Spectrophotometry

Question 1

Analytic techniques

Answer 1

1. Spectrophotometry ( spectrophotometry, atomic absorption, mass spectrometry)
2. Luminescence ( fluorescence, chemiluminescence)
3. Electroanalytic ( electrophoresis, potentiometry, amperometry)
4. Chromatography ( gas, liquid, thin layer)

Question 2

What is light

Answer 2

A type of radiant energy- it travels in waves

Question 3

What is a cycle

Answer 3

One complete wavelength

Question 4

What is frequency (v)

Answer 4

cycles / sec ( hertz)

Question 5

What is the relationship between frequency and wavelength

Answer 5

Inverse relationship
V= 1/ wavelength

Short wavelengths = high energy & high frequency
Long wavelengths= low energy & low frequency

Question 6

Velocity (c)

Answer 6

Distance travelled by a wave in one second

Question 7

Wavelength

Answer 7

The distance from a point on a wave to the corresponding point on the next, measured in nm( 10^-9m)

Question 8

Amplitude

Answer 8

The magnitude of the peak of the wave

Question 9

Radiation from low wavelengths to high ( aka high frequency to low)

Answer 9

Gamma
X ray
UV
Visible 
Infared 
Radiowaves

Question 10

Visible light and corresponding wavelengths

Answer 10

Violet ( 380-440)
Indigo( 420-450)
Blue( 440-500)
Green(500-580)
Yellow(580-600)
Orange(600-620)
Red( 620-750)

Question 11

What is the average range of visible light wavelengths

Answer 11

400-700nm

Question 12

Dispersion

Answer 12

When light is passed through a prism and separated into its component colours

Question 13

absorption/transmittance of light

Answer 13

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.

Question 14

how is electromagnetic radiation absorbed

Answer 14

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.

Question 15

absorption or emission spectra

Answer 15

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

Question 16

beers law 4 parts

Answer 16

1. equal thickness of an absorbing material will absorb a constant fraction of energy incident upon it
2. 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

4. the greater the number of absorbing molecules the greater the absorbance
   i. e the deeper the color of solution the greater the absorbance

Question 17

beers law equation

Answer 17

C(t) = A(t) / A(s) xC(s)

Question 18

photometry vs spectrophotometry

Answer 18

photometry is a measurement of the intensity of light, independent of wavelength

spectrophotometry is the measurement of the intensity of light at selected wavelengths

Question 19

what is a filter photometer and what are the parts

Answer 19

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)

Question 20

what is a spectrophotometer

Answer 20

instruments that use prism ratings to select the wavelength

Question 21

reagent blank

Answer 21

corrects for the color contribution of the reagents

contains all the reagents used in procedure but doesn’t contain the substance being measured

Question 22

single beam vs double beam spectrophotometer

Answer 22

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

Question 23

absorbance definition

Answer 23

expression of the amount of light absorbed by a solution. values directly proportional to the concentration

Question 24

percent transmittance definiton

Answer 24

amount of light that passes through a coloured solution. as concentration increases %T decreases

Question 25

photometer scale and equation

Answer 25

reads transmittance and absorbance, expressed as %T to avoid fractions A= 2-log%T

Question 26

standard curve

Answer 26

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

Question 27

plotting and using a standard curve

Answer 27

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

Question 28

light source in a spec

Answer 28

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

Question 29

monochromator in a spec

Answer 29

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

Question 30

bandwidth

Answer 30

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

Question 31

monochromator- types of filters

Answer 31

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)

Question 32

monochromator- diffraction gratings(2)

Answer 32

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)

Question 33

monochromator- prisms

Answer 33

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

Question 34

cuvettes( sample cells)

Answer 34

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

Question 35

photodetectors (radiant energy detectors )

Answer 35

change light energy to electrical energy includes: barrier layer cell( photocell) phototube photomultiplier tube photodiode

Question 36

Photodetector- barrier layer cell ( photocell)

Answer 36

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 )

Question 37

Photodetector - phototube

Answer 37

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

Question 38

Photodetector- photomultiplier tube

Answer 38

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

Question 39

Galvanometer

Answer 39

Measures current Light hits the radiant energy detector, current is produced Current is directly proportional to the amount of light transmitted

Question 40

What quality assurance measures need to be checked for spectrophotometry

Answer 40

Wavelength accuracy Stray light Linearity

Question 41

Quality assurance of wavelength accuracy

Answer 41

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

Question 42

Quality assurance stray light

Answer 42

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

Question 43

Quality assurance linearity

Answer 43

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