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Fundamentals of Gas Chromatography

Customer Support Centre Shimadzu Asia Pacific Pte. Ltd. Singapore 2005

Gas Chromatography (1)


Chromatography

Separated, individual compounds Mixture of compounds Data analysis

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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Gas Chromatography (2)

Mixture

Stationary phase

Mobile (moving) 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

Instrument used for gas chromatographic analysis is known as Gas Chromatograph

Construction of a Gas Chromatograph (GC-2010)


Sample in microsyringe
Detector gas(es) flow controller (APC, 1 per detector) Carrier gas flow controller (AFC, 1 per injector)

Detector Injector

Carrier gas supply (e.g. Helium, Nitrogen, Hydrogen)

Data System

Oven

Column

= flow of carrier gas

Instrument control Data acquisition Data processing Data storage

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

If analytes travel through the column at different rates, separation is achieved


the fastest moving analyte exits (elutes) the column first, followed by the remaining analytes in corresponding order

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

GC Sample Introduction Techniques


Liquid and gas samples can be introduced to gas chromatograph. Most common technique: liquid injection Sample is diluted in solvent and injected to GC Injection Port by a syringe (volume of injection normally 1-2 ul). Other techniques:
Introduction of gas sample by gas tight syringe or gas Valve Headspace SPME (Solid Phase Microextraction) Pyrolysis

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

AOC-20i for liquid injection


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Automated Sampling

AOC-20i+s for liquid injection (higher throughput)

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Automated Sampling

AOC-5000: - Liquid - Headspace - SPME

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Main GC components & Their Functions


Gas flow controllers Injector Column Detector Data system (PC w/ GCsolution software)

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Gas Flow Controllers


Control the supply of gas(es) to the GC Electronic flow controller
Convenient, precise Electronic flow controllers:
GC-2010 & GC-2014 carrier gas GC-2010 detector gases GC-2014 detector gases (option)

Manual flow controller


More economical
Carrier Gas Flow Controller Injector Detector

Manual flow controllers:


GC-2014 detector gases (standard)
Detector Gas(es) Flow Controller

Hydrogen

Carrier Gas

GC

Oven

Air

Column

Make up

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Injection Port (Injector)


Where sample is introduced to GC Turns or keep sample in vapor form Mixes sample vapor with carrier gas

Injector
Sample Flow Controller Detector

Carrier Gas supply

GC

Column

Data System Oven


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Column
Separate mixture of compounds into individual components

Carrier Gas Flow Controller

Column Injection Port Detector Analyst

Carrier Gas supply

Data System

GC

Oven
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GC Column
Capillary Column
Carrier gas

Packed Column

Carrier gas Tubing - Fused silica - Metal Stationary phase

Glass or stainless steel Stationary phase (thin film) Solid support material
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Separation in column
From Injection Port

Retention Time = time spent by a compound inside the column

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

Carrier Gas Flow Controller

Detector
Injector

Detector Gas(es) Flow Controller

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

TCD (Thermal Conductivity Detector)


Detects inorganic gases and organic compounds General detector

ECD (Electron Capture Detector)


Selective detector For higher sensitivity detection of electrophilic compounds
Example of analysis: analysis of PCBs (polychlorinated biphenyls) analysis of PBBs (polybrominated biphenyls) analysis of PBDEs (polybrominated diphenyl ethers) analysis of organochlorine pesticides (DDT, BHC, etc.)
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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

FTD 2010 (Flame Thermionic Detector)


Selective detector For analysis of organic nitrogen and phosphorus containing organic compounds Example of analysis:
analysis of nitrosamines analysis of organophosphorous and nitrogen-containing pesticides
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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

Flow Controller Injection Port Detector

Data System

Carrier Gas supply

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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Does this sample contain pesticides???

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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.

Chromatogram of Standard Sample


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

Standard Sample

Chromatogram of Unknown Sample


uV (x10,000) 1.50 Chromatogram 1.25 1.00

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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How much pesticides are contained in this sample???

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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

M = mobile phase (carrier gas) S = stationary phase


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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

The process is repeated many many times inside the column

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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

Poor chromatography Coeluting peaks

Analysis time

Analysis time

Make each sample band travel at a different rate Minimize the width of the sample band
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Migration rates of compounds in column (1)


Different migration rates of compounds can be achieved if these compounds have different interaction strengths with the stationary phase

Stronger interaction

Weaker interaction

Stationary phase
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Migration rates of compounds in column (2)

Migration rate of compounds in column depend on:


Compound chemical structure Stationary phase chemical structure Column temperature

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Effect of compound chemical structure on migration rate

M S

M S

GC Column

GC Column
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Effect of stationary phase on migration rate

M S

M S

GC Column 1

GC Column 2
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Effect of column temperature on migration rate

M S
Lower T Higher T

M S

GC Column

GC Column

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Sample Band Width

Sample band width depends on:


Operating conditions Dimensions of the 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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Retention Time (tR)


The time an analyte takes to travel through the column A measure of the amount of time an analyte spends in the column Sum of the time spent in the stationary phase and the mobile phase

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Retention time of an unretained compound (tM)


The time an unretained compound to travel through the column Unretained compound travels down the column at the same rate as the mobile phase (carrier gas) Equivalent to the time a compound spends in the mobile phase

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Retention factor (k)


Another measure of retention Ratio of the amount of time a compound spends in the stationary and mobile phases A measure of retention by the stationary phase Previously called capacity factor, or partition factor

tR - tM k= tM

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Distribution constant (K)


Ratio of analyte concentration in the stationary phase and mobile phase K is constant for a given compound, stationary phase, and column temperature
cS K= cM

cS = concentration in stationary phase cM = concentration in mobile phase

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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

r = column radius (m) df = film thickness (m)


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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

tR2 tR1 wh1 + wh2

R= 2

tR2 tR1 wb1 + wb2

wh = peak width at half peak height wb = peak width at base


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Number of theoretical plates (N) or Column Efficiency


Theoretical plates is a concept Theoretical plates numbers are an indirect measure of peak width for a peak at a specific retention time Columns with high N are considered to be more efficient than those with lower N A column with a high N will have a narrower peak at a given retention time Column efficiency is a function of:
Column dimensions Type of carrier gas and its average linear velocity Compound and its retention
N = 5.545 tR wh N = 16 tR wb
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Height equivalent to a theoretical plate (HETP or H)


Another measure of column efficiency Small plate heights indicate higher efficiency
L N L = column length (mm) N = theoretical plates number

H=

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Carrier Gas Linear Velocity (v)


Affects the chromatographic resolution (i.e. separation) For each gas there is a linear velocity where optimum separation can be achieved (minimum HETP)
Van Deemter Curve

Linear Velocity(cm/s)

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uV (x10,000) 7.5 Chromatogram

Analyses of Vegetable Oils


7.5 uV (x10,000)

5.0

2.5

0.0 5.0

uV(x10,000) 7.5Chromatogram

12.5

15.0

17.5

20.0

min

Soya Bean Oil

2.5

5.0

0.0 12.5 15.0 17.5 20.0 min

2.5

Canola Oil
uV (x100,000) 1.5 Chromatogram

0.0 12.5 15.0 17.5 20.0 min

1.0

Sunflower Oil
Column: Rtx-65TG (30m x 0.25 mm x 0.1um) Carrier gas: Helium

0.5

0.0 12.5 15.0 17.5 20.0 min

Linear velocity: 40cm/s (constant linear velocity) Injector temp: 3650C

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

Aromatic Components of Tea by Headspace-GC

Analysis of Refined Green Tea

Analysis of Coarse Green Tea

Analysis of Black Tea


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Analysis of Spearmint Oil Analysis of Peppermint Oil

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Analyses of Kerosene and Light Oil

Kerosene

Light Oil

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Headspace-GC Analysis on Phenols in Electrical Wiring Surfaces

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Chromatograms of two different swimming pools

Chromatograms of a swimming pool consisting of sea water


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Determination of Trihalogenated Methanes by ECD Detector

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Analysis of Anti-Epylepsy Drug

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Analysis of Cold Medicine

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Analysis of Solvents by Headspace-GC


Instrument: GC equipped with Headspace Sampler Injector temp:1800C Pressure: 24.9 kPa Split Ratio: Column: 3 Rtx-624, 30m x 0.53mm x 3um

Carrier gas: Helium FID temp: 2200C

Column flow: 4.89 ml/min

Injection volume: 1 ml

Column temp:380C(2min) 10C/min 4000C(6min) 200C/min 2000C(7min)


uV(x10,000) Benzene Chromatogram 2.0 Dichloromethane Dichloromethane Trichloroethylene Hexane Toluene 15.0

1.5

1.0

Acetone Isopropanol

Chloroform

0.5

0.0 5.0 7.5 10.0 12.5 min

n-Butanol

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