Introduction to High Performance Liquid Chromatography and Gas Chromatography

Sicences and Reseach campus Islamic Azad University Faculty Of sciences and engineer of Food Renderers: Mohsen Zolmajdi Ali akbar Hasas Alireza Mehrdadfar Professor: Afshin Mirzai Ph.D

In This Section, You Will Learn About :

GC and HPLC The differences between High Performance Liquid Chromatography and Gas Chromatography

The components of the high performance liquid chromatography (HPLC)

The separation process The chromatogram The most common modes of HPLC
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You are Chemical Engineer in Factory and Youve Got a Problem to Solve

I need a quantitative separation of carbohydrates in some of our products as soon as possible.

Ill get on it!

Ill need a separation technique.

Separation Techniques

I Know two separation techniques That common, High Performance Liquid Chromatography and Gas Chromatography. Which should I use?

What is HPLC?
High Performance Liquid Chromatography High Pressure Liquid Chromatography (usually true) Hewlett Packard Liquid Chromatography (a joke) High Priced Liquid Chromatography (no joke) HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time!

What is GC?
Gas Chromatograph GC is premier technique for separation and analysis of volatile compounds Gases, liquids, dissolved solids Organic materials A gas chromatograph is a chemical analysis instrument for separating chemicals in a complex sample. A gas chromatograph uses a flow-through narrow tube known as the column, through which different chemical constituents of a sample pass in a gas stream (carrier gas, mobile phase) at different rates depending on their various chemical and physical properties and their interaction with a specific column filling, called the stationary phase. As the chemicals exit the end of the column, they are detected and identified electronically

Comparison of HPLC and GC

Sample Volatility HPLC

No volatility requirement Sample must be soluble in mobile phase

Sample Polarity HPLC

Separates both polar and non polar compounds PAH - inorganic ions

GC
Sample must be volatile

GC
Samples are nonpolar and polar

Comparison of HPLC and GC

Comparison of HPLC and GC

Sample Thermal Lability HPLC

Analysis can take place at or below room temperature

Sample Molecular Weight HPLC

No theoretical upper limit In practicality, solubility is limit.

GC
Sample must be able to survive high temperature injection port and column

GC
Typically < 500 amu

Comparison of HPLC and GC

Sample Preparation HPLC

Sample must be filtered Sample should be in same solvent as mobile phase

Sample Size HPLC

Sample size based upon column i.d.

GC
Solvent must be volatile and generally lower boiling than analytes

GC
Typically 1 - 5 L

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Comparison of HPLC and GC

Separation Mechanism HPLC

Both stationary phase and mobile phase take part

Detectors HPLC
Most common UV-Vis 3-dimensional detectors Sensitivity to pg

GC
Mobile phase is a sample carrier only

GC
Most common FID, universal to organic compounds

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How can We Analyze the Sample?

Carbohydrates
1. 2. 3. 4. 5. 6. fructose Glucose Saccharose Palatinose Trehalulose isomaltose
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mAU

3 4 6

Zorbax NH2 (4.6 x 250 mm) 70/30 Acetonitrile/Water 1 mL/min Detect=Refractive Index

time

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Separations
Injector

Mixer

Separation in based upon differential migration between the stationary and mobile phases. Mobile Phase - carries the Stationary Phase the stationary sample through - the phase which remains fixed in the phase as it moves through column, e.g. C18, Silica the column.

Pumps

Column

Detector

Solvents

Waste

High Performance Liquid Chromatograph

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

High Performance Liquid Chromatograph

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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How can We Analyze the Sample?

Carbohydrates
1. 2. 3. 4. 5. 6. fructose Glucose Saccharose Palatinose Trehalulose isomaltose
2

mAU

3 4 6

Zorbax NH2 (4.6 x 250 mm) 70/30 Acetonitrile/Water 1 mL/min Detect=Refractive Index

time

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The Chromatogram

to - elution time of unretained peak tR- retention time - determines sample identity tR

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

Injection

HPLC Analysis Parameters

Solvent Reservoirs

Mobile Phases Flow Rate Composition

Autosampler

Degasser
Pump

Column Compartment

Injection Volume Column Oven Temperature Wavelength Time Constant

Detector

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Modes of High Performance Liquid Chromatography

Types of Compounds

Mode

Stationary Phase

Mobile Phase

Neutrals Weak Acids Weak Bases

Ionics, Bases, Acids

Reversed Phase

C18, C8, C4 cyano, amino

Water/Organic Modifiers

Ion Pair Normal Phase Ion Exchange Size Exclusion

C-18, C-8

Water/Organic Ion-Pair Reagent Organics

Compounds not soluble in water Ionics Inorganic Ions

Silica, Amino, Cyano, Diol Anion or Cation Exchange Resin Polystyrene Silica

Aqueous/Buffer Counter Ion Gel FiltrationAqueous Gel PermeationOrganic

High Molecular Weight Compounds Polymers

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HPLC Applications

Bioscience Chemical
polystyrenes dyes phthalates proteins peptides nucleotides

tetracyclines Pharmaceuticals corticosteroids antidepressants barbiturates

Consumer Products
lipids antioxidants sugars

Environmental
polyaromatic hydrocarbons Inorganic ions herbicides

Clinical
amino acids vitamins homocysteine 34

 . ( ) . . HPLC GC

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Refrebces:
http://192.215.107.101/ebn/942/tech/techfocus/1071main.html http://www.chem.usu.edu/~sbialk/Classes/565/opamps/opamps.html Skoog, Holler, and Neiman. Principles of Instrumental Analysis. 5th ed. Orlando: Harcourt Brace & Co., 1998. http://weather.nmsu.edu http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLCHP1090/HPLCIN J.HTM http://testequipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analytical_Instruments/ Chromatographs/HPLC_Columns http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-col.htm

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The End

Thank for Your attention

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