Review_of_Analytical_Methods_1

Review_of_Analytical_Methods_1 - Review of Analytical...

Info iconThis preview shows pages 1–16. Sign up to view the full content.

View Full Document Right Arrow Icon
Review of Analytical Methods Part 1: Spectrophotometry Roger L. Bertholf, Ph.D. Associate Professor of Pathology University of Florida Health Science Center/Jacksonville
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Analytical methods used in clinical chemistry Spectrophotometry Electrochemistry Immunochemistry Other Osmometry Chromatography Electrophoresis
Background image of page 2
Introduction to spectrophotometry Involves interaction of electromagnetic radiation with matter For laboratory application, typically involves radiation in the ultraviolet and visible regions of the spectrum. Absorbance of electromagnetic radiation is quantitative.
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Electromagnetic radiation E H A Wavelength ( λ ) Velocity = c
Background image of page 4
Wavelength, frequency, and energy λ ν hc h E = = E = energy h = Plank’s constant ν = frequency c = speed of light λ = wavelength
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
The Electromagnetic Spectrum γ x-ray UV visible IR μ Rf 10-11 10-9 10-6 10-5 10-4 10-2 102 Wavelength ( λ , cm) Frequency ( ν , Hz) 108 1012 1014 1015 1016 1019 1021 Nuclear Inner shell electrons Outer shell electrons Molecular vibrations Molecular rotation Nuclear Spin
Background image of page 6
Visible spectrum 390 780 450 520 590 620 Wavelength (nm) IR UV Increasing Energy Increasing Wavelength “Red-Orange-Yellow-Green-Blue”
Background image of page 7

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Molecular orbital energies σ or π molecular orbital s or p atomic orbital σ * or π * molecular orbital non-bonding orbital σ→ n π→ n n →σ * n →π * σ→σ * π→π * Energy
Background image of page 8
Molecular electronic energy transitions E 0 E 4 E 3 E 2 E 1 Singlet Triplet A VR F IC P 10-6-10-9 sec 10-4-10 sec
Background image of page 9

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Absorption of EM radiation I 0 (radiant intensity) I (transmitted intensity) abc A bc k T kN I I dn k I dI kI dn dI I I N = - = - = - = - = ; log ; ln ; ; 0 0 0 0
Background image of page 10
Manipulation of Beer’s Law ) 2 ( 10 % , ) log(% 2 ) log(% 2 ) log(% ) 100 log( % 100 log % 100 log 1 log log A T and T A T T T T T T abc A - = - = - = - = = = - = = Hence, 50% transmittance results in an absorbance of 0.301, and an absorbance of 2.0 corresponds to 1% transmittance
Background image of page 11

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Absorbance Error ( dA/A) 0.0 2.0 Beer’s Law error in measurement 0.434
Background image of page 12
Design of spectrometric methods The analyte absorbs at a unique wavelength (not very common) The analyte reacts with a reagent to produce an adduct that absorbs at a unique wavelength (a chromophore) The analyte is involved in a reaction that produces a chromophore
Background image of page 13

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Measuring total protein All proteins are composed of 20 (or so) amino acids. There are several analytical methods for measuring proteins: Kjeldahl’s method (reference) Direct photometry Folin-Ciocalteu (Lowery) method Dye-binding methods (Amido black; Coomassie Brilliant Blue; Silver) Precipitation with sulfosalicylic acid or trichloracetic acid (TCA) Biuret method
Background image of page 14
Kjeldahl’s method Specimen Hot H 2 SO 4 digestion Correction for non-protein nitrogen NH 4 + Titration or Nessler’s reagent (KI/HgCl 2 /KOH) Protein nitrogen Total protein Multiply by 6.25 (100%/16%)
Background image of page 15

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 16
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 09/18/2011 for the course MED 6566 taught by Professor Staff during the Summer '11 term at University of Florida.

Page1 / 74

Review_of_Analytical_Methods_1 - Review of Analytical...

This preview shows document pages 1 - 16. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online