THE APPLICATION NOTEBOOK – MARCH 2007 ADVERTISING SUPPLEMENT Molecular Spectroscopy | 21

Quantitative Absorbance Spectrophotometry

Ocean Optics, Inc.

bsorbance spectrophotometry can be used as a qualitative Table I: Absorption wavelength bands of assorted chromophores tool to identify or “fingerprint” substances, and as a Maximum Absorbance A Chromophore quantitative tool to measure the concentration of a colored Absorption (in nm) Intensity 251 66,000 substance (chromophore) in a transparent solvent. In some Phenanthrene situations, the absolute absorbance, or extinction coefficient 292 14,000 272 180,000 of the unknown substance is desired. In other situations, the Naphthacene 473 12,500 concentration is related empirically to standard solutions of 310 300,000 Pentacene known concentration. In either case, the derivation of ab- 585 12,000 sorbance is called the Beer–Lambert Law (1,2,3). 174 80,000 Pyridine 195 6000 Experimental Conditions 257 1700 Absorbance is measured in a spectrophotometer by passing a 227 37,000 collimated beam of light at wavelength ␭ through a plane par- Quinoline 270 3600 allel slab of material that is normal to the beam (4). For liq- 314 2750 uids, the sample is held in a cuvette. The light energy incident 218 80,000 Isoquinoline 266 4000 on the sample (I0) is more intense than the light that makes it through the sample (I) because some of the energy is absorbed 317 3500 by the molecules in the sample. ple. The definition of the blank sample thus becomes an im- portant qualifier of the measurement. Results You can account for the absorbance of a blank solution ei- Transmission (T), expressed as a percentage, is the ratio: ther by measuring it and subtracting it from the sample ab- T = I/ I0 sorbance measurements, or by setting the instrument to start Transmission ranges from 0% for an opaque sample to 100% at 100%T or 0 absorbance. This is much like setting the tare on for a transparent sample. If there are absorbing molecules in a balance. the optical path, the transmission will be <100%. The number Many factors can affect the validity of Beer’s Law. It is usual of molecules that interact with the light will depend on the to check for the linearity of Beer’s Law for a chromophore (see pathlength (l) and the concentration of the molecules (c). The sampling of chromophores in Table I) by measuring the ab- attenuation will also depend on the ability of the molecule to sorbance of a series of standards. This “calibration” can also absorb light at that wavelength, expressed as the extinction co- remove errors in the experiment, the equipment, and the efficient or molar absorptivity ⑀. batch of reagents (such as cuvettes of unknown pathlength). -⑀lc T = I/ I0 = e ␭ Absorbance (A ) is the negative log10 of transmission. This References mathematical transformation is used to make the relationship (1) T.L. Brown, H.E. LeMay, and B.E. Bursten, General Chemistry: The Cen- between concentration and absorbance linear: tral Science, Prentice Hall: Englewood Cliffs, NJ, 1997, pgs. 921–924. ⑀ A ␭ = -log (I/I0) = cl (2) D. Ebbing, Experiments in General Chemistry, Houghton Mifflin Com- Absorbance ranges from 0 for perfectly transparent samples pany, Boston, 1987. (T = 100%) to infinity for perfectly opaque samples (T = 0%). (3) D. A. Skoog and D. M. West, Fundamentals of Analytical Chemistry, The value of ⑀ depends on the units of c. Typically, c is in mo- Saunders College Publishing: Philadelphia, PA, 1982, pgs. 790–791. larity (moles literϪ1), l is measured in cm, and ⑀ has units of (4) Qhobosheane, M., Introduction to Spectroscopy in the Teaching Lab absorbance (cmϪ1 liters moleϪ1). Using Ocean Optics Spectrometers, pp. 13–14 (2006).

Conclusions In an absorbance experiment, light is attenuated not only by the chromophore, but also by reflections from the interface between air and the sample and between the sample and the Ocean Optics, Inc. cuvette, and by absorbance by the solvent. These factors can be 830 Douglas Ave., Dunedin, FL 34698 quantified separately, but are often removed by defining I as 0 Tel. (727) 733-2447, Fax (727) 733-3962 the light passing through a sample “blank” or reference sam- [email protected], www.oceanoptics.com

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