1. Instrumental Analysis
Applications of spectrophotometry
3rd Lecture
5 Biological PHCM561
Dr. Nermin Salah

2. Objectives Methods of Standardization
Direct method  Calibration curve method
For absorbing analytes
For non-absorbing analytes
Serum iron determination as an example
For mixtures
Spectrophotometric titration
Standard addition method

3. Beer’s Law Ax =  b cx Methods of Analysis (Standardization methods)
I – Direct method (calibration curve method)
a. Applications to absorbing species
The most straightforward way to use Beer’s law for quantitative analysis is to measure the absorbance of the sample solution at a wavelength at which the species in solution is known to absorb radiation.
Knowing the value of molar absorptivity, , for the analyte at the given wavelength, you can directly substitute in Beer’s law to get the value of the unknown concentration of the analyte.
Beer’s Law Ax =  b cx
where : molar absorptivity,
b: pathlength, and
c: molar concentration

4. Single-point calibration Standard with measured absorbance
If the value of molar absorptivity is not available at the selected wavelength:
Single-point calibration
Standard with measured absorbance
Astd =  b cstd
Unknown with measured absorbance
Aunk =  b cunk
Ratio the two equations
Solve for cunk
Note that, the standard solution of the species under test should be prepared and measured under the same conditions of the analyte (in the same cell and at the same )

5. But, conclusions based upon a single measurement are not statistically sound, because it is easy to make a measurement error that can not be detected with a single measurement
Calibration Curve
Series of standard solutions containing known concentrations of the analyte are prepared and introduced into the instrument.
Series of standard solutions of the analyte is prepared by successive (serial) dilutions.
x10-4 M x10-4 M
Highest A
Lowest A
Coriginal = Cdiluted X dilution factor
Cdiluted = Coriginal / dilution factor
dilution factor = final volume / original volume
Remember that

6. Absorbance is recorded.
Absorbance is corrected for the blank reading.
Resulting data is then plotted to give a linear graph of corrected absorbance readings vs. analyte concentration.
Absorbance of the analyte is measured at the same conditions and concentration of the analyte can be found:
By calculating the slope of the linear curve (b), and substituting in Beer’s law to find the concentration of the analyte
Aunk = (b) cunk where b is the slope of the calibration
curve

7. By direct extrapolation
Aunk b
Concentration of unknown
iii. By substitution in the straight line equation.
Y= mx + b where y = absorbance
m = slope = b
b = intercept
For example
Y = 10800x

8. b. Applications to non-absorbing species
Many non-absorbing analytes can be determined spectrophotometrically by causing them to react with chromophoric reagents to give products that absorb strongly in the UV-Vis regions (products of high ).
Serum Iron Determination
Human blood usually contains 45 vol% cells and 55 vol% plasma (liquid)
If blood is collected without an anticoagulant, the blood coagulates, and the liquid that remain is called serum. Serum normally contain Fe attached to transferrin.
For spectrophotometric determination of iron in blood serum, the following steps should be followed:
Reduce Fe3+ in transferrin to Fe2+, which is released from the protein. The reducing agent are hydroxyl amine, thioglycolic acid or ascorbic acid
2Fe3+ + 2HSCH2COOH Fe2+ + HOOCCH2S-SCH2COOH + 2H+
Thioglycolic acid

9. protein(aq) protein(s)
Add trichloroacetic acid (Cl3CH2COOH) to precipitate proteins, leaving Fe2+ in solution. Centrifuge the mixture to remove the precipitate. If protein were left in the solution it will cause light scattering which would result in an error in the absorbance reading.
protein(aq) protein(s)
Transfer a measured volume of supernatant liquid (colorless solution containing Fe2+) from step 2 to a fresh test tube. Add buffer and excess ferrozine (chromophoric reagent) where a purple complex is formed. Measure the absorbance of at 562 nm (max for the complex)
Fe ferrozine [(ferrozine)3Fe]4-
colorless purple complex
It is important to prepare a reagent blank containing all the reagents, but with the analyte is replaced by distilled water. Any absorbance of the blank is due to the color of uncomplexed ferrozine plus the color caused by the iron impurities in the reagents or glassware. Subtract the absorbance of the blank from the absorbance of the sample before doing any calculations.

10. Prepare a series of standard Fe2+ solutions
Prepare a series of standard Fe2+ solutions. Standards should be prepared following the same procedure as unknown. Ferrous ammonium sulfate (Fe(NH4)2(SO4)2.6H2O) can be a suitable standard.
Measure the absorbance of the standard solutions at 562 nm and plot a calibration graph between the absorbance readings and concentrations of standards.
If unknown and standards are prepared in the same way with identical volumes, then the quantity of iron in the blood serum can be determined through extrapolation.
(ferrozine)3Fe(II)
complex
max = 562 nm

11. Stable complex with little absorbance at 562 nm
In the preceding iron determination, the results would be about 10% high because serum copper also form colored complex with ferrozine.
Spectral interference of copper is eliminated if neocuproine or thiourea is added. These reagents are said to mask copper because they form strong complexes and prevent Cu+ from reacting with ferrozine.
2neocuproine + Cu [(neocuproine)2Cu]+
Stable complex with little absorbance at 562 nm

12. Analysis of a Mixture
Sample may contain multiple analytes that absorb light.
Analyte A
Mixture of
Analytes A and B
Analyte B

13. Analysis of a Mixture
Detector does not distinguish among absorbance readings of individual analytes
Absorbance of analytes are additive.
For our example:
and more generically:
mix.

14. A mixture contains two analytes, X and Y
Measure absorbance of the mixture at max for X
Measure absorbance of the mixture at max for Y
(l)
(l) X l
l A Y
By knowing the values of molar absorptivities at the two wavelengths, the above two equations can be solved simultaneously for the two unknowns [X] and [Y]. l

15. ’ ’’ Determination of , , and Prepare standard solutions of pure X.
Measure the absorbance of these solutions at ’ and ’’.
Plot a calibration graph between absorbance readings and concentrations of the standards at both s. From the slopes of the two graphs, the values of and can be determined. The values of and can be determined in a similar way. ’
’’

16. II-Spectrophotometric titration
If the analyte (At), the titrant (T), or the reaction product (P) absorbs radiation in the UV-Vis. regions, absorbance measurements at fixed wavelength can be used to locate the endpoint of the titration:
At + T P
The absorbance of the analyte solution is measured after each addition of the titrant and the end point is located from the plot of the absorbance readings and the volume of the titrant added (Titration curve)

17. Let’s consider the analysis of hydrogen peroxide (At) with potassium permanganate (T) in an acidic solution.
The potassium permanganate or MnO4 (T) is the only colored substance in the reaction.
How would the absorbance change as titrant was added?

18. 5H2O2 + 2MnO4- + 6H+  5O2 (g) + 2Mn2+ + 8H2O
(Analyte) (Titrant)
purple (colorless products)
Note that you do not need to have
data points at the equivalence point.
Equivalence point located by extrapolation of the two lines.
absorbance
Equivalence point
MnO4- reacting,
color disappears
MnO4-
accumulates
Volume of titrant (mL KMnO4)

19. III- Standard Addition Method in Spectrophotometry
Direct calibration curve method can be applied for analyzing unknown sample only and only if the standard solutions and the unknown solution are prepared and measured under exactly the same conditions.
However, there are frequently what are known as matrix effects where substances other than the analyte affect the absorbance reading of the sample solution at the wavelength chosen for the analysis.
We can avoid the complication of matching the matrix of the standards to the matrix of the sample by conducting the standard addition method.
In this method, the matrix, that present in the sample, will affect the absorbance readings for both the analyte in the standard and the sample in the same way at the same time. Thus, it is possible the analyze the analyte accurately even in the presence of matrix.

20. Standard addition usually involves the addition of an increasing increments of a standard solution to sample aliquots of the same size (spiking).

21. Example of Standard Addition Data
Glucose in blood serum is determined spectrophotometrically by the formation of a colored complex with o-toludine.
Six identical 50.0 L samples of blood serum were treated with increasing
amount of known standards of
glucose.
The o-toludine reagent is added, and the absorbance is measured at the analytical wavelength.
The following results were obtained:
What is the glucose concentration in the blood serum samples?
g Glucose
(standard added)
Absorbance
0.230 10
0.272 25
0.340 40
0.416 60
0.507 75
0.568

22. A plot is constructed of Absorbance (y-axis) vs. amount of std
A plot is constructed of Absorbance (y-axis) vs. amount of std. (x-axis) g of standard glucose added in this example.
m (slope) =
Y= X
Amount of glucose in sample
Value of X when Y = 0
b (intercept) = 0.229

23. The value of glucose in the original sample is found by determining the x-intercept by extrapolation, i.e., finding the value of x when y = 0.
Or by using the values shown by the Linear Fit and the form of the linear equation
y = mx + b
0 = x
Solving for x,
x = / = - 50 g.
The amount of glucose in unknown glucose sample, that had no standard glucose was added, is 50 g.
Since the volume was 50.0 L, the [Glucose] = 50 g / 50 L or 1 g/L. This could also be expressed as [Glucose] = 1 mg/mL

24. Resources and references
Textbook: Principles of instrumental analysis, Skoog et al., 5th edition,
chapter 14.
Quantitative chemical analysis, Daniel C. Harris, 6th edition , chapter
19.
Lecture slides partially adopted from Dr. Raafat Aly slides.
Useful links
Self evaluation questions
Extra resources are available on the intranet.