GAS CHROMATOGRAPHY

used for separation of volatile substances, or

substances that can be made volatile, from one
another in a gaseous mixture at high
temperatures
Mobile phase just as carrier – no interaction
between MP and Analyte (compared it with
HPLC)
Substance should be thermostabile
GAS CHROMATOGRAPHY

To Waste or Flow
Meter
Two-Stage
Regulator Syringe Detector
Injector flame ionization

Flow Controller

Carrier Gas Column

nitrogen or helium
Cylinder
 GAS CHROMATOGRAPHY

top view
Flame
Injection Port Ionization
Detector

Column

Oven

front view
 GAS CHROMATOGRAPHY

 Injection Port - Sample introduction

Manual - Direct Injection

Automated - Autosampler
- Purge and Trap
 Oven

Temperature Control
• Isothermal • Gradient

240

200

Temp (deg C) 160

120

80

40

0
0 10 20 30 40 50 60
Time (min)
 GAS CHROMATOGRAPHY

Mobile phase just as carrier – no interaction between MP and Analyte (compared it with HPLC)
GAS CHROMATOGRAPHY
GAS CHROMATOGRAPHY
GAS CHROMATOGRAPHY
GAS CHROMATOGRAPHY
 Comparison of HPLC and GC

Sample Volatility Sample Polarity Sample Thermal lability

HPLC HPLC HPLC
•Separates both polar and •At room or higher
•No need to be volatility non polar compounds temperature

•Sample must be soluble

in mobile phase

GC GC GC
•Samples usually nonpolar •Must be thermostable
•Sample must be volatile
(if not?)
 Comparison of HPLC and GC
Sample MW Separation mech. Detector
HPLC HPLC HPLC
• Both stationary phase • Non destructive
• No theoretical upper limit and mobile phase take • Most common : UV
part
• In practicality, solubility is •
limit.

GC GC GC
• Mobile phase is a • Destructive
• Typically < 500 amu
sample carrier only

13
 Comparison of HPLC and GC

14
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

High Performance Liquid Chromatograph

15
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

16
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

17
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

18
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

19
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

20
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

21
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

22
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

23
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

24
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

25
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

26
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

27
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

28
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

29
 DETECTION AND CHROMATOGRAM
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

30
 Instrumentation - Detectors
Destructive
• Mass Spectral (CI/EI)
• Flame Ionization (FID)
• Nitrogen-Phosphorus (NPD)
• Flame Photometric (FPD)
• Electrolytic Conductivity (Hall/ELCD)
Non-Destructive
• Thermal Conductivity (TCD)
• Electron Capture (ECD) [551.1]
• Photo Ionization (PID) [502.2]
 CHROMATOGRAM OF GC
tR- retention time - determines sample identity QUALITATIVE
to - elution time of unretained peak

tR
QUANTITATIVE

tR
mAU Area is proportional
to the quantity of
analyte.
to

Time (minutes)
 QUALITATIVE ANALYSIS
Melamine Determination in several Milk

a : Melamin Standard 0,5 ppm Look at the retention time of standard and
b : Strawbery Milk compare it with the retention time of
c : strawberry Milk spike 1 ppm sample
d : strawberry Milk spike 2 ppm
 QUALITATIVE ANALYSIS
How if my sample contain a lot of peaks? Which one should I take?
Look at the retention time of standard and
compare it with the retention time of
sample
 QUANTITATIVE ANALYSIS
Using AUC (Area Under Curve)  make calibration curve

Type of calibration curve :

1. External Standard Calibration Curve
2. Internal Standard Calibration Curve
3. Standard Addition Calibration Curve
 QUANTITATIVE ANALYSIS
External Standar Calibration Curve

1. Inject several concentration (minimum

5) into HPLC/GC
2. Record the AUC of each concentration
3. Plot graph (in excel) where the axis is
concentration and the y is AUC
4. Find the regression linear equation from
the graph
5. Inject the sample into HPLC/GC and
AUC

record the AUC in defined Retention

time
Y = mx + h
6. Input the AUC into the equation and
AUC = slope x concentration + h
calculate the concentration

concentration
 QUANTITATIVE ANALYSIS
Using AUC (Area Under Curve) 

4500

4000 C (ppm) AUC RT

3500 5 334.8 15.932
3000 15 1010.3 15.608
2500
25 1670.5 15.588
2000
35 2389.5 15.484
1500
45 3006.4 15.257
1000 y = 69.838x - 46.223
R² = 0.9979 55 3882 15.187
500

0
Sample 2200 15.129
0 10 20 30 40 50 60 Sample 5000 20.000

y = 69.838x - 46.223 2200 = 69.838x – 46.223 X = …..

 QUANTITATIVE ANALYSIS
Internal Standar Calibration Curve

• substance different from the analyte added

in a constant amount to all samples, blanks,
and standards
• Internal standards are used when the
detector response varies slightly from run
to run because of hard to control
parameters. (e.g. Flow rate in a
RATIO

chromatograph)
• Even if absolute response varies, as long as
the relative response of analyte and
Y = mx + h standard is the same, we can find the
AUC = slope x concentration + h analyte concentration.
• Plotting the ratio of analyte to internal-
concentration standard
• Internal standard must not produce signals
that interfere with analyte Usually they are
separated by wavelength or time.
 QUANTITATIVE ANALYSIS
Internal Standar Calibration Curve

• Plotting the ratio of analyte to internal-

standard.
• Internal standard must not produce signals
that interfere with analyte Usually they are
separated by wavelength or time.
• A mixture is prepared with a known amount
of analyte and standard. The detector
RATIO

usually has a different response for each

species

Y = mx + h
AUC = slope x concentration + h

concentration
Area of analyte signal  area of standard signal 
 F 
Concentration of analyte  Concentration of standard 
 QUANTITATIVE ANALYSIS
Internal Standar Calibration Curve
 QUANTITATIVE ANALYSIS
Internal Standar Calibration Curve