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Clinical Chemistry (CC 1) > (P) Lec 5: Analytical Techniques in CC > Flashcards

(P) Lec 5: Analytical Techniques in CC Flashcards

1
Q

The 4 analytical techniques MAINLY used in the laboratory, except:
A. Electrophoresis
B. Osmometry
C. Turbidity
D. Nephlometry
E. NOTA

A

B

colorimetry, volumetric, turbidimetry, nephelometry, and electrophoresis

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2
Q

Transmitted via EM waves characterized by its frequency and wavelength

A

Energy

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3
Q

Analytes when placed in a machine are converted into?

A

energy

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4
Q

Wavelength

The distance between 2 successive peaks and is expressed in?

A

nanometer (nm)

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5
Q

TOF. Some analytes require specific wavelengths for their measurement.

A

T

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6
Q

3 regions where measurements are done

A

UV, Visible and Infrared region

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7
Q

Wavelength

less than 400 nm

A

UV Region

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8
Q

Wavelength

400 nm to 700 nm; majority of the analytes are measured here

A

Visible Region

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9
Q

Wavelength

TOF. Infrared (IR) Region has more than 800 nm.

A

F (700)

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10
Q

The Relationship Between Wavelength and Energy is Described by what formula?

A

E = hv

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11
Q

No. of vibrations of waves per second created during analysis

A

Frequency

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12
Q

TOF. Frequency is DIRECTLY proportional to wavelength and energy.

A

F.

Frequency is inversely proportional to wavelength and energy

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13
Q

Frequency

TOF. The lower the wave frequency, the longer the wavelength and energy.

A

T

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14
Q

Frequency

TOF. Wavelength and energy are directly proportional with each other.

A

T

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15
Q

Represent the wavelength in nm at peak transmittance of the analyt

A

Nominal Wavelength

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16
Q

Light that completely passed through the sample is called?

A

Peak transmittance

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17
Q

TOF. A slight error in adjustment can introduce significant errors in absorbance readings.

A

T

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18
Q

wavelength indicated on the control dial being the actual wavelength of light that has passed through (transmittance) the monochromator

A

Wavelength Accuracy

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19
Q

Wv accuracy

wavelength indicated on the control dial being the actual wavelength of light that has passed through (transmittance) the?

A

monochromator

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20
Q

Used to check for wavelength accuracy and proper calibration (quality control)

A

Didymium or Holmium Oxide Filter

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21
Q
  • Verifies the absorbance accuracy on linearity
  • Ensures correct readings on the samples in machines
A

Neutral Density Filters and Dichromate Solution

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22
Q

→ the color of the solution has an effect on the reading of the results

A

Colorimetry

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23
Q

This instruments measure light intensity without considering the wavelength (simple)

A

Photoelectric Colorimetry

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24
Q

this uses the isolation of discreet portions of the spectrum or wavelength for measurement purposes

A

spectrophotometry or filter photometry

photoelectric Colorimetry

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25
Q
A
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26
Q

Photoelectric Colorimetry

Two types of measurement:

A
  1. Spectrophotometric Measurment
  2. Photometric measurement
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27
Q
A
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28
Q
  • The measurement of light intensity in a narrower wavelength (narrow range)
  • May be on the UV, visible, or IR region
A

Spectrophotometric Measurement

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29
Q

methods of measurement

Measurement of light intensity

A

Photometric measurement

it’s also actually applicable to spectro also

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30
Q

TOF. Photoelectric measures the amount of light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution

A

F (Spectrophotometry)

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31
Q

Spectrophotometry

The light that passes through the sample

A

Light transmitted

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32
Q

Spectrophotometry

Follows what principle?

A

principle of Beer’s Law

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33
Q

Beer’s Law

TOF. The concentration of an unknown substance is Directly proportional to the absorbed light (absorbance or optical density.

A

T

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34
Q

Spectrophotometry

TOF. The concentration of the unkown substance is directly proportional to the amount of transmitted light (transmittance)

A

F (indirectly)

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35
Q

TOF. Beer’s law mathematically established the relationship between CONCENTRATION and ABSORBANCE.

A

T

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36
Q

Spectro

TOF. a sample that is darkly colored/turbid, has higher concentration

A

T

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37
Q

Spectrophotometry:

TOF. If you introduce the light on it, the lightt absorbance is also?

A

increased

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38
Q

TOF. As light absorbance is increased, the transmitted light is low.

A

T

If majority of the light is already absorbed, the amount of light that passes through it will be much lower

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39
Q

TOF. A sample that is lightly colored/clear has a high concentration and high light transmittance.

A

F (low concentration; high light transmisttance.

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40
Q

→ aka optical density
→ amount of light absorbed by the solution
→ proportional to the inverse logarithm of transmittance (reflected light)

A

Absorabnce A

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41
Q

TOF. Absorbance (A) is proportional to the DIRECT log of transmittance (reflected light)

A

F (inverse)

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42
Q

Absorbance (A)

mathematically derived from?

A

%T (Percent transmittance)

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43
Q

Absorbance

FORMULAE (3)

A

A = abc
A = 2 - log%T
A = -logT

A - absorbance
a - molar absorptivity (compound absorptivity under standard conditions)
b - length of light through the solution
c - concentration of absorbing molecules or solutions

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44
Q

Formula for the concentration of the unknown

A

Cu = Au/As x Cs

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45
Q

TOF. The absorbance portion is provided by the machine using the abc formula.

A

T

Absorbance standard; ang need lang natin hanapin is Cu, la ka na pake sa iba

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46
Q

TOF. In the laboratory, we receive the concentration of the known derived from the absorbance formula.

A

F (unknown)

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47
Q

Formula of Percent Transmittance (%T)

A

%T = It/Io x 100

ratio of the radiant energy transmitted (T) divided by the radiant energy incident in the sample (I)

kahit ‘wag na kabisa kasi sabi galing lang rin naman sa machine ‘to makukuha

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48
Q

Percent Transmittance (%T)

It

A

transmittance of light through the sample

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49
Q

Percent Transmittance (%T)

Io

A

transmittance of light striking the sample

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50
Q

TOF. if the sample is blank/clear, light can pass through it completely, assuring a 90% transmittance.

A

F (100%)

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51
Q

Single Beam

A. the simplest type of absorption spectrometer
B. designed to make one measurement at a time at one specified wavelength
C. absorption maximum or wavelength of the analyte must be known in advance when used
D. AOTA

correct where

A

D

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52
Q

Double beam spectrophotometer

  • An instrument that splits the monochromatic light into 2 components: beam for a sample and a beam for a reference solution
  • the sample absorbance can be recorded directly as the electrical output of the sample beam
A

Regular double beam

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53
Q

Regular Double Beams

This beam corrects for the variation in the light source intensity

A

The second beam (that passes through the reference solution or blank)

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54
Q

→ uses 2 photodetectors for the sample beam and reference beam

A

Double Beam in Space

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55
Q

→ uses 1 photodetector and alternately passes the monochromatic light through the sample cuvette and then through the reference cuvette using a chopper or rotating sector mirror

A

Double Beam in Time

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56
Q

THE 6 COMPONENTS OF A SINGLE OR DOUBLE BEAM CONFIGURATION SPECTROPHOTOMETER

A

Stable source of radiant energy (light source)
Filter that will isolate a specific region of the EM spectrum
Sample holder
Radiation detector
Signal processor
Read-out device

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57
Q

PARTS OF THE SPECTROPHOTOMETER

→ provides polychromatic light and must generate sufficient power to measure the analyte of interest
→ an intense beam of light is directed through the monochromator in the sample

A

Light/Radiant Energy

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58
Q

Light/Radiant Energy

to give accurate absorbance measurements throughout its absorbance range, the response to a change in light intensity should always be?

A

linear

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59
Q

Light/Radiant Energy

TOF. If the light changes and the intensity continuously increase, it should decrease after some time.

A

F (kapag increase, increase lang, same for decreasing)

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60
Q

Light/Radiant Energy

→ emits radiation that **changes in intensity (is flexible) **which should be linear
→ the most widely used in the laboratory

A

Continuum Source

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61
Q

most commonly used light source in the visible and near IR region

A

Tungsten Light Bulb

62
Q

Continuum Source

→ routinely used to provide UV radiation in analytic spectrometers

A

Deuterium Lamp

63
Q

Continuum Source

→ produces a continuous source of radiation which covers both the UV and visible range

A

Xenon Discharge Lamp

64
Q

ALTERNATIVES FOR TUNGSTEN

Visible and UV

A

Mercury Arc

65
Q

ALTERNATIVES FOR TUNGSTEN

UV

A

Deuterium Lamp
(165 nm)
Hydrogen Lamp
Xenon Lamp

66
Q

ALTERNATIVES FOR TUNGSTEN

IR

A

Merst Glower
Globar (Silicone Carbide)

67
Q

Line Source

An example used to measure UV and visible regions

A

Mercury Sodium Vapor Lamps

68
Q

Line source

light source used for AAS

A

Hollow Cathode Lamps

69
Q

FACTORS TO CONSIDER WHEN CHOOSING A LIGHT SOURCE

A
  • Range
  • Spectral distribution within the range
  • Source of radiant production
  • Stability of the radiant energy
  • Temperature
70
Q

FACTORS TO CONSIDER WHEN CHOOSING A LIGHT SOURCE:

  • if it can measure the UV, visible, or IR region
71
Q

FACTORS TO CONSIDER WHEN CHOOSING A LIGHT SOURCE:

For proper storage

A

Temperature

72
Q

FACTORS TO CONSIDER WHEN CHOOSING A LIGHT SOURCE:

if it’s a continuum or line source

A

Source of radiant production

73
Q

→ minimizes unwanted or stray light; prevents entrance of scattered light into the monochromator system

A

Entrance slit

74
Q

Stray Light

TOF. Does not originate from the polychromatic light source.

75
Q

Stray Light

TOF. Any wavelength INSIDE the band transmitted by the monochromator.

A

F (outside)

76
Q

Stray Light

TOF. The most common cause of loss of linearity at a high-analyte concentration.

77
Q

Stray light

TOF: It LIMITS the maximum absorbance that a spectrophotometer can achieve

78
Q

this isolates specific or individual wavelengths of light; each one has their own band pass

A

Monochromator

79
Q
  • The total range of wavelengths transmitted
  • Defines the range of wavelengths transmitted and is calculated as width at more than half the maximum transmittance
80
Q

TYPES OF MONOCHROMATORS

→ simple, least expensive, not precise, but useful

81
Q

TYPES OF MONOCHROMATORS

→ usually passes a relatively wide band of radiant energy and has a low transmittance of the selected wavelength

82
Q

TYPES OF MONOCHROMATORS

→ made by placing semi-transparent silver films on both sides of a dielectric (magnesium fluoride)

83
Q

→ produces monochromatic light based on the principle of constructive interference of waves wherein light waves enter one side of the filter and are reflected at the second surface

A

Interference Filters

84
Q

TYPES OF MONOCHROMATORS

→ wedge-shaped pieces of glass, quartz, or sodium chloride
→ a narrow beam focused on a prism is refracted as it enters the more dense glass (advantage over filters)
→ the prism can be rotated, allowing only the desired wavelength to pass through an exit slit

85
Q

refracted more than long wavelengths, resulting in a dispersion of white light into a continuous spectrum

A

short wavelengths

86
Q

TYPES OF MONOCHROMATORS

→ most commonly used; gives a better resolution compared to prisms
→ consists of many parallel/cutting grooves (15k or 30k per inch) or slits into an aluminized surface of a flat piece of crown glass etched onto a polished surface

A

Diffraction Gratings

87
Q

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

A

Diffraction

88
Q

TYPES OF MONOCHROMATORS

→ much more expensive than diffraction gratings

A

Holographic Gratings

89
Q

→ controls the width of the light beam (bandpass)
→ allows only a narrow fraction of the spectrum to reach the sample cuvette (becomes more select on the fraction that is needed)
→ accurate absorbance measurement requires a bandpass of less than ⅕ of the natural bandpass of the spectrophotometer

90
Q

Exit slit

→ accurate absorbance measurement requires a bandpass of less than _____ of the natural bandpass of the spectrophotometer

91
Q

Exit slit

TOF. spectral purity is reflected by the bandpass wherein: the NARROWER the bandpass, the greater the resolution

92
Q

→ aka absorption, analytical, or sample cell
→ it holds the solution whose concentration is to be measured later on

A

Cuvette/Cuvet

93
Q

Cuvette

→ most commonly used in 350-2,000 nm (wide range)

A

Alumina Silica Glass

94
Q

Cuvette

→ for measuring solutions requiring visible and UV spectra

A

Quartz/Plastic

95
Q

Types of Cuvette

A
  • Alumina Silica Glass
  • Quartz/Plastic
  • Borosilicate Glass
  • Soft Glass
96
Q

TOF. Cuvettes with scratches on their optical surface produce scattered light (should be discarded).

97
Q

Silica cuvettes can transmit light effectively at a wavelength of?

A

more than 220 nm

98
Q

TOF. Alkaline solutions can be left in cuvettes for prolonged periods.

A

F

it can damage the cuvette along with the readings

99
Q

The path of length of the cuvette is

100
Q

TOF. Much LONGER path lengths are used in automated systems

101
Q

TOF. To increase sensitivity, some are designed to have a path length of 20cm.

102
Q

→ detects and converts transmitted light into photoelectric energy
→ detects the amount of light that passes through the sample in the cuvette

A

Photodetector

103
Q

Kinds of photodetector:

A
  • Photocell/Barrier Cell/Photovoltaic Cell
  • Phototube
  • Photomultiplier Tube (PMT)
  • Photodiode
104
Q

Photodetector

→ the simplest and least expensive
→ is temperature sensitive (disadvantage)
→ used in filter photometers with a wide bandpass
→ a basic phototransducer used for detecting and measuring radiation in the visible region
→ composed of selenium on a plate of iron covered with a transparent layer of silver
→ advantage: it requires no external voltage source but utilizes internal electron transfer for the production of a low internal resistance current (advantage)

A

Photocell/Barrier Cell/Photovoltaic Cell

105
Q

Photodetector

→ contains a cathode and anode enclosed in a glass case (positive and negative poles)
→ has a photosensitive material that gives off electrons when light energy strikes it which is converted into energy
→ requires an external voltage for operation

106
Q

Photodetector

→ most commonly used and most sensitive; has a rapid response and can detect very low levels of light (highly concentrated solution)
→ measures both the visible and UV region
→ the response begins when incoming photons strike a photocathode
→ prone to breakage when exposed to room light (disadvantage)
→ are limited to measuring low power radiation because intense light can cause irreversible damage to the surface (another disadvantage)

A

Photomultiplier Tube (PMT)

107
Q

→ not as sensitive as the PMT but has excellent linearity
→ measures light at a multitude of wavelengths (lesser amounts of light)
→ has a lower dynamic range and higher noise compared to PMT
→ most useful as a simultaneous multi channel detector

A

Photodiode

108
Q

→ displays the amount of the detection system
→ examples: galvanometers, ammeters, and LED displays

A

Meter or Read-Out Device

109
Q

Color absorbed and complementary color

350-430

A

Violet, Yellow-Blue

110
Q

Color absorbed and complementary color

431-475

A

Blue, Yellow

111
Q

Color absorbed and complementary color

476-495

A

green-blue, orange

112
Q

Color absorbed and complementary color

496-505

A

blue green, red

113
Q

Color absorbed and complementary color

506-555

A

Green, purple

114
Q

Color absorbed and complementary color

556-575

A

yellow-green, violet

115
Q

Color absorbed and complementary color

576-600

A

yellow, blue

116
Q

Color absorbed and complementary color

601-650

A

orange, green-blue

117
Q

Color absorbed and complementary color

651-700

A

red, blue-green

118
Q

Read-Out Device

color detected by our naked eye

A

Color absorbed

119
Q

Read-Out Device

how the machine reads the sample

A

Complementary color

120
Q

Amount of light absorbed at a particular wavelength depends on?

A

molecular and ion type present

This amount may also vary with concentration, pH, and temperature

121
Q

TOF. The light path must be kept constant to have an absorbance proportional to the concentration.

122
Q

A change in instrument or chemical reactions, will cause a deviation in?

A

Beer’s law

123
Q

commonly a result of a finite bandpass of the filter or monochromator

A

Instrument deviations

124
Q

Turbidity readings on spectrophotometers are greater in the?

A

blue region

125
Q

TOF. The linearity of the spectrophotometer is determined using **UV filters or solutions **that have known absorbance values for a given wavelength

A

F (optical filters or solution)

126
Q

a blank solution contains serum WITHOUT a reagent to complete the assay

A

BLANKING TECHNIQUE

127
Q

Which is false about BLANKING TECHNIQUE
A. Effective for high turbidity serum
B. Helps to check in absorbance
C. Correct for artifactual absorbance readings or dual-wavelength methods may be used

128
Q

Blanking technique

what is necessary to clear the serum of plasma and chylomicrons

A

ultracentrifugation

129
Q

Blanking Technique

Scattering light (turbidity) is increased because of this interference

130
Q

measures light emitted by a single atom burned in a flame

A

Flame Emission Photometry (FEP) or Spectrophotometry

131
Q

Flame Emission Photometry (FEP) or Spectrophotometry

Which is false:
A. measurement of excited ions (e.g. electrolytes such as Na, K, Cl, Ca, and Mg)
B. excitation of electrons from their higher state to lower energy state
C. corrects variations in flame and atomizer characteristics
D. requires an indirect internal standard method

132
Q

FEP

this requires an indirect internal standard method

A

Lithium/cesium

133
Q

Migration of charged particles in an electric field

A

Electrophoresis

134
Q

Atomic Absorption Spectrophotometry (AAS)

measures light absorbed by atoms dissociated by?

135
Q

AAS

TOF. the element is not excited but dissociated from its chemical bond and placed in an unionized, unexcited, and ground state.

136
Q

the light source ni AAS

A

hollow-cathode lamp

138
Q

Atomic Absorption Spectrophotometry (AAS)

Which is false:
A. measure of unexcited ions
B. less sensitive, accurate, and specific compared to FEP
C. internal standard is not needed

139
Q

ADDITIONAL PARTS OF THE AAS

this converts ions into atoms

A

Atomizer/Nebulizer/Graphite Furnace

140
Q

ADDITIONAL PARTS OF THE AAS

→ modulates and controls the light source

141
Q

ADDITIONAL PARTS OF THE AAS

→ added to samples to form stable complexes with phosphate
→ this reduces interferences because of the chemical bond created with phosphate

A

Anthanum/Strontium Chloride

142
Q

VOLUMETRIC (TITRIMETRIC)

A. It determines the amount of scattered light by a particulate matter suspended in a turbid solution
B. It determines the amount of light blocked (how much light is reduced) by a particulate matter in a turbid solution
C. The unknown sample is made to react with a known solution is the presence of an indicator

143
Q

The volumetric is mainly used for electrolyte measurement of?

A

chloride and calcium

144
Q

Turbidmetry

A. It determines the amount of scattered light by a particulate matter suspended in a turbid solution
B. It determines the amount of light blocked (how much light is reduced) by a particulate matter
C. The unknown sample is made to react with a known solution is the presence of an indicator

145
Q

Turbidmetry

TOF. Measurement of abundant small particles (proteins) and bacterial suspensions.

A

F LARGE PARYICLES

146
Q

Turbidimetry

TOF. Is not independent on the specimen concentration and particle size.

147
Q

Turbidimetry

applications include, except:
A. Protein measurement
B. Detects bacterial growth in broth cultures
C. Antimicrobial test
D. Detects clot formations
E. A, B C
F. A, D
G. ABCD

A

All are correct

148
Q

NEPHELOMETRY

A. It determines the amount of scattered light by a particulate matter suspended in a turbid solution
B. It determines the amount of light blocked (how much light is reduced) by a particulate matter
C. The unknown sample is made to react with a known solution is the presence of an indicator

149
Q

NEPHELOMETRY

TOF. Light scattering is dependent on the specimen concentration and particle size.

A

F (that’s for turbidimetry, nephelometry is dependent on the wavelength and particle size)

150
Q

NEPHELOMETRY

WHICH IS MALI:
A. Light is scattered forward
B. for measuring the amount of antigen-antibody complexes
C. the wavelengths used are 320-650 nm
D. detector (PMT) output is inversely proportional to the concentration

151
Q

THE 6 COMPONENTS OF A NEPHELOMETER

A

Light source
Collimator
Monochromator
Sample cuvette
Stray light trap
Photodetector

152
Q

COMPONENTS OF A NEPHELOMETER

narrows the beam of particles with waves (sample) for easy measurement; direction of scattered light becomes more aligned

A

Collimator

Clinical Chemistry (CC 1) (33 decks)

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