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Fundamentals of

UV-visible

spectroscopy

A Workbook

Fundamentals of

UV-visible spectroscopy

A Workbook

Preface

UV-visible spectroscopy is a well-established analytical technique with mature methods and equipment. It is commonly used in both research and science as well as in industry. Applications are found in classic analytical fields such as in the chemical industry (mainly petrochemical and dyestuff industry), the pharmaceutical industry or in environmental analyses. Other fields of application are gaining more and more importance such as biochemistry and bioscience. UV-visible spectrophotometers are available in almost all laboratories that do chemical or physical measurements. In general, they are simple to operate and modern spectrophotometers with built-in or external computer systems deliver processed results very rapidly. However, the apparent simplicity and speed of the technique can mean that spectrophotometers are used without sufficient understanding and erroneous results can be all too common.

This workbook is intended to provide information for practical training and instruction of school groups or students of engineering or natural sciences. It is also meant for self-study and independent learning of laboratory personnel and professional users. It is a companion to the primer "Fundamentals of Modern UV-visible Spectroscopy" (Hewlett-Packard publication number 12-5965-5123E) which can be used in teaching and learning the theory of UV-visible spectroscopy and instrumentation. With this workbook users can deepen the theoretical knowledge they may already have gained and complement it with practical exercises. The experiments and the results that are achieved help to better understand the theoretical background of UV-visible spectroscopy. The workbook will enable users to understand the possibilities, but also the sources of error, pitfalls and the limits when working with UV-visible spectroscopy. Soon users will develop a better understanding of their equipment and will be able to appreciate the advantages of diode array spectrophotometers compared to conventional scanning spectrophotometers.

Little prior knowledge of the equipment and of laboratory work is required before starting with the experiments. Nevertheless all users are asked to read and thoroughly follow the safety instructions for laboratory work and especially for the chemical substances they deal with. Unqualified persons have to be guided and supervised by authorized personnel. Make yourself familiar with the handbooks and users’ guides of your equipment. Only if the experiments are carried out carefully according to the Good Laboratory Practice (GLP) regulations best results can be achieved.

We would be very pleased to receive feedback on this workbook, especially with suggestions for improvements or additional experiments that could be included.

The primer "Fundamentals of modern UV-visible Spectroscopy", is available from Hewlett-Packard as publication number 12-5965-5123E.

General Equipment

o UV-visible spectrophotometer. With a few exceptions all experiments described in this workbook were performed on an HP 8453 diode-array UV-visible spectrophotometer but, in principle, any good quality UV-visible spectrophotometer may be used. The times given (for the experiments including evaluation) are based on the use of the HP 8453 which scans full spectra in about 1. seconds. If a scanning spectrophotometer is used, the time for experiments that require spectral measurement will be significantly longer.

o Calculator or personal computer for evaluation of data.

o Analytical balance for accurate preparation of test samples.

o Spatula for handling powders.

o Pipette bulb (to avoid mouth pipetting).

o Weighing papers.

o Cuvettes. When using a single beam spectrophotometer only one cuvette is needed. If a double-beam spectrophotometer is used two, preferably matched, cuvettes are required.

General Aspects of Sample Handling and Preparation

To minimize possible sources of error always consider the following general aspects of good sample handling:

- Use a pipette to empty and fill the cells. - Rinse the cell with the solvent or solution to be measured at least three times before measurement. - Take care not to dirty the optical surfaces of the cell with fingerprints or with any other substances. - In order to verify the quality and cleanliness of the cell used, measure a reference on air and a sample on the cell filled with distilled water before sample measurement. - When using a conventional scanning spectrophotometer, measure the references in the same wavelength range as the sample measurements. - Use fresh samples only. Storing the samples, especially in light and warm rooms, may create multiple sources of error due to unwanted reactions such as decomposition and sample contamination.

Caution: Carefully read, understand and follow the safety instructions for the substances you deal with. Chemical substances must always be handled according to legal regulations for hazardous materials that are in force in your country and at your place of work. All substances used must be handled using exactly the concentrations and compositions described in this workbook.

• Basic Principles and Applications Part 1.
• 1.1. Basic Principles—What is a UV-visible Spectrum?...............................................
• 1.2. Chromophores
• 1.3. Environment of Chromophores
• 1.4. The Effect of Solvents on UV-visible Spectra......................................................
• 1.5. The Meaning of Color in Spectroscopy
  • Path Length Dependence of Absorbance Values 1.6. Quantitative Analysis — Lambert-Beer’s Law
• 1.7. Concentration Dependence of Absorbance Values
• 1.8. Possible Sources of Error — Influence of Impurities
• 1.9. Influence of Temperature — Potassium Chloride
• 1.10. Influence of Temperature — Methyl Orange.......................................................
• 1.11. Influence of pH — Buffered Methyl Orange Solutions.......................................
• 1.12. Influence of pH — Potassium Dichromate Solution
• 1.13. Effect of Concentration
• 1.14. Principle of Additivity.............................................................................................
• Measuring Instrument Performance Part 2.
• 2.1. Wavelength Accuracy
• 2.2. Wavelength Accuracy Using the Deuterium Lines..............................................
• 2.3. Photometric Accuracy Using Potassium Dichromate
• 2.4. Photometric Accuracy Using Neutral Density Glass Filters..............................
• 2.5. Stray Light
• 2.6. Spectral Resolution...............................................................................................
• 2.7. Noise
• 2.8. Baseline Flatness...................................................................................................
• 2.9. Stability...................................................................................................................
• Sample Handling and Measurement Part 3.
• 3.1. Sample Handling
• 3.2. Cell Types...............................................................................................................
• 3.3. Cleanliness
• 3.4. Influence of Instrumental Parameters................................................................
• 3.5. Properties of Solvents
• 3.6. Background Absorbance......................................................................................
• 3.7. Sample Decomposition.........................................................................................
• Applications Part 4.
• 4.1. Single Component Analysis
• 4.2. Multicomponent Analysis.....................................................................................
• 4.3. Derivative Spectroscopy
• 4.4. Kinetic Analysis — Studying Melting Temperatures of DNA
• 4.5. Isosbestic Points
• 4.6. Biochemical Spectroscopy...................................................................................
  • Appendix

Part 1

5 Basic Principles and

Applications

Basic Principles and Applications

Evaluation

1 Enter the wavelengths of the main absorbance maxima of caffeine and erythrosine in the table below.

2 If the color of light and wavelength are related as follows, which colors of light do caffeine and erythrosine absorb?

Color Wavelength Range [nm]

violet 380 – blue 435– green 480– yellow 560– orange 595– red 650–

3 Explain the relationship between the color of light absorbed by matter and the color of light that you can observe when looking at it.

4 The following equation shows the relationship between absorbance and transmittance:

A = 2 – log T [%]

Calculate the transmittance values at the main absorbance maximum of each sample and enter the values in the table below.

Evaluation Table 1.1. Measured Wavelengths and Absorbance Values of the Absorbance Maxima

Compound λ max [nm] Absorbance [AU]

Caffeine

Erythrosine

Evaluation Table 1.2. Calculated Transmittance Values of the Absorbance Maxima

Compound λ max [nm] Absorbance [AU] Transmittance [%T]

Caffeine

Erythrosine

Basic Principles and Applications

5 Which compound absorbs light of higher energy (per photon)? Use the following equations to calculate the energy of light in Joule that is absorbed at the maxima of both compounds.

where:

E = energy [J] h = Planck’s constant (6.62 × 10 -34^ Js) c = speed of light (3 × 108 ms-1) v = frequency [s-1] λ = wavelength [m]

E =h v

c =λ v

Evaluation Table 1.3. Calculated Energies of Light at the Absorbance Maxima

Compound λ max [nm] Energy [J]

Caffeine

Erythrosine

Basic Principles and Applications

4 Transmittance values calculated from the absorbance values are shown in the table below.

An absorbance of 1 is measured when the sample absorbs 90 % of the incident light.

5 The calculated energies of the light at the maxima are shown in the table below.

Caffeine absorbs light of higher energy than erythrosine. The lower the wavelength, the higher the energy per photon.

Results Table 1.2. Calculated Transmittance Values of the Absorbance Maxima

Compound λ max [nm] Absorbance [AU] Transmittance [%T]

Caffeine 205 0.699 20. Erythrosine 526 0.798 15.

Results Table 1.3. Calculated Energies of Light at the Absorbance Maxima

Compound λ max [nm] Energy [J]

Caffeine 205 9.69^ ⋅^10 -

Erythrosine 526 3.78^ ⋅^10 -

Basic Principles and Applications

1.2. Chromophores

Introduction

Chromophores are parts of a molecule that have electronic bands with energy differences comparable to the energies of the UV-visible light which is then absorbed by them. Chromophores, for example, dienes, nitriles, carbonyl, or carboxyl groups often contain π bonds. Other types of chromophores are transition metal complexes and their ions.

Molecules without chromophores, such as water, alkanes or alcohols, should be ideal solvents for UV-visible spectroscopy because they hardly show any absorbance themselves.

The following experiment shows that small variations in the structure of molecules can lead to significant differences in the resulting absorbance spectra.

Reagents and Equipment

o acetone

o acetaldehyde

o 2-propanol

o distilled water

o three 20-ml volumetric flasks

o 0.1-ml pipette or syringe

o disposable glass pipettes (minimum 4)

o 10-mm path length quartz cell

Experiment

Time: about 45 min 1 Prepare the following solutions: a) 0.1 ml acetone in 20 ml distilled water b) 0.2 ml acetaldehyde in 20 ml distilled water c) 0.1 ml 2-propanol in 20 ml distilled water 2 Measure a reference on distilled water. 3 Measure the spectra of the acetone, acetaldehyde and 2-propanol solutions in the range from 200 to 350 nm.

Basic Principles and Applications

Example Results & Discussion

Samples: acetone in distilled water (5 ml/l) acetaldehyde in distilled water (10 ml/l) 2-propanol in distilled water (5 ml/l)

Cell: 10-mm path length quartz cell

Instrument Parameters: wavelength range: 200–350 nm absorbance range: 0.0–1.5 AU

Diagram: Measured Absorbance Spectra of the Different Chromophores

1 The wavelengths and absorbance values at the maxima for each compound are shown in the table below.

1 2

3

Absorbance [AU]

200 225 250 275 300 325 350

1 Acetone 2 Acetaldehyde 3 2-Propanol

Wavelength [nm]

Results Table 1.4. Wavelengths and Absorbance Values at the Absorbance Maxima

Compound λ max [nm] Absorbance [AU]

Acetone 265 1. Acetaldehyde 277 1. 2-Propanol no maximum found not applicable

Basic Principles and Applications

2 The molecular structures of the three compounds are:

The absorbance bands of acetaldehyde and acetone are caused by the presence of the C=O chromophore. Note that the position and intensity of the absorbance bands are similar. If either of these solvents has to be used for making sample solutions for UV-visible measurements, the data in the range from 225 to 325 nm will be affected by the absorbance of the solvent itself.

3 Propanol has no chromophore and shows no significant absorbance band in the UV-visible range. It is an almost ideal solvent for UV-visible measurements.

Acetone Acetaldehyde 2-Propanol

C

O CH 3 CH 3 C

OH CH 3 CH 3 H

C

O CH 3 H

Basic Principles and Applications

Evaluation

1 Discuss the differences in the measured spectra of naphthalene, anthracene and phenanthrene solution. Take the wavelengths of absorbance maxima and the structure of bands into consideration.

2 Enter the wavelength ranges of the absorbance maxima of the compounds in the table below.

Evaluation Table 1.5. Wavelength Ranges of the Absorbance Maxima

Compound λ max [nm]

Naphthalene

Anthracene

Phenanthrene

Basic Principles and Applications

Example Results & Discussion

Samples: naphthalene in 100 ml ethanol (24 mg/l) anthracene in 100 ml ethanol (16 mg/l) phenanthrene in 100 ml ethanol (50 mg/l)

Cells: 2-mm path length quartz cell for naphthalene solution 10-mm path length quartz cell for the other two solutions

Instrument Parameters: wavelength range: 200–400 nm absorbance range: 0.0–1.2 AU

Diagram: Measured Absorbance Spectra of the Different Chromophores

1 The spectra show that not only the kind of chromophore is important. In our example we have the benzene-rings that are chromophores with π–π*^ transitions because of conjugated double bonds. The environment of the chromophores or the combination with other chromophores also have a strong influence on the position and values of absorbance bands.

The molecular structures of the three compounds are:

Absorbance [AU]

200 250 300 350 400

1.00 (^) 1 Naphthalene 2 Anthracene 3 Phenanthrene

Wavelength [nm]

1

2

3

Naphthalene Anthracene^ Phenanthrene