Let’s build a spectrophotometer
Light source sample
1) Measure I o
(100% T)
2) Measure I (T of sample)
3) Calculate A detector

Scanning an absorbance spectrum
Light source monochromator sample
Change the monchromator to measure
A versus wavelength
Resolution will be defined by dispersion and slit width, bandpass ≤ 2nm is sufficient
A detector
λ (nm)

Measure all wavelengths at once
Light source sample
Detector array monochromator
Diode array spectrometer
1) Sample receives broad irradiation (i.e., white light)
2) Dispersion element comes after the sample
3) Project spectrum onto an array of detectors

Some applications of spectrophotometry
1) Concentration
Proteins
DNA
2) Environmental effects
Effect of solvent pH
Ligand or protein interactions
3) Time – resolved measurements
Measure time dependent changes
Timescales

Measuring concentration
Suppose we are studying an enzyme that consumes NADH as part of its catalytic reaction, we can use the long wavelength absorbance band of the pyridine chromophore to measure NADH concentration, and thereby activity.
A + H + + NADH → NAD + + AH
2
We know the K
M of the enzyme for NADH is 100 μM (and for A, K
M
=5μM)
We wish to measure the total activity present in a series of extracts.
Measure V max at saturating S, (i.e., NADH & A)
[NADH] = 1 mM (10-fold> K
M
)
Measure A at 340 nm, A = ε c l
A= 6.23 x 10 3 M -1 cm -1 (1 x10 -3 M) = 6.23

Measuring concentration (contd)
A > 6 means % T < 0.0001 % , 99.9999 % of I o absorbed
Most spectrophotometers are only linear up to A= 3
Stray light
How can we fix the problem?
Dilute the NADH.
A
3
2
What about the pathlength?
1
Measure off the peak.
[x] (M)

2.0
A
1.0
The spectrum of NADH
NAD +
20
ε (mM -1 cm -1 )
10
NADH
0
220 300 400
Wavelength (nm)
0

2.0
Environmental effects-Solvent perturbation a) Trp in aqueous buffer
A
1.0
b) + co-solvent e.g., 10% DMSO
E
2
Red- shift
Longer λ
0
260 280 300
Wavelength (nm)
E
1
Blue-shift to
Shorter λ

0.1
ΔA
Difference spectroscopy- small changes
Using trp in aqueous buffer as reference, i.e., 100 % T
0.05
0
-0.05
-0.1
260 280 300
Wavelength (nm)

Split-beam spectrophotometer mirror
Reference
Trp in buffer
Beam splitter
Light source monochromator
Sample
Trp + DMSO detectors

Split- beam or split cuvette
In the two compartments (1 and 2)we have two proteins A and B that we suppose form a complex,
A + B AB
Place equal volumes of the two protein solutions in the two sides,
Measure this as reference, i.e., 100 %T, then mix and record again,
The difference spectrum is generated
1 2
Light source monochromator sample detector

Ligand binding
Cyanide binding to the respiratory enzyme- cytochrome c oxidase
What we know, cyanide is a potent poison of respiration
CN inhibition to respiring mitochondria is instantaneous
[O
2
]
Blocks electron transfer to O
2
S
CN
M
Time (min)

1
A
Cyanide reaction with cytochrome c oxidase
A
430 nm
A=εcl
A
CN
=ε
CN cl
E-CN
E
0.5
+CN
0
400 420 440 460
Wavelength (nm)
0 1 10 100 1000
Time

ΔA
0.4
0.2
0
-0.2
-0.4
Time-resolved spectroscopy
Time (m)
1000
1) Isosbestic points
700
400
100
1
2) E + CN
E-CN
3) Reactivity of the enzyme
in vitro is different from enzyme in vivo
4) Oxidase exists in multiple states
400 420 440 460
Wavelength (nm)