Assumptions
Circular sample well with depth of 6.5mm and height of 7mm
Concentration of Hemoglobin is 20 mg/mL
Quantum yield of Tryptophan is 0.2
UV LED
Voltage is 7 volts
Current is 40 mA
http://www.digikey.com/product-detail/en/MTE280F13-UV/1125-1262-ND/4965442
----------------------------------------------Power = IV = 0.28 Watts
● Power required to turn on UV LED. EE's have approved this value of 0.28 Watts.
Power Output = 1.5 mW (from product datasheet)
Intensity = Power/Area = (1.5mW)/(38.5mm^2) = 3.9 mW/cm^2
● Based on power output of light and the sample well area subjected to the light
Tryptophan Absorbance
----------------------------------------------Beer-Lambert Law: A=Ecl (absorbance=molar absorptivity*concentration*pathlength)
● Molar absorptivity (E): 5600 cm^-1/M
○ Intrinsic property of tryptophan, based on literature tryptophan attenuates light at
280nm at 5,579 cm^-1/M
○ Fasman, G. D., Editor (1976) Handbook of Biochemistry and Molecular Biology,
3rd Edition, Proteins, Volume I, pp. 183-203, CRC Press, Cleveland, Ohio.
● Concentration (c): 1.86 mol/m^3
○ Calculated by finding the concentration of hemoglobin and then multiplying by six
○ Assumed hemoglobin concentration of 20 mg/mL in our 0.25mL sample well
○ Molar mass of hemoglobin is 64500 Daltons
○ Six moles of tryptophan per mole of hemoglobin
■ Based on six tryptophan residues on every molecule of hemoglobin
■ http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1977.tb11676.x/pdf
○ (20 mg/mL)(1/64500 moles/g)(6) = 1.86 mol/m^3
● Pathlength (l): 0.65cm
○ Based on the depth of the sample well (distance the light will shine through)
● A = (5600 cm^-1/M)(1.86 mol/m^3)(0.65cm) = 6.77
● A = log10(I0/I)
●
10^6.77 = I0/I ⇒ 5,888,436/(3.9 mW/cm^2) = 1,509,855 mW/cm^2
Quantum Yield: Φ=(# of photons emitted)/(# of photons absorbed) = 0.2
● Tryptophan will emit 20 percent of the photons it absorbs.
●
Assume tryptophan absorbs all 3.9 mW/cm^2 of 280 nm light ⇒ Emits (3.9*.2)=7.8
mW/cm^2
Fluorescence Spectroscopy
● Energy absorbed by a molecule is equal to the sum of energies absorbed by electronic,
vibrational, and rotational energy levels
● Molecules that absorb photons are called chromophores
Fluorometer Options
● Filter fluorometer - Produces specific excitation wavelengths (filter blocks other light)
● Spectrofluorometer
Intensity based on wattage of bulb- incident
■ I= Power/Area
■ Laser: I= 10mW/ 4*pi*(7mm)^2= 16 W/m^2
■ UV-LED: I= 0.28W/ 4*pi*(0.007mm)^2=455 W/m^2
Beer-Lambert Law
Assumptions
Hemoglobin Molecular Weight=64,500 grams
NOTE: 6nm Deviation Between Observed and Predicted Wavelengths
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1301402/)
Energy Equations
Energy =
ℎ∗𝑐
𝜆
Glossary of Terms
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●
●
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Maximum excitation and emission wavelength (nm): corresponds to the peak in the excitation
and emission spectra (usually one peak each)
Molar extinction coefficient (1/(M*cm)): measure of light attenuation by a chemical species.
Quantum yield: efficiency of the energy transferred from incident light to emitted fluorescence (#
of emitted photons/# absorbed)
Lifetime (in picoseconds): duration of the excited state of a fluorophore before returning to its
ground state. It refers to the time taken for a population of excited fluorophores to decay to 1/e
(≈0.368) of the original amount.
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Stokes shift: difference between the max excitation and max emission wavelengths.
Fluorophore: chemical compound that absorbs light at a specific wavelength and emits light at a
longer wavelength.