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Discover the use of naturally colored molecules, synthetic compounds, and theory of color in chemistry. Learn about spectrophotometry, absorption, and the perception of colors. Explore the origins of color, measurement techniques, and impacts on absorbance. Gain insights into the complexities of spectrometry and the role of light sources. Enhance your knowledge of colored tracers and their applications in scientific research.
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Chromogen Tracers • Use of colored compounds as tracers includes at least 4 primary approaches: • Use of naturally colored molecules as endogenous indices of biological status • Attachment of colored molecules to compounds of interest to make them visible • Chemical reactions that produce dyes or change their colors • Conjugation of naturally colored molecules to binder- ligand components
Naturally Colored Molecules Proteins: Heme proteins(hemoglobin, myoglobin, cytochrome C, hemocyanin) Redox & photoproteins(phytochromes, phycoerythrin, phycocyanin, chlorophylls, phycobiliproteins, rhodopsin, ferredoxins) Pigments: Melanins(black, brown) Xyanthophylls, flavinoids(yellow) Cyanins(blue),anthocyanins(red – blue) Carotinoids, lycopene(yellow, red) Minerals, metal ions, metallic chelates
Synthetic Colored Molecules Metallic ions & chelates Aromatics & polyaromatics (especially heterocycles; quinoids, terpenes; extended π – bonded structures) Crystalline arrays
Theory of Color The origins of color in minerals: http://www.minsocam.org/msa/collectors_corner/arc/color.htm Origins of color : http://www.chemistry.nmsu.edu/studntres/chem435/Lab5/ Color perception & mixing: http://www.handprint.com/HP/WCL/color14.html; http://www.csun.edu/~hchum001/bookcase/light/spectrum.html
Measurement of Color Color indicates effective absorbance of electromagnetic radiation in or near the range of visual perception (~300 - 800 nm). If the endpoint is the presence of color, e.g., qualitative staining of a microscopic specimen, it can be directly observed or recorded on film or by digital means. If a quantitative test is done, color is usually measured with a spectrophotometer.
Spectrometry The Joy of Visual Perception: http://www.yorku.ca/eye/spectru.htm
Spectrometry http://www.irvingcrowley.com/cls/back.htm http://bouman.chem.georgetown.edu/S02/lect10/lect10.htm Single – beam Spectrophotometer Double – beam Spectrophotometer
Atomic Absorption, Flame Photometry www.thebritishmuseum.ac.uk/.../ sr-tech-aas.html
Spectrometry ULTRAVIOLET SPECTROSCOPY, ORIGINS OF COLOR: http://www.uwplatt.edu/~sundin/354-7/l547-38a.htm The Joy of Visual Perception: http://www.yorku.ca/eye/spectru.htm
Spectrometry: Basic Equations %Transmittance = T = (I x100)/I0 where I = observed intensity & I0 = incident intensity T ranges from 0 – 100%; no radiation absorption = 100%; total radiation absorption = 0% Absorbance = A = - Log10(T) = Log10 (1/T) = Log10 (I0/I) A, or OD, optical density, ranges from 0 to ∞; no radiation absorption = 0; total radiation absorption =∞ A is specific for wavelength, compound, solvent, pH, & temperature
Spectrometry: Beer/Lambert Law A = ελx b c where ελx = A for a 1 M/L solution of specific compound x in a specific solvent, pH, & temperature at wavelength λ; b is the pathlength of the observation cuvette or cell (usually in cm); & c is the concentration of the solution of x being measured (in M/L) If ελx& b are constant across a series of samples, A is proportional to c & may be used to estimate c in unknowns by comparison to standards or from published values of ελxif measurements are done on purified material in a known solvent. www.rx.uga.edu/.../phrm4120/ spectrophotometry.htm
Impacts of pH on Absorbance Titration of phenol www.biomedcentral.com/1472-6793/ 2/5/figure/F3 wps.prenhall.com/.../724/ 741576/chapter_08.html
Impacts of Temperature on Absorbance Melting DNA Thermosensing polymer wps.prenhall.com/.../724/ 741576/chapter_08.html reaktor.ch.pw.edu.pl/~dybko/ csrg/patents/thermo/
Impacts of Solvent on Absorbance www.photobiology.com/photoiupac2000/ karapire/
Complications for Spectrophotometry • Light scattering, A is not directly proportional to c • Can be used: nephelometry (scattered light) & turbidimetry (decrease in absorbance) • Photochemical reactions, the system is dynamic • Quenching, multiple compounds absorb light energy & alter spectral intensities acting on other compounds present • Nonlinearity or spectral impurity of light sources, illumination intensity varies by wavelength
www.physics.montana.edu/.../ emissionspectra.html Light Source Intensity Variation by Wavelength www.pti-nj.com/ obb_spectra.html Other examples:www.firstrays.com/ spectra.htm
Lasers: Coherent Light Sources Laser Theory: www.physics.ubc.ca/.../p420_95/ mark/htheory.html www1.union.edu/.../ laser_theory.htm webphysics.davidson.edu/.../ laserlab/theory.htm people.deas.harvard.edu/.../ ls2_unit_6.html www.people.fas.harvard.edu/.../ chemlaser.htm Infrared & Optical Masers:www.coseti.org/ schawlow.htm www.phy.davidson.edu/.../ diode_laser.htm
Histochemical Staining www.udel.edu/.../illuspage/ lec1/lec1.htm Histochemical Society: www.histochem.org/ main.htm; www.lab.anhb.uwa.edu.au/mb140/ MoreAbout/stains.ht
Histochemical Staining (cont.) www.udel.edu/.../illuspage/ lec1/lec1.htm
Histochemical Staining (cont. 2) www.udel.edu/.../illuspage/ lec1/lec1.htm
Chemistry of H&E Staining Eosin www.gspchem.com/ eosin.htm www.histology.to/ hemx.html Hematoxylin
Link catalogs of histological microscopy images: turing.bear.uncw.edu/.../ vertebrate.asp www2.uerj.br/~micron/atlas/ atlasenglish/lymph/