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 shift®p p* bathochromic/red shift
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…
max 253 239 256 248
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
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.
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
(-1
-1)
eM
cm
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
(-1
-1)
eM
cm
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
(-1
-1)
eM
cm
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
k
LR
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 RLBL
RLKa
])[(1
])[(][
][
][
][maxmax RLB
KRLBK
L
RL
free
bound
da
Measuring the equilibrium constant (The Scatchard Plot)
])[(1
])[(][
][
][
][maxmax RLB
KRLBK
L
RL
free
bound
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
V
MVxM
1
)1(