Week 2 Flashcards
what happens to radiant energy (light) when it passes through an object
-it is reflected, absorbed and transmitted
-signal is directly or inversely proportional to analyte concentration
what is constructive interference
-when two waves go through the same point they interact constructively or destructively
-when crest and trough are in phase with same frequency get added together to produce a wave with large amplitude and intensity
-the 2 short waves get added to make one tall wave
what is destructive interference
-when two waves that are not in the same phase interact with each other producing smaller amplitude with low intensity or a flat like
-this is used to isolate light from specific wavelength
what is light
-both wave and particle
-a wave because it can move in space like a wave
-a particle because it is a stream of particles made up of photons with no mass or charge moving at speed of light. The energy the photon carries varies with color
-energy known as electromagnetic radiation. Maxwells electromagnetic theory mentions that light is energy in waves with electrical and magnetic fields
What is the visible light spectrum?
-400 (violet) to 700 (red) nm
-gamma, xray, UV, THEN Visible
how do you select the appropriate wavelength to measure absorption
-chose a wavelength with the greatest sensitivity. So one with the max difference in Absopr readings with a small concentration change
-the wavelength chosen has to show a broad peak so if there is a slight error in calibration it will not create a large change in absorbance and loss of sensitivity
-a wavelength selector isolates the wavelength for measurement
-it seperates the light emitted from the lamp into its colors and isolates the wavelength you need .
-you want high intensity of the wavelength you need and low transmission of other ones
what is amplitude and cycle
Amplitude:
-wave height
-determines intensity of light which is also determined by the light source which must emit a constant source
Cycle
-one wave made up of one crest and one trough
=cps = CYCLES PER SECOND
what is frequency F or v
-number of waves/cycles per second that pass a point in one second
-constant no matter the medium
-controlled by light source
-Hertz HZ
-frequency and wavelength are inversely proportional so f=1/lamda. so if one increases the other decreases.
-wavelength = speed of light (c)/ frequency
Velocity (c)
-how fast an object moves through space (m/s)
-in a constant vacuum speed travels in a straight line
–related to wavelength and frequency
-dependent on media - slow in dense media
-shorter wavelengths have lower velocity
c=wavelength x f
what is Energy
-directly proportional to frequency and inversely proportional to wavelength
E=hf E - hc/lamba
h is Planks constant = relationship between energy and frequency
for example
Violet light - shorter wave, high f = high E
Red light -longer wave, low f = low E
What is reflection and what are the types of surfaces
-when light is returned from an illuminated surface which is not the source of light
-the angle of incidence = angle of reflection
-Vitros 350 measures reflected light
Smooth surface like a mirror
-when light is reflected it is in equal angle perpendicular to the surface called specular reflection
-makes an identifiable image
Rough surface
-light is scattered/diffused
-makes unidentifiable image
-The amount of light and its reflection is dependent on the nature of reflecting body and the shape and nature of the reflecting surfacew
what is refraction
-light bending between two different medias. When light passes from one to an another - air into glass, air into water
-Speed of light varies based on nature of the medium and the wavelength of light
-Refractometers are used to measure RI which indirectly measures total dissolved solids
Formula for refraction (n)
-the amount light bending between two media is dependent on the RI
-it is the ratio of velocity of light (c) in a vacuum vs velocity of light in the medium (v)
n=c/v n>1
-light entering a dense medium (air to water) will bend toward normal so the angle of refraction is less than the angle of incidence
-light entering a less dense medium (water to air), the ray is refracted away from normal making the angle of refraction greater then the angle of incidence
-velocity decreases in a denser medium
what is the dispersion of light
-when white light is spread into a full spectrum after passing through a prism - nonlinear
-as the white light seperates into different colors the associated wavelengths travel with a different velocities and refract at varying degrees
-shorter wavelength = smaller angle of refraction entering the prism and greater angle of refraction of light coming out of the prism ?
what is the diffraction of light
-how light bends around corners and obstables
-diffraction varies with wavelength - shorter waves bend less
-causing constructive and destructive interference
-diffraction gratings disperse white light into different wavelengths
how does diffraction grating work and what are the types
-ruled, reflective surface disperse polychromatic light into a uniform spectrum
-light is dispersed into colors using diffraction and wave interference
2 types are reflectance and transmittance
simple spec: wavelength is changed by pivoting the grating
-angle of the grating to the fixed exit slit causes different wavelengths of light to pass through the slit
what are the advantages of diffraction grating
-linear dispersion
-can produce less than 1nm of light in some analyzer
-better than prisms for isolation
what does the absorption of light depend on
-chemical structure of molecules in sample
-concentration of light absorbing molecules
-wavelength of light passing through the sample
what is absorption
-when white light passes through a colored solution some is
1.absorbed selectively by molecules in the solution
2. and rest is transmitted (these wavelengths are seen as color)
-blue solution absorbs yellow transmitting wavelength to appear blue (both complementary colors)
-ref absorbs green and transmits red
what is transmittance
-light traveling through a substance
-any light that is not transmitted is absorbed
-ratio of radiant energy transmitted through a medium divided by radiant energy shined on the medium
T= I/I0
-there are no units so T is expressed as % T of a 100
what is the relationship between A and %T
100%T= 0 absorbance
-100%T is set with a blank reagent with all reagents but not the specimen
-as A increases, %T decreases, therefore A and T are inversely related. They are inversely and logarithmically related A=2-log%T
-on LINEAR graph paper %T is inversely proportional to concentration
what is Beers law A= abc
=relationship between absorption of light by a solution and concentration of that solution
-A is directly proportional to concentration and distance light travels through a solution (b) which is consistent
A = absorbance
a = molar absorptivity (constant for solution of any concentration if measured at a specific wavelength). light absorbing ability of a substance under specific conditions. absorbance per unit concentration and unit path length
b=light path length through solution (cm)
c = concentration [M] in mol/L
what is molar absorptivity
a is absorptivity when concentration is 1 mol/L [M] and light path is 1 cm = M^-1 cm ^-1
a is the characteristic of a substance and can be used to check purity and calculate concentration
how to calculate the concentration of an unknown analyte
-standard graph As vs Cs (knowns to find unknown Au and Cu) - graph must be linear
-using absorptivity value of analyte = known - used to calculate enzyme activity in serum/plasma c=A/ab
-use Beers Law however the Absorbance and concentration must be linear because if the graph is curve then it shows that Beers Law was not followed at high concentrations
what is the most common light source for visible radiation
incandescent tungsten or tungsten iodide lamp - common for visible and near IR
-emits continuous spectrum 320-700 nm
-not used for measurement over 700 nm because the heat filter absorbs the infrared light emitted
What are the disadvantages of using a standard graph to find unknowns
-you cant make a graph for each analyte
-graphing and reading errors
now mostly used for new test or assay development to determine if its linear
what is a spectrophotometer
-measure the light transmitted by a substance to determine concentration of the light absorbing solution
how do we check purity with absorptivity
- make a solution of known concentration and read absorbance at a specific wavelength
-calculated absorptivity needs to be within a certain range if the sample is pure - aka a bilirubin standard
what is the quartz halogen lamp
-used as light source for visible radiation
-tungsten in quartz envelope filled with low pressure iodine or bromine vapor
-high intensity light over wide spectrum
-last very long through very high heat hence high energy output
-best for measuring near UV region
why are dilutions analyzed on specimens
-to ensure absorbance readings in the sensitive range are detected
-if a sample has an absorbance higher than the highest standard the instrument will not be able to calculate the result
-so we dilute the sample and reanalyze with a lower absorbance allowing the instrumentation to make accurate calculations
-when you get a result make sure you multiply it by the inverse of the dilution factor
what is the most important when it comes to light source
Range
-continuous -all wavelengths of light emitted at same range in with equal strength
-discontinuous - only some wavelengths are emitted
-light source should be able to produce visible radiation, UV or both
-Source of radiant energy
-stability of radiant energy
-temperature
what are the parts of the optical system in the spectrophotometer
light source
filter
monochromator - dispersing element (prism or grating) and slits
cuvette
light energy detectors
-the power source needs to be regulated because it can affect the stability of the instrument. you can use a constant voltage transformer or electronic power regulator
what is a LASER
Light Amplification by Stimulated Emission of Radiation
-narrow intense beam of light - helium and neon laser540-640nm
-uses helium and neon gases between 2 mirrors with one that reflects fully and other partially
-needs external energy source
-gaseous atoms get excited by light reflecting between the two mirrors and emit photons
-energy is released as short intense beam
-used in cell counter and nephelometers
What is Ultraviolet Radiation and the different types of tubes
–radiation produced by causing electrical discharge in a seal tube containing gas. The electrical energy excites atoms causing them to emit UV radiation
Hydrogen discharge tube
-continuous spectrum 200-360 nm if over 360nm a discontinuous spectrum is emitted for UV region
Deuterium Discharge Lamp
-continuous spectrum down to 165 nm
-light intensity UV light
-more stable and longer life than hydrogen
-UV region measurements
Mercury Vapour Lamp
-more powerful than deuterium
-emits discontinuous spectrum 200-1000nm
-intense at specific wavelengths - best for wavelength calibration
Xenon flash lamp
-xenon gas
-high intensity in both UV and visible region
-needs a cooling mechanism
-high caliber instruments
what problems can arise with light sources
light intensity variation
-measurement errors
-need voltage regulator to control current
heat production
-filters or thermal insulation helps to minimize
Lamp positioning or dirt/accumulation
-reduced light intensity
-can change spectrum emitted
-need PM - like lamp positioning procedure Vitros 350
dont handle lamps directly they can leave fingerprints and burn you
when in automated systems
-light source + wavelength selector+ detector =instrument optical system
THE OPTICAL SYSTEM RESPONSIBLE FOR THE MEASURMENT
What is the monochromator system
-found in a spectrophotometer
-allows you to select different wavelengths for testing
contains:
entrance slit:-limits light into narrow beam
lense/mirror; focuses and directs beam into dispersing element
dispersing element: the prism or diffraction grating
exit slit: isolates the chosen wavelength can be variable to fixed width
what are the colored glass filters
-absorbs unwanted wavelengths
-not a true wavelength selector as it cannot produce monochromatic light
what is an interference filter
-light waves enter on one side of the filter and are reflected on 2nd surface
-cause constructive and destructive interference
-able to produce monochromatic light
-wavelengths that are larger than the space between the filters will reflect back and forth while the other wavelengths phase out
-gives a narrower range of wavelengths than glass filters
what is a simple glass prism
-type of monochromator
-light beam is focused on a prism and is refracted as it enters the denser glass
-shorter wavelengths are refracted more than long ones causing dispersion of white light into a spectrum
-shorter violet wavelengths travel slowest with greater angle of refraction while longer red wavelengths travel fastest and are refracted the least
-a prism can be rotated so the desired wavelength can pass through the exit slit
what is a cuvette and what are the types
-found between monochromator and the detector
-holds the reagent
-can be glass (visible range) , quartz (UV range), or plastic (single use and disposable)
-need to be clean, no scratches as they can cause scatter
-position is critical as it contains two frosted sides
round
-needs to be a set and needs to be aligned
-as diameter of cuvette increases Absorbance increases because A is directly proportional to light path
square:
-plane-parallel surface
-uniform path length
-less likely to have optical aberrations
-no issues with alignment
-expensive
what is a flow through vs fixed
flow through
-built into instrument
-lots of samples flow into the same cuvette
-in instrument permanently and washed between samples
Fixed
-continuously washed and reused only replaced when damaged (AU480)
-needs high quality external water/reagents for washing
what is a photodetector
-able to convert transmitted radiant energy into an equivalent amount of electrical energy
-contains cathode with a photo emissive substance that can emit electrons when it is struck by transmitted light
-then those electrons become attracted to a positively charged anode which is then measured as a current
current will be proportional to the intensity of the light hitting the cathode
phototube, photomultiplier tube, photodiode
what is a phototube
-type of photodetector
-has negatively charged cathode and positively charged anode in a glass case
-when it is exposed to lights it emits electrons
-a dark current
-even without light it produces a small amount of current due to electron seepage between electrodes
what is a photomultiplier tube PMT and what are its dis/advantages
type of photodetector
-detects and amplifies radiant energy
-light transmitted onto photoemissive cathode to positively charged anode
-electrons are produce at a primary anode called dynode 1 then they move to different dynodes and each time there are more secondary electrons
-successive dynodes allow for amplification of electron signal x10^7
advantages:
-sensitive to low intensity levels
-instant reponse
-last longer
disadvantages
-needs stable power supply
-expensive
-must protect from stray light - due to amplification
what is a photodiode
-semiconductor device that detects light
-when light strikes the semiconductor the electrons or current flow through it
-the current measure is proportional to the intensity of light
types: PIN silicone, photodiode
3 layers of silicone: Positive, Intrinsic, Negative types
what is photodiode array PDA
-Two dimensional matrix of light sensitive semiconductors (photodiodes)
-dispersion device is placed after the cuvette and light of all wavelengths (polychromatic) is dispersed into the photodiode array
-each diode is calibrated for a specific wavelength
the entire spectrum can be recorded in milliseconds
what is band pass or band width (half band width)**
-range of wavelengths at the exit slit
-allowing frequencies in a certain range but rejecting others
-number of wavelengths transmitted at a point equal to 1/2 peak transmittance; shows how efficient a wavelength selector is
-the smaller the band pass the more efficient the monochromator
what are signal processing/read out devices
-converting electrical signal to a readable display
-converts %T to A
-monitors rate of reaction
-subtracts reagent blanks
-calculates concentrations
-measures area under a peak
Types of read out devices
Meter:
Galvanometer- measures current by magnetic effect
Digital - LED display
Printed report
Recorder- draws peaks on chart paper
Automated - uses micro processors for data management with results on monitors
What is instrument blanking?
-used to set an instruments measuring limits
set 0 %T - no light transmitted
set 100%T - no light absorbed all light transmitted
use a blank solution water or reagent in new cuvette but it can be specific depending on the type of instrument, test or kit
-Blanking also compensates for unwanted absorbance from reagents or sample
what are two types of instrument blanks
Reagent blank
-has everything but the analyte being measured
-distilled water
-set at 100%T or 0 A
Sample Blank
this type of blank is used when the specimen absorbs light unrelated to the analyte
-may occur when there is interfering substances - RBC which absorbs at same wavelength of analyte being measured
-HAS ALL components of a reaction
-before you do the chemical readings a blank absorbance must be done so you can subtract this from your final reading
what are the anayltical principle in AU480
Spectrophotometry and potentiometry
what are the analytical types in AU480
endpoint
fixed end point and indirect ISE
what are the analytical methods of the AU480
Colorimetry
turbidimetry
latex agglutination
homogenous EIA
indirest ISE
what does the AU480 use to analyze
-diode array spectrophotometer, monochromator isnt needed as the polychromatic light passes through the cuvette
-light is dispersed by a grating that is directed onto an individual photodiode
-quick gets results faster
what does the VITROS350 use to analyze**
-dry slide technology - slides should be brought to room temperature before use
-uses a colorimetric and potentiometric slide
-uses spec to measure color density that corresponds to analyte concentration
-tests done on the the Vitros are end point chemistries, rate and two point rate
at the end point - a colored complex is formed and the density of reflection corelates to the concentration. Reflectance readings are taken at end of incubation period.
at the rate: -many reflectance readings are taken as reaction progresses and rate of color change in the slide is related to enzyme activity
Two point rate: two reflectance readings are taken and rate of change is related to concentration
what is a colorimeteric slide
-measures the concentration or activity of an analyte by light reflectance.
-intensity of light reflected is proportional (NOT DIRECTLY) to color formation
-consists of four layers :Top down -
Spreading- porous so the sample can spread traps protein, lipids and cells acts as filtrate, reflects light eliminating interference from pt
sample
Reagent- where chemical reactions occur
Indicator - forms colored complex with reagent layer which can be measured and has a concentration proportional to the analyte. Read with reflectance photometry.
what is a potentiometric slide
-determines electrolyte concentrations by measuring potential difference between sample and reference electrodes
-uses reference fluid with known concentration of ions
what is reflectance spectrophotometry
-instead of the traditional 0 and 100%T being set reflectance spec needs 100% reflectance set with white ceramic plug and 0% reflectance set with black plug
-light from desired wavelength goes through clear support layer and is absorbed by the indicator layer. Any light that isnt absorbed is reflected back to the photodetector; the intensity of this light is measured as concentration. There is minimum interference from large molecules as they are trapped in the spreading layer
