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
Beer’s Law equation
A=abc
A- absorbance at a specific wavelength (in a spectrophotometer)
a- molar absorptivity constant
b- cuvette light path in cm
c- Moles/Liter of light-absorbing product
Beer’s Law- explained
- the number of light-absorbing molecules in a solution are proportional to the amount of color of that solution (absorbance)
- absorbance is proportional to the analyte concentration (within certain limits)
- if he molar absorptivity constant is known for a given analyte, then that analyte can be measured in an unknown specimen without using a standard
- there are many applications in industry for this
- we use standards in the clinical lab
Beer’s Law proportionality calculation
- assume that the method is linear (actually follows Beer’s Law)
(Abs unknown/Abs standard)x conc standard= concentration of unknown
- you just have to include the measurement of one standard with the controls and patients
- it works because Beer’s Law says:
(Abs unknown/Abs standard)= (Conc unknown/Conc standard)
- the ratio of the two absorbances is equal to the ratio of the two concentrations
- do not use calculation if it does not follow Beer’s Law
Analytical Measurement Range (AMR)
- AKA linear, reportable, or dynamic range
- perform linearity study and plot absorbance vs concentration
- straight line is the range of analyte concentration that follows Beer’s Law with at least 90% accuracy
- must be verified every 6 months if nothing has changed
Absorbance curves
- run at least 6 standards
- plot absorbance vs concentration of each standard
- look to see if method follows Beer’s Law (linear?)
- if not linear then, this graph can be used as a calibration curve
Absorbance vs %Transmittance
%T=% of Pi hitting the detector
Absorbance=the amount of Po NOT hitting the detector
Conversions
%T to Absorbance
Abs=2.000-Log(%T)
Conversions
Absorbance to %T
Log(%T)= 2.000- Abs
- 000 Abs = ? %T
1. 000 Abs = ? %T
100%T
10%T
Absorbance curves to choose best wavelength for assay
- our reaction mixture (reagent+specimen) containing product to be measured in a cuvette
- zero spectrophotometer on water
- scan wavelength from 400-700nm
- complimentary wavelength gives the highest absorbance curve(reading)
Zeroing the Spectrophotometer (blanks)
- blank: solution used to tare or zero the absorbance reading
- water blank: used to zero the spectrophotometer when the reagent does not absorb light
- reagent blank: used to zero the spectrophotometer when the reagent does absorb light
Reagent Blanks
- reagent blanks are done by zeroing the instrument with the reagent in the cuvette
- now the instrument will subtract the absorbance of the reagent from every solution that you measure
- sometimes the reagent is too dark to zero the spectrophotometer on the reagent, so you need to do “manual” reagent blanking
Manual reagent blanking
- zero using a water blank
- place the reagent blank in the spectrophotometer and see how much light it absorbs by itself
—> if your sample uses 1mL reagent with 25uL patient sample, mimic formula for the blank but use water in place of patient sample - manually subtract the absorbance value of he reagent blank from all tube results (standards, patients) for the true, adjusted values
Strange blanks
- specimen blanks: used when a specimen has color due to hemolysis, lipemia, icterus
- this will falsely elevate test values because the color of the specimen also absorbs light