Introduction for
Chromatography
1
Chromatography
Analysis of complex mixtures (Organic Compounds)
Separation & Determination
(a) Qualitative
(b) Quantitative
Sample
Mixture of organic compounds dissolved in an organic solvent.
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Chromatography Theory
The components to be separated are distributed
between two phases:
Mobile Phase Stationary Phase
Gas Liquid Solid Liquid
Gas Liquid GSC LSC GLC LLC
Chromatography Chromatography
(GC) (LC)
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Gas Chromatography
(GC)
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Gas Chromatography (GC)
Separates the volatiles organic mixtures into individual components.
used to
Identify Components Measure their concentrations
by by
Appearance time Peak size
(Retention Time) (Height or Area)
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6890N GC Overview
In this module, you will be familiarized with the basic functional areas of the
6890N GC system.
Gas Chromatography consists of:
• Carrier Gas
• Inlets
• Column
• Oven
• Detectors
• Data Handling Device
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1. Carrier Gas
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Carrier Gas
must be inert (free of oxygen and moisture)
A high-purity gas with
traps for
Water Hydrocarbons Oxygen
(Moisture)
A purity of at least
99.999% is recommended
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Carrier Gas
is responsible for
(a) Carrying the vaporized sample through the inlets, column, and the detectors.
(b) Sample component desorption during the sorption-desorption process inside
the column.
Carrier Gas
There are many types of carrier gases used in GC analysis.
such as
Helium Nitrogen Argon/5%Methane Hydrogen
Note:
For capillary applications, some methods recommend using Hydrogen as a carrier gas.
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Carrier Gas
Contamination in carrier gas
may react with
Sample Column
create
Spurious Load the Raise
Peaks detector baseline
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2. Inlets
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Inlets
It is the most critical heated zone where the sample must be
introduced as a vapor into the carrier gas stream.
The most common
Inlets are
Injection Ports Sampling Valves
Operating Procedures
Liquid samples, in micro-liter volume, are usually injected by a special syringe
through a silicon rapper septum onto the heated block, the sample is vaporized as a
''plug'' and carried into the column by the carrier gas stream.
As for gas samples, the injector may be used, but only for qualitative analyses not for
quantitative analyses, a special gas tight syringe is used for such a kind of this
analysis. For quantitative identifications, a Gas Sample Valve (GSV) must be used.
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A. Injection Ports
(a) Handle gas or liquid samples.
(b) Often heated to vaporize liquid samples.
(c) The design and choice of injection ports
depends on the column
Diameter Type
Note:
Liquid or gas syringes are used to inject the sample through a septum into the
carrier gas stream.
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Inlet Types
Inlet Type Gas Control
1. Split/Splitless Injector EPC and Non-EPC
2. Purged Packed Injector EPC and Non-EPC
3. Cool On-Column Injector EPC only
4. Programmed Temperature EPC only
Vaporization (PTV)
5. Volatiles Interface (VI) EPC only
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Inlet Types
Inlet Type Description
1. Split/Splitless Injector It is called capillary injector as it is
used only with capillary columns.
Note
There are four injection modes:
(a) Split Mode Major component analysis (High Concentration)
(b) Splitless Mode Trace component analysis (Low Concentration)
(c) Pulsed-Split Mode Allows larger injection volume
(d) Pulsed-Splitless Mode Allows larger injection volume
Faster sample transfer to column
Less breakdown/Adsorption
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Split/Splitless Injector
Septum Nut
Septum
Liner O-Ring
Cartridge split vent trap
Liner
Washer/Seal
EPC
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Inlet Types
Inlet Type Description
2. Purged-Packed Injector It is called packed injector
as it is used for packed
columns analysis.
Note
During injection, the sample (1ul or 2ul) is vaporized inside a glass liner (insert) in a
very short period of time (milliseconds), thus, it may be called “Flash Injector”.
The type of injection mode used in a purged-packed injector is Splitless Mode.
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Purged-Packed Injector
Septum Purge Weldment
Septum
Glass Liner Sleeve
Heater/Sensor
Assembly Column Adapter
“O” Ring 1/4” Vespel Seal
Insulation Cup
Ferrule
Column Nut 18
Inlet Types
Inlet Type Description
3. Cool On-Column It is called on-column injector as
Injector the sample is injected and
deposited directly into the column
as liquid. When the sample
components are thermally unstable
and high sensitive is required.
Note
Most widely used applications in Environmental.
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Cool On-Column Injector
Automated
Automated
Injection
Injection Manual 200 uM
530
250/320 Fused silica needle
Heater/Sensor
Assembly Septum Ferrule
Column Nut
Column Positioning Inserts
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Inlet Types
Inlet Type Description
4. Programmable Temperature It is a temperature
Vaporization Injector (PTV) programmable split/splitless
inlet.
Note
Injection into hot or cold inlet - rapid heating/cooling.
Large volume injection capability through solvent venting for lower MDL's
Less thermal breakdown.
Fully integrated into 6890.
EPC pneumatics.
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Programmable Temperature Vaporization Injector
Septumless Head Septum Head
Cryo
Connection
Split Vent
Silver Seal
Liner
Heater/Thermocouple
Column Adapter
Split Nut for
Graphpack Inlet Adapter
Column Ferrule
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Inlet Types
Inlet Type Description
5. Volatiles Interface Injector It is an ideal inlet for Purge
(VI) & Trap, Headspace,
Thermal Desorption, or
other gas injection devices.
Note
• Very Inert.
• Very small internal volume (35ul)
• Low split ratio or direct injection for high sensitivity.
• EPC control from GC keyboard or ChemStation.
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Volatiles Interface Injector
Sampler Input
Split Vent
Trickle Flow
Column Connection
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GC Electronic Pneumatic Control (EPC)
Capillary Inlet EPC Module
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Electronic Pneumatic Control
Capillary column (Constant Column + Makeup Flow Mode)
Inlet Pressure (measured/controlled)
Makeup Gas Flow Rate
Constant Inlet Oven Temperature
Pressure
Actual Column Flow
Makeup gas is programmed during run to compensate for decreasing column flow
Net carrier flow (Column + Makeup) is then constant during the GC analysis
Important for detectors that are flow dependant - such as TCD/NPD/ECD
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To Convert To Multiply By
1. Psi Bar 0.0689476
kPa 6.89476
2. Bar Psi 14.5038
kPa 100
3. kPa Psi 0.145038
Bar 0.01
Septum Purge
The septum purge line is near the septum where the sample is injected.
A small amount of carrier gas exists through this line to sweep out any bleed.
Each inlet has a different septum purge flow. The GC automatically sets the purge flow
for EPC inlets, but you can measure it from the septum purge vent at the flow manifold if
you like.
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Septum Purge Flows
Inlet Carrier Septum Purge
(ml/min)
1. Split/Splitless He, N2, Ar/5%Me 3
H2 6
1. Purged-Packed All 1 to 3
2. Cool On-Column He, N2, Ar/5%Me 15
H2 30
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Septum Purge Flows
Inlet Carrier Septum Purge
(ml/min)
4. Programmed Temp. He, N2, Ar/5%Me 3
Vaporization (PTV) H2 6
5. Volatiles Interface (VI) He, N2, Ar/5%Me 3
H2 6
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B. Valves
Used for quantitative analyses of gas mixtures. Different gas sample loops
are available and range from 250ul to 50ml.
There are many valving configurations to accommodate each kind of gas
analyses requirements.
Four-Port Six-Port Eight-Port Ten-Port
Valves Valves Valves Valves
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3. Columns
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Columns
The separation takes place here.
Column’s types
Packed Columns Capillary Columns
(Open Tubular) 6850 Column
6890 Column
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A. Packed Columns
Packed columns contain a finely divided, inert, solid support material
(Commonly based on diatomaceous earth) coated with liquid
stationary phase.
Packed columns are 1.5 – 10m in length and have an internal diameter
of 2 – 4mm.
Packed columns have high sample capacity and are still useful for gas
samples, but capillary columns offer better resolution for most liquid
samples.
There are two types of packed columns
Glass Packed Column Stainless Steel Packed Column
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B. Capillary Columns
Capillary columns is an open tube with the stationary phase coated on its
inside surface. There is no packing.
Capillary columns have an internal diameter of a few tenths of a
millimeter.
Capillary columns require smaller samples than packed columns.
There are two types of capillary columns
Wall-Coated Open Support-Coated Open
Tubular (WCOT) Tubular (SCOT)
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I. Wall-Coated Open Tubular (WCOT)
Consists of a capillary tube whose walls are coated with liquid stationary
phase.
II. Support-Coated Open Tubular (SCOT)
The inner wall of the capillary is lined with a thin layer of support material
such as diatomaceous earth, onto which the stationary phase has been
absorbed.
Note
• SCOT columns are generally less efficient than WCOT columns.
• Both types of capillary columns are more efficient than packed columns.
• In 1979, a new type of WCOT column was devised, the Fused Silica Open Tubular
(FSOT) column.
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Fused-Silica Open Tubular (FSOT)
These columns have thinner walls than the glass capillary columns, and are
given strength by the polyimide coating. These columns are flexible and can
be wound into coils.
Advantages
1. Physical strength
2. Flexibility
3. Low reactivity
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The Column
The purpose of a column is to produce narrow, well-separated peaks from a
multi-component sample.
The Column Efficiency
A high-efficiency column produces narrow peaks.
Efficiency is determined by
The Column Construction The Carrier Gas Flow Rate
(Small tubing diameter
and thin stationary phase
layer is best)
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The Column Selectivity
This is less clearly defined property of the stationary phase.
Essentially, it is how well a phase differentiates between two compounds.
Low Selectivity High Selectivity
(They elute together) (The peaks separates)
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Column Temperature
The stationary phase in the column has a preferred temperature range.
The minimum temperature The maximum temperature
(is usually a melting point) (is usually related to a boiling or
degradation point)
Below this, you are doing Gas/Solid Chromatography.
Above this, you are doing Gas/Liquid Chromatography.
Gas Control
Flow in packed columns is usually controlled using mass flow controllers.
Capillary columns, because of the very low flow rates, are usually pressure-
controlled.
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4. Oven
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Oven
Columns are mounted in a temperature controlled oven because separations are
highly temperature dependent.
The oven temperature
Isothermal Programmed
Oven Features Oven
• Temperature range -80C (Liquid N2) or -60C (CO2) to the configured limit.
• Maximum temperature 450C.
• Temperature programming up to six ramps.
• Maximum run time 999.99 minutes.
• Temperature ramp rates 0 to 120C/min, depending on instrument configuration.
• Oven is configured as fast 2250W or regular 1600W.
• AC driven via triac control.
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A. Isothermal Oven
This is the simplest way to run the oven.
The oven remains at the same temperature throughout the analysis.
Advantages
1. The oven is always ready for a sample analysis.
2. There is no recovery time between analysis.
Disadvantages
1. Samples with a wide range of component times take a long time
to run.
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B. Programmed Oven
The oven temperature changes, usually upward during the analysis.
Advantages
1. Analysis time is reduced.
2. Peak shapes are constant throughout the run, making detection and
measurement easier.
Disadvantages
1. Components are subjected to higher temperatures than with an isothermal
oven. This
could cause degradation of sensitive components.
2. The oven must cool to the starting temperature.
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5. Detectors
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Detectors
It is a heated zone, which is located at the exit of the separation column, which
senses the presence of the individual components as they leave the column.
Detectors are classified as
Universal Detectors Selective Detectors
There are three major response characteristics of detectors:
1. Sensitivity
2. Selectivity
3. Dynamic Range
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Sensitivity
It is the response per amount of sample, that is the slope of the response/amount
curve.
Selectivity
It is a measure of which categories of compounds will give a detector response.
Dynamic Range
It is the range of sample concentrations for which the detector can provide accurate
quantization.
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The Gas Chromatography (GC) has several detector systems available:
1. Flame Ionization Detector (FID)
2. Thermal Conductivity Detector (TCD)
3. Nitrogen-Phosphorous Detector (NPD)
4. Flame Photometric Detector (FPD)
5. Electron Capture Detector (ECD)/ Micro-Cell Electron Capture Detector (uECD)
6. Mass Spectrometer Detector (MSD)
Multiple Detectors
Detectors can be classified also as:
Destructive Non-destructive
Detectors Detectors
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Detector Selectivity Delectability Dynamic Range
1. FID Most organic 100pg 10^7
compounds
2. TCD Universal 1ng 10^7
3. NPD Nitrogen &
10pg 10^6
Phosphorous in
compounds
4. FPD Sulphur, Phosphorous,
tin, boron, arsenic, 100pg 10^3
germanium, selenium,
chromium
5. ECD/uECD Halides, nitrates, nitriles,
peroxides, anhydrides, 50fg 10^5
organometallics
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Common non-destructive detectors are:
1. Thermal Conductivity Detector (TCD)
2. Infra Red Detector (IRD)
3. Photo Ionization Detector (PID)
Common destructive detectors are:
1. Flame Ionization Detector (FID)
2. Nitrogen-Phosphorous Detector (NPD)
3. Flame-Photometric Detector (FPD)
4. Mass Spectrometer Detector (MSD)
Comparison of GC Detectors
FID
ECD
NPD (N)
NPD (P)
FPD (S)
(SIM)
MSD (SCAN)
10-15 10-12 10-9 10-6 10-3
Sensitivity fg pg ng ug mg grams
1 ppt 1 ppb 1 ppm 0.1 % 100 %
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Makeup Gas Flow
Makeup gas enters the detector close to the end of the column.
Its purpose is to speed the peaks through the detector, especially with capillary
columns, so that the peak separation achieved by the column is not lost
through remixing in the detector.
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6. Data Handling System
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Data Handling
The signal of a detected peak which comes out from the detector can be
measured in volts (or millivolts), so we can use a voltmeter to see that signal,
of course this kind of measurements is not practical because we need a
documented data sheet includes the plotted chromatogram and the area counts
(integration) of each peak in that chromatogram in order to make handling
with these data. The data handling device is responsible for that.
The data handling device output may be a plotted chromatogram only
(Recorder), or a plotted chromatogram with accurate counts of retention time
(RT) and area for each peak (Integrator).
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ChemStation Overview
The Agilent ChemStation is a set of programs that allow control of up to
four GCs, LCs, UV-VIS, LC-MS, and CE instruments acquiring two
dimensional data.
ChemStation features include user control of instrument set-points,
acquisition of chromatographic data, data analysis (Integration),
calibration and reporting of results.
The instruments can also be automated for multiple sample analysis.
The instrument communication is via GPIB or LAN, depending on the
instrument configuration.
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Definitions
1. Instrument…
A set of all Analytical devices that will be used together to make a
chromatographic analysis.
2. Analysis...
All the steps performed by the Chemstation controlling a Chromatographic
system to identify and quantify a sample.
Major Steps in performing an Analysis:
1) Load Instrument parameters.
2) Acquire Data.
3) Analyze Data.
4) Report Results.
3. Method…
A group of Chemstation files that contain all the instructions and parameters
needed for total automation of a single Chromatographic analysis.
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The END
Prepared By
Mohamed Salama
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