CHEM-C127 Bioanalytical Spectroscopy Notes
CHEM-C127 Bioanalytical Spectroscopy Notes Chemistry C127
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This 2 page Class Notes was uploaded by Kathryn Brinser on Thursday March 10, 2016. The Class Notes belongs to Chemistry C127 at Indiana University taught by Dr. Norman Dean in Fall 2016. Since its upload, it has received 814 views. For similar materials see Principles of Chemistry and Biochem I Lab in Chemistry at Indiana University.
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Date Created: 03/10/16
C127 Lecture 8 Notes- Bioanalytical Spectroscopy 3-10-16 Learning Goals o Learn principles of UV-Visible absorption spectroscopy o Learn how absorption of light is related to concentration (Beer’s Law) o Use spectroscopy to determine concentration of a chemical (whey protein) in milk without precipitating anything Add stain/dye to protein to cause color change; whey is colorless by itself o For this dye, has different color in polar surroundings than nonpolar Safety o Wear gloves Protein standard has small amount of toxic chemical (sodium azide) Bradford reagent contains phosphoric acid, which is corrosive, and will stain skin blue o Ethanol flammable- no open flames Spectroscopy- study of how light interacts with atoms/molecules o Chemists “talk” to molecules using light o Provides quantitative measurement of amount of chemical present in solution o Different wavelengths correspond to different colors, energies, wavelengths, and frequencies o Different amplitudes correspond to different brightness (bigger = brighter) o Recall that interaction of light with molecules is quantized- photons contain discrete amounts of energy depending on wavelength About Spectrometers o Light goes from source (bulb) → slit in barrier (directs light) → monochromator (lets us pick 1 wavelength to pass through sample) → sample (absorbs some light) → detector (generates small amount of voltage when light hits it; looks for any photons of any wavelength) → computer/electronics in spectrometer o Measures transmittance (T)- ratio of intensities of light at detector with and without sample present ???? ???? = ????0 where ???? = intensity with sample0 ???? = intensity without anything o Absorbance (A)- related to transmittance, more favored by chemists ???? = −log ???? 10 Beer’s Law- relates absorbance of light to concentration of chemical compound o ???? = ???????????? ???? = absorbance ???? = absorptivity (“chance” that light will be absorbed; light that will be absorbed more readily will have high value, light that is less likely will have low value; tells how strongly a sample absorbs a certain wavelength) ???? = path length (how much sample, usually in mm, the light is passing through) ???? = concentration of absorbing compound o Direct relationship- as concentration increases, so does absorption More solution light has to pass through = higher absorption of light Plotting ???? vs. ???? gets straight line with slope ???????? and y-intercept 0 Ex. Different wavelengths of light have different ???? values o For particular molecule: o Starts absorbing at 300 nm, maximum around 425 nm, stops around 580 o Concentration/path length constant o Only absorptivity changes, causing absorbance to change Ex. Plotting a known concentration vs. Absorbance (calibration graph): o Can set y-intercept at 0 o Should give straight line, Excel gives equation o Once we have this graph, can put other samples in spectrometer, measure absorbance, and calculate concentration