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MEDL 210 > Module 8 > Flashcards

Module 8 Flashcards

1
Q

What does flame emission photometry measure?

A

Sodium, potassium and lithium.

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2
Q

Why is the wavelength used for the emission photometry chosen for a particular element?

A

Has sufficient intensity to provide adequate sensitivity.

Free from other interfering lines.

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3
Q

What percentage of atoms are excited by flame photometry?

A

5%

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4
Q

What are standards used for?

A

Making a calibration curve.

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5
Q

How is a flame emission calibration made?

A

Concentration vs emission

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6
Q

What are the components of a flame photometer?

A

Atomizer/burner assembly- aspirates a constant amount of solution into the flame

Wavelength selector- interference filters with narrow bandpass

Photodetector

Signal processor- extracts and amplifies the difference between the sample and reference signals

Display

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7
Q

What is the aspiration of solution into the flame during flame emission called?

A

Nebulization

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8
Q

What keeps the flame temperature constant during emission photometry?

A

Fuel type and oxidant- usually propane and air (1925°C), compressed air is filtered

Fuel to oxidant ratio- pressure regulators

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9
Q

What types of interference are there in flame photometry?

A

Spectral

Ionization

Physical interferences

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10
Q

What causes spectral interference?

A

Other substances emit light- use low temp and filters

Self absorption- emitted energy is absorbed by atoms of the element, reduce by diluting

Mutual excitation- transfer of energy from one atom to one of another element, compensate by using standards with concentrations in the physiological range or an internal standard

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11
Q

When does ionization normally take place?

A

At higher temps

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12
Q

What is physical interference due to?

A

Surface tension- use a wetting agent/detergent

Viscosity- dilute the sample

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13
Q

What is the function of an internal standard?

A

Compensates for variations in air pressure (flame temp), aspiration rate and mutual excitation.

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14
Q

What is an internal standard?

A

Solution of known concentration, structurally similar to analytes of interest.

Added to all samples.

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15
Q

What characteristics are desired for internal standards?

A

High emission intensity

Normally not in biological fluids

Emission at a wavelength removed from sodium and potassium

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16
Q

How does compensation occur with an internal standard?

A

Variations in signal readings of analytes and the standard affect the signal readings, any deviations in the standard can be applied to the analyte.

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17
Q

How does an internal standard act as a radiation buffer?

A

Minimizes mutual excitation effects.

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18
Q

What is scatter dependent on?

A

The wavelength used and the relation to the size of particles.

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19
Q

What types of scatter are there?

A

Rayleigh

Rayleigh-Debye

Mie

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20
Q

Why is a monochromator used?

A

To select a specific wavelength and produce consistent scatter patterns.

21
Q

What does turbidity measure?

A

The decreasing intensity of incident light due to scattering.

22
Q

What is the relationship between light measured in turbidity and concentration?

A

Inverse

23
Q

What is turbidimetry used for?

A

Bacterial growth and antimicrobial sensitivity

Hematology analyzers to measure clot formation

24
Q

What does nephelometry measure?

A

The amount of light scattered.

25
Q

What type of relationship does the amount of light scattered have to concentration?

A

Direct

26
Q

Where is the photodetector placed in nephelometry?

A

At an angle to the light path, usually 90°

27
Q

What are the uses of nephelometry?

A

Measuring plasma proteins by forming immune complexes

Hematology analyzers to count and identify blood cells

28
Q

How does flame emission photometry work?

A

Heat excites the electrons, when they return to ground state them emit energy in the form of a characteristic wavelength which is related to the initial concentration.

29
Q

What are the types of sources of error in light scattering methods?

A

Variation in particle size- try to reproduce and control conditions

Matrix effects- other substances scatter light, use sample blanks

Debris

Fluorescence- can increase in measured light

Settling of particles- mix prior to reading

30
Q

What is fluorescence?

A

Absorption of light energy excites an electron, energy is lost during radiation vibrationless deactivation, when it drops to the ground state light is emitted.

31
Q

How do the wavelengths absorbed and emitted differ in fluorescence?

A

The wavelength is emitted is longer.

Wavelength absorbed is UV.

32
Q

What is Stoke’s shift?

A

Loss of energy due to RVD.

The difference between Max A wavelength and max wavelength of the emitted light.

33
Q

What is fluorescence used for?

A

Tags or markers

Hematology analyzers and flow cytometers- blood cell ID

Microbiology analyzers that measure bacterial growth

Microscopy methods for cell marker or organism ID

Immunoassay analyzers- detects drugs

34
Q

What are the advantages of fluorometry over flame emission spectrophotometry?

A

More specific- only one wavelength is measured

More sensitive

35
Q

What are the differences between fluorometers and spectrofluorometers?

A

Fluorometers- use glass or interference filters

Spectrofluorometers- use prisms or diffraction gratings

36
Q

Where are measurements made in fluorometry?

A

90°to the incident beam

Reduces interference

37
Q

How many monochromatic devices are needed for fluorometry and what are their purposes?

A

Two

Primary- isolates the excited wavelength

Secondary- isolates the emitted wavelength

38
Q

What are the components of a fluorometer?

A

Light source- intense radiant energy

Wavelength detector- diffraction gratings

Sample holder- glass (plastic can absorb UV light)

Photodetector- photomultiplier tubes

39
Q

What are the limitations of fluorescence?

A

Inner filter effect

Quenching- self absorption at high concentrations

Light scattering- background interference

Solvent effects- impurities may fluoresce

Sample matrix effects- background fluorescence

Temperature effects- fluorescence intensity decreases with increasing temp

40
Q

What happens to fluorescence when concentration increases?

A

Excitation intensity is lost as molecules absorb the light.

Only see a linear relationship if

41
Q

When does light scattering occur?

A

With a small Stoke’s shift, excitation and emission spectra overlap.

Reduce with narrow bandpass filters or using wavelengths far apart.

42
Q

What are the biggest contributors to sample matrix effects but why are they normally not a problem?

A

Bilirubin and proteins

Excitation maxima are >300nm

43
Q

What does reflect since photometry measure?

A

Reflected light, depends on the analyte concentration.

44
Q

What is the test sample for reflect acne photometry?

A

Dry reagent pad/slide with a reflective surface.

45
Q

What are the layers of the dry reagent pad?

A

Spreading layer- sample applied to top, underside reflective

Reagent/indicator layer- contain reagent for reaction, products absorbs light

Support layer- clear, allows light through

46
Q

What happens to light that isn’t absorbed by the spreading layer?

A

It’s reflected and measured.

47
Q

How is the amount of light reflected in reflect acme photometry related to concentration?

A

Inversely

48
Q

What are the standards used for reflectance photometry?

A

Zero concentration- white surface, 100% reflectance

High concentration- black surface, 0% reflectance

MEDL 210 (12 decks)

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