Kovat’s retention IndexKovat’s retention Index

The retention index, I, was first proposed by The retention index, I, was first proposed by Kovats in 1958 as a parameter for identifying Kovats in 1958 as a parameter for identifying solutes from chromatograms. The retention solutes from chromatograms. The retention index for any given solute can be derived from index for any given solute can be derived from a chromatogram of a mixture of that solute a chromatogram of a mixture of that solute with at least two normal alkanes (chain length with at least two normal alkanes (chain length >four carbons) having retention times that >four carbons) having retention times that bracket that of the solute. bracket that of the solute.

That is, normal alkanes are the standards upon which That is, normal alkanes are the standards upon which the retention index scale is based. By definition, the the retention index scale is based. By definition, the retention index for a normal alkane is equal to 100 retention index for a normal alkane is equal to 100 times the number of carbons in the compound times the number of carbons in the compound regardless of the column packing, the temperature, or regardless of the column packing, the temperature, or other chromatographic conditions. The retention other chromatographic conditions. The retention index system has the advantage of being based upon index system has the advantage of being based upon readily available reference materials that cover a wide readily available reference materials that cover a wide boiling range. The retention index of a compound is boiling range. The retention index of a compound is constant for a certain stationary phase but can be constant for a certain stationary phase but can be totally different for other stationary phases. totally different for other stationary phases.

In finding the retention index, a plot of the number of In finding the retention index, a plot of the number of carbons of standard alkanes against the logarithm of carbons of standard alkanes against the logarithm of the adjusted retention time is first constructed. The the adjusted retention time is first constructed. The value of the logarithm of the adjusted retention time value of the logarithm of the adjusted retention time of the unknown is then calculated and the retention of the unknown is then calculated and the retention index is obtained from the plot. The adjusted index is obtained from the plot. The adjusted retention time, tretention time, tRR’, is defined as:’, is defined as:

ttRR’ = t’ = tRR - t - tMM

Kovat’s retention Index PlotKovat’s retention Index Plot

Log tR’

Number of Paraffinic Carbons

4

5

6

7

8

GC/MSGC/MS

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

t0

t1

t2

t3

t4

Distance Across the Column

PumpsPumps

InjectorsInjectors

Inject

Pump

To Column

Inject

Waste Waste

Pump

To Column

(a) (b)

ColumnsColumns

DetectorsDetectors

UV-VisUV-Vis

Po P

From Column

To Waste

Refractive IndexRefractive Index

Source

Detector

Sample

Reference

Mirror

TermsTerms Bonded phase chromatographyBonded phase chromatography Types pf stationary phasesTypes pf stationary phases Reverse phase LCReverse phase LC Normal phase LCNormal phase LC Ion ChromatographyIon Chromatography Ion-pairing chromatographyIon-pairing chromatography Size exclusion (gel permeation) chromatographySize exclusion (gel permeation) chromatography EfficiencyEfficiency SelectivitySelectivity Mobile phase strengthMobile phase strength

LC/MSLC/MS

Thin layer chromatographyThin layer chromatography

Ismailov and Schraiber introduced the first report on Ismailov and Schraiber introduced the first report on thin layer chromatography (TLC) in 1938. After this thin layer chromatography (TLC) in 1938. After this report, extensive work was conducted were report, extensive work was conducted were revolutionary expansion of the technique started in revolutionary expansion of the technique started in the early 1950's. TLC theory can be viewed as a the early 1950's. TLC theory can be viewed as a special case of LSC in which a flat sheet of adsorbent special case of LSC in which a flat sheet of adsorbent is used instead of the column. Stationary phase is is used instead of the column. Stationary phase is usually an active adsorbent with very high surface usually an active adsorbent with very high surface area. This material is fixed to a glass plate or other area. This material is fixed to a glass plate or other flat support flat support

The mobile phase is a liquid or a mixture of liquids The mobile phase is a liquid or a mixture of liquids that can dissolve the sample in order to effect that can dissolve the sample in order to effect separation. In some cases a mobile phase is modified separation. In some cases a mobile phase is modified by addition of some salts in order to achieve better by addition of some salts in order to achieve better separations. The TLC plate is placed in a special tank separations. The TLC plate is placed in a special tank called "developing tank" which contains the mobile called "developing tank" which contains the mobile phase. The sample is spotted just above the level of phase. The sample is spotted just above the level of the mobile phase so that it can be carried up by the the mobile phase so that it can be carried up by the solvent. solvent.

Adsorption of solutes on the active solid Adsorption of solutes on the active solid stationary phase and its affinity to remain in stationary phase and its affinity to remain in the solvent are the two major factors leading to the solvent are the two major factors leading to selectivity in TLC separations. This is selectivity in TLC separations. This is sometimes referred to as sorption desorption sometimes referred to as sorption desorption mechanism.mechanism.

TLC tankTLC tank

Qualitative AnalysisQualitative Analysis

TLC methods utilize a calculated factor as the basis for TLC methods utilize a calculated factor as the basis for qualitative analysis. This is called the retardation factor (Rqualitative analysis. This is called the retardation factor (R ff) ) which has a specific value for a specific solute using specific which has a specific value for a specific solute using specific mobile and stationary phases. Rf is defined asmobile and stationary phases. Rf is defined as

Distance traveled by spotDistance traveled by spot

RRff = _____________________________ = _____________________________

Distance traveled by solvent frontDistance traveled by solvent frontWhere, distance is measured from the center of the spot. Where, distance is measured from the center of the spot. Solutes that are strongly adsorbed to the solid stationary phase Solutes that are strongly adsorbed to the solid stationary phase

exhibit smaller Rexhibit smaller Rff values than others which are less strongly values than others which are less strongly adsorbed.adsorbed.

Mass SpectrometryMass Spectrometry

IonSource

Mass Analyzer

IonDetector

Inlet DataSystem

VacuumPumps

SampleIntroduction

DataOutput

Source – IonizationSource – Ionization

Once we have produced gas phase molecules, we can start to look at the ionization process.

With some techniques, ionization occurs in conjunction with the vapourization step (we’ll discuss them later). If it does not, then we need to create the ions.

• Electron Impact ionization (EI)

• Chemical ionization (CI)

• Thermal ionization (TI)

Electron Impact IonizationElectron Impact Ionization

Neutral gas phase molecules drift into ionization region. Energetic electron beam (50 - 100 eV) intersects molecules. Collisions create cations. Field extracts ions from ionizer and focuses and accelerates (500 - 1000 eV) ion beam towards mass analyzer.

~70 Volts

+

_

+

_

e- e-e-

++ ++++

_

Electron Collector (Trap)

Repeller

ExtractionPlate

Filament

toAnalyzer

Inlet

Electrons

NeutralMolecules

PositiveIons

Electron ImpactIonization Source

EI FragmentationEI Fragmentation

It only takes about 5 to 15 eV of energy to ionize the molecule. The rest of the electron energy is available to induce molecular bond rupture.

Fragmentation with EI is extensive.

This is useful since by studying the fragmentation pattern, one can discern the likely structural components of the original molecule and develop a model for its structure.

Fragmentation with EI is so extensive, however, that the parent ion – the ion arising from the original molecule – is often not detectable. EI really “blows apart” the target molecules.

Chemical IonizationChemical Ionization

Chemical Ionization (CI) is a means of “softening” the fragmentation effect of EI. A buffer gas, often methane CH4, is added in a large excess (~x100 fold) over the analyte. The electron beam ionizes the methane, gas phase reactions produce molecular ions such as CH5

+ which collide with analyte molecules (M) to gently produce the analyte ions (MH+).

Can perform CI in same unit with EI except for much higher pressure.

Must provide additional pumping speed to move extra buffer gas out of ionizer so as to not degrade vacuum in analyzer.

Ionization process much gentler and it tends to produce ions with an extra proton mass.

Electrospray IonizationElectrospray Ionization

The most gentle ionization technique. Produces multiply charged ions. Based ion Ion Evapouration process.

Rubinson 544

Mass AnalyzersMass Analyzers

At the heart of a mass spectrometer is the mass analysis system. The selection is always based upon a mass-to-charge ratio, rather than on absolute mass. In many techniques, it is common to produce multiply charged species so that they show up at a much lower m/z value. The principal devices for mass selection are

• quadrupole

• magnetic sector

• double focusing (sector)

• ion trap

• ion cyclotron

• time-of-flight

Mass ResolutionMass Resolution

A principal measure of the performance of a mass spec is its resolution. It is usually given as

A spectrometer with a resolution of 2500 is needed to be able to distinguish between carbon monoxide (CO) and nitrogen (N2). Both are nominally with a mass of 28 amu, but a more precise calculation has m(CO) = 27.9949 amu and m(N2) = 28.0061 amu.

A resolution of 50 is all that is needed to distinguish ammonia (NH3) from methane (CH4) whose nominal masses differ by about 1 amu (17 amu vs. 16 amu).

Commercial units with resolution from 500 to 500,000 are available.

R m

m

QuadrupoleQuadrupole

Perhaps the most common mass spectrometer. Not the highest resolution. Opposite rods are polarized in phase with each other and out of phase with the other two rods.

DC voltage drives ions to negative rods

RF signal reverses sign in time to push ions away.

Only specific m/z has right velocity to match with RF signal.

Scan masses by scanning RF and DC but keep ratio constant.

Detector

IonSource

+

_

_

DC and ACVoltages

Quadrupole Ion Filter

resonant ion

non-resonant ion

+

Magnetic SectorMagnetic SectorA charged particle (an ion) experiences a force that bends its path when moving through a magnetic field. The balance between magnetic force and centripetal force brings an ion of a particular m/z to the entrance slit of a detector.

Skoog p. 515

ion trajectory not in register

(too heavy)

IonSource

Detector

ion trajectory not in register

(too light)

ion trajectory in register

S

N

Magnetic Sector Mass Analyzer

Electromagnet

Double FocusingDouble Focusing

A single magnetic sector instrument’s resolution is limited by the spread of translational energy of the ions coming from the source. A double focusing instrument uses an electrostatic field to narrow the energy spread before the ions enter the magnetic sector. Resolutions of 105 are achievable with these instruments.

Skoog p. 517

Time-of-FlightTime-of-Flight

Ions accelerated to same energy. Masses differences mean different velocities. They travel a long path (1 - 2 m) and arrive at the detector at different times. Time of arrival calculates m/z. Newer instruments are Reflectrons, with ion mirror.

Rubinson p.562

ttransit L2V ezm

12

194

67 109

5582

42

16513694

40 60 80 100 120 140 160 180 200

Abundance

Mass (amu)

Mass Spectrum

NC

CNH

CO

C

O

N

N

C H

C 3H

C3H

MassSpectrometer

A Typical Mass Spectrum

Typical sample: isolatedcompound (~1 nanogram)