Professional Documents
Culture Documents
Chromatography
1
Chromatography
Analysis of complex mixtures (Organic Compounds)
(a) Qualitative
(b) Quantitative
Sample
2
Chromatography Theory
The components to be separated are distributed
between two phases:
3
Gas Chromatography
(GC)
4
Gas Chromatography (GC)
Separates the volatiles organic mixtures into individual components.
used to
by by
5
6890N GC Overview
In this module, you will be familiarized with the basic functional areas of the
6890N GC system.
6
1. Carrier Gas
7
Carrier Gas
must be inert (free of oxygen and moisture)
A purity of at least
99.999% is recommended
8
Carrier Gas
is responsible for
(a) Carrying the vaporized sample through the inlets, column, and the detectors.
Carrier Gas
There are many types of carrier gases used in GC analysis.
such as
Note:
For capillary applications, some methods recommend using Hydrogen as a carrier gas.
9
Carrier Gas
Contamination in carrier gas
Sample Column
create
10
2. Inlets
11
Inlets
It is the most critical heated zone where the sample must be
introduced as a vapor into 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.
12
A. Injection Ports
(a) Handle gas or liquid samples.
(b) Often heated to vaporize liquid samples.
(c) The design and choice of injection ports
Diameter Type
Note:
Liquid or gas syringes are used to inject the sample through a septum into the
carrier gas stream.
13
Inlet Types
14
Inlet Types
Note
There are four injection modes:
(a) Split Mode Major component analysis (High Concentration)
Liner O-Ring
Washer/Seal
EPC
16
Inlet Types
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”.
17
Purged-Packed Injector
Septum Purge Weldment
Septum
Heater/Sensor
Assembly Column Adapter
Insulation Cup
Ferrule
Column Nut 18
Inlet Types
19
Cool On-Column Injector
Automated
Automated
Injection
Injection Manual 200 uM
530
250/320 Fused silica needle
Heater/Sensor
Assembly Septum Ferrule
Column Nut
20
Inlet Types
21
Programmable Temperature Vaporization Injector
Cryo
Connection
Split Vent
Silver Seal
Liner
Heater/Thermocouple
Column Adapter
Note
• Very Inert.
• Very small internal volume (35ul)
• Low split ratio or direct injection for high sensitivity.
• EPC control from GC keyboard or ChemStation.
23
Volatiles Interface Injector
Sampler Input
Split Vent
Trickle Flow
Column Connection
24
GC Electronic Pneumatic Control (EPC)
25
Electronic Pneumatic Control
Capillary column (Constant Column + Makeup Flow Mode)
Makeup gas is programmed during run to compensate for decreasing column flow
Net carrier flow (Column + Makeup) is then constant during the GC analysis
26
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.
27
Septum Purge Flows
H2 6
1. Purged-Packed All 1 to 3
H2 30
28
Septum Purge Flows
Vaporization (PTV) H2 6
H2 6
29
B. Valves
Used for quantitative analyses of gas mixtures. Different gas sample loops
are available and range from 250ul to 50ml.
30
3. Columns
31
Columns
The separation takes place here.
Column’s types
6890 Column
32
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
33
B. Capillary Columns
Capillary columns is an open tube with the stationary phase coated on its
inside surface. There is no packing.
34
I. Wall-Coated Open Tubular (WCOT)
Consists of a capillary tube whose walls are coated with liquid stationary
phase.
35
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
36
The Column
The purpose of a column is to produce narrow, well-separated peaks from a
multi-component sample.
Efficiency is determined by
37
The Column Selectivity
This is less clearly defined property of the stationary phase.
38
Column Temperature
The stationary phase in the column has a preferred temperature range.
Capillary columns, because of the very low flow rates, are usually pressure-
controlled.
39
4. Oven
40
Oven
Columns are mounted in a temperature controlled oven because separations are
highly temperature dependent.
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.
41
A. Isothermal Oven
This is the simplest way to run the oven.
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.
42
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.
43
5. Detectors
44
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.
45
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.
46
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
Destructive Non-destructive
Detectors Detectors
47
Detector Selectivity Delectability Dynamic Range
1. FID Most organic 100pg 10^7
compounds
48
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)
49
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.
50
6. Data Handling System
51
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.
52
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.
53
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.
54
The END
Prepared By
Mohamed Salama
55