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Let There Be LightLet There Be Light

ExploringExploring ChemicalChemical AnalysisAnalysis

Fourth Edition

1818

歐亞書局

Let there be light and there was light.

Spectrophotometry-the use of electromagnetic radiation to measure chemical concentrations

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18-1 Properties of Light

Light can be described both as waves and as particles.

Light waves consist of perpendicular, oscillating electric and magnetic fields.

Wavelength, λ.

Frequency, ν, is the number of oscillations per second.

One oscillation per second is also called 1 hertz (Hz).

 Relation between frequency and wavelength: λν=c

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Figure 18-1

Figure 18-1 Plane-polarized electromagnetic radiation.

Ordinary, unpolarized light has electric and magnetic field components in all planes.

Polarized light processes in one plane.

Example:Example: Relating Wavelength and FrequencyRelating Wavelength and Frequency What is the wavelength of radiation in your

microwave oven, whose frequency is 2.45 GHz?

SOLUTION:SOLUTION:

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C =

h: Planck’s constant ( = 6.626×10-34 J s).‧

: (=1/λ) is called the wavenumber 波數 .

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Light can also be thought of as particles called photons.

The energy, E (measured in joules, J), of a photon is proportional to its frequency ：

~

能量 ~~ 正比於波數 頻率 ~~ 反比於波長

Figure 18-2

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Figure 18-2 Electromagnetic spectrum, showing representative molecular processes that occur when radiation in each region is absorbed.

The visible spectrum spans the wavelength range 380 to 780 nanometers (1 nm= 10 － 9 m).

High energy low energy

Figure 24-3 in Skoog’s analytical Chemistry, 8th edition

low energy high energy

vibration

能量 ~~ 正比於波數 頻率 ~~ 反比於波長

Example:Example: Photon EnergiesPhoton Energies

By how many joules is the energy of a molecule increased when it absorbs (a) visible light with a wavelength of 500 nm(b) infrared radiation with a wavenumber of 1 251 cm-1?

SOLUTION:SOLUTION:

(a)

(b)

Figure 18-3

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• Absorption of light increases the energy of a molecule.

• Emission of light decreases its energy.

The lowest energy state of a molecule is called the ground state. When a molecule absorbs a photon its energy increases the molecule is promoted to an excited state.

吸收能量 釋放能量

18-2 Absorption of Light

Figure 18-4

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Figure 18-4 Schematic representation of a single-beam spectrophotometric experiment.

A spectrophotometer measures transmission of light.

Light with a very narrow range of wavelength is said to be monochromatic.

In Figure 18-4, light passes through a monochromator ( 單色器 ,單光器 ) , a device that selects a narrow band of wavelengths.

只有一個波長之光線的 ,單色光的

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Transmittance Transmittance 穿透率

Transmittance, T, is the fraction of incident light that passes through a sample.

P0 ≧ P

0 T < 1≦

The most useful quantity for chemical analysis is absorbance, A:

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Absorbance Absorbance 吸光率

When no light is absorbed, P = P0 and A = 0. The sample does not absorb the light.

P0 ≧ P

Figure 24-7 in Skoog’s analytical Chemistry, 8th edition

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Example:Example: Absorbance and TransmittanceAbsorbance and Transmittance

• What absorbance corresponds to 99% transmittance? • What absorbance corresponds to 0.10% transmittance?

SOLUTION:SOLUTION:

Transmittance Transmittance 穿透率 ↗ Absorbance Absorbance 吸光率↘

Absorbance is proportional to the concentration of light-absorbing molecules in the sample.

Figure 18-5 shows that the absorbance of KMnO4 is proportional to concentration over four orders of magnitude (fro 0.6μM to 3 mM).

Peak absorbance at 555 nm is proportional to concentration fro

m 0.6μM to 3 mM.

A : absorbance

ε: molar absorptivity 莫耳吸光係數

b: the pathlength that the light travels through a substance

c : concentration

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Beer’s LawBeer’s Law

b

Color Plate 13

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COLOR PLATE 13

Fe(phenanthrolines)32+ Standards for Spectrophotometric

Analysis (Section 18-2) Volumetric flasks containing Fe(phenanthroline)3

2+ solutions with iron concentrations ranging from 1 mg/L (left) to 10 mg/L (right).

Color Plate 13 shows that color intensity increase as the concentration of the absorbing molecule increases. Absorbance is a measure of the color.

(a) Find the molar absorptivity and percent transmittance of this solution.

(b) What would be the absorbance if the pathlength were 0.100 cm?

(c) What would be the absorbance in a 1.000-cm cell if the concentration were decreased by a factor of 4?

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Example: Using Beer’s Law

The peak absorbance of 3.16×10-3 M KMnO4 at 555 nm in a 1.000-cm-pathlength cell in Figure 18-5 is 6.54.

SOLUTION:SOLUTION:

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The peak absorbance of 3.16×10-3 M KMnO4 at 555 nm in a 1.000-cm-pathlength cell in Figure 18-5 is 6.54.

(a) Find the molar absorptivity and percent transmittance of this solution.

Percent transmittance is 100T=2.88×10-5 %. When the absorbance is 6.54, transmittance is very tiny.

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If we decrease pathlength by a factor of 10, we decrease absorbance by a factor of 10 to 6.54/10=0.654.

(b) What would be the absorbance if the pathlength were 0.100 cm?

A = (2.07×10-3 M-1 cm-1) (0.1cm) (3.16×10-3 M) = 0.654

If we decrease concentration by a factor of 4, we decrease absorbance by a factor of 4 to 6.54/4 to 1.64.

A = (2.07×10-3 M-1 cm-1) (10cm) (3.16×10-3 M/4) = 1.64

(c) What would be the absorbance in a 1.000-cm cell if the concentration were decreased by a factor of 4?

Example: Finding Concentration from Absorbance

Gaseous ozone has a molar absorptivity of 2700 M-1cm-1 at the absorption peak near 260 nm in the spectrum at the beginning of this chapter.

Find the concentration of ozone (mol/L) in air if a sample has an absorbance of 0.23 in a 10.0-cm cell. Air has negligible absorbance at 260 nm.

SOLUTION:

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Absorption SpectraAbsorption SpectraAn absorption spectrum is a graph showing how A (or ε) varies with wavelength (or frequency or wavenumber).

Figure 18-6 Absorption spectrum of typical sunscreen lotion shows absorbance versus wavelength in the ultraviolet region.

320-400 nm

280-320 nm

SPF = 1/T

SPF T A ↗ ↘ ↗

See Problem 18-12

From the spectrum, the absorbance A is about 0.35 at 300 nm.

The transmittance is T=10 － A = 10 － 0.35 = 0.45 = 45%.

Just over half the ultraviolet radiation (55%) is absorbed by sunscreen and does not reach your skin.

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Example:Example: How Effective Is Sunscreen?How Effective Is Sunscreen?

What fraction of ultraviolet radiation is transmitted through the sunscreen in Figure 18-6 at the peak absorbance near 300 nm?

SOLUTION:SOLUTION:Absorption spectrum of typical sunscreen lotion

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The substance absorbs certain wavelengths of white light, and our eyes detect wavelengths that are not absorbed.

White light contains all colors of the rainbows.

A substance that absorbs visible light appear colored, when white light is transmitted through it or reflected from it.

18-3 Practical Matters

The instrument represented in Figure 18-4 is called a single-beam spectrophotometer because it has only one beam of light.

Figure 18-7

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Sample is usually contained in a cell called a cuvet, which has flat, fused-silica faces.

Common cuvets for ultraviolet and visible measurements.

• Simple glasses are fine in the visible region.

• Fused silica or quartz is necessary in the UV region (< 380 nm).

• Halide salts (KBr, NaCl, AgCI) are often used in the IR region but have the discadvantages of being expensive and somewhat water soluble.

Transmittance ranges for various optical materials.

Figure 25-2, Skoog

Silicate glass is completely adequate for use in the visible region In the UV region, fused silica or quartz must be substituted.

In the IR region, glass, quartz, and fused silica all absorb at wavelengths longer than about 2.5 μm.

Optical elements for IR spectrometry are typically made from halide salts or, in some cases, polymeric materials. Figure 25-2, Skoog

In a single-beam spectrophotometer, we do not measure the power of incident beam P0, directly.

Skoog, chapter 24

Rather, the radiant power passing through a reference cuvet containing pure solvent is measured and defined as P0.

P0 和 Pe( 或 P) 之間的差異未必是被樣品吸收 而是以其他方式如散射 (scattering)或反射 (reflection)

In a single-beam spectrophotometer(1) Measure P0 (reference: pure solvent)(2) Measure P (sample: solvent + solute)

P0: radiation power passing through cuvet filled with solvent

P: radiation power passing through cuvet filled sample (solvent + solute)

Transmittance = P/P0

P0: radiation power passing through cuvet filled with solvent

P: radiation power passing through cuvet filled sample (solvent + solute)

Transmittance = P/P0

Double-beam spectrophotometer

With a double-beam system, you can measure P and P0 simultaneously!

Good operating techniques

Cuvets should be held with a tissue to avoid putting fingerprints on the cuvet faces and must be kept scrupulously clean.

Cuvet cleaning reagent

Do not touch the clear face of a cuvet.

18-4 Using Beer’s Law

Spectrophotometric analysis with visible radiation is called colorimetric analysis.

For a compound to be analyzed by spectrophotometry, it must absorb electromagnetic radiation, and this absorption should be distinguishable from that of other species in the sample.

Biochemists assay proteins in the ultraviolet region at 280 nm because the aromatic amino acids tyrosine, phenylalanine, and tryptophan have maximum absorbance near 280 nm.

tyrosine phenylalanine tryptophan

Example:Example: Measuring Benzene in HexaneMeasuring Benzene in Hexane

(a) A solution prepared by dissolving 25.8 mg of benzene (C6H6, FM 78.11) in hexane and diluting to 250.0 mL has an absorption peak at 256 nm, with an absorbance of 0.266 in a 1.000-cm cell. Hexane does not absorb at 256 nm.

Find the molar absorptivity of benzene at this wavelength.

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Molarabsorptivity

=ε

= A/bc

= 0.266/(1.000m)(1.321×10-3 M)

= 201.3 M-1cm-1.

SOLUTION:SOLUTION:

(b) A sample of hexane contaminated with benzene has an absorbance of 0.070 at 256 nm in a cell with a 5.000-cm pathlength.

Find the concentration of benzene.

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SOLUTION:SOLUTION:

BOX 18-2

Pyrocatechol violet indicator bound to metal is blue

Free pyrocatechol violet indicator is yellow

A designed ligand that binds to two Zn2+ ions

The distance between the two Zn2+ ions is just right to bind to the metal ion indicator pyrocatechol

Near neutral pH, phosphate binds tightly to the two Zn2+ ions

The pyrocatechol is displaced

and is free

Designing a colorimetric reagent to detect phosphate

The change in adsorption provides a quantitative measure the amount of phosphate.

UV/Vis spectra of the [Zn2(H-bpmp)]3+ -pyrocatechol violet mixture (50 m in a pH 7.0 aqueous solution) in the presence of various anions (250 m).

UV/Vis spectra obtained by additions of [Zn2(H-bpmp)]3+ solution (final concentrations: 0, 10, 20, 30, 40, and 50 m) to the pH 7.0 aqueous buffer (HEPES, 10 m) containing pyrocatechol violet (50 m); b) UV/Vis spectra obtained by additions of HPO4

2- solution (final concentrations: 0, 25, 50, 100, 150, 200, 250, and 500 mm) to the pH 7.0 aqueous buffer (HEPES, 10 m) containing [Zn2(H-bpmp)(pyrocatechol violet)]t (50 m).

The aquarium nitrite analysis is based on a reaction whose colored product has an absorbance maximum at 543 nm (Figure 18-9):

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Using a Standard Curve to Measure NitriteUsing a Standard Curve to Measure Nitrite

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To measure the amount of nitrite –

the nitrite analysis is based on a reaction whose colored product has an absorbance at 543 nm.

Figure 18-9

For quantitative analysis, we prepare a standard curve (also called a calibration curve) in which absorbance at 543 nm is plotted against nitrite concentration in a series of standards.

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Reagents1. Color-forming reagent2. Standard nitrite (~0.02 M)

Calibration curve for nitrite analysis

• standard curve• calibration curve

Table 18-2

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Calibration curve for nitrite analysis

(0.4575, 0.082)

(0.9150, 0.164)

(1.830, 0.325)

(?, 0.278)

Procedure1. Construct a standard curve from known nitrite

solutions.

2. Analyze duplicate samples of unknown aquarium water that has been filtered prior to dilution to remove suspended solids.

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Example:Example: Preparing Nitrite StandardsPreparing Nitrite Standards

How would you prepare a nitrite standard containing approximately 2 ppm nitrite nitrogen from a concentrated standard containing 0.018 74 M NaNO2?

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SOLUTIONSOLUTION

Example:Example: Using the Standard CurveUsing the Standard Curve

From the data in Table 18-2, find the molarity of nitrite, with an average absorbance 0.276, in the aquarium.

SOLUTIONSOLUTION

---- by the method of least square

代入所觀測之吸收值入直線方程式 得到濃度值

由重量濃度單位改為 M

Nitrate (NO3-) in natural waters is derived from sources

such as fertilizers and undertreated animal and human waste.

NO3- is commonly analyzed by reducing to NO2

-, followed by colorimetric assay of NO2

-.

Metallic Cd has been the most common reducing agent for NO3-. However the use of toxic Cd should be reduced.

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Enzyme-Based Nitrate Analysis-A Green IdeaEnzyme-Based Nitrate Analysis-A Green Idea

P.408-Nicotinamide adenine dinucleotide

The enzyme nitrate reductase catalyzes the reduction: 

Enzyme-Based Nitrate Analysis - A Green IdeaEnzyme-Based Nitrate Analysis - A Green Idea

colorimetric assay

NADH is derived from niacin