner
wall of the capillary coated with the stationary phase.
GC columns vary in length from less than 2 m to 50 m or more.
In order to fit into the column oven, they are usually formed as coils.
The control of column’s temperature is critical to attain a good separation in
GC, thus the column is located inside a thermostated oven to control the
temperature.

GC Detectors Thermal conductivity detector (TCD)

Flame ionization detector (FID)
Nitrogen phosphorous detectors (NPD)
GC Applications:

Food Analysis
Analysis of foods is concerned with confirming the presence
and determination the quantities of the analytes (lipids,
proteins, carbohydrates, preservatives, flavours, colorants,
and also vitamins, steroids, and pesticide residues).

Drug Analysis
GC is widely applied to identification of the active
components, possible impurities as well as the metabolites.
Environmental Analysis
The environmental contaminants; e.g. (DDT) is present
in the environment at very low concentrations and are found among
many of other compounds. GC, with its high sensitivity and high
separating power, is mostly used in the analysis of environmental
samples.

Forensic Analysis
In forensic cases, very little sample is available, and the
concentration of the sample components may be very low.
GC is a useful due to its high sensitivity and separation efficiency.
 Comparison of HPLC and
GC
Sample Volatility Sample Polarity

HPLC HPLC
• No volatility requirement • Separates both polar and
non polar compounds
• Sample must be soluble
in mobile phase

GC GC
• Samples are nonpolar
• Sample must be volatile
and polar
Comparison of HPLC and
GC
Sample Preparation Sample Size

HPLC HPLC
• Sample must be filtered
• Sample size based upon
• Sample should be in column.
same solvent as mobile
phase

GC GC

• Solvent must be volatile • Typically 1 - 5 L

and generally lower
boiling than analytes
 Comparison of HPLC and
Separation Mechanism
GC Detectors

HPLC HPLC
• Both stationary phase • Most common UV-Vis
and mobile phase take • Wide range of non-
part destructive detectors
• 3-dimensional detectors

GC GC
• Most common FID,
•Mobile phase is a universal to organic
sample carrier only compounds
 Our GC System
(Limited to volatile chlorine containing organic compounds.)

Gas Supply: propane line gas

Injector: 0.3 ml of vapor through latex tubing
Column: 5 ml pipet filled with Tide detergent
Detector: based on Beilstein reaction of chlorinated
hydrocarbons with hot Cu metal to give bright
blue/green flame coloration.
 GC
Construction
TOP
Ring Stand
CdS Photocell
Clamps Straw/Stopper
mounted in bracket
5ml pipet
Packed with Tide Attach leads to computer

1 ml Syringe Black paper

cylinder

SIDE
Buret Valve
Latex coupling Align photocell with
midpt. of flame
Cu coil
Gas inlet Fiber plugs
 Wrapping the detector coil
(Detail of Cu coil
*Do not
winding.)
Pipet leave any
gaps
between
1. Hold here with thumb while winding 18-20
turns.
turns around the pipet.
Do NOT use the Coil Adjusting Tool for this step.
Bend so mounting post is centered in coil
using the Coil Adjusting Tool**

Side End View

Do NOT try to adjust coil
View in pipet tip!

**Coil Adjusting Tools are located in the Hood.

(Please return them to the Hood as soon as your are finished with them.)
 Filling the Column
1. Add about 1 tsp of dry/sifted Tid
to fill pipet within 1/4” of top
(Tide has been sifted
and dried,
2. Tap so keep with
column lid closed on
a pencil
tocontainer.)
settle the powder.

3. Use a plug of fiberfill

to hold Tide in place
*Do not compact Tide into column.
**Do not leave any dead space at head of column.
 Burner Adjustment Parameters

Adjustment of
height
above pipet tip
Length of coil will affect
will
flame stability.
affect the fuel /
air ratio.
r best results, flame should be 1/4” - 3/8”
n-luminous (blue), and non-flickering.
Detail of Sensor (CdS Photoresistor)

Check that leads

are not shorted face of sensor should
inside straw. be 1/8” back from
end of straw
foam plug
Straw covered
with electrical
tape
Note: The sensor should be tested by
connecting to computer and checking
voltage in light and with sensor face
covered with your finger.
 Sensor Alignment
Top View Flame Shield

Sensor/Stopper

Column Flame

1. Remove sensor from stopper and sight through tube.

2. Adjust clamp so that base of flame can be seen.
3. Carefully replace sensor in tube.
 Sensor Response Curve
1.1 M Ω
(dark)

R (Ω)

350 Ω
(bright)
Brightness

The sensor has the largest change in

resistance in the low light region.
(Blue flame is best.)
Too much light will ‘saturate’ the
sensor.
 Sample Injection
. Fill sample vapor only in syringe (NOT liquid!).
. Overfill syringe then adjust to desired amount.
. Do not let the sample remain in the syringe long before
injecting to avoid vapor loss into the rubber plunger of
the syringe.
. Rotate the syringe when piercing the latex tubing to avoid
a pressure surge which may blow out the flame.
. Inject as close as possible to the column head.
. Push the plunger fairly rapidly during injection.
. Chloroform (CHCl3 ) may require a larger amount
than suggested to get adequate sensor response
 General Settings for GC Startup program

Y axis: 1-5 v (0-1 volt is in bright light, 4-5 volt is dark)

X axis: 0-400 seconds
 Plot of GC Elution Data for
Dichloromethane and Chloroform
On 25 cm Tide Column

Peaks are smooth, well separated and elute

 Plot of GC Elution Data for
Dichloromethane and Chloroform
On 25 cm Tide Column

Poor: peaks are noisy, due to flickering

flame, and elute slowly.
To fix: Adjust sensor so that it is looking
 Printing Elution Curves

From File menu in GC program choose Export Data A

1. Give the file a name.
2. Choose text option.
3. Save to desktop.
4. Send it to your email accounts* via minermail
using web.
* Don’t forget to cc Dr. Bolon bolonc@umr.edu
5. Open file, highlight text, copy and paste to your
favorite graphing program (e.g. Excel).
 Determination of the
Amount
of Sample Components
eak height is proportional to the amount
Present
terial eluting from the column at any given

rea under the peak is a measure of the total

t of material that has eluted from the colum
tronic integrators are used for area measure
ommercial GCs. We will be using ALGEBRA. 
 The Gaussian curve can be
approximated as triangular in shape,
to simplify area measurement.

Area = 1/2 wb h

wb

the height is measured to the top of the tan

s above the actual curve peak.
 If voltage data is very noisy,
resulting in poor peak shape,
some peak parameters may be estimated
from visual
observations, however areas cannot be
calculated. So have the TA verify your
data before you take apart your GC.

retention time

peak
widt
h

Blue Flame Green Flame Blue Flame

onset of green end of green

 Experiments
1. Test run of CH2Cl2 without sensor check for visible
color, reasonable width and retention time on column.
2. Run of Pure Compounds: (1 good run of each)
CH2Cl2 (Dichloromethane aka Methylene Chloride)
CHCl3 (Chloroform)
3. Mixture: CH2Cl2:CHCl3 (2:3 mix)
t voltage vs time data and also note visual
sappearance of green flame color.
 Data and
Calculations
(There is a separate handout available with this information.)

A. Graphs (3) from computer

1. Elution data for pure CH2Cl2
2. Elution data for pure CHCl3
3. Elution data for mixture

B. Calculations (handwritten, for

each graph)
1. Peak Area = 1/2 (W x H)
2. Number of theoretical
plates, N = 16 (TR / Wb)2
Checkout
1-pipet (5 ml graduated, for GC column)
1-flow regulator (buret valve)
1-clothespin
1-pair forceps
1-1cc syringe
1-flame shield (black construction paper cylinder)
1-sensor/stopper
1-coil adjusting tool
1-pc Cu wire (discard after use)
In Hoods:
Tide (Replace lid to prevent moisture absorption)
teaspoons
plastic funnels
fiberfill (looks like cotton)
CH2Cl2 - dichloromethane or methylene chloride
(clear septum vial)
CHCl3 - chloroform
(brown septum vial)

Notes:
Work in groups of four. 
Hazards
Needles are sharp.
Detector coil is hot.
Carrier gas is flammable.
CH2Cl2 and CHCl3 are toxic.
Waste
Empty Tide from columns into solid waste.
Do NOT use water to clean column.
Stockroom will clean stuck columns.
This Week
Review Session – November 29, 8:30-10:00pm in G3.
Next Week (December 3-6)
*Final Exam – 1-2 Hour Exam during regularly
scheduled class time. You will need a calculator.
**Checkout after exam. $35 fine for not checking out.
(This means NO Chem 2 Final during Finals Week.)