1.4: Spectral Techniques

1. If one switched from a large cuvette to a smaller one, the %T readings on the spectrophotometer would:
   1. decrease
   2. increase
   3. fluctuate
   4. remain unchanged
   5. have equal absorbance readings
2. A solution is measured in a spectrophotometer with a resulting %T of 10%. Calculate the absorbance.
   1. 0
   2. 0.5
   3. 1.0
   4. 1.5
   5. 2.0
3. Compound Y has been determined to have a molar absorptivity (extinction coefficient) of 1000 liters/ mole • cm at 390 nm. An unknown solution of this compound has an absorption of 0.750 when placed in a cell with a light path of 1 cm. The concentration of this compound is:
   1. 375 mole/L
   2. 750 mole/L
   3. 750 \(\mu\)mole/L
   4. 0.375 mmole/L
   5. 750 mmole/L
4. The %T scale on a spectrophotometer is a logarithmic one while the absorbance scale is a linear scale. Because of this relationship, very high absorbance readings are inaccurate.
   1. true
   2. false
5. A standard solution of Compound X at 10 mg/L gives A₄₅₀ = 1.200 when used in a specific colorimetric assay. A serum sample analyzed for Compound X gives the following results: run undiluted: A₄₅₀ = 2.400, run diluted 1:10: A₄₅₀ = 0.300. The concentration of Compound X in serum is:
   1. 2.5 mg/L
   2. 2.0 mg/L
   3. 20 mg/L
   4. 25 mg/L
   5. 200 mg/L
6. Compounds A and B both have a chemical formula of C₉H₁₈N₂O; therefore, both compounds must have the same molar absorptivity coefficient (\(\epsilon\) or a) and the same spectral curve:
   a. true
   b. false
7. Refer to the following spectral-transmission curve made on the same colored solution using two different instruments. All of the following statements are true except: (Note:one instrument had a bandpass of 35 nm, the other 5 nm)
   1. A is the curve which was obtained with the 35 nm instrument
   2. B is the curve obtained with the 5 nm instrument
   3. reading the color intensity at point C would yield the greatest sensitivity
   4. 550 nm would be an appropriate wavelength at which to read the reaction
   5. a and b

8. To detect transmitted light, either barrier tubes or photomultiplier tubes can be
   used. Both work on the principle that incident photons of light interact with a thin
   metallic layer resulting in the generation of an electric current.
   1. true
   2. false

Instruments - Spectrophotometers

Match each of the following instrument components with its most accurately described function — Questions: 53 thru 55

1. slit(s)
2. barrier layer cell
3. interference filter
4. photomultiplier
5. potentiometer

9. Converts electromagnetic energy to electricity
   1.  
   2.  
   3.  
   4.  
   5.  
10. Renders light rays parallel
    1.  
    2.  
    3.  
    4.  
    5.  
11. Converts light to electricity and amplifies the electron flow
    1.  
    2.  
    3.  
    4.  
    5.  
12. Which of the following combinations are not consistent?:
    1. tungsten lamp, diffraction grating, glass cuvette, barrier layer cell
    2. Xenon arc lamp, diffraction grating, quartz cuvette, photomultiplier tube
    3. Deuterium lamp, quartz prism, quartz cuvette, photomultiplier tube
    4. Deuterium lamp, glass prism, glass cuvette, photomultiplier tube
    5. B and D
13. The principle of atomic absorption spectrophotometry is best stated as:
    1. ground state (unexcited) atoms absorb light energy of a characteristic wavelength
    2. excited atoms emit light of a characteristic wavelength after absorbing energy
    3. excited atoms emit light of a longer wavelength after absorption of energy of
       a shorter wavelength
    4. ground state (unexcited) atoms absorb light energy of a characteristic wavelength, become ionized, and emit light
    5. excited atoms absorb light
14. The most common light source in atomic absorption instruments is a:
    1. Xenon arc
    2. flame
    3. hollow cathode lamp
    4. grating
    5. deuterium lamp
15. In atomic absorption spectrophotometry, the purpose of the light source is:
    1. to provide a chamber to house the flame
    2. to produce a wavelength of light characteristic for the metal in the cathode
    3. to act as a detector of light energy transmitted by the flame
    4. to transmit argon or neon gas into the flame as a source of fuel
    5. to act as a detector of light energy allowed to be transmitted by the chopper
16. If one desired to measure calcium by atomic absorption spectrophotometry, the light source would have to be made of which component(s)?:
    1. magnesium
    2. deuterium
    3. tungsten and quartz
    4. calcium
    5. zinc
17. The heat of the atomic absorption (A.A.) flame is not critical for accurate and precise measurement of Ca⁺⁺ since the procedure uses a calibration curve.
    1. True
    2. False
18. A single source for the hollow-cathode lamp is used for Ca⁺⁺, Mg⁺⁺, and Li⁺, while a different lamp must be used to measure Cu⁺⁺.
    1. True
    2. False
19. Most atomic absorption instruments use filters for their monochromometers.
    1. True
    2. False
20. The purpose of the flame in atomic absorption is to excite the atoms so that they can absorb light from the hollow cathode lamp.
    1. True
    2. False
21. Nephelometers measure light at:
    1. a different wavelength than the input light
    2. the wavelength of maximum
    3. the wavelength of maximum fluorescence
    4. the same wavelength as the input light
    5. the point of longest scatter
22. Turbidity measurement is equivalent to:
    1. fluorescence
    2. nephelometry
    3. absorbance
    4. phosphorescence
    5. quenching
23. Most spectrophotometric analyses use absorbance measurements because there is no direct relationship between %T and concentration.
    1. True
    2. False

Match the principle which best describes the effect of each of the following (questions 24 thru 28):

Principle

1. spectrophotometry
2. fluorometry
3. turbidity
4. nephelometry

Effect

24. Increase in light absorption caused by particulate interaction with absorbent compound.
    1.  
    2.  
    3.  
    4.  
25. Absorption and emission of light at different wavelengths.
    1.  
    2.  
    3.  
    4.  
26. Decrease in observed light intensity caused by light scattering.
    1.  
    2.  
    3.  
    4.  
27. Increase in observed light intensity caused by light scattering.
    1.  
    2.  
    3.  
    4.  
28. Absorption of incident light by compound resulting in a decrease in observed light intensity
    1.  
    2.  
    3.  
    4.  
29. Which of these reactions can occur in a flame? (M = metal)
    1. M⁺ + electron → M°
    2. M* → M° + photon
    3. M° + photon → M*
    4. a and c
    5. all of the above
30. A spectral transmittance curve for compound X is shown below. The wavelength at which measurement should be made is:
    1. 400
    2. 450
    3. 500
    4. 550
    5. 600

31. When an atom, ion or molecule absorbs a photon of light, this is termed:
    1. atomic transition
    2. fluorescence
    3. radiationless transition
    4. electronic transition
    5. Beer’s Law
32. A beam of white light is passed through a green filter, and the transmitted light is passed into a green solution. The transmitted light from this solution was measured and the %T and absorbance recorded. This experiment is repeated. The filter remains green but now a red solution is used. Which of the following responses for %T and absorbance would be expected?
    1. %T and absorbance both increase
    2. %T increases, absorbance decreases
    3. %T decreases, absorbance increases
    4. %T and absorbance both decrease
    5. %T and absorbance values remain unchanged
33. Both atomic absorption and flame photometers use filters as the monochrometers:
    1. true
    2. false
34. In sunlight, what color is the solution of the compound whose spectral curve is shown below:
    1. blue
    2. green
    3. yellow
    4. purple
    5. colorless

35. 10 mL of a solution is read spectrophotometrically in a 2 cm cuvette. The
    absorbance obtained after correcting for dilution is 30. The molar absorptivity of
    this compound, whose M.W. is 1000, is 150. What is the concentration of this
    compound in mg/mL?
    1. 0.1 mg/mL
    2. 1 mg/mL
    3. 10 mg/mL
    4. 100 mg/mL
    5. 1000 mg/mL
36. A 0.01 M solution of a compound is prepared and its absorption at 260 nm is
    measured in a 20 mm cell and found to be 0.500. What is molar absorptivity of
    this compound?
    1. 50
    2. 25
    3. 12.5
    4. 2.5
    5. none of the above
37. You are performing a manual procedure in which you need 14 10 mm cuvettes. Only 13 10 m cuvettes are available so you read one of the standards in a 20 mm cuvette. Upon calculating the concentration of this solution, you find the standard appears:
    1. more concentrated than it should be
    2. less concentrated than it should be
    3. the same concentration as expected
38. A linearity check is performed on the spectrophotometer using a solution that follows Beer’s Law up to a concentration of 2.5. The following values are obtained:

Concentration (mol/L)	Absorbance
1.01	1.60
0.75	1.35
0.50	0.90
0.25	0.45

You would conclude:

1. the instrument is linear
2. the instrument is non-linear

Use the following code to answer questions 39 thru 41:

1. If A is greater than B
2. B is greater than A
3. A and B are about equal

39. A. U.V. light absorbed by glass
    B. U.V. light absorbed by fused silica
40. A. linearity of spectrum produced by a prism
    B. linearity of spectrum produced by a diffraction grating
41. A. transmission of blue light by a cuvette filled with a red solution
    B. transmission of yellow light by a cuvette filled with a red solution

Use the following Key to answer questions 42 through 56:

1. 1,2 and 3 are correct
2. 1 and 3 are correct
3. 2 and 4 are correct
4. only 4 is correct
5. all are correct

42. The following can affect the linear response of concentration vs. absorbancy and thus result in a deviation from Beer’s Law:
    1. stray light
    2. use of photometer instead of a spectrophotometer
    3. very high analyte concentration
    4. Using only one standard for calculation
43. On linear graph paper, a straight line through zero with three plotted standards indicates which of the following?:
    1. the concentration of the standard vs. the absorbance is linear
    2. the test is in compliance with Beer’s Law’
    3. the concentration of the standard vs. the log %T is linear
    4. the test is not in compliance with Beer’s Law.
44. Deviations from Beer’s Law are caused by
    1. stray light
    2. polychromatic light
    3. very high concentrations of substance being measured in a colorimetric reaction
    4. very low concentration of absorbing material
45. Which of the following are preferred for checking the wavelength calibration of a spectrophotometer?:
    1. cobalt sulfate
    2. didymium jilter
    3. potassium dichromate
    4. helium oxide filter
46. The concept of thermal excitation as used in emission photometry involves:
    1. the absorption of light by certain alkali metals
    2. characteristic line spectra being given off by excited orbital electrons as they return to the ground state
    3. orbital electrons absorbing radiant energy which is converted to electrical energy as measured by a gal manometer
    4. the heating of alkali metal salts to produce a colored light of characteristic wavelength
47. In flame photometry the measured light comes from:
    1. burning fuel
    2. light produced when atoms go from ground state (M°) to excited state (M*)
    3. light from a tungsten bulb
    4. light produced when atoms go from excited state (M*) to ground state (M°)
48. The purpose of the nebulizer in atomic absorption is to:
    1. break chemical bonds
    2. excite atoms
    3. measure the sample flow rate
    4. disperse aqueous sample into fine droplets
49. Lanthanum diluents is used in atomic absorption measurements to:
    1. precipitate interfering proteins
    2. bind Mg⁺⁺ so it will not interfere with Ca⁺⁺ measurements
    3. release Ca⁺⁺
    4. release Ca⁺⁺ complexes
50. Which of the following statement(s) concerning the use of cesium (Cs) as an internal standard for flame photometry is (are) true? Cesium:
    1. is normally present in very low concentrations in body fluids
    2. has an emission line easily distinguished from those of Na and K
    3. is used to correct for minor variations of reaction conditions
    4. can be replaced by other elements such as magnesium
51. Turbidimetry and nephelometry measure:
    1. fluorescence in turbid solution
    2. light absorbance by particles in suspension
    3. light absorbance by a colored substance
    4. light scatter by particles in suspension
52. Components of a nephelometer include:
    1. light source
    2. detector
    3. filter
    4. chopper
53. In fluorometry:
    1. the compound measured absorbs light of a shorter wavelength and emits light of a longer wavelength
    2. quenching occurs when a substance other than the one being measured emits fluorescence
    3. fluorometery is a relative measurement of light signal
    4. the compound measured absorbs light of a longer wavelength and emits light of a shorter wavelength
54. Which of the following types of lamps would be most appropriate for the entire UV range?
    1. mercury vapor
    2. tungsten iodide with quartz envelope
    3. tungsten with glass envelope
    4. deuterium
55. An acceptable means of achieving spectral isolation in the UV range is (are):
    1. reflectance diffraction grating
    2. glass filter
    3. quartz prism
    4. glass prism
56. The correct wavelength for reading a sample on a spectrophotometer is:
    1. the wavelength of the highest absorbance maxima
    2. the wavelength at which the most light passes through the solution
    3. the wavelength at which a straight line (percent transmittance vs. concentration) is obtained on a semi-log paper
    4. the wavelength at which the photo detector has optimum response.
57. When a fluorescent molecule absorbs polarized excitation light, the emitted light loses polarization when:
    1. the the light intensity decreases
    2. light intensity increases
    3. the excited state changes
    4. the fluorescent molecule is held rigid
    5. the fluorescent molecule
58. To retain a linear relationship between fluorescence intensity and the amount of light absorbed by a solution, the absorbance of the solution should not exceed:
    1. 0.001
    2. 0.01
    3. 0.1
    4. 1.0
    5. 2.0
59. Fluorescence polarization assays are different from those that measure only fluorescence intensity because:
    1. those which measure intensity can tolerate unstable light sources.
    2. those which measure intensity can tolerate less intense light.
    3. the light intensity is greater.
    4. polarization measurements are less affected by variations in fluorescence intensity.
    5. polarization measurements are more affected by variations in fluorescence intensity.
60. Florescence attenuation assays are used in dedicated fluorometric instruments such as the Abbott TDx. In these assays, the fluorescent dye concentration is:
    1. Constant
    2. linearly proportional to analyte Concentration
    3. logarithmically proportional to analyte concentration
    4. proportional to analyte formation
    5. inversely proportional to analyte formation
61. Chemiluminescent reactions are those in which one of the products is
    1. Luminol
    2. oxidized luminol
    3. reduced luminol
    4. heat
    5. light
62. Time delayed fluorescence is a term that refers to:
    1. fluorescence which starts to occur only after one millisecond following exposure to exciting light
    2. fluorescence which is delayed by solution chemistry
    3. fluorescence which is measured after a time delay
    4. the time it takes for a photon to jump from a ground state to an excited state
    5. the delayed time required before the incident light excites the fluor
63. Atomic absorption can be considered the opposite of flame emission because in atomic absorption:
    a. thermal energy applied to the sample causes formation of excited atoms
    b. a specific light source is necessary for the determination of each element
    c. electron’s movement to a higher energy level is measured.
    d. the amount of light absorbed by an electron moving to a higher energy level is measured.
    e. light is given off by excited atoms.
64. In a fluorometer, when the primary excitation source produces fluorescence, the secondary light emission by the sample has:
    a. a longer wavelength than the primary light
    b. a shorter wavelength than the primary light
    c. the same wavelength as the primary light
    d. no specific wavelength
    e. any of the above
65. Which of the following describes a physical property that distinguishes luminescence from fluorescence?
    a. fluorescence is at longer wavelengths.
    b. in luminescence a photon goes from an excited state to a ground state.
    c. in fluorescence a photon goes from an excited state to a ground state.
    d. luminescence has a longer transition time to the ground state.
    e. fluorescent light is always polarized.
66. Chemiluminescence can be induced by
    1. light
    2. flame ionization
    3. a chemical reaction
    4. firefly luciferase
    5. atomic absorption

Answer

1. b (p. 88)
2. c (p. 88,38)
3. c (p. 88,38)
4. b (p. 88)
5. d (p. 88,38)
6. b (p. 86-88)
7. c (p. 89,90,92)
8. a (p. 91)
9. b (p. 91)
10. a (p. 91)
11. d (p. 91)
12. d (p. 91-93)
13. a (p. 95)
14. c (p. 95)
15. b (p. 95)
16. d (p. 95-96)
17. b (p. 97)
18. a (p. 96)
19. b (p. 96)
20. b (p. 95)
21. d (p. 101-103)
22. c (p. 101-102)
23. a (p. 88)
24. c (p. 101-102)
25. b (p. 97-98)
26. c (p. 101-102)
27. d (p. 101-103)
28. a (p. 88-89)
29. e (p. 97)
30. c (p. 92-93)
31. d (p. 86)
32. c (p. 86-88)
33. b (p. 95-97)
34. d (p. 86)
35. d (p. 88-89)
36. b (p. 88-89)
37. a (p. 88-89)
38. b (p. 88-89)
39. 1 (p. 91)
40. 2 (p. 90)
41. 3 (p. 88)
42. b (p. 88-89)
43. a (p. 88-89)
44. a (p. 88-89)
45. c (p. 93-94)
46. c (p. 87, 97)
47. d (p. 97)
48. d (p. 96)
49. d (p. 96)
50. a (p. 97)
51. d (p. 101-102)
52. a (p. 102)
53. b (p. 97-98)
54. d (p. 89-90)
55. b (p. 90)
56. b (p. 92-93)
57. e (p. 100-101)
58. c (p. 99)
59. d (p. 100-101)
60. a (p. 99)
61. e (p. 99-100)
62. c (p. 99)
63. d (p. 99)
64. a (p. 98)
65. d (p. 98-100)
66. c (p. 99-100)