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Some important points to remember!1.) Typical response obtained by chromatography (i.e., a chromatogram):

Where:tR= retention timetM= void timeWb = baseline width of the peak in time units

Wh = half-height width of the peak in time units

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2.) Solute Retention:

A solutes retention time or retention volume in chromatography

is directly related to the strength of the solutes interaction withthe mobile and stationary phases.

Retention on a given column pertain to the particulars of thatsystem:

- size of the columnflow rate of the mobile phase

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3.) Efficiency:

Efficiency is related experimentally to a solutes peak width.- an efficient system will produce narrow peaks

- narrow peaks

smaller difference in interactions in order to separate twosolutesEfficiency is related theoretically to the various kinetic processes that areinvolved in solute retention and transport in the column

- determine the width or standard deviation (s) of peaks

Wh

Estimate s from peak widths,assuming Gaussian shaped peak:

Wb = 4s

Wh = 2.354s

Dependent on the amount of time that a solute spends in the column (k or tR)

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Number of theoretical plates (N): compare efficiencies of a system forsolutes that have different retention times

N = (tR/s

)2

or for a Gaussian shaped peak

N = 16 (tR/Wb)2

N = 5.54 (tR/Wh)2

The larger the value of N is for a column, the better the column will beable to separate two compounds.

- the better the ability to resolve solutes that have small differencesin retention

- N is independent of solute retention- N is dependent on the length of the column

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Plate height or height equivalent of a theoretical plate (H or HETP): compareefficiencies of

columns with different lengths:H = L/N

where: L = column lengthN = number of theoretical plates for the column

Note: H simply gives the length of the column that corresponds to one theoreticalplate

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 diffusionb. Mobile phase mass transfer

c. Stagnant mobile phase mass transferd. Stationary phase mass transfer

e. Longitudinal diffusion

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Van Deemter equation: relates flow-rate or linear velocity to H:

H = A + B/m + Cm

where:

m = linear velocity (flow-rate x Vm/L)H = total plate height of the columnA = constant representing eddy diffusion &

mobile phase mass transferB = constant representing longitudinal

diffusionC = constant representing stagnant mobilephase & stationary phase masstransfer

One use of plate height (H) is to relate these kinetic process toband broadening to a parameter of the chromatographic system(e.g., flow-rate).

This relationship is used to predict what the resulting effect wouldbe of varying this parameter on the overall efficiencyof the chromatographic system.

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m optimum

Plot of van Deemter equation shows how H changes with the linear velocity (flow-rate) of the mobile phase

Optimum linear velocity (mopt) - where H has a minimum value and thepoint of maximum column efficiency.

mopt is easy to achieve for gas chromatography, but is usually too small forliquid chromatography requiring flow-rates higher than optimal to separate

compounds

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4.) Measures of Solute Separation:

separation factor (a) parameter used to describe how well two solutes areseparated by a chromatographic system:

a = k2/k1 k = (tRtM)/tMwhere:

k1 = the capacity factor of the first solutek2 = the capacity factor of the second solute,

with k2~ k

A value of a ~ 1.1 is usually indicative of a good separation

Does not consider the effect of column efficiency or peak widths, only retention.

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Resolution (RS) resolution between two peaks is a second measure of howwell two peaks are separated:

RS =

where:tr1, Wb1 = retention time and baseline width for the first eluting peaktr2, Wb2 = retention time and baseline width for the second eluting peak

tr2 tr1

(Wb2 + Wb1)/2

Rs is preferred over a sinceboth retention (tr) and columnefficiency (Wb) are consideredin defining peak separation.

Rs ~1.5 represents baselineresolution, or completeseparation of two neighboringsolutes ideal case.

Rs ~1.0 considered adequatefor most separations.

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Gas ChromatographyA.) Introduction:

Gas Chromatography (GC) is a chromatographic technique in whichthe mobile phase is a gas.

GC is currently one of the most popular methods for separating and

analyzing compounds. This is due to its high resolution, low limits ofdetection, speed, accuracy and reproducibility.

GC can be applied to the separation of any compound that is eithernaturally volatile (i.e., readily goes into the gas phase) or can be

converted to a volatile derivative. This makes GC useful in theseparation of a number of small organic and inorganic compounds.

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Gas Chromatography

B.) Equipment:

A simple GC system consists of:1. Gas source (with pressure and flow regulators)

2. Injector or sample application system3. Chromatographic column (with oven for temperature

control)4. Detector & computer or recorder

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A typical GC system used is shown below (a gas chromatograph)

Carrier gas: He (common), N2, H2Pinlet 10-50 psigFlow = 25-150 mL/min packed columnFlow = 1-25 mL/min open tubular column

Column: 2-50 m coiled stainless steel/glass/TeflonOven: 0-400 C ~ average boiling point of sample

Accurate to

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Schematic Diagram of Gas Chromatograph

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C.) Mobile Phase:

GC separates solutes based on their different interactions with themobile and stationary phases.

-solutes retention is determined mostly by its vapor pressureand volatility

- solutes retention is controlled by its interaction with thestationary phase

- gas mobile phase has much lower density

decreased chance for interacting with soluteincreased chance that solid or liquid stationary phase

interacts with solute

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Carrier gas main purpose of the gas in GC is to move the solutesalong the column, mobile phase is often referred to as carriergas.

Common carrier gas: include He, Ar, H2, N2

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Carrier Gas or Mobile phase does not affect solute retention, butdoes affect:

1.) Desired efficiency for the GC System- low molecular weight gases (He, H2) largerdiffusion coefficients

- low molecular weight gases faster, more efficientseparations2.) Stability of column and solutes

- H2 or O2 can react with functional groups on solutesand stationary phase or with surfaces of the injector,connections and detector

3.) Response of the detector- thermal conductor requires H2 or He- other detectors require specific carrier gas

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D.) Stationary Phases:

Stationary phase in GC is the main factor determining theselectivity and retentionof solutes.

There are three types of stationary phases used in GC:Solid adsorbentsLiquids coated on solid supportsBonded-phase supports

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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] and clay)silicaactive carbon

Magnified Pores in activated carbon

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Gas-solid chromatography (GSC):

advantages:- long column lifetimes- ability to retain and separate some compounds not easily

resolved by other GC methods geometrical isomers permanent gases

disadvantage:- very strong retention of low volatility or polar solutes- catalytic changes that can occur on GSC supports- GSC supports have a range of chemical and physical

environments different strength retention sites non-symmetrical peaks variable retention times

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2.) Gas-liquid chromatography (GLC)

- stationary phase is some liquid coated on a solid support

- over 400 liquid stationary phases available for GLC many stationary phases are very similar in terms of

their retention properties

- material range from polymers (polysiloxanes, polyesters,polyethylene glycols) to fluorocarbons, molten salts and liquidcrystals

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Based on polarity, of the 400 phases available only 6-12 are needed formost separations. The routinely recommended phases are listed below:

Name

Chemical nature of

polysiloxane

Max.

temp.

McReynolds constants

x y z m sSE-30 Dimethyl 350 14 53 44 64 41

Dexsil300 Carborane-dimethyl 450 43 64 111 151 101

OV-17 50% Phenyl methyl 375 119 158 162 243 202

OV-210 50% Trifluoropropyl 270 146 238 358 468 310

OV-225 25% Cyanopropyl-

25% phenyl

250 238 369 338 492 386

Silar-SCP 50% Cyanopropyl-

50% phenyl

275 319 495 446 637 531

SP-2340 75% Cyanopropyl 275 520 757 659 942 804

OV-275 Dicyanoallyl 250 629 872 763 1106 849

McReynolds constants based on retention of 5 standard probe analytesBenzene, n-butanol, 2-pentanone, nitropropanone, pyridine

Higher the

numberthe highertheabsorption.

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Preparing a stationary phase for GLC:

- slurry of the desired liquid phase and solvent is madewith a solid support

solid support is usually diatomaceous

earth (fossilized shells of ancient aquaticalgae (diatoms), silica-based material)

- solvent is evaporated off, coating the liquid stationaryphase on the support

- the resulting material is then packed into the column

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disadvantage:- liquid may slowly bleedoff with time

especially if high temperatures are used contribute to background change characteristics of the column with

time

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3.) Bonded-Phase Gas chromatography

- covalently attach stationary phase to the solid supportmaterial

- avoids column bleeding in GLC- bonded phases are prepared by reacting the desiredphase with the surface of a silica-based support

reactions form an Si-O-Si bond between the stationary phaseand support

or

reactions form an Si-C-C-Si bond between the stationary phaseand support

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- many bonded phases exist, but most separations can be formed with thefollowing commonly recommended bonded-phases:

Dimethylpolysiloxane Methyl(phenyl)polysiloxane

Polyethylene glycol (Carbowax 20M) Trifluoropropylpolysiloxane Cyanopropylpolysiloxane

advantages:- more stable than coated liquid phases- can be placed on support with thinner and more uniform

thickness than liquid phases

Si

CH3

CH3

O

n

Si

CH3

CH3

O

n

Si

C6H5

C6H5

O

m

C CHO O

H

H

H

H

H

n

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E.) Support Material:

There are two main types of supports used in GC:Packed columns

large sample capacity preparative work

Capillary (open-tubular) columns higher efficiency smaller sample size

analytical applications

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F.) Elution Methods:A common problem to all chromatographic techniques is that in any onesample there may be many solutes present, each retained by thecolumn to a different degree:

Best separation and limitsof detection are usuallyobtained with solutes withk values of 2-10

Difficult to find onecondition that elutes allsolutes in this k rangegeneral elution problem

Gradient elution - change column condition with time which changes

retention of solutes to overcome general elution problem

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Temperature Programming changing the temperature on the column with time tosimulate gradient elution in GC since a solutes retention in GC is related to its

volatility.

ISOTHERMALColumn temp. 120oC

Programmed temp.(30oC to 180oC) (5o/min)

Comparison of a GC separation using isothermal conditions and temperatureprogramming is shown below

Temperature programming is usually done either in a stepwise change, a linear change or acombination of several linear changes. A single linear change or ramp is the most common

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G.) GC Detectors:

The following devices are common types of GC detectors:

1. Thermal Conductivity Detector (TCD)2. Flame Ionization Detector (FID)3. Nitrogen-phosphorus Detector

4. Electron Capture Detector (ECD)5. Mass Spectrometers (discussed later in the course)

The choice of detector will depend on the analyte andhow the GC method is being used (i.e., analytical or

preparative scale)

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1.) Thermal Conductivity Detector (TCD)

- katherometer or hot-wire detector- first universal detector developed for GC

Process

- measures a bulk property of the mobile phase leaving thecolumn.

- measures ability to conduct heat away from a hot-wire (i.e.,thermal conductivity)

- thermal conductivity changes with presence of othercomponents in the mobile phase

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Design- based on electronic circuit known as a Wheatstone bridge.- circuit consists of an arrangement of four resistors with a fixed current applied to

them.- thermal conductivity changes with presence of other 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

as solute emerge from column:change in thermal conductivity change in amount of heat removed fromresistor change in resistors temperature and resistance change in voltagedifference between points (+) and (-).

-one resistor in contact withmobile phase leavingcolumn

-another in contact withreference stream of puremobile phase

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Considerations

- mobile phase must have very different thermal conductivitythen solutes being separated.

- most compounds separated in GC have thermal conductivityof about 1-4X10-5.

- H2 and He are carrier gases with significantly differentthermal conductivity values.- H2 reacts with metal oxides present on the resistors, so not

used

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advantages:- truly universal detector

applicable to the detection of any compound in GC- non-destructive

useful for detecting compounds frompreparative-scale columns

useful in combination with other types of GC

detectors

disadvantage:- detect mobile phase impurities- sensitive to changes in flow-rates

- limit of detection ~ 10-7 M

much higher then other GC detectors

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2.) Flame Ionization Detector (FID)

- most common type of GC detector- universal detector capable of measuring the presence of almost any

organic and many inorganic compound

Process- measures the production of ions when

a solute is burned in a flame.

- ions are collected at an electrode tocreate a current

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advantages:

- universal detector for organicsdoesnt respond to common inorganic

compounds

- mobile phase impurities not detected- carrier gases not detected- limit of detection: FID is 1000x better than TCD- linear and dynamic range better than TCD

disadvantage:

- destructive detector

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3.) Nitrogen-Phosphorus Detector (NPD)

- used for detecting nitrogen- or phosphorus containing compounds

- also known as alkali flame ionization detector or thermionic detector

Process- same basic principal as FID- measures production of ions when a solute

is burned in a flame- ions are collected at an electrode to

create a current- contains a small amount of alkali metal

vapor in the flame- enhances the formation of ions from

nitrogen- and phosphorus- containing compounds

Alkali Bead

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3.) Nitrogen-Phosphorus Detector (NPD)

advantages:- useful for environmental testing

detection of organophosphate pesticides- useful for drug analysis

determination of amine-containing or basicdrugs

- Like FID, does not detect common mobile phase impurities

or carrier gases- limit of detection: NPD is 500x better than FID in detecting

nitrogen and phosphorus- containing compounds- NPD more sensitive to other heterocompounds, such assulfur-, halogen-, and arsenic- containing molecules

disadvantage:- destructive detector- NPD is less sensitive to organic compounds compared to

FID

4 ) El C D (ECD)

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4.) Electron Capture Detector (ECD)

- radiation-based detector- selective for compounds containing

electronegative atoms, such as halogens

Process- based on the capture of electrons byelectronegative atoms in a molecule

- electrons are produced by ionization of thecarrier gas with a radioactive source3H or 63Ni

- in absence of solute, steady stream ofthese electrons is produced

- electrons go to collector electrode where

they produce a current- compounds with electronegative atoms

capture electrons, reducing current

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advantages:

- useful for environmental testingdetection of chlorinated pesticides or herbicidesdetection of polynuclear aromatic carcinogens

detection of organometallic compounds

- selective for halogen- (I, Br, Cl, F), nitro-, and sulfur-containing compounds

- detects polynuclear aromatic compounds, anhydridesand conjugated carbonyl compounds

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Gas Chromatography Application

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Gas Chromatography can only be used to

analyze volatile components (i.e. compoundsthat can be volatilized at 250oC without

decomposition).

Non-volatile components have to bederivatized before GC analysis.

Retention Times

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Response

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Response

GC Retention Time on SE-30

Hexane

RT= 4.0 min on SE-30

Retention Times

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TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDSResponse

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

OctaneDecane

1.6 min = RT

Response

Unknown compound may be Hexane

1.6 min = RT

Retention Time on Carbowax-20 (min)

SEMI QUANTITATIVE ANALYSIS OF FATTY ACIDS

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SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS

C

C

C

Detector 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

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GLC ADVANTAGES

1. Very good separation

2. Time (analysis is short)

3. Small sample is needed - ml4. Good detection system

5. Can be used for both qualitative

and quantitative analysis

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DISADVANTAGES OF GASCHROMATOGRAPHY

Material has to be volatilized at 250oCwithout decomposition. If not the analyte hasto be derivatized.

e.g. Fatty acids Methyl esters

Phenols Silanized

Sugars Silanized

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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

+ +

Gas Chromatogram of Methyl Esters of Fatty

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Gas Chromatogram of Methyl Esters of Fatty

Acids

Derivation of Glucose with Trimethylchlorosilan

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OH

O

OH

OHHO

CH2OH

1

23

45

6

+ Si

CH3

CH3

CH3

5Cl

O-Si(CH3)3

O

O-Si(CH3)3

O-Si(CH3)3(CH3)3-Si-O

CH2O-Si(CH3)3

1

23

45

6

5HCl+

Derivation of Glucose with Trimethylchlorosilan

Glucose Trimethylchlorosilane

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Effects of Derivation

1. Time consumption

2. Side reaction

3. Loss of sample

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STKK2723 - GC