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Customer Support Centre Shimadzu Asia Pacific Pte. Ltd. Singapore 2005
Identification & quantification Chromatography is an analytical technique for separating compounds in a mixture Chromatography uses two phases to separate the compounds:
Mobile phase Stationary phase
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Mixture
Stationary phase
t1
t2
t3
In Gas Chromatography (GC), a gas is used as the mobile phase (normally called carrier gas)
GC Samples
Samples suitable for GC analysis:
Thermally stabile Sufficiently volatile (can be vaporized at 400-4500C or below)
Examples of GC analyses:
Analysis of pesticides in vegetables, drinking water, waste water Analysis of alcohol in cooking sauces Analysis of hydrocarbons in petroleum products Analysis of solvents in packaging materials, paint, photographic film, drugs Analysis of components in food flavor and fragrances Analysis of triglycerides in oils
Etc.
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Gas Chromatograph
Detector Injector
Data System
Oven
Column
GC Analysis
CARRIER GAS flows into the injector, through the column, and then into the detector Sample is introduced into the INJECTOR Sample is turned into vapor and mixed with the carrier gas Sample vapor is transferred into the COLUMN by the carrier gas Analytes travel through the column at certain rates
mainly determined by their physical properties, the temperature of the column, and the composition of the column
When an analyte elutes from the column, it enters the DETECTOR Electric & electronic signal is generated if analyte interacts with the detector The size of the signal is recorded by the DATA SYSTEM and plotted against elapsed time to produce a plot called chromatogram
Headspace (HS)
Headspace
Incubation / Extraction
Equilibrium
Sampling
Injection
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SPME
Vial Injection Extraction Injection Desorption
Fiber
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Pyrolysis
For analysis of samples which cannot be vaporized by GC injector, usually polymers How it works:
Sample is decomposed by heating (pyrolyzed) Decomposition products are analyzed by GC
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Automated Sampling
Automated Sampling
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Automated Sampling
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Hydrogen
Carrier Gas
GC
Oven
Air
Column
Make up
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Injector
Sample Flow Controller Detector
GC
Column
Column
Separate mixture of compounds into individual components
Data System
GC
Oven
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GC Column
Capillary Column
Carrier gas
Packed Column
Glass or stainless steel Stationary phase (thin film) Solid support material
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Separation in column
From Injection Port
To Detector
Column
From Injection Port
To Detector
Column
From Injection Port
To Detector
Column
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Detector
Detects the compound(s) eluted from column Generates electric & electronic signal Transmit signal to Data System for recording & processing Signal is a function of time Signal is proportional to the amount of the compound
Detector
Injector
Hydrogen
Carrier Gas
GC
Oven
Air
Column
Make up
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Shimadzu GC Detectors
FID (Flame Ionization Detector)
Detects most organic compounds General detector
Shimadzu GC Detectors
FPD (Flame Photometric Detector)
Selective detector For higher sensitivity of detection for sulfur, phosphorous, or tin containing organic compounds Example of analysis:
Analysis of organophosphorous pesticides (malathion, dichlorvos, chlorpyrifos, etc.) analysis of H2S gas analysis of SO2 gas
Data System
Converts signal from detector into human-readable format Process and analyze data Controls GC system (for the more advanced, software-based systems) E.g.
GCsolution Software (PC based) Chromatopac
Data System
Column GC Oven
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GC Data : Chromatogram
uV (x10,000) 1.50 Chromatogram 1.25 1.00 0.75 0.50 0.25 0.00 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
Time where peak is detected is used to identify the compound in the sample
Retention time: time required for a compound to travel through the column
Size (area or height) of peak is used to measure the amount of the compound in the sample
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Purpose of GC Analysis
Qualitative Analysis
Quantitative Analysis
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Qualitative Analysis
Retention times of compounds in standard sample is compared to the retention times of peaks in unknown sample. Under the same analysis condition, the same compounds have the same retention times.
Standard Sample
Unknown Sample
Same Compound
0.75 0.50 0.25 0.00 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
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Quantitative Analysis
The peak areas or heights of compounds in standard sample (with known concentration) are compared to that of the sample of unknown concentration
Calibration curve
Y (Area or height)
Sample Standard 2 Standard 1
Standard 3
X (Concentration)
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Separation of compounds
When analytes are introduced into the column, the molecules distribute between the stationary and mobile phases
M S
The molecules in the mobile phase are carried down the column Those in the stationary phase are temporarily immobile and do not move down the column
Separation of compounds
The molecules in the mobile phase are carried down the column
M S
Those in the stationary phase are temporarily immobile and do not move down the column
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Separation of compounds
Molecules in the mobile phase reenter the stationary phase when they collide with the stationary phase
M S
At the same time span, molecules leave the stationary phase and enter the mobile phase
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Separation of compounds
The molecules in the mobile phase are carried down the column
M S
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Separation of compounds
All molecules of the same compound travel through the column at nearly the same rate and appear as a band of molecules (called sample band) Compound which is less soluble in the stationary phase moves faster
M S
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Separation of compounds
Goal of gas chromatography :
No overlap between adjacent sample bands as they exit the column
Good chromatography
Analysis time
Analysis time
Make each sample band travel at a different rate Minimize the width of the sample band
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Stronger interaction
Weaker interaction
Stationary phase
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M S
M S
GC Column
GC Column
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M S
M S
GC Column 1
GC Column 2
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M S
Lower T Higher T
M S
GC Column
GC Column
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Basic GC parameters
Retention time (tR) Retention time of unretained compound (tM) Retention factor (k) Distribution constant (K) Phase ratio () Separation factor () Resolution (R) Number of theoretical plates (N) Height equivalent to a theoretical plate (HETP) Carrier gas linear velocity (v)
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tR - tM k= tM
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Phase Ratio ()
The change in the phase ratio can be used to calculate the change in a compounds retention, provided that the same stationary phase and column temperature (program or isothermal) are maintained An increase in phase ratio results in a decrease in retention (k), since K is constant; and vice versa
r 2df
K = k
Separation Factor ()
A measure of the time or distance between the maxima of two peaks = 1 means the two peaks have the same retention and co-elute
k2 k1
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Resolution (R)
A measure of overlap between two peaks; the higher the resolution, the less the overlap Separation () is only the distance between two peak maxima; resolution takes both and the width of the peaks into account Baseline resolution usually occurs at R = 1.50
R = 1.18
R= 2
H=
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Linear Velocity(cm/s)
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5.0
2.5
0.0 5.0
uV(x10,000) 7.5Chromatogram
12.5
15.0
17.5
20.0
min
2.5
5.0
2.5
Canola Oil
uV (x100,000) 1.5 Chromatogram
1.0
Sunflower Oil
Column: Rtx-65TG (30m x 0.25 mm x 0.1um) Carrier gas: Helium
0.5
Olive Oil
Injection method: Split (Split Ratio = 40) Column temp: 500C(0.5min) 500C/min 3500C(0min) 10C/min 3650C(5min) FID temp: 3650C 57
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Kerosene
Light Oil
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Injection volume: 1 ml
1.5
1.0
Acetone Isopropanol
Chloroform
0.5
n-Butanol
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