Unit 2 Flashcards
Reflectance Photometry
- vitros analyzers use this principle
- amount of light reflected is proportional to amount of analyte
Absorption Spectroscopy
- the darkness of a colored solution quantitatively relates to the Molar concentration of the chromosphere molecules in solution
- darkness of the solution partly determines how much light that solution absorbs
Principle of absorption spectroscopy
- the number of light-absorbing molecules in solution are proportional to the amount of color of that solution (absorbance)
- absorbance is proportional to the analyte concentration (within certain limits)
6 parts of a spectrophotometer
1- light source (exciter lamp) 2- monochromator 3- primary exit slit 4- cuvette 5- photomultiplier tube (light detector) 6- readout device
Electro-magnetic radiation (EMR)
- exists in wave forms and photons
- wave form has 2 parts
——1- magnetic field (z)
——2- electric field (y)
Electromagnetic Spectrum (nm from crest-crest)
- visible spectrum from 380-725nm
- the smaller the wavelength(nm), the greater the light energy
- we do a lot of assays at 340nm because NADH absorbs the light but NAD+ does not
Light spectrum and their colors
>/= 725nm= infrared, low energy, not visible 600-725nm= oranges & reds 580-600nm= yellows 500-580nm= greens 440-500nm= blues 380-440nm= violets <380nm= ultraviolets, not visible
Light source- types of exciter lamps
- Tungsten-halide
——most common, emits wavelengths from 335-750nm - Hydrogen, Deuterium, or Mercury Arc Lamps
——limited UV use, emits wavelengths from 180-380nm
Types of monochromators
- prisms (very inefficient)
- diffraction gratings (most efficient)
- interference filters (most common)
Prisms
- rarely used
- resolving power= B(dn/d¥)
Diffraction grating
Most efficient
Interference filter
- most commonly used
- not true monochromators because they don’t break up white light into a spectrum of colors
Spectrophotometer vs. colorimeter
- spectrophotometer uses a true monochromator
- colorimeter uses an interference filter
- many times the terms are used interchangeably
Wavelength accuracy
- now that we have a light source and the wavelength is set, is it correct?
- test and document with:
—-holmium oxide: narrow bandwidth
—-didymium: broad bandwidth
Bandpass
The range of light waves that pass through the primary slit
Spectral bandwidth
- the range of the bandpass that has enough energy to potentially interact with the test solution
Also known as the Full Width Half Maximum (FWHM)
Spectral bandwidth documentation (using Holmium Oxide filter)
- find the peak at 361 no, record the absorbance (maybe 0.300A)
- move the wavelength to the left until the absorbance is half of the starting absorbance (0.150A) and record the nm reading
- repeat on other side of curve
- pretend nm reading on left was 357nm and right was 365nm
- difference between them=8nm= spectral bandwidth
Cuvettes
- made of optically perfect quartz glass or sometimes a good grade of plastic
- round cuvettes: inferior, curvature will scatter light
- square cuvettes: superior to round, lights hits 90* angle
Detection devices
Photomultiplier tube
- light energy from the sample hits the cathode and bounces back and forth, loses electrons along the way until it releases an electron to the anode at the other end of the tube
Photometric accuracy
-NBS (NIST) transmittance standards
—colored solutions with known absorbances e.g. nickel sulfate, ammonium molybdate
-NBS (NIST) SRM-930 & 931 series of neutral gray filters
—each filter has a known absorbance
—no colors to fade, just gray screens
Photometric linearity (example)
- remove cuvettes and set readout to 0.000A
- place 0.400 A filter in place of cuvettes and document absorbance reading
- remove filter, readjust air to 0.400 A, replace filter, document absorbance
- continue to 1.600 A
Stray light
- light not passing through the cuvette, but hitting the photodetector
- always causes falsely low absorbance readings
- affects high absorbances more than low
- causes of stray light: defects in cuvette, dust in bowels of instrument, degrading diffraction grating
- documented using cut-off filters at 380 & 680nm
- 680 filter will not transmit light above 680nm
—remove cuvette, dial in 700nm, put filter where cuvette goes
—if any light hits the detector, it’s stray light
Spectrophotometer QC- daily/weekly
Daily
- document wavelength calibration accuracy using Holmium Oxide or Didymium (1st)
- document photometric accuracy using SRM-930,931 neutral gray filters with a known absorbance reading
Weekly - must also document 1- photometric linearity 2- stray light 3- spectral bandwidth
Readout devices
- LED/LCD meter
- analog meter
- strip chart recorder
- printer