Module 8: Other Photometric Methods

Question 1

Flame emission photometry

Answer 1

certain elements (alkali metals) can emit characteristic wavelengths of light when headed in a flame
Na, K, Li most commonly evaluated
Sample introduced into a flame, atoms of elements are excited by heat energy (move into higher energy orbitals)
standards of known concentration are measured to make cal curve

Question 2

when excited electrons return to the ground state,

Answer 2

they emit light energy at specific wavelengths characteristic for that element

Question 3

Flame emission photometry reaction

Answer 3

A+ + e- = Ao
Ao + heat = A*
A* = Ao + hv

A* = excited atom
Ao = atom in ground state
hv = photon of light energy

Question 4

light vs atoms vs concentration in flame photometry

Answer 4

amount of light = number of atoms = concentration of element

Question 5

Na, K, Li wavelengths for flame photometry

Answer 5

Na measured at 589nm
K measured at 766nm
Li measured at 670nm

Question 6

Components of flame photometer

Answer 6

Atomizer/burner assembly: acts as cuvet or sample holder; aspirates constant amount of solution into the flame as fine droplets (nebulization)
Wavelength selector: interference filter with narrow bandpass. Isolate wavelength characteristic of the element to be measured
Photodetector: photomultiplier tubes
Signal processor: amplify signal
Display

Question 7

Factors affecting the flame in flame photometry

Answer 7

1) type of fuel and oxidant (usually propane and air are used, flame is 1925degC, must be pure propane)
2) Fuel to oxidant ratio: determined by pressure regulation, required to produce constant thermal output

Question 8

Flame photometry interference: Spectral

Answer 8

other substances may emit light as well (controlled by using low flame temp and narrow bandpass filter)
Self-absorption when energy emitted is absorbed by other atoms of the same element (controlled by diluting samples to be measured)
Mutual excitation when transfer of energy from an atom of one element to an atom of another (controlled by using an internal standard that acts as a radiation buffer)

Question 9

Flame photometry interference: ionization

Answer 9

if atoms are provided with sufficient energy they will ionize and emit different wavelengths of light (fewer atoms in the flame emitting the measured wavelength)
controlled by keeping flame temp low

Question 10

Flame photometry interference: physical interferences

Answer 10

Surface tension reduced by addition of wetting agent

Viscosity reduced by dilution of the sample (usually 1/100 or 1/200)

Question 11

Internal standard

Answer 11

a solution with a known concentration of a substance that is structurally similar to the analytes of interest that is added to all samples
Used to account for variations in the system
Li or Cesium often used
Added to all samples and any variations in the system will affect the signal reading of both analytes and the internal standard

Question 12

Variation that an internal standard can account for

Answer 12

fluctuations in air/gas pressure causing changes in flame temp and stability
changes in aspiration rate affecting size and # of droplets
Mutual excitation of K atoms by Na atoms

Question 13

desirable characteristics of an internal standard element

Answer 13

high emission intensity
normally absent from biological fluids
emission at a wavelength sufficiently removed from Na and K to permit spectral isolation

Question 14

Types of scattering

Answer 14

Rayleigh scatter
Raleigh-Debye scatter
Mie scatter (aka Tyndall or Tyndall-Mie)

Question 15

Rayleigh scatter

Answer 15

particle size (d) is smaller than wavelength of incident light (d

Question 16

Raleigh-Debye scatter

Answer 16

particle size (d) is approximately equal to wavelength of incident light (d=λ)
as particle size increases, the scatter loses its symmetry and shows an increase in forward scattering

Question 17

Mie scatter (AKA Tyndall or Tyndall-Mie)

Answer 17

particle size (d) is larger than wavelength of incident light (d>λ)
large particles produce a predominance of scattered light in a narrow angular region in the forward direction

Question 18

What is used to produce consistent scatter patterns

Answer 18

a monochromator used to select specific wavelength of light

Question 19

2 photometric methods to measure scattered light

Answer 19

turbidimetry

nephelometry

Question 20

Turbidimetry

Answer 20

measurement of the decrease in intensity of the incident light beam that is caused by scattering
Inverse relationship between the light measured and the concentration of the sample
Same setup as photometer, with photodetector directly in line with the path of the light beam

Question 21

Nephelometry

Answer 21

measurement of the amount of light scattered
Direct relationship between the light measured and the concentration
Photodetector is NOT in the direct path of the light beam (instead placed at an angle to the light path, typically 90Degrees)
Multiple photodetectors at different angles may be used

Question 22

Sources of error for light scattering methods

Answer 22

Variations in particle size
Matrix effects (other substances in the solution that scatter light)
Debris within the system (dust)
Fluorescence (fluorescing compounds can be excited if short wavelength is used - false increase in light measured)
Settling of particles (mix prior to reading)

Question 23

Fluorescence

Answer 23

once the molecule is in excited state, radiationless vibrational deactivation (RVD) occurs until it is in its lowest excited state (some energy is lost)
As electron drop to ground state, light energy is emitted
Because some has already been lost to RVD, light energy emitted is less than and of a longer wavelength than the excitation energy
**Compounds that fluoresce absorb light of one wavelength and emit light of a longer wavelength

Question 24

Fluorescence excitation wavelength

Answer 24

in the UV range

Question 25

Fluorescence emission wavelength

Answer 25

in the visible range

Question 26

Fluorescence: Stokes shift

Answer 26

difference between the max wavelength of excitation light and max wavelength of emitted fluorescent light
Constant measure of the energy lost in the excited state due to RVD

Question 27

Clinical uses of fluorescence

Answer 27

often used as tags or cell markers
Hematology analyzer/flow cytometry to identify blood cells
Micro to measure bacterial growth
Immunoassay analyzer that use antibodies to detect specific drugs/substances

Question 28

Advantages to fluorometry

Answer 28

1000-10 000x more sensitive than photometry

More specific because only the wavelength emitted by the compound is measured

Question 29

Fluorometer

Answer 29

Fluorometer: use glass or interference filters
Spectrofluorometer: used prisms/diffraction gratings

measurement is made at 90deg to incident light (no interference from light transmitted by sample)
2 monochromatic devices: 1 to isolate the excitation wavelength (primary), one to isolate the emission wavelength (secondary)

Question 30

Components of fluorometers

Answer 30

Light source: 300-500nm, very high energy (xenon, quartz halogen, mercury), lasers may be used
Wavelength selector: filters, prisms, diffraction grating
Sample Holder: cuvet made of silica or quartz
Photodetector: photomultiplier tube (wide range of spectral responses, rapid response time, sensitive)

Question 31

Limitations of Fluorescence measurements

Answer 31

Inner filter effect: as concentration increases, relationship becomes nonlinear. Caused by loss of excitation intensity across the cuvet path length
Quenching: self absorption
Light scattering: scattered light reaching emission photodetector causes background interference (controlled with narrow bandpass filters)
Solvent effects: impurities in the solvent that may have fluorescent properties
Sample matrix effects: serum and urine may contain compounds that can fluoresce
Temp effects: fluorescence intensity decreases with increasing temp

Question 32

Reflectance photometry

Answer 32

incident beam of light is directed at test sample and reflected light is measured.
Amount of light reflected depends on concentration of analyte
Amount of light reflected back is inversely related to concentration

Question 33

Dry chemical systems

Answer 33

disposable dry reagent slides that also function as the cuvet
Spreading Layer: sample applied
Reagent/indicator layer: contain reagent required for the reaction. Analyte reacts with reagent to produce color change
Support layer: clear plastic support that allows incident light to pass through the indicator layer
Incident light is directred from underneath
Light strikes underside of spreading layer and is reflected back (reflected light measured by photodetector)

Question 34

Standardizing a dry chemical reflectance photometry system

Answer 34

0 concentration standard = white surface (100% reflectance)

high concentration standard = black surface (0 reflectance)