Module 4: Photometry

Question 1

When atoms absorb radiant energy (heat or light)

Answer 1

some valence electrons move from their normal position (ground state) to a new orbital position (excited state)

Question 2

Wavelength and color properties

Answer 2

wavelengths of light that a substance transmits and absorbs are characteristic of the substance (results in unique color properties)
These properties allow for detection and quantitation of the substance in samples

Question 3

Complimentary color

Answer 3

the wavelengths of light that are maximally absorbed are the complimentary color to the one transmitted
ex. light transmitted and observed = blue
complimentary color/max light absorbed = yellow

Question 4

Spectral Transmittance curve

Answer 4

plot of all the wavelengths used versus the observed transmittance of the solution (amount of light allowed to pass through the solution at each wavelength)
U shaped graph

Question 5

Spectral Absorbance curve

Answer 5

plot of all the wavelengths used versus the observed absorbance of the solution (amount of light absorbed by the solution at each wavelength) ∩ shaped

Question 6

Absorbance Max (Max A)

Answer 6

the wavelength that is absorbed the most (spectral absorbance curve)
best wavelength to observe this solution at

Question 7

Transmittance

Answer 7

transmittance of a substance in solution is the proportion of the incident light that is transmitted (often as %)

%T = [transmitted (Is)/incident (Io) ] x100

Io = original intensity of light

Question 8

Transmittance vs concentration

Answer 8

as concentration increases, more light is absorbed and less is transmitted. %T varies inversely and logarithmically with concentration (neg slope on a graph, non linear/curved line)

Question 9

Transmittance vs concentration on semi-log graph paper

Answer 9

%T on log axis, a straight line with negative slope will be demonstrated
Concentration is inversely proportional to the log of %T

Question 10

Absorbance

Answer 10

cannot be measured directly but can be calculated from transmittance
Absorbance is directly related to concentration
Absorbance and T are inversely and logarithmically related
A=2-log%T
No units, often reported to 3 decimal points

Question 11

Lambert’s Law

Answer 11

Absorbance is directly proportional to the distance the light must travel through (path length, b). The Greater the path length the light must travel through, the greater amount of light absorbed
A∝b (proportional)
Diameter of cuvet represents path length

Question 12

Beer’s Law (original)

Answer 12

The amount of radiant energy of a particular wavelength absorbed varies directly with he concentration of the absorbing molecules
The greater the concentration (c), the more light will be absorbed
A∝c
Absorbance vs concentration is a linear relationship (straight line)
Wavelengths that are absorbed the most (max A) produce the best linearity

Question 13

Combined Beer-Lambert Law (AKA Beer’s Law)

Answer 13

It states that the absorbance (A) of a solution is directly proportional to the product of the concentration (c) times the depth of the solution that the light must travel (b) times a constant (a)
A=a x b x c
a=absorptivity, absorbance of the solute in a concentration of 1g/L measured in a 1cm light path at a specific wavelength
b=light path in cm
c= concentration of substance in g/L (g/L when a is in equation)
a and b stay the same, so A will vary with c only

Question 14

Molar absorptivity (ε) instead of a in combined beers law

Answer 14

ε is the absorbance of a solute in a concentration of 1mol/L of solution measured in 1cm light path at specific wavelength, temp, pH and solvent. Expressed in L/mol cm
A=ε x b x c
A has no units
ε in L/(mol x cm)
b in cm

Question 15

Graphing combined beers law

Answer 15

independent variable, c, always on x axis
dependent variable, A or %T always on y axis
Direct relationship (straight line) only observed when beers law is obeyed

Question 16

Failure to follow or obey beers law results in

Answer 16

a loss of linearity on an A vs concentration graph

Question 17

Conditions affecting beers law

Answer 17

Operator: incorrect wl
high concentration of analyte
light path not kept constant
failure to set 0%T and 100%T
failure to maintain pH and temp
Impurity of solution
presence of an interfering substance

Instrument:
Stray light (common)
inability to select narrow band of wl
line voltage fluctuations
unstable light source
nonlinear detector response

Question 18

Applying beers law (2)

Answer 18

To calculate concentration of the unknowns by comparing unknown A to that of a known solution (a standard)
A calibration curve to plot A vs concentration of a number of known standards that can be used to determine the concentration of unknowns

Question 19

Applying beers law to calculate c of unknowns by comparing unknown A to a standard - conditions

Answer 19

Beers law is obeyed
Using A (not %T)
The blank (0 standard) is set at 0A
The standard is close in concentration to the unknown
The standard was analyzed at the same time as the unknown

Question 20

Procedure to apply beers law to calculate c of unknown

Answer 20

1) A measured of a single standard (with a known concentration)
2) A is measured of the clinical sample (with unknown concentration)
3) The A values obtained along with known concentration of standard can be used to calculate the unknown concentration

(Au/As)=(Cu/Cs)