DAISY SHANE L. ATAYAN Molecular orbital–Describes the distribution Ion-exchange chromatography.
tion Ion-exchange chromatography. Anions such
MS 1
CHEMISTRY 220 – CHEAT SHEET 1
MARCH 1, 2018
CHAPTER 18
Spectrophotometry–measure chemical
concentrations using light(in dilute solution)
Colorimetry –absorption of visible light
Wave-Particle Duality of Light:
ℎ𝑐
𝑐 = 𝜈𝜆 𝐸 = ℎ𝜈 =
𝜆 multiplicity (e.g., triplet S singlet). It’sslower
Molarabsorptivityamt light absorbed at𝜆
Chromophore responsible for light absorption
𝝀𝒎𝒂𝒙 Color absorbed Color
observed
380–420Violet Green-yellow
420–440Violet-blue Yellow
440–470Blue Orange
470–500Blue-green Red
500–520Green Purple
520–550Yellow-green Violet
550–580 Yellow Violet-blue
580–620 Orange Blue
620–680Red Blue-green
680–780Red Green
Chemiluminescence - emission from
chemical reaction
Basic components of a spectrophotometer:
include a radiation source, a monochromator,
a sample cell, and a detector.
Minimize errors:if the absorbance falls in the
range A < 0.3–2
In spectrophotometric titration:absorbance is
monitored as titrant is added.In many
reactions, there is an abrupt change in slope
when the equivalence point is reached. When
a molecule absorbs light, it is promoted to an
excited state from which it returns to the
ground state by nonradiative processes or by
fluorescence (singlet S singlet emission) or
phosphorescence (triplet S singlet emission).
Emission intensity - proportional to concat
low conc; at high conc, emission decreases
because of selfabsorption by the analyte.
Excitation spectrum - graph of emission
intensity vs excitation wavelength; observed
at lower energy than the absorption spectrum
and tends to be the mirror image of the
absorption spectrum.
***A molecule that is not fluorescent can be
analyzed by attaching a fluorophore to it or by
its ability to enhance or diminish the
fluorescence of a sensor compound. Light
emitted by a chemical reaction—
chemiluminescence—is also used for
quantitative analysis.
Absorbance, A = log (P0/P), P0radiant power
also called optical density
Absorption spectrum - graph of absorbance
vs𝜆, frequency, or wavenumber
Beer’s law- Relates the absorbance, A, of a
sample to its concentration, c,pathlength, b,
and molar absorptivity, ε: A =εbc. More
correctly calledBeer-Lambert-Bouguer law.
Derivatization – Chemical alteration to attach
a group to a molecule sothat the molecule can
be detected. Thiscan alter volatility or
solubility to allow easier separation.
Electromagnetic spectrum –whole range of
electromagnetic rad: visible, radio, X-rays, IR
Electronic transition –an electron goes from
one energy level to another.
Excitation spectrum - graph of luminescence
(measured at fixed 𝜆) versus excitation
frequency or wavelength. Closely corresponds
to an absorption spectrum bc luminescence is
generally proportional to the absorbance.
Excited state - state of an atom or a molecule
having more than minimum possible energy.
Fluorescence -emits a photon10-8 to 10-4 s
after absorbing a photon. Results from a
transition between states of thesame spin
multiplicity (e.g., singlet S singlet).
Frequency - The number of cycles per unit
time for a repetitive event.
Hertz - SI unit of f, s-1, or reciprocal seconds
Irradiance- Power per unit area (W/m2) EM
rad beam akaradiant power or intensity.
Luminescence- emission of light by molecule
Masking - Adding a masking agent to sample
to prevent other components from interfering
in a chemical analysis.
Molar absorptivity - tells how much light is
absorbed at particular𝜆 by a particular
substance aka extinction coefficient.
components with a constant total concentra-
of an e- within a molecule.
Monochromatic light - very narrow range of
wavelengths (“one color”).
Monochromator - device (usually a grating
or prism) that disperses light into its
component wavelengths and selects a narrow
band of wavelengths to pass through the exit
slit.
Phosphorescence- Emission of light during a
transition bet states of different spin
than fluorescence, with emission occurring
~104 to 102 s after absorption of a photon.
Photon - “particle” of light with energy hv,
where h is Planck’s constant & v is the f.
s detector response as a function of elution
Quenching- Process in which emission from an
excited molecule isdecreased by energy
transfer to another molecule called a
quencher.
Raman scattering - Scattering of light in
which the wavelength of scattered light is
changed from that of incident light by an
energy correspondingto vibrational energy of
the molecule responsible for scattering.In
Stokes Raman scattering, the molecule gains
vibrational energy andscattered light has less
a Retention factor: 𝑘 =
energy than incident light. In anti-Stokes
Ramanscattering, an excited molecule loses
vibrational energy and scatteredlight has
more energy than incident light.
Reagent blank - A solution prepared from all
of the reagents except analyte. The blank
measures the response of the analytical
method to impurities in the reagents or any
other effects caused by any component other
than the analyte. The reagent blank, unlike
the method blank, is not subjected to all
sample preparation steps before analysis.
Refractive index - The speed of light in any
medium is c/n, where c isthe speed of light in
vacuum and n is the refractive index of the
medium.The refractive index also measures
the angle at which a light ray is bentwhen it
passes from one medium into another.
LASER - Light Amplification thru Stimulated
Emission of Radiation
Stray light wavelengths outside the
bandwidth expected from the monochromator
Electrons emitted from the photosensitive
surface strike a second surface, called a
dynode, which is positive with respect to the
photosensitive emitter.
A thermocouple is a junction between two
different electrical conductors.
A ferroelectric material, such as deuterated
triglycine sulfate, has a permanent electric
polarization because of alignment of the
molecules in the crystal.
Snell’s (refraction) Law: n1sinϴ1 = n2sin ϴ2
Interferenceis any effect that changes the
signal while analyte concentration remains
unchanged. Interference can be corrected by
removing the source of interference or by
preparing standards that exhibit the same
interference.
Chemical interference is caused by any
component of the sample that decreases the
extent of atomization of analyte.
Ionization interference can be a problem in
the analysis of alkali metals at relatively low
temperature and in the analyses of other
elements at higher temperature.
MALDI: matrix-assisted laser
desorption/ionization
Distribution coefficient:
𝑡𝑜𝑡𝑎𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑝ℎ𝑎𝑠𝑒 2
𝑡𝑜𝑡𝑎𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑝ℎ𝑎𝑠𝑒 1
Chromatography operates on the same
principle as extraction, but one phase is held
in place while the other moves past it.
The mobile phase (the solvent moving through
the column) in chromatography is either a
liquid or a gas. The stationary phase (the one
that stays in place inside the column) is most
commonly a viscous liquid chemically bonded
to the inside of a capillary tube or onto the
surface of solid particles packed in the
column.
Fluid entering the column is called eluent.
Adsorption chromatography. A solid
stationary phase and a liquid or gaseous
mobile phase are used.
Partition chromatography. A liquid
stationary phase is bonded to a solid surface,
which is typically the inside of the silica (SiO2)
chromatography column in gas
chromatography.
as —SO_3 or cations such as —N(CH3)_3
are covalently attached to the stationary solid
phase, usually a resin.
Molecular exclusion chromatography. Also
called size exclusion, gel filtration, or gel
permeation chromatography, this technique
separates molecules by size, with the larger
solutes passing through most quickly.
Affinity chromatography. This most selective
kind of chromatography employs specific
interactions between one kind of solute
molecule and a second molecule that is
covalently attached (immobilized) to the
stationary phase.
A chromatogram is a graph showing the
time.
The retention time, tr, for each component is
the time that elapses between injection of the
mixture onto the column and the arrival of
that component at the detector.
Retention volume, Vr, is the volume of mobile
phase required to elute a particular solute
from the column.
Adjusted retention time: t’R = tR - tM
𝑡𝑟−𝑡𝑚
𝑡𝑚
time solute spends in stationary phase
𝑘=
time solute spends in mobile phase
Resolution:
2Δ𝑡𝑟
𝑅=
w𝑏 + w𝑎
Number of plates on column:
L 16𝑡𝑟 2
𝑁= =
H 𝑤2
CHAPTER 19
Flow injection analysis - sample is injected
into flowing liquid carrier containing a
reagent that reacts with the
analyte.Additional reagents might be added
farther downstream. As sample flowsfrom
injector to detector, the sample zone
broadens and reacts with reagentto form a
product to which the detector responds.
Immunoassay - An analytical measurement
using antibodies.
Method of continuous variation - Procedure
for finding the stoichiometryof a complex by
preparing a series of solutions with different
metal-toligandratios. The ratio at which the
extreme response (such as spectrophotometric
absorbance) occurs corresponds to the
stoichiometry of the complex. Also called
Job’s method.
Quantum yield - In photochemistry, the
fraction of absorbed photons thatproduce a
particular result. For example, if a molecule
can isomerize froma cis to a trans isomer
when light is absorbed, the quantum yield for
isomerization is the number of molecules that
isomerize divided by thenumber that absorb
photons. Quantum yield is in the range 0 to 1.
Quenching - Process in which emission from
an excited molecule is decreased by energy
transfer to another molecule called a
quencher.
Scatchard plot - A graph used to fi nd the
equilibrium constant for a reactionsuch as X 1
P Δ PX. It is a graph of [PX]/[X] versus [PX] or
anyfunctions proportional to these quantities.
The magnitude of the slope ofthe graph is the
equilibrium constant.
Sequential injection - Analytical technique
related to flow injection. Sample and
reagents are taken into a holding coil through
a multiportvalve. After a suitable reaction
time, flow is reversed and the zones of
reagent,product, and sample are pushed
through a detector to measure theamount of
product. Flow is not continuous, so sequential
injection consumesless reagents than does flow
injection.
The absorbance of a mixture is the sum of
absorbances of the individualcomponents. At
a minimum, you should be able to fi nd the
concentrationsof two species in a mixture by
writing and solving two simultaneousequations
for absorbance at two wavelengths. This
procedure ismost accurate if the two
absorption spectra have regions where they
donot overlap very much. With a
spreadsheet, you should be able to usematrix
operations to solve n simultaneous Beer’s law
equations for ncomponents in a solution, with
measurements at n wavelengths. Youshould be
able to use Excel Solver to decompose a
spectrum into a sumof spectra of the
components by minimizing the function
.Isosbestic (crossing) points are observed when
a solution containsvariable proportions of two
tion. A Scatchard plot is used to measure an
equilibrium constant. Youshould be able to
apply Excel Solver to fit nonlinear
experimental measurementssuch as
absorbance or fluorescence to the equilibrium
expression to find an equilibrium constant. The
method of continuous variation(Job’s plot)
allows us to determine the stoichiometry of a
complex. In flow injection analysis, sample
injected into a flowing carrierstream is mixed
with a color-forming reagent and passed into
a flow-through detector. Analyte disperses
and reacts withreagent without reaching
equilibrium. Precision depends on carryingout
the process reproducibly. In sequential
injection, sampleand reagent are separately
aspirated into the carrier by acomputer-
controlled syringe pump. After being
stationary in thereaction coil, analyte,
product, and reagent are pushed throughthe
detector.Immunoassays use antibodies to
detect the analyte of interest. Inan enzyme-
linked immunosorbent assay, signal is
amplified by coupling analyte to an enzyme
that catalyzes many cycles of a
reactionproducing a colored or fl uorescent
product. Time-resolved fl
uorescencemeasurements provide sensitivity
by separating analyte fl uorescencein time
and wavelength from background
fluorescence.Luminescence intensity is
proportional to the concentration of
the emitting species if the concentration is low
enough.
CHAPTER 20
Attenuated total reflectance - An analytical
technique based on passage oflight through a
waveguide or optical fi ber by total internal
refl ection. Theabsorption of the cladding is
sensitive to the presence of analyte. Some
ofthe evanescent wave is absorbed in the
cladding during each reflection inthe presence
of analyte. The more analyte, the more signal
is lost.
Bandwidth- Usually, the range of
wavelengths or frequencies of anabsorption
or emission band, typically measured at a
height equal to halfof the peak height. Also,
the width of radiation emerging from the exit
slitof a monochromator.
Beam chopping - technique using a rotating
beam chopper to modulatethe signal in a
spectrophotometer at a frequency at which
noise is reduced. In atomic absorption,
periodic blocking of the beam allows a
distinction tobe made between light from the
source and light from the flame.
Blackbody radiation - Radiation emitted by a
blackbody. The energy andspectral
distribution of the emission depend only on the
temperature of theblackbody.
Charge coupled device - An extremely
sensitive detector in which lightcreates
electrons and holes in a semiconductor
material. The electronsare attracted to
regions near positive electrodes, where the
electrons are“stored” until they are ready to
be counted. The number of electronsin each
pixel (picture element) is proportional to the
number of photonsstriking the pixel.
Diffraction - The bending of light rays by a
grating. This process occurswhen
electromagnetic radiation passes through or is
reflected from slitswith a spacing comparable
to the wavelength. Interference of waves
fromadjacent slits produces a spectrum of
radiation, with each wavelength
emerging at a different angle.
Dispersion - A measure of the ability of a
monochromator to separatewavelengths
differing by Dl through the angle Df. The
greater the dispersion,the greater the angle
separating two closely spaced wavelengths.
Fora prism, dispersion refers to the rate of
change of refractive index withwavelength,
dn/dl.
Ferroelectric material - A solid with a
permanent electric polarization(dipole) in the
absence of an external electric field. The
polarization resultsfrom alignment of
molecules within the solid.
Fourier analysis - Process of decomposing a
function into an infi nite
series of sine and cosine terms. Because each
term represents a certain frequency
or wavelength, Fourier analysis decomposes a
function into itscomponent frequencies or
wavelengths.
Grating - Either a refl ective or a transmitting
surface etched with closelyspaced lines; used
to disperse light into its component
wavelengths.
Greenhouse gas - A component of Earth’s
atmosphere that absorbs infraredradiation
from the ground and reradiates some of it
back to the ground, therebykeeping Earth
warmer than it would be in the absence of the
greenhouse gas.
Laser- Source of intense, coherent
monochromatic radiation. Light is producedby
stimulated emission of radiation from a
medium in which anexcited state has been
pumped to a high population. Coherence
means thatall light exiting the laser has the
same phase.
Monochromator - A device (usually a grating
or prism) that disperseslight into its component
wavelengths and selects a narrow band of
wavelengthsto pass through the exit slit.
Optical fiber - Fiber that carries light by total
internal reflection becausethe transparent
core has a higher refractive index than the
surroundingcladding.
Optode - A sensor based on an optical fi ber.
Also called optrode
Photoconductive detector - A detector whose
conductivity changes whenlight is absorbed by
the detector material.
Photodiode array - An array of
semiconductor diodes used to detect light.The
array is normally used to detect light that has
been spread into itscomponent wavelengths.
One small band of wavelengths falls on each
detector.
Photomultiplier tube - One in which the
cathode emits electrons whenstruck by light.
The electrons then strike a series of dynodes
(plates thatare positive with respect to the
cathode), and more electrons are
releasedeach time a dynode is struck. As a
result, more than 106 electrons mayreach the
anode for every photon striking the cathode.
Phototube - A vacuum tube with a
photoemissive cathode. The electric current
flowing between the cathode and the anode
is proportional to theintensity of light striking
the cathode.
Photovoltaic detector - A photodetector with
a junction across which thevoltage changes
when light is absorbed by the detector
material
Polychromator - A device that spreads light
into its component wavelengthsand directs
each small band of wavelengths to a different
regionwhere it is detected by a photodiode
array.
Refraction - Bending of light when it passes
between media with differentrefractive
indexes.
Resolution - How close two bands in a
spectrum or a chromatogram canbe to each
other and still be seen as two peaks. In
chromatography, it is defined as the
difference in retention times of adjacent
peaks divided bytheir width. In mass
spectrometry, resolution is the smallest
difference inm/z values that can be detected
as separate peaks. Report the m/z value
atwhich resolution is measured.
Root-mean-square (rms) noise - Standard
deviation of the noise in a region
where the signal is flat:
where Ai is the measured signal for the ith
data point, A is the mean signal,and n is the
number of data points.
Signal averaging - Improvement of a signal
by averaging successivescans. The signal
increases in proportion to the number of scans
accumulated.The noise increases in proportion
to the square root of the number ofscans.
Therefore, the signal-to-noise ratio improves
in proportion to thesquare root of the number
of scans collected.
Snell’s law -
Stray light -Unintentional light that reaches
thedetector but has not passed through the
sample or is not part of the narrow set of
wavelengths expected from the
monochromator.
Surface plasmon resonance - A sensitive
means to measure the bindingof molecules to
a thin gold layer on the underside of a prism.
Lightdirected through the prism is reflected
from the gold surface. There is onenarrow
range of angles at which reflection is nearly 0
because the goldabsorbs the light to set up
oscillations (called plasmons) of the electron
cloud in the metal. When a thin layer of
material (such as protein or DNA)binds to the
side of the gold away from the prism, the
electrical propertiesof the gold are changed
and the reflectivity changes.
Thermocouple - An electrical junction across
which a temperaturedependentvoltage exists.
Thermocouples are calibrated for
measurementof temperature and usually
consist of two dissimilar metals in contact with
each other.
Waveguide - A thin layer or hollow structure
in which electromagneticradiation is totally
reflected.
. Tungsten and silicon carbide lamps
behaveapproximately as blackbodies, which
are objects that absorb alllight striking them
and reradiate a characteristic spectrum of
radiation.Emission of radiant energy from the
surface of the blackbodyis proportional to the
fourth power of temperature and shifts to
shorter wavelengths as temperature increases.
Lasers provide highintensity,coherent,
monochromatic radiation by stimulated
emissionfrom a medium in which an excited
state has been pumped to ahigher population
than that of a lower state. Sample cells must
betransparent to the radiation of interest. A
reference sample compensatesfor reflection
and scattering by the cell and solvent. A
gratingmonochromator disperses light into its
component wavelengths.The finer a grating is
ruled, the higher the resolution and the
greaterthe dispersion of wavelengths over
angles. Narrow slits improveresolution but
increase noise, because less light reaches the
detector.A bandwidth that is one-fi fth of the
width of the spectral peak isa good
compromise between maximizing signal-to-
noise ratio andminimizing peak distortion.
Stray light introduces absorbance errorsthat
are most serious when the transmittance of a
sample is verylow. Filters pass wide bands of
wavelength and reject other bands.A
photomultiplier tube is a sensitive detector of
visible and ultravioletradiation; photons cause
electrons to be ejected from a metallic
cathode. The signal is amplified at each
successive dynode on whichthe photoelectrons
impinge. Photodiode arrays and charge
coupleddevices are solid-state detectors in
which photons create electrons andholes in
semiconductor materials. Coupled to a
polychromator, thesedevices can record all
wavelengths of a spectrum simultaneously,
withresolution limited by the number and
spacing of detector elements. An image
intensified charge coupled device converts
incoming photons to electrons, amplifies the
electrons with a microchannel plate, converts
electrons back to photons with a fluorescent
screen, and then detectsthe photons. Common
infrared detectors include thermocouples,
ferroelectric materials, and photoconductive
and photovoltaic devices.Photoacoustic
spectroscopy detects infrared absorption by
convertingsome absorbed energy to sound
waves that are detected. Cavity
ringdownspectroscopy measures very low
absorption by gases or liquidsby circulating a
laser beam through the sample thousands of
times tomultiply the pathlength. The decay
time of the circulating laser poweris a
measure of absorption by the sample.
CHAPTER 21
Ablation - Vaporization of a small volume of
material by a laser pulse.
Aerosol - A suspension of very small liquid or
solid particles in air or gas.Examples include
fog and smoke.
Atomic absorption - A technique in which the
absorption oflight by free gaseous atoms in a
flame or furnace is used to measure
theconcentration of atoms
Atomic emission - A technique in which the
emission oflight by thermally excited atoms in
a plasma, flame, or furnace is used tomeasure
the concentration of atoms.
Atomic fluorescence - A technique in which
electronic transitionsof atoms in a flame,
furnace, or plasma are excited by light, and
the fluorescence is observed at a right angle
to the incident beam.
Atomization - Process in which a compound is
decomposed into its atomsat high
temperature.
Background correction - In atomic
spectroscopy, a means of distinguishingsignal
due to analyte from signal due to absorption,
emission, or scattering by the flame, furnace,
plasma, or sample matrix.
Boltzmann distribution - Relative population
of two states at thermalequilibrium:
where Ni is the population of the state, gi is
the degeneracy of the state, Eiis the energy
of the state, k is Boltzmann’s constant, and T is
temperaturein kelvins. Degeneracy refers to
the number of states with the same energy.
Chemical interference - In atomic
spectroscopy, any chemical reactionthat
decreases the efficiency of atomization.
Detection limit -The smallest quantity of
analyte that is “significantlydifferent” from a
blank. The detection limit is often taken as the
concentrationof analyte that gives a signal
equal to three times the standard deviationof
signal from a blank. Also called lower limit of
detection
Doppler effect -A molecule moving toward a
source of radiation experiencesa higher
frequency than one moving away from the
source.
Graphite furnace - A graphite tube that can
be heated electrically to about2 500 K to
decompose and atomize a sample for atomic
spectroscopy
Heisenberg uncertainty principle - Certain
pairs of physical quantitiescannot be known
simultaneously with arbitrary accuracy. If dE is
the uncertaintyin the energy difference
between two atomic states and dt is the
lifetimeof the excited state, their product
cannot be known more accuratelythan dEdt $
h/(4p), where h is Planck’s constant. A similar
relation holdsbetween the position and the
momentum of a particle. If position is
knownvery accurately, then the uncertainty in
momentum is large, and vice versa.
Inductively coupled plasma - A high-
temperature plasma that derives itsenergy
from an oscillating radio-frequency fi eld. It is
used to atomize asample for atomic emission
spectroscopy.
Ionization interference - In atomic
spectroscopy, loss of signal intensityas a result
of ionization of analyte atoms.
Ionization suppressor - An element used in
atomic spectroscopy todecrease the extent of
ionization of the analyte.
Isobaric interference - In mass spectrometry,
overlap of two peaks withnearly the same
mass. For example, 41K1 and 40ArH1 differ
by 0.01atomic mass unit and appear as a
single peak unless the spectrometerresolution
is great enough to separate them.
Laser-induced breakdownspectroscopy -
Semiquantitative measurementof elements in a
surface by vaporizing a small patch with a
short laserpulse and measuring atomic
emission from the plasma above the surface
matrix - The medium containing analyte (that
is, everything in the sampleother than
analyte). For many analyses, it is important
that standards beprepared in the same
matrix as the unknown.
Matrix modifier - Substance added to sample
for atomic spectroscopy tomake the matrix
more volatile or the analyte less volatile so
that the matrixevaporates before analyte
does.
Nebulization - In atomic spectroscopy, this
device breaks the liquid sample into a mist of
fine droplets.
Piezoelectric crystal - A crystal that deforms
when an electric field isapplied.
Plasma - A gas that is hot enough to contain
free ions and electrons, aswell as neutral
molecules.
Pressure broadening - In spectroscopy, line
broadening due to collisionsbetween
molecules.
Releasing agent - In atomic spectroscopy, a
substance that prevents chemicalinterference.
Self-absorption - In a luminescence
measurement, a high concentration ofanalyte
molecules can absorb energy from excited
analyte. Also calledinner filter effect. If the
absorbed energy is dissipated as heat
instead of light, fluorescence does not
increase in proportion to analyte
concentration. Analyte concentration can be so
high that fluorescence decreases with
increasing concentration. In flame emission
atomic spectroscopy, there is alower
concentration of excited-state atoms in the
cool, outer part of the flame than in the hot,
inner flame. The cool atoms can absorb
emission from the hot ones and thereby
decrease the observed signal.
Spectral interference - In atomic spectroscopy,
any physical process thataffects light intensity
at the analytical wavelength. Created by
substancesthat absorb, scatter, or emit light of
the analytical wavelength.
X-ray fluorescence - Emission of X-rays from
a material following theabsorption of X-rays
by the material.
In atomic spectroscopy, absorption, emission,
or fl uorescence from gaseous atoms is
measured. Liquids may be atomized by a
plasma, a furnace, or a flame. Flame
temperatures are usually in the range2 300–
3 400 K. The choice of fuel and oxidant
determines the temperature of the flame and
affects the extent of spectral, chemical, or
ionization interference that will be
encountered. Temperature instabilityaffects
atomization in atomic absorption and has an
even largereffect on atomic emission, because
the excited-state population isexponentially
sensitive to temperature. An electrically
heated graphitefurnace requires less sample
than a flame and has a lower detectionlimit.
In an inductively coupled plasma, a radio-
frequency inductioncoil heats Ar1 ions to 6
000–10 000 K. At this high
temperature,emission is observed from
electronically excited atoms and ions.There is
little chemical interference in an inductively
coupled plasma,the temperature is very
stable, and little self-absorption is observed.
Plasma emission spectroscopy does not
require a light source andis capable of
measuring |70 elements simultaneously with a
chargeinjection device detector. Background
correction for a given emissionpeak is based
on subtracting the intensity of neighboring
pixels in thedetector. The lowest detection
limits are obtained by directing theplasma
into a mass spectrometer that separates and
measures ions from the plasma. In flame and
furnace atomic absorption spectroscopy,a
hollow-cathode lamp made of the analyte
element providesspectral lines sharper than
those of the atomic vapor. The
inherentlinewidth of atomic lines is limited by
the Heisenberg uncertaintyprinciple. Lines in a
fl ame, furnace, or plasma are broadened by
a
factor of 10–100 by the Doppler effect and
by atomic collisions. Correctionfor background
emission from the flame is possible by
electricallypulsing the lamp on and off or
mechanically chopping thebeam. Light
scattering and spectral background can be
subtracted bymeasuring absorption with a
deuterium lamp or by Zeeman background
correction, in which the atomic energy levels
are alternatelyshifted in and out of resonance
with the lamp frequency by a magnetic field.
Chemical interference can be reduced by
addition ofreleasing agents, which prevent the
analyte from reacting with interferingspecies.
Ionization interference in flames is suppressed
by addingeasily ionized elements such as
Cs.In inductively coupled plasma–mass
spectrometry, isobaricinterference occurs
between species with the same mass and
charge.Interference can be eliminated if mass
spectral resolution is suffi -
ciently great, but this is not often the case. A
dynamic reaction cellreduces isobaric
interference by using an exothermic reaction
of agas such as NH3, N2O, or CO to remove
interfering molecular ionssuch as ArO1 or to
transform analyte into a molecular ion that
canbe measured without interference.For
qualitative and semiquantitative analysis,
solids and liquidscan be sampled by laser
ablation. Ablated material is swept throughan
inductively coupled plasma to detect multiple
elements by atomicemission or mass
spectrometry. When emission is measured, the
technique is called laser-induced breakdown
spectroscopy. Matrixmatchedstandards are
necessary for semiquantitative analysis.X-ray
fl uorescence is the emission of X-rays by a
material afterit has absorbed X-rays.
Handheld spectrometers enable
qualitativeand semiquantitative measurements
of elemental composition in the field. Each
element emits characteristic, narrow X-ray
lines, whoseenergies identify the element and
whose integrated intensities areproportional
to the concentration of the element. Many
elementscan be measured at levels below
0.01 wt% with |10% accuracywhen suitable
care is exercised. X-rays from the
spectrometersource ionize electrons from the
inner (K and L) shells of atoms.When outer
electrons fall into the vacancies, characteristic
X-rayscalled Ka, Kb, La, and Lb are emitted.
You must verify that bothexpected lines for
each element are observed in the spectrum
beforeyou conclude that the element is
present.
CHAPTER 22 Ions are created or desorbed in the ion
source of a mass spectrometer. Neutral molecules are
converted into ions by electron ionization (which
produces a molecular ion, M1?, and many fragments)
or by chemical ionization (which tends to create MH1
and few fragments). A magnetic sector mass
spectrometer separates gaseous ions by accelerating
them in an electric field and defl ecting ions of
different mass-to-charge (m/z) ratio through different
arcs in a magnetic field. Ions are detected by an
electron multiplier, which works like a photomultiplier
tube. A conversion dynode may be necessary prior to
the electron multiplier to ensure that all ions produce
a similar electrical response at the detector. The mass
spectrum is a graph of detector response versus m/z
value. A double-focusing mass spectrometer attains
high resolution by employing an electric sector with
the magnetic sector to select ions with a narrow range
of kinetic energy. Other mass separators include the
transmission quadrupole mass spectrometer, the time-
of-flight mass spectrometer, the three-dimensional
quadrupole ion trap, the linear ion trap, and the
orbitrap. The time-of-fl ight instrument is capable of
high acquisition rates and has a nearly unlimited
upper mass range. Resolving power is defi ned as
m/Dm or m/m1/2, where m is the mass being
measured, Dm is the difference in mass between two
peaks that are separated with a 10% valley
between them, and m1/2 is the width of a peak at
half-height. Time-of-flight and orbitrap spectrometers
can provide high-resolution spectra with part per
million mass accuracy. In a mass spectrum, the
molecular ion is found from the highest m/z value of
any “signifi cant” peak that cannot be attributed to
isotopes or background signals. For a given
composition, you should be able to predict the
relative intensities of the isotopic peaks at M11, M12,
and so on. Among common elements, Cl and Br have
particularly diagnostic isotope patterns. From a
molecular composition, the rings 1 double bonds
equation helps us propose structures. An organic
compound with an odd number of nitrogen atoms will
have an odd mass. Fragment ions arising from bond
cleavage and rearrangements provide clues to
molecular structure. Gas emerging from a capillary
gas chromatography column can go directly into the
ion source of a well-pumped mass spectrometer to
provide qualitative and quantitative information
about the components of a mixture. For capillary
liquid chromatography with a flow rate of |500
nL/min, liquid can be admitted directly to a well
pumped electron ionization source. For liquid
chromatography with wider columns and higher fl ow
rates, electrospray ionization employs high voltage at
the exit of the column, combined with coaxial N2 gas
fl ow, to create a fi ne aerosol of charged droplets
containing ions that were already present in the liquid
phase. Analyte is often associated with other ions to
give species such as [MNa]1 or [M(CH3CO2)]2.
Control of pH helps ensure that selected analytes are
in anionic or cationic form. Another interface between
liquid chromatography and mass spectrometry is
atmospheric pressure chemical ionization, which
utilizes a corona discharge to create a variety of
gaseous ions from aerosol droplets. Both atmospheric
pressure chemical ionization and electrospray tend to
produce unfragmented ions. Collisionally activated
dissociation to produce fragment ions is controlled by
the cone voltage at the mass spectrometer inlet.
Electrospray of proteins typically creates an array of
highly charged ions such as MHn1n. Matrix-assisted
laser desorption/ionization is a gentle way to
produce predominantly singly charged, intact protein
ions for mass spectrometry. A reconstructed total ion
chromatogram shows the signal from all ions above a
chosen m/z emerging from chromatography as a
function of time. An extracted ion chromatogram
shows the signal for one ion taken from the complete
mass spectrum. Selected ion monitoring of one or a
few values of m/z improves signal-to-noise because
all of the time is spent measuring just that one or a
few ions. In selected reaction monitoring, a precursor
ion isolated by one mass fi lter passes into a collision
cell in which it breaks into products. One (or more)
product ion is then selected by a second mass fi lter
for passage to the detector. This process is extremely
selective for just one analyte and vastly increases the
signal-to-noise ratio for this analyte. Electrontransfer
dissociation is employed in protein sequencing to
break amide bonds in a polypeptide without
breaking other bonds. Several methods can ionize
molecules from the surface of an object in ambient
atmosphere. Direct analysis in real time (DART) and a
low-temperature plasma use electronically excited
helium or nitrogen to ionize analytes. Desorption
electrospray ionization (DESI) directs electrosprayed
solvent onto a surface to dislodge ions. An ion
mobility spectrometer separates gas-phase ions by
their different mobilities in an electric field at
atmospheric pressure. Field asymmetric waveform
spectrometry separates ions based on differences of
mobility in a high electric field at which mobility is not
a linear function of electric field. Ion mobility can add
another dimension to mass spectrometry by
separating isobaric ions according to differences in
mobility. Atmospheric pressure chemical ionization
- A method to interface liquid chromatography to
mass spectrometry. Liquid is nebulized into a fine
aerosol by a coaxial gas flow and the application of
heat. Electrons from a high-voltage corona discharge
create cations and anions from analyte exiting the
chromatography column. The most common species
observed with this interface is MH1, the protonated
analyte, with little fragmentation. Base peak - Most
intense peak in a mass spectrum. Chemical ionization
- gentle method of producing ions for a mass
spectrometer without extensive fragmentation of the
analyte molecule, M. A reagent gas such as CH4 is
bombarded with electrons to make CH15, which
transfers H1 to M, giving MH1. Collisionally
activated dissociation - Fragmentation of an ion in a
mass spectrometer by high-energy collisions with gas
molecules. In atmospheric pressure chemical ionization
or electrospray interfaces, collisionally activated
dissociation at the inlet to the mass filter can be
promoted by varying the cone voltage. In tandem
mass spectrometry, dissociation occurs in a collision
cell between the two mass separators. Also called
collision-induced dissociation. Desorption
electrospray ionization (DESI) - A solvent is
electrosprayed onto a surface to dissolve analyte
from the surface into aerosol microdroplets, which can
be analyzed with a mas spectrometer. Direct
analysis in real time (DART) - A DART source
produces excited He or N2, which is directed at the
surface of an object to be sampled in ambient
atmosphere. The excited species react with ambient
moisture to create protonated water clusters that
react with analyte M to make MH1. The MH1 is
measured by mass spectrometry. Double-focusing
mass spectrometer - A spectrometer that uses electric
and magnetic sectors in series to obtain high
resolution. Electron ionization - used to create ions
from gaseous molecules in the inlet of a mass
spectrometer by bombardment of the gas with high
energy electrons. Electrospray ionization - method
for interfacing liquid chromatography to mass
spectrometry. A high potential applied to the liquid at
the column exit creates charged droplets in a fi ne
aerosol. Gaseous ions are derived from ions that
were already in the mobile phase on the column. It is
common to observe protonated bases (BH1), ionized
acids (A2), and complexes formed between analyte,
M (which could be neutral or charged), and stable
ions such as NH14, Na1, HCO22, or CH3CO22 that
were already in solution. Extracted ion
chromatogram - Chromatogram made by collecting
consecutive full-range mass spectra, but selecting just
one value of m/z for display. Most time is spent
monitoring values of m/z that will not be displayed. A
selected ion chromatogram provides greater signal-
to-noise ratio than an extracted ion chromatogram
because all of the time is taken to monitor just one or
a few values of m/z in the selected ion
chromatogram. Ion mobility spectrometer - An
instrument that measures the drift time of gaseous ions
migrating in an electric fi eld against a flow of gas.
The “spectrum” of detector current versus drift time is
really an electropherogram of a gas. Linear
quadrupole ion trap - An instrument that separates
gaseous ions by trapping them in stable trajectories
inside a linear quadrupole by use of radio-frequency
fields. Ions can be expelled from the trap in order of
increasing m/z for mass spectrometry. Magnetic
sector mass spectrometer - A device that separates
gaseous ions that have the same kinetic energy by
passing them through a magnetic field perpendicular
to their velocity. Trajectories of ions with a certain
mass-to-charge ratio are bent exactly enough to
reach the detector. Other ions are deflected too much
or too little. Mass spectrometry - A technique in which
gaseous molecules are ionized, accelerated by an
electric field, and then separated according to their
mass-to-charge ratio. Mass-to-charge ratio, m/z -
The mass of an ion in daltons divided by the charge
of the ion measured in multiples of the elementary
charge. For 23Na1, for example, m/z ¯ 23/1 5 23.
Matrix-assisted laser desorption/ionization (MALDI)
- A gentle technique for introducing predominantly
singly charged, intact macromolecular ions into the
gas phase. An intimate solid mixture of analyte plus a
large excess of a small, ultraviolet-absorbing
molecule is irradiated by a pulse from an ultraviolet
laser. The small molecule (the matrix) absorbs the
radiation, becomes ionized, evaporates, and expands
in a supersonic jet that carries analyte into the gas
phase. Matrix ions apparently transfer charge to the
analyte. Molecular ion - In mass spectrometry, an ion
that has not lost or gained any atoms during
ionization. Nitrogen rule - A compound with an odd
number of nitrogen atoms—in addition to C, H,
halogens, O, S, Si, and P—will have an odd nominal
mass. A compound with an even number of nitrogen
atoms (0, 2, 4, …) will have an even nominal mass.
Nominal mass - Integer mass of the species with the
most abundant isotope of each of the constituent
atoms. For C, H, and Br, the most abundant isotopes
are 12C, 1H, and 79Br. Therefore, the nominal mass
of C2H5Br is (2 3 12) 1 (5 3 1) 1 (1 3 79) 5 108.
Orbitrap mass spectrometer - A device that traps
ions in stable orbits around a central electrode. Ions
oscillate from one end of the trap to the other,
inducing image currents in the outer electrodes.
Fourier analysis of the image currents gives m/z for
the oscillating ions. Precursor ion - In tandem mass
spectrometry (selected reaction monitoring), the ion
selected by the fi rst mass separator for
fragmentation in the collision cell. Product ion - In
tandem mass spectrometry (selected reaction
monitoring), the fragment ion from the collision cell
selected by the fi nal mass separator for passage
through to the detector. Reconstructed total ion
chromatogram - In chromatography, a graph of the
sum of intensities of all ions detected at all masses
(above a selected cutoff) versus time. Resolving
power - In mass spectrometry, resolving power can
be defined as m/Dm, where Dm is the separation of
two peaks when the overlap at the base is 10% of
the peak height and m is the smaller of the two m/z
values. Alternatively, resolving power can be taken as
m/m1/2, where m1/2 is the width of a peak at half
the maximum height. In this case, the dip between two
barely resolved peaks is 8% below the peak heights.
Rings 1 double bonds formula - The number of rings
1 double bonds in a molecule with the formula
CcHhNnOx is c 2 h/2 1 n/2 1 1, where c includes all
Group 14 atoms (C, Si, Ge, Sn, Pb, which all make
four bonds), h includes H 1 halogens (which make one
bond), and n is the number of Group 15 atoms (N, P,
As, Sb, Bi, which make three bonds). Group 16 atoms
(which make two bonds) do not affect the result.
Selected ion chromatogram - A graph of detector
response versus time when a mass spectrometer
monitors just one or a few species of selected mass-
to-charge ratio, m/z, emerging from a
chromatograph. Also called mass chromatogram.
Selected ion monitoring - Use of a mass
spectrometer to monitor species with just one or a few
mass-to-charge ratios, m/z. Selected reaction
monitoring - A technique in which the precursor ion
selected by one mass separator passes through a
collision cell in which the precursor breaks into several
fragment ions (product ions). A second mass separator
then selects one (or a few) of these ions for detection.
Selected reaction monitoring improves
chromatographic signal-to-noise ratio because it is
insensitive to almost everything other than the
intended analyte. Also called mass spectrometry–
mass spectrometry (MS–MS) or tandem mass
spectrometry. Three-dimensional quadrupole ion
trap – An instrument that separates gaseous ions by
trapping them in stable three dimensional trajectories
inside a metal chamber to which a radio-frequency
electric field is applied. Application of an oscillating
electric field between the ends of the chamber
destabilizes the trajectories of ions with a particular
mass-to-charge ratio, expelling them from the cavity
and into a detector. Time-of-flight mass
spectrometer - Ions of different mass accelerated
through the same electric fi eld have different
velocities. The lighter ions move faster than the
heavier ions. The time-of-flight spectrometer finds the
mass-to-charge ratio by measuring the time that each
group of ions requires to travel a fixed distance to
the detector. Transmission quadrupole mass
spectrometer – A mass spectrometer that separates
ions by passing them between four metallic cylinders
to which are applied direct current and oscillating
electric fields. Resonant ions with the right mass-to-
charge ratio pass through the chamber to the detector
while nonresonant ions are deflected into the
cylinders and are lost. CHAPTER 23 A solute can be
extracted from one phase into another in which it is
more soluble. The ratio of solute concentrations in
each phase at equilibrium is called the partition
coefficient. If more than one form of the solute exists,
we use a distribution coefficient instead of a partition
coefficient. We derived equations relating the
fraction of solute extracted to the partition or
distribution coefficient, volumes, and pH. Many small
extractions are more effective than a few large
extractions. A metal chelator, soluble only in organic
solvents, can extract metal ions from aqueous
solutions, with selectivity achieved by adjusting pH.
Crown ethers and salts containing a hydrophobic ion
act as phase transfer agents to bring a hydrophilic
ion from an aqueous phase into an organic phase. In
adsorption and partition chromatography, a
continuous equilibration of solute between mobile and
stationary phases occurs. Eluent goes into a column
and eluate comes out. Columns may be packed with
stationary phase, or may be open tubular with
stationary phase bonded to the inner wall. In ion-
exchange chromatography, the solute is attracted to
the stationary phase by coulombic forces. In molecular
exclusion chromatography, the fraction of stationary
phase pore volume available to solute decreases as
the size of the solute molecules increases. Affinity
chromatography relies on specific, noncovalent
interactions between the stationary phase and one
solute in a complex mixture. The relative retention of
two components is the quotient of their adjusted
retention times. The retention factor for a single
component is the adjusted retention time divided by
the elution time for solvent. Retention factor gives the
ratio of time spent by solute in the stationary phase
to time spent in the mobile phase. When a separation
is scaled up from a small load to a large load, linear
velocity is held constant and the crosssectional area
of the column should be increased in proportion to the
loading. Plate height (H 5 s2/x) is related to the
breadth of a band emerging from the column. The
smaller the plate height the sharper the band. The
number of plates for a Gaussian peak is N 5
5.55t2r/w21/2. Plate height is approximately the
length of column required for one equilibration of
solute between mobile and stationary phases.
Resolution of neighboring peaks is the difference in
retention time divided by the average width
(measured at the baseline, w54s). Resolution is
proportional to 1N. Doubling the length of a column
increases resolution by 12. Some retention (k < 0.5 to
20) is essential to achieve resolution, but greater
retention increases separation time without substantial
improvements in resolution. Relative retention (a)
strongly influences chromatographic resolution. The
standard deviation of a diffusing band of solute is
s512Dt, where D is the diffusion coefficient and t is
time. The van Deemter equation describes band
broadening on the chromatographic column: H < A 1
B/ux 1 Cux, where H is plate height, ux is linear
velocity, and A, B, and C are constants. The first term
represents irregular flow paths, the second,
longitudinal diffusion, and the third, the finite rate of
mass transfer of solute between mobile and
stationary phases. The optimum flow rate, which
minimizes plate height, is faster for gas
chromatography than for liquid chromatography. The
number of plates and the optimal flow rate increase
as the stationary phase particle size or open tubular
column diameter decreases. In gas chromatography,
open tubular columns can provide higher resolution or
shorter analysis times than packed columns. Bands
spread during injection, during detection, and in
connecting tubing, as well as during passage through
the separation column. The observed variance of the
band is the sum of the variances for all mechanisms of
spreading. Fronting and tailing can be corrected by
injecting less sample and by masking strong
adsorption sites on the stationary phase. Adjusted
retention time = Tr’ = tr - tm, where tr is the retention
time of a solute and tm is the time needed for mobile
phase to travel the length of the column. Adsorption
chromatography - solute equilibrates between the
mobile phase and adsorption sites on the stationary
phase. Affinity chromatography - a particular solute
is retained by a column by virtue of a specifi c
interaction with a molecule covalently bound to the
stationary phase. Asymmetry factor – In C, the
parameter describing the shape of a peak. B is the
distance from the peak apex to the back of the peak
measured at 10% of peak height. A is the distance
from the peak apex to the front of the peak at 10%
of peak height. B/A 5 1 is a symmetrical peak. B/A .
1 is tailing and B/A , 1 is fronting Baseline resolution
– In C, when two adjacent peaks are suffi ciently
resolved that the signal between the peaks returns to
the baseline (resolution . 1.5). Allows determination of
accurate peak areas. Chromatogram - graph
showing chromatography detector response as a
function of elution time or volume. Diffusion - Net
transport of a solute from a region of high
concentration to a region of low concentration caused
by the random movement of molecules in a liquid or
gas (or, very slowly, in a solid). Diffusion coefficient
- Defined by Fick’s first law of diffusion: J = -
D(dc/dx), where J is the rate at which molecules
diffuse across a plane of unit area and dc/dx is the
concentration gradient in the direction of diffusion
Distribution coefficient - For a solute partitioned
between two phases, the distribution coeffi cient is the
total concentration of all forms of solute in phase 2
divided by the total concentration in phase 1. Eluate -
What comes out of a chromatography column. Also
called effluent. Eluent - Solvent applied to the
beginning of a chromatography column. Elution -
Process of passing a liquid or a gas through a
chromatography column. Extraction - Process in which
a solute is transferred from one phase to another.
Analyte is sometimes removed from a sample by
extraction into a solvent that dissolves the analyte.
Gel permeation chromatography - See molecular
exclusion chromatography. Laminar flow - Motion
with a parabolic velocity profi le of fl uid through a
tube. Motion is fastest at the center and zero at the
walls. Linear velocity - In chromatography, the
distance per unit time travelled by the mobile phase.
Also called linear fl ow rate, in contrast to volume
flow rate Longitudinal diffusion - Diffusion of solute
molecules parallel to the direction of travel through a
chromatography column. Mass transfer - Net
movement of a molecule from one location to another
by mechanisms such as diffusion, convection, and
deliberate mixing. In chromatography, mass transfer
refers to movement of solute between the mobile and
stationary phases. Molecular exclusion
chromatography - technique in which the stationary
phase has a porous structure into which small
molecules can enter but large molecules cannot.
Molecules are separated by size, with larger
molecules moving faster than smaller ones. Also called
size exclusion, gel filtration, gel permeation, or size
exclusion chromatography. Open tubular column - In
chromatography, a hollow capillary column whose
walls are coated with stationary phase Partition
chromatography - A technique in which separation is
achieved by equilibration of solute between two
phases Partition coefficient K- The equilibrium
constant for the reaction in which a solute is
partitioned between two phases: solute (in phase 1) Δ
solute (in phase 2). Relative retention - In
chromatography, the ratio of adjusted retention times
for two components. If component 1 has an adjusted
retention time of T’r1 and component 2 has an
adjusted retention time of alpha = T’r2/T’r1, the
relative retention is a 5 t¿r2/t¿r1. Also called
separation factor. Resolution - How close two bands
in a spectrum or a chromatogram can be to each
other and still be seen as two peaks. In
chromatography, it is defined as the difference in
retention times of adjacent peaks divided by their
width. In mass spectrometry, resolution is the smallest
difference in m/z values that can be detected as
separate peaks. Report the m/z value at which
resolution is measured. Retention factor - In
chromatography, the adjusted retention time for a
peak divided by the time for the mobile phase to
travel through the column. Retention factor is also
equal to the ratio of the time spent by the solute in
the stationary phase to the time spent in the mobile
phase. Also called capacity factor, capacity ratio, and
partition ratio. Retention time - The time from
injection for an individual solute to reach the detector
of a chromatography column. Retention volume - The
volume of solvent needed to elute a solute from a
chromatography column. Silanization - Treatment of
a chromatographic solid support or glass column with
hydrophobic silicon compounds that bind to the most
reactive Si—OH groups. It reduces irreversible
adsorption and tailing of polar solutes. Stationary
phase – phase that does not move through the
column. Van Deemter equation - Describes the
dependence of chromatographic plate height, H, on
linear velocity, ux: H = A +B/ux +Cux. The constant A
depends on band-broadening processes such as
multiple fl ow paths that are independent of flow
rate. B depends on the rate of diffusion of solute in
the mobile phase. C depends on the rate of mass
transfer (that is, the equilibration time) between the
stationary and mobile phases. CHAPTER 24 In gas
chromatography, a volatile liquid or gaseous solute is
carried by a gaseous mobile phase over a stationary
phase on the inside of an open tubular column or on a
solid support. Long, narrow, fused-silica open tubular
columns have low capacity but give excellent
separation. They can be wall-coated or porous layer.
Packed columns provide high capacity but poor
resolution. Solutes must be partially in the vapour
phase to be eluted from a gas chromatography
column. Solute partial pressure and retention are
governed by thermodynamics, meaning that column
temperature controls retention. Each liquid stationary
phase most strongly retains solutes in its own polarity
class (“like dissolves like”). Solid stationary phases,
including porous carbon, alumina, and molecular
sieves, are used to separate permanent gases. The
retention index measures elution times in relation to
those of linear alkanes. Temperature or pressure
programming reduces elution times of strongly
retained components. Without compromising
separation effi ciency, the linear velocity may be
increased when H2 or He, instead of N2, is used as
carrier gas. Split injection provides high-resolution
separations of relatively concentrated samples.
Splitless injection of very dilute samples requires
solvent trapping or cold trapping to concentrate
solutes at the start of the column to give sharp bands.
On-column injection is best for quantitative analysis
and for thermally unstable solutes. Quantitative
analysis is usually done with internal standards in gas
chromatography. Coelution of an unknown peak with
a spike of a known compound on several columns with
different retention mechanisms is useful for qualitative
identifiation of the unknown peak. Mass spectral
detection provides qualitative information to help
identify unknowns. The mass spectrometer is more
sensitive and less subject to interference when
selected ion monitoring or selected reaction
monitoring is employed. Thermal conductivity
detection has universal response but is not very
sensitive. Flame ionization detection is sensitive
enough for most columns and responds to most
organic compounds. Electron capture, nitrogen-
phosphorus, flame photometry, photoionization,
chemiluminescence, and atomic emission detectors are
specific for certain classes of compounds or individual
elements. You need to define the goal of an analysis
before developing a chromatographic method. One
key to successful chromatography is a clean sample.
Solid-phase microextraction, stir-bar sorptive
extraction, purge and trap, and thermal desorption
can isolate volatile components from complex
matrices. After sample preparation the remaining
decisions for method development are to select a
detector, a column, and the injection method, in that
order. Carrier gas - Mobile phase gas in gas
chromatography Cold trapping - Splitless gas
chromatography injection technique in which solute is
condensed far below its boiling point in a narrow
band at the start of the column. Electron capture
detector - Gas chromatography detector that is
particularly sensitive to compounds with halogen
atoms, nitro groups, and other groups with high
electron affi nity. Makeup gas (N2 or 5% CH4 in Ar)
is ionized by b-rays from 63Ni to liberate electrons
that produce a small, steady current. High-electron-
affi nity analytes capture some of the electrons and
reduce the detector current. Flame ionization
detector - A GC detector in which solute is burned in
an H2-air flame to produce CHO1 ions. The current
carried through the flame by these ions is
proportional to the concentration of susceptible
species in the eluate. Gas chromatography - A form
of chromatography in which the mobile phase is a
gas. General elution problem - In chromatography,
the inability of a single isothermal or isocratic
condition to provide adequate separation in
reasonable time for solutes with a wide range of
retention. Guard column - In high-performance liquid
chromatography, a short column packed with the
same material as the main column and placed
between the injector and the main column. The guard
column removesnimpurities that might irreversibly bind
to the main column and degrade it. Also called
precolumn. In gas chromatography, the guard column
is a length of empty, silanized capillary ahead of the
chromatography column. Nonvolatile residues
accumulate in the guard column. Headspace - Gas
phase above a solid or liquid sample within a sealed
vial. The concentration of a volatile component in the
headspace is proportional to its concentration in the
solid or liquid. Molecular sieve - crystalline solid
particle with pores the size of small molecules.
Zeolites (sodium aluminosilicates) are a common type.
On-column injection - Technique used in gas
chromatography to place a thermally unstable
sample directly on the column without excessive
heating in an injection port. Solute is condensed at the
start of the column by low temperature, and then the
temperature is raised to initiate chromatography.
Open tubular column - a hollow capillary column
whose walls are coated with stationary phase Packed
column - column filled with stationary phase particles.
Phase ratio - the relative volume of mobile phase to
stationary phase in the column. In older literature, the
phase ratio may be given as Phi which is the
reciprocal of Beta m. Purge and trap - A method for
removing volatile analytes from liquids or solids,
concentrating the analytes, and introducing them into
a gas chromatograph. A carrier gas bubbled through
a liquid or solid extracts volatile analytes, which are
then trapped in a tube containing adsorbent. After
analyte has been collected, the adsorbent tube is
heated and purged in the reverse direction to desorb
the analytes, which are collected by cold trapping in
a gas chromatography column. Retention gap - In
gas chromatography, a 3- to 10-m length of empty,
silanized capillary ahead of the chromatography
column. The retention gap improves the peak shape
of solutes that elute close to solvent when large
volumes of solvent are injected or when the solvent
has a very different polarity from that of the
stationary phase. Sample preparation - Transforming
a sample into a state that is suitable for analysis. This
process can include concentrating a dilute analyte
and removing or masking interfering species. Selected
ion monitoring - Use of a mass spectrometer to
monitor species with just one or a few mass-to-charge
ratios, m/z. Selected reaction monitoring - A
technique in which the precursor ion selected by one
mass separator passes through a collision cell in which
the precursor breaks into several fragment ions
(product ions). A second mass separator then selects
one (or a few) of these ions for detection. Selected
reaction monitoring improves chromatographic signal-
to-noise ratio because it is insensitive to almost
everything other than the intended analyte. Also
called mass spectrometry–mass spectrometry (MS–
MS) or tandem mass spectrometry. Septum - A disk,
usually made of silicone rubber, covering the injection
port of a gas chromatograph. The sample is injected
by syringe through the septum. Solid-phase
microextraction - Extraction of compounds from
liquids or gases into a coated fiber dispensed from a
syringe needle. After extraction, the fiber is
withdrawn into the needle and the needle is inserted
through the septum of a chromatograph. The fiber is
extended inside the injection port and adsorbed
solutes are desorbed by heating (for gas
chromatography) or solvent (for liquid
chromatography). Solvent trapping - Splitless gas
chromatography injection technique in which solvent is
condensed below its boiling point at the start of the
column. Solutes dissolve in a narrow band in the
condensed solvent Spiking - Addition of a known
compound (usually at a known concentration) to an
unknown. In isotope dilution mass spectrometry, the
spike is the added, unusual isotope. Spike is a noun
and a verb. In co-chromatography, spiking is the
simultaneous chromatography of a known compound
with an unknown. If a known and an unknown have
the same retention time on several columns, they are
probably identical. Also called fortifi cation. Split
injection - used in capillary gas chromatography to
inject a small fraction of sample onto the column; the
rest of the sample is blown out to waste. Splitless
injection - used in capillary gas chromatography for
trace analysis and quantitative analysis. The entire
sample in a low-boiling solvent is directed to the
column, where the sample is concentrated by solvent
trapping (condensing the solvent below its boiling
point) or coldntrapping (condensing solutes far below
their boiling range). The column is then warmed to
initiate separation. Stir-bar sorptive extraction -
Sample preparation method similar to solid phase
microextraction, except the sorptive layer is coated
on the outside ofba stirring bar. Coating volume is
greater than the fi ber volume in solidphase
microextraction, so it provides ~102 times greater
sensitivity for traces of analyte. Analyte is removed
from the coating by thermal desorption for
chromatography. Temperature programming -
Raising the temperature of a gas
chromatographyncolumn during a separation to
reduce the retention time of late eluting components.
Thermal conductivity - Rate at which a substance
transports heat (energy per unit time per unit area)
through a temperature gradient (degrees per unit
distance). Energy flow [J/(s x m2)] = –k(dT/dx),
where k is the thermal conductivity [W/(m x K)] and
dT/dx is the temperature gradient (K/m). CHAPTER
25 In high-performance liquid chromatography
(HPLC), solvent is pumped at high pressure through a
column containing stationary phase particles with
diameters of 1.5–5 mm. The smaller the particle size,
the more effi cient the column, but the greater the
resistance to fl ow. Microporous silica particles with a
covalently bonded liquid phase such as octadecyl
groups (—C18H37) are most common. Polarity index
measures the ability of a solvent to dissolve polar
molecules. Mobile phase strength measures the ability
of a given solvent to elute solutes from the column. In
normal-phase chromatography and hydrophilic
interaction chromatography, the stationary phase is
polar and a less polar solvent is used. Mobile phase
strength increases as the polarity of the
solventincreases. Reversed-phase chromatography
employs a nonpolar stationaryphase and polar
solvent. Mobile phase strength increases as the
polarity of the solvent decreases. Most separations of
organic compounds can be done on reversed-phase
columns. Polar compounds that are not retained on
reversed-phase columns can be separated by
hydrophilic interaction chromatography. Polar
compounds that only dissolve in organic solvents can
be separated by normal-phase chromatography.
Normal-phase chromatography or porous graphitic
carbon is good forseparating isomers. Chiral phases
are used for optical isomers. If a solution of organic
solvent and water is used in reversed phase
chromatography, mobile phase strength increases as
the percentage of organic solvent increases. If the
solvent has a fixed composition, the process is called
isocratic elution. In gradient elution, mobile phase
strength is increased during chromatography by
increasing the percentage of strong (less polar)
solvent. A short guard column containing the same
stationary phase as the analytical column is placed
before the analytical column to protect it from
contamination with particulate matter or irreversibly
adsorbed solutes. A high-quality pump provides
smooth solvent flow. The injection valve allows rapid,
precise sample introduction. The column is best housed
in an oven to maintain a reproducible temperature.
Column efficiency increases with smaller particles and
at elevated temperature because the rate of mass
transfer between phases is increased. Mass
spectrometric detection provides quantitative and
qualitative information for each substance eluted
from the column. Ultraviolet detection is most common,
and it can provide qualitative information if a
photodiode array is used to record a full spectrum of
each analyte. Refractive index detection has universal
response but is not very sensitive. Evaporative light
scattering and the charged aerosol detectors respond
to the mass of each nonvolatile solute. Electrochemical
and fl uorescence detectors are extremely sensitive,
but selective. In supercritical fluid chromatography,
nonvolatile solutes are separated by a process whose
efficiency, speed, and detectors more closely
resemble those of gas chromatography than those of
liquid chromatography. Steps in method development:
(1) determine the goal of the analysis, (2) select a
method of sample preparation, (3) choose a detector,
and (4) use a systematic procedure to select the
mobile phase for isocratic or gradient elution.
Aqueous acetonitrile, methanol, and tetrahydrofuran
are customary solvents for reversed-phase
separations. Retention is optimized by varying the
amount of organic solvent in the mobile phase.
Relative retention is adjusted by fine-tuning the
organic solvent concentration, altering the
temperature, and by choice of a new organic solvent
or column. Column length and particle size can be
changed to improve efficiency or speed of analysis.
Criteria for a successful separation are 0.5 # k # 20,
resolution $ 2.0, operating pressure # 15 MPa (for
conventional equipment), and asymmetry factor in the
range 0.9–1.5. Retention of weak acids and bases is
governed by their ionization state, which is controlled
by the mobile phase pH. In selecting a mobile phase
buffer, consider the desired pH, buffering capacity,
solubility, and compatibility with detection. A wide
gradient is a good first choice to determine whether
to use isocratic or gradient elution. If it is not possible
to separate all components with 0.5 # k # 20,
gradient elution is needed. Retention factors
measured at a few solvent compositions can be fi t to
the linear solvent-strength model to enable computer
optimization of a separation. Bonded stationary
phase - In high-performance liquid chromatography,
a stationary liquid phase covalently attached to the
solid support Charged aerosol detector - Sensitive,
nearly universal detector for liquid chromatography
in which solvent is evaporated from eluate to leave
an aerosol of fi ne particles of nonvolatile solute.
Aerosol particles are charged by adsorption of N1 2
ions and fl ow to a collector that measures total
charge reaching the detector versus time. Dead
volume - Volume of mobile phase within a
chromatography column, equal to the entire space
accessible to solvent and small solutes. Includes pores
in the particles and volume between particles. If time
required for solvent or unretained solute to travel
through the column is tm, the dead volume is Vm =
tmF, where F is the volume flow rate. Derivatization -
Chemical alteration to attach a group to a molecule
so that the molecule can be detected conveniently.
Alternatively, treatment can alter volatility or
solubility to allow easier separation. Dwell volume -
Volume in chromatography between the point of
mixing solvents and the start of the column.
Electrochemical detector - LC detector that measures
current when an electroactive solute emerges from the
column and passes over a working electrode held at
a fixed potential with respect to a reference
electrode. Also called amperometric detector. Eluent
strength - A measure of the ability of a solvent to
elute solutes from a normal-phase chromatography
column. It is a measure of the solvent adsorption
energy on bare silica. Evaporative light-scattering
detector - LC detector that makes a fine mist of eluate
and evaporates solvent from the mist in a heated
zone. The remaining particles of liquid or solid solute
fl ow past a laser beam and are detected by their
ability to scatter the light. Gradient elution -
Chromatography in which the composition of
thenmobile phase is progressively changed to
increase the eluent strength of the solvent. High-
performance liquid chromatography - using very
small stationary phase particles and high pressure to
force solvent through the column. Hydrophilic
interaction chromatography - Chromatographic
separation of polar solutes with a hydrophilic
stationary phase using mixed organicaqueous eluent.
Eluent strength increases with decreasing organic
solvent. Commonly called HILIC. Isocratic elution -
Chromatography using a single solvent (or single
solvent mixture) for the mobile phase. Microporous
particles - Chromatographic stationary phase
consisting of porous particles 1.5–10 mm in diameter,
with high efficiency and high capacity for solute.
Mobile phase strength - Refers to the ability of a
mobile phase to elute a particular compound from a
liquid chromatography column. Also called solvent
strength. Normal-phase chromatography - A
chromatographic separation utilizing a polar
stationary phase and a less polar mobile phase
Polarity index - In partition liquid chromatography,
refers to the ability of a solvent to dissolve polar
compounds. Polarity arises from dipole, hydrogen-
bond-donating, and hydrogen-bond-accepting
interactions. Refractive index detector - Liquid
chromatography detector that measures the change in
refractive index of eluate as solutes emerge from the
column. Reversed-phase chromatography - Liquid
chromatography in which the stationary phase is less
polar than the mobile phase. Supercritical fluid - fluid
whose temperature is above its critical temperature
and whose pressure is above its critical pressure. It
has properties of both a liquid and a gas.
Superficially porous particle - A stationary phase
particle for liquid chromatography containing a thin,
porous outer layer and a dense, nonporous core.
Mass transfer is faster in the superfi cally porous
particle than in a fully porous particle of the same
diameter. Also called fused-core particle. Ultra-high-
performance liquid chromatography – Liquid
chromatography with stationary phase particles in the
1.5–2 microm range. Higher pressure is required to
force liquid through a UHPLC column than through
columns with larger particle size. Ultraviolet detector
– LC detector that measures ultraviolet absorbance of
solutes emerging from the column CHAPTER 25 Ion-
exchange chromatography employs resins and gels
with covalently bound charged groups that attract
solute counterions (and that exclude ions having the
same charge as the resin). Ion-exchange binding
increases with increased ion charge and
polarizability, and it depends inversely on the radius
of the hydrated ion. Ion binding results in release of
an equivalent ionic charge from the resin. Polystyrene
resins are useful for small ions. Greater cross-linking
of the resin increases the capacity, selectivity, and
time needed for equilibration. Ion-exchange gels
based on cellulose and dextran have large pore sizes
and low charge densities, suitable for the separation
of macromolecules. Certain inorganic solids have ion-
exchange properties and are useful at extremes of
temperature or radiation. Ion exchangers operate by
the principle of mass action, with a gradient of
increasing ionic strength most commonly used to effect
a separation. Zwitterionic hydrophilic interaction
chromatography can separate anions and cations on
the same column. In suppressed-ion chromatography,
a separator column separates ions of interest, and a
suppressor membrane converts eluent into a nonionic
form so that analytes can be detected by their
electrical conductivity. Eluent and suppressor can be
generated continuously by electrolysis. Alternatively,
nonsuppressed ion chromatography uses an ion-
exchange column and low-concentration eluent. Ion-
pair chromatography employs an ionic surfactant in
the eluent to make a reversed-phase column function
as an ion-exchange column. Molecular exclusion
chromatography is used for separations based on
size and for molecular mass determinations of
macromolecules. Molecular exclusion is based on the
inability of large molecules to enter pores in the
stationary phase. Small molecules enter these pores
and therefore exhibit longer elution times than large
molecules. In affinity chromatography, the stationary
phase retains one particular solute in a complex
mixture. After all other components have been eluted,
the desired species is liberated by a change in
conditions. In hydrophobic interaction
chromatography, high concentrations of ammonium
sulfate induce proteins to adhere to a hydrophobic
stationary phase. A gradient of decreasing salt
concentration is applied to increase the solubility of
proteins in water and to elute them from the column.
In capillary zone electrophoresis, ions are separated
by differences in mobility in a strong electric field
applied between the ends of a silica capillary tube.
The greater the charge and the smaller the
hydrodynamic radius, the greater the electrophoretic
mobility. Normally, the capillary wall is negative, and
solution is transported from anode to cathode by
electroosmosis of cations in the electric double layer.
Solute cations arrive first, followed by neutral species,
followed by solute anions (if electroosmosis is stronger
than electrophoresis, which typically is true). Apparent
mobility is the sum of electrophoretic mobility and
electroosmotic mobility (which is the same for all
species). Zone dispersion (broadening) arises mainly
from longitudinal diffusion and the finite length of the
injected sample. Stacking of solute ions in the
capillary occurs when the sample has a low
conductivity. Electroosmoti flow is reduced at low pH
because surface SiOO2 groups are protonated.
SiOO2 groups can be masked by polyamine cations,
and the wall charge can be reversed by a cationic
surfactant that forms a bilayer along the wall.
Covalent coatings reduce electroosmosis and wall
adsorption. Hydrodynamic sample injection uses
pressure; electrokinetic injection uses an electric field.
Ultraviolet absorbance is commonly used for
detection. Micellar electrophoretic chromatography
uses micelles as a pseudostationary phase to
separate neutral molecules and ions. Capillary gel
electrophoresis separates macromolecules by sieving.
In contrast with molecular exclusion chromatography,
small molecules move fastest in gel electrophoresis.
Microfluidic devices (“lab-on-a-chip”) use
electroosmotic or hydrodynamic flow in
lithographically fabricated channels to conduct
chemical reactions and chemical analysis. Affinity
chromatography - A technique in which a particular
solute is retained by a column by virtue of a specific
interaction with a molecule covalently bound to the
stationary phase. Anion exchanger - An ion
exchanger with positively charged groups covalently
attached to the support. It can reversibly bind anions.
Capillary electrophoresis - Separation of a mixture
into its components by a strong electric fi eld imposed
between the two ends of a narrow capillary tube fi
lled with electrolyte solution. Unlike chromatography,
there is no stationary phase in electrophoreses.
Solutes are separated by differences in mobility.
Capillary gel electrophoresis - A form of capillary
electrophoresis in which the tube is fi lled with a
polymer gel that serves as a sieve for
macromolecules. The largest molecules have the
slowest migration through the gel. Also called
capillary sieving electrophoresis. Capillary zone
electrophoresis - A form of capillary electrophoresis
in which ionic solutes are separated because of
differences in their electrophoretic mobility. Cation
exchanger - An ion exchanger with negatively
charged groups covalently attached to the support. It
can reversibly bind cations. Cross-linking - Covalent
linkage between different strands of a polymer.
Donnan exclusion - Ions of the same charge as those
fixed on an ion exchange resin are repelled from the
resin. Thus, anions do not readily penetrate a cation-
exchange resin, and cations are repelled from an
anion exchange resin. Electrokinetic injection - In
capillary electrophoresis, the use of an electric field
to inject sample into the capillary. Because different
species have different mobilities, the injected sample
does not have the same compositionnas the original
sample. Electroosmosis - Bulk flow of fluid in a
capillary tube induced by an electric field. Mobile
ions in the diffuse part of the double layer at the wall
of the capillary serve as the “pump.” Also called
electroendosmosis Electrophoresis - Migration of ions
in solution in an electric fi eld. Cations move toward
the cathode and anions move toward the anode. Ions
can be separated from one another by their differing
rates of migration in a strong electric field.
Equivalent - redox reaction, amount of reagent that
can donate or accept one mole of electrons. For an
acid-base reaction, the amount of reagent that can
donate or accept one mole of protons. For ion
exchange, the amount of reagent that will exchange
with one mole of monovalent cation or anion. Gel -
Chromatographic stationary phase particles, such as
Sephadex ormpolyacrylamide, which are soft and
pliable. Gradient elution - Chromatography in which
the composition of the mobile phase is progressively
changed to increase the eluent strength of the solvent.
Hydrodynamic injection - In capillary
electrophoresis, the use of a pressure difference
between the two ends of the capillary to inject
sample into the capillary. Injection is achieved by
applying pressure on one end, by applying suction on
one end, or by siphoning. Hydrophobic interaction
chromatography - Chromatographic separation
based on the interaction of a hydrophobic solute with
a hydrophobic stationary phase. Indirect detection –
C detection based on the absence of signal from a
background species. For example, in ion
chromatography, a light-absorbing ionic species can
be added to the eluent. Nonabsorbing analyte
replaces an equivalent amount of light-absorbing
eluent when analyte emerges from the column,
thereby decreasing the absorbance of eluate.
Interstitial volume - The volume of mobile phase
outside of the stationary phase in a molecular
exclusion chromatography column. The total volume of
mobile phase is the interstitial volume plus the volume
of solvent inside the stationary phase particles.
Formerly called void volume. Ion chromatography -
HPLC ion exchange separation of ions. See also
suppressed-ion chromatography and single-column
ion chromatography. Ion-exchange chromatography
- A technique in which solute ions are retained by
oppositely charged sites in the stationary phase Ion-
pair chromatography - Separation of ions on
reversed-phase highperformance liquid
chromatography column by adding to the eluent a
hydrophobic counterion that pairs with analyte ion
and is attracted to stationary phase. Micellar
electrokinetic chromatography - A form of capillary
electrophoresis in which a micelle-forming surfactant is
present. Migration times of solutes depend on the
fraction of time spent in the micelles. Micelle - An
aggregate of molecules with ionic headgroups and
long, nonpolar tails. The inside of the micelle
resembles hydrocarbon solvent, whereas the outside
interacts strongly with aqueous solution. Mobility -
The terminal velocity that an ion reaches in a field of
1 V/m. Velocity = mobility * field. See also apparent
mobility and electrophoretic mobility. Molecular
exclusion chromatography - the stationary phase
has a porous structure into which small molecules can
enter but large molecules cannot. Molecules are
separated by size, with larger molecules moving
faster than smaller ones. Also called size exclusion,
gel filtration, gel permeation, or size exclusion
chromatography.