Spectrophotometry Flashcards
Analytic techniques
- Spectrophotometry ( spectrophotometry, atomic absorption, mass spectrometry)
- Luminescence ( fluorescence, chemiluminescence)
- Electroanalytic ( electrophoresis, potentiometry, amperometry)
- Chromatography ( gas, liquid, thin layer)
What is light
A type of radiant energy- it travels in waves
What is a cycle
One complete wavelength
What is frequency (v)
cycles / sec ( hertz)
What is the relationship between frequency and wavelength
Inverse relationship
V= 1/ wavelength
Short wavelengths = high energy & high frequency
Long wavelengths= low energy & low frequency
Velocity (c)
Distance travelled by a wave in one second
Wavelength
The distance from a point on a wave to the corresponding point on the next, measured in nm( 10^-9m)
Amplitude
The magnitude of the peak of the wave
Radiation from low wavelengths to high ( aka high frequency to low)
Gamma X ray UV Visible Infared Radiowaves
Visible light and corresponding wavelengths
Violet ( 380-440) Indigo( 420-450) Blue( 440-500) Green(500-580) Yellow(580-600) Orange(600-620) Red( 620-750)
What is the average range of visible light wavelengths
400-700nm
Dispersion
When light is passed through a prism and separated into its component colours
absorption/transmittance of light
the colours seen on the visible spectrum are of the wavelengths that are transmitted and not absorbed. all other colours that are absorbed are not seen.
the greater the # of absorbing molecules the more light is absorbed therefore the less that is transmitted.
how is electromagnetic radiation absorbed
for a ray of electromagnetic radiation to be absorbed it must have the same frequency as a rotational or vibrational frequency in the atom or molecule it hits.
absorption or emission spectra
absorption of energy by atoms results in a line spectra
molecules emit a bank of energy over a large region resulting in a band spectra
incandescent solids emit light in a continuous spectra
beers law 4 parts
- equal thickness of an absorbing material will absorb a constant fraction of energy incident upon it
- each equal layer will absorb a constant fraction and the absorption energy is logarithmic.
i. e 20% each layer would follow as 100%-80%-64%-51.2%-41% etc..
3.thicker absorbing material = greater absorbance
- the greater the number of absorbing molecules the greater the absorbance
i. e the deeper the color of solution the greater the absorbance
beers law equation
C(t) = A(t) / A(s) xC(s)
photometry vs spectrophotometry
photometry is a measurement of the intensity of light, independent of wavelength
spectrophotometry is the measurement of the intensity of light at selected wavelengths
what is a filter photometer and what are the parts
an instrument that uses filters to select the wavelength
parts : light source( provides white light), filter( selects the wavelength that will give the greatest absorbance for the coloured solution), cuvette( hold the test sample, radiant energy detector( converts light energy into electrical energy) , meter( displays the current as absorbance or %T)
what is a spectrophotometer
instruments that use prism ratings to select the wavelength
reagent blank
corrects for the color contribution of the reagents
contains all the reagents used in procedure but doesn’t contain the substance being measured
single beam vs double beam spectrophotometer
single beam has a light source, monochromator, cuvette, detector, and meter
it uses a reagent blank
double beam has everything in duplicate except for the light source
it doesn’t require a reagent blank bc it allows fo automatic correction of the sample and reference absorbance
absorbance definition
expression of the amount of light absorbed by a solution. values directly proportional to the concentration
percent transmittance definiton
amount of light that passes through a coloured solution. as concentration increases %T decreases
photometer scale and equation
reads transmittance and absorbance, expressed as %T to avoid fractions
A= 2-log%T
standard curve
s graph with absorbance or % T plotted on the y axis (vertical) and increasing concentrations on the x axis (horizontal)
conc. vs %T is on semi-log
conc. vs A is on linear paper
straight line = beer’s law
plotting and using a standard curve
using absorbance or %T from a standard ( with known concentration) is plotted on graph them test sample absorbance or %T is read on spectrophotometer, use that value to compare to standard curve and find concentration
light source in a spec
provides wavelength of light in the visible near infrared range
- incandescent tungsten lamp
- tungsten- iodide lamp
- gives a range of 300nm-1000nm
provides wavelength of light in the UV range
- deuterium discharge lamp
- mercury arc lamp
monochromator in a spec
eliminates unwanted wavelength of light and allow the desired light to pass through sample
includes : filters, prisms, and diffraction gratings
the small the bandwidth the better the monochromator
bandwidth
the range of wavelengths where %T is 1/2 the peak of transmittance
to determine draw a horizontal line at 1/2 %T
then draw 2 vertical lines where the horizontal line intersects the curve
monochromator- types of filters
Glass: thin layer or coloured glass containing metals ( bandwidth 25-50nm)
Wratten : layer of coloured gelatin, doesn’t withstand heat well, prone to leakage
( bandwidth 25-50nm)
INTERFERNCE ( best)
two metallic layers, two glass layers and a dielectric layer ( glass or vacuum), use cut off filters to absorb undesired wavelengths
( bandwidth 10-17nm)
monochromator- diffraction gratings(2)
flat glass plates of an aluminum and copper alloy with thousands of etched grooves
two types:
1. reflection; reflects spectrum from the surface
2. transmission; creates a spectrum as light passes through
both give a linear spectrum ( equal space between colours/ wavelengths)
monochromator- prisms
separates light into a spectrum by refraction
longer wavelengths are bent less than short ones as they pass through
produce a non-linear spectrum
rotating prism allows desired wavelength to be selected
cuvettes( sample cells)
vessel to hold solution to be measured
can be : glass( used for work in visible range)
silica (quartz)- used for wavelengths below 340nm
plastic - problems with tolerance, cleaning, etching by solvents and temperature, disposable plastic cuvettes are usually used
photodetectors (radiant energy detectors )
change light energy to electrical energy
includes: barrier layer cell( photocell)
phototube
photomultiplier tube
photodiode
Photodetector- barrier layer cell ( photocell)
Not very sensitive
Not good for high or low levels ( not easily amplified)
Used with wide band pass
Light sensitive layer ( selenium) on a plate of iron, with a layer of silver on top
Electrons in selenium layer are excited when exposed to light and are released to the conductive silver layer ( no external voltage source needed )
Photodetector - phototube
Cathode acts as a resistor in the dark but emits electrons when exposed to light
Electrons are attracted to the positively charged anode
Transfer of electrons produces a current which is measured by galvanometer
An outside voltage is required
Photodetector- photomultiplier tube
Can amplify
Signal increase 200x
Detects and amplifies low levels of light
Electrons are attracted to the anodes called dynodes*
Each dynode has a successively increasing positive charge which gives an increasing number of secondary electrons
Accumulation of electrons produces a current
Galvanometer
Measures current
Light hits the radiant energy detector, current is produced
Current is directly proportional to the amount of light transmitted
What quality assurance measures need to be checked for spectrophotometry
Wavelength accuracy
Stray light
Linearity
Quality assurance of wavelength accuracy
Set wavelength on the spec should match the actual wavelength that is passed through the monochromator
Check by using standard absorbing solutions or filters of known maximum absorbance, didymium and holimium oxide in glass are commonly used filters
Another method involves substituting a mercury lamp for the usual light source
*If the wavelength was inaccurate, the optics would be adjusted to calibrate monochromator correctly
Quality assurance stray light
Scratched and dust in the light path are most common causes of stray light
Cutoff filters eliminate all wavelengths other than the one of interest and can be used to check for stray light
Quality assurance linearity
A calibration curve should result in a straight line filling beers law
Linearity is checked by using coloured solutions that are diluted and ensuring that beers law is followed
