Laboratory Activity Two UV/Visible Absorption Spectroscopy

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.

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 & 250-300 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 250-300nm. Aromatic AA’s absorb maximally from 210-235 nm & 250-300 nm. Potassium permanganate Conjugated double bonds: alternate with single bonds Pigment molecules for photosynthesis- lots of double bond

What Wavelengths ? Mostly . . . Ultra-violet (190 – 380 nm). Visible (380 – 700 nm). Near infrared (700 – 1000 nm). (UV-A = 320-380 nm) (UV-B = 280-320 nm) (UV-C = 190-280 nm) 400 500 600 700 Wavelength (nanometers) (IR) (UV) UV-A = long wavelengths of UV (320-380nm); UV-B = (280-320nm); UV-C = (190-280 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. 

What Wavelengths ? But . . . Not all wavelengths are equally absorbed. Some wavelengths are transmitted or reflected. UV-A = long wavelengths of UV (320-380nm) UV-B = (280-320nm) UV-C = (190-280 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)

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.

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  400-800 nm. Dueterium  200-360 nm. Xenon  190-1100 nm. White fluorescent  400-600 (blue-yellow, no orange/red). Sodium vapor lamp  550-650 (mainly yellow) Sunlight (at sea level) 300-2000 nm.

Usefulness of Possible Light Sources for Absorption Spectroscopy (All Light Sources Are Not Created Equal) Sunlight (at sea level)  300-2500 nm (UV – Yellow – IR). White fluorescent  400-600 (blue-yellow; no orange/red). Sodium vapor lamp  550-650 (mainly yellow). Incandescent / tungsten  400-2500 nm (Blue – Far Red – IR). Deuterium / hydrogen  200-360 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 ********

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

*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

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 = = 8.04 x 10-5 moles liter = 80 µM = 80 nanomoles/mL

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.

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

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

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

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

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

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).

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