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Clinical Chemistry > Module 3a: Photometry and Spectrophotometry > Flashcards

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

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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

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3
Q

Light energy is a form of ________ radiation

A

Electromagnetic

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4
Q

light travels in packets of radiant energy called _____ or _____

A

photons or quanta

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5
Q

light energy is characterized by _______ and _______

A

frequency and wavelength

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6
Q

definition: FREQUENCY

A

number of vibrations of wave per second, has a direct relationship with energy

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7
Q

definition: WAVELENGTH

A

distance between two successive peaks in nanometers, has an inverse relationship with energy and frequency

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8
Q

light is used to describe radiant energy from the spectrum _____ to ____ nm

A

290 to 750 nm (ultraviolet to visible light)

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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

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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

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11
Q

basic components of a spectrophotometer according to the path of light

A
  1. Light source
  2. Entrance slit
  3. Monochromator
  4. Exit slit
  5. Cuvette with sample
  6. Photodetectors
  7. Meter or Readout devices
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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.

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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)
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14
Q

light that has already passed through sample

A

transmitted light

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15
Q

light that hasn’t passed through a sample

A

incident light

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16
Q

percent transmittance is the ratio of ____

A

sample beam signal/ blank beam signal

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17
Q

% transmittance when all light is absorbed

A

0%

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18
Q

% transmittance when no light is absorbed

A

100%

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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

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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

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21
Q

Tungsten/Tungsten Iodide Lamp does not supply sufficient radiant energy or measurements below ____ nm

A

320 nm

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22
Q

Provide sources of continuous spectra in the UV region with some sharp
emission lines.

A

Hydrogen and deuterium lamps

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23
Q

Deuterium lamps provide continuous emission down to ___ nm.

A

165 nm

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24
Q

Advantage of deuterium lamp over Hydrogen lamp

A

more stable and has longer life

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25
Q

incandescent lamp which is useful for calibration purposes but is not practical or absorbance measurements because it is used only at certain wavelengths.

A

Low Pressure Mercury Arc Lamp

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26
Q

Low Pressure Mercury Arc Lamp discontinuous or sharp line spectrum with both ____ and ___ lines

A

UV and visible lines.

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27
Q

Low Pressure Mercury Arc Lamp is commonly used in

A

high-pressure liquid chromatographic (HPLC)

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28
Q

Incandescent lamp that has a very-narrow-wavelength intense source.

A

Hollow cathode lamp

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29
Q

incandescent lamp used to measure peptides and proteins due to its giving a line of _____ nm, which is close to ______

A

zinc hollow cathode lamp, 214 nm, 206 nm

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30
Q

LASER meaning

A

light amplification by stimulated emission of radiation

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31
Q

laser transforms light of various frequencies into an _____

A

> extremely intense
focused
nearly nondivergent beam of
monochromatic light

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32
Q

LED meaning

A

Light Emitting Diodes

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33
Q

LED lights are made of _______ and _____

A

semiconductor ( aluminum gallium arsenide) and insulator.

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34
Q

monochromators are devices for ______

A

Spectral Isolation

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35
Q

system for isolating radiant energy at a desired wavelength and excluding
that of other wavelengths

A

monochromators.

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36
Q

defines the range of wavelengths transmitted

A

bandpass

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37
Q

calculation of bandpass

A

width at more than half the maximum transmittance

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38
Q

three types of light source

A

Incandescent Lamps
Lasers Sources
LED

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39
Q

three types of incandescent lamps

A

Tungsten/Tungsten Iodide Lamp
Hydrogen and deuterium lamps
Low Pressure Mercury Arc Lamp

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40
Q

three types of monochromators

A

Filters, prisms and diffracting gratings

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41
Q

Simplest type of filter but not considered a true monochromator because it transmits light over a relatively wide range of wavelengths.

A

Glass filter (thin layer of colored glass)

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42
Q

Glass filters are also known as

A

wide bandpass filter

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43
Q

refers to the spectral purity of a filter or other monochromators.

A

Spectral bandwidth/ Spectral bandpass

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44
Q

used to eliminate light below a given wavelength

A

Sharp-cutoff filter

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45
Q

uses dielectric material of controlled thickness sandwiched between two thinly
silvered pieces of glass

A

Narrow-bandpass or interference filters

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46
Q

the width of the transparent spacer in interference filters is______

A

one-half the desired wavelength

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47
Q

separates white light into a continuous spectrum through refraction

A

prism

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48
Q

____wavelengths are bent to a greater extent as they pass through a prism

A

shorter

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49
Q

most commonly used monochromator which has extreme accuracy and low light scatter

A

Diffraction grating:

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50
Q

separation of light into component wavelengths based on the principle that wavelengths bend as they pass a sharp corner.

A

Diffraction

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51
Q

used to hold a liquid sample to be analyzed in the light path of a spectrometer

A

cuvette

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52
Q

Cuvets suitable for measurements in the visible portion of the spectrum

A

Ordinary borosilicate glass cuvets

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53
Q

Cuvettes used for readings below 340 nm

A

Quartz cells

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54
Q

devices which convert the transmitted radiant energy into an equivalent amount of electrical energy

A

Photodetectors

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55
Q

It is the most commonly used monochromator. It is extremely accurate and have low light scatter.

A

diffraction grating

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56
Q

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.

A

aluminum-copper alloy

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57
Q

the separation of light into component wavelengths based on the principle that wavelengths bend as they pass a sharp corner.

A

difrraction

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58
Q

Gratings with very fine line rulings produce a widely dispersed spectrum– a linear spectra, which are called __________

A

orders

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59
Q

HPLC detectors frequently use a _____________ in their optical system

A

concave holographic reflective grating

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60
Q

3 types of diffraction gratings

A

a. plane transmission gratings
b. reflective gratings
c. echelette

61
Q

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.

A

plastic cuvets

62
Q

Cuvets may be cleaned though soaking in a mixture of

A

concentrated HCl/water/ethanol (1:3:4)

63
Q

cuvets should never be soaked in

A

dichromate cleaning solution

64
Q

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.

A

Photomultiplier tube

65
Q

advantages of PMT photodetector

A
  1. extremely rapid response times
  2. sensitive
  3. slow to fatigue
66
Q

2 types of solid state detectors

A

photodiodes

charge-coupled detectors

67
Q

solid state detctor that fabricated from photosensitive semiconductor materials which is capable of measuring light at a multitude of wavelengths

A

photodiodes

68
Q

Multi- channel devices with good dynamic ranges and signal to noise ratios that are superior to those of PM tubes.

A

Charge coupled detectors

69
Q

Charge coupled detectors operates like:

A

a large number of photo detecting shift registers read horizontally and vertically

70
Q

Main application of of charge coupled detectors

A
  • molecular flourescence measurement of very low concentration fluorophores
71
Q

photocell is also known as

A

barrier-layer cell
selenide cell
photovolataic cell

72
Q

Photodetectors used in older instruments and is the least expensive

73
Q

Photocells are composed of:

A

thin layer of silver over a film of selenium on a plate of iron

74
Q

Adavantages of Photocell

A

inexpensive and durable

75
Q

disadvantages of photocell

A

temperature sensitive

non linear at very and very low illumination

76
Q

similar to photocell but requires and outside voltage to operate and is more sensitive

77
Q

phototube contains:

A

an anode and a cathode in a glass case

78
Q

Displays the electrical energy from a detector

A

readout devices

79
Q

Readout devices can be classified into:

A

direct reading

null point system

80
Q

readout device where the output of the photocell is used to drive a sensitive meter directly, without further amplification

A

direct reading system

81
Q

readout device where the output of the detector is balanced against the output of a reference circuit and direct digital readouts are obtained

A

null point system

82
Q

readout device that has faster response, is easier to read and decreases operator fatigue

A

digital readout devices

83
Q

digital readout devicds operate on the principle of

A

selective illumination of LED

84
Q

Types of spectrophotometers

A

Single beam spectrophotometer
Double beam in space spectrophotometer
Double beam in time spectrophotometer

85
Q

Composed of one of each of the basic components of the spectrophotometer, and a single measurement is derived

A

single beam spectrophotometer

86
Q

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.

A

Double beam spectrophotometer

87
Q

In double beam in space spectrophotometer, all components are doubled except:

A

the light source and the readout device

88
Q

Spectrophotometer that has two detectors which allow simultaneous measurement of the sample and the standard.

A

Double beam in space spectrophotometer

89
Q

In double beam in time spectrophotometer, all components are single except the:

90
Q

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.

91
Q

Spectrophotometer where in the detector alternates between measuring the sample beam and the reference beam in synchronism with the chopper.

A

Double beam in time spectrophotometer

92
Q

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.

A

Flame Emission Photometry

93
Q

Flame emission photometry os based on the principle:

A

excited atoms returning to ground state emit light energy

94
Q

In flame photometry, ____________ is being measured

A

emission of light

95
Q

Flame emission photometry is widely used in determining the concentration of

A

Sodium (Na)
Potassium (K)
Lithium (Li)

96
Q

Six basic components of Flame photometer

A
  1. aspirator
  2. atomizer
  3. flame
  4. lense
  5. monochromator
  6. detector
97
Q

Process of flame photometry

A
  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
Q

fuel source of the flame in flame emission photometry

A

propane and compressed air

99
Q

Flame colors and monochromators used for Na, K, Li

A

Na - yellow - 589 nm
K - violet - 767 nm
Li - red - 671 nm

100
Q

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

A

High concentration of Lithium or Cesium

101
Q

Advantages of FEP

A

accuracy and precision of +/- 1-5% in aqueous solution
suitable for metallic ions
fast, simple
free from interference

102
Q

Limitations of FEP

A

can only measure pure metals

other variables affecting instensity of light

103
Q

FEP in routince clinical laboratory are used in the determination of

A

lithium levels in serum of manic-depression patients on lithium therapy

104
Q

Ions that give detectable signs for FEP but cannot be used due to insufficient reliabilty and sensitivity

A

Magnesium, Calcium

105
Q

spectrophotometry that is considered the inverse of FEP

A

Atomic absorption spectrophotometry

106
Q

Principle of Atomic Absorption spectrophotometry

A

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
Q

Components of an Atomic Absorption Spectrophotometer

A
  1. Hollow cathode lamp corresponding to metal being measured
  2. Flame as the sample cell
  3. Nebulizer
  4. Monochromator
  5. Light detector
  6. Data processor
108
Q

The evacutaed gas tight chamber of a hollow cathode lamp in Atomic Absorption spectrophotometer contains (3)

A

anode
cylindrical cathode
imert gas such as He or Ar

109
Q

The most common flame burner for Atomic Absorption Spectrophotometer

A

Premix long-path burner

110
Q

Function of flame in atomic absorption spectrophotometer

A

breaks the chemical bonds amd forms free unexcited atoms

111
Q

Possible alternatives for flame in Atomic Absorption Spectrophotometer

A

Carbon rod or graphite surface

112
Q

This is where the sample is mixed with air and fuel and produces fine droplets which reach the flame in AAs

113
Q

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.

A

Monochromator

114
Q

Most commonly used light detector for the Atomic absorption spectrophotometer

A

Photomultiplier tube

115
Q

It is where data is sent and processed for the generation of results in AAS

A

Data processor

116
Q

Operation or Process in AAS

A
  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
Q

Advantages of AAS

A
  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
Q

Disadvantages of AAS

A
  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
Q

how to prenvent ionization of atoms in AAS

A

reducing flame temperature

120
Q

how to reduce matrix interference in AAS

A

pretreatment of sample by extraction

121
Q

In this photometry, the reaction mixture is illuminated with diffused light, and the intensity of reflected light is compared to a reference surface.

A

reflectance photometry

122
Q

The relationship between the reflected light in reflectance photometry and the analyte concentration is

A

non linear

123
Q

Commonly used to convert data from reflectance photometry into linear format

A

Kubelka Munk equation

Clapper Williams transformation

124
Q

Applications of Reflectance Photometry

A
  1. automated therapeutic drug monitoring analyzer like Vitros
  2. handheld POCT device for bedside testing
  3. urinalysis
125
Q

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.

A

Mass spectrophotometry

126
Q

Mass spectrophotometry measures the:

A

mass to charge ratio of ions

127
Q

Principle of mass spectrophotometry

A

A sample is volatilized, ionized, and separated according to m/z.
The instensity of the ion current is detected.

128
Q

Components of Mass spectrophotometer

A
  1. Sample Inlet
  2. Ionization source ( Gas phase ions)
  3. Mass analyzer ( Ion sorting)
  4. Iom detector
  5. Data system
129
Q

Process of Mass spectrophotometer

A
  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
Q

Mass Spectrophotometry is often applied in tandem tests with:

A

GC and HPLC

131
Q

Most common form of ionization used in GC/MS

A

Electron Ionization

132
Q

Most common form of ionization in LC/ MS

A 
Electrospray Ionization (ESI)
Atmopsheric Pressure Chemical Ionization
133
Q

Advanatages of Mass Spectrophotometry

A
  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
Q

Disadvantage of Mass Spectrophotometry

A

difficult to quantitaively interpret absolute magnitude of detector responses

135
Q

Applications of Mass Spectrophotometry

A
  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
Q

Most common light source for visible region & near-infrared

Visible region: Only 15% of light emitted

137
Q

two forms of tungsten

A

tungsten iodide

Incandescent tungsten

138
Q

Increases lifetime of tungsten

A

presence of iodine or bromine vapor

139
Q

Inserted between lamp & sample to absorb of infrared radiation

A

heat absorbing filter

140
Q

light source used in measurement of the UV region

A

Deuterium discharge lamp

141
Q

light source used to measure UV region but is less stable and has shorter life than deuterium discharge

A

hydrogen lamp

142
Q

light source used to measure UV region but is less stable and has shorter life than deuterium discharge

A

hydrogen lamp

143
Q

used in measurement of the UV region and has 3 forms

A

mercury arc lamp

144
Q

mercurh lamp that emits a sharp line for UV and visible regions, but is used only for calibration purposes

A

Low pressure mercury arc lamo

145
Q

mercury lamp for ciontinuum from UV to midvisible region

A

medium ans high presseure mercury lamp

146
Q

minimizes and prevents stray light into monochromator

A

entrance slit

147
Q

value of accurate absorbance

A

less than 1/5 of ntural absorbance of substance

148
Q

value of accurate absorbance

A

less than 1/5 of ntural absorbance of substance

149
Q

Allows passage of the isolated wavelength to the sample

Clinical Chemistry (14 decks)

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