Absorbance Spectroscopy/Reflectance Photometry Flashcards
Differentiate photometers from spectrophotometers
Both instruments measure light intensity, but they isolate wavelengths in incident light in different ways. Photometers use coloured filters to isolate a specific range of wavelengths, whereas spectrophotometers use “devices” (prisms, diffraction grating) to split the incident light into a spectrum. The specific wavelengths are angled in such a way as to isolate them for use during analysis.
List the basic components of a spectrophotometer
Light source
Collimator
Monochromator
Wavelength Selector
Sample Solution vessel (cuvette)
Detector
Read out display
List ideal properties of a light source
Provide high-intensity stable radiation at the wavelength of interest
Ideally able to provide a constant intensity
List common visible light sources
Tungsten Filament
Quartz Haliade
LED
LASER
Discuss how a tungsten filament lamp produces light
A tungsten filament is encased with a glass bulb, as the filament is heated it releases energy in the form of light. “Output is a function of filament temperature”. Most of the output is released as IR radiation which is perceived as heat. Heat evaporates the filament, causing tungsten to deposit on the glass, turning it black.
Discuss how a quartz halide lamp produces light
AKA tungsten-halogen lamp. It is a tungsten filament encased in a quartz bulb that is filled with halogen gas (inert). As the filament is heated it produced light and evaporates the filament, the halogen gas causes the tungsten to deposit back onto the filament.
Describe the emission spectrum of a tungsten filament lamp
Tungsten filament lamps emit a continuous nonlinear spectrum, most output is in the IR region (we perceive it as heat).
Define LED
Light Emitting Diode
Discuss how an LED produces light
A semiconductive material is used, and the LED is given two legs. Short leg = cathode, long leg = anode. Electrons flow through the semiconductive material (diode) from the anode to the cathode, releasing photons.
Define LASER
Light Amplification by Stimulated Emission Radiation.
Discuss how a LASER produces light
A flash tube is held within a mirrored cylindrical compartment filled with gas. The flash bulb will emit high-intensity light, and the electrons within the gas will absorb the light energy. Once the electrons move from their excited state to their ground state and emit the light that they absorbed. This emitted light is extremely intense and can be monochromatic.
List common UV light sources
Deuterium Lamp
Xenon Arc Lamp
Mercury Vapour Lamp
Identify light sources that produce a continuous emission spectrum
Tungsten Filament
Quartz Haliade
LED
LASER
Deuterium Lamp
Xenon Arc Lamp
Identify light sources that produce a non-continuous emission spectrum
Mercury Vapour Lamp
Define bandwidth (bandpass)
Measured in units of frequency (Hz) it refers to the range of frequencies in a signal. (range of light wavelengths transmitted). As bandwidth increases, more wavelengths are transmitted.
Calculate the wavelengths transmitted by a filter when given the nominal wavelength and bandpass
Divide the bandpass by 2, then add and subtract that number from the wavelength.
Bandpass = 10. Wavelength = 40.
10/2 = 5. 40+5=45, 40-5=35.
Wavelengths transmitted are 35-45nm.
Discuss how absorption filters work
These are made of coloured glass or plastic and are designed to transmit certain wavelengths, the rest of the wavelengths are absorbed by the filter. The colour of the filter corresponds to the colour of the visible light it will transmit.
Discuss how interference filters work
These are formed of multiple very thin layers of material. The thickness of the layer determines which wavelengths are transmitted. Wavelengths that aren’t designed to transmit through the filter are removed by destructive interference. (competing wavelengths that cancel each other out).
Describe Bandpass filters
Transmit a specific range of wavelengths. Used to select desired wavelengths for analysis.
Describe Shortpass filters
shortpass = Cut-off wavelength.
Transmit shorter wavelengths
Describe longpass filters
Longpass = Cut-on wavelengths
Transmit longer wavelengths
Describe Dichroic filters
Certain wavelengths are transmitted, and the rest are reflected.
Describe neutral density filters
Used to reduce light intensity across a wide range of wavelengths, not to isolate a specific range of wavelengths.
List two types of monochromators
Prisms
Diffraction Gratings
Discuss how a prism splits white light
Uses refraction to disperse white light into a continuous spectrum of monochromatic light. Short (blue) wavelengths are bent the most, and long (red) are bent the most.
Discuss how a diffraction grating works
Uses diffraction to produce a spectrum of monochromatic light from white light. They are a series of parallel lines etched onto a surface, when white light hits the etchings it is diffracted. The spectrum will have white light emitted from the centre, surrounded by the “first-order rainbows”, which are surrounded by the “second-order rainbow”.
First-order rainbows are more intense than second.
Two types:
Transmission grating: light is passed through the grating to produce a spectrum.
Reflection grating: Material is reflected, so the spectrum is produced by the reflection of the incident light.
List wavelength selectors based on their bandpass
Diffraction grating (0.5nm)
Prisms (1nm)
Interference filters (1-15nm)
Absorption filters (25-50nm)
Discuss methods of wavelength calibration:
Chemical standards
Chemical solutions have characteristic spectra with specific peaks, these are purchased in sealed cuvettes. The solution will be scanned across a range of wavelengths, and the peaks are examined to sure they are the correct size and place.
Crystalline substances
crystalline substances have characteristic spectra with specific peaks, these are purchased in sealed cuvettes. The solution will be scanned across a range of wavelengths, and the peaks are examined to sure they are the correct size and place.
Mercury lamp
This is the most accurate method of wavelength calibration. Mercury lamps only emit certain wavelengths, and in between the wavelengths, there will be no emission.
List the functions of a cuvette
Holds the sample
Sets the path length
Discuss the different types of cuvette material
Quartz or fused silica - Visible and UV
Borosilicate Glass - Visible
Plastic - Visible and certain UV, cheap and disposable.
Discuss sources of error when using cuvettes and how they will affect absorbance readings
Scratched or dirty, increased absorbance
Absorbance readings will be increased or decreased depending on if the cuvette position makes the path length longer or shorter.
Discuss the function of a photon detector
Converts the light energy transmitted by the sample into an electrical signal that can be recorded.
Define “transducer”
A device that converts one form of energy into a different form of energy.
Differentiate photoelectric effect and photoconductive effect
Photoelectric: Metals will release electrons when struck with EM radiation.
Photoconductive: Electrons are not released from the photosensitive material, and inside “jiggle” inside of the material.
Discuss ideal properties of photon detectors
Can respond to light energy over a broad range of wavelengths.
Are sensitive to low levels of light.
Exhibit a fast response time.
Produce an electrical signal that is proportional to the amount of light detected.
Produce minimal dark current.
Vacuum/gas phototube
Photoemssive, the flow of electrons from the emitter (cathode) to the collector (anode) generates a current proportional to the intensity of the light.
PMT
Highly sensitive photon detector that consists of photosensitive cathode emitter, dynodes, and anode. A photon passes through a series of dynodes that amplify the number of electrons available. The number of electrons emitted is proportional to the intensity of light that hits the cathode.
Photodiode
Convert light into electricity. Photoconduction. The current generated is proportional to the intensity of light striking the p-n junction.
Discuss limitations of photon detectors
Detectors are not equally sensitive to all wavelengths and are impacted by dark currents.
Define stray light
The light that hits the detector, but is not the light transmitted through the cuvette.
Define dark current
Current that is generated when there is no light hitting the detector.
List examples of functions performed by a signal processor
to receive a signal from a photon detector and convert it into an output that can be understood by the read-out display.
Filtering
Scaling (changing the amplitude of a signal)
combining multiple signals.
State the purpose of a read-out device
Takes the output form the signal processor and converts it into a form that is useful for humans.
List examples of read-out devices
Meters
Printers
Digital Displays
Single-beam
Light follows a single continuous path.
Double-beam
Has two light beams from one light source. One light beam will pass through the “blank” and the other passes through the sample cuvette.
Discuss the limitations of single-beam instrument design
Erratic readings due to light source intensity fluctuations.
Must blank the cuvette whenever the wavelength is changed.
Differentiate the two types of double-beam instruments
Double-beam in space: two separate detectors
Double-beam in time: one detector
Discuss the advantages of double-beam instruments
Don’t need to blank the instrument
Can measure reactions over a period of time, so fluctuations in light have a lessened effect.
Describe a diode array instrument
A diode array allows the instrument to measure multiple wavelengths simultaneously.
Discuss advantages of diode array instruments
Lower stray light.
Better at wavelength accuracy because the wavelength selector does not need to move.
Define wet chemistry and dry chemistry
Wet Chemistry: Chemical reaction occurs in a liquid phase.
Dry Chemistry: Analysis is done on “dry” slides which contain the required reagents in a non-liquid form.
List advantages of dry chemistry
No reagent prep
No water, plumbing, or drains
No pumps or tubing
Reagents typically have a longer shelf life
List the layers and a Vitros colourimetric MicroSlide and state the function of each layer
Spreading layer - distributes the sample
Filtering layer - removes interfering substances
Reagent layer - Analyte reacts with chemicals
Indicator layer - coloured product is formed
Support layer - optical interface
Define specular reflectance and give an example of how it can be used in lab instrumentation
Occurs when light hits a reflective surface and bounces off. There is no absorption of energy by the surface. The angle of reflection is equal to the angle of incidence.
Mirror to direct light.
Define diffuse reflectance
Occurs when light hits a dull or rough surface. The light is reflected in many directions, and some light is absorbed by the surface.
State how %reflectance is calculated
%R = (Is/Ir)x100
Is= Intensity of light reflected from the sample slide
Ir= Intensity of light reflected from the reference slide
Represents the light that is not absorbed by the slide, and is reflected off of the slide. As analyte concentration increases, %R decreases.
Define reflectance density
Dr is an indication of the amount of light absorbed by the coloured indicator layer, and is proportional to analyte concentration (non-linear relationship).
Dr= - logR
State the relationship between analyte concentration, reflectance, and reflectance density
As the concentration of the analyte increases reflectance decreases non-linearly, and the DR increases non-linearly.
State the two light sources that are typically found in reflectance instruments
Tungsten halogen
LED
Describe the orientation of the light source with respect to the photon detector
The light source is typically perpendicular to the surface of the slide, and the reflectance at 45° is measured by the photon detector.
State the wavelength selector device that is typically found in reflectance instruments
Interference filters
LED light sources remove the need for a filter.
State the photon detector that is typically found in reflectance instruments
Single photodiode
Discuss how reflectance measurements are made on the Vitros instrument
The reflectance for a white standard slide is determined
The reflectance for a black standard slide is determined
The reflectance for the sample is determined
The corrected reflectance value is compared to standard curve data by the instruments computer
List some sources of error associated with dry slide technology and reflectance instrumentation
Reagent variation
Light source fluctuations
Filter Quality
Stray light
List some clinical applications of dry chemistry and reflectance instrumentation
POC instruments
Urinalysis
Clinical chemistry analyzers
Discuss why many POC instruments use reflectance
The components for reflective instruments can be very small, the light source can be a LED, and only a single photodiode is needed. Liquid reagents do not need to be pumped through the instrument, so no probes are needed.
