Ch4BioanalyticalApplicationsofElectronicSpectroscopy - Ch 4...

Info icon This preview shows pages 1–3. Sign up to view the full content.

View Full Document Right Arrow Icon
4-1 Ch. 4 Bioanalytical Applications of Electronic Spectroscopy Objective To use two methods for determining the concentration of protein in milk whey from buttermilk and in solutions of gelatin based on UV-Vis spectroscopy. To evaluate which method is better suited for the analysis. Principles of Spectroscopy Spectroscopy is the study of the interaction of light with matter and is often used to deduce molecular structure or measure the amount of a substance in a sample. You are already familiar with atomic spectra from last week’s lab. Two things are important to remember: the atomic spectra you observed were emission spectra , which resulted from the release of a photon from the atom when an excited state electron relaxed to a lower energy state, and the spectra were line spectra , meaning only certain energies of light were emitted. (What is the physical basis of non- continuous spectra?) Today you will use a much more commonly used form of spectroscopy in which we measure the absorbance of light rather than emission, and we will observe a continuous spectrum of absorption over the UV/visible range (approximately 200-800nm) due in part to the fact we are looking at molecules rather than atoms. All organic compounds are capable of absorbing electromagnetic radiation, because all contain valence electrons that can be excited to higher energy levels. To understand electrons in molecules, a bonding theory called Molecular Orbital Theory (MO theory) makes an extension from atomic orbital calculations. (This will be discussed in class more later.) Just as electrons occupy atomic orbitals in an atom, electrons occupy molecular orbitals in molecules. Exactly analogous to an electron in the ground state of an atom absorbing energy to be promoted to an excited state, the electrons in the ground state molecular orbitals can be excited to higher energy molecular orbitals. In molecules that contain only single bonds, the energy difference between the ground state MO and the excited state MO is large. The excitation energies associated with electrons involved with single bonds are sufficiently high that absorption is restricted to the
Image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
4-2 vacuum UV region ( λ <185 nm). It is difficult to perform absorption experiments in this region because components of the atmosphere absorb strongly in this range and interfere with the absorbance of the analyte . Absorption of longer wavelength ultraviolet and visible radiation is more practical for analysis of molecules because atmospheric components do not interfere. Absorption in this region is restricted to molecules that contain ground state MOs and excited MOs that are relatively close in energy. From the bonding of molecules, we can predict arrangements of bonds that are likely to absorb in the UV/vis range. These arrangements are called chromophores . Chromophores can be recognized by the fact that they contain double bonds.
Image of page 2
Image of page 3
This is the end of the preview. Sign up to access the rest of the document.