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Chemical Effects |
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Important Parameters Specific rotation is a very useful parameter for assessing differences in structure at, or near, a compound’s chiral center, or centers. Specific rotation is affected by temperature, probing wavelength, solvent, pH and concentration. It is important to keep these five parameters in mind when comparing specific rotation measurements. Temperature variations can be adequately controlled during an analysis by the use of a column oven. The probing wavelength is fixed at 670 nanometers with the PDR-Chiral Detector. However, as the next few examples will show, it is very important to consider the effects of solvent, pH and concentration when doing quantitative measurements. Concentration In order for specific rotation to be a useful and precise measure of the structural features associated with the chiral centers in an optically active molecule, it must be measurable in a standardized, repeatable manner.
The above data show that optical activity is affected by both temperature and concentration. With respect to concentration, the optical activity of a molecule is influenced by the chemical environment surrounding it. At concentrations below 10 mM, molecule-molecule interactions are negligible and the specific rotation will be influenced by the solvent. Measurements should be made at low concentration in order to use specific rotation as a sensitive probe of structure, or structural changes at, or near, the chiral center. The PDR-Chiral Detector has the requisite sensitivity to allow operation under these conditions. Solvent As mentioned, specific rotation is affected by the chemical environment surrounding the analyte. At concentrations below approximately 10 mM, molecule-solvent interactions dominate. The polarity of the solvent in which the optically active molecule is dissolved, as well as its pH and ionic strength, influence the magnitude and sign of [a ].
The PDR-Chiral Detector can operate in solvents of greatly differing polarity and pH, from very hydrophobic solvents (e.g. hydrocarbon), to aqueous solutions at the extremes of the pH scale. Thus, the measurement protocol can be developed to match the unique challenges of the problem under investigation, rather than to match the limitations of the instrument. With the enhanced sensitivity of laser polarimeter technology, new experiments and experimental configurations are possible. Flow injection analysis (FIA) can be used to reproducibly introduce the sample into the PDR-Chiral Advanced Laser Polarimeter. The FIA configuration provides precise control over the analyte distribution, the amount of analyte injected, and the characteristics of the mobile stream. Thus, specific rotation can now be easily studied as a function of solvent or pH. Such studies were previously difficult or impossible to carry out due to the poor sensitivity of conventional polarimeters, or to the increased noise resulting from flow in the polarimetric detection cell. pH Due to the insensitivity of the conventional polarimeter used to obtain the values given below, high concentrations of amino acid were commonly required. It is interesting to note that this wide range of values reflects differences in the arrangement of atoms surrounding the alpha carbon in the amino acid. This underscores the sensitivity of specific rotation to subtle structural variations.
These subtle, structural variations can include changes in the state of ionization at the compound’s chiral center. Differences in specific rotation are observed as a function of pH for all of the optically active amino acids. Glycine does not contain a chiral center. However, at these high concentrations, molecule-molecule interactions cannot be assumed negligible. More precise and accurate measurements are possible with the PDR-Chiral Detector, which can make similar measurements at concentrations 2 to 3 orders of magnitude lower. Using experiments similar to that depicted in the figure below, the specific rotation for the amino acid phenylalanine (Phe) was measured as a function of pH. The concentration of Phe used for these studies was 0.5 mM, a level where molecule-molecule interactions can be assumed negligible. Thus, any changes observed can be attributed solely to the effect of pH. Several interesting trends are evident.
First, there are clear changes in the specific rotation as one goes from the free amino acid, to the di- and tri-peptide. For this series, not only does the magnitude of the specific rotation change as a function of the number of amino acid units, but the sign changes as one goes from the amino acid, to the di- and tri-peptide. Further, clear differences are noted for all three species as one moves from acid solution, to the basic side of the pH scale. The sensitivity of specific rotation to the environment around the chiral center, either due to structural variations or ionization differences, underscores its potential as a means to monitor changes at the optically active portion of a complex molecule. Similar experiments were performed on the amino acid alanine (Ala), an amino acid which does not have a chromophore assessable in the UV or visible region of the spectrum. As with Phe, several interesting trends are evident. 
First, a sign change is observed in the specific rotation as one goes from the amino acid to the di- and tri-peptides, although in the case of Ala, the change is from positive to negative. Second, the variation of specific rotation with pH follows a similar trend for all three species with the magnitude decreasing with increasing pH. Other studies (not shown) further probed these unique trends. In studies of mixed peptides, for example, Phe-Ala and Ala-Phe, clear differences were noted in the magnitude of the specific rotation, and in the change as a function of pH, suggesting that order is important. Additional mixed di-peptides were studied including Gly-Phe, Phe-Gly, Gly-Ala and Ala-Gly. Since glycine is not optically active, only its partner contributes to the innate optical activity of the di-peptide. Interesting differences were noted between each matched pair. For example, the specific rotation for Gly-Phe was found to be significantly different from that of Phe-Gly. Similar results were obtained for the Ala-Gly and Gly-Ala pair. Recent work has shown that small peptides are critical to such important physiological processes as intracellular communication and nerve transduction. It is clear that polarimetric detection may have great potential in both the analysis and study of these types of important compounds. Although these results are very preliminary, they do show the sensitivity of specific rotation to structural features at, or even near the chiral center. Also, with the experimental sensitivity of the PDR-Chiral Detector, additional studies may allow some systematic organization of the rules governing optical activity to be developed. Last, these studies show the potential of polarimetric detection as a probe of structural integrity. Measurement of specific rotation for larger molecules, (proteins, for example) may be used in basic studies of denaturization processes, or as part of a quality control protocol for protein-based therapeutic agents. For example, the protein lysozyme is known to undergo denaturization at low pH. Using FIA for sample introduction, the specific rotation of lysozyme (globulin G1), a 129 amino acid protein (MW, 14,400 Daltons) was measured as a function of pH. The specific rotation experienced a large change below pH’s of 5, which corresponded with a large change in the heat of denaturization at this pH and below. Although more research is needed, it does appear that specific rotation can be used to assess changes in the tertiary structure of complicated biochemical systems. The polarimetric study was experimentally simple and the method appears to be more sensitive than CD to these important structural changes. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
