Applications of Spectrophotometry(Chapter 19)

Red shift of lmax with increasing conjugationCH2=CHCH2CH2CH=CH2 lmax =185 nm

CH2=CHCH=CH2 lmax =217 nm

vs.

Red shift of lmax with # of ringsBenzene lmax =204 nmNaphthalene lmax =286 nm

Polar solvents more likely to shift absorption maximaShifts of lmax with solvent polarityn®p* hypsochromic/blue shiftp®p* bathochromic/red shift

heptanemethanol

Hypsochromic shiftO

Generally, extending conjugation leads to red shift

“particle in a box” QM theory; bigger box

Substituents attached to a chromophore that cause a red shift are called “auxochromes”

Strain has an effect…

lmax 253 239 256 248

Generally, extending conjugation leads to red shift

“particle in a box” QM theory; bigger box

Determination of Concentration – Multicomponent System

CB

l1 l2

Abso

rban

ce

C+BAbso

rban

ce

Wavelength (nm)

Wavelength (nm)

l2l1

Determination of Concentration – Multicomponent System

CB

l1 l2

Abso

rban

ce

Wavelength (nm)

(1) Measure eB and eC at l1 and l2 (pure substances)

Determination of Concentration – Multicomponent System

C+BAbso

rban

ce

Wavelength (nm)l2l1

(2) Measure A at l1 and l2 (mixture)

Determination of Concentration – Multicomponent System

C+BAbso

rban

ce

Wavelength (nm)l2l1

A1 = xeB1 + yeC1

A2 = xeB2 + yeC2

x = molarity By = molarity C

Isobestic Points

Brocresol green

HIn (acid form) In- (base form)

When one absorbing species is converted to another, it is apparent in the absorption spectrum.

Isobestic Points

[OH-]pH[H+]

pH

I see an isobestic point!!!

Isobestic Points

The Total concentration of Bromocresol Green is constant throughout the reaction

HIn (acid form) In- (base form)

[Hin] + [In-] = F bromocresol green

A = b (exCx + eyCy)

Cx + Cy = C

But at the isosbestic point both molar absorptivities are the same!

ex + ey = e

Isobestic Points

HIn (acid form) In- (base form)

ex + ey = e Therefore, the absorbance does not depend on the extent of reaction (i.e. on the particular concentrations of x and y)

A = b (exCx + eyCy) = b e (Cx + Cy) = b e C

An isobestic point is good evidence that only two principal species are present in a reaction.

Isobestic Points - Application

Oximetry

600 650 700 750 800 850 900 950 10000.0E+00

1.0E+03

2.0E+03

3.0E+03

4.0E+03

5.0E+03

6.0E+03

Hb02Hb

l (nm)

e (M

-1cm

-1)

400 500 600 700 800 900 10000.0E+00

2.0E+04

4.0E+04

6.0E+04

8.0E+04

1.0E+05

1.2E+05

1.4E+05

1.6E+05

1.8E+05

2.0E+05 Hb02Hb

l (nm)

e (M

-1cm

-1)

250 350 450 550 650 750 850 9500.0E+00

1.0E+05

2.0E+05

3.0E+05

4.0E+05

5.0E+05

6.0E+05

Hb02Hb

l (nm)

e (M

-1cm

-1)

Hb HbO2

O2

deoxyhemoglobin Oxyhemoglobin

100 % O2

0 % O2

Measuring the equilibrium constant (The Scatchard Plot)

Biochemistry example: The cellular action of a hormone begins when the hormone (L) Binds to it’s receptor protein (R) in a tight and specific way. The binding thus gives rise to conformational changes which change the biological activity of the receptor (an enzyme, an enzyme regulator, an ion channel, or a regulator of gene expression.

R + L RL

The binding depends of the concentration of the concentration of the components.

dr

fa Kk

kLR

RLK 1]][[

][

Ka is the association constant and Kd is the dissociation constant

Measuring the equilibrium constant (The Scatchard Plot)

When binding has reached equilibrium, the total number of binding sites, Bmax = [R] + [RL]whereas, the number of unbound sites would be [R] = Bmax – [RL].

The equilibrium constant would then be

This expression can then be rearranged to find the ratio of bound to unbound (free) hormone

])[]([][

max RLBLRLKa -

])[(1])[(][][

][][

maxmax RLBK

RLBKLRL

freebound

da --

Measuring the equilibrium constant (The Scatchard Plot)

])[(1])[(][][

][][

maxmax RLBK

RLBKLRL

freebound

da --

The method of continuous variation (for isomolar solution)(Job’s method)

A + n B ABn

Call this C

Abso

rban

ce

Mole fraction (c = nb / na + nb)0 1

Allows for the determinationof the stoichiometry of thePredominant product.

The method of continuous variation (for isomolar solution)(Job’s method)

Requirements:

The system must conform to Beer's law.

Only single equilibrium

Equimolar solutions MA = MB

Constant volume 1 = VT = VA + VB

K reasonably greater than 1

pH and ionic strength must be maintained constant

The method of continuous variation (for isomolar solution)(Job’s method)

A + n B C

Start

Change

Equil.

M(1-x) Mx 0

-C -nC +C

M(1-x)-C Mx-nC C

M(1-x) is a concentrationT

A

VMVxM

-

1)1(

Cor

rect

ed A

bsor

banc

e

Mole fraction (c = nb / na)

0 1

A3X AX AX2

0.50.25 0.66