Analytical Instrumentation Principles (Unit 1- Part 1)

Cards

A red block reflects what color? Red
What is transmittance? The proportion of light that penetrates the solution
What color of light is a red soluntion of liquid transmitting? Red light
What does the darkness of a solution quantitatively relate to? The molar concentration of the chromaphore molecules in solution
What can the darkness of a solution be called? The absorbance of light by that solution
What are the number of light absorbing molecules in a solution proportional to? The amount of color of that solution (absorbance)
What is absorbance proportional to? analyte concentration (within certain limits)
What are the 6 parts of a spectrophotometer? light source, monochromator, primary exit slit, cuvette, photomultiplier tube (light detector), readout device
What are the 2 main types of exciter lamps? 1. Tungsten Halide 2. Hydrogen, Deuterium, or Mercury Arc Lamps
What is the most common exciter lamp? What wavelengths does it emit? Tungsten Halide; 325-750nm
Which exciter lamp has limeted UV use? What wavelenths of light does it emit? Arc Lamps; 180nm to 380nm
What are the three types of monochromators? Which one is the most efficient? Least efficient? prisms (very inefficient), diffraction gratings (most efficient) , interference filters
Which monochromator is not a true monochromator and why? interference filters bc they don't really break up white light into a spectrum of colors, but they are the most commonly used on current instrumentation
What are cuvettes made of? optically perfect quartz glass or a good grade of plastic
What are the two types of cuvettes? Which is superior to the other and why? Round and square cuvettes. Square cuvettes are better bc they don't inherently scatter light. Unless light hits a round cuvette dead on, some of it will be scattered.
What is the difference between a micro vs a macro cuvette? Micro cuvettes hold less volume than macro cuvettes. This effects the path length.
Describe briefly how a photomultiplier tube works. Light from the sample travels down the photomultiplier tube going from the cathode to the anode. This light is them amplified.
what are the 4 read out devices of a spectrophotometer? 1. Led or LCD meter 2. analog meter 3. strip chart recorder 4. printer
What is the difference between a spectrophotomer and a colorimeter? A spectrophotometer uses a true monochromator (prism or diffraction grating). A colorimeter does not use a true monochromator. Instead it uses an interference filter.
What does beers law state? 1. The number of light-absorbing molecule in solution are proportional to the amount of color of that solution(absorbance). 2. Absorbance is proportional to the analyte concentration within certain limits.
What is the formula for beers law? A=Elc
What does each variable in beers law represent? A= Absorbance at a specific wavelength (no units), E= molar absorptivity constant, l= cuvette path length in cm, c= moles/liter of light-absorbing product
If the molar absorptivity constant is known for a given analyte, do you need a standard? No, the anaylte can be measured in an unknown specimen without using a standard
Describe the beers law porpotionality calculation. 1. Assume that the method is linear (actually does follow beers law) 2. then use the equation ((abs. unknown)/(absorbance std))*concentration of the standard = concentration of the unknown note: you just have to include the measurements of one standard with the controls and patients
Explain why the shortcut method works. It works bc beers law says that the ratio of the two absorbances is equal to the ratio of the two concentrations
What are the 4 steps to making an absorbance cure? run at least 6 standards, plot the absorbance vs. concentration of each standard, look to see if the method follows beers law, if not linear, then the graph can be used as a calibration curve
What is the difference between absorbance and % T? absorbance is the amount of Po not hitting the detector. % T is the % of P1 hitting the detector.
What equation do you use to convert %T to absorbance? absorbance= 2.0-log%T
zero absorbance =__________ 100 %T
1.0 Absorbance =___________ 10 %T
How do you find the reportable or dynamic range? perform a linearity study and plot absorbance vs. concentration. The straight line is the range of analyte concentration that follows beers law. If the lower limit is 20mg/dL, then anything lower than 20, must be reported as less than 20mg/dL. If the upper limit is 750, then any sample's value greater than 750, must be diluted and then have the result multiplied by the dilution factor
When must a linear range be studied and identified? must be done for every new test and again when changing lot # of reagents, and/or calibrating the instrument. It mst also be done every 6 months if nothing has changed.
How do you choose the best wavelength for an assay? put the reaction mixture (reagent + specimen) containing the product to be measured in a cuvette. Zero the spectrophotometer on water blank. Scan wavelength from 400-700nm. The complimentary wavelenth gives the highest absorbance curve (reading).
What is considered one wavelength? crest to crest or trough to trough
The smaller the nm, the _______ greater the light energy
Which has higher energy, 340nm or 700nm? 340nm
Why do we do many assays at 340nm? because NADH absorbs the light, but NAD+ does not
Describe the wavelengths of colors in the electromagnetic spectrum. 600-725 oranges and reds, 580-600 yellows, 500-580 greens, 440-500 blues, 380- 440 violets
What is above 725 nm and what is below 380nm? above 725nm is infared (low energy and not visible), below 380nm = ultra-violets- not visible
What are the two types of blanks and when should you use them? water blanks and reagent blanks. Water blanks are used to zero the spectrophotometer when the reagent does not absorb light. A reagent blank is used to zero the spectrophotometer when the reagent does absorb light.
Why would a specimen have color? Why is this bad? due to hemolysis, lipemia, or icterus; This will cause falsely elevated test values because the color of the specimen also absorbs light
What do you use to get valid results from a patient sample if the specimen has color? Use a specimen blank. Take an absorbance reading on the specimen and then take an absorbance reading on the specimen blank. Deduct the absorbance from the original absorbance reading
If you assay 2mL of color reagent and 50uL of specimen, what will your blank have? 2mL of water and 50uL of specimen
Explain how to blank out the absorbance from the specimen alone. zero on a water blank, then read the absorbance of the specimen blank. Subtract the specimen blank's absorbance from that patient's original absorbance of the test.
When do you need specimen blanks? hemolysis, bilirubin, or turbidity
From what range does hymolysis absorb light? 400-600nm, so below 600nm Hgb will interfere, but above 600 nm there will be no effect
From what range does bilirubin absorb light? 400-530nm, so below 530nm Bilirubin will interfere and above 530nm there will be no effect