Topic 6 Spectrochemistry

Spectrochemical Methods: the most widely used tools for the elucidation of molecular structure as well as the quantitative and qualitative determination of both inorganic and organic compounds.

Properties of light

1)

2) Electromagnetic Spectrum 3) Absorption vs Emission of light

ν~hcλ1hchνE

Properties of light

ν~hcλ1hchνE

Electromagnetic spectrum

Absorption vs Emission of light

• Emission– Chemiluminescence– Photoluminescence

• Absorption

Photoluminescence

• fluorescence• phosphorescence

Absorption of light2) .

Mmolcmmolcm

εbcA :Law sBeer'

-logTA AbsorbancePPT nceTransmitta

0

When no light is absorbed, P=P0 and A=0

For mix:Atotal = A1 + A2 + …+ An = 1 bc1 + 2 bc2 + …+ n bcn

Absorbance is proportional to the concentration

• A = bc• : molar absorptivity (M-1cm-1)

Absorption of light

(ex) How effective is sunscreen ? at the peak absorbance near 300 nm ?

A ~ 0.35T = 10-A

= 10-0.35

= 0.45 = 45%

55% UV-B is absorbed.

Why is a red solution red?

Electronic transitionsAn electron moves from one orbital to another.

• The total energy E associated with a molecule– E = Eelectronic + Evibrational + Erotational

Vibrational and rotational transitions

• Rotate & vibrate freely in gas phase

• Unable to rotate freely in nonpolar solvent.

• Collisions & interactions cause the spectrum as a single broad peak in H2 O.

UV-visible spectra

Limits to Beer’s Law

• Real deviations– At concentrations exceeding about 0.01 M,

the average distances between ions or molecules of the absorbing species are diminished to the point where each particle affects the charge distribution and thus the extent of absorption of its neighbors.

• Instrumental deviations• Chemical deviations

Chemical deviations

•

Polychromatic radiation & stray light•

select

λmax to reduce

Instrumental deviations

Three types of spectra: line, band, and continuum

Emission of electromagnetic radiation

Three types of spectra

• The line spectrum consists of a series of sharp, well-defined spectral lines caused by excitation of individual atoms that are well separated, as in a gas.

• The band spectrum, marked bands, is comprised of several groups of lines so closely spaced that they are not completely resolved.

• The source of the bands is small molecules or radicals in the source flame.

• Finally, the continuum spectrum is responsible for the increase in the background that appears above about 350 nm.

Energy level diagram

homework

• 24.3, 24.13, 24.18, 24.29

Instruments for optical spectrometry

A Instrument components

A Instrument components

Sample is usually contained in a cell called a cuvet

Optical materials

Spectroscopic sources

• Continuum sources• Line sources

a. entrance slitb. collimating

mirror or lensc. a prism or

gratingd. focal planee. exit slit

Monochromator: disperses light into its component wavelengths and selects a narrow band of wavelengths to pass through the sample.

Wavelength selectors

Choosing the bandwidth: exit slit width

Resolution

Signal

Monochromator

trade-off

Wavelength selectors

A detector produces an electric signal when it is struck by photons, i.e. convert radiant energy (photons) into an electrical signal.

An ideal detector : high sensitivity, high signal/noise, constant response for λs, and fast response time.

Detector

a) Single-beam

b) Double-beam

Spectrophotometer

Molecular Absorption Spectrometry

Ultraviolet and visible molecular absorption spectroscopy

Absorbing Species: Organic molecules

• Wavelength region between 180 ~ 780 nm

• Electrons in double and triple bonds of organic molecules.

• Chromophores: unsaturated organic functional groups that absorb in the ultraviolet or visible regions.

Saturated organic compounds containing such heteroatoms as oxygen, nitrogen, sulfur, or halogens have nonbonding electrons that can be excited by radiation in the 170 nm ~ 250 nm range.

Absorbing Species: Inorganic Species

Qualitative application of UV/Vis• Spectrophotometric measurements with UV

radiation are useful for detecting chromophoric groups.

• Ultraviolet spectra do not have sufficient fine structure to permit an analyte to be identified unambiguously.

• UV qualitative data must be supplemented with other physical or chemical evidence such as infrared, nuclear magnetic resonance, and mass spectra as well as solubility and melting- and boiling-point information.

Solvents

• Usually measured using dilute solutions of the analyte

• Solvent must be transparent in the region of the spectrum where the solute absorbs (Table 26.3).

• For qualitative analysis, analyte spectra should thus be compared to spectra of known compounds taken in the same solvent.

The Effect of Slit Width

• Small slit widths are used for qualitative studies to preserve maximum spectral detail.

• Peak heights and peak separation are distorted at wider bandwidths.

Effect of stray radiation

• Stray radiation occasionally causes false peaks to appear when a spectrophotometer is being operated at its wavelength extremes.

Quantitative applications

The important characteristics of spectrophotometric and photometric methods are:

– Wide applicability– High sensitivity– Moderate to high selectivity– Good accuracy– Ease and convenience

Molecular absorption measurements are applicable in every area

1. Applications to absorbing species2. Applications to nonabsorbing species : react

with chromophoric reagents to produce products that absorb strongly in the ultraviolet and visible regions.

Chelating reagents

• (a) Proteins at 280 nm: tyr, phe, trp. • (b) A colorimetric reagent to detect phosphate

Application in biosystem

Ex.: Nitrite in an aquarium(using a standard curve)

543 nm

Application in environment

Wavelength selection

(toxic when > 1 ppm) NH3 animals & plant

(toxic when > 1 ppm)

[O]

[O]

NO3-

Application in environment

Wavelength selection

Standard Nitrite

ACorrected / Aobserved =Vt / Vi

Calibration curve

from least square

A = 0.1769 [ppm] + 0.0015

ACorrected / Aobserved =Vt / Vi

The multiple-additions method• Assume that several identical aliquots Vx of the unknown

solution with a concen-tration cx are transferred to volumetric flasks having a volume Vt.

• To each of these flasks is added a variable volume Vs mL of a standard solution of the analyte having a known concentration cs.

xxsst

xx

t

sss ckVckV

VcbV

VcbV

A

xxs

ss

ckVbkcm

bmVA

Analysis of a mixture

1) Absorbance of a mixture :

The total absorbance of a solution at any given wavelength is equal to the sum of the absorbances of the individual components in the solution.

A1 = M1 bcM + N1 bcNA2 = M2 bcM + N2 bcN

Analysis of a mixture

2) Isosbestic points : for rxn: X Y, every spectrum recorded during chemical reaction will cross at the same point. Good evidence for only two principle species in rxn.

Ex: HIn

In- + H+

Analysis of a mixture

Why isosbestic point?

HInIn bε

In bεHIn bεA :mixture aFor

εεεInHIn when

In εA

HIn εA

465

465In

465HIn

465

465465In

465HIn

465In

465

465HIn

465

Ex: The metal ion indicator xylenol orange is yellow at pH 6 (max = 439 nm). The spectral chages that occur as VO2+ is added to the indicator at pH 6 are shown in the figure. The mole ratio (VO2+)/(xylenol orange) as each point is shown in the table. Suggest a sequence of chemical reactions to explain the spectral changes.

Trace Mole ratio

Trace Mole ratio

0 0 9 0.90

1 0.10 10 1.0

2 0.20 11 1.1

3 0.30 12 1.3

4 0.40 13 1.5

5 0.50 14 2.0

6 0.60 15 3.1

7 0.07 16 4.1

8 0.80

Titration Curve is a plot of absorbance (corrected for volume change) as a function of titrant volume.

Photometric & Spectrophotometric Titrations

s: : substance, : p : product, t : titrate

M + L solutions with identical analytical concentrations are mixed such that the total volume and the total moles of reactants in each mixture are constant but the mole ratio of reactants varies systematically.

The corrected absorbance is plotted against the volume fraction of one reactant, that is, VM /(VM + VL )

Continuous variations

the analytical concentration of one reactant (usually the metal ion) is held constant while that of the other is varied.

If the formation constant is reasonably favorable, two straight lines of different slopes that intersect at a mole ratio that corresponds to the combining ratio in the complex are obtained.

The mole-ratio method

Spectrophotometric Titrations

Ferric nitrilotriacetate

[used to avoid Fe(OH)3 ]

Spectrophotometric Titrations

125 μL ferric nitrilotriancetate2 mL apotransferrin A = 0.260

A corrected = ? 0.276

Spectrophotometric Titrations

The absorbance measured after adding 125 L of ferric nitrilotriacetate to 2.000 mL of apotransferrin was 0.260. Calculate the coorected absorance.

Corrected A for the effect of dilutionCorrected A = (Vt / Vi ) (observed A) (Beer’s law)

Infrared absorption spectroscopy

• A powerful tool for identifying pure organic and inorganic compounds because almost all molecular species absorb infrared radiation.

• However, it is a less satisfactory tool for quantitative analyses than its ultraviolet and visible counterparts because of lower sensitivity and frequent deviations from Beer’s law.

• Additionally, infrared absorbance measurements are considerably less precise.

• The energy of infrared radiation can excite vibrational and rotational transitions, but it is insufficient to excite electronic transitions.

Homework (chapter 26)

• 26.1 (b), 26.16, 26.19, 26.20 (a), 26.21(a), 26.22 (A), 26.23

Molecular Fluorescence

What happens when a molecule absorbs light ?

1) Absorbing species :M + hν M* (lifetime : 10-8 ~ 10-9 sec)

Relaxation processes :a) M* M + heat (most common)b) M* new species (photochemical reaction)c) M* M + h

(fluorescence, phosphorescence)

1. Molecular fluorescence is measured by exciting the sample at an absorption wave-length, also called the excitation wavelength, and measuring the emission at a longer wavelength called the emission or fluorescence wavelength.

2. Usually photoluminescence emission is measured at right angles to the incident beam to avoid measuring the incident radiation.

3. The short-lived emission that occurs is called fluorescence, while luminescence that is much longer lasting is called phosphorescence.

4. Fluorescence emission occurs in 10-5 s or less. 5. In contrast, phosphorescence may last for several minutes

or even hours.

A Theory of molecular fluorescence

Molecular fluorescence

• Two most important processes after excited to E1 or E2:

– Nonradiative relaxation, – Fluorescence emission.

• Two most important nonradiative relaxation:– Vibrational relaxation

• transfer of the excess energy of a vibrationally excited species to molecules of the solvent. This process takes place in less than 10-15 s and leaves the molecules in the lowest vibrational state of an electronic excited state.

– Internal conversion• transfer of the excess energy of a species in the lowest

vibrational level of an excited electronic state to solvent molecules and conversion of the excited species to a lower electronic state.

Molecular fluorescence

Electron spin states

What happens to absorbed energy

Luminescence procedures : emission spectrum of M* provides information for qualitative or quantitative analysis.

Photoluminescence :1. Fluorescence : S1 S0, no change in electron

spin. (< 10-5 s)2. Phosphorescence : T1 S0,

with a change in electron spin. (10-4~102 s)Chemiluminescence : (not initiated by light)

release energy in the form of light. ex : firefly.

A molecule absorbs light

Relationship between Excitation Spectra & Fluorescence Spectra

This shift to longer wavelength is called the Stoke’s shift.

Luminescence instrumentation

.hνout (photon)

heat

breaking a chemical bond

hνin

Luminescence

2) I = kPo C

incident radiationsensitivity

by P0

or C

3) more sensitive than Absorption

Fluorimetric Assay of Selenium in Brazil Nuts– Se is a trace element essential to life: destruct

ROOH (free radical)

– Derivatized:

– Self-absorption: quench

Luminescence: application 1

why is the figure curved? why does it reach a maximum?

Luminescence: application 2

Immunoassarys

employ anitbody

to detect analyte.

Ex: ELISA (enzyme-linked immunosorbent assay)

Luminescence

a. pregnancy test. sensitive to < 1 ng of analyteb. Enviromental Analysis. (ppm) or (ppt)

pesticides, industrial chemicals, & microbialtoxins.

homework

• 27-3, 27-4, 27-6

Mass Spectrometry

The ions formed are separated on the basis of their mass-to- charge ratio (m/z) and directed to a transducer that converts the number of ions (abundance) into an electrical signal.

Principles of mass spectrometry

a mass spectrum

An instrument that produces ions, separates them according to their m/z values, detects them, and plots the mass spectrum.

Mass spectrometers

magnetic sector analyzer

Quadrupole analyzers are mass filters that only allow ions of a certain m/z to pass.

Quadrupole analyzers

Atomic mass spectrometry

Interference effects: 1. spectroscopic interferences: an ionic species

in the plasma has the same m/z value as an analyte ion.

2. matrix effects become noticeable when the concentrations of matrix species ex-ceed about 500 to 1000 µg/mL.

These effects cause a reduction in the analyte signal, although enhancements are sometimes observed.

Interferences effects

Molecular mass spectrometry

Ethyl benzene (C6 H5 -CH2 -CH3 )

A gas-phase source, the sample is first vaporized and then ionized. A desorption source, the sample in a solid or liquid state is converted directly into gaseous ions. Desorption sources are applicable to nonvolatile and thermally unstable samples.

Ion Sources

• Electron ionization• Chemical ionization

Ionization

1) Electron ionization

M + e-

M+ + e- + e-

70 eV -55 eV 0.1eV

Molecular ion break into fragments.

Base peak: most intense peak.

2) Chemical ionization

CH4 + e-

CH4+ + 2e-

CH4+ + CH4 CH5

+ + CH3

CH5+ + M CH4 + MH+

CH4+ CH3

+ + H

CH3+ + CH4 C2 H5

+ + H2

• Total ion Chromatograms21-16a is a reconstructed total ion chromatogram showing all ions from seven opium alkaloids found in street heroin.

• Selected ion Chromatograms:– Simplify analysis – improve S/N

Information in a Mass Spectrum

Nominal Mass : CNominal Mass : C44 HH99 Br is 136 Br is 136

Information in a mass spectrum

Rxn : CH3 (CH2 )2 CH2 –OH + Br-

CH3 (CH2 )2 CH2 –Br1–Butanol 1–Bromobutane

CH3 15

CH2 14

Br 79

C4 H979Br+ 50.0%

C4 H981Br+

Information in a mass spectrumFragmentation Patterns

Information in a mass spectrumIsotope PatternsCn Hx Oy Nz12C/13CIntensity = n x 1.1%Ex: C6 H6

(M+1)/M+ = 6 x 1.1 %

Nitrogen Rule:A compound: odd nominal mass / odd number of N atoms; even nominal mass/ even number of N atoms