You Gotta Know These Chemistry Lab Techniques
- Flame Tests detect the presence of elements by dipping a wooden splint or nichrome wire in a sample of the element or its salt, then placing the sample over a Bunsen burner. The unique emission spectrum of the element present then causes the flame to briefly change color. The D lines of sodium produce one of the strongest flame test colors: a bright yellow. Because of this, sodium can contaminate samples, so flames are usually viewed through a cobalt blue glass to filter out the yellow. Other notable flame test colors include red, produced by lithium, calcium and strontium; lilac, produced by potassium; green, produced by barium; and blue, produced by copper, selenium, arsenic, and indium.
- Titrations calculate the concentration of a solution by adding in small volumes of a reactant of known concentration until a chemical change, like a pH indicator changing color, occurs. Acid-base titrations are usually performed in a thin glass tube called a buret and use pH indicators like phenolphthalein and bromothymol blue. The Henderson-Hasselbalch equation can be used to calculate the pH at any point during a titration. The equivalence point is the point at which equal amounts of acid and base have been mixed and there is a sharp inflection point in the pH curve. Redox titrations use an oxidation-reduction reaction instead of an acid-base reaction. In complexometric titrations, the analyte forms a coordination complex with the titrant. Karl Fischer titrations use electrolysis to determine the amount of water in a substance.
- Distillation separates a mixture of liquids based on their boiling point by heating, causing the more volatile component to vaporize and condense in a different container while the other components remain in the original vessel. In the laboratory, the vapors are usually cooled with a water-based Liebig condenser or Vigreux condenser, and the product is collected in a round-bottomed receiving flask. In industry, multiple rounds of distillation are performed in a single “column” packed with trays, each of which can be modeled as a “theoretical plate.” Oil refining relies on fractional distillation, in which different products are pulled out of the mixture at various trays along the column. Azeotropes are mixtures that cannot be separated because at the specified pressure and composition, both components boil at the same temperature.
- Calorimetry calculates the heat or enthalpy change of a chemical or physical process by using specialized vessels to measure a change in temperature. A very simple calorimeter can be made by placing a thermometer in an insulating polystyrene coffee cup and sealing it with a lid, causing the reaction inside to occur at constant pressure. Bomb calorimeters are very sturdy containers used to conduct combustion reactions at constant volume. Isothermal titration calorimetry (ITC) is a variant which can be used to determine the binding affinites and stoichiometry of proteins and enzymatic reactions. Differential scanning calorimetry (DSC) is another variant that measures how a compound’s heat capacity changes with temperature; it is commonly used to determine properties of polymers such as their melting point or glass transition temperature.
- Chromatography separates a complex mixture into its individual components, commonly illustrated by the separation of pen ink into many colors. Chromatography involves two components: a mobile phase, which moves, and a stationary phase, which interacts differently with different components of the mobile phase to produce a separation. For example, in thin-layer chromatography, a mixture is spotted on one end of a plate of silica gel (the stationary phase), then a solvent (the mobile phase) carries the components across the plate and separates them based on their polarity—polar substances will strongly interact with the polar silica gel and not move very far, while nonpolar substances will move very far. In gas chromatography (GC), substances are vaporized and run through a packed column, where the time it takes each component to travel through—the retention time—is determined. High-performance liquid chromatography (HPLC) is like gas chromatography, but the sample remains in the liquid phase and is pushed through using pressures of up to 400 atmospheres. Ion-exchange chromatography uses stationary phases with acidic or basic functional groups to remove charged compounds; it can be used for water softening.
- Infrared spectroscopy (IR) acquires information about the chemical groups present in a compound based on which wavelengths of infrared light the bonds in those groups absorb. When IR-active bonds absorb infrared light, they undergo a change in dipole moment and are excited to a higher-energy vibrational mode, which have names like “stretching,” “wagging,” and “scissoring.” The output of an IR experiment (called an “IR spectrum”) is a graph of absorbance on the y-axis against wavenumbers, measured in cm−1 (inverse centimeters), on the x-axis. The most distinguishable IR peak is that of a carbonyl group, which displays a very strong absorbance at 1700 cm−1, while peaks below 1500 cm−1 produce a complex pattern unique to the compond being analyzed, called the fingerprint region. Samples are typically prepared by grinding them into a potassium bromide pellet or by creating a mull with the oil Nujol.
- Nuclear Magnetic Resonance (NMR) uses magnetic fields to determine the arrangement of nuclei in a molecule. Typical NMR methods only work on nuclei that have nonzero spin, such as 1H and 13C—the nonzero spin means that the nuclei oscillate between two spin states in a phenomenon called Larmor precession. NMR measures the frequency at which each nucleus oscillates, whose deviation from a reference nucleus (such as tetramethylsilane) depends on the local electron density and is called the chemical shift. Nuclei that have more electron density (due to proximity to electron-donating groups like alkyl groups) are said to be “shielded” and have lower chemical shifts, while those that have less electron density (due to proximity to electron-withdrawing groups like halogens) are said to be “deshielded” and have higher chemical shifts. Peaks in NMR can be “split” into several peaks due to J-coupling if they are adjacent to identical nuclei, like the three hydrogens of a methyl group. NMR is the theoretical basis for magnetic resonance imaging (MRI) in medicine.
- Mass Spectrometry (mass spec or MS) identifies an unknown compound by ionizing it, fragmenting it into pieces, then passing it through electromagnetic fields to separate the pieces based on their mass-to-charge ratio (m/z). A mass spectrum plots the abundance of each fragment aginst the m/z—the spectrum features a base peak (the peak of highest intensity) and a series of peaks whose spacing tells you what elements are present—for instance, a peak spacing of 14 typically indicates a CH2 or methylene unit. The different isotopes of elements can also produce characteristic peak patterns—for instance, two equal-intensity peaks spaced 2 units apart and 79 units away from the next peak indicates bromine. When analyzing proteins, fragmentation is often undesirable, so “soft” ionization methods such as matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) are used. In forensics, mass spectrometry is often “coupled” to gas chromatography in GC-MS; gas chromatography first separates the unknown mixture, then mass spec identifies the individual components of the mixture.
- Ultraviolet-Visible Spectrophotometry (UV-Vis) quantifies the presence of compounds by shining light in the ultraviolet-to-visible range on a molecule, then measuring which wavelengths are absorbed. Parts of a molecule which absorb visible light are referred to as chromophores; since different molecules have different chromophores, the wavelength of maximum absorbance, or lambda-max, can be used to identify a compound. Beer’s law is then used to quantify the concentration of the present compound. Samples are held in small square plastic or quartz tubes called cuvettes. The Woodward-Fieser rules empirically estimate the lambda-max from the types of bonds and functional groups in a molecule. UV-Vis has many uses in biology—for example: calculating the OD600 to measure bacterial growth rate, determining nucleic acid quality with the 260/280 ratio, measuring protein concentration in the Lowry and Bradford protein assays, and monitoring protein folding by measuring tryptophan or tyrosine absorbance.
- Liquid-Liquid Extraction separates mixtures based on their relative solubilities in two immiscible solvents, such as oil and water. It is commonly performed by placing the mixture and solvents into a separatory funnel, shaking, then using the stopcock to remove one of the two phases. The partition coefficient quantifies the desired compound’s relative solubility in the two phases. The material left over after the desired material has been extracted is called the raffinate. A variant of liquid-liquid extraction that uses phenol and chloroform as the two solvents is used to isolate DNA from cells.
This article was contributed by NAQT writer Rohith Nagari.