CHEM 2303 Lecture 2 Jan 16 2015 A

Topics: Protein, Fluorescence, Spectroscopy Pages: 99 (5160 words) Published: January 13, 2015
CHEM 2303

ANALYTICAL CHEMISTRY II
LECTURE 2 – APPLICATIONS OF SPECTROPHOTOMETRY

TENTATIVE LECTURE SCHEDULE
Date

Topics
• Presentation / discussion of syllabus
• Tips for success
• Laboratory
• Introduction to the analytical process
• Sample preparation
Jan 9th, 2015
• Spectrophotometry
• Properties of light
• Absorption of light
• Excited states
• Luminescence
• Applications of spectrophotometry
• UV-Vis spectroscopy
• Fluorimetry
Jan 16th, 2015
• Fluorescent staining, green fluorescent protein
• Immmunoassays
• Discussion on lab test
• Optional tutorial on Ch. 1, 2, 3, 4-1 and 4-2 (8:359:20am) Jan 23rd, 2015 - LAB
• Error calculations (not on lab test)
TEST
• 10 minute break
• Test on Ch 1, 2, 3, 4-1 and 4-2 (9:30-11:25am)

- Lecture and test / Midterm schedule is subject to change at the discretion of Instructor

Chapters (N = Notes)
Lecture Slides
Lecture Slides
See lab maunal
Ch. 0
Ch. 27-2
Ch. 17
Ch. 17-1
Ch. 17-2
Ch. 17-6
Ch. 17-7
Ch. 18, 19
Ch. 19-1,-2,-3, N
Ch. 17-7, N
N
Ch. 18-5, N
Ch. 1, 2, 3, 4-1, 4-2
Ch. 1, 2, 3, 4-1, 4-2
Ch. 4-8
N/A
Ch. 1, 2, 3, 4-1 and 4-2

UV-Vis spectroscopy – Ch. 17-3

A = εbc = -logT

T=

𝑃𝑃
𝑃𝑃0

http://www.cofc.edu/~kinard/221LCHEM/CHEM221L%20Spectrophotometric%20Determination%20of%20Iron.htm

A single beam instrument
P0 (or I0 above) is not measured directly – must use a
“blank”

UV-Vis spectroscopy – Ch. 17-3

Figure 17-5
Cuvets must be kept clean!!

UV-Vis spectroscopy – Ch. 19
Double beam scanning spectrophotometer

Figure 19-1

Again, cuvets must be kept clean!!
Cuvets must be identical to each other.

UV-Vis spectroscopy – Ch. 19

UV-Vis spectroscopy – Ch. 19
Deuterium arc
lamp

Disperses light
into various λ
Quartzhalogen (like a
headlight on a
car)

Again, cuvets
must be kept
clean and
must be
identical to
each other.

Selects narrow
band of λ
Figure 19-2

Sources of light – Ch. 19-1
Tungsten lamp:
• excellent source of
visible and near-IR
radiation
• operates at
temperature near
3000 K
• produces useful
radiation in the
range of 320 to
2500 nm
Deuterium arc lamp:
• electric discharge
causes D2 to
dissociate and emit
Figure 19-4
UV radiation from
200 to 400 nm
In a typical UV-Vis spectrophotometer, the lamps are switched when passing through 360 nm so that the source with the highest intensity is always employed

Monochromators – Ch. 19-2

http://chemtrails.cc/blog/wp-content/uploads/2009/01/grating_spec-br.jpg

Diffraction Grating

Monochromators – Ch. 19-2

Diffraction Grating

Colour plate 20a in QCA 8th Edition

Monochromators – Ch. 19-2

Diffraction Grating

Colour plate 20b in QCA 8th Edition

Monochromators – Ch. 19-2

Figure 19-7

Diffraction Grating

Monochromators – Ch. 19-2

Diffraction Grating

Colour plate 12 in QCA 8th Edition

Monochromators – Ch. 19-2

Measure of
resolution…
= λ / ∆λ
= ability to
separate two
closely spaced
peaks

Measure of
bandwidth

Figure 19-6
…disperses light into its component wavelengths and selects a narrow band of wavelengths to pass on to the sample or detector

Monochromators – Ch. 19-2 Bandwidth should be as large as possible, but small compared with the width of the peak being measured

Larger bandwidth =
greater sensitivity and
signal to noise…
However, can cause
problems in some cases
(well resolved light
source (i.e. high
resolution) can be
valuable to an analysis)

Figure 19-10

Monochromators – Ch. 19-2

Stray light

• wavelengths outside the
bandwidth expected from the
monochromator
• can come from outside the
instrument if the sample
compartment, or entry holes for
tubing and wires, are not
perfectly sealed
• high quality spectrometers may
have 2 monochromators to
Figure 19-11
reduce stray light
Error from stray light is highest when the sample absorbance is high (above figure) because the stray light...
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