Nuclear Magnetic Resonance (NMR) Spectroscopy Overview

Hi, I'm Alicia Luers, Chemistry Research Associate at Emory Pharma. Today I'd like to tell you a little bit more about our chemistry services. Our Bruker 400 MHz NMR is a critical component in our chemistry lab and it also happens to be my favorite instrument. Our instrument has a tunable broadband probe which means we can test almost any nucleus. This includes proton, carbon, phosphorus, chlorine, fluorine, silicon and more. We also have a variable temperature unit. This allows us to run experiments from negative 130 to positive 130 degrees Celsius. We routinely perform full structural characterization by one and two dimensional NMR for API submissions to regulatory agencies such as the FDA or European Reach. We use these same techniques along with data from our other instrumentation to tackle complete structure elucidation of known or unknown impurities. Let's take a closer look at some of the more common experiments we perform during structure elucidation. The first step is to figure out the molecular formula of the compound. This is done by acquiring an accurate mass, elemental analysis and other experiments to determine the abundance of each element in the molecule. At this point we narrow it down to the most likely molecular formula. Later NMR experiments will confirm the correct formula. Armed with the likely molecular formula, we start the NMR portion of structure elucidation with a simple one dimensional proton spectrum. We use the chemical shift, multiplicity, coupling constants and integration to begin to understand the molecule. From there we perform a two dimensional cosy experiment. A cozy experiment shows correlations between protons that are coupled to each other by plotting the proton spectrum on both the horizontal and vertical axes. Next, we begin to look at the carbons in the molecule with a carbon 13 NMR experiment. Distortionless enhancement of polarization transfer, also known as DEPT experiments, can be used to determine the number of hydrogens attached to each carbon atom. Once the basic proton and carbon experiments are complete, we run an HSQC experiment. Heteronuclear single quantum coherence spectroscopy determines which hydrogens are attached to each carbon atom. The proton spectrum is shown on the horizontal axis and the carbon spectrum is shown on the vertical axis. Finally, we conduct an HMBC experiment. Just like in hsqc, the proton spectrum is shown on the horizontal axis and the carbon spectrum is shown on the vertical axis. Unlike hsqc, heteronuclear multiple bond correlation spectroscopy shows the correlations between protons and carbons that are separated by multiple bonds three, four or even five bond separations. HMBC is incredibly useful for assigning tricky atoms such carbons with no attached protons, carbons that are nearly chemically equivalent, or protons that are not directly attached to a carbon. HMBC supports all previous proton and carbon assignments and confirms the hypothesized structure. If it's necessary to figure out absolute stereochemistry, NMR can help in a number of ways. One way is through NOE NMR experiments. Nuclear Overhauser effect is the transfer of nuclear spin polarization via cross relaxation, which gives information about distances between hydrogen atoms. With dimensions of the molecule known, stereochemistry can be determined. Another way is through the use of a chiral derivatizing agent. In this method, the compound is coupled with both the R and S forms of the chiral derivatizing agent. The differences in chemical shift of the two analogs are analyzed by proton nmr. From this we can determine stereochemistry. At Emory Pharma, we are experts in one and two dimensional NMR characterization and structure elucidation. We provide our clients with a fully annotated report and support our results with high resolution mass spectrometry and elemental analysis. For a detailed description of our NMR techniques and tips on structure elucidation, check out my blog. For more information on how we can help you, please Visit us@emorypharma.com.