Instrum. Pharm. Analys.€¦ · 1. Gas-solid chromatography (GSC): Same material is used as both...

Lecture 5a

by

Sherif S. Ebada, Ph.D.

Instrum. Pharm. Analys.

Contact information:

Assoc. Prof. Dr. Sherif S. Ebada

Pharmaceutical Chemistry Dept.

Office hours:

Mondays and Tuesdays: 12:00–14:00 pm

E-mail: [email protected]

1

3

Course outline

Introduction.

Adsorption (CC & TLC), Flash CC, Chromatotron etc.

Partition (Paper).

Gel Chromatography.

Ion exchange Chromatography.

Affinity Chromatography.

Electrophoresis.

HPLC.

GC.

Spectroscopy.

4

Chromatography

Lecture 5a

GC

5

Course outline Introduction to Gas Chromatography (GC).

History of GC.

Principle of GC.

GC vs. HPLC.

Uses of GC.

Sample Preparation:

• Headspace method.

• Distillation method.

• Solvent extraction method.

• Solid-phase microextraction.

Instrument Overview:

• Carrier gas.

• Injectors.

• Column & Stationary phases.

• Detectors.

• Operating conditions.

Applications of GC.

INTRODUCTION TO

GAS CHROMATOGRAPHY

6

Gas Chromatography: partition between an inert carrier gas (He, N2, or

H2) mobile phase and solid/liquid stationary phase.

Samples (Analytes) can be gases or volatile liquids (only gases can go

through the column).

GC replaced distillation as the preferred method for separating volatile

materials.

GC is most widely used analytical technique in the world.

GC is premier technique for separation and analysis of volatile

compounds.

Gases, liquids, and dissolved solids.

Organic and inorganic materials.

MW from 2 to > 1,000 Daltons.

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INTRODUCTION TO GAS CHROMATOGRAPHY (GC)

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Carrier Gas or Mobile phase doesn’t affect solute retention, but does

affect:

1.) Desired efficiency for the GC System:

↓ M. wt. gases (He, H2) → ↑ diffusion coefficients.

↓ M. wt. gases (He, H2) → faster, more efficient separation.

2.) Stability of column and solutes:

H2 or O2 can react with functional groups on solutes and stationary phase or

with surfaces of the injector, connections and detector.

3.) Response of the detector:

Thermal conductivity detector requires H2 or He.

Other detectors require specific carrier gas → compatibility.

9


Stationary phases in GC is the main factor determining the selectivity &

retention of solutes.

There are three types of stationary phases used in GC:

Solid adsorbents (GSC).

Liquids coated on solid supports (GLC).

Bonded-phase supports.

1. Gas-solid chromatography (GSC):

Same material is used as both the stationary phase and support material.

Common adsorbents include alumina, molecular sieves (crystalline

aluminosilicates [zeolites]), silica, active carbon.

Advantages:

• Long column lifetimes.

• Ability to retain and separate some compounds not easily resolved by

other GC methods, e.g. geometrical isomers and permanent gases.

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OH

Si

O

OH

Si

OO

O

OH

Si

OO

OH

Si

OO

OH

Si

OO

O

Si

OO

Si

O

OSi

OO

Si

OO

O

Si

OO

Si

OO

Si

OO

O



Silica (SiO2)

O

Al

O O

Al

O

OH

Al

O

OH

Al

O

OH

Al

OH

O

AluminaAcidic: -Al-OH

Neutral: -Al-OH + -Al-O-

Basic: -Al-O-

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Disadvantages:

• Very strong retention of low volatility or polar solutes.

• Catalytic changes that can occur on GSC supports.

• Non-symmetrical peaks.

• Variable retention times.

2. Gas-liquid chromatography (GLC): LIKE DISSOLVES LIKE!

Stationary phase is a liquid coated on a solid support.

Over 400 liquid stationary phases available for GLC.

Material range from polymers (polysiloxanes, polyesters, polyethylene

glycols) to fluorocarbons, and liquid crystals etc.

Based on polarity, of the 400 phases available only 6-12 are needed for most

separations.

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2. Gas-liquid chromatography (GLC): LIKE DISSOLVES LIKE!

Preparing a stationary phase for GLC:

Slurry of the desired liquid phase and solvent is made with a solid support.

Solid support is usually diatomaceous earth (fossilized shells of ancient

aquatic algae, diatoms, silica-based material).

Solvent is evaporated off, coating the liquid stationary phase on the support

the resulting material is then packed into the column.

Disadvantages:

• Liquid may slowly bleed off with time

especially if high temperatures are used.

• Contribute to background (noise).

• Change characteristics of the column with time.

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3. Bonded-Phase Gas chromatography:

Covalently attach stationary phase to the solid support material.

Avoids column bleeding in GLC.

Bonded phases are prepared by reacting the desired phase with a silica-

surface. Reactions form Si-O-Si or Si-C-C-Si bonds between the stationary

phase and support.

Advantages:

• More stable than coated liquid phases.

• It can be placed on support with thinner and more uniform thickness

than liquid phases.

Many bonded phases exist, but most separations can be formed with the

following phases:

Dimethylpolysiloxane Methyl(phenyl)polysiloxane Polyethylene glycol

(Carbowax 20M)

O Si

CH3

CH3 n

O Si

CH3

CH3 n

O Si

C6H5

C6H5 m

HO C C

H

H

H

H

O H

n

14


Advantages of GC:

Fast analysis.

Typically minutes (even sec.).

Can be automated.

Small samples (μL or μg needed).

High resolution.

Reliable, relatively simple and

cheap (~ $20,000).

Non-destructive.

Allows on-line coupling, e.g. to MS.

Sensitive detectors (easy ppm,

often ppb).

Highly accurate quantification.

Disadvantages of GC:

χ Only Volatile samples.

T of column ~ 380 °C

Analytes, boiling point < 500 °C.

χ Not suitable for thermally labile

samples.

χ Some samples may require

intensive preparation (soluble &

inert to the column).

χ Requires spectroscopy (usually

MS) to confirm the peak identity.

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Comparison of GC vs. HPLC:

High Performance Liquid

Chromatography (HPLC):

• Non-volatile samples.

• Thermally unstable

compounds.

• Macromolecules.

• Inorganic and ionic

samples.

• More complex interface to

Mass Spectrometer.

Gas Chromatography (GC):

• Volatile samples.

• Thermally Stable

compounds.

• Rapid analysis.

• Good resolution.

• Easily interfaced to Mass

Spectrometer.

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Uses of Gas Chromatography (GC):

• Separation and analysis of organic compounds.

• Testing purity of compounds.

• Determine relative amounts of components in mixture.

• Compound identification.

• Isolation of pure compounds (microscale work).

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SAMPLE PREPARATION

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SAMPLE PREPARATION

I. Headspace method:

Typically the sample is placed in a closed container and a flow of

inert gas such as nitrogen is used to purge the headspace onto an

adsorbent or a cryogenic trap.

The trap can be eluted with solvent, the solvent can be concentrated

and injected.

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SAMPLE PREPARATION

II. Distillation method:

Produces a dilute aqueous sample which is not the best for GC.

Sample can be re-extracted with solvent.

Food

Sample

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SAMPLE PREPARATION

III. Solvent extraction method:

• Blend sample and mix with non-miscible solvent. (a solvent that

doesn’t mix with water).

• Centrifuge to unmix solvent and food sample.

• Separate the solvent containing the analytes of interest and

concentrate by evaporation for injection into GC.

1 2 3 4

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SAMPLE PREPARATION

IV. Solid phase microextraction (SPME):

Sampling devices (manual and autosampler) consist of a coated

silica fiber inside a hollow needle. The coating on the fiber

adsorbs and concentrates volatiles from the headspace inside

the sample container. The needle protects the fiber which can be

inserted through the septa of the GC injector for direct analysis.

INSTRUMENT OVERVIEW

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INSTRUMENT OVERVIEW

1

2

3

4

51. Carrier gas.

2. Injectors.

3. Column.

4. Detectors.

5. Operating conditions.

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INSTRUMENT OVERVIEW

I. Carrier gas:

Chemically inert gas as mobile phase.

Flows continuously throughout instrument.

Carries the sample vapor through the column to detector.

He (most common), N2 and H2 in pressured tank.

Pressure regulator, gauges and flow meter.

Pressure: 10~50 psi → 25~150 mL/min with Packed column,

or 1~25 mL/min with Capillary column.

Flow rates must be precisely controlled → Reproducible tr values.

Flow rate ↓ as column T ↑, i.e. Viscosity of carrier gas increases with T.

Necessary properties:

INERT: Does not chemically interact with sample, SP or detectors.

COMPATIBLE with detector: No noise or explosions.

HIGHLY PURIFIED: Impurities will degrade column and cause noise in

detector.

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INSTRUMENT OVERVIEW

II. Injectors:

Properties:

Versatile, rapid, quantitative.

Introduce sample to column as a sharp, symmetric band.

Heated injection port:

Vaporize sample (50°C > analyte b.p.).

Low enough to avoid degradation.

Packed columns:

Flash vaporizer or on-column.

Capillary columns:

Split: 1-2%, Higher resolution.

Splitless: ~100%, Trace analysis.

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INSTRUMENT OVERVIEW

II. Injectors:

1-2% ~ 100% 100%

Lecture 5b

by

Sherif S. Ebada, Ph.D.

Instrum. Pharm. Analys.

Contact information:

Assoc. Prof. Dr. Sherif S. Ebada

Pharmaceutical Chemistry Dept.

Office hours:

Mondays and Tuesdays: 12:00–14:00 pm

E-mail: [email protected]

27

29

Course outline

Introduction.

Adsorption (CC & TLC), Flash CC, Chromatotron etc.

Partition (Paper).

Gel Chromatography.

Ion exchange Chromatography.

Affinity Chromatography.

Electrophoresis.

HPLC.

GC.

Spectroscopy.

30

Chromatography

Lecture 5b

GC

31

INSTRUMENT OVERVIEW

III. Column & stationary phases:

Gas Chromatography Columns

Packed Opened-Tubular (OT)

GSC

Wall-Coated OT(WCOT)

Support-Coated OT(SCOT)

Porous-Layer OT(PLOT)

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INSTRUMENT OVERVIEW


Packed GC columns:

Easy to make and use.

Limited resolution (N < 8,000).

Outside: Solid tubing usually

made of stainless steel.

Because of strength. Glass when

more inert substrate is needed.

Inside: Tightly packed with

inert support.

Solid supports should be inert

and have high surface area.

Typically diatomaceous earth or

fluorocarbon polymer.

Open (Capillary) GC columns:

Most common and efficient.

High resolution (N > 100,000).

Outside: Solid tubing made

from fused silica.

Inert, flexible, strong, and easy to

use.

Inside: Column is an open tube.

Very low resistance to flow.

Long lengths possible ( L > 100

m).

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INSTRUMENT OVERVIEWIII. Column & stationary phases:

Packed GC columns:

Stationary liquid phase is

coated on the solid support.

3-10% by weight of the solid

support.

Open (Capillary) GC columns:

Stationary phase is a thin, uniform

liquid film coated on the wall of the

tubing Column or covalently bound

to the solid support.

N; number of theortical plates in the

column. Length α N α Resolution.

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INSTRUMENT OVERVIEW


The critical parameters for GC columns:

Dimensions: Internal diameter, Column length, Film thickness.

Conditions: Temperature, Flow rate.

Composition: Stationary phase composition, Carrier gas.

Given a sample, you will need to first choose the what stationary phase will

work best:

1. First pick the type of column, then think about dimensions.

2. Conditions can be optimized for given column dimensions.

3. Choice of stationary phase is very important. It determines what kind of

sample you can run. Critical for packed columns, but less so for OT columns

because of high efficiency.

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INSTRUMENT OVERVIEW


Comparing results from different columns:

How do you know if two columns are the same?

How do you compare tr on different columns?

How do you express relative polarity?

1. Kovats retention index (for different analytes).

2. McReynolds constant (for different columns).

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INSTRUMENT OVERVIEW


1. Kovats retention index (for different analytes).

I = Kovats retention index, n = the number of carbon atoms in the

smaller n-alkalne, N = the number of carbon atoms in the larger n-

alkane, tr = the retention time.

Retention time (tr) is directly proportional to the number of carbon

atoms.

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INSTRUMENT OVERVIEW


2. McReynolds constant (for different columns).

ΔI = I – Istandard

Compare the change in the Kovats retention index, I, as a function of the type of

column.

Provides a measure of the relative polarity of one column to a standard

(Squalane).

Example: Kovat‘s index values of toluene is 773 on SP squalene (non-polar) and

860 on SP dioctylphatalate. Define McReynolds constant.

McReynolds constant (ΔI) = 860 – 773 = 87

Results:

Dioctylphatalate is a more polar stationary phase.

The larger the McReynolds constant, the greater the retention time than

compared to the non-polar column.

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INSTRUMENT OVERVIEW

IV. Detectors:

1. Thermal conductivity detector (TCD): Universal detector.

2. Flame ionization detector (FID): Hydrocarbons.

3. Analyte-specific detector:

Flame photometric detector (FPD): Phosphorous & sulphur.

Electron capture detector (ECD): Halogenated compounds.

4. Mass spectrometry: Tunable for any species, i.e. universal.

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INSTRUMENT OVERVIEW

IV. Detectors:


Hot-wire detector.

First universal detector developed for GC.

Process:

Measures a bulk property of the mobile phase leaving the column.

Measures ability to conduct heat away from a hot-wire (i.e.,

thermal conductivity).

Thermal conductivity changes with presence of other components

of the mobile phase.

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INSTRUMENT OVERVIEW

IV. Detectors:


Design:

• Based on electronic circuit known as a Wheatstone bridge.

• Circuit consists of four resistors with a fixed current applied to them.

• Thermal conductivity changes with components in the mobile phase.

• The voltage between points (+) and (-) will be zero as long as the

resistances in the different arms of the circuit are properly balanced.

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INSTRUMENT OVERVIEW

IV. Detectors:


Considerations:

Mobile phase must have very different thermal conductivity than solutes.

H2 and He are carrier gases with significantly different thermal

conductivity values (6-10 times greater than those of organic

compounds).

H2 reacts with metal oxides present on the resistors, so not used.

Advantages:

• Truly universal detector.

• Non-destructive.

Disadvantages:

• Detect mobile phase impurities.

• Sensitive to changes in flow-rates.

• Limit of detection ~10-7 M, i.e. much higher then other GC detectors.

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INSTRUMENT OVERVIEW

IV. Detectors:

2. Flame ionization detector (FID):

Hydrocarbons.

Most common type of GC

detector.

“Universal” detector for any

organic compound.

Principle of operation:

Measures the production of

ions when a solute is burned in

a flame. Ions are collected at an

electrode to create a current.

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INSTRUMENT OVERVIEW

IV. Detectors:

2. Flame ionization detector (FID): Hydrocarbons.

Advantages:

- Universal detector for organics.

- Doesn’t respond to common inorganic compounds.

- Mobile phase impurities not detected.

- Carrier gases not detected.

- Limit of detection: FID is 1000x better than TCD.

Disadvantage:

- Destructive detector

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INSTRUMENT OVERVIEW

IV. Detectors:


Flame photometric detector (FPD): Phosphorous & sulphur.

Principle of Operation:

Decompose of analytes using H2/Air flame.

Products emit bands of radiation, i.e. S2* (394 nm) and HPO*

(526 nm).

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INSTRUMENT OVERVIEW

IV. Detectors:


Electron capture detector (ECD): Halogenated compounds.

Radioactive decay-based detector.

Principle of Operation:

Based on the capture of electrons by electronegative atoms in

a molecule. Electrons are produced by ionization of the carrier

gas with a radioactive source such as 63Ni.

In absence of solute, steady stream of these electrons is produced

going to collector electrode where they produce a current.

Compounds with electronegative atoms capture electrons and

reducing current

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INSTRUMENT OVERVIEW

IV. Detectors:


Electron capture detector (ECD):

Halogenated compounds.

Advantages:

Useful for environmental testing.

Detection of chlorinated pesticides

or herbicides, aromatic carcinogens,

organometallic compounds.

Selective for halogen- (I, Br, Cl, F),

nitro-, and sulfur-containing

compounds.

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INSTRUMENT OVERVIEW

IV. Detectors:

4. Mass spectrometry: Tunable for any species, i.e. universal.

• Ionizes samples and separates the ions by mass.

• Peak pattern characteristic of compound.

• Universal.

• Provides both qualitative and quantitative analysis.

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INSTRUMENT OVERVIEW

V. Operating condition and temperature (T) control:

• Oven temperatures:

Set by user depending on analytes and type of column.

Should NOT be higher than b.p. of analyte.

Column temperature should be just above “dew point” of analyte.

• Isothermal:

Simplest mixtures and cheapest GCs.

Temperature of oven remains constant.

• Temperature programming:

Analytes with wide range of volatilities.

Best separation in shortest time.

Gas chromatography (GC) separates components based on Volatility

(Oven T < B.P. of analyte) and Polarity (Column, Like dissolves Like).

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ISOTHERMAL

Column temp. 120oC

Programmed temp.

(30oC to 180oC) (5o/min)

INSTRUMENT OVERVIEW

V. Operating condition and temperature (T) control:

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APPLICATIONS OF GC

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APPLICATIONS OF GC

• Analysis of foods is concerned with the assay of lipids, proteins,

carbohydrates, preservatives, flavors, colorants, vitamins, steroids,

drugs, and pesticide residues.

• Most of the components are non-volatile (thus the use of HPLC) but

with modification (?), GC can be effectively used.

• Derivatization of lipids and fatty acid to their methyl esters.

• Proteins are acid hydrolyzed followed by esterification (N-propyl

esters).

• Carbohydrates derivatized by silylation to produce a volatile

compound.

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◙ Species volatile and thermally stable.

◙ Qualitative analysis: purity determination.

Confirm the presence or absence of a suspected

compounds.

Determine an unknown: hyphenated GC with other methods.

Retention index: Normal alkanes.

◙ Quantitative analysis:

Area (preferred) of a peak vs. Conc. linearly.

Calibration of standards: straight line through origin.

Internal standard method.

APPLICATIONS OF GC

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APPLICATIONS OF GC

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