hl 17.7.g.7. chromatography
TRANSCRIPT
HL Chemistry - Option A :HL Chemistry - Option A : Modern Analytical Modern Analytical
ChemistryChemistry
ChromatographyChromatography
CHROMATOGRAPHY
Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distribution) of sample components between 2 different phases.
One of these phases is a mobile phase and the other is a stationary phase.
Stationary Phase: AluminaStationary Phase: Alumina
O
AlO O
AlO
OH
AlO
OH
AlO
OH
Al
OH
O
Acidic: -Al-OHNeutral: -Al-OH + -Al-O-
Basic: -Al-O-
Stationary Phase: Silica (SiOStationary Phase: Silica (SiO22))
OH
Si
O
OH
SiO
OO
OH
Si
OO
OH
Si
OO
OH
SiO O
O
Si
OO
Si
OO
Si
OO
SiO O
OSi
OO
Si
OO
SiO O
O
Definition:
Different affinity of these 2 components to stationary phase causes the separation.
Concentration of component A in stationary phase
Concentration of component A in mobile phase
Distribution Coefficient (Equilibrium Distribution )
Some Types of Chromatography
1. Liquid Column Chromatography (Reverse Phase too)
2. High Pressure (performance) Liquid Chromatograph (HPLC)
3. Paper Chromatography4. Thin-layer Chromatography (TLC)5. Gas Liquid Chromatography
LIQUID COLUMN CHROMATOGRAPHY
A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.
With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.
A + B + C OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO
Sample (A+B+C)
Column
Solid Particles(packing material- stationary phase)
Eluant (eluate)
DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY
A
B
C
Solvent(mobile or moving phase)
Diagram of Simple Liquid Column ChromatographyDiagram of Simple Liquid Column Chromatography
The 4 basic liquid chromatography modes are named according to the mechanism involved:
1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)
BASIC LIQUID CHROMATOGRAPHY
LIQUID SOLID CHROMATOGRAPHY
Si - O - H
Normal phase LS Reverse phase LS
Silica Gel
The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel.
LIQUID SOLID CHROMATOGRAPHY
Si - OH
HEXANE
OH
C-CH3
CH3
CH3- CCH3
CH3
OH
OH
CH3
CH3
WATER-SOLUBLE VITAMINS
1. Niacinamide 2. Pyridoxine
N
CONH2
N
CH2OHCH2OH
HO
H3C
3. Riboflavin
N
NNH
NCH2
HOCHHOCHHOCH
CH2OH
O
OH3C
H3C
ClN
S
N
NH3C
CH2
NH2
CH3
CH2CH2OH
4. Thiamin
WATER-SOLUBLE VITAMINS
0 5 10 15 20
Column: u Bondapak C18 Solvent: MeOH Sample: Water-Soluble Vitamins
Inject1
2
3
4
LIQUID-LIQUID CHROMATOGRAPHY
ODPN(oxydipropionylnitrile)
Normal Phase LLC Reverse Phase LLC
NCCH3CH2OCH2CH2CN(Normal)CH3(CH2)16CH3 (Reverse)
The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible in the solvent (Mobile) phase. Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation.
MOBILE PHASE LIQUID
Liquid-LiquidChromatography (Partition)
Liquid-SolidChromatography (Adsorption)
Liquid Solid
Normal Phase Reverse Phase Normal Phase Reverse Phase
Mobile Phase - NonpolarStationary phase - Polar
Mobile Phase - PolarStationary phase - Nonpolar
FORMAT
STATIONARYPHASE
Chromatography SchematicChromatography Schematic
ION-EXCHANGE CHROMATOGRAPHY SO3
- Na+
Separation in Ion-exchange Chromatography is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing).
REMEMBER…REMEMBER… The stationary phase is The stationary phase is POLARPOLAR The more polar component interacts The more polar component interacts
more strongly with the stationary more strongly with the stationary phasephase
The more polar component moves The more polar component moves more slowlymore slowly..
The non-polar component moves The non-polar component moves more more rapidly.rapidly.
MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS
SO3-
SO3-
Na+
COO-
H3N+
Na+
COOHH3N
+
pH2
pH4.5
Ion-exchange Resin
H3N+
SO3-
SO3-
SO3-
SO3-
SO3-
SO3-
H3N+
COOH
OH
COOH
COOHH3N+
H3N+OH
COO-Na+
H3N+
COO-
Na+
Na+
H+ OH- = H2O
H+ OH- = H2O
Na+
Na+
pH3.5
Mobile PhaseStationary Phase
Exchange Resin
pH4.5
Chromatography of Amino AcidsChromatography of Amino Acids
GEL-PERMEATION CHROMATOGRAPHY
Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution. Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules.
SOLVENTS
Polar Solvents
Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane > Cyclohexane
SELECTING AN OPERATING MODE
Sample Type LC Mode Positional isomers LSC or LLC
Moderate Polarity Molecules LSC or LLC
Compounds with Similar Functionality LSC or LLC
Ionizable Species IEC
Compounds with Differing Solubility LLC
Mixture of Varying Sized Molecules GCC
1.1. Ultraviolet DetectorUltraviolet Detector
200-400nm 200-400nm 254 nm254 nm
2.2. Reflective Index DetectorReflective Index Detector
Universal DetectorUniversal Detector
DetectorsDetectors
Liquid Chromatography Set Liquid Chromatography Set UpUp
HPLC ChromatographyHPLC Chromatography1.1. Pump System.Pump System. Mobil phase pressures up to 6000 psi are Mobil phase pressures up to 6000 psi are
necessary to achieve reasonable column elution times (~ necessary to achieve reasonable column elution times (~ minutes). Typical flow rates are 0.1 to 10 mL/minute.minutes). Typical flow rates are 0.1 to 10 mL/minute.
2.2. Injection System.Injection System. Used to introduce small samples (0.1 Used to introduce small samples (0.1 to 500 µL) into the carrier stream under high pressure. to 500 µL) into the carrier stream under high pressure.
3.3. Reservoirs (Solvents).Reservoirs (Solvents). Multiple solvents are necessary Multiple solvents are necessary for performing gradient elution's (i.e. changing the polarity for performing gradient elution's (i.e. changing the polarity of the mobil phase during a run). of the mobil phase during a run).
4.4. Chromatographic ColumnChromatographic Column. Typically 10-30 cm in length . Typically 10-30 cm in length containing a packing of 5-10 µm diameter. Many types of containing a packing of 5-10 µm diameter. Many types of columns are available, depending on the type of liquid columns are available, depending on the type of liquid chromatography desired.chromatography desired.
5.5. Detector.Detector. Many types are available including UV, IR, Many types are available including UV, IR, refractive index, fluorescence, conductivity, mass refractive index, fluorescence, conductivity, mass spectrometry, and electrochemical. Diode array detectors spectrometry, and electrochemical. Diode array detectors are used when wavelength scans are desired.are used when wavelength scans are desired.
Schematic of an HPLC Schematic of an HPLC SystemSystem
HPLC SystemHPLC System
Pump SystemPump SystemDesirable Features:Desirable Features: Must generate pressures Must generate pressures
up to 6,000 psiup to 6,000 psi To allow for separation in To allow for separation in
reasonable time framesreasonable time frames Flow-rates range from Flow-rates range from
0.1 to 10 mL/minute0.1 to 10 mL/minute Limited pulsing in the Limited pulsing in the
systemsystem Many HPLC systems have Many HPLC systems have
a dual pump system to a dual pump system to minimize pulsingminimize pulsing
Flow control and Flow control and reproducibility < 0.5%reproducibility < 0.5%
Corrosion resistanceCorrosion resistance
Sample Injection SystemSample Injection SystemUsed to Used to introduce introduce small samples small samples (0.001 to 0.5 (0.001 to 0.5 mL) into the mL) into the carrier stream carrier stream under high under high pressurepressure
HPLC DetectorsHPLC Detectors No universal or versatile detectorNo universal or versatile detector TypesTypes
GeneralGeneral – respond to mobil phase bulk – respond to mobil phase bulk properties which vary in the presence of properties which vary in the presence of solutes (e.g. refractive index)solutes (e.g. refractive index)
SpecificSpecific – respond to some properties of – respond to some properties of the solute (not possessed by the mobil the solute (not possessed by the mobil phase (e.g. UV adsorption)phase (e.g. UV adsorption)
““Hyphenated”Hyphenated” detector – LC-MS detector – LC-MS
Absorbance DetectorsAbsorbance Detectors The UV/Vis source usually comes The UV/Vis source usually comes
from a monochromator so the from a monochromator so the wavelength can be selected, or wavelength can be selected, or scanned.scanned.
Absorbance increases as eluate Absorbance increases as eluate passes through the cell.passes through the cell.
If wavelength scanning is desired, If wavelength scanning is desired, the flow is stopped long enough for the flow is stopped long enough for the scan to take place.the scan to take place.
It’s possible to have the same It’s possible to have the same setup using IR light, although not setup using IR light, although not as common since many useful as common since many useful solvents are not IR transparentsolvents are not IR transparent..
Diode Diode Array Array
DetectoDetectorr
HPLC DetectorsHPLC Detectors
HPLC ColumnHPLC Column Must operate in high Must operate in high
pressurepressure Usually constructed of metalsUsually constructed of metals
Typical dimensionsTypical dimensions 10-30 cm long10-30 cm long 1-3 cm ID1-3 cm ID
Contains packing material Contains packing material which holds the stationary which holds the stationary phasephase Many types existMany types exist Typical packing materials are 5-Typical packing materials are 5-
10 µm in diameter10 µm in diameter Guard column used to Guard column used to
extend life of main columnextend life of main column
Type of Type of HPLC HPLC
Depends Depends on:on:1.1. Molecular Molecular
weight of weight of solutesolute
2.2. Water solubility Water solubility of the soluteof the solute
3.3. Polarity of the Polarity of the solutesolute
4.4. Ionic/non-ionic Ionic/non-ionic character of character of the solutethe solute
Separation Principles in Separation Principles in HPLCHPLC
General Rule of Thumb:General Rule of Thumb:Polarity of analytes ≈ polarity of stationary Polarity of analytes ≈ polarity of stationary phase ≠ polarity of mobile phasephase ≠ polarity of mobile phase
To achieve good separation, the analytes To achieve good separation, the analytes should interact with the stationary phase, should interact with the stationary phase, but not too strongly or the retention time but not too strongly or the retention time will become very longwill become very long
Increasing Mobil phase Polarity, Decreases Elution Time
Reversed orderof elution
Typical Applications of Typical Applications of HPLC ChromatographyHPLC Chromatography
Field of ApplicationField of Application SeparationSeparationPharmaceuticalsPharmaceuticals Antibiotics, Sedatives, Steroids, Antibiotics, Sedatives, Steroids,
AnalgesicsAnalgesicsBiochemicalBiochemical Amino acids, Proteins, Carbohydrates, Amino acids, Proteins, Carbohydrates,
LipidsLipidsFood ProductsFood Products Artificial Sweeteners, Antioxidants, Artificial Sweeteners, Antioxidants,
PreservativesPreservativesIndustrical ChemicalsIndustrical Chemicals Condensed Aromatics, Surfactants, Condensed Aromatics, Surfactants,
Propellants, DyesPropellants, DyesForensic ChemistryForensic Chemistry Drugs, Poisons, Blood Alcohol, Drugs, Poisons, Blood Alcohol,
narcoticsnarcoticsClinical MedicineClinical Medicine Bile Acids, Drug Metabolites, Urine Bile Acids, Drug Metabolites, Urine
Extracts, EstrogensExtracts, EstrogensPollutantsPollutants Pesticides, Herbicides, Phenols, PCBsPesticides, Herbicides, Phenols, PCBs
HPLC of Orange Juice Compounds
How to Increase HPLC Resolution
1. Increase column length2. Decrease column diameter3. Decrease flow-rate4. Pack column uniformly5. Use uniform stationary phase (packing material)6. Decrease sample size7. Select proper stationary phase8. Select proper mobile phase9. Use proper pressure10. Use gradient elution
Separating Proteins from MixturesIn order to understand and study proteins it is essential to separate them from the biological fluid.
Proteins can be separated from each other based on differences in physical properties
Due to different amino acid sequences proteins differ in solubility, size, charge, and binding affinity and can be separated on either of these properties.
The inside of a cell. White shapes are proteins (several 10s of thousands per cell).
Water, Chemical bonds and groups Amino acids, pH dependence
C
COO-
H R
H3N+
Protein primary sequence, peptide bonds, secondary structures
Protein studies: Understanding protein structure and function relationships
All proteins have a distinctive 3D structural conformation
This unique structure enables its function
Amino acid sequence determines structure
A major goal of biochemistry is to determine how amino acid sequences specify the 3D conformations of proteins and to catalogue all proteins in cells.
Characterization
cell
Protein purification: general experimental setup
Homogenize
Centrifugation
Column chromatography
Characterization
Gel permeation chromatography: separating on basis of size
Mixture of proteins1. A mixture of proteins in a small volume
is applied to a column filled with porous beads
2. Because large proteins cannot enter the beads, they emerge sooner than do small ones
3. A detector (e.g. UV) is used to detect protein fragments
4. Fragments are collected separately
UV
time
Affinity Chromatography: separating on the basis of affinity
XX X
X
XX
XX
To separate proteins that recognize a chemical group X
1. X is covalently attached to beads that are packed in a column
2. Sample of proteins is added3. Washed with buffer to remove non
specifically bound protein4. Eluted with high concentration of
soluble X
XXX
X X
X X X
X
Separation on the basis of charge
All proteins are charged. Their charges depend on the relative number of acid and basic amino acids in their primary structure.
All proteins have a pH value where they are uncharged: the isolelectic point (pI)
H2N- Met Ala Asn Cys His Glu Ser Thr Glu Arg-COOH
Ionic amino acids
Separation on the basis of charge (continued)
H2N- Met Ala Asn Cys His Glu Ser Thr Glu Arg-COOH
His: 6.0Glu: 4.1Arg: 12.5N-terminal amine: 8.0C-terminal acid: 3.1
For this peptide:pI=pKa/N= 6.3
Positively charged at pH < 6.3
Negatively charged at pH > 6.3
Ion Exchange Chromatography: separation on basis of net charge
++ +
+
++
++
---+
---+ ---- +
+
+
-
---
--
--
---
--
-
--
--
- --
----
1. Positive or negatively charged resin can be used for separation of positive or negatively charged proteins
2. Sample of proteins is added
3. Washed with buffer to remove non specifically bound protein
4. Elute with increasing concentration of salt
5. Proteins with highest net charge come of last
Why hydrogels are used for protein separations
1. Correct protein folding in aqueous environment2. Proteins can denature on surfaces3. Hydrogels are >90% water, good environment for proteins
1. 2.
3.
In Normal Phase Liquid Chromatography:
The column packing in the column is very polar!
Polar compounds are going to be attracted to the polar column packing by hydrogen bonding or dipole-dipole attractions. Polar compounds are going to move slowly!
Non-polar compounds are going to come off the column first, while the polar compounds are going to come off column last.
Usually, one starts will a less polar solvent to removethe less polar compounds, and then you slowly increase the polarity of the solvent to remove the more polar compounds.
Compare Reverse Phase toCompare Reverse Phase to Normal Phase Column Normal Phase Column
ChromatographyChromatography
Reverse Phase Reverse Phase Column ChromatographyColumn Chromatography
The stationary phase (column packing) The stationary phase (column packing) is nowis now NON-POLARNON-POLAR
Non-polar compounds will move more Non-polar compounds will move more slowly because they are attracted to the slowly because they are attracted to the column packing.column packing.
The more polar component moves The more polar component moves more more quickly down the columnquickly down the column. .
Polar solvents, such as water and Polar solvents, such as water and methanol are used in reverse phase methanol are used in reverse phase chromatographychromatography
Used mainly in columns, such as HPLCUsed mainly in columns, such as HPLC
Reverse phase Reverse phase chromatographychromatographySilica is alkylated with long chain hydrocarbon groups, using 18
carbons long. This is usually referred to as C-18 silica.
O
Si
O
O
SiO
OO
O
Si
OO
O
Si
OO
O
SiO O
O
Si
OO
Si
OO
Si
OO
SiO O
OSi
OO
Si
OO
SiO O
O
CH2
CH3
17Si
CH3
CH3
CH2
CH3
17Si
CH3
CH3SiCH3)3
SiCH3)3SiCH3)3
Summary of Methodology
One of the main aims of biochemistry is to characterize and catalogue all proteins in the cell
We have discussed some important tools for separating proteins based on physical properties such as size, affinity, charge.
Chromatography methods: ion exchange, affinity, gel permeation chromatography
Electrophoresis: iso electric focusing, SDS PAGE, 2D gels (in the Biochemistry lecture series)
Overview of Paper Overview of Paper ChromatographyChromatography
Works on principle of Works on principle of Partition. Partition.
Separates dried liquid Separates dried liquid samples.samples.
Mobile phase is solvent Mobile phase is solvent used.used.
Stationary phase is water Stationary phase is water bound to surface of paper.bound to surface of paper.
Advantage : its cheap!Advantage : its cheap!
Important Concepts in Important Concepts in Paper ChromatographyPaper Chromatography
Capillary ActionCapillary Action – – the movement of liquid within the the movement of liquid within the spaces of a porous material due to the forces of adhesion, spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquidcohesion, and surface tension. The liquid is able to move up is able to move up the filter paper because its attraction to itself is stronger than the filter paper because its attraction to itself is stronger than the force of gravity. the force of gravity.
SolubilitySolubility – the degree to which a material (solute) dissolves – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents. solutes to be separated by different combinations of solvents.
Separation of components depends on both their solubility in Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile the mobile phase and their differential affinity to the mobile phase and the stationary phase.phase and the stationary phase.
Paper/TLCChromatographyAnimation
Simple Example of Paper Simple Example of Paper Chromatography using “Sharpie” Chromatography using “Sharpie”
PensPens
Dye Separation in a Black Dye Separation in a Black “Sharpie”“Sharpie”
Concentration of Isopropanol
0% 20% 50% 70% 100%
1. Dyes separated – purple and 1. Dyes separated – purple and blackblack
2. Not soluble in low 2. Not soluble in low concentrations of isopropanolconcentrations of isopropanol
3. Partially soluble in 3. Partially soluble in concentrations of isopropanol concentrations of isopropanol >20%>20%
Thin Layer ChromatographyThin Layer Chromatography Works mainly on Works mainly on
principle of principle of adsorption.adsorption.
Mobile phase is the Mobile phase is the solventsolvent
Stationary phase is Stationary phase is the solid on the plate.the solid on the plate.
TLC vs. Column ChromatographyTLC vs. Column Chromatography
Thin-layer chromatography and column Thin-layer chromatography and column chromatography and are different types chromatography and are different types of of liquid chromatographyliquid chromatography. .
The mobile (moving) phase is a liquid. The mobile (moving) phase is a liquid. The stationary phase is usually silica or The stationary phase is usually silica or alumina. This phase is alumina. This phase is very polar.very polar.
The principle of operation is the same!The principle of operation is the same!
Thin Layer ChromatographyThin Layer ChromatographyThe surface of a plate consists of a very thin layer of silica on a plastic or aluminum backing. The silica is very polar. This is the stationary phase. Material is spotted at the origin (bottom) of the TLC plate.
The plate is placed into a glass jar with a small amount of a solvent in a glass jar. This solvent acts as the moving phase.
The plate is removed from the bottle when the solvent is close to the top of the plate.
The spots are visualized (explanation to follow).
Non-polar compounds will be less strongly attracted to the plate and will spendmore time in the moving phase. This compound will move faster and will appear closer to the top of the plate.
Polar compounds will be more strongly attracted to the plate and will spend lesstime in the moving phase and appear lower on the plate.
Thin-Layer Chromatography: A Thin-Layer Chromatography: A Two-Component MixtureTwo-Component Mixture
More polar!
Less polar!
solvent frontorigin mixture
solvent front
component B
component A
origin
solvent front
component B
component A
origin
Increasing Development Time
Thin-Layer Chromatography: Thin-Layer Chromatography: Determination of Determination of RRff Values Values
solvent front
component B
component A
origin
dSdB
dA
Rf of component A =
dA
dS
Rf of component B =
dB
dS
The Rf value is a decimal fraction, generally only reported to two decimal places
Thin-Layer Thin-Layer ChromatographChromatography: Qualitative y: Qualitative
AnalysisAnalysis
A B unknown
Visualization MethodVisualization Method The previous slide shows colored spots. The previous slide shows colored spots.
Most of the time, the spots won’t show Most of the time, the spots won’t show unless they are visualized!unless they are visualized!
Vizualization is a method that is used to Vizualization is a method that is used to render the TLC spots visible.render the TLC spots visible.
A visualization method can be:A visualization method can be: Ultraviolet lightUltraviolet light Iodine vapors to stain spotsIodine vapors to stain spots Colored reagents to stain spotsColored reagents to stain spots Reagents that Reagents that selectively selectively stain spots while stain spots while
leaving others unaffected.leaving others unaffected.
TLC AdvantagesTLC AdvantagesAdvantages over paper:Advantages over paper: Its fasterIts faster It gives a better separation.It gives a better separation. It is more versatile as the solid on the It is more versatile as the solid on the
plate can be varied.plate can be varied.
Uses of TLCUses of TLC To determine how many components To determine how many components
there are in a mixture (is it really pure?)there are in a mixture (is it really pure?) To determine the best solvent conditions To determine the best solvent conditions
for separation on a columnfor separation on a column To identify the substances being studiedTo identify the substances being studied To monitor the composition of fractions To monitor the composition of fractions
collected from column chromatographycollected from column chromatography To monitor the progress of a reactionTo monitor the progress of a reaction
Gas-Liquid ChromatographyGas-Liquid Chromatography Works on principle of Partition.Works on principle of Partition. Mobile phase is the carrier gas.Mobile phase is the carrier gas. Stationary phase is oil bound to Stationary phase is oil bound to
surface of beads within the column.surface of beads within the column.
Most Common Stationary Phases
1. Separation of mixture of polar compoundsCarbowax 20M (polyethylene glycol)
2. Separation of mixtures of non-polar compoundsOV101 or SE-30 (polymer of methylsilicone)
3. Methylester of fatty acidsDEGS (diethylene glycol succinate)
Gas-Liquid ChromatographyGas-Liquid Chromatography
Gas-Liquid ChromatographyGas-Liquid Chromatography Retention time is used to identify a Retention time is used to identify a
component of a mixture. It depends component of a mixture. It depends on:-on:-
The temperature of the column.The temperature of the column. The length of the column.The length of the column. The material used to pack the column.The material used to pack the column. The gas pressure.The gas pressure.
Gas –Liquid Gas –Liquid ChromatographyChromatography
The area under a peak is proportional to The area under a peak is proportional to the amount of substance present.the amount of substance present.
Filters/Traps
Air
Hydrogen
Gas C
arrier
Column
Gas ChromatographyGas Chromatography
gas systemgas system inletinlet columncolumn detectordetector data data
systemsystem
Data system
Syringe/Sampler
Inlets
Detectors
Regulators
H
RESET
Schematic Diagram of Flame Ionization Schematic Diagram of Flame Ionization DetectorDetector
Collector
Jet
Flame
Detector electronics
- 220 volts
Column
Chassis ground
Signal output
Gas-Liquid ChromatographyGas-Liquid Chromatography Gas-Liquid Chromatography is often combined Gas-Liquid Chromatography is often combined
with mass spectroscopy. The GC separates with mass spectroscopy. The GC separates the components then the MS analyses them.the components then the MS analyses them.
One possible Use of GC:
SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS
C
C
CDetector Response
Retention Time
14
16
18 Peak Area (cm )
Sample Concentration (mg/ml)
2
4
6
8
10
0.5 1.0 1.5 2.0 2.5 3.0
2
The content % of C fatty acids =C
C + C + C
= the content % of C fatty acids14
14
Gas Chromatogram of Methyl Esters of Fatty Acids
Another GC Use:
TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS Response
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane1.6 min = RT
Response
Unknown compound may be Hexane
1.6 min = RT
Retention Time on Carbowax-20 (min)
GLC ADVANTAGES
1. Very good separation
2. Time (analysis is short)
3. Small sample is needed - l
4. Good detection system
5. Quantitatively analyzed
DISADVANTAGES OF GAS CHROMATOGRAPHY
Material has to be volatilized at 250º C without decomposition! R C OH CH3OH H2SO4
O
R C O CH3
O
CH2 O C R
CH O C R
CH2 O C R
O
O
O
CH3OH
O
R C O CH3
CH3ONa
Fatty Acids Methylester
Reflux
+ 3
Volatile in Gas Chromatography
Volatile in Gas Chromatography
+ +
Summary of Some Chromatographic TechniquesSummary of Some Chromatographic TechniquesTechniqueTechnique Stationary PhaseStationary Phase Mobile PhaseMobile Phase Typical ApplicationTypical Application
PaperPaper Trapped water Trapped water in the paperin the paper Organic SolventOrganic Solvent
amino acid amino acid mixturesmixtures food colors or food colors or dyesdyes
Thin LayerThin Layer Oxide CoatingOxide CoatingOrganic SolventOrganic Solvent
detect amino detect amino acidsacids composition of composition of dyes and food dyes and food colorscolors
ColumnColumn Oxide packing Oxide packing or resinor resin Organic SolventOrganic Solvent
preparativepreparative separation of separation of plant pigmentsplant pigments
Gas-LiquidGas-Liquid Oxide or Oxide or volatile liquid volatile liquid on a solid on a solid supportsupport
GasGas analysis of oil analysis of oil mixturesmixtures detect drugs & detect drugs & steroidssteroids fruit estersfruit esters
High Performance High Performance LiquidLiquid
Oxide Packing Oxide Packing or Resin or or Resin or Molecular SieveMolecular Sieve
LiquidLiquid analyze foods, analyze foods, pesticides, etcpesticides, etcdetect iron in detect iron in body fluidsbody fluidsdetect blood detect blood alcoholalcohol