1. Laboratory Activity Two
UV/Visible
Absorption Spectroscopy

2. Purpose(s) of Lab 02
Introduce you to the theory and application of absorption spectroscopy in biochemistry.
Mechanism & wavelengths of light absorption.
Spectrophotometers & the Biomate 3 Spectrophotometer.
The Beer/Lambert Law in absorption sepectroscopy.
Applications:
Concentration determinations.
Absorbance spectra.
Enzyme kinetics.

3. Introduction to Light Absorption
Light absorption is . . .
A fundamental property of biomolecules.
Mediated by valence electrons.
Dependent on the wavelength of light.
Facilitated by (conjugated) double bonds.
Strong
Absorbances
≤ 240 nm & nm
Valence electrons = electrons in the outermost orbital (energy level) that are most frequently involved in chemical reactions.
Absorbance of organic compounds with conjugated double bonds involves pi electrons, and extends absorbance into the visible wavelengths.
Linoleic acid is off scale at <240 nm, with 3 peaks from nm.
Aromatic AA’s absorb maximally from nm & nm.
Potassium permanganate
Conjugated double bonds: alternate with single bonds
Pigment molecules for photosynthesis- lots of double bond

4. What Wavelengths ? Mostly . . . Ultra-violet (190 – 380 nm).
Visible (380 – 700 nm).
Near infrared (700 – 1000 nm).
(UV-A = nm)
(UV-B = nm)
(UV-C = nm)
400
500
600
700
Wavelength (nanometers)
(IR)
(UV)
UV-A = long wavelengths of UV ( nm); UV-B = ( nm); UV-C = ( nm).
Titanium dioxide & Zinc oxide (are both opaque & white, & used as sunblocks).
IR Spectroscopy ranges from about 1000 nm – 20,000 nm+.
Ozone (O3) is a gas produced naturally in the stratosphere where it strongly absorbs incoming UV radiation. 

5. What Wavelengths ? But . . . Not all wavelengths are equally absorbed.
Some wavelengths are transmitted or reflected.
UV-A = long wavelengths of UV ( nm)
UV-B = ( nm)
UV-C = ( nm)
Organic molecules don’t absorb wavelengths equally
Chlorophyll is green because its reflecting or transmitting green light
Absorbs blue, reds, and oranges
400
500
600
700
Wavelength (nanometers)
(IR)
(UV)

6. An Absorbance Spectrum is . . .
A graphical plot of the amount of light absorbed (i.e. its “absorbance”) by a substance across a continuous range of different wavelengths.
Wavelength, nm
Relative Absorbance
Chlorophyll a
Chlorophyll b
Experimental proof (or explanation why) chlorophyll is green.
Absorbance spectra for chlorophylls a & b.

7. A Spectrophotometer is . . .
A device that measures how much light a substance* absorbs at various wavelengths.
*Substance must be in solution.
Basic components of a spectrophotometer include . . .
Incandescent  nm.
Dueterium  nm.
Xenon  nm.
White fluorescent  (blue-yellow, no orange/red).
Sodium vapor lamp  (mainly yellow)
Sunlight (at sea level)  nm.

8. Usefulness of Possible Light Sources for Absorption Spectroscopy (All Light Sources Are Not Created Equal)
Sunlight (at sea level)  nm (UV – Yellow – IR).
White fluorescent  (blue-yellow; no orange/red).
Sodium vapor lamp  (mainly yellow).
Incandescent / tungsten  nm (Blue – Far Red – IR).
Deuterium / hydrogen  nm (UV).
Xenon  190 – 1100 nm (UV – Yellow – Far Red).
Deuterium nucleus contains one proton + one neutron (H has only a proton); A deuterium lamp is a low-pressure gas-discharge light source
A xenon lamp is a type of gas discharge lamp, an electric light that produces light by passing electricity through ionized xenon gas at high pressure.
Halogen lamps are very similar to tungsten lamps except the halogen gas (iodine) that allows the tungten filament to be hotter, & thus give off brighter light. The filament is enclosed in quartz to prevent failure due to melting at high temperatures.
Which of the following lamps is used for all wavelengths on a spectrometer? Xenon lamp ********

9. Some Important Expressions
% Transmitted = x 100 I I0
% Absorbed = x 100
I0-I I0
Incident
Beam
(I0)
Light Source
Transmitted
(I)
Sample Holder (cuvette)
Detector
I/I0= transmitted (not percent)
Absorbance (“A”) =
Log10 I0 I

10. *The Beer/Lambert Law is often referred to a “Beer’s Law”.
The Beer/Lambert* Law: A Mathematical Expression that Relates Absorbance, Concentration and Path Length
A = ecl
where: e is the molar extinction coefficient (liters/molecm)
c = the concentration (moles/liter)
l = the path length (1 cm)
Importance: since e and l are both constants, absorbance is directly proportional to concentration.
*The Beer/Lambert Law is often referred to a “Beer’s Law”.
All units cancel out
No mathematical units
Optical density units
Absorbance is directly related to concentration

11. Example in the Use of Beer’s Law
Consider a solution of NADH:
Unknown concentration
OD340 = 0.50
340 = 6220 (liters/mole)(cm-1)
Beer’s Law: A = ecl
Solve for “c”: c = A/l
Substitute in:
What’s the concentration of NADH ?
NADH- nictotino amide
0.50
liters
molecm
(6220
) (1 cm)
c =
= x 10-5
moles
liter
= µM
= nanomoles/mL

12. Absorbance/Transmittance Data for a Substance with e = 1.0
Some Important Trends
Absorbance/Transmittance Data for a Substance with e = 1.0
Conc. (M) %T
% Absorbed
I0/I
Absorbance
(log [I0/I])
0.046
0.097
0.301
1.0
2.0
3.0
100 90 80 50 10 1
0.1 20 99
99.9
100/100
100/90
100/80
100/50
100/10
100/1
100/0.1
Io/I = reciprocal of transmittance.
Keep absorbance below 1.5
Between 2-3 don’t even bother recording
Take Home Messages:
Keep absorbance measurements low (most accurate between 0 – 1).
Keep absorbance of blanks low (< 0.100).
Absorbances from 2 – 3 are almost meaningless or nonsense.

13. Importance of Blanks
“Blanks” are reference samples used to estimate and/or subtract out background/interfering absorbance due mainly to . . .
The solvent in which the sample is dissolved.
Non-sample solutes.
The walls of the cuvette that contains the sample.
The air between the source & the detector.
Remember - Its really important to keep the absorbance of blanks low.
Air
Light Source
cuvette
Detector
solvent
Measuring change of absorbance of NADH as it gets oxidized

14. Choice of Cuvettes is Extremely Important
Strong absorbance in UV.
Soluble in organic solvents.
Volume to contain.
Extremely expensive.
“Proprietary”
Acrylic
Poly-
Styrene
Glass
Quartz cuvettes cost $150; glass $50, Proprietary $1 each.
Cuvettes have different properties
Glass can’t use below 320
Poly-styrene is the cheapest
Quartz is the best
Using acrylic this week (second cheapest)
Glass 20 bucks a piece
Quartz

15. Main Activities for Lab 02
VII. Use the Biomate 3 Spectrophotometer to . . .
Determine the concentration of an absorbing substance (KMnO4) in solution three different ways.
Graphically.
Using a linear regression.
Using Beer’s Law.
Create a duplicate standard curve for KMnO4.
Determine the absorbance and transmittance spectrum of KMnO4 (480 – 580 nm).
Measure the activity of Lactate Dehydrogenase.
KMnO4 absorbs strongly from 480 – 580 nm (green to yellow), leaving blues, orange & red = purple.
C1V1=C2V2
Try to duplicate standard curve
Transmittance should be almost reflective but negative of the absorbance

16. Main Activities for Lab 02
VII. Use the Biomate 3 Spectrophotometer to . . .
Determine the concentration of an absorbing substance (KMnO4) in solution three different ways.
Graphically.
Using a linear regression.
Using Beer’s Law.
Create a duplicate standard
Curve for KMnO4.
Y = absorbance
Solve for A

17. A Closer Look at Lactate Dehydrogenase
Present in almost all animal tissues.
Catalyzes the reversible oxidation of lactic acid to pyruvic acid.
Uses NAD+/NADH as the electron acceptor/donor.
CH3-C-COOH + NADH + H+ ↔ CH3-C-COOH + NAD+ OH I O II H
(Pyruvic Acid)
(Lactic Acid)
= -35
kcal/mole
Reaction Time
ABS340
G of LDH = -35 kcal/mole.
Diagram is of reduced form (NADH).
Lactate dehydrogenase: associated with lactate fermentation
NADH- nicodine …..
340 nm- NADH absorbs very strongly (NAD zero absorbance)
ABS
Time
A = ecl

18. Take Care of My Spectrophotometers !
$3,500 ea.
Keep them clean:
Cuvettes must be clean & dry outside.
Do not add reagents to cuvette while in sample holder.
Do not spill reagents on spectrophotometer or inside sample compartment.
Keep lid closed, except to insert or remove sample; don’t slam the lid.
Insert sample correctly (triangle toward you).
Turn on  5 min. before use; leave on until done.
Do not bump, jar or move the spectrophotometer during use.
Use cuvettes sparingly (two per application).

19. Questions or Comments