PHAR229 – Analytical Chemistry 2 Final Notes

Introduction to Optics
 Spectroscopy: Study of the interaction of electromagnetic radiation (based on wave or particles) with matter this interaction produce
,

 Based on wave description: characterized by wavelength (Λ) or frequency (f) current

 Period (P): Time for 1Λ to pass fixed point

 Frequency (f): Λ passing per second (1/P)
 Wavenumber: Λ per cm (1/Λ)
 Velocity (V) = Λ x f = Λ / P &
 Vvacuum = c = 2.99782x108 m/s =,i stat assis - jj
 For a given frequency of light, the wavelength must decrease as the velocity decreases oi·

Vgas X V liquid)" solid

The energy associated with a given segment of the spectrum is proportional to its frequency.

 Spectroscopy: Study of the interaction of electromagnetic radiation (wave or particles) with matter
 Certain electromagnetic radiation phenomena:

I
 Transmission: Propagation of electromagnetic waves through a medium without significant absorption
or scattering, maintaining its original direction and frequency
 Reflection: Bouncing back of electromagnetic waves when they encounter a boundary between two
different media
 Refraction: Occurs when waves pass from one medium to another, causing a change in direction and
Based on wave
a
speed due to the change in the medium's refractive index
description  Diffraction: Bending and spreading of electromagnetic waves as they encounter an obstacle or pass
through a narrow opening, resulting in interference patterns
 Interference: Occurs when two or more electromagnetic waves superpose and interact with each other
 Constructive interference, where the waves reinforce each other
 Destructive interference, where the waves cancel each other out
 Scattering: Redirection of electromagnetic waves in different directions when they interact with
particles or irregularities in a medium, causing waves to scatter in various angles, changes in intensity,
frequency, and polarization
 Polarization: Orientation of the electric field vector of an electromagnetic wave (can be linearly
polarized, circular or elliptical)
 Based on quantum mechanics (photon):
 ΔE = h x f = (h x c) / Λ
1) Atoms, ions and molecules exist in discrete energy states only
 E0 (grounded), E1-E2-E3… (excited) states
 2) When an atom, ion or molecule changes energy state, it absorbs or emits energy equal to the
energy difference (ΔE = E1 - E0)
 3) Wavelength or frequency of radiation absorbed or emitted during a transition proportional ΔE
  Sub-electronic energy levels (Vibrational and Rotational energy levels)

because we have different internations between molecules

 Spectrochemical information is provided by measuring the electromagnetic radiation emitted by the

species as it returns to the ground state from an excited level or by measuring the radiation absorbed
in the excitation process
 Absorption: Energy of photon is taken up by the matter (electrons of the atom), then electromagnetic energy
is transformed to other forms of energy (like heat) Adsorption takes place with the adhesion of particle onto the surface of substance
:

 Emission: Energy of photon is released (sending out gas, heat, light etc.) by the atoms whose electrons make a
transition between two energy levels
 Chemiluminescence: Emissions of species excited by chemical reactions
 Photoluminescence: Emissions of photons in excited states produced by radiational activation such as
light source (fluorescence, phosphorescence)
 Luminescence: emission from cool bodies or emission from hot bodies that is not due to thermal
excitation (e.g. electricity, laser etc.)
 Spectroscopic Measurements
 A= Absorbance Each chemical has different absorption
 𝜀= Molar absorptivity coefficient (M-1cm-1)
 b= cell width (cm)
 c= concentration (M)
 T= fraction of light transmitted
 I= Transmitted intensity of light beam
 Io= Initial intensity of light beam

Po > P1
Absorbing
of
solution
concentration C A

 For more substance  𝑨=𝜺𝟏𝒃𝑪𝟏+𝜺𝟐𝒃𝑪𝟐+𝜺𝟑𝒃𝑪𝟑+⋯+𝜺𝒏𝒃𝑪𝒏

 Absorptivity is the fundamental property of a substance that contains the observable spectroscopic
information which can be linked to quantum mechanics
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 3 spectrum types: sharp well-defined spectra
,

 1) Line spectrum: Radiating species are individual atoms/ions that are well separated
 2) Band spectrum: Produced in spectral sources due to presence of gaseous radicals or small molecules,
arising from numerous vibrational levels superimposed on the ground state electronic energy level
 3) Continuum spectrum: Produced when solids such as carbon or tungsten heated to incandescence
Firsis
 Blackbody radiation: Produced by innumerable atomic and molecular oscillations excited in
the condensed solid due to thermal energy
 ΔE = ΔEelectronic + ΔEvibrational + ΔErotational
For each electronic state , vibrational state
. For each vibrational state rotational state
many ,
many

 Emission by Fluorescence and Phosphorescence

 Processes in which atoms/molecules are excited by absorption of a beam of electromagnetic radiation
 Excited species then relax to the grounds state, giving up excess energy as photons
 Fluorescence takes place more rapidly than Phosphorescence
 Spectrophotometer components: Source  Wavelength selector (Monochromator)  Sample cell  Detector
 Important: Light source should be perpendicular to the sample in Fluorescence (emission method)
 Optical instruments:
 Cuvette: Holds the sample, transparent, must be of fixed size (1 cm is very common)
 For Visible light: Ordinary Glass or Silica
 For UV region: Fused Silica or Quartz
 For IR system: KCl, NaCl, IRTRAN
 Tungsten filament lamp: distribution of wavelengths from 320 to 2500 nm
 Deuterium lamp: to provide continuum radiation in UV region (160 to 400 nm)
 Monochromators: wavelength selectors that make use of concave mirrors
 Polychromatic radiation enters from the “entrance slit”
 Light is collimated (light beams made parallel) at the “first concave mirror”
 Then, “reflection grating” diffracts different wavelengths at different angles
 “Second concave mirror” focuses each wavelength at different points on “focal plane”
 Only one narrow band of wavelengths can go out from the “exit slit”
 Selection of slit width determines resolution and signal to noise ratio:
 Large slit width: more energy reaches the detector  higher signal/noise ratio
 Small slid width: less energy reaches the detector  BUT better resolution
 Measurement applications (2 types): Single Beam Instruments vs. Double Beam Instruments

Atomic Spectroscopy
 Principle: Interaction of electromagnetic radiation with “free” atoms (isolated atoms in gas phase) to observe
specific “spectral lines” (discrete lines of light) produced after an atom undergoes excitation/de-excitation
 Aim: Measuring wavelength and intensities of radiation absorbed or emitted by atoms to identify chemical
elements in a sample and their concentrations
 Atomic excitation: Absorbed energy from the light causes atom’s electrons to move to higher energy levels
 Atomic de-excitation: Atom releases its energy and electrons move to lower energy states
 Types of Atomic Spectroscopy:
 1) Atomic Absorption Spectroscopy (AAS)
 2) Atomic Emission Spectroscopy (AES)
 3) Atomic Fluorescence Spectroscopy (AFS)
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 Atomic Spectroscopy Experiments: each step influences accuracy and precision of result.
 Sample intro  Forming gas phase atoms  Excitation/Emission  Detecting photons  Readout
 Light source: Hollow Cathode Lamp
Advantage sharp lines specific for element of interest  Ionizes inert gas to high potential
:

Disadvantage expensive need to

:
, use different larp  Gas cations are attracted to “hollow cathode” and hit the surfaces
for
each element tested
 After hitting, some of the deposited elements dislodge into gas phase (“sputtering”)
 Excited elements relax to ground state and emit characteristic radiation
 Methods of Sample Introduction:
 Pneumatic nebulizers (solution or slurry)
 Ultrasonic nebulizers (solution)
 Electro-thermal vaporizer (solid, liquid, solution)
 Hydride generation techniques (hydride solutions of certain elements)
 Sample atomization: (1) Flame, (2) Electrothermal and (3) Specialized atomization techniques
For metals atomic
spectroscopy optimum height
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 1) Types of flame: Selection of right region in flame is important for optimal performance
 Primary combustion zone: Not in thermal equilibrium, not used
 Interconal region: Highest temperature; often used in spectroscopy be , can narrower

 Outer cone: Cooler region rich in oxygen due to surrounding air; gives metal oxide formation
 2) Electrothermal atomization: For really small
samples

 Sample is contained in a heated, graphite furnace which is heated by passing an electrical current
 To prevent oxidation of the furnace, it is sheathed in gas (Argon usually)
 No nebulization, sample is introduced as a drop
 3) Plasma atomization:
 Higher temperature values can be reached, which allows efficient atomization
 Samples can be introduced via nebulization, electrothermal methods or ablation (removal)
 Then, samples are transported into plasma with argon

Molecular Spectroscopy
 1) UV-Vis spectroscopy
 Analytical signal originates due excitation of an atom/molecule by UV or visible radiation (190-700 nm)
 Absorption spectrums obtained, showing which particular wavelengths (Λ) of light the chemical species
can absorb
 Atoms and molecules can absorb electromagnetic radiation, but only at certain energies and thus Λ
 Electrons in bonding/non-bonding (lower energy) molecular orbitals undergo a transition to anti-
V bonding (higher energy) molecular orbitals

 σ  σ* transitions (alkanes):
 Energy required is large
 Absorption max. around 125 nm which are never observed in ordinarily accessible UV region
 n  σ* transitions (O, N, S, halogens):
 Saturated compounds containing atoms with unshared electrons
 Require less energy than σ  σ* type
 Radiation between 150 – 250 nm (most absorption peaks appear below 200 nm)
 Molar absorptivities (𝜀) for peaks in n  σ* are low to intermediate (100 – 3,000 M-1cm-1)
 n  π* & π  π* transitions (carbonyls & unsaturated FGs):
 Mostly preferred, because energies required for these processes bring absorption peaks into
experimentally convenient spectral region (200 – 700 nm)
 Molar absorptivities (𝜀) for peaks in n  π* are generally low (10 – 100 M-1cm-1)
 Molar absorptivities (𝜀) for peaks in π  π* fall in between 1,000 – 10,000 M-1cm-1
 Absorption with d and f electrons:
 Most of the transition metal ions absorb in UV or visible region of the spectrum (200-700 nm)
 This type of transitions create very bright colors that can be observed under UV-VIS
 Transition metal ions such as Ni2+, Cr2O72-, Cu2+, Co2+
  Charge-Transfer absorption:
 Molar absorptivities of these species are very large (𝜀max > 10,000)
 Complexes that exhibit charge-transfer absorption are called “charge-transfer complexes”
 Fe(SCN)2+, Fe(phenyl)32+, Starch-I5-
 Solvent effect: selection of appropriate solvent is very important
 Intensely absorbing compounds must be examined in dilute solutions, so that meaningful light
energy can be received by the detector, and this requires the use of completely transparent
(non-absorbing) solvents
 Most commonly used solvents are water, ethanol, hexane and cyclohexane
 Min. wavelength limits are especially important in UV-Vis, HPLC, CE etc. (used in detectors)
 Qualitative/Quantitative analysis by UV-Vis Spectro:
 Λmax is used where the UV absorbance is greatest
 Different standards with different C values are prepared which provide different A values
 y=mx+b equation is obtained from A vs. C graph and used to find concentration of the
unknown sample
 2) IR spectroscopy the measurement of infrared interaction with matter by absorption
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identify chemical substances
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 Every molecular compound has a unique infrared absorption spectrum, thus exact match between the liquid Form.

spectrum of known compound with the spectrum of unknown analyte directly identifies the analyte
 No two molecules give exactly the same IR spectrum (except enantiomers)
 Less satisfactory in quantitative analysis than UV-Vis due to lower sensitivity and deviations from Beer’s
Law, and absorbance measurements are less precise
 Looking for presence/absence of functional groups:
 Polar bond is usually IR-active
 Non-polar bond in a symmetrical molecule will absorb weakly or not at all
 IR does not cause electronic transitions, but induce transitions in vibrational and rotational states -M

within the ground electronic state of the molecule (stretching & bending vibrations)
 UV-Vis  related to wavelength, whereas IR  related to wave number
 Each IR absorption peak defines different type of stretching or bending vibrations

 Instruments for IR spectroscopy: Dispersive instruments, FT-IR (mostly used), Filter Photometers; all
of which are different devices with different designs
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*  3) Fluorescence Spectroscopy
 Used for identification of species having fluorescence property *
 Following of the analyte is easier in FS Aug> Hong
 Molecular fluorescence is measured by exciting the sample at “absorption/excitation wavelength”, and
by measuring the emission at a longer wavelength called “emission/fluorescence wavelength”
 Fluorescence emission is usually measured at right angles to the incident beam
 The short lived emission that occurs is called “fluorescence” whereas luminescence that is much longer
lasting is called “phosphorescence” (continuous process, not preferred)
 Analysis: similar approach
 Emitted electromagnetic wavelength is proportional to concentration
 Calibration curve is constructed by the analysis of standard solutions
 Finally, sample solution property is measured and unknown material is determined

Introduction to Analytical Separation

 Masking: Reagent is added to sample solution to chemically bind to interferent as complex so that it no longer
contributes to the signal of the analyte; masking agent must not affect the behaviour of the analyte significantly
 Precipitation and Filtration: Analyte or interferent is removed from solution selectively as insoluble species
 Distillation: Separating volatile analytes from non-volatile interferents
 Extraction: Separation by extent to which a solute (both inorganic and organic) distributes itself between two
immiscible liquids differently
 K (Distribution constant) = [Solute]organic phase / [Solute]aqueous (partition of a solute between two
immiscible phases – the distribution law)
 Ion Exchange: Process to separate ions by which ions held in a porous insoluble solid are exchanged for ions in
a solution that is brought into contact with the solid
 Synthetic ion-exchange resins: High molecular-weight polymers containing large # of ionic functional
groups per molecule; cation-exchange resins contain acidic (SO3-H+), whereas anion-exchange resins
contain basic (N(CH3)3+OH-) groups in column this reaction
During passing
occurs.
,

 Ion exchange resins are used to eliminate ions that would otherwise interfere with analysis
 Ion exchange resins are particularly useful for chromatographic separation of ionic species
from solution
concentrating ions
very
dilute .

Chromatography
 Definition: Components of a mixture are carried through the stationary phase by the flow of the mobile phase,
and separations occur based on differences among migration rates of the mobile phase components
 Stationary Phase: Fixed in place either in a column or on a planar surface
shouldn't interact with each
Mobile Phase: Moves over the stationary phase, carrying with it the analyte mixture They
other

 Elution: Process in which solutes are washed through the stationary phase by the movement of mobile phase
 Eluent: Solvent used (carrier) that moves chemicals of the mixture through the stationary phase
 Eluate: Mobile phase that exists the column (material emerging from the column)
 Classification of Chromatography
 According to shape: Planar vs. Column
 According to mobile phase: Liquid vs. Gas vs. Supercritical fluid
 According to separation mechanism:
 1) Adsorption Chromatography: Type of liquid chromatography in which chemicals are
retained based on their adsorption at the surface of the support (stationary phase)
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 2) Ion Exchange Chromatography: ions held in a porous insoluble solid are exchanged for ions
in a solution that is brought into contact with the solid
 3) Size Exclusion Chromatography: separates molecules based on their size by filtration
through a gel
 Large particles do not enter the cell and eluted earlier; need less volume for elution
 Small particles enter the cell and eluted later; need more volume for elution
 4) Partition Chromatography: Separation of components between two liquid phases which
are original solvent and another film of solvent used in the column liquid-liquid chromatography
 Plate Theory:
-15903 .2 , &ni  Chromatographic column contains large number of separate layers called theoretical plates
·is so i sti  Separate equilibrations of the sample between stationary and mobile phase occur in these plates
,

 The thinner the plate, the higher the number of plate, the more effective the separation
 Variables that influence the Plate Height (H in mm)
 Particle size: Less sized column packing material  less plate height
 Film coat thickness: Less film coat thickness  less plate height
control it
by adding organic solvent  Viscosity: Mobile phase viscosity can be decreased to change interactions of the substances
 Temperature: Increased temperature  Reduced band broadening effect interaction of the substance with mobile and

solid phase
 Flow rate: H increases with increased linear flow rate (cm/s) .

 Distribution Constants:
 All chromatographic separations are based on differences
in extent to which solutes are distributed between mobile
and stationary phase ( A-mobile  A-stationary)
 Distribution constant value is used to understand the
analysis system, but determining the concentarion of
each matter in both phases is very hard
 Retention Time (tR): Time between injection of a sample and appearance of a solute peak at the detector of the
chromatographic column
 Dead Time (tM): Time it takes for non-retained species to pass through the column It doesn't affect the separation but should keep
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Retention factor (k): Compares the migration rates of the solutes on columns ( k=[ tR - tM ] / tM ) should be higher than 2
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 Selectivity factor (α): α = KB / KA where B is the more strongly retained species, whereas A is the more rapidly
eluted species, thus α is always greater than unity
 Number of theoretical plates (N): N= L / H where L is the length of the column packing and H is plate height;
efficiency increases as N increases and It decreases
,

 Resolution (RS): Quantitative measure of the ability to separate two analytes; better when retention time
differences are higher and peak widths are smaller
 Tailing factor (S): Coefficient that shows the degree of peak symmetry at 10% peak height (AS = B / A)
*
 Relative Standard Deviation (RSD): Used to investigate reproducibility of the results

Gas Chromatography
 Principle: Partition of molecules between mobile phase and stationary phase
 Applications:
 Characterization of unformulated drugs, with regards to detection of process impurities
 Limit tests for impurities in drug substances
 Quantification of drugs in formulations, particularly if the drug lacks chromophore
 Characterization of raw materials used in synthesis of drug molecules
 Characterization of volatile oils which may be used as excipients
 Measurement of drugs and their metabolites in biological fluids
 Strengths:
 Quantitative accuracy and precision like HPLC
 Much greater separation power than HPLC when used with capillary columns
 Readily automated
 Determines the compounds that lack chromophores
 Mobile phase does not vary and not require disposal and cheap compared to organic solvents in HPLC
 Limitations:
 Only thermally stable and volatile compounds can be analyzed
 Sample introduction is more difficult because of the small volumes of sample injected
 Aqueous solutions and salts cannot be inject into the instrument
 Column Oven: controls the temperature change
 Isothermal: Keeps oven at one temperature, only useful for series of very similar compounds differing
in boiling points such as alcohols (not very useful in general)
 Gradient: Changing temperature profile which provides better resolution and analysis time
 Column types:
 Packed: Filled with a coated inert solid that are chemically bonded to the support such as fire brick,
alumina and graphite
 Longer column life and less bleeding (source of background noise)
 Higher capacity/concentration
 Low resolution and low S/N
 Capillary: Coated on inside diameter of the capillary wall with film thickness of 0.1 – 5μ, where ticker
film provides better resolution but also more bleeding (typically fused silica coated with polyamide)
 High resolution and high S/N
 Low capacity and higher cost
 Detectors: (1) Flame ionization, (2) Thermal conductivity, (3) Electron capture
 Derivatization: When the analyte is not a volatile material, chemical reaction with special reagents changes the
non-volatile substance into a volatile substance
 Loss of sample
 Side reaction
 Time consumption

High Performance Liquid Chromatography (HPLC)

&

 Normal-phase (Liquid-solid) chromatography:

 Polar stationary phase is used
 Non-polar compounds elute out first, increasingly polar compounds elute later
 Eluents (carrier) used: hexane, CH2Cl2, CHCl3 (from low to moderate polarity)
 Water (highly polar) is a strong solvent in normal phase but a weak one in reversed phase
 Ideal for compounds with low to moderate polarity, water insoluble and non-ionic
 Reversed-phase chromatography:
 Stationary phase is non-polar and mobile phase is polar (opposite of normal-phase)
 C18 columns are used
 Retention time is higher for non-polar molecules, and they elute later than polar solutes
 Eluents used: aqueous solutions of organic solvents such as MeOH, Acetonitrile, THF
& Eluting power of the solvent is higher if the compound is less polar
 Applications: Amino acids, homologs, herbicides
 Mobile phase:
 Viscosity: Low viscosity solvents reduce the pressure to achieve a given flow rate (preferred)
 Boiling point: Lower BP facilitate solvent removal from collected fractions
 Common modifiers:
 Buffers: Stabilize pH of the mobile phase (phosphate, acetate, citrate)
 Acidifiers: To suppress ionization of acidic analytes (phosphoric acid, acetic acid)
 Ionic strength: To control elution of ionic analytes (e.g. NaCl)
 Ion-pair reagents: For separation of ionic compounds (hexane sulfonate)
 Amine modifiers: To reduce tailing of basic analytes (triethylamine)
 Guard column: Protects the analytical column from particles, interferences, and prolongs its life
 Column packing: Smaller the particles, higher the plate number (N) and higher pressure is needed to move
eluent through the column
 Support type: Silica or polymer
 Bonded groups: C18, C8, amino, cyano, phenyl
 Particle size (dp): 3, 5, 7, 10 or 20 μm; efficiency and column pressure is inversely proportional to d p
 Pore size: 60-300A; wider pore materials are used for biomolecules or polymers
 Surface area: 90-400 m2/g; high surface area maximizes solute interaction with bonded groups
 Column length (L): common length is 10-25 cm
 Column efficiency (n) is proportional to L
 Columns can be connected together to increase efficiency
 Column back pressure is proportional to L
 Column diameter (dc):
 Sample capacity is proportional to (dc)2
 Flow rate is proportional to (dc)2
 Detection limits is inversely proportional to (dc)2
 Isocratic Elution: Using constant-composition mobile phase, only if a single composition solvent mixture or pure
solvent is used
 Gradient Elution: Mobile phase composition changes over time during the separation; gradients can be
continuous or step-wise (e.g. binary, ternary and quaternary solvent gradients)
 Detector types:
 UV (single, variable, multiple wavelengths)
 Fluorescence
 Electrochemical
 Mass spectrometric
 UPLC (Ultra Performance LC)
 Chromatography columns with very small particles
 Improved resolution, speed and sensitivity
 Appropriate for developing new and improving existing methods
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