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Bioanalytical Science > Spec > Flashcards

Spec Flashcards

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1
Q

What is the principle of spec?

A

To measure the intensity of light energy absorbed by an analyte solution at a selected wavelength.

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2
Q

What is the visible spectrum?

A

390 - 770nm

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3
Q

What is the UV spectrum?

A

~100 - 400nm

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4
Q

What is spec in terms of analytical?

A

it is a quantitative and qualitative analytical tool.

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5
Q

What are the 2 basic concepts of spec?

A
  • That light absorption is exponentially related to the no. of molecules in the absorbing solute (conc.
  • Light absorption is exponentially related to the the length of the light path through the absorbing solution.
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6
Q

What is the intensity of the transmitted light defined as?

A

T = I/Io

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7
Q

What are some of the errors of the incident light of the spec?

A
  • Some of the incident light may be reflected by the surface of the cell or absorbed by the cell or solvent.
    Elimination = use of a reference cell identical to the sample cell except compound of interest is blank
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8
Q

What is Abs defined by?

A

A = -log IS / IR = -log T

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9
Q

What are the 5 components of a spec?

A
  • light source
  • monochromator (spectral isolation
  • cuvette
  • photodetector
  • readout device
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10
Q

What is a type of light source that is used in a spec?

A

Incandescent lamps ex: tungsten

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11
Q

What is the role of the monochromator?

A

To isolate radiant energy of a desired wavelength -

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12
Q

What is an example of a monochromator?

A

Prisms, diffraction gratings and or filters

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13
Q

What is the main type of cuvette required for readings below 340nm?

A

Quartz

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14
Q

What is the role of photodetectors?

A

To convert light into an electrical signal - proportional to the number of protons striking its surface.

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15
Q

What is a multiplier tube and which part of the spec is is associated with?

A

This produces rapid response times, very sensitive and slow to fatigue.
- Photodetector

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16
Q

What are the 2 types of specs?

A

Single beam and double beam

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17
Q

What do double beam instruments allow for?

A

‘real time’ referencing

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18
Q

What is a double beam in time spec composed of?

A

Beam is split in two, but measured by the same detector.

19
Q

What is a double beam in space spec composed of?

A

Beam is split into two paths and measured by matched detectors.
Split between reference and sample

20
Q

What is the Beer Lambert Law?

A

Demonstrates that the conc of a substance is directly proportional to the amount of light absorbed or inversely proportional to the log of the transmitted light.

21
Q

Beer lambert equation?

A

A = ecl

22
Q

What is an absorption spectrum?

A

This is the recording of the absorbance of a molecule as a function of wavelength.

23
Q

What is the absorption maximum?

A

This is the wavelength of highest light absorption.

24
Q

How can the linear limitation (abs vs conc) be used to calculate the conc of an unknown target?

A
  1. Calib curve

A single purified substance can be quantified using Beer Lambert - given absorptivity is known at lambda max

25
Q

What is molar absorptivity?

A

This is the abs of a solution with a conc of 1molL-1 of the compound in a light path of 1 cm = M-1cm-1

26
Q

What is the equation used to find the conc of an unknown solution?

A

(abs of unknown/ abs of standard) x glucose standard

27
Q

Why are reference blanks important?

A
  • calibration
  • document the baseline response of the environment - instrument sample system.
  • documented the influence of the cuvette
  • using the same solvent, any abs, fluorescent or scattering due to the solvent phase itself are documented.
28
Q

Why are control samples important?

A
  • quality control, ensure that the results obtained are reliable
29
Q

Why are calibrating solutions important?

A

Minimise uncertainty by ensuring the accuracy of testing equipment.
calibration quantifies and controls errors or uncertainties within measurement processes to an acceptable level

30
Q

What can an assay design be based on?

A
  • the analyte absorbing at a unique wavelength
  • the analyte reacting with a reagent producing a new compound absorbing at a unique wavelength.
  • analyte is involved in a reaction producing a chromophore
31
Q

What is a chromophore?

A

This is the part of a molecule that is responsible for colour - absorbing light.

32
Q

What is the Biuret Reagent composed of?

A
  • potassium hydroxide
  • copper sulfate
  • potassium tartrate
33
Q

What is the principle of the colorimetric biuret assay?

A

Presence of peptides, copper ions form a violet coloured coordination complexes in an alkaline solution - more peptides present = more intense the colour

34
Q

What is an example of a dye used in the formation of coloured compounds and chromophores?

A

Quinoneimine dye - abs max @500nm

35
Q

What has two peaks ? NAD or NADH

A

NADH

36
Q

What is fluoresence?

A

This is the emission of light by a substance that has absorbed light or other electromagnetic radiation.

37
Q

What does the emitted light of fluorescence have?

A

The emitted light has a longer wavelength and lower energy, than the absorbed radiation.

38
Q

What is the stokes shift?

A

This is the difference between the abs exc and abs em.

39
Q

What are the 2 spectrums which fluorescent molecules have?

A
  • absorption

- emission

40
Q

What is the principle components of fluroescence specs?

A
  • 2 monochromators
41
Q

What is an example of an intrinsic fluorescent molecule?

A

aspirin

pollutants

42
Q

What are the 2 advantages of fluorescence spec?

A

enhanced sensitivity and specificity

43
Q

What is a disadvantage of fluorescence spec?

A
  • not all cpds show intrinsic fluorescence, limited application but can couple to fluorescent dyes or fluorophores.
  • ‘quenching’ - oxygen can interfere with measurements

Bioanalytical Science (3 decks)

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