Chromatography

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Mar 18, 2009 - chromatography provides a way to identify unknown compounds ... Chromatography is a laboratory technique that .... Ion-exchange chromatography ..... 1 ppt. 1 ppb. 1 ppm. 0.1 %. 100 %. Sensitivity. (SIM). (SCAN). ELCD. (X).
Chromatography

What is Chromatography? 2

Derived from the Greek word Chroma meaning colour, chromatography provides a way to identify unknown compounds and separate mixtures

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What is Chromatography? 3

Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components.

• Analyze Separate

• Identify • Purify

Mixture HPRC

Components

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Uses for Chromatography Chromatography is used by scientists to: • Analyze – examine a mixture, its components, and their relations to one another

• Identify – determine the identity of a mixture or components based on known components

• Purify – separate components in order to isolate one of interest for further study

• Quantify – determine the amount of the a mixture and/or the components present in the sample

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Uses for Chromatography Real-life examples of uses for chromatography: • Pharmaceutical Company – determine amount of each chemical found in new product

• Hospital – detect blood or alcohol levels in a patient’s blood stream

• Law Enforcement – to compare a sample found at a crime scene to samples from suspects

• Environmental Agency – determine the level of pollutants in the water supply

• Manufacturing Plant – to purify a chemical HPRC needed to make a product

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Applications of Chromatography 6

separating mixtures of compounds

Forensics

Research

Pharmaceutical industry

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identifying unknown compounds establishing the purity or concentration of compounds monitoring product formation in the pharmaceutical and biotechnology industries 3/18/2009

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Definition of Chromatography Detailed Definition:

Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass.

Terminology: • Differential – showing a difference, distinctive • Affinity – natural attraction or force between things • Mobile Medium – gas or liquid that carries the components (mobile phase)

• Stationary Medium – the part of the apparatus that does © RGR not move with the sample (stationary phase)

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Introduction to Chromatography 8

Definition Chromatography is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium. Components: mobile phase: a solvent that flows through the supporting medium stationary phase: a layer or coating on the supporting medium that interacts with the analytes supporting medium: a solid surface on which the stationary phase is bound or coated HPRC

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CHROMATOGRAPHY

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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.

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10 Distribution Coefficient (Equilibrium Distribution )

Definition: Concentration of component A in stationary phase Concentration of component A in mobile phase

Different affinity of these 2 components to stationary phase causes the separation.

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Definition of Chromatography 11

Simplified Definition: Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase.

Explanation: • • • •

Compound is placed on stationary phase Mobile phase passes through the stationary phase Mobile phase solubilizes the components Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases © RGR

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Illustration of Chromatography 12 Stationary Phase

Separation

Mobile Phase

Mixture

Components

Components

Affinity to Stationary Phase

Affinity to Mobile Phase

Blue

----------------

Insoluble in Mobile Phase

Black





Red





Yellow



        

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Milestones in Chromatography

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1903 Tswett - plant pigments separated on chalk columns 1931 Lederer & Kuhn - LC of carotenoids 1938 TLC and ion exchange 1950 reverse phase LC 1954 Martin & Synge (Nobel Prize) 1959 Gel permeation 1965 instrumental LC (Waters) HPRC

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Types of Chromatography… 14

Thin layer

Paper

© RGR

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Gas

Column

Chromatographic methods classification

(1) Phases involved

1.) 16 The primary division of chromatographic techniques is based on the type of mobile phase used in the system: Type of Chromatography  Gas chromatography (GC)  Liquid chromatograph (LC)

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Type of Mobile Phase gas liquid

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Classification based on Mobile Phase Liquid chromatography (LC)

Column (gravity flow)

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High performance (pressure flow)

Thin layer (adsorption)

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(2) Geometry of the system 18

 In column chromatography the stationary phase is contained in a tube called the column.  Planar chromatography. In this geometry the stationary phase is configured as a thin two-dimensional sheet. (i) In paper chromatography a sheet or a narrow strip of paper serves as the stationary phase. (ii) In thin-layer chromatography a thin film of a stationary phase of solid particles bound together for mechanical strength with a binder, such as calcium sulfate, is coated on a glass plate or plastic or metal sheet. HPRC

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(3) Mode of operation 19  Development chromatography. In terms of operation, in development chromatography the mobile phase flow is stopped before solutes reach the end of the bed of stationary phase. The mobile phase is called the developer, and the movement of the liquid along the bed is referred to as development. Example of planar development chromatography – TLC or PC

 Elution chromatography. This method, employed with columns, involves solute migration through the entire system and solute detection as it emerges from the column. The detector continuously monitors the amount of solute in the emerging  mobile-phase stream—the eluate—and transduces the signal, most often to a voltage, which is registered as a peak on a strip-chart recorder.

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(4) Retention mechanism 20

Classification in terms of the retention mechanism is approximate, because the retention actually is a mixture of mechanisms. The main interactions are: RF and RT.

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2.) Further divisions can be made based on the type of 21 stationary phase used in the system: Gas Chromatography Name of GC Method Type of Stationary Phase Gas-solid chromatography solid, underivatized support Gas-liquid chromatography liquid-coated support Bonded-phase gas chromatography chemically-derivatized support

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Liquid Chromatography

22 Name of LC Method Adsorption chromatography Partition chromatography Ion-exchange chromatography Size exclusion chromatography Affinity chromatography

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Type of Stationary Phase solid, underivatized support liquid-coated or derivatized support support containing fixed charges porous support support with immobilized ligand

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i.

Adsorption chromatography. 23

5) Principle of separation

Chromatography in which separation is based mainly on differences between the adsorption affinities of the sample components for the surface of an active solid.

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Molecular exclusion chromatography. 24 A separation technique in which separation mainly according to the hydrodynamic volume of the molecules or particles takes place in a porous non-adsorbing material with pores of approximately the same size as the effective dimensions in solution of the molecules to be separated.

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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. HPRC

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Diagram of Simple Liquid Column Chromatography 26 DIAGRAM OF SI MPLE LIQUI D COLUM N CHROM ATOGRAPH Y Solvent(mobil e or movi ng phase) A +B +C

Sampl e (A+B+C)

OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO Column OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO Solid Particl es OOOOOOOOOOO (packing materialOOOOOOOOOO stationary phase) OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO

OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOA OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOB OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOC OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO

El uant (eluate) © RGR

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i.

Partition chromatography. 27

Chromatography in which separation is based mainly on differences between the solubility of the sample components in the stationary phase (gas chromatography), or on differences between the solubilities of the components in the mobile and stationary phases (liquid chromatography).

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Ion-exchange chromatography.

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Chromatography in which separation is based mainly on differences in the ionexchange affinities of the sample components. Anions like SO3- or cations like N(CH3)3+ are covalently attached to stationary phase, usually a resin

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Affinity29chromatography. The particular variant of chromatography in which the unique biological specificity of the analyte and ligand interaction is utilized for the separation.

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The affinity chromatography 30 Common for all types of affinity chromatography is that an affinity ligand specific for a binding site on the target molecule, is coupled to an inert chromatography matrix. Under suitable binding conditions this affinity matrix will bind molecules according to its specificity only. All other sample components will pass through the medium unadsorbed After a wash step the adsorbed

molecules are released and eluted by changing the conditions towards dissociation or by adding an excess of a substance that displaces the target molecule from the affinity ligand.

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The development of an affinity chromatography method boils down to: 1.Finding a ligand specific enough to allow step elution. 2.Finding conditions for safe binding and release within the stability window of the target molecule and the ligand.

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(A) uses charge, (B) uses pores, and (C) uses covalent bonds to create the differential affinities among the mixture components for the 32 stationary phase.

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Chromatography of Amino Acids Stationary Phase

M obile Phase H3 N

-

SO3 Na+

+

COOH +

Na SO3

OH

-

H3 N

+

COOH Exchange Resi n -

SO3 H3N+ COOH SO3

pH3.5 OH

-

H3 N +

+ -

COO

Na

H

+

-

OH = H 2 O

+

Na SO3

-

H3 N

+ -

COO -

H

+

OH = H 2 O

+

SO3Na

pH4.5 © RGR

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Detectors 34  UV

Single wavelength (filter) [610, 8330]  Variable wavelength (monochromator) [8316, 8325]

Multiple wavelengths (PDA) [555]  Fluorescence [610]  Electrochemical [605]  Mass Spectrometric [8325]

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Properties of a Good Detector  High sensitivity - ∆Response/ ∆Conc’n  Universal or selective response  selectivity - ability to distinguish between species

 Rapid response  Linearity - concentration range over which signal proportional to concentration  Stability with respect to noise (baseline noise) and time (drift)

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Theory of Chromatography 1.) Typical 36 response obtained by chromatography (i.e., a chromatogram): chromatogram - concentration versus elution time

Wh

Wb

Inject Where: tR = retention time tM = void time Wb = baseline width of the peak in time units Wh = half-height width of the peak in time units

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Plate height or height equivalent of a theoretical plate (H or HETP): compare efficiencies of columns with different lengths:

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H = L/N where: L = column length N = number of theoretical plates for the column

Note: H simply gives the length of the column that corresponds to one theoretical plate H can be also used to relate various chromatographic parameters (e.g., flow rate, particle size, etc.) to the kinetic processes that give rise to peak broadening: Why Do Bands Spread? a. Eddy diffusion b. Mobile phase mass transfer c. Stagnant mobile phase mass transfer d. Stationary phase mass transfer e. Longitudinal diffusion

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Application of chromatography  Qualitative analysis  Quantitative analysis - Analysis based on peak height - Analysis based on peak area

• Calibration and standards • The internal standard method • The area normalization method HPRC

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Gas Chromatography (GC)

Principle The organic compounds are separated due to differences in their partitioning behavior between the mobile gas phase and the stationary phase in the column.

Gas Chromatography (GC) 43 Separates the volatiles organic mixtures into individual components. used to

Identify Components

Measure their concentrations

by

by

Appearance time (Retention Time)

Peak size (Height or Area)

6890N GC Overview 44 In this module, you will be familiarized with the basic functional areas of the 6890N GC system.

Gas Chromatography consists of: • Carrier Gas • Inlets • Column • Oven • Detectors • Data Handling Device

45

1. Carrier Gas

Carrier Gas 46 must be inert (free of oxygen and moisture) A high-purity gas with traps for

Water (Moisture)

Hydrocarbons

A purity of at least 99.999% is recommended

Oxygen

Carrier Gas 47

is responsible for (a) Carrying the vaporized sample through the inlets, column, and the detectors. (b) Sample component desorption during the sorption-desorption process inside the column.

Carrier Gas There are many types of carrier gases used in GC analysis. such as

Helium

Nitrogen

Argon/5%Methane

Hydrogen

Note: For capillary applications, some methods recommend using Hydrogen as a carrier gas.

Carrier Gas 48 Contamination in carrier gas may react with

Sample

Column

create

Spurious Peaks

Load the detector

Raise baseline

49

2. Inlets

Inlets 50

It is the most critical heated zone where the sample must be introduced as a vapor into the carrier gas stream. The most common Inlets are

Injection Ports

Sampling Valves

Operating Procedures Liquid samples, in micro-liter volume, are usually injected by a special syringe through a silicon rapper septum onto the heated block, the sample is vaporized as a ''plug'' and carried into the column by the carrier gas stream. As for gas samples, the injector may be used, but only for qualitative analyses not for quantitative analyses, a special gas tight syringe is used for such a kind of this analysis. For quantitative identifications, a Gas Sample Valve (GSV) must be used.

A. Injection Ports 51 (a) Handle gas or liquid samples. (b) Often heated to vaporize liquid samples. (c) The design and choice of injection ports depends on the column

Diameter

Type

Note: Liquid or gas syringes are used to inject the sample through a septum into the carrier gas stream.

Inlet Types

Inlet Type

Gas Control

1.

Split/Splitless Injector

EPC and Non-EPC

2.

Purged Packed Injector

EPC and Non-EPC

3.

Cool On-Column Injector

4.

Programmed Temperature Vaporization (PTV)

5.

Volatiles Interface (VI)

EPC only EPC only EPC only 52

Inlet Types

Inlet Type

Description

1. Split/Splitless Injector

It is called capillary injector as it is used only with capillary columns.

Note There are four injection modes: (a) Split Mode Major component analysis (High Concentration) (b) Splitless Mode

Trace component analysis (Low Concentration)

(c) Pulsed-Split Mode

Allows larger injection volume

(d) Pulsed-Splitless Mode

Allows larger injection volume Faster sample transfer to column Less breakdown/Adsorption

53

Split/Splitless Injector Septum Nut Septum

Liner O-Ring

Cartridge split vent trap Liner Washer/Seal

EPC

54

Inlet Types

Inlet Type

Description

2. Purged-Packed Injector

It is called packed injector as it is used for packed columns analysis.

Note During injection, the sample (1ul or 2ul) is vaporized inside a glass liner (insert) in a very short period of time (milliseconds), thus, it may be called “Flash Injector”. The type of injection mode used in a purged-packed injector is Splitless Mode.

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Purged-Packed Injector 56 Septum Purge Weldment

Septum Glass Liner Sleeve

Heater/Sensor Assembly

Column Adapter

“O” Ring

Insulation Cup Ferrule Column Nut

1/4” Vespel Seal

Inlet Types

Inlet Type 3. Cool On-Column Injector

Description It is called on-column injector as the sample is injected and deposited directly into the column as liquid. When the sample components are thermally unstable and high sensitive is required.

Note Most widely used applications in Environmental.

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Cool On-Column Injector

58 Automated Injection 250/320

Heater/Sensor Assembly

Automated Injection 530

Septum

Manual 200 uM Fused silica needle

Ferrule Column Nut

Column Positioning Inserts

Inlet Types

Inlet Type Description 4. Programmable Temperature It is a temperature Vaporization Injector (PTV) programmable split/splitless inlet. Note Injection into hot or cold inlet - rapid heating/cooling. Large volume injection capability through solvent venting for lower MDL's Less thermal breakdown. Fully integrated into 6890. EPC pneumatics.

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Programmable Temperature Vaporization Injector 60 Septumless Head

Septum Head

Cryo Connection

Split Vent Liner

Silver Seal

Heater/Thermocouple Column Adapter Graphpack Column Ferrule

Split Nut for Inlet Adapter

Inlet Types

Inlet Type 5. Volatiles Interface Injector (VI)

Description It is an ideal inlet for Purge & Trap, Headspace, Thermal Desorption, or other gas injection devices.

Note • Very Inert. • Very small internal volume (35ul) • Low split ratio or direct injection for high sensitivity. • EPC control from GC keyboard or ChemStation.

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Volatiles Interface Injector 62

Sampler Input

Split Vent Trickle Flow

Column Connection

6890N GC Electronic Pneumatic Control (EPC) 63

6890N Capillary Inlet EPC Module

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Electronic Pneumatic Control Capillary column (Constant Column + Makeup Flow Mode)

Inlet Pressure (measured/controlled) Makeup Gas Flow Rate Constant Inlet Pressure

Oven Temperature Actual Column Flow

Makeup gas is programmed during run to compensate for decreasing column flow Net carrier flow (Column + Makeup) is then constant during the GC analysis Important for detectors that are flow dependant - such as TCD/NPD/ECD

1.

To Convert

To

Psi

Bar

0.0689476

kPa Psi kPa Psi Bar

6.89476 14.5038 100 0.145038 0.01

2.

Bar

3.

kPa

Multiply By

Septum Purge The septum purge line is near the septum where the sample is injected. A small amount of carrier gas exists through this line to sweep out any bleed. Each inlet has a different septum purge flow. The GC automatically sets the purge flow for EPC inlets, but you can measure it from the septum purge vent at the flow manifold if you like. 65

Septum Purge Flows Inlet

Carrier

Septum Purge (ml/min)

1.

Split/Splitless

2.

Purged-Packed

3.

Cool On-Column

He, N2, Ar/5%Me

3

H2

6

All

1 to 3

He, N2, Ar/5%Me

15

H2

30 66

Septum Purge Flows

Inlet

Carrier

Septum Purge (ml/min)

4.

Programmed Temp. Vaporization (PTV)

5. Volatiles Interface (VI)

He, N2, Ar/5%Me H2

He, N2, Ar/5%Me H2

3 6

3 6

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B.68Valves Used for quantitative analyses of gas mixtures. Different gas sample loops are available and range from 250ul to 50ml. There are many valving configurations to accommodate each kind of gas analyses requirements.

Four-Port Valves

Six-Port Valves

Eight-Port Ten-Port Valves Valves

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3. Columns

Columns

70

The separation takes place here. Column’s types

Packed Columns

6890 Column

Capillary Columns (Open Tubular)

6850 Column

A. Packed Columns 71

Packed columns contain a finely divided, inert, solid support material (Commonly based on diatomaceous earth) coated with liquid stationary phase. Packed columns are 1.5 – 10m in length and have an internal diameter of 2 – 4mm. Packed columns have high sample capacity and are still useful for gas samples, but capillary columns offer better resolution for most liquid samples. There are two types of packed columns

Glass Packed Column

Stainless Steel Packed Column

B. Capillary Columns 72

Capillary columns is an open tube with the stationary phase coated on its inside surface. There is no packing. Capillary columns have an internal diameter of a few tenths of a millimeter. Capillary columns require smaller samples than packed columns.

There are two types of capillary columns

Wall-Coated Open Tubular (WCOT)

Support-Coated Open Tubular (SCOT)

I.73Wall-Coated Open Tubular (WCOT) Consists of a capillary tube whose walls are coated with liquid stationary phase.

II. Support-Coated Open Tubular (SCOT) The inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been absorbed. Note • SCOT columns are generally less efficient than WCOT columns. • Both types of capillary columns are more efficient than packed columns. • In 1979, a new type of WCOT column was devised, the Fused Silica Open Tubular (FSOT) column.

Fused-Silica Open Tubular (FSOT) 74 These columns have thinner walls than the glass capillary columns, and are given strength by the polyimide coating. These columns are flexible and can be wound into coils.

Advantages 1. Physical strength 2. Flexibility 3. Low reactivity

The Column 75

The purpose of a column is to produce narrow, well-separated peaks from a multi-component sample.

The Column Efficiency A high-efficiency column produces narrow peaks. Efficiency is determined by

The Column Construction (Small tubing diameter and thin stationary phase layer is best)

The Carrier Gas Flow Rate

The Column Selectivity 76

This is less clearly defined property of the stationary phase. Essentially, it is how well a phase differentiates between two compounds.

Low Selectivity (They elute together)

High Selectivity (The peaks separates)

Column Temperature 77

The stationary phase in the column has a preferred temperature range.

The minimum temperature (is usually a melting point)

The maximum temperature (is usually related to a boiling or degradation point)

Below this, you are doing Gas/Solid Chromatography. Above this, you are doing Gas/Liquid Chromatography.

Gas Control Flow in packed columns is usually controlled using mass flow controllers. Capillary columns, because of the very low flow rates, are usually pressurecontrolled.

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4. Oven

Oven 79 Columns are mounted in a temperature controlled oven because separations are highly temperature dependent. The oven temperature

Isothermal

Oven Features

Programmed

Oven

• Temperature range -80C (Liquid N2) or -60C (CO2) to the configured limit. • Maximum temperature 450C. • Temperature programming up to six ramps. • Maximum run time 999.99 minutes. • Temperature ramp rates 0 to 120C/min, depending on instrument configuration. • Oven is configured as fast 2250W or regular 1600W. • AC driven via triac control.

A. Isothermal Oven 80 This is the simplest way to run the oven. The oven remains at the same temperature throughout the analysis.

Advantages 1. The oven is always ready for a sample analysis. 2. There is no recovery time between analysis.

Disadvantages 1. Samples with a wide range of component times take a long time to run.

B. Programmed Oven 81 The oven temperature changes, usually upward during the analysis.

Advantages 1. Analysis time is reduced. 2. Peak shapes are constant throughout the run, making detection and measurement easier.

Disadvantages 1. Components are subjected to higher temperatures than with an isothermal oven. This could cause degradation of sensitive components. 2. The oven must cool to the starting temperature.

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5. Detectors

Detectors 83

It is a heated zone, which is located at the exit of the separation column, which senses the presence of the individual components as they leave the column. Detectors are classified as

Universal Detectors

Selective Detectors

There are three major response characteristics of detectors: 1. Sensitivity 2. Selectivity 3. Dynamic Range

Sensitivity

84 It is the response per amount of sample, that is the slope of the response/amount curve.

Selectivity It is a measure of which categories of compounds will give a detector response.

Dynamic Range It is the range of sample concentrations for which the detector can provide accurate quantitation.

The Gas Chromatography (GC) has several detector systems available: 1. Flame Ionization Detector (FID) 2. Thermal Conductivity Detector (TCD) 3. Nitrogen-Phosphorous Detector (NPD) 4. Flame Photometric Detector (FPD) 5. Electron Capture Detector (ECD)/ Micro-Cell Electron Capture Detector (uECD) 6. Mass Spectrometer Detector (MSD)

Multiple Detectors Detectors can be classified also as:

Destructive Detectors

Non-destructive Detectors

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Detector

Selectivity

Delectability

Dynamic Range

1. FID

Most organic compounds

100pg

10^7

2. TCD

Universal

1ng

10^7

10pg

10^6

100pg

10^3

50fg

10^5

Nitrogen & Phosphorous in compounds Sulphur, 4. FPD Phosphorous, tin, boron, arsenic, germanium, selenium, chromium 5. ECD/uECD Halides, nitrates, nitriles, peroxides, anhydrides, organometallics 3. NPD

86

Common non-destructive detectors are: 1. Thermal Conductivity Detector (TCD) 87Infra Red Detector (IRD) 2. 3. Photo Ionization Detector (PID)

Common destructive detectors are: 1. Flame Ionization Detector (FID) 2. Nitrogen-Phosphorous Detector (NPD) 3. Flame-Photometric Detector (FPD) 4. Mass Spectrometer Detector (MSD) Comparison of GC Detectors

ELCD ELCD

(SorN) (

AED

X)

TCD FID ECD NPD (N) NPD (P) FPD (S) PID MSD

Sensitivity

10-15 fg 1 ppt

(SIM)

10-12 pg 1 ppb

IRD (SCAN) 10-9 ng 1 ppm

10-6 ug 0.1 %

10-3 mg 100 %

grams

Makeup Gas Flow 88

Makeup gas enters the detector close to the end of the column. Its purpose is to speed the peaks through the detector, especially with capillary columns, so that the peak separation achieved by the column is not lost through remixing in the detector.

TCD Detector A TCD detector consists of an electrically-heated wire.The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it. Changes in thermal conductivity, such as when organic molecules displace some of the carrier gas, cause a temperature rise in the element which is sensed as a change in resistance. The TCD is not as sensitive as other detectors but it is non-specific and nondestructive.

ECD Detector Uses a radiactive Beta emitter (electrons) to ionize some of the carrier gas and produces a current between a biased pair of electrodes. When an org. mol. that contains electornegative functional gr., such as halojens, phosphorous and nitro groups, pass by the detector, they capture some of the electrons and reduce the current.

FID Detector Consists of a hydrogen/air flame and a collector plate. The eff. from the GC column passes through the flame, shich breaks down org. mol. and produces ions. The ions are collected on a biased electrode and produce an elec. sig. Extremely sensitive, large dynamic range.

MS Detector Uses the difference in mass-to-charge ratio (m/e) of ionized atoms or molecules to separate them from each other. Molecules have distinctive fragmentation patterns that provide structural information to identify structural components. The general operation of a mass spectrometer is: 1. create gas-phase ions 2. separate the ions in space or time based on their mass to charge ratio 3. Measure the quantity of ions of each mass-to-charge ratio.

MS Detector Cont’d The ion separation power of an MS is described by the resolution: R = m/∆m Where m is the ion mass and ∆m is the difference in mass between two resolvable peaks in a mass spectrum. E.g., an MS with a resolution of 1000 can resolve an ion with a m/e of 100.0 from an ion with an m/e of 100.1.

MS Components  Ionization source  Analyzer  Detector

Ionization Methods  Electron capture (EC)  70 eV e-  neutral molecule  energetic molecular ion  hard; fragmentation

 Chemical ionization (CI)  Reagent ion + molecule  molecular ion + reagent ion  Reagent ion = He, OH- (water), CH5+ or CH3+ (CH4)  soft; less fragmentation

Ionization Methods  Electrospray (ESI)  generation of ions by desolvation or desorption of charged liquid droplets

 Matrix Assisted Laser Desorption (MALDI)  ionization facilitated by laser irradiation of sample dissolved in an organic matrix  EX: sinapinic acid

Types of MS Analyzers  Quadrupole - most common  Ion trap  Time of Flight (TOF)

Two Operational Modes  Scan  Collect mass data over known range  Slow

 Selective ion monitoring (SIM)  Sample mass at predetermined values  Fast

Total Ion Chromatogram tr

Detector Response

time of injection

Retention Time

Mass Spectrum - GC-MS  x-axis  GC-MS - m/z  LC - retention time or volume

 y-axis - detector response  GC-MS - % abundance  LC - Abs

Analysis of Organic Mass Spectral Data Analytical Chemistry

Mass Spectrum  X - axis: m/z  mass - based on 12C ≡ 12.0000  Y - axis: relative abundance  usually normalized wrt largest line (base peak)  0 - 100 %

Molecular Ion  Ion whose mass equals that calculated from the molecular formula using the masses for each element which have the highest natural abundance; often tallest peak in highest m/z group  Base peak - most intense peak in spectrum; not necessarily the molecular ion peak!

28

6.3

29

64

30

3.8

31

100.

32

66.

33

0.98

34

0.14

m/z

CH3OH + e-  CH3OH+ + 2eCH3OH +  CH2OH+ + H CH3OH +  CH3+ + OH CH2OH +  H2 + CHO+

40

1.0

37

17

15

34

0.21

31

16

28

13.

25

15

22

2.4

19

14

16

0.72

13

13

10

0.33

7

12

31

100 90 80 70 60 50 40 30 20 10 0

4

Rel. Abundance

1

m/z

Rel. Abundance, %

Example: Mass Spectrum of Methanol (CH3OH)

Example 2: Mass spectra for cyclophosphamide  Method of sample ionization may also change molecular ion  EI: M +  CI: MH+

Figure taken from Rubinson, K.A. Chemical Analysis Boston: Little, Brown, 1987.

GC Detectors After the components of a mixture are separated using gas chromatography, they must be detected as they exit the GC column. Thermal-conduc. (TCD) and flame ionization (FID) detectors are the two most common detectors on commercial GCs. The others are 1. Atomic-emmision detector (AED) 2. Chemiluminescence detector 3. Electron-capture detector (ECD) 4. Flame-photometric detector (FPD) 5. Mass spectrometer (MS) 6. Photoionization detector (PID)

GC Detectors Cont’d The requirements of a GC detector depend on the separation application. E.g. An analysis may require a detector selective for chlorine containing molecules. Another analysis might require a detector that is nondestructive so that the analyte can be recovered for further spectroscopic analysis. You can not use FID in that case because it destroys the sample totally. TCD on the other hand is non-destructive.

Interpreting the trace

 Calibration – known compounds are added to the column and conditions kept constant.  Amount of substance – area under peak / peak height.  Relative proportions can be determined.

Uses of G.l.c.  Very sensitive - small quantities of substances detected, explosives, drugs etc.  Separation of pure substances for collection.  Can be connected to mass spectrometer for direct identification of substances.

The GC-MS Process

111

The END Produced By Chemist Ahmed said