Laboratory Techniques

separations - answer-a variety of lab techniques that use intermolecular forces to separate a mixture into its component parts; can be separated according to solubility, melting point, boiling point, or any other physical property main types: extraction, distillation, crystallization, and chromatography extraction - answer-a separation technique based on solubility; involves two phases, most commonly the aqueous layer and the less dense organic mixture What are the steps to performing an extraction? - answer-1. Add weak acid; acid protonates strong bases; makes them polar and they move to aqueuos layer and strong bases are removed. 2. Add strong acid; weak bases removed. 3. Add weak base; strong acids removed. 4. Add strong base; weak acids removed. distillation - answer-technique used to separate compounds that have significantly different boiling points; liquids with boiling point differences of at least 20C can be separated by slow boiling; the compound with lower BP boils off first fractional distillation - answer-more precise method of distillation that can be used to separate liquids whose boiling points are fairly close together vapor is run through glass beads, allowing the compound with the higher boiling point to repeatedly condense and fall back into the solution crystallization - answer-based on the principle that pure substances form crystals more easily than impure substances; very inefficient method of separation difficult to arrive at a pure substance; exothermic process chromatography - answer-used to purify a compound from a mixture and/or to identify the ratio of compounds in a mixture; separation of a mixture by passing it over or through a matrix that absorbs (binds) different compounds more or less strongly according to their properties mobile phase - answer-mixture is dissolved into a solution that passes through stationary phase - answer-matrix surface (solid surface usually) that adsorbs compounds from the mixture Which compounds move more slowly? - answer-those that have a greater affinity for the surface; usually polar compounds column chromatography - answer-a solution containing the mixture is dripped down a column containing the solid phase (usually glass beads); more polar compounds travel slowly, creating separate layers high pressure liquid chromatography (HPLC) - answer-the column and solution use an apparatus that puts the system under high pressure paper chromatography - answer-a small portion of the sample to be separated is spotted onto paper; one end of the paper is then placed into a nonpolar solvent; as the solvent moves up the paper, more polat components of the sample move more slowly because they are attracted to the polar paper -- less polar components dissolve more easily to move up the paper with the solvent Rf factor - answer-can be calculated for each component in paper chromatography -- divide the distance traveled by the component by the distance traveled by the solvent non-polar components -- Rf close to 1 polar components -- lower Rf factor thin-layer chromatography - answer-similar to paper chromatography; coated glass or plastic plate is used instead of paper; the results are visualized via an iodine vapor chamber gas-liquid chromatography - answer-the liquid phase is the stationary phase; the mixture is dissolved into a heated carrier gas and passed over a liquid phase bound to a column; compounds in the mixture equilibrate with the liquid phase at different rates and pass through an exit port as individual components size-exclusion chromatography - answer-well suited for peptides and proteins; molecules are separated by their SIZE and sometimes their MOLECULAR WEIGHT, often through gel filtration ion-exchange chromatography - answer-molecules are separated based on their net surface charge; utilizes cationic or anionic "exchangers" that slow down the movement of charged molecules affinity chromatography - answer-uses highly specific interactions to slow down select molecules; can make use of receptor-ligand, enzyme-substrate, and antigen-antibody interactions gel electrophoresis - answer-mixtures of amino acids (DNA and RNA fragments) or mixtures of proteins/polypeptides can be separated based on size and charge; the molecular mixture is placed in a gel and an electric field is applied larger particles move more slowly distinct bands form according to protein size How else can proteins be separated via gel electrophoresis? - answer-based on their isoelectric points ladder (in gel electrophoresis) - answer-a mixture of DNA, RNA, or polypeptide fragments of known sizes or quantities; the migration distances of the experimental mixture's bands can be compared against those of the ladder's bands to determine apprx. size blotting - answer-technique by which molecules are transferred from the gel onto a membrane, maintaining the same spatial relationship of bands based on size; used to visualize nucleic acids or proteins Southern blotting - answer-used to identify target fragments of a known DNA sequence in a large population of DNA 1. denature DNA fragments in basic solution -- obtain single-strands 2. place a membrane on top or below the gel, transfer fragments to membrane 3. labeled probe added to membrane 4. prove marks the target fragment 5. membrane is visualized to reveal location of probe Northern blot - answer-just like a Southern blot, but identifies RNA fragments Western blot - answer-used to detect particular protein in a mixture of proteins What is most commonly used to identify proteins in a Western blot? - answer-antibodies! primary antibody specific to the protein of interest is place on the membrane and then a secondary antibody-enzyme conjugate is added. this binds to the primary and marks it with an enzyme for subsequent visualization What are the 3 ways to resolve enantiomers from a racemic mixture? - answer-1. use differences in crystallization of the enantiomers 2. stereospecific enzymes can be added and will react with only one enantiomer 3. enantiomers can be converted into diastereomers, which have different physical and chemical properties that can be used for separation spectroscopy - answer-the study of the interaction between matter and electromagnetic radiation (light) Nuclear Magnetic Resonance (NMR) spectroscopy - answer-the study of the action between atomic nuclei and radio waves *most commonly used to study hydrogen nuclear spin - answer-property possessed by nuclei with ODD atomic or mass numbers; generates a magnetic field The ______ the magnetic field, the greater the difference in energy between the a and B spin states. - answer-stronger! What does an NMR spectrum show? - answer-it is a graph of the magnetic field strengths absorbed by the hydrogen atoms of a specific compound at a single frequency; measured in ppm (parts per million) What does each peak represent in NMR? - answer-each peak represents chemically equivalent hydrogens What is splitting of peaks caused by? - answer-neighboring hydrogens chemically equivalent hydrogens - answer-hydrogens whose positions on the compound are indistinguishable by NMR; have the same chemical shifts chemical shift - answer-the difference between between the resonance frequency of the chemically shifted hydrogens and the resonance frequency of hydrogens on a reference compound integral trace - answer-a line drawn above the peaks that rises each time it goes over a peak; the rise of the integral trace is proportional to the number of chemically equivalent hydrogens in the peak beneath it Hydrogens adjacent to electron-withdrawing groups tend to have peaks _______. - answer-downfield (to the left) splitting of a peak - answer-(also called spin-spin splitting) caused by neighboring hydrogens that are not chemically equivalent The number of peaks due to splitting for a group of chemically equivalent hydrogens is given by the simple formula: - answer-n + 1; where n is the number of neighboring hydrogens that are not chemically equivalent **the number of neighboring hydrogens for a given peak can be determined by subtracting 1 from the number of smaller peaks into which the peak is split steps to interpreting proton NMR spectroscopy - answer-1. Identify chemically equivalent hydrogens. 2. Identify and count neighboring hydrogens that are not chemically equivalent. Use n+1 to determine the number of peaks created by splitting for the chemically equivalent hydrogens. 3. If necessary, identify electron withdrawing/donating groups near the chemically equivalent hydrogens. IR spectroscopy - answer-uses molecular dipoles to find information about *functional groups* an infrared spectrometer slowly changes the frequency of infrared light shining on a compound and records the frequencies of absorption in number of cycles per cm What happens to bonds when exposed to radiation in the infrared region? - answer-polar bonds within a compound stretch and contract, causing *intramolecular vibrations and rotations* What is the most predictable section of the IR spectrum? - answer-1600 to 3500 cm^-1 most likely spectra tested - answer-C=O, sharp dip around 1700 (carbonyl) O-H, broad dip around C-H, N-H, 3300 shallow dip fingerprint region - answer-where many of the complex vibrations that distinguish one compound from a similar compound are found in the 600 to 1400 ultraviolet region - answer-much shorter than infrared light, between 200 and 400 nm and at a much higher energy level ultraviolet (UV) spectroscopy - answer-detects *conjugated systems* (double bonds separated by one single bond) by comparing the intensities of two beams of light from the same monochromatic light source rule of thumb for UV absorption - answer-30 to 40 nm increase for each additional conjugated double bond, and a 5 nm increase for each additional alkyl group visible regio