Introduction to Spectrophotometric Measurements
Introduction
The solutions of many compounds have characteristics colors, and the intensity of such a color is
proportional to the concentration of the compound. During our last lab, you had the opportunity
to use various colorimeters to determine the concentrations of a number of compounds in Cape
Fear River water.
Spectrophotometers measure the absorption of light by various compounds in solution. By using
the spectrophotometer to measure light passed through a treated sample, scientists can arrive at
the concentration of a material in solution. This is done extensively for nutrients such as Nitrite
-3
(NO2 ), Phosphate (PO4 ), or Silicate (SiO3-2). Many times, like for these ions, the material of
interest is colorless and to get a meaningful and measurable absorption of light, reagents are
added and then the light absorbed by the subsequent colored, chemically-created, compound is
directly measured. The actual nutrient's concentration is calculated or found from a constructed
Calibration or Standardization Graph.
In this activity you shall investigate how the proper spectrophotometric wavelength for a
material is found AND use a Standardization Graph (Absorbance vs Concentration plot) from
which the concentrations for various "Unknowns" will be determined. Being an application of
Beer's Law, this plot will be a straight line.
A. Finding the characteristic wavelength for Cobalt Chloride (CoCl2)
The characteristic wavelength or “Optimum wavelength” is the one with the highest absorbance
and will be the wavelength at which the spectrophotometer is set for all subsequent absorbance
measurements when using cobalt chloride. The following absorbances were recorded at different
wavelengths:
Wavelength (nm)
420
430
440
450
460
470
480
490
500
505
510
515
520
525
530
540
550
560
570
580
590
Absorbance
0.069
0.101
0.149
0.218
0.280
0.330
0.370
0.410
0.452
0.468
0.478
0.482
0.469
0.440
0.410
0.320
0.220
0.140
0.100
0.070
0.040
Construct an Absorbance vs. Wavelength graph in Excel using the above data.
Absorbance should be on the Y-axis. What is the optimum wavelength of cobalt chloride?
CoCl2 Optimum Wavelength (nm) = ______________
B. Standardization Graph:
During the last lab, you learned how to make two dilutions of a solution of copper II sulfate
(CuSO4 · 5H2O). As a check on your technique, we could have analyzed those samples to
determine the concentration of the dissolved substance. According to Beer’s law, the absorbance
of a solute in solution is a function of its concentration at a particular wavelength, and therefore,
absorbance measurements can be used to determine the concentration of solutions.
A stock solution of cobalt chloride and a blank of deionized water were measured using the
optimum wavelength (found in Part A above) on the spectrophotometer. The following
absorbances were recorded:
Standards
100%
0%
Absorbance
0.500
0.000
An Absorbance vs. Concentration (%) graph from Excel is attached. Connect the 100%
and the 0% points to make a line. To use the standardization graph go into the graph with the
unknown's absorbance, read across to the line, and its concentration is directly below!
Let’s say we made 3 dilutions of cobalt chloride. A stock solution of cobalt chloride (100%) was
diluted to a 75%, a 50%, and a 25% solution. Using your Standardization graph, what is the
absorbance of each of these dilutions?
Standards
75%
50%
25%
Absorbance
Use your Standardization Graph to determine the concentration (%) of the following
unknowns:
Unknown #
1
2
3
4
5
6
7
8
9
10
Absorbance
0.100
0.178
0.240
0.300
0.410
0.480
0.070
0.275
0.400
0.460
Concentration (%)
This assignment is due as homework at the beginning of our next class!!!!!
Absorbance vs. Concentration (%) of CoCl 2
0.550
0.500
Absorbance
0.450
0.400
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0
20
40
60
% Concentration
80
100
120