7.

Beer’s Law and It’s Implications for

Instrument Construction
 1. Derive Beer’s Law

ASSUMPTIONS
1. No light is emitted
*
M  h   M  kT
2. dx infinitesimal

3. Monochromatic light uniform on the surface, S

4. dn molecules in a section volume

V  dx S
5. Capture cross sectional area is

dS  dn
 Set up Derivation

P hotons im pinging ( capture area ) P0 dS 

P hotons captured  
total area S

dS  dn
Consider a large # of boxes

Po P1 P2

P0 dn 
 dP 
S
This is an integration

P dPx n dn
 
P0 Px 0 S
 dPx n dn

P
 P n
A   lo g     lo g T 
P0 Px 0 S  P0  2 .3 0 3 S
P n  n 0 
 ln Px P0
 Substitutions
S
V  1L 
S  n  M N a  3 3 V
n b  1 0 cm 
 ln P  ln P0  
S  1L 
 M N a  3 3 V
 P  n P  1 0 cm 
 ln    A   lo g   
 P0  S  P0  V 
2 .3 0 3  
P b
n
 lo g      1L  
 P0  2 .3 0 3 S  N a  1 0 3 cm 3  
A   M b  bM
 2 .3 0 3 
 
 A   lo g T  bM

What is the absorbance when the

light transmitted is 50% of the initial
beam in a 2 cm path length cell for a
concentration of 10-3 M?
Deviations

1. Assumed each molecule was independent of the other

When will the assumptions fail?

Molecules not independent when:
Neighbors experience each other

1. High concentrations

2. High electrolyte

3. Large local fields due to large absorption probability (alpha)

Apparent Instrumental Deviations

**polychromatic radiation***

What is the source of polychromatic radiation?

 1
2 Similarly

 P1   P2 
A 1   lo g     1 bC A 2   lo g     2 bC
 P0 1   P0  2 

Rearrange
  1 bC P2  P0 2 1 0   2 bC
P1  P0 1 1 0

Total absorbance  P1  P 2   P0  2  P0  2 

A m easured   log    log  
 P0  2  P0  2   P1  P 2 

 P0  2  P0  2 
A m easured  log    bC   bC

 P0 1 10 1  P0  2 1 0  2 

Consider several cases using this equation

  P0  2  P0  2 
1. Monochromatic light A m ea su red  log    1bC

2 b C 
 P0 1 10  P0  2 1 0 
 1   2  

 P0  2  P0  2 
A m easu red  log  

 0 1 
 P  P 1 0 bC
02



 1 
A m ea sured  lo g  bC 
 10 

 
A m easured  lo g 1 0 b C  bC Reality check ok
  P0  2  P0  2 
2. Case 2 A m ea su red  log  
  1bC 2 b C 
 P0 1 10  P0  2 1 0 
Po ,  1  P0 ,  2  P0
 
 2 
 P0  P0  A m ea sured  lo g  
A m easu red  log  2 b C    10    
 1 0    
3 3
  1 b C  20 00 1 10  2 0 0 1 10
P
 0 1 0  P0 1 0 
 
 2 P0 
A m ea sured  log  
 
 0 
 P 10  1bC  1 0   2 bC  
 A m easured  lo g   2
2   0 .4 9 4
 
 10  1 0  0 . 2  

 2 
A m easu red  log   bC 
 
 1 0 1  1 0   2 bC  


Example Calculation .494

B=1
M=0.001
Molar absorptivity at 1=2000
at 2 = 200 M
When would this situation apply?
  P0  2  P0  2 
3. Stray Light A m ea su red  log  
  1bC 2 b C 
 P0 1 10  P0  2 1 0 
Po ,  1  P0 ,  2 and  2  0

 P0  1  P0  2 
A m ea sured  log  Example Calculation
  1bC  0 bC 
P
 0 1 10  P02 1 0 
Stray light is 0.5% of total light
 P0  1  P0  2  P0  2  0 .00 5 P0  1
A m ea sured  lo g    1bC 
P
 0 1 1 0  P02 

 P0  1  0 .00 5 P0  1 
A m easured  log  
What happens when light at 1 is strongly absorbed?  0 .00 5 P0  1 

P0  1 10  1bC  P0  2
 1.0 05 
A m easured  log    2 .30 3
 0 .00 5 
 P0  1  P0  2 
A m easured  lo g  
 P0  2  The maximum absorbance the
Instrument is capable of measuring is
2.303
 Comparison of Instruments
Instrument %stray light maxA
Spect 20 0.5 2.3
McPherson 0.1 3
McPherson +filter 0.01 4
Double monochromator 0.001 5
 Physical Dimensions: 89.1 mm x 63.3 mm x 34.4 mm
Weight: 190 grams
Detector: Sony ILX511 linear silicon CCD array
Detector range: 200-1100 nm
Pixels: 2048 pixels
Pixel size: 14 μm x 200 μm
Pixel well depth: ~62,500 electrons
Sensitivity: 75 photons/count at 400 nm; 41 photons/count at 600 nm
Design: f/4, Symmetrical crossed Czerny-Turner Czerny-Turner
Focal length: 42 mm input; 68 mm output
construction
Entrance aperture: 5, 10, 25, 50, 100 or 200 µm wide slits or fiber (no slit)
Grating options: 14 different gratings, UV through Shortwave NIR
Detector collection lens option: Yes, L2
OFLV filter options: OFLV-200-850; OFLV-350-1000
Other bench filter options: Longpass OF-1 filters
Collimating and focusing mirrors: Standard or SAG+
UV enhanced window: Yes, UV2
Fiber optic connector: SMA 905 to 0.22 numerical aperture single-strand optical fiber
Spectroscopic Wavelength range: Grating dependent
Optical resolution: ~0.3-10.0 nm FWHM
Signal-to-noise ratio: 250:1 (at full signal)
A/D resolution: 12 bit
Dark noise: 3.2 RMS counts
Dynamic range: 2 x 10^8 (system); 1300:1 for a single acquisition
Integration time: 3 ms to 65 seconds
Stray light: <0.05% at 600 nm; <0.10% at 435 nm
What would be Corrected linearity: >99.8%

The maximum Electronics Power consumption: 90 mA @ 5 VDC

Data transfer speed: Full scans to memory every 13 ms with USB 2.0 or 1.1 port, 300 ms with serial port
A this could measure?
What is the maximum amount of absorbance
you can measure if the stray light in an
instrument is 8%?

If it is 0.05% at 600 nm as for the Ocean

Optics?
1. Where does stray light come from?

2. Is stray light likely to be more important for 200 or

for 900 nm light?

3. Is stray light likely to be more or less important

near a region where solvent interferes?
Double Dispersion Reduces the Stray Light
 Comparison of Instruments
Name $ ∆ range Ps/Po%

Spect 20 2-4k 2-8 190-1000 0.5

Double Beam 4-15k 195-850 0.1
PE-57 >5k 0.2 190-750 <0.1
Double dispersive 0.07 185-3125 0.0008
Multichannel Array 7-9k 200-920
Beer’s Law and Standard Additions
 QUANTITATION

1. Wide chromophore range (universality)

-extended by color forming reactions
for example complexation

2. Good sensitivity

3. Selectivity

4. Accuracy

5. Ease
1. Standard Curves
Choose a wavelength where the molar absorptivity does not change
where would this be?
why choose this wavelength region?

Need clear cells and no greasy fingers. Why?

Need to control: temperature; pH; electrolyte/solvent. Why?

2. Standard addition method

is useful when matrix (the solution containing the
sample analyte) effects complicate matters
 Overcoming Matrix Effects in Calibration Curves

Solvent
Signal
Matrix

Sample
Signal
Matrix Effect Matrix
If we don’t have a Example: Flame
Clear idea what Atomic Absorption for
The matrix effect is Pb in SeaWater, PbCl2
Then we drastically Is lost lowering the signal
Misjudge the conc
Of the sample from Ppm Metal
The measured signal
Our standards Standards made
Suggest this Up in the matrix of
Sample conc. The sample would
Suggest this sample
Conc.
 Overcoming Matrix Effects in Calibration Curves
0

 total m oles 
A m ea sured  b   10
 total volu m e 
20

 n un know n  n a dded 
A new , m ea su red  b   30

V sam ple of un know n  V added 

40

 V un kow n M unkn ow n  V stam d ard M s tan da rd 

A m ea su red  b   50

 V un kow n  V s tan dard 

V M  V sta m dard M s tan d ard 

A m ea sured  b  unkow n un know n 
 V total 

 V unko w n M un kn ow n   M s tan da rd 
A m easured  b    b  V stam da rd
 V total   V total 
 intercept slope
y x
 V u nkow n M unknow n   M s tan dard 
A m easured  b    b  V stam da rd
 V to tal   V total 

V M 
int ercept  b  u nkow n u nknow n 
 V to tal 

 M 
slop e  b  s tan d ard 
 V total 

V M 
b  un kow n unkn ow n 
in t ercept V tota l   V unko w n M u nkno w n
 
slop e  M  M s tan dard
b  s tan d ard 
 V tota l 

 int ercep t   M s tan dard 

    M u nkno w n
 slope   V unko w n 
 M=slope=0.03912

B=intercept=0.2422

Vunknown= 10 ml

Mstandard=11.1ppm

 total m oles 
A m easured  b 
 int ercept   M s tan dard 
     M unknow n
 total volu m e   slop e   V unkow n 

 0 .24 22   1 1.1 ppm 

    7 .0 1 ppm
 0 .03 81 2   1 0 
You did standard addition for the flame lead
analysis. You found:

A  0 0 .05 2 1  0 .4 3 3 ( ppb )

Your unknown volume is 10 mL and the standard

you added is 20 ppb.

What is the unknown concentration?

 Two Component Spectra

A 1   M 1 bC M   N 1 bC N

A 2   M 2 bC M   N 2 bC N

measure

Must be known

Result is two equations in two unknowns – can be solved