Spectrophotometry

Rob Morris1

1 Ocean Optics, Dunedin, Florida
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 2.1
DOI:  10.1002/9780470089941.et0201s11
Online Posting Date:  November, 2015
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Abstract

Spectroscopy is the study of the interaction of light with matter. By observing how light interacts with matter—through reflection, refraction, elastic scattering, absorbance, inelastic scattering, and emission—it is possible to (1) identify the wavelengths of light that interact with atoms and molecules, and (2) quantify the amount of light being absorbed, reflected, scattered, or emitted at a particular wavelength. This unit describes the background and basic principles of spectrophotometry (the study of the reflection or transmission properties of a substance as a function of wavelength), in particular, absorbance spectrophotometry. Also included is discussion of key spectrophotometer components and their functions, the relationship between absorbance and transmittance, experimental considerations, and the steps necessary in preparing a standard curve for determining absorbance concentration. © 2015 by John Wiley & Sons, Inc.

Keywords: light; matter; spectroscopy; absorbance; fluorescence; reflection; refraction; scattering; emission; solutions

     
 
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Table of Contents

  • Overview and Principles
  • Components of a Spectrophotometer
  • How a Spectrophotometer Works
  • Strategic Questions
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Preparation of a Standard Curve for Dipicolinic Acid (DPA)
  • Basic Protocol 2: Determination of a Standard Curve for Dipicolinic Acid (DPA) and Measurement of DPA Concentration in Unknown Sample
  • Support Protocol 1: Optimizing Spectrophotometer Settings
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of a Standard Curve for Dipicolinic Acid (DPA)

  Materials
  • Sodium hydroxide (NaOH)
  • Calcium chloride (CaCl 2)
  • Dipicolinic acid (DPA, Sigma cat. no. D‐0759)
  • 1‐liter volumetric flask(s) with stopper
  • Weighing paper or weighing boat
  • Balance (unit 1.2; Guzy, )
  • Pipets
NOTE: There are dozens of suppliers of DPA, including Sigma, Spectrum Chemicals and Laboratory Products, and Alfa Aesar. Labware is available from a number of suppliers. In particular, Ocean Optics is a good source for cuvettes, cells, and reference materials for spectroscopy.

Basic Protocol 2: Determination of a Standard Curve for Dipicolinic Acid (DPA) and Measurement of DPA Concentration in Unknown Sample

  Materials
  • Reference solution
  • Sample solutions
  • Spectrophotometer
  • Light source
  • Spectroscopy software
  • Optical fiber
  • Sample compartment (cuvette holder)
  • 1‐cm cuvettes
NOTE: There are hundreds of models of spectrophotometers (a good directory of spectrometer companies is available at http://www.spectroscopynow.com). Details of their use will vary. To optimize results, consult the technical documentation provided by the manufacturer. The following lists some general guidelines on spectrophotometer operation, as well as details for use of an Ocean Optics Flame Spectrometer equipped with a DH‐2000 Deuterium Tungsten Halogen Light Source, fiber optics, and a 1‐cm pathlength CUV‐UV cuvette holder (sample compartment).
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Figures

Videos

Literature Cited

Literature Cited
  Brown, L., LeMay, H.E., and Bursten, B.E. 1997. General Chemistry: The Central Science, pp. 921‐924. Prentice Hall, Englewood Cliffs, N.J.
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  Pace, C.N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. 1995. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4:2411‐2423. doi: 10.1002/pro.5560041120.
  Samuels, A.C., DeLucia, F.C. Jr., McNesby, K.L., and Miziolek, A.J. 2003. Laser‐induced breakdown spectroscopy of bacterial spores, molds, pollens, and protein: Initial studies of discrimination potential. Appl. Opt. 42:6205‐6209. doi: 10.1364/AO.42.006205.
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