Chromatography basics

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ANALYTICAL CHEMISTRYAND
INSTRUMENTATION
3(2-1)
Course Contents:
Principal, instrumentation, applications
Chromatography including paper, thin layer, gel filtration, ion-
exchange, affinity, HPLC, gas chromatography, GC-MS and LC–MS;
Spectroscopy types including NMR, visible, ultraviolet, luminescence,
flame, atomic absorption, fluorescence
Flow cytometry
X-ray diffraction
Dialysis
Ultra-filtration
Lyophilization
Ultracentrifugation
Amino acid analyzer

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Chromatography
History
The Russian botanist Mikhail
Tswett coined the term
chromatography in 1906 to
describe his experiments in
separating different colored
constituents of leaves by passing
an extract of the leaves through a
column

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HISTORY
● Chromatography
(from Greek :chromatos -- color ,
"graphein" -- to write)
● 1903 Tswett - plant pigments separated on
chalk columns
● 1931 Lederer & Kuhn - LC of carotenoids
● 1938 TLC and ion exchange
● 1950 Reverse phase LC
● 1954 Martin & Synge (Nobel Prize)
● 1959 Gel permeation
● 1965 instrumental LC (Waters)
Chromatography
Is a technique used to separate and identify the
components of a mixture.
Works by allowing the molecules present in the
mixture to distribute themselves between a
stationary and a mobile medium.

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Purpose of Chromatography
● Analytical - determine
chemical composition of a
sample
● Preparative - purify and
collect one or more
components of a sample
Uses of Chromatography
Real-life examples of uses for
chromatography:
• Pharmaceutical Company – determine amount of
each chemical found in new product
• Hospital – detect blood or alcohol levels in a
patient’s blood stream
• Law Enforcement – to compare a sample found at
a crime scene to samples from suspects
• Environmental Agency – determine the level of
pollutants in the water supply
• Manufacturing Plant – to purify a chemical
needed to make a product

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Gas Chromatography
Used to determine the chemical composition of
unknown substances, such as the different
compounds in gasoline shown by each separate
peak in the graph below.
Paper
Chromatography
Can be used to separate the
components of inks, dyes, plant
compounds (chlorophyll), make-up,
and many other substances
Liquid
Chromatography
Used to identify unknown plant
pigments & other compounds.
Thin-Layer
Chromatography
Uses thin plastic or glass trays to identify
the composition of pigments, chemicals,
and other unknown substances.
Examples of Chromatography
Classification of Methods
There are two classification schemes:
– mobile phase
– attractive forces

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Mobile Phase
● Gas (GC)
● Water (LC)
● Organic solvent (LC)
● Supercritical fluid (SCFC)
Classification based on Mobile Phase
Gas Chromatography
Gas - solid Gas - liquid

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Classification based on Mobile Phase
Liquid chromatography (LC)
Column
(gravity flow)
High performance
(pressure flow)
Thin layer
(adsorption)
Classification based on Attractive
Forces
● Adsorption
● Ion Exchange
● Partition
● Size Exclusion

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Adsorption Chromatography
➢ Separation based on their
adsorption onto the surface of
solid (stationary phase).
➢ Normal phase-like separation
– Nonpolar mobile phase
➢ for polar non-ionic compounds
➢ Ex; Column chromatography (CC)
TLC, HPLC
Partition Chromatography
➢ solute are separated based on their partition
between a liquid mobile phase and a liquid
stationary phase coated on a solid support.
– Paper Chromatography

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Ion Exchange Chromatography
➢ Uses ionic stationary phase
– ions separated on the basis of their tendency to
displace counter ions adsorbed on stationary phase
(Depends on charge, hydration, “solubility”…)
➢ Anionic stationary phases: used for cation separation
➢ Cationic stationary phases : for anion separation
➢ for ionic compounds
➢ - Ex : CC, HPLC
Size Exclusion Chromatography
➢ Separation is a result of “trapping”
of molecules in the pores of the
packing material
● Very large molecules can’t get into
the pores – unretained
● Very small molecules get hung up in
to pores for a long time - most
retained – longest retention time
● stationary phase is a porous matrix
● Ex: CC, HPLC

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STATIONARY PHASE
Type of
chromatography
Material
Paper chromatography Filter paper, cellulose
Thin Layer Chromatography Silica gel, alumina,
polyamide
Gas chromatography
(GC)
Squalene, apezion,
carbowax M
High Performance Liquid
Chromatography
C-8, C-18, Licosorb,
Silicone
MOBILE PHASE
Type of chromatography Solvent
Paper chromatography Combination of different
solvents
Thin Layer
Chromatography
Hexane, ether petroleum,
alcohol.
Gas chromatography
(GC)
He, Ar, N2
High Performance Liquid
Chromatography
Cyclohexane, n-hexane, carbon
tetrachloride, ethanol,
methanol, air

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PAPER
CHROMATOGRAPHY
DEFINITION
A chromatographic analytical
separation technique for
complex mixtures involving the
progressive adsorption of the
dissolved component onto a
special grade of paper.

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PRINCIPLE
• Certain solvents are used to separate a mixture
ex: water, alcohol.
• Due to capillary action the solvent will move up
to filter paper.
• Movement of a solvent will carry components of
mixture along.
• Each component will move up at characteristic
velocity
● The retention factor, or Rf, is defined as the distance
traveled by the compound divided by the distance
traveled by the solvent
For example, if a compound travels 2.1 cm and the
solvent front travels 2.8 cm, the Rf is 0.75:

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• Beakers or jars
• Covers or lids
• Solvent (Distilled H2O,
Isopropanol)
• Graduated cylinder
• Filter paper
• Sample (Different colors
of pins, plant extract)
• Pencil
• Ruler
• Scissors
• Tape
Materials List
• Prepare the solvent solution in various concentration:
- 0%, 5%, 10%, 20%, 50%, and 100%
Preparing the solvent solution

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Preparing the Chromatography Strips
1. Cut filter paper
2. Draw a line 1 cm above
the bottom edge of the
strip with the pencil
3. Label each strip with its
corresponding solution
4. Place a spot from each
pen on your starting line
Developing the Chromatograms
● Place the strips in the beakers
● Make sure the solution does not
come above your start line
● Keep the beakers covered
● Let strips develop until the
ascending solution front is about
2 cm from the top of the strip
● Remove the strips and let them
dry

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Developing the Chromatograms
Spot Detection
- Color spot  observed by naked eye
- Non – color spot  color reagent will give
specific colors for different compound.
Example :
➢ Ninhydrin – a.amino
➢ Iodin dalam etanol –
➢ AgNO3

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THIN LAYER
CHROMATOGRAPHY
Learning Objective
1. State the definition of TLC
2. Explain the phases used in TLC
3. List the materials & methods
used in TLC
4. List the application of TLC
5. List the advanteges &
disadavantages of TLC

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Thin Layer Chromatography
One of analysis method that is
used to identify the unknown
compounds and to determine the
purity of mixture.
● This method is simple, rapid and
cheap
● Widely used in pharmaceutical &
food stuff industry.
-A plate of TLC can be made from aluminium or
glass which is coated by a solid matter as a
stationary phase.
- The coated material has 0.1-0.3mm in thickness
-some of them has been added by fluorescent
indicator that will make it florescence during the
UV light exposure.

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STATIONARY PHASE
● Silica is commonly used as
stationary phase
● The separation of sample mixture
will be dependent on the polarity
of sample.
● Some modified silica is also used
in certain purposes.
Stationery phase Description Application
Silica gel G Silica gel with average
particle size 15µm
containing ca 13%
calcium sulfate binding
agent
Used in wide range
pharmacopoeial test
Silica gel G254
Silica gel G with
fluorescence added
Same application with
Silica gel G where
visualization is to be
carried out under UV
light.
Cellulose Cellulose powder of
less than 30µm
particle size.
Identification of
tetracyclines

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MOBILE PHASE
● The ability of mobile phase to
move up is dependent on the
polarity itself
● Volatile organic solvents is
preferably used as mobile phase.
MOBILE PHASE
SOLVENT POLARITY INDEX
Heksana 0
Butanol 3.9
Chloroform 4.1
Methanol 5.1
Ethanol 5.1
Acetonitrile 5.8

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MATERIALS
• TLC plate
• ‘Developing container’
- chamber/ jar/ glass beaker
• Pencil
• Ruler
• Capillary pipe
• Solvents / mobile phase
- organic solvents
• UV lamp
METHOD

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1.Developing Container Preparation
Solvent is transferred
into the container with
0.5-1cm in dept from the
bottom
➢ Commercially obtained with 5cm
x 20cm in size
➢ Prepare your size when
necessary
➢ Line 1 cm from the bottom with
a pencil as a part should be
spotted.
2. TLC Plate Preparation

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3.Spotting’ TLC plates
➢ Make sure that your sample is
liquified already.
➢ stick it using capillary pipe &
spott onto the line you have made
➢
4.‘Develop the plate’
➢ after spotting, put the plate inside
the chamber in the standing position
➢ Make sure that the dept of solvent
doesn't touch the spots
➢ Let it develop up to the 1cm from
the top of plate
➢ After that, pull out the plate from
the chamber and let the solvent be
vaporized

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5. Detection of spots
- The color samples are easy to
be seen and no need to use UV
lamp to detect them
6. DETECTION OF SPOT
1) Iodination-put the plate in which the spots face to
the iodine crystal and see what is the spot color
changing
2) Ninhydrin:
-specific identification of amino acid compounds.
- Ninhydrin solution will show a purple spot when it is
sprayed to the amino acid spot.
3) KMnO4
used to identify a reducing agent such as glucose,
fructose, vitamin C and others.
4) Alkaline tetrazolium blue
specifically used for corticosteroid identification

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The use of Rf as separation parameter
- The distance taken through by the solvent to move up will be assigned as
solvent front
- The distance taken through by the sample to move up will be assign as
sample front
- Rf value is obtained by dividing the sample front toward solvent front
Rf = sample front
solvent front
-
Thin-Layer Chromatography: Determination of Rf
Values
solvent front
component B
component A
origin
dS
dB
dA
Rf of component A =
dA
dS
Rf of component B =
dB
dS
The Rf value is a
decimal fraction,
generally only
reported to two
decimal places
More polar!
Less polar!

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7. Quantitative determination of known sample
- Done by scratching the spot using spatula, and
extract the compound using the suitable solvent
- The liquid extract can be determined its content
using other method such as spectroscopy.
Problems commonly occur in TLC and how to solve
a. The spot shape is too broad
- Diameter is supposed to be < 1-2mm
b. The movement of solvent
- should be straight up
- unproportionality in stationary phase surface
will inhibit the movement of solvent
c. streaking formation
- caused by too concentrated sample

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TLC Compared to Paper Chromatography
1. Precise and effective
2. More stable toward various organic
solvents
Advantages
● Cheap
● Simple
● The developing can be monitored
visually
● Able to use various chemical as a
detector

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Identification unknown drugs using standard Reference

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Rf = distance moved by substance
distance moved by solvent front
For substances that are very soluble in the liquid
Rf will be close to ....
For substances that are rather insoluble in the liquid
Rf will be close to ....
1
0
Gas Liquid Chromatography
Here the mobile phase is an non-reactive gas ( eg
Nitrogen) flowing through a tube.
And the stationary phase is a non-volatile liquid
held on particles of a solid support.

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In the animation below the red molecules are more soluble
in the liquid (or less volatile) than are the green molecules.
In practice the Column is contained in a thermostatic oven.
About 1μL of liquid is injected into one end of the column.
As each component reaches the other end it is detected
and registered on a chart recorder.
The Retention Time is characteristic of a particular
substance. (for the same column, temperature, gas flow etc.)
The area under each peak indicates the relative quantities.

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Oven
Detector
Injection
port
Nitrogen
cylinder
Column
Recorder

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Chromatogram of petrol
Suggest identities of some of the unlabelled peaks.
Gas Chromatography
Introduction
1.) Gas Chromatography
➢ Mobile phase (carrier gas) is a gas
- Usually N2, He, Ar and maybe H2
➢ Requires analyte to be either naturally volatile or can be converted to a volatile
derivative
- GC useful in the separation of small organic and inorganic compounds
➢ Stationary phase:
- Gas-liquid partition chromatography – nonvolatile liquid bonded to solid support
- Gas-solid chromatography – underivatized solid particles
- Bonded phase gas chromatography – chemical layer chemically bonded to solid
support
Magnified Pores in activated carbon
Zeolite molecular sieve
Bonded phase

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Gas Chromatography
Introduction
2.) Instrumentation
➢ Process:
- Volatile liquid or gas injected through septum into heated port
- Sample rapidly evaporates and is pulled through the column with carrier gas
- Column is heated to provide sufficient vapor pressure to elute analytes
- Separated analytes flow through a heated detector for observation
Gas Chromatography
Instrumentation
1.) Open Tubular Columns
➢ Commonly used in GC
➢ Higher resolution, shorter analysis time, and greater sensitivity
➢ Low sample capacity
➢ Increasing Resolution
- Narrow columns  Increase resolution
- Resolution is proportional to , where N increases directly with column lengthN
Easy to generate long (10s of meters)
lengths of narrow columns to maximize
resolution

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Gas Chromatography
Instrumentation
1.) Open Tubular Columns
➢ Increasing Resolution
Decrease tube diameter
Increase resolution
Increase Column Length
Increase resolution
Gas Chromatography
Increase Stationary Phase Thickness
Increase resolution of early eluting compounds
Also, increase in
capacity factor and
reduce peak tailing
But also decreases
stability of stationary
phase
Instrumentation
1.) Open Tubular Columns
➢ Increasing Resolution

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Gas Chromatography
Instrumentation
2.) Choice of liquid
stationary phase:
➢ Based on “like dissolves like”
➢ Nonpolar columns for
nonpolar solutes
➢ Strongly polar columns for
strongly polar compounds
➢ To reduce “bleeding” of
stationary phase:
- bond (covalently attached)
to silica
- Covalently cross-link to
itself
Gas Chromatography
Instrumentation
3.) Packed Columns
➢ Greater sample capacity
➢ Broader peaks, longer retention times and less resolution
- Improve resolution by using small, uniform particle sizes
Packed column
Open tubular column

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Gas Chromatography
Instrumentation
3.) Packed Columns
➢ The major advantage and use is for large-scale or
preparative purification
➢ Industrial scale purification maybe in the kilogram or
greater range
500 L chromatography
column
Oil refinery – separates
fractions of oil for
petroleum products
Gas Chromatography
Retention Index
1.) Retention Time
➢ Order of elution is mainly determined by volatility
- Least volatile = most retained
- Polar compounds (ex: alcohols) are the least volatile and will be the most
retained on the GC system
➢ Second factor is similarity in polarity between compound and stationary
phase

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Gas Chromatography
Retention Index
2.) Describing Column Performance
➢ Can manipulate or adjust retention time by changing polarity of stationary
phase
➢ Can use these retention time differences to classify or rate column
performance
- Compare relative retention times between compounds and how they change
between columns
➢ Can be used to identify unknowns
Gas Chromatography
Retention Index
2.) Describing Column Performance
➢ Retention index based on the difference in the number of carbons (N, n) for
linear alkane and corresponding retention times (tr’(unknown), tr’(N),tr’(N)):
➢ Provides a means to compare the performance of different columns











)n(tlog)N(tlog
)n(tlog)unknown(tlog
)nN(nI
'
r
'
r
'
r
'
r100
IncreaseinPolarity

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Gas Chromatography
Temperature and Pressure Programming
1.) Improving Column
Efficiency
➢ Temperature programming:
- Temperature is raised
during the separation
(gradient)
- increases solute vapor
pressure and decrease
retention time
Temperature gradient
improves resolution
while also decreasing
retention time
Gas Chromatography
Temperature and Pressure Programming
1.) Improving Column Efficiency
➢ Pressure Programming:
- Increase pressure  increases flow of mobile phase (carrier gas)
- Increase flow  decrease retention time
➢ Pressure is rapidly reduced at the end of the run
- Time is not wasted waiting for the column to cool
- Useful for analytes that decompose at high temperatures
Van Deemter curves indicate
that column efficiency is
related to flow rate

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Gas Chromatography
Carrier Gas
1.) N2, He and H2 are typical carrier gases
➢ He:
- Most common and compatible with most
detectors
- Better resolution (smaller plate heights)
- Solutes diffuse rapidly  smaller mass
transfer term
➢ N2:
- Lower detection limit for a flame ionization
detector
- Lower resolution and solute diffusion rates
➢ H2:
- Fastest separations
- Can catalytically react with unsaturated
compounds on metal surfaces
- Can not be used with mass spectrometers
Forms explosive mixtures with air
- Better resolution (smaller plate heights)
- Solutes diffuse rapidly  smaller mass
transfer term
Flow rate increases N2 < He < H2
Diffusion coefficients follow: H2 > He > N2
Gas Chromatography
Sample Injection
1.) “Sandwich” Injection
➢ Separate sample with air bubbles and solvent
- Solvent is used to push out sample, but bubble prevents mixing
- Final air bubble pushes out solvent
- Gas-tight syringe is required for gas samples
- Injection volume is typically 0.1-2 mL

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Sample Injection
1.) “Sandwich” Injection
➢ Injection port
- Inject rapidly ( < 1s) through septum into evaporation zone
- Injector temperature is kept high (350oC) for fast evaporation
- Rapid gas flows carries sample to mixing chamber for complete vaporization
and complete mixing before entering column
Gas Chromatography
Gas Chromatography
Sample Injection
2.) On-column Injection
➢ Delivers ~100% of sample to the column
➢ For samples that decompose above their boiling points
➢ Solution injected directly on column
- Warming column initiates chromatography
Raise temperature to volatize
sample and start separation
Lower initial column temperature
to prevent sample decomposition

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Gas Chromatography
Detectors
1.) Qualitative and Quantitative Analysis
➢ Mass Spectrometer and Fourier Transform Infrared Spectrometers can identify
compounds as part of a GC system
- Compare spectrum with library of spectra using a computer
➢ Compare retention times between reference sample and unknown
- Use multiple columns with different stationary phases
- Co-elute the known and unknown and measure changes in peak area
➢ The area of a peak is proportional to the quantity of that compound
2
10641 wtpeak heigh. peakGaussianofArea
PeakArea
Concentration of Standard
Peak area increases proportional
to concentration of standard if
unknown/standard have the
identical retention time  same
compound
Gas Chromatography
Ohm’s Law: V =IR
Based on Ohm’s law, monitored
potential (V) or current (I) Changes
as resistance (R) of filament changes
due to presence of compound
Detectors
2.) Thermal Conductivity Detector
➢ Measures amount of compound leaving column by its ability to remove heat
- He has high thermal conductivity, so the presence of any compound will lower
the thermal conductivity increasing temperature of filament
➢ As heat is removed from filament, the resistance (R) of filament changes
- Causes a change in an electrical signal that can be measured
➢ Responds to all compounds (universal)
- Signal changes in response to flow rate of mobile phase and any impurities
present
- Not very sensitive

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Gas Chromatography
Detectors
3.) Flame Ionization Detector
➢ Mobile phase leaving the column is mixed with H2 and air and burned in a flame
- Carbon present in eluting solutes produces CH radicals which produce CHO+ ions
- Electrons produced are collected at an electrode and measured
➢ Responds to almost all organic compounds and has good limits of detection
- 100 times better than thermal conductivity detector
- Stable to changes in flow rate and common mobile phase impurities (O2, CO2,H2O,NH3)
Burn sample and measure
amount of produced electrons
Gas Chromatography
Detectors
4.) Mass Spectrometry
➢ Detector of Choice  But Expensive!
➢ Sensitive and provides an approach to identify analytes
➢ Selected ion monitoring – monitor a specific mass/charge (mz) compared to
scanning over the complete spectra
- Simplifies complex chromatogram
- Increases sensitivity by 102-103

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Gas Chromatography
Sample Preparation
1.) Transform sample into form suitable for analysis
➢ Extraction, concentration, removal of interfering species or chemically
transforming (derivatizing)
2.) Solid-phase microextraction
➢ Extract analytes from complex
mixture without solvent
➢ Uses a fused-silica fiber coated
with stationary phase
- Stationary phase similar to
those used in GC
➢ Expose Fiber to sample to
extract compounds and then
inject fiber into GC to evaporate
analytes
Gas Chromatography
Sample Preparation
3.) Purge and Trap
➢ Removes volatile analytes from liquids or solids,
concentrates sample and transfer to GC
➢ Goal is to remove 100% of analyte
Bubble purge gas (He)
through heated sample to
evaporate analytes
Analytes are captured
on adsorbent column
Connect port to GC
Heat column to
200oC to transfer
analytes to GC

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Gas Chromatography
Method Development in GC
1.) How to Choose a Procedure for a Particular Problem
➢ Many Satisfactory Solutions
➢ The order in which the decision should be made should consider:
1. Goal of the analysis
2. Sample preparation
3. Detector
4. Column
5. Injection
➢ Goal of the analysis
- Qualitative vs. quantitative
- Resolution vs. sensitivity
- Precision vs. time
- Interest in a specific analyte
➢ Sample preparation
- Cleaning-up a complex sample is essential
- Garbage in  garbage out
➢ Choosing the Detector
- Detect a specific analyte(s) or everything in the sample
- sensitivity
- Identify an unknown (MS, FTIR)
Gas Chromatography
Method Development in GC
1.) How to Choose a Procedure for a Particular Problem
➢ Selecting the Column
- Consider stationary phase, column diameter and length, stationary phase
thickness
- Match column polarity to sample polarity
- To improve resolution, use a:
a. Longer column
b. Narrower column
c. Different stationary phase
➢ Choosing the Injection Method
- Split injection is best for high concentrated samples
- Splitless injection is best for very dilute solutions
- On-column injection is best for quantitative analysis and thermally instable
compounds

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Biotron Gas Chromatography instrument