LIGHT MEASURING SYSTEMS

Question 1

Formula for Absorbance

Answer 1

2 - log%T

Question 2

What is a bandpass ?

Answer 2

• range of wavelengths that reach the exit slit of a monochromator (max wavelength - min wavelength)
• transmitted at a point equal to half the peak intensity transmitted

Question 3

Range of wavelengths for UV light or Visible light

Answer 3

UV light: 200 to 300 nm
Visible light: 300 to 700 nm

Question 4

How do spectrophotometers measure transmittance ?

Answer 4

By comparing the signal generated by the transmitted light (Is) to the signal of the incident light (Io)

Question 5

The relationship between absorbance and %T is __

Answer 5

The relationship between absorbance and %T is logarithmic

Question 6

Formula for Absorbance under Beer-lambert’s law

Answer 6

A = εbc

ε = molar absorptivity (L/ mol x cm)
b = light path (cm)
c = concentration of analyte (mol/L)

Question 7

State the limitations of the Beer-Lambert law

Answer 7

• monochromatic light (Io)
• solvent has insignificant absorbance to analyte
• analyte follows Beer-lambert’s law
• standard and unknown are measured in matched cuvettes
• the concentration range obeys Beer-lambert’s
• absorbance of unknown < absorbance of standard; a dilution can be done
• no optical interferences
• no side reactions between analyte and other molecules
• sides of cuvette are parallel
• no stray light

Question 8

What are spectral interferences ?

Answer 8

• observed when a compound causes a response similar to that of the analyte
  ie. HIL

Question 9

Explain the principle of reflectance spectrophotometry

Answer 9

• polychromatic light is directed to a diffraction grating or light-emitting diodes to produce monochromatic light
• monochromatic light hits the test surface, light is absorbed and reflected
• color intensity increases with [analyte], absorption increases, and diffuse reflection decreases
• a single wavelength of the diffuse reflected light is selected and directed onto a photodetector

Question 10

Define reflection density (DR)

Answer 10

A measure of the amount of light absorbed by the chromophore on the test surface:

DR = log (R0 / Rtest)

where,
R0 = light reflected by the white standard
Rtest = light reflected by the surface

Question 11

In reflectance spectrometry, the relationship between analyte concentration and reflectance is not __, it is __.

Answer 11

In reflectance spectrometry, the relationship between analyte concentration and reflectance is not linear, it is logarithmic

Question 12

What is Bichromatic Analysis ?

Answer 12

• used to correct for spectral interferences when the MAX absorbance of the analyte = MAX absorbance of interference
• two readings are made: one at MAX absorbance (A1) and one where analyte minimally absorbs light (A2)
• the standard curve is based on A1 = A2 or the ratio (A1/A2)

Question 12

The Beer-lambert law states that the __ of a substance is __ to the amount of radiant energy __

Answer 12

The Beer-lambert law states that the concentration of a substance is directly proportional to the amount of radiant energy absorbed

Question 13

What is Allen Correction ?

Answer 13

• multi-chromatic analysis of a rxn to correct for background absorbance
• two wavelengths, in addition to the MAX absorbance are monitored to subtract average background absorbance

ie. A(510nm) = 0.100, A(520) = 0.800, and A(530nm) = 0.300
Corrected A = A520 - [(A510 + A530) / 2]

Question 13

The Beer-lambert law states that the __ of a substance is __ to the amount of radiant energy __

Answer 13

The Beer-lambert law states that the concentration of a substance is directly proportional to the amount of radiant energy absorbed

Question 13

What is Bichromatic analysis ?

Answer 13

• monitors a reaction at two wavelengths
• used to correct for background interference

Question 14

What is a bandpass ?

Answer 14

• range of wavelengths that reach the exit slit of a monochromator
• transmitted at a point equal to half the peak intensity transmitted

Question 14

Define icterus

Answer 14

refers to the orange color imparted to a sample because of the presence of bilirubin

Question 15

What is Kinetic Analysis ?

Answer 15

• change in the monitored parameter over time related to concentration
• measurements usually made very early in the rxn period

Question 15

What is line spectrum ?

Answer 15

a discontinuous emission spectrum of elements in which emitted light bands cover very narrow (0.1 nm) range of wavelengths

Question 16

What is Allen Correction ?

Answer 16

• multi-chromatic analysis of a rxn to correct for background absorbance
• two wavelengths, in addition to the MAX absorbance are monitored to subtract average background absorbance

Question 16

The Beer-lambert law states that the __ of a substance is __ to the amount of radiant energy __

Answer 16

The Beer-lambert law states that the concentration of a substance is directly proportional to the amount of radiant energy absorbed

Question 16

Define icterus

Answer 16

refers to the orange color imparted to a sample because of the presence of bilirubin

Question 16

What is a bandpass ?

Answer 16

• range of wavelengths that reach the exit slit of a monochromator
• transmitted at a point equal to half the peak intensity transmitted

Question 17

What is Bichromatic analysis ?

Answer 17

- monitors a reaction at two wavelengths - used to correct for background color

Question 17

What is line spectrum ?

Answer 17

a discontinuous emission spectrum of elements in which emitted light bands cover very narrow (0.1 nm) range of wavelengths

Question 17

What is Kinetic Analysis ?

Answer 17

- change in the monitored parameter over time related to concentration - measurements usually made very early in the rxn period

Question 18

Define Molar Absorptivity

Answer 18

the absorbance of light, at a specific wavelength, divided by the product of the concentration of moles per litre and the sample path (cm)

Question 19

Units for Molar Absorptivity

Answer 19

L/(mol x cm)

Question 20

What is reflectance spectrophotometry ?

Answer 20

- a quantitative spectrophotometric technique in which light reflected off a surface of a colorimetric reaction is used to measure the amount of the reaction product

Question 21

Define stray light

Answer 21

- Radiant energy reaching the detector that consists of wavelengths other than those defined by the filter/ exit slit of monochromator - Causes false decrease in absorbance measurements/ deviation from Beer-lambert’s

Question 22

How can stray light be detected ? How is it reduced ?

Answer 22

- detected by inserting a cutoff filter that does not transmit emission the light If %T > 0 = stray light is present - reduced by changing light source, sealing light leaks, realigning instrument components, cleaning optical surfaces, removing dust

Question 23

Define turbidity (as an interferent)

Answer 23

- scatter of light in a liquid that contains suspended particles ie. lipemia can make a sample turbid

Question 24

What errors can occur with low %T ?

Answer 24

- greater imprecision - substantial error when absorbance is calculated bc of compressed nature of the log scale

Question 25

What errors can occur with low %T ?

Answer 25

- greater imprecision - substantial error when absorbance is calculated bc of compressed nature of the log scale

Question 26

What errors can occur with high %T ?

Answer 26

- detection system may have limited capacity to measure small differences between high levels of absorbance accurately - can be overcome by diluting the sample and using a sample blank

Question 26

How can spectral interferences be resolved ?

Answer 26

- using a sample blank - lipemia can be cleared using ultracentrifugation or extraction - turbidity can be reduced by dilution

Question 27

List the 6 components of a spectrophotometer

Answer 27

1. Polychromatic light source 2. Entrance slit 3. Monochromator 4. Exit slit 5. Cuvette 6. Detector

Question 28

List 2 methods used to correct for spectral interferences

Answer 28

1. Bichromatic analysis 2. Allen correction

Question 28

How can spectral interferences be mitigated ?

Answer 28

- using a sample blank - lipemia can be cleared using ultracentrifugation or extraction - turbidity can be reduced by dilution

Question 28

How can spectral interferences be mitigated ?

Answer 28

- using a sample blank - lipemia can be cleared using ultracentrifugation or extraction - turbidity can be reduced by dilution

Question 28

What errors can occur with high %T ?

Answer 28

- detection system may have limited capacity to measure small differences between high levels of absorbance accurately - can be overcome by diluting the sample and using a sample blank

Question 28

List the 6 components of a spectrophotometer

Answer 28

1. Polychromatic light source 2. Entrance slit 3. Monochromator 4. Exit slit 5. Cuvette 6. Detector

Question 28

What errors can occur with low %T ?

Answer 28

- greater imprecision - substantial error when absorbance is calculated bc of compressed nature of the log scale

Question 28

List 2 methods used to correct for spectral interferences

Answer 28

1. Bichromatic analysis 2. Allen correction

Question 28

What 3 materials can Cuvettes be made of ?

Answer 28

1. Borosilicate glass; 320 - 950nm 2. Silica quartz; < 320nm 3. Plastic; all wavelengths

Question 29

What is the difference between photomultiplier tubes and photodiodes ?

Answer 29

Photomultiplier tubes: - detect and amplify photons of light that hit its surface - composed of photon sensitive cathode, series of DYNODES that amplify signal, and an anode Photodiodes: - semiconductors that change their charged voltage when hit by a photon - generate a proportional current - less sensitive; DOES NOT AMPLIFY signal

Question 30

Describe how photodiode arrays work

Answer 30

- prism/ grating disperses light after the cuvette, and onto the array - each photodiode is calibrated for a specific wavelength - when a photon hits surface of a charged coupled device (CCD), electrons are released and quantitated

Question 31

How can wavelength accuracy be confirmed ?

Answer 31

Using standard absorbing solutions OR filters with absorbance maxima of known wavelengths

Question 32

How can linearity of detector be confirmed ?

Answer 32

- Using solid glass filters OR a solution known to follow Beer-lambert’s law ie. Copper sulphate - Non-linear response can be due to faulty detector or stray light

Question 33

How is a reflectance spectrophotometer calibrated ?

Answer 33

Using a white surface = reflects all incident (Io) light Black surface = calibrates for zero reflectance

Question 34

Dry slide chemistry is an example of __. Dry slides are composed of __ layers.

Answer 34

Dry slide chemistry is an example of reflectance spectrophotometry. Dry slides are composed of multiple layers.

Question 35

Describe dry slide chemistry

Answer 35

- patient sample is applied on a spreading layer - as the sample moves down through the layers, it can become altered - the sample is impregnated with dry chemical reagents in the reagent layer - coloured product - light is directed at the reagent/ indicator layer and the amount of diffuse light measured = [analyte]

Question 36

“The detection and measurement of a decrease in the intensity of an incident beam of light as it passes through a solution of particles”

Answer 36

Turbidimetry

Question 37

“The measurement of emitted light that occurs when a molecule absorbs light at one wavelength and reemits it light at a longer wavelength”

Answer 37

Fluorometry

Question 38

What is an advantage of using fibre optics in spectrophotometers?

Answer 38

instruments can be miniaturized

Question 39

Which light source can provide light in the visible spectrum?

Answer 39

Tungsten

Question 40

Which light source can provide light in the UV spectrum?

Answer 40

Deuterium

Question 41

Which of the following best describes photomultiplier tubes? a. PMTs are made of small silicon cells that when light enters release a proton. b. PMTs are made of small silicon cells that when light enters release an electron. c. PMTs are made of a cathode that can absorb radiant energy and release electrons in proportion to the radiant light. d. PMTs are semiconductor that change their charged voltage upon being struck by light; the change is converted to current and measured.

Answer 41

c. PMTs are made of a cathode that can absorb radiant energy and release electrons in proportion to the radiant light. cross out

Question 42

Which of the following substances is suitable to use as a 100% reflectance standard? a. cobalt II sulfate b. barium sulfate c. silver I oxide d. silica dioxide

Answer 42

b. barium sulfate

Question 43

Which of the following actions may reduce stray light? a. reset 100% absorbance b. verifying linearity c. reset percent transmission (%T) to 100 d. realign instrument components

Answer 43

d. realign instrument components

Question 44

How many wavelengths must be checked to certifying the accuracy of spectrophotometer with a grating?

Answer 44

2 wavelengths

Question 45

Which of the following wavelength selectors may be found in a photometer? a. diffraction grating cross out b. Wratten filter cross out c. prism cross out d. echelette

Question 46

Which cuvette type is best for use below 320 nm? a. quartz b. polystyrene c. polyacrylate d. glass

Answer 46

a. quartz

Question 47

What is the function of an exit slit? a. to determine the band pass b. focus the light on the detector c. disperses light into its spectral form d. to make light parallel

Answer 47

a. to determine the band pass

Question 48

Which wavelengths of light (nm) will be transmitted from a interference filter designated for 550 nm? a. 275, 550 b. 275, 550, 1100 c. 550, 1100 d. 550

Answer 48

b. 275, 550, 1100

Question 49

Which of the following describes the function of a Wratten filter? a. selectively reflects the desired wavelength and absorbs all other wavelengths. b. reflects some radiant energy but allows other wavelengths to penetrate and reflect off a second surface. c. selectively transmits the desired wavelength and absorbs all other wavelengths. d. disperses radiant energy from the light source into a spectrum.

Answer 49

c. selectively transmits the desired wavelength and absorbs all other wavelengths.

Question 50

Which of the following substance is suitable for a 100% reflectance standard? (multiple answers) a. cobalt II sulfate b. barium sulfate c. silica dioxide d. silver I oxide e. magnesium carbonate

Answer 50

b. barium sulfate AND e. magnesium carbonate

Question 51

Which is the best description of electronic transition? a. the movement of an electron from a lower energy state to a higher energy state b. the movement of an proton from one element to another c. the movement of an electron from one element to another d. the movement of an electron from a higher energy state to a lower energy state

Answer 51

a. the movement of an electron from a lower energy state to a higher energy state

Question 52

Which best describes the composition of a Wratten filter? a. one or more layers of colored glass b. colored gelatin sealed between two glass plates c. a glass plate coated with silver d. a glass plate coated with gold

Answer 52

b. colored gelatin sealed between two glass plates