Kovat

’

s retention Index

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

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

  In finding the retention index, a plot of the number of carbons of standard alkanes against the logarithm of the adjusted retention time is first constructed. The value of the logarithm of the adjusted retention time of the unknown is then calculated and the retention index is obtained from the plot. The adjusted retention time, t R ’ , is defined as:

t R ’ = t R - t M

Kovat

’

s retention Index Plot

Log t R ’ 4 5 6 7 Number of Paraffinic Carbons 8

GC/MS

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

t 2 t 3 t 0 t 1 t 4 Distance Across the Column

Pumps

Injectors

Waste Waste Inject (a) To Column Pump Inject To Column (b) Pump

Columns

 UV-Vis

Detectors

From Column P o P To Waste

Refractive Index

Sample Source Mirror Reference Detector

Terms

          Bonded phase chromatography Types pf stationary phases Reverse phase LC Normal phase LC Ion Chromatography Ion-pairing chromatography Size exclusion (gel permeation) chromatography Efficiency Selectivity Mobile phase strength

LC/MS

Thin layer chromatography

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

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

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

TLC tank

Qualitative Analysis

TLC methods utilize a calculated factor as the basis for qualitative analysis. This is called the retardation factor (R f ) which has a specific value for a specific solute using specific mobile and stationary phases. Rf is defined as Distance traveled by spot R f = _____________________________ Distance traveled by solvent front Where, distance is measured from the center of the spot. Solutes that are strongly adsorbed to the solid stationary phase exhibit smaller R adsorbed.

f values than others which are less strongly

Mass Spectrometry

Sample Introduction

Inlet

Data Output

Data System Ion Source Ion Detector Mass Analyzer Vacuum Pumps

Source

–

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 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 Repeller Electron Impact Ionization Source Electron Collector (Trap) Positive Ions

+

Neutral Molecules

+

Inlet

+ + + + e e _ e -

Electrons Filament Extraction Plate

+ +

to Analyzer

EI 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 Ionization

Chemical Ionization (CI) is a means of “softening” the fragmentation effect of EI. A buffer gas, often methane CH produce molecular ions such as CH 5 4 + , is added in a large excess (~x100 fold) over the analyte. The electron beam ionizes the methane, gas phase reactions 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 Ionization

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

Rubinson 544

Mass 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 Resolution

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

R

 

m m

A spectrometer with a resolution of 2500 is needed to be able to distinguish between carbon monoxide (CO) and nitrogen (N m(N 2 ) = 28.0061 amu.

2 ). Both are nominally with a mass of 28 amu, but a more precise calculation has m(CO) = 27.9949 amu and A resolution of 50 is all that is needed to distinguish ammonia (NH 3 ) from methane (CH 4 ) whose nominal masses differ by about 1 amu (17 amu

vs.

16 amu).

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

Quadrupole

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.

Ion Source

Quadrupole Ion Filter non-resonant ion resonant ion

+ _ _ +

Detector

DC and AC Voltages

Magnetic Sector

A 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 Source

Magnetic Sector Mass Analyzer ion trajectory in register ion trajectory not in register (too light)

S N Electromagnet Detector

ion trajectory not in register (too heavy)

Double 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 10 5 are achievable with these instruments.

Skoog p. 517

Time-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.

t

transit 

L

  2

V e z m

  1 2 Rubinson p.562

A Typical Mass Spectrum O C H 3 H 3 C C N N C C H C C O N H N Typical sample: isolated compound (~1 nanogram)

Mass Spectrometer

194

Mass Spectrum

67 109 55 82 42 40 60 80 94 136 100 120 Mass (amu) 140 165 160 180 200