S5. DESIGN OF A SPECTROPHOTOMETRIC ANALYSIS
PURPOSE
1. To determine the appropriate series of standards for a visible absorption calibration graph.
2. To determine the concentration of a sample by absorption spectrophotometry.
INTRODUCTION
To determine the concentration of an unknown solution by spectroscopy involves analysing a
series of solutions of known concentration, the standards, and comparing the value obtained
for the unknown.
Beer’s Law is generally linear over the range of 0.1 – 1.0 absorbance units hence it is
advisable to have all solutions fall within this range. When the appropriate standards are not
already prepared the chemist will need to devise a procedure that will provide standards that
fall within the appropriate range. The following method is one way of determining the
standards required (Note the method uses the premise that the absorptivity coefficient is a
constant as per Beer’s Law):
Example
A 1000mg/L standard solution has an absorbance of 3.811. Diluted 10 times to 100 mg/L, it
gives an absorbance of 1.128.
Step 1- Determining the A/c relationship
Example
Calculate the constant (k=ab) from the absorbance and
concentration of the standard. The absorbance of this
solution must be less than 2.0
k = 1.128/100
= 1.128 x 10-2
k = A/c
Step 2 – Determining the Concentration of the 0.2
Absorbance standard
Calculate the concentration of a solution of the analyte
with an absorbance of 0.2 using the value of k calculated
in Step 1.
c = 0.2/k
c = 0.2 / 1.128 x 10-2
= 17.73 mg/L
Step 3 – Determining a suitable concentration range Conc (mg/L)
17.73 20
for standards
Round the concentration from step 2 to a manageable
40
value (nearest 5 or 10). The other standards are 2, 3 and 4
60
times the concentration
80
Approx Abs
0.2
0.4
0.6
0.8
Step 4 – Preparation of the standards
The 0.2, 0.4, 0.6 and 0.8 absorbance standards can be
most conveniently and accurately prepared by dilution of
5, 10, 15 and 20 mL aliquots, respectively, of a more 5 mL of X mg/L diluted to
concentrated stock solution (X).
100 mL of 20 mg/L.
To calculate the concentration of X, assume that 100 mL
of the final standards are being prepared. Therefore, 5
X = 20 x 20 mg/L
mL of X is diluted to produce 100 mL of 0.2 Abs std.
= 400 mg/L
Using C1V1 = C2V2 then C2 = 100 x conc 0.2 std
5
= 20 x conc. of 0.2 std
Step 5 – Preparation of X
If X is greater than 500 mg/L, than it can be prepared
directly. Otherwise, it should be prepared by diluting a
more concentrated standard. Using pipettes, the most
convenient and accurate dilutions are 2 (50 100),
4(25 100), 5 (20 100), 10 (10100) or 20 (5100).
A burette could, however, be used. You need at least 100
mL of stock standard ( 5+10+15+20).
400 mg/L is too low to prepare
directly (unless you make at least
500mL of it). Given that
1000mg/L standards are very
often available, a suitable method
would involve a 2.5 dilution of
the 1000 mg/L stock would give
400 mg/L.
40 mL of 1000 mg/L diluted to
100mL of 400 mg/L
PROCEDURE
1. Obtain two coloured solutions: a 1000 mg/L stock solution and a sample solution of
unknown concentration.
2. Using a measuring cylinder, dilute the stock solution to 100mg/L by taking 10 mL of the
1000 mg/L and making it up to 100 mL. Run an absorption spectrum of this solution.
3. Determine the wavelength of maximum absorbance, and the absorbance value for the
solution prepared in Step2.
4. Using the data obtained in step 3, determine the range of concentrations suitable for
preparation of a calibration graph. Your final dilutions should use 5, 10, 15 and 20 mL
aliquots diluted quantitatively to 100 mL.
5. Measure the absorbance of your sample. Determine the factor by which it needs to be
diluted to bring the absorbance into the range of your standards.
6. Prepare, quantitatively 100 mL of diluted unknown sample.
7. Record the absorbance of your standards and the diluted sample.
CALCULATIONS
1. Graph the absorbance of the standards against their concentration. .
2. Determine the concentration of the diluted sample.
3. Determine the concentration of the concentrated unknown sample.
QUESTIONS
1. What is the optimum range of absorbance for Beer’s Law calibration graphs?
2. What errors arise at absorbance below the optimum range?
3. What errors arise at absorbance above the optimum range?
4. When determining the absorbance of the stock solution, why could you carry out dilution
of the solution approximately?
5. Why is it convenient to prepare the calibration graph standards using the 5, 10, 15 and 20
mL series of aliquots?
S5. RESULTS SHEET
Date of analysis
Identity of instrument:
Sample solution code:
Wavelength of maximum absorbance:
Absorbance of solutions
Solution
Maximum
absorbance
100 mg/L (from stock)
Sample solution
Step
1. Determining the A/c constant
Calculation
2. Determining the concentration
of 0.2 Abs standard
3. Determining a suitable
concentration range
4. Preparation of the standards
5. Preparation of the sample
Dilution factor for unknown:
Solution
Blank
Standard 1
Standard 2
Standard 3
Standard 4
Diluted sample
Conc (mg/L)
0
Abs
Have you?
Completed the instrument log
Completed the sample register
Completed the standard register
Teachers signature
Date
Signature