Beer’s Law and Concentration: Determination of Allura Red in Mouthwash Experiment 9

#9 Beer’s Law and Concentration: Determination of Allura Red in Mouthwash Goal: To employ spectroscopic quantitative analysis of Allura Red concentrations using Beer’s Law Method: Perform serial dilutions to vary concentration Measure %transmittance measurements Determine absorbance Relate absorbance to concentration

Absorption and Emission

Molecular Absorption and Emission Atoms: Electronic states only Molecules: Vibrational states within electronic states

Molecular Spectra Bands vs. Lines (atoms) Absorption Emission 400 500 600 700 l (nm) Absorption Emission Intensity Bands vs. Lines (atoms)

Absorbed vs. Observed Color absorbed → Complementary color observed R BG B YG BV V Y RV O G Color absorbed → Complementary color observed

Allura Red 2Na+ Formula: C18H14N2Na2O8S2 Molar mass: 496.42 g/mol

During Absorption… Starting e- arrangement: After photon absorption:

Absorption is random Photons collide with molecules Certain probability of absorption

Transmittance, T I0 I1 T: ratio of light “in” vs. “out” = fraction of light passing through Depends on: # of molecules b & c molecules’ identity e Pathlength, b I0 I1 Concentration,c

Transmittance and Pathlength For cell with same pathlength: constant T I0 I1 I2

Transmittance and Pathlength For cells with same pathlength: constant T Double pathlength: square T #1 #2 I0 I2

… T and Pathlength I0 Ib For b cells (b pathlengths = 1cm)  Tb #1 #b b in power

Concentration # photons absorbed depends on # molecules in path I0 I1

Transmittance and Concentration 1.0 M reference solution gives I0 I1 I2 … 2.0 M: Double concentration,c→“Double pathlength, b”… bc in power pathlength and concentration

Molar Extinction Coefficient,  T: concentration c & pathlength b What about: molecular identity?  Probability of absorption Specific to molecule Function of l lmax Most efficient absorption Peak of absorption curve “Best l” for experiment

b, c, and  Therefore: bc in power in power pathlength and concentration in power molar extinction coeff. Therefore:

A: Directly proportional to concentration Absorbance Beer’s Law: Defines absorbance, A A: Directly proportional to concentration high A ≡ low T

Part 1 Spectral Profile of Allura Red, lmax Use stock solution (record conc.) cstock Record %T, 400 – 700 nm %T 10 nm intervals near lmax 20 nm intervals elsewhere Record cell width, b = pathlength b Calculate A A Plot A vs. l Determine lmax lmax

Part 1: lmax Find l where %T is lowest Absorption A is highest 400 500 600 700 l (nm) Absorption Transmittance Intensity Find l where %T is lowest A is highest This is not necessarily Allura Red (but would appear red)

Spectral Profile l (nm) Absorbance 400 0.078 420 0.130 440 0.268 460 0.555 480 0.966 490 1.189 500 1.383 510 1.485 520 1.515 530 1.459 540 1.217 560 0.395 580 0.063 600 0.019 620 0.015 640 0.010 660 0.009 680 0.006 700 0.000

Part 2 Absorbance for Various Concentrations Stock solution, 4 dilutions, and blank n1 = n2 M1 . V1 = M2 . V2 Determine %T at lmax for each %T Calculate A A Plot A vs. conc (Beer’s Law plot) Slope = eb eb

Part 2 Concentrations 1 blank: pure DI water M0 = 0.00 M allura red 1 stock: 0.002% allura red M5 = 4×10-5 M 4 dilutions, each by ½: (4×10-5 M)(25.00 mL) = (M1)(50.00 mL) M1 = 2×10-5 M (2×10-5 M)(25.00 mL) = (M2)(50.00 mL) M2 = 1×10-5 M (1×10-5 M)(25.00 mL) = (M3)(50.00 mL) M3 = 5×10-6 M (5×10-6 M)(25.00 mL) = (M4)(50.00 mL) M4 = 2×10-6 M

Beer’s Law e,b constant c varied

Beer’s Law Plot y = 1.9 x

Beer’s Law Plot example Dilution Factor M (mol/L) T A = -logT 1.00 0.000 1/16 2.E-06 0.87 0.060 1/8 5.E-06 0.73 0.137 1/4 1.E-05 0.58 0.240 1/2 2.E-05 0.32 0.495 1 4.E-05 0.10 1.000 Stock M 4E-05 mol/L A = e×b×c   Slope = DA/Dc Here: e×b = 24943 A 1-cm cell: e = 24943 cm-1M-1

Part 3 Allura Red Concentration in Mouthwash Use 1:25 dilution Determine %T at lmax %T Calculate A A Measure b b Find concentration, c c

Part 3 Example Mouthwash trials (1:25 dilution): T A Mdilute   T A Mdilute Mconcentrated 0.68 0.167 7E-06 1.7E-04 0.70 0.155 6E-06 1.6E-04 0.65 0.187 8E-06 1.9E-04 0.69 0.161 Mdilute= A/(b×e) if cell length b is constant Average Mconcentrated = 1.7×10-4 M = 2×10-4 M

Report Abstract Data/Results Sample calculations including: Absorbance from transmittance Dilution Slope and extinction coefficient [Allura red]cuvette and [Allura red]mouthwash # allura red molecules in 1 mL mouthwash Discussion/review questions