UV-Vis Molecular Absorption Spectrometry

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UV-Visible Molecular Absorption Spectrometry

Mainly Chapters 7 and 13, a little of Chapter 6 and 14 also.

Based on an increase in electronic energy (Eelectronic) from the absorption of

a photon in the UV or visible region, as previously discussed.

Absorption spectrophotometry is the single most common method for

quantitative analysis of molecules, but

Beers Law has its limitations (Section 13B).

1. Already know about linear range, for any instrumental method. In

the derivation of Beers Law, it is assumed that solutes act

independently. If the solute conc. is too high, then that is not a good

assumption and the absorptivity changes.2. In the derivation of Beers Law monochromatic incident radiation is

assumed to irradiate the sample. If a source is used that puts out

light of all wavelengths (a continuous source), then Beers Law does

not apply.

To illustrate this 2nd point, consider a beam of incident radiation on an

absorbing sample consisting of 2 wavelengths

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3. Stray Light: Minimize by

closing the cover! Cannot eliminate.

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Noise in spectrophotometric measurements

UV-Vis Molecular Absorption Instrumentation (Section 13D + parts of

Chapters 6 & 7)

First sources.

A continuous source that emits all wavelengths of radiation in the

region is used.

Mostly Blackbody radiators. When a conducting solid is heated, it

will emit electromagnetic radiation (incandescence).

1. The total amount of lightenergy increases with

increasing temperature.

2. The spectral intensity

shifts to higher energies

with increasing

temperature.

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The W filament lamp is the most common source for visible (and

near-infrared) spectrometers. (~2900K)

The W/halogen lamp can operate at a higher temperature, and can be

used as a UV source (if housed in quartz which does not absorb UV

light, rather than glass which does). ~3500K. They are also superior

visible sources.

The continuous spectral output of a blackbody radiator can beunderstood qualitatively from the MO diagram of a solid conductor.

Compare this to the Atomic orbital diagram of Na discussed earlier.

A more common UV source is the D2 lamp which provides a continual

spectral output from 190-400 nm.

LED for smaller instruments like the OOI spectrometer we have often

been using. LED does not produce a continuous output, so how does it

put out a continuous spectrum of white light?

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If the spectrometers source emits a continuous range of wavelengths

of light, and the goal is to obtain a spectrum of absorbance as a

function of wavelength, there must be a way to select a wavelength, or

range of wavelengths, for sample irradiation.

Second Wavelength selectors. Section 7C

Ideally output from a wavelength selector is a single wavelength.

Reality output from a wavelength selector is a band of wavelengths.

The narrower the band, the better the wavelength selector, the greaterthe spectral resolution attainable.

Wavelength selectors can be

either filters or

monochromators. We will

discuss monochromators

exclusively. In any case the

effective bandwidth is one

important defining factor of

a wavelength selectors

performance, defining its

resolution.

Monochromators can be

based on the use ofprisms, which work on the

principal of refraction, or

gratings, which work on

the principal of

diffraction.

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You can see that a traditional monochromator consists of more

than a grating or a prism, but these are the parts of a wavelength

selector most important to selecting a band of wavelengths from a

continuous source. The next figure shows why diffraction gratings are

superior to prisms for most applications.

Now a few specifics about grating monochromators.

Function to disperse different wavelengths of light at different angles.

Angular dispersion: dr/d

Remember from earlier:

This would be a transmission grating,

where it was shown that the following

conditions result in constructiveinterference:

n = d sin

where d is the distance between the holes

in the grating

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Monochromators invariably use a reflection grating, where closely

spaced graves are cut out of a mirrored face.

In this case constructive

interference occurs whenn = d(sin i + sin r)

i is the angle of incidence, r

is the angle of reflection. d

is defined at left, and n is an

integer value (diffractionorder)

Differentiation of the above

equation at a constant angle of incidence:

While the angular dispersion is important, the linear dispersion D is

more relevant, since it refers to the variation in wavelength along the

focal plane (AB in figure of grating monochromator)

Most important is the inverse of the linear dispersion, the reciprocal

linear dispersion D-1

This to a large extent determines the spectrometers resolution. The

ability to distinguish absorbances at different wavelengths close to one

another.

A grating monochromators resolving power depends largely on

d the space between grating blazes

f the monochromators focal length

n the diffraction order

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Say a conventional grating has 1200 blazes/mm and a focal length of

0.5 m. (Both of these numbers are pretty standard).

Find D-1 for n = 1

Find D-1 for n = 2

Find D-1 for a grating with 600 blazes/mm

Will a large D-1 or a small D-1 provide better resolution?

Why is spectral resolution important? The higher the resolution the lower the S/N

The higher the resolution the greater the information content

(potentially)

Here is an illustration of vapor phase UV spectra of benzene at 2

different resolutions

Resolution will be revisited soon. First, a discussion of the last major

instrument component of UV-Visible molecular absorbance

spectrometers.

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Portions of Section 7E, detectors.In the UV-Vis region we discuss 3 types, the first 2 are very similar in

principle but have very different performance characteristics.

1. Vacuum Phototube

2. Photomultiplier tube

Both of these are photoemissive devices. Shine light on them, and

they emit electrons.

3. Multichannel photon transducers. Shine light on these, and they

conduct electricity. Advantage of smaller size and can constructinstruments with a different design as a result.

The photoemissive detectors work on the principle of Einsteins

photoelectric effect (Section 6C).

Here is a vacuum phototube.

The cathode is

coated with a low

ionization energy

material.

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117 = K-Cs-Sb, S11 = Cs3Sb

Vacuum phototube: 1 photon > ionization energy of photocathode => 1photoelectron emitted.

Photomultiplier tube: 1 photon > ionization energy of photocathode =>

~106 photoelectrons emitted.

PMT much greater sensitivity. Much lower light levels give

measurable signals.

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In a PMT there is still a photocathode, but between that and the anode

are a series of dynodes.

If at each dynode 5 e- are emitted for each electron that strikes it, for

the 9 diode arrangement above the PMT current gain = 59

Dynodes are irreversibly damaged by high intensity light, which is why

we are paranoid when using the fluorescence spectrometer.

These 2 photoemissive detectors are fine, but they are large. The

usefulness of a very small detector will be shown after a brief

discussion of the basic principles of how one works.

Small photoconductive detectors are based on semiconductors.

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Since each detector is about 25m, a small instrument can fit many

detectors. This allows for a different type of instrument.

Types of Instruments Section 13D-2

Single Channel instruments use a single large detector like a vacuum

phototube or a photomultiplier.

These single channel instruments can be single beam

These single channel instruments can also be double beam

The small semiconductor detectors afford the capability of building a

multichannel instrument.

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like the Ocean Optics single beam Array Spectrometer.

Now as promised, spectral resolution revisited.

First single channel instruments (Section 7-C3), then multichannel

instruments.

Spectral resolution is determined by the monochromator.

Thus far only the grating has been

discussed, and its D-1. To

understand resolution we must alsothink about entrance and exit slits.

For a single channel instrument almost invariably:

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The size of the entrance slit width = size of exit slit width

Different wavelengths are brought to the exit slit by rotating the

grating

Consider the following scenario if we have monochromatic source input

into the monochromator, say from a Na vapor lamp.

1. Monochromator illuminated with line source o = 589 nm

2. Entrance slit width = exit slit width = 1 mm

3. Monochromator D-1 = 20 nm/mm

D-1 = d/dy = range of wavelengths spread over the distance dy along the

exit slit focal plane.

1. When monochromator set to 589 nm, entrance slit image fills exit

slit maximum signal intensity.

2. When monochromator set to 579 or 599 nm, entrance slit image half

fills exit slit half of maximum signal intensity.

3. When monochromator set to 569 or 609 nm, entrance slit image

misses exit slit no signal intensity.

This is the origin of the triangular slit function shown above.

Little sense in illuminating a monochromator with monochromatic light

Consider the more realistic scenario

1. Monochromator illuminated with polychromatic light

2. Entrance slit width = Exit slit width = 1 mm

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3. Monochromator D-1 = 20 nm/mm

Now For every present there is a triangular distribution of

energies exiting the monochromator.

When the monochromator is set to 589 nm what range of wavelengths

are passed through the exit slit?

Setting the monochromator to 589 nm:

100% of source power at 589 nm passes through

50% of source power at 579, 599 nm passes through

etc.

The sample is illuminated with a polychromatic band of light of various

wavelengths each with varying intensity!

To define the width of the wavelength band, we go back to the effective

bandwidth, FWHH, now also called the spectral slit width (S)

S = W x D-1 where W = physical slit width

Spectral slit width or effective bandwidth

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defines spectrometer resolution

affects the applicability of Beers Law

affects S/N

affects the ability to acquire detailed spectral information

Earlier the deviations from Beers law due to polychromatic radiation

were addressed. If the monochromators effective bandwidth < 10% of thewidth of the FWHH of the absorption band, then Beers law is obeyed.

Since absorption bands in UV-Vis absorption in liquids are so broad, this

is usually not a problem.

Spectral resolution with a polychromator (i.e. a multichannel instrument)

must be looked at somewhat differently.

Here the entrance slit width is fixed such that the image from the

continuous source illuminates the entire detector array.

Commonly, 1024 detectors are lined up in a 1-D array. [If the detectors

are 0.025 mm apart, this many detectors fits along a line of 25.6 mm

(about 1 inch)!]

Since there is no exit slit there is no slit function as with a single channel

instrument. If there are 1024 detectors over a given spectral range , say

800 nm (200 nm 1000 nm) then the spectral resolution is given by the

range of wavelengths that a single detector is sensing.

Res. = 800 nm/1024 detectors = 0.78 nm/detector

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The range of wavelengths distributed in a given distance along the focal

plane is still dependent on D-1.

Advantages of multichannel instruments: No moving parts, rapid spectral

acquisition (signal averaging), enhanced source throughput.

Disadvantage of multichannel instruments: spectral resolution is notvariable.

Finally a little on applications of UV-visible absorption spectrometry (Ch.

14).

The absorption of UV/visible light generally results from excitation of

bonding electrons. In organic compounds, useful transitions are n *

and *. Compounds with useful transitions are said to containchromophores. Transitions below 200 nm are not useful (vacuum UV).

You would think that the wavelengths of absorption bands could be

correlated with the types of bonds and functional groups in a compound.

In theory that is correct.

In practical terms, UV-Vis molecular absorption spectrometry is almost

totally useless for qualitative analyses. By far the most important

application for this type of spectroscopy is for quantitative analysis.

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Chapter 6 Wave & particle properties of EM radiation.

Diffraction. Interaction of radiation with matter, absorption,

emission. Line spectra, continuous spectra, atomic/molecular

absorption and emission. Quantitative aspects of absorption and

emission measurements. Problems/Questions 1e,f,g,h,l, 2, 3, 7, 8,

9a, 13, 14, 15 Chapter 7 General designs of optical instruments, UV-Vis

continuous sources, grating monochromators and performance

characteristics and resolution, UV-Vis detectors (radiation

transducers), optical Fourier transform spectroscopy.

Problems/Questions 1, 3, 4, 5, 8, 19a, 20,

Chapter 13 Transmittance, absorbance, Beers Law and

limitations/deviations, slit widths and effects on spectra,

instrumentation (sources, types of instruments).Problems/Questions 1, 2, 5, 7, 8, 9, 13b,g,h, 17, 18, 22, 23

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UV-Vis Molecular Absorption Spectrometry