Light Measuring Systems Flashcards
Absorbance equation
2-log%T
Absorbance
directly proportional to the concentration of the absorbing species if Beer-Lambert law is followed
Absorptivity
the absorbance of an analyte dived by the product of the concentration of a substance and the sample path length
Allen Correction
multi-chromatic analysis of a reaction to correct for background absorbance. Two wavelengths in addition to the absorbance maximum are monitored to subtract the average background absorbance
Bandpass
the range of wavelengths that reach the exit slit of a monochromator usually referred to as the range of wavelengths transmitted at a point equal to half the peak intensity transmitted
Beer-Lambert Law
an equation that states the concentration of a substance is directly proportional to the amount of radiant energy absorbed
Bichromatic analysis
monitors a reaction at two wavelengths. It is used to correct background colour
Blank
a solution that consists of all components including solvents and solutes except for the compound of interest measured. This solution is used to set Io (original light intesity)
Chemical Interferent
a compound that ither produces an endogenous colour or interferes directly with the reaction or process that is being monitored
Chromogen
a coloured compound
Cuvette
the receptacle in a spectrophotometer in which the sample is held
Endpoint Reaction
a reaction is monitored at a single time point when the reaction is virtually complete
Grating
an optical device consisting of a reflecting ruled surface that disperses polychromatic light into a uniform continuous spectrum
Hemolysis
the breakage of red blood cells either in vitro or in vivo, gives plasma a red colour
Icterus
refers to the orange colour imparted to a sample because of the presence of bilirubin
Interferent
any chemical or physical phenomenon that interferes in or disrupts a reaction or process
Kinetic Analysis
analysis in which change in the monitored parameter over time is related to concentration measurements are usually made very early in the reaction period
Light Scattering
the interaction of light with particles that cause light to bend away from its original path
Line spectrum
a discontinuous emission spectrum of elements in which emitted light bands cover very narrow (0.1nm) range of wavelengths
Lipemia
the presence of lipid particles (usually VLDL) in a sample it can five the sample a turbid appearance
Molar Absorptivity
absorbance of light at a specific wavelength divided by the product of the concentration of moles per litre and the sample path in cm expressed as L/mol cm
Monochromatic Light
one colour in practice this refers to radiant energy composed of a very narrow range of wavelengths
Photodetector
a device that responds to light usually in a manner proportional to the number of photons striking its light-sensitive surface commonly a current that is proportional to the incident light intensity is generated
Polychromatic Light
is composed of many colours, usually referred to as white light
Reagent Blank
reaction mixture minus sample, used to subtract endogenous reagent colour from the absorbance of the complete reaction
Reflectance Spectrophotometry
a quantitative spectrophotometric technique in which light reflected off a surface of a colorimetric reaction is used to measure the amount of the reaction product
Sample Blank
sample plus diluent used to correct absorbance of a complete reaction mixture for endogenous sample colour
Spectral Interference
interference observed when a compound causes a response in the spectrophotometer similar to that of the analyte of interest
Spectrophotometer
an instrument that measures light intensity it is composed of a radiant energy source, entrance slit, monochromator, exit slit, cuvette holder, detector and read-out device
Stray Light
radiant energy reaching the detector that consists of wavelengths other than those defined by the filter or monochromator
Transmitted Light
the portion of radiant energy that passes through an object
Turbidity
scatter of light in a liquid that contains suspended particles, lipemia in a sample can make a sample turbid
Ultraviolet Radiation
the regions of the electromagnetic spectrum from 220-360 nm
Visible Light
the radiant energy in the electromagnetic spectrum visible to the human eye approximately 390-780 nm
Window
a term used to denote a specific time during which reactions are monitored, a phenomenon can be observe or a procedure can be initiated
Beer-Lambert Law Equation
A=Ebc
What are the conditions of the Beer-Lambert Law
- the incident light is monochromatic
- the solvent absorption is insignificant to the analyte of interest
- the analyte must follow Beer-Lambert Law
- the standard and unknown must be measured in the same cell
- the concentration range that obeys Beer-Lambert law must be established
- the absorbance of the unknown must be less than the standard
- an optical interferent is not present
- a chemical reaction does not occur between the molecule of interest and another molecule
- the sides of the cuvette are parallel
- there is no stray light
%T Equation
%T=[Is/Io]x100%
What are possible errors in spectrophotometry
- low %T causes greater imprecision when transmitted light is measured, this results in substantial error when A is determined due to the logarithmic scale
- high %T causes absorbance to be relatively large to the absolute value of absorbance
- at high absorbance the detection system may have a limited capacity to measure slight differences
- spectral interference may occur
When is hemoglobin a significant interference
500-600 nm
When is bilirubin a significant interference
similar to hemolysis (500-600 nm)
How is bichromatic analysis done
a reading is taken at the maximum absorbance wavelength and a second measurement is taken near the wavelength where the analyte minimally absorbs light. The curve is then based on A1-A2 or A1/A2
How is the allen correction done
it uses absorbance readings at three wavelengths, Amax and two additional wavelengths equal distance from Amax to calculate the corrected absorbance the sum of the two peripheral absorbance measurements is divided and subtracted from the maximum absorbance
What are sources of visible light
tungsten filament and tungsten halogen
What are sources of UV light
hydrogren/deuterium and mercury vapour
What are sources of visible and UV light
light emitting diodes and lasers
What are borosilicate glass cuvettes used for
measurements between 320-950 nm
What are silica quartz cuvettes used for
measurements less than 320 nm
What are plastic cuvettes used for
all wavelengths
How is wavelength accuracy checked
using standard absorbing solutions of filters with absorbance maxima of known wavelengths
How can the linearity of the detector be checked
using solid glass filters or by using a solution known to follow the Beer-Lambert law
What is stray light
a radiant energy that reaches the detector at wavelengths other than those indicated by the exit slit of the monochromator
How can the linearity of the detector be checked
using solid glass filters or by using a solution known to follow the Beer-Lambert law
What is dry slide chemistry
a way of using reflectance spectrometry to quantify colorimetric reactions, patient sample is applied through an opening in the housing onto a spreading layer as the sample moves through the slide it may pass through one or more inner layers that alter the sample.
