c28 spectrometry

8/2/2019 C28 Spectrometry

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SPECTROMETRICMETHODS OF ANALYSIS:

UV-VIS AND FTIRSPECTROSCOPYCHEM 28

Prof. Kurt W. E. Sy Piecco



The Rise of Spectroscopy

Before the beginning of the twentieth century most quantitative chemical

analyses used gravimetry or titrimetry as the analytical method. With these

methods, analysts achieved highly accurate results, but were usually limited to

the analysis of major and minor analytes.

Other methods developed during this period (1856) extended quantitativeanalysis to include trace level analytes.

One such method was colorimetry – where the sample’s color is compared

against the colors of a range of standards to determine the analyte’s

concentration.

Colorimetry, in which a sample absorbs visible light, is one example of aspectroscopic method of analysis.

During the twentieth century, spectroscopy has been extended to include other

forms of electromagnetic radiation; X-rays, microwaves, radio waves, and

energetic particles (e.g. electrons and ions).



Electromagnetic Radiation (EM)

EM radiation consists of electric and magnetic components, which are

perpendicular to each other.



EM radiation is a form of energy.

Energy has both wave-like and particle-like properties.

Wavelike properties:

Propagates (moves) through space in a straight line at constant speed

(the speed of light). Oscillations in the electric and magnetic fields are

perpendicular to each other, and to the direction of the wave’s

propagation.

Can be refracted, reflected, or diffracted

Can be described by these parameters; frequency, wavelength,

amplitude, etc.



Particle-like properties:

EM radiation can also be pictured as being made up of energetic

particles, called photons.

Photons have momentum ( p = mv)

p is momentum, m is mass, v is velocity The energy of a photon is related to its frequency (E = hγ)

E is energy, h is Plank’s constant, γ is frequency

h = 6.626 x 10 – 34 J · s

De Broglie’s equation

Relates the wave and particle properties of EM radiation



The EM Spectrum



Interactions of EM Radiation with Matter

When a sample absorbs electromagnetic radiation it undergoes a change in

energy.

When a photon is absorbed by a sample, the photon is “destroyed,” and the

photon’s energy acquired by the sample.

Substances can only absorb discrete amounts of energy. The amount differs

from one substance to another.

After absorption of energy, the sample’s atoms, molecules, ions or radicals

become excited. When these excited particles return to their ground states, the

absorbed energy is released (as heat or light, or both).



Different parts or motions of the atoms are affected depending on the energy

(which is proportional to the frequency) of the incident EM radiation.



Two Broad Classes of Spectroscopy

1. Involves transfer of energy between photons and the sample: absorption and

emission of radiation.

In absorption spectroscopy the energy carried by a photon is absorbed by

the analyte, promoting the analyte from a lower-energy state to a higher-

energy, or excited , state. The source of the energetic state depends on the photon’s energy.

The intensity of photons passing through a sample containing the analyte is

attenuated because of absorption.

The measurement of this attenuation, which we call absorbance, serves

as our signal. Absorption only occurs when the photon’s energy matches the difference

in energy, Δ E , between two energy levels.

A plot of absorbance as a function of the photon’s energy (proportional to

its frequency or wavelength) is called an absorbance spectrum



Emission of a photon occurs when an analyte in a higher-energy state

returns to a lower-energy state.

The higher-energy state is produced after the analyte has absorbed of

photon, or after the analyte has undergone a chemiluminescent reaction.

2. Involves changes in amplitude, polarization, phase angle or direction of

propagation of the incident radiation due to refraction, reflection,

scattering, diffraction, or dispersion by the sample.



Measuring EM Radiation

Absorbance

Attenuation (lessening) of

radiation due to absorption by

the analyte.

Absorbance only at the λmax

(quantitative analysis)

Full absorption spectrum

(qualitative analysis)



Emission

Occurs when a photon is ejected

(emitted) as the excited particle

relaxes to a lower energy state.

Photoluminescence – emissionoccurs after absorption of a

photon.

Chemiluminescence – emission

occurs due to a chemical

reaction. The wavelength of the emitted

radiation is usually longer than the

wavelength of the absorbed

radiation.



Basic Components of Spectrometers

1. Energy source

Source of EM radiation or thermal energy

2. Wavelength/frequency selector

Selects a very narrow band from the continuum radiation spectrum to

interact with the sample.

3. Detector

Senses changes in the intensity, polarization, phase angle or direction of

propagation of the incident radiation.

4. Signal processor Electronic component that calculates and converts the electric signals to

numerical or graphical data.



The Energy Source

1. Electromagnetic radiation (see

table on the next slide)

Continuum source – emits a

limited spectrum of EM

radiation

Line source – emits only a few

selected, narrow wavelength

ranges

2. Thermal energy

Flame (2,000-3,400K) Plasma (6,000-10,000K)

3. Chemical sources of energy

Exothermic reactions

Chemiluminescent reactions



The choice of lamp to use depends on the properties

of the sample and on the type of analysis needed (that

is, on the frequency required by the type of analysis).



Each kind of lamp listed in the table in the

previous slide has its own emission (continuum or

line) spectrum.

Observe that the intensity at each wavelength

(figures on the right) is different. When the lamp

is new, the intensities are high and the

spectrometer is accurate even at high sample

concentrations.

However, as the lamp wears out, the

intensities get weaker. This is one reason why spectrometers have

to be calibrated often.

Some spectrometer models are equipped with

a feature that tests the lamp’s performance.



Wavelength Selection

To eliminate or minimize interferences from

other substances that also absorb radiation, the

spectrometer has to be set at a wavelength range

where (ideally) only the analyte will absorb.

A high nominal wavelength produces a better

signal-to-noise ratio.

A narrow effective bandwidth provides better

resolution.

Improving one of these two parameters,

however, results in the deterioration of the other.Therefore, the two parameters must be balanced.

A high nominal wavelength is better for

quantitative analysis.

A narrow effective bandwidth is better for

qualitative analysis.



Wavelength Selectors

Monochromators

Unlike filters in which the selected band is fixed, monochromators can be adjusted to any band needed .

Two types: fixed wavelength (for quantitative analyses only) and scanning.

Interferometers

Instead of filtering or dispersing the electromagnetic radiation, an interferometer

simultaneously allows source radiation of ALL wavelengths to reach the detector.

Filters

Absorption filter – absorbs radiation from

a narrow band of the EM Spectrum (e.g.

colored glass)

Interference filter – uses constructive anddestructive interference to isolate a narrow

band of the EM spectrum (better, but more

costly than absorption filters)



The Monochromator

Functions of each part

Collimating mirror – reflects a

parallel beam of radiation

Diffraction grating – optically

reflecting surface with a large

number of parallel grooves.

Diffraction by the grating disperses

the radiation in space.

Wavelength selection is done by

rotating the diffraction grating.

In some, a prism is used instead.

Focusing mirror – combines

radiation of the same wavelength.

Exit slit – improves the resolution

by narrowing the bandwidth.

Polychromatic

EM radiation

source



Observe the effect of narrow and broad

bandwidths (can be controlled by adjusting the

width of the opening of the exit slit) on the noise

and resolution of a sample’s spectrum.

Too much noise

Bad resolution – some spectral features almost gone

OK

OK



The Interferometer

The beam splitter splits the radiation beam in two. Each beam is reflected on either of

the two mirrors. Then both beams are recombined on the sample/detector.

The recombination of the two beams creates an interference pattern (an

interferogram) for ALL WAVELENGTHS SIMULTANEOUSLY.

The amount of radiation absorbed by the sample, at every wavelength, is calculated

(Fourier Transformations) with the aid of a modern computer.

Advantages over monochromators:

Jacquinot’s advantage: higher throughput of source radiation because of

interferometers have fewer parts (no slits, and other components from radiation is

scattered or lost).

Resulting to a very high nominal wavelength (that is, better signal-to-noise

ratio)

Fellgett’s advantage: much faster because all wavelengths are analyzed

simultaneously



Detectors

Modern detectors use sensitive transducers that convert light or heat into

electrical signals.

Ideally the detector’s signal should be a linear function of the electromagnetic

radiation’s power.

Photon transducers Silicon photodiodes can be miniaturized and arranged in an array

Thermal transducers

Frequently used for IR spectrometers (because IR radiation has

insufficient energy to activate photon transducers)



Photon detectors with photosensitive coating: phototubes and photomultipliers

Photon detectors that are semiconductors: silicon photodiodes, photoconductors

and photovoltaic cells



Absorption of EM Radiation

When radiation passes through a sample, most of it is transmitted without loss

of intensity.

However, intensity is attenuated at certain frequencies due to absorption of

the incident radiation by the sample.

Two requirements for absorption:

There must be a mechanism by which the radiation’s electric field or

magnetic field interacts with the analyte

The energy of the electromagnetic radiation must exactly equal the

difference in energy, ΔE, between two of the analyte’s quantized energy

states.



Vibrational

states

Electronic states



Transmittance

Transmittance is defined as the ratio of the electromagnetic radiation’s power

exiting the sample, PT , to that incident on the sample from the source, P0.

Transmittance is related to Absorbance;



Beer’s Law

Relates Absorbance to analyte concentration;

A = abC or A = εbC

where A is absorbance, b is the path length or the diameter of the sample cell, C

is the analyte concentration and, a and ε are the absorptivity and molar

absorptivity (respectively) of the sample.

For non-reacting multicomponent samples;



Fundamental limitations of Beer’s Law:

Beer’s law is a limiting law that is valid

only for low concentrations of analyte.

At high concentrations,

intermolecular interactions becomesignificant which changes ε.

Higher concentrations also changes

the refractive index of the sample,

which changes ε.

Chemical limitations:

Chemical reactions and ionization

changes ε.



Instrumental limitations:

Errors due to narrow bandwidth

Beer’s Law is strictly valid only for monochromatic spectrometry.

Polychromatic spectrometry always gives a negative deviation from Beer’s

Law, but is minimized if ε over the wavelength range selected. Therefore, it is

advisable to choose the λ max from broad peaks.

When measurements must be made on the slope, linearity is improved by

choosing a narrower effective bandwidth (that is, a narrower exit slit).

Stray radiation

Due to imperfections in the wavelength selector that allows some radiation to

“leak” and reach the detector.

Stray radiation causes negative deviations from linearity if the analyte

concentration is too high. Therefore, sample concentrations must be kept

low.



A Quick Look at Emission Spectroscopy

e n e r g y



Standardization methods

Calibrating signals – determines the mathematical relationship between the

absorbance and the concentration of the analyte solution.

Weight correction for buoyancy in air:

Primary reagents

high purity solids used to precisely determine the concentration of

secondary standard solutions (which is used to quantitatively react

directly/indirectly with the analyte)

External standards

Solutions containing known quantities of the analyte (usually used for

constructing calibration curves)

Method of Standard additions (see next slides)

Useful for minimizing matrix errors



The concentration of the analyte can be

calculated from the x-intercept



Internal standards

Substances added to all samples and standards, whose signal is

measured in addition to the signal produced by the analyte.

Calibration curves (using statistical methods)

Slope: Y-intercept: = − −

− 2 = −



Blank corrections

Total Youden Blank:



UV-Vis Spectroscopy

When a molecule or ion absorbs ultraviolet or visible radiation it undergoes achange in its valence electron configuration.

Instrument Designs

Filter Photometer

Single-Beam Fixed-Wavelength Spectrophotometer

Double-Beam In-Time Scanning Spectrophotometer

Diode Array Spectrophotometer

Instruments using monochromators for wavelength selection are calledspectrometers.

In absorbance spectroscopy, where the transmittance is a ratio of two radiantpowers, the instrument is called a spectrophotometer.



The Filter Photometer

A single-beam instrument.

Portable, rugged, easy to maintain and

inexpensive. Calibrated to 0%T with the shutter closed, then

to 100%T with the “blank sample”.

Must be recalibrated every time the filter is

changed.

Either an absorption or

interference filter

The Single Beam Fixed Wavelength



The Single-Beam Fixed-WavelengthSpectrophotometer

Calibrated and used in the same way as photometers.

More appropriate for a quantitative analysis than for a

qualitative analysis.

Fixed-wavelength single-beam spectrophotometers are not

practical for recording spectra since manually adjusting the

wavelength and recalibrating the spectrophotometer is

awkward and time-consuming.

The accuracy of a single-beam spectrophotometer is

limited by the stability of its source and detector over time.

The Double Beam In Time Scanning



The Double-Beam In-Time ScanningSpectrophotometer

The chopper controls the radiation’s path, alternating it between the sample, the blank, and a shutter. The

signal processor uses the chopper’s known speed of rotation to resolve the signal reaching the detector

into that due to the transmission of the blank (P0) and the sample (PT). By including an opaque surface as

a shutter it is possible to continuously adjust the 0%T response of the detector.



The limitations of fixed-wavelength,single-beam spectrophotometers are

minimized by using the double-beam in-

time spectrophotometer.

The effective bandwidth of a double-beam

spectrophotometer is controlled by means

of adjustable slits at the entrance and exit

of the monochromator.

A scanning monochromator allows for the

automated recording of spectra.

Double-beam instruments are more

versatile than single-beam instruments,

being useful for both quantitative and

qualitative analyses; they are, however,

more expensive.



The Diode Array Spectrophotometer

A linear photodiode array consists of multiple detectors, or channels, allowing an

entire spectrum to be recorded.

Source radiation passing through the sample is dispersed by a grating. The linear

photodiode array is situated at the grating’s focal plane, with each diode recording the

radiant power over a narrow range of wavelengths.

One advantage of a linear photodiode array is the speed of data acquisition, which

makes it possible to collect several spectra for a single sample. Individual spectra are

added and averaged to obtain the final spectrum. Signal averaging improves the

signal-to-noise ratio.



The Sample Compartment

Sample compartments of modern spectrophotometers prevent loss of radiation

due to scattering and reflection. The addition of stray radiation is likewise

prevented.

Liquid or dissolved samples are placed in UV-Vis transparent cells made of

fused-silica, quartz, glass or plastic. For analyses performed below 300nm

wavelengths, quartz or fused-silica cells must be used.

Commonly, cells have 1cm internal diameters (path-lengths). Cells with longer

path-lengths are used for very dilute solutions and gaseous samples.

High quality cells have rectangular shapes to reduce radiation losses to

reflection. These cells are also usually “matched” (having identical opticalproperties). Cylindrical cells are of lower quality and are usually used for

single-beam instruments.

Fiber optic probes are used for real time monitoring or for remote

measurements of a sample’s spectrum.



Sample cells used for

UV-Vis spectroscopy

Examples of fiber optic probes

Sample Preparation for UV Vis



Sample Preparation for UV-VisSpectrophotometry

The solvent must not absorb in the same region as the analyte. The most

common solvents used are listed in the table on the next slide.

The way the solvent influences the shifts in the wavelength of absorbed

radiation must also be noted.

In choosing the right solvent, one must also consider its possible interactionswith the analyte (such as, H-bonding).

The figure on the next slide shows the absorption spectra of phenol in two

different solvents: ethanol and isooctane. There is a loss of resolution

when ethanol is the solvent due to H-bonding with the analyte, phenol.

Other interactions that must be considered include complex formation,ionization and possible chemical reactions.



Chromophores

Chromophores are groups of atoms that absorb in the UV-Vis region of the

EM spectrum.

Auxochromes are groups that increase the absorption intensity or that shift the

absorption wavelength. Bathochromic (red) shift – shift to longer (lower energy) wavelengths

Hypsochromic (blue) shift – shift to shorter (higher energy) wavelengths

Hypochromic effect – decrease in absorption intensity

Hyperchromic effect – increase in absorption intensity

The greater the conjugation of π -bonds, the greater the shift of spectra to

longer (lower energy) wavelengths

Lone pairs shift spectra to shorter (higher energy) wavelengths



UV-Vis Spectrum Generalizations

These generalizations only serve as a guide and could be more useful if combined with

FTIR and NMR data:

1. A single band of low to medium intensity at < 220nm usually indicates an n→σ*

transition. Possibilities are amines, alcohols, ethers, thiols and cyano (weak n→π*)

groups provided these are not part of conjugated systems.

2. A single band of low intensity within 250 and 360nm with no absorption bands

within 200-250nm usually indicates n→π* transition. A simple (unconjugated)

chromophore, generally that contains an O, N or S atom is present. Examples are

azides, nitriles, amides, esters, carboxylic acids, nitrates, aldehydes, and ketones.

3. Two bands of medium intensity both with λmax above 200nm generally indicate the

presence of an aromatic system. A third band near 200nm generally means a

polynuclear aromatic system is present. In non-polar solvents, fine spectral structure

is found in longer wavelengths.



4. High intensity bands above 210nm generally represent an α,β-unsaturated ketone, a

diene or a polyene. The longer the conjugated system, the longer the observed

wavelength.

5. Simple ketones, acids, esters, amides, and other compounds containing both π

systems and unshared electron pairs show two absorptions: an n→π* transition atlonger wavelengths (>300 nm, low intensity) and a π→π* transition at shorter

wavelengths (<250 nm, high intensity).

6. Compounds that are highly colored (have absorption in the visible region) are likely

to contain a long-chain conjugated system or a polycyclic aromatic chromophore.However, some simple nitro, azo, nitroso, α-diketo, polybromo, and polyiodo

compounds may also exhibit color, as may many compounds with quinoid structures.



Infrared Spectroscopyand Chemical Bonding



Note: Infrared spectra of compounds are

often complicated by overtones,

combination bands and difference bands

– all on top of the fundamental absorption

vibrations.



IR Spectrophotometers

IR spectrophotometers have similar block diagrams as UV-Vis spectrophotometers

Filter photometers

portable, dedicated analyzers for gaseous samples.

Single-beam

Double-beam

preferred over single-beam optics because IR sources and detectors are less

stable than those for UV-Vis. Also, error corrections for IR absorption by CO2

and H2O are easier for double-beam optics.

Fourier-transform (with interferometer)

Single-beam instrument, therefore, a background spectrum has to be taken and

subtracted from the sample spectrum to correct for atmospheric CO2 and H2O

absorbance.

Rapid data acquisition, which allows improvement of the signal-to-noise ratio

through signal averaging.



IR instruments are generally

classified into two groups:

Dispersive (those thathave gratings, prisms or

monochromators) and

Fourier Transform

(those with

interferometers)

FTIR A l i M h d



FTIR Analysis Methods

Transmission

Gaseous samples

Placed in a small chamber with NaCl or KBr windows

Path-length is usually 10cm (longer, if mirrors are used)

Liquid samples

A thin film of a non-aqueous non-volatile sample is prepared by placing a drop

between two NaCl plates.

Volatile samples and sample solutions (both non-aqueous) must be placed in

sealed cells; two NaCl plates separated by a teflon tube. The tube length is equal

to the pathlength.

Solid samples

Transparent samples can be placed directly in the path of the IR beam using the

appropriate sample holder.

Opaque samples can be dissolved in a suitable solvent (e.g. Nujol or CCl4) and

analyzed as described above. Alternatively, the sample can be mixed with KBr

and pressed into a transparent pellet.



Attenuated Total Reflectance (ATR)

Aqueous samples must be prepared by applying a thin film on an ATR

crystal (e.g. ZnSe, which is an IR transparent crystal with a high

refractive index).

Solid samples can also be analyzed with ATR. ATR spectra are similar (but, not identical) to transmission spectra.

Radiation from the source enters the ATR crystal, where it undergoes a

series of total internal reflections before exiting the crystal. During each

reflection, the radiation penetrates into the sample to a depth of a few

microns. The result is a selective attenuation of the radiation at thosewavelengths at which the sample absorbs.



Diffuse Reflectance

Powdered samples are mixed with KBr. Diffuse reflection occurs when

light penetrates the solid and is scattered by refraction and reflection

towards neighboring crystals or towards the detector, where it is analyzed.

The spectra are similar (but, not identical) to transmission spectra.

The disadvantage of using KBr is the possibility of moisture

contamination. (Also likely in pelletization).

Th IR B k d S



The IR Background Spectrum

CO2

H2O



The two absorptions at 2350 cm−1 are due to the asymmetric stretching modesof carbon dioxide.

The groups of peaks centered at 3750 cm−1 and 1600 cm−1 are due to the

stretching and bending modes of atmospheric (gaseous) water molecules (that

is, water vapor).

The bell-shaped curve is due to differences in the output of the IR source -

corrected by a feature called, autobaseline.

I f d S t



Infrared Spectra

Continued on next slide



Main References:

Harvey, D. Modern Analytical Chemistry. McGraw-Hill, 2000.

Pavia, D. et al. Introduction to Spectroscopy, 4 th ed. Brooks/Cole,

Cengage Learning 2009. Skoog, D. et al. Fundamentals of Analytical Chemistry, 8th edition.

Thomson-Brooks/Cole, 2004.

Silberberg. Chemistry: The Molecular Nature of Matter and Change, 4th

ed

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