hl 17.7.g.7. chromatography

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