Module 3a: Photometry and Spectrophotometry Flashcards
1
Q
majority of analytic techniques fall into one of four basic disciplines within the field of analytic chemistry which are
A
> spectrometry
luminescence
electroanalytic methods
chromatography
2
Q
five types of Spectrophotometry/Photometry/Colorimetry
A
a. Spectrophotometry
b. Flame Emission Spectrophotometry
c. Atomic Absorption Spectrophotometry
d. Reflectance Spectrophotometry
e. Mass Spectrophotometry
3
Q
Light energy is a form of ________ radiation
A
Electromagnetic
4
Q
light travels in packets of radiant energy called _____ or _____
A
photons or quanta
5
Q
light energy is characterized by _______ and _______
A
frequency and wavelength
6
Q
definition: FREQUENCY
A
number of vibrations of wave per second, has a direct relationship with energy
7
Q
definition: WAVELENGTH
A
distance between two successive peaks in nanometers, has an inverse relationship with energy and frequency
8
Q
light is used to describe radiant energy from the spectrum _____ to ____ nm
A
290 to 750 nm (ultraviolet to visible light)
9
Q
differentiate: PHOTOMETRY and SPECTROPHOTOMETRY
A
both are measurements of the luminous intensity of light, but spectrophotometry measures only light at a selected wavelength
10
Q
define: Planck’s theory
A
theory that energy is transmitted through units of quanta or photons
where the more waves there are (higher frequency), the more light energy
11
Q
basic components of a spectrophotometer according to the path of light
A
- Light source
- Entrance slit
- Monochromator
- Exit slit
- Cuvette with sample
- Photodetectors
- Meter or Readout devices
12
Q
define: Beer’s Law
A
The concentration of a substance is directly proportional to the amount of light absorbed, or is inversely proportional to the log of the transmitted light.
13
Q
Beer’s law formula
A
A = abc A (absorbance) a (proportionality constant defined as absorptivity) b (light path in cm) c (concentration in g/l)
14
Q
light that has already passed through sample
A
transmitted light
15
Q
light that hasn’t passed through a sample
A
incident light
16
Q
percent transmittance is the ratio of ____
A
sample beam signal/ blank beam signal
17
Q
% transmittance when all light is absorbed
A
0%
18
Q
% transmittance when no light is absorbed
A
100%
19
Q
used to measure the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution.
A
spectrophotometer
20
Q
Most common light source in the visible portion of the spectrum, with most emitted in the infrared region.
A
Tungsten/Tungsten Iodide Lamp
21
Q
Tungsten/Tungsten Iodide Lamp does not supply sufficient radiant energy or measurements below ____ nm
A
320 nm
22
Q
Provide sources of continuous spectra in the UV region with some sharp
emission lines.
A
Hydrogen and deuterium lamps
23
Q
Deuterium lamps provide continuous emission down to ___ nm.
A
165 nm
24
Q
Advantage of deuterium lamp over Hydrogen lamp
A
more stable and has longer life
25
incandescent lamp which is useful for calibration purposes but is not practical or absorbance measurements because it is used only at certain wavelengths.
Low Pressure Mercury Arc Lamp
26
Low Pressure Mercury Arc Lamp discontinuous or sharp line spectrum with both ____ and ___ lines
UV and visible lines.
27
Low Pressure Mercury Arc Lamp is commonly used in
high-pressure liquid chromatographic (HPLC)
28
Incandescent lamp that has a very-narrow-wavelength intense source.
Hollow cathode lamp
29
incandescent lamp used to measure peptides and proteins due to its giving a line of _____ nm, which is close to ______
zinc hollow cathode lamp, 214 nm, 206 nm
30
LASER meaning
light amplification by stimulated emission of radiation
31
laser transforms light of various frequencies into an _____
> extremely intense
> focused
> nearly nondivergent beam of
> monochromatic light
32
LED meaning
Light Emitting Diodes
33
LED lights are made of _______ and _____
semiconductor ( aluminum gallium arsenide) and insulator.
34
monochromators are devices for ______
Spectral Isolation
35
system for isolating radiant energy at a desired wavelength and excluding
that of other wavelengths
monochromators.
36
defines the range of wavelengths transmitted
bandpass
37
calculation of bandpass
width at more than half the maximum transmittance
38
three types of light source
Incandescent Lamps
Lasers Sources
LED
39
three types of incandescent lamps
Tungsten/Tungsten Iodide Lamp
Hydrogen and deuterium lamps
Low Pressure Mercury Arc Lamp
40
three types of monochromators
Filters, prisms and diffracting gratings
41
Simplest type of filter but not considered a true monochromator because it transmits light over a relatively wide range of wavelengths.
Glass filter (thin layer of colored glass)
42
Glass filters are also known as
wide bandpass filter
43
refers to the spectral purity of a filter or other monochromators.
Spectral bandwidth/ Spectral bandpass
44
used to eliminate light below a given wavelength
Sharp-cutoff filter
45
uses dielectric material of controlled thickness sandwiched between two thinly
silvered pieces of glass
Narrow-bandpass or interference filters
46
the width of the transparent spacer in interference filters is______
one-half the desired wavelength
47
separates white light into a continuous spectrum through refraction
prism
48
____wavelengths are bent to a greater extent as they pass through a prism
shorter
49
most commonly used monochromator which has extreme accuracy and low light scatter
Diffraction grating:
50
separation of light into component wavelengths based on the principle that wavelengths bend as they pass a sharp corner.
Diffraction
51
used to hold a liquid sample to be analyzed in the light path of a spectrometer
cuvette
52
Cuvets suitable for measurements in the visible portion of the spectrum
Ordinary borosilicate glass cuvets
53
Cuvettes used for readings below 340 nm
Quartz cells
54
devices which convert the transmitted radiant energy into an equivalent amount of electrical energy
Photodetectors
55
It is the most commonly used monochromator. It is extremely accurate and have low light scatter.
diffraction grating
56
Diffraction grating is prepared by depositing a thin layer of _______________ on the surface of a flat glass plate; then, many small parallel grooves are made in the metal coating.
aluminum-copper alloy
57
the separation of light into component wavelengths based on the principle that wavelengths bend as they pass a sharp corner.
difrraction
58
Gratings with very fine line rulings produce a widely dispersed spectrum-- a linear spectra, which are called __________
orders
59
HPLC detectors frequently use a _____________ in their optical system
concave holographic reflective grating
60
3 types of diffraction gratings
a. plane transmission gratings
b. reflective gratings
c. echelette
61
Cuvets that have good clarity in both visible and UV ranges but often present problems related to tolerance, cleaning, etching by solvents, and temperature deformation.
plastic cuvets
62
Cuvets may be cleaned though soaking in a mixture of
concentrated HCl/water/ethanol (1:3:4)
63
cuvets should never be soaked in
dichromate cleaning solution
64
Commonly used photodetector for measurement of light intensity in the UV and visible regions of the spectrum and is capable of significantly amplifying a current.
Photomultiplier tube
65
advantages of PMT photodetector
1. extremely rapid response times
2. sensitive
3. slow to fatigue
66
2 types of solid state detectors
photodiodes
| charge-coupled detectors
67
solid state detctor that fabricated from photosensitive semiconductor materials which is capable of measuring light at a multitude of wavelengths
photodiodes
68
Multi- channel devices with good dynamic ranges and signal to noise ratios that are superior to those of PM tubes.
Charge coupled detectors
69
Charge coupled detectors operates like:
a large number of photo detecting shift registers read horizontally and vertically
70
Main application of of charge coupled detectors
- molecular flourescence measurement of very low concentration fluorophores
71
photocell is also known as
barrier-layer cell
selenide cell
photovolataic cell
72
Photodetectors used in older instruments and is the least expensive
Photocell
73
Photocells are composed of:
thin layer of silver over a film of selenium on a plate of iron
74
Adavantages of Photocell
inexpensive and durable
75
disadvantages of photocell
temperature sensitive
| non linear at very and very low illumination
76
similar to photocell but requires and outside voltage to operate and is more sensitive
phototube
77
phototube contains:
an anode and a cathode in a glass case
78
Displays the electrical energy from a detector
readout devices
79
Readout devices can be classified into:
direct reading
| null point system
80
readout device where the output of the photocell is used to drive a sensitive meter directly, without further amplification
direct reading system
81
readout device where the output of the detector is balanced against the output of a reference circuit and direct digital readouts are obtained
null point system
82
readout device that has faster response, is easier to read and decreases operator fatigue
digital readout devices
83
digital readout devicds operate on the principle of
selective illumination of LED
84
Types of spectrophotometers
Single beam spectrophotometer
Double beam in space spectrophotometer
Double beam in time spectrophotometer
85
Composed of one of each of the basic components of the spectrophotometer, and a single measurement is derived
single beam spectrophotometer
86
Spectrophotometer in which light splits into two beams, one beam passes through a reference material, the other through the sample. The measurement displayed comes from the ratio of the two beam intensities.
Double beam spectrophotometer
87
In double beam in space spectrophotometer, all components are doubled except:
the light source and the readout device
88
Spectrophotometer that has two detectors which allow simultaneous measurement of the sample and the standard.
Double beam in space spectrophotometer
89
In double beam in time spectrophotometer, all components are single except the:
cuvet
90
A rotating wheel with alternate silvered and cut out sections founf after the exit slit in double beam in time spectrophotometers to split the beam into two.
Chopper
91
Spectrophotometer where in the detector alternates between measuring the sample beam and the reference beam in synchronism with the chopper.
Double beam in time spectrophotometer
92
Photometry which is based on the principle that excited atoms when returning to ground state emits light energy that is characteristic for the atomic species.
Flame Emission Photometry
93
Flame emission photometry os based on the principle:
excited atoms returning to ground state emit light energy
94
In flame photometry, ____________ is being measured
emission of light
95
Flame emission photometry is widely used in determining the concentration of
Sodium (Na)
Potassium (K)
Lithium (Li)
96
Six basic components of Flame photometer
1. aspirator
2. atomizer
3. flame
4. lense
5. monochromator
6. detector
97
Process of flame photometry
1. sample is diluted and aspirated through suction in the form of spray
2. atomkzer dispenses the sample as fine mist
3. mist interacts with flame to excite ions
4. as electrons drop back to lower energy level, light passes through slit to eleminate stray radiation
5. monochromator determines wavelength
6. detected by phototube
98
fuel source of the flame in flame emission photometry
propane and compressed air
99
Flame colors and monochromators used for Na, K, Li
Na - yellow - 589 nm
K - violet - 767 nm
Li - red - 671 nm
100
Used as an internal standard in FEP to compensate for a possible fluctuation in the light source caused by changes in fuel or air pressure
High concentration of Lithium or Cesium
101
Advantages of FEP
accuracy and precision of +/- 1-5% in aqueous solution
suitable for metallic ions
fast, simple
free from interference
102
Limitations of FEP
can only measure pure metals
| other variables affecting instensity of light
103
FEP in routince clinical laboratory are used in the determination of
lithium levels in serum of manic-depression patients on lithium therapy
104
Ions that give detectable signs for FEP but cannot be used due to insufficient reliabilty and sensitivity
Magnesium, Calcium
105
spectrophotometry that is considered the inverse of FEP
Atomic absorption spectrophotometry
106
Principle of Atomic Absorption spectrophotometry
element is disassociated from its chemical bonds and placed in ground state where it is capable of absorbing radiation at a very narrow bandwidth
107
Components of an Atomic Absorption Spectrophotometer
1. Hollow cathode lamp corresponding to metal being measured
2. Flame as the sample cell
2. Nebulizer
3. Monochromator
4. Light detector
5. Data processor
108
The evacutaed gas tight chamber of a hollow cathode lamp in Atomic Absorption spectrophotometer contains (3)
anode
cylindrical cathode
imert gas such as He or Ar
109
The most common flame burner for Atomic Absorption Spectrophotometer
Premix long-path burner
110
Function of flame in atomic absorption spectrophotometer
breaks the chemical bonds amd forms free unexcited atoms
111
Possible alternatives for flame in Atomic Absorption Spectrophotometer
Carbon rod or graphite surface
112
This is where the sample is mixed with air and fuel and produces fine droplets which reach the flame in AAs
Nebulizer
113
In AAS, this is used to isolate the desired emission line from other lamp emission lines and serves to protect the photodetector from excessive light.
Monochromator
114
Most commonly used light detector for the Atomic absorption spectrophotometer
Photomultiplier tube
115
It is where data is sent and processed for the generation of results in AAS
Data processor
116
Operation or Process in AAS
1. The sample in solution is aspirated as a spray into the chamber
2. Large drops fall as it passes into baffles and fine droplets reach the flame
3. Atoms become excited due to collision and releases specific wavelength as it returns to ground state.
4. Light passes through the ground state atoms and is absorbed.
5. Excited atoms are produced and returns to ground state.
6. There will be a steady emission due to dynamic population of absorbing and emitting atoms.
7. The Light detector distinguishes the light emitted from hollow cathode and light emitted from excited atoms.
117
Advantages of AAS
1. accurate, precise, specific
2. used for trace ,etals not ewsly excited
3. more sensitive than flame emissoom because atoms in propane or air - acetylene flame remains in ground state
118
Disadvantages of AAS
1. flame cannot dissociate sample into free atom
2. ionization of atoms
3. matrix interference
4. absorption of close species
5. scattering by salt particles or oxide
6. background emission
119
how to prenvent ionization of atoms in AAS
reducing flame temperature
120
how to reduce matrix interference in AAS
pretreatment of sample by extraction
121
In this photometry, the reaction mixture is illuminated with diffused light, and the intensity of reflected light is compared to a reference surface.
reflectance photometry
122
The relationship between the reflected light in reflectance photometry and the analyte concentration is
non linear
123
Commonly used to convert data from reflectance photometry into linear format
Kubelka Munk equation
| Clapper Williams transformation
124
Applications of Reflectance Photometry
1. automated therapeutic drug monitoring analyzer like Vitros
2. handheld POCT device for bedside testing
3. urinalysis
125
Spectrophotometry which is both a qualitative and quantitative technique generally used to find the composition of a physical sample by generating a mass spectrum representing the masses of sample components.
Mass spectrophotometry
126
Mass spectrophotometry measures the:
mass to charge ratio of ions
127
Principle of mass spectrophotometry
A sample is volatilized, ionized, and separated according to m/z.
The instensity of the ion current is detected.
128
Components of Mass spectrophotometer
1. Sample Inlet
2. Ionization source ( Gas phase ions)
3. Mass analyzer ( Ion sorting)
4. Iom detector
5. Data system
129
Process of Mass spectrophotometer
1. production of Ion
2. separation of ions according to mass
3. Number of ions of each mass
4. Data collection
5. Generation of mass spectrum
130
Mass Spectrophotometry is often applied in tandem tests with:
GC and HPLC
131
Most common form of ionization used in GC/MS
Electron Ionization
132
Most common form of ionization in LC/ MS
```
Electrospray Ionization (ESI)
Atmopsheric Pressure Chemical Ionization
```
133
Advanatages of Mass Spectrophotometry
1. large number of components in a single analysis (peptides)
2. analyzing small peptides bc provides better resolutiom, smaller number of charge state, sensitivity
3. analyze components without developing antibodies
4. large number of measurements
5. post translational modifications
134
Disadvantage of Mass Spectrophotometry
difficult to quantitaively interpret absolute magnitude of detector responses
135
Applications of Mass Spectrophotometry
1. protein concentration
2. genetic variant of pre albumom and carbohydrate deficient transferrin
3. small molecule analysis
4. GC/MS for urine toxicology drug abuse
5. LC/MS for low level and mixed polarity analytes (vitamim S, testosterone, immunosuppresants)
6. proteomics and pathogen identification
136
Most common light source for visible region & near-infrared
| Visible region: Only 15% of light emitted
tungsten
137
two forms of tungsten
tungsten iodide
| Incandescent tungsten
138
Increases lifetime of tungsten
presence of iodine or bromine vapor
139
Inserted between lamp & sample to absorb of infrared radiation
heat absorbing filter
140
light source used in measurement of the UV region
Deuterium discharge lamp
141
light source used to measure UV region but is less stable and has shorter life than deuterium discharge
hydrogen lamp
142
light source used to measure UV region but is less stable and has shorter life than deuterium discharge
hydrogen lamp
143
used in measurement of the UV region and has 3 forms
mercury arc lamp
144
mercurh lamp that emits a sharp line for UV and visible regions, but is used only for calibration purposes
Low pressure mercury arc lamo
145
mercury lamp for ciontinuum from UV to midvisible region
medium ans high presseure mercury lamp
146
minimizes and prevents stray light into monochromator
entrance slit
147
value of accurate absorbance
less than 1/5 of ntural absorbance of substance
148
value of accurate absorbance
less than 1/5 of ntural absorbance of substance
149
Allows passage of the isolated wavelength to the sample
exit slit
