Chem 222

#12 Ch18Feb 22, 2005

Announcement

• Advice: KH 3-14 Exp (Nicotine in Tobacco) smells quite nasty. So wear lab coat.

• KH 3-14 Exp Handout Website Report Cover

Sheets and HandoutsHANDOUT KH 3-14

• Don’t be late for the class!

Q3

• 2b When you decrease pH, how does the concentration of free Ca2+ change? Does it increase or decrease? Explain the reason.

• Kf’ = αY4Kf = [CaY2-]/[Ca2+][EDTA]

Nicotine

H—+

H—+

H+

B BH+ BH2+

(Read P234)

KH 3-14

Nicotine1 mol

H+ (in Acetic Acid Solvent)2 moles

From HClO4

Warning:•Glacial Acetic Acid Very Corrosive• Perchloric Acid Strong Oxidant

(May Explode with Organic Solvent)

Wear Nitrie Gloves and Goggles

(b) Titration of 10.0 mL of 0.100 M nicotine(pKb1 =6.15, pKb2 =10.85) with 0.100 M HCl

P232 (Ch 12)

The second H+ does not completely react with Nicotine in H2O

Ch. 18 Fundamental Spectrophotometry

How ozone is formed

How ozone is decomposed by Freon-12 (CCl2F2)

Ozone layer absorb UV light

Applications of Spectrophotometry

♦ Measurement of concentration♦ Detector of molecules in HPLC

(19-4)♦ Cell imaging with probe dyes♦ Immunoassays (19-5)♦ Conformation analysis by IR

18-1 Properties of Light

• The wave length λ is the crest-crest distance between waves

The frequency ν is the number of complete oscillations that the wave makes each second

Relation between λ and νλ ×ν = c (18-1)

Light has characters as both of a wave and a particle

A light particle called photon has energy of

E =hν, (18-2)where h is Plunck’s constant 6.626 ×10-34 J-s

Wave length, Energy, Frequency, Wave Number

Higher Energy Lower Energy

E = hν = hc/λ = hc ν (18-3)↑

ν = c/λ

~

Wave Number: ν = 1/ λFor wave number, a unit of cm-1 is still commonly used

~

(Memorize this)

Example (p409)

A single photon has energy of

∆E = hc/[Q1] =(6.626×10-34 J/s)(2.998×108

m/s)/(147×10[Q2]m)= 1.35 × 10-18 [Q3 unit? ]

For 1 moles of molecules,

∆Emoles = (1.35 × 10-18) ×(6.022 × 1023)

= 814

• When light is absorbed by a sample, the irradiance of the beam of light is decreased.

• Irradiance P: the energy per second per unit area of the light beam--• --• --•--• --• --• P = Nhν

--• --• --• ☺ ☺ --• --•--• --• --• ☺ --•

P0 P

Molecules Absorb Photons

Transmittance: T = P/P0

hv

Sample Cells or Cuvets

Absorbance & Transmittance

• T = P/P0 ( 0 ≤ T ≤ 1)Percentage transmittance %T = 100 T

Absorbance is defined• A = -log(P/P0) = -log(T)

Q. How much is A for the following P(a) P = 0.001P0(b) P = 0.1P0

Beer’s LawA = εbCC: Concentration b: Path length (Thickness of your sample)[cm]ε: Molar Absorptivity [M-1cm-1]

Answer See Box 18-1

↑Different depending on molecules and λ

Usable to characterize molecules

Absorption spectra of Chlorophyll a & b

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/ActionSpectrum.html

Examples (p412)• Find the absorbance and transmittance of

a 0.00200 M solution of a substance with a molar absorptivity of 250 M-1cm-1 in a cell with 2.00 cm path length.

A = (250 M-1cm-1) [Q1] [Q2] = 1.00

A = -logT T = 10 –A

A = 10 [Q3] = [Q4]

Note:Beer’s law usually work for a dilute solution (≤ 0.01 M) When c is high, molecules start to interact.

Ex. Monomer to Dimmer ε changes

Absorption Spectrumwave length dependence of ε

• A = εbc

Visible absorption spectrum of (ferrozine)3Fe(II)

λ (nm)

ε(M

-1cm

-1)

Sweep λ and measure A

Serum Iron Determination (p416)

• Serum normally contains 1 µg of Fe in 1 mL of serum in a form of complex with transferrin(protein).

Step1 ) Reduce Fe3+ (use thiglycolic acid or vitamin C)

Step 2 ) Add trichloroacetic acids to remove proteins

Step 3) Transfer supernutant, and add ferrozine

Human Blood: 45 % cells, 55 % plasmaIf you remove anticoagulutant (blood clot) Serum

Calibration curve for A = kC

↑

562 nm

Try example in p418