Chapter 29 – Summary
29.1 Chromatography Basics
Chromatography is an efficient way for chemists to separate and analyze mixtures. It is a method by which a mixture is separated by distributing its components between two phases. The stationary phase remains fixed in place while the mobile phase carries the components of the mixture through the medium being used. The stationary phase acts as a constraint on many of the components in a mixture, slowing them down to move slower than the mobile phase. The movement of the components in the mobile phase is controlled by the significance of their interactions with the mobile and/or stationary phases. Because of the differences in factors such as the solubility of certain components in the mobile phase and the strength of their affinities for the stationary phase, some components will move faster than others, thus facilitating the separation of the components within that mixture.
29.2 Thin Layer (TLC) and Paper Chromatography (PC)
In paper chromatography (PC), samples are separated using paper as the stationary phase. The mobile phase is typically water. The procedure for using paper chromatography is described and can be completed with household items. Paper chromatography is a visible qualitative separation method that can be quantified using Rf values (comparison of distance component travelled compared to the distance the solvent travelled). Two-way paper chromatography helps to separate components that have similar Rf values. Rf values remain consistent under consistent conditions but changing temperature or solvent will change the values.
Thin layer chromatography (TLC) is a similar technique that uses a thin layer of silica gel or alumina on a rigid surface as the stationary phase. It also involves a mobile phase of liquid solvent. In cases where components are not visible, UV fluorescence can be used to “see” the spots. This requires TLC plates that include a UV compound.
29.3 Chromatographic Columns
Chromatography is an analytical technique that separates components in a mixture. The same principles used in thin layer chromatography can be applied on a larger scale to separate mixtures in column chromatography. Column chromatography is often used to purify compounds made in the lab. Chromatographic columns are part of the instrumentation that is used in chromatography. Five chromatographic methods that use columns are gas chromatography (GC), liquid chromatography (LC), Ion exchange chromatography (IEC), size exclusion chromatography (SEC), and chiral chromatography. The basic principles of chromatography can be applied to all five methods.
29.4 Chromatography Technology
Gas chromatography is a term used to describe the group of analytical separation techniques used to analyze volatile substances in the gas phase. In gas chromatography, the components of a sample are dissolved in a solvent and vaporized in order to separate the analytes by distributing the sample between two phases: a stationary phase and a mobile phase. The mobile phase is a chemically inert gas that serves to carry the molecules of the analyte through the heated column.
High Performance Liquid Chromatography (HPLC) is a powerful analytical technique used for the separation of compounds soluble in a particular solvent. This separation occurs based on the interactions of the sample with the mobile and stationary phases. Because there are many stationary/mobile phase combinations that can be employed when separating a mixture, there are several different types of chromatography that are classified based on the physical states of those phases.
29.5 Spectroscopy Basics
Spectroscopy is an experimental method used by chemists to elucidate structural information. The interaction between a compound or sample and a selected region of the electromagnetic spectrum can be measured both qualitatively and quantitatively. Absorption of an appropriate quantity of energy can raise the atoms and bonds of molecules from a lower to a higher energy level, while emission of electromagnetic radiation corresponds to a change from a higher to a lower energy level. The resulting spectrum can be used to determine structural information about the molecule.
29.6 Infrared (IR) Spectroscopy
The infrared region of the electromagnetic spectrum causes asymmetric bonds to stretch, bend, and/or vibrate. This interaction can be measured to help elucidate chemical structures. Asymmetry and polarity increase the strength of IR absorption (infrared active). Symmetrical carbon-carbon double and triple bonds will not absorb IR light and are called “infrared inactive”. The analysis and interpretation of the IR spectra for several compounds are explained. One of the most common applications of infrared spectroscopy is the identification of organic compounds. The IR spectra for the major classes of organic molecules are shown and can be used to help determine present functional groups. Numerous examples are provided.
29.7 Mass Spectrometry (MS)
Mass spectrometry is an analytic method that employs ionization and mass analysis of compounds in order to determine the mass, formula and structure of the compound being analyzed. A mass spectrometer creates charged particles (ions) from molecules. It then analyzes those ions to provide information about the molecular weight of the compound and its chemical structure. A mass analyzer is the component of the mass spectrometer that takes ionized masses and separates them based on charge to mass ratios and outputs them to the detector where they are detected and later converted to a digital output. Fragmentation patterns are formed when organic molecules are fed into a mass spectrometer. When interpreting fragmentation patterns, the weakest carbon-carbon bonds are the ones most likely to break. The relative formula mass (relative molecular mass) of an organic compound can be determined from its mass spectrum. Mass spectrometry is widely used in industry through the coupling Gas Chromatography (GC) with Mass Spectrometry (MS).
29.8 Nuclear Magnetic Resonance
Nuclear Magnetic Resonance (NMR) Spectroscopy uses the electromagnetic radiation of radio waves to probe the local electronic interactions of a nucleus. The chemical shift is the resonant frequency of a nucleus relative to a standard in a magnetic field (often TMS). The position and number of chemical shifts provide structural information about a molecule.
29.9 1H NMR Spectroscopy
Proton NMR finds use for both qualitative analyses and quantitative analyses. In an applied, external magnetic field, protons in different locations of a molecule have different resonance frequencies, because they are in non-identical electronic environments. Equivalent protons experience the same electronic environment. An approximate idea of the chemical shifts of the most common types of protons is helpful when interpreting 1H NMR spectra. Tables of values are available to help with determining the molecular location of protons. The ratio of proton signal areas correlates with the proton ratio of a compound providing useful structural information. The peaks can be split into multiplets when the magnetic field experienced by the protons of one group is influenced by the spin arrangements of the protons in an adjacent group. Splitting occurs primarily between non-equivalent hydrogens that are separated by three bonds.
29.10 13C NMR Spectroscopy
The 12C isotope of carbon – which accounts for up about 99% of the carbons in organic molecules – does not have a nuclear magnetic moment, and thus is NMR-inactive. Fortunately for organic chemists, however, the 13C isotope, which accounts for most of the remaining 1% of carbon atoms in nature, has a magnetic moment just like protons. Most of what we have learned about 1H-NMR spectroscopy also applies to 13C-NMR, although there are several important differences. 13C chemical shifts are analogous to proton chemical shifts and are influenced by the electro-magnetic environment of the carbon atoms. Tables of values are available for chemical shifts of carbon atoms and can be useful in determining the carbon skeletal structure of molecules.
29.11 Visible and Ultra-Violet Spectroscopy (UV-Vis)
As light passes through a sample, its power decreases as some of it is absorbed. This attenuation of radiation is described quantitatively by two separate, but related terms: transmittance and absorbance. Beer’s law connects absorbance to the concentration of the absorbing species. The determination of an analyte’s concentration based on its absorption of ultraviolet or visible radiation is one of the most common quantitative analytical methods. In addition, if an analyte does not absorb UV/Vis radiation—or if its absorbance is too weak—we often can react it with another species that is strongly absorbing.
The conjugated double bonds are characterized by alternating carbon-carbon bonds separated by carbon-carbon single bonds. The stability of conjugated dienes can be explained using the delocalization of charge through resonance. The ultraviolet absorption maximum of a conjugated molecule is dependent upon the extent of conjugation. The ultraviolet (UV) region of the electromagnetic spectrum corresponds to conjugated bond energies. The most useful UV region of the electromagnetic spectrum has a wavelength between 200 and 400 nm. Absorption in the visible region of the electromagnetic spectrum results in coloured compounds.
Attribution & References
- 29.1 Summary adapted from “Chromatography” In Instrumental Analysis, CC BY-NC-SA 4.0 AND 10: Solids, Liquids and Solutions by Ed Vitz, John W. Moore, Justin Shorb, Xavier Prat-Resina, Tim Wendorff, & Adam Hahn, Chemical Education Digital Library (ChemEd DL) In ChemPRIME (Moore et al.) , CC BY-NC-SA 4.0
- 29.2 Summary written by Samantha Sullivan Sauer, CC BY-NC 4.0.
- 29.3 & 29.4 Summaries adapted from “Chromatography” In Instrumental Analysis, CC BY-NC-SA 4.0 and “V. Chromatography” by Jim Clark In Instrumental Analysis, CC BY-NC 4.0
- 29.5 Summary is adapted from “11: Infrared Spectroscopy and Mass Spectrometry” In Map: Organic Chemistry (Wade), Complete and Semesters I and II , CC BY-NC-SA 4.0. and “21: Spectra and Structure of Atoms and Molecules” In by Ed Vitz, John W. Moore, Justin Shorb, Xavier Prat-Resina, Tim Wendorff, & Adam Hahn, Chemical Education Digital Library (ChemEd DL) In ChemPRIME (Moore et al.), CC BY-NC-SA 4.0.
- 29.6 summary adapted from “11: Infrared Spectroscopy and Mass Spectrometry” In Map: Organic Chemistry (Wade), Complete and Semesters I and II , CC BY-NC-SA 4.0.
- 29.7 Summary adapted from “Mass Spectrometry” by David Harvey In Instrumental Analysis, CC BY-NC-SA 4.0 and “11: Infrared Spectroscopy and Mass Spectrometry” In Map: Organic Chemistry (Wade), Complete and Semesters I and II , CC BY-NC-SA.
- 29.8 Summary adapted from “12: Nuclear Magnetic Resonance Spectroscopy” In Map: Organic Chemistry (Wade), Complete and Semesters I and II , CC BY-NC-SA 4.0.
- 29.9 Summary adapted from “19: Nuclear Magnetic Resonance Spectroscopy” by David Harvey In Instrumental Analysis, CC BY-NC-SA 4.0 and “12: Nuclear Magnetic Resonance Spectroscopy“In Map: Organic Chemistry (Wade), Complete and Semesters I and II , CC BY-NC-SA 4.0.
- 29.10 Summary adapted from “12: Nuclear Magnetic Resonance Spectroscopy” In Map: Organic Chemistry (Wade), Complete and Semesters I and II , CC BY-NC-SA 4.0.
- 29.11 Summary adapted from “13: Introduction to Ultraviolet/Visible Absorption Spectrometry” and “14: Applications of Ultraviolet/Visible Molecular Absorption Spectrometry” by David Harvey In Instrumental Analysis, CC BY-NC-SA 4.0 and “16: Conjugated Systems, Orbital Symmetry, and Ultraviolet Spectroscopy” In Map: Organic Chemistry (Wade), Complete and Semesters I and II , CC BY-NC-SA 4.0.
