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Konstantin Bespalov
Konstantin Bespalov

Basic 1H- And 13C-NMR Spectroscopy

L-methionine (Met) oxidation products in tripeptides, in Met-enkephalin and in the bovine basic pancreatic trypsin inhibitor have been identified by 1H and 13C NMR spectroscopy. The oxidation of Met residues by stoichiometric amounts of chloramine-B, H2O2 and I2 yields a mixture of L-methionine sulfoxide (Met (O)) and dehydro-L-methionine (DH-Met), Met (O) and DH-Met, respectively, at pH 7.0 and 25.0 degrees C. The formation of DH-Met occurs only if the amino-group of Met is not derivatized. The analysis of 1H and 13C NMR spectra allows us to quantitate Met oxidation products, to ascertain the relative proportion of the R and S forms of Met (O) and DH-Met, and to reveal the presence of a Met residue at the N-terminal position in peptides and proteins.

Basic 1H- and 13C-NMR Spectroscopy

The liquid hydrolysate contains sugars used for microbial fermentation, such as glucose and xylose, and also contains fermentation inhibitors, such as 5-hydroxymethylfurfural (5-HMF) and formate [24]. Previous research showed a positive correlation between the glucose concentration in the liquid hydrolysate and the starch content of various types of rice straw [25]. 2D 1H-13C HSQC-NMR spectroscopy can elucidate the relationship between the lignin and polysaccharide components in rice straw and the concentrations of various components of the liquid hydrolysate (e.g., glucose, xylose, 5-HMF, and formate) and the relationship between the components in rice straw and the amount (weight) of acid-insoluble residue.

The primary aim of the present study was to characterize changes in lignin and polysaccharide components resulting from dilute acid pretreatment. To achieve this aim, 2D 1H-13C HSQC-NMR spectroscopy was used to examine samples of 13 cultivars of rice straw before and after dilute acid pretreatment (i.e., raw biomass and acid-insoluble residues). These results from multiple numbers of rice cultivars could reveal the common effects of dilute acid pretreatment on lignin and polysaccharide components. A secondary aim was to analyze the relationships between 2D 1H-13C HSQC-NMR data and the concentrations of glucose, xylose, 5-HMF, and formate in the liquid hydrolysate and acid-insoluble residue yield. The results of these relationships revealed that starch in the rice straw was one of important factors to determine the amount of these compounds in the liquid hydrolysate.

1. BackgroundOver the past fifty years nuclear magnetic resonance spectroscopy, commonly referred to as nmr, has become the preeminent technique for determining the structure of organic compounds. Of all the spectroscopic methods, it is the only one for which a complete analysis and interpretation of the entire spectrum is normally expected. Although larger amounts of sample are needed than for mass spectroscopy, nmr is non-destructive, and with modern instruments good data may be obtained from samples weighing less than a milligram. To be successful in using nmr as an analytical tool, it is necessary to understand the physical principles on which the methods are based.

Strong magnetic fields are necessary for nmr spectroscopy. The international unit for magnetic flux is the tesla (T). The earth's magnetic field is not constant, but is approximately 10-4 T at ground level. Modern nmr spectrometers use powerful magnets having fields of 1 to 20 T. Even with these high fields, the energy difference between the two spin states is less than 0.1 cal/mole. To put this in perspective, recall that infrared transitions involve 1 to 10 kcal/mole and electronic transitions are nearly 100 time greater.For nmr purposes, this small energy difference (ΔE) is usually given as a frequency in units of MHz (106 Hz), ranging from 20 to 900 Mz, depending on the magnetic field strength and the specific nucleus being studied. Irradiation of a sample with radio frequency (rf) energy corresponding exactly to the spin state separation of a specific set of nuclei will cause excitation of those nuclei in the +1/2 state to the higher -1/2 spin state. Note that this electromagnetic radiation falls in the radio and television broadcast spectrum. Nmr spectroscopy is therefore the energetically mildest probe used to examine the structure of molecules. The nucleus of a hydrogen atom (the proton) has a magnetic moment μ = 2.7927, and has been studied more than any other nucleus. The previous diagram may be changed to display energy differences for the proton spin states (as frequencies) by mouse clicking anywhere within it.

Unlike infrared and uv-visible spectroscopy, where absorption peaks are uniquely located by a frequency or wavelength, the location of different nmr resonance signals is dependent on both the external magnetic field strength and the rf frequency. Since no two magnets will have exactly the same field, resonance frequencies will vary accordingly and an alternative method for characterizing and specifying the location of nmr signals is needed. This problem is illustrated by the eleven different compounds shown in the following diagram. Although the eleven resonance signals are distinct and well separated, an unambiguous numerical locator cannot be directly assigned to each.

iv) Intramolecular hydrogen bonds, especially those defining a six-membered ring, generally display a very low-field proton resonance. The case of 4-hydroxypent-3-ene-2-one (the enol tautomer of 2,4-pentanedione) not only illustrates this characteristic, but also provides an instructive example of the sensitivity of the nmr experiment to dynamic change. In the nmr spectrum of the pure liquid, sharp signals from both the keto and enol tautomers are seen, their mole ratio being 4 : 21 (keto tautomer signals are colored purple). Chemical shift assignments for these signals are shown in the shaded box above the spectrum. The chemical shift of the hydrogen-bonded hydroxyl proton is δ 14.5, exceptionally downfield. We conclude, therefore, that the rate at which these tautomers interconvert is slow compared with the inherent time scale of nmr spectroscopy.

Test your ability to interpret 1H nmr spectra by analyzing the seven examples presented below. The seven spectra may be examined in turn by clicking the "Toggle Spectra" button. Try to associate each spectrum with a plausible structural formula. Although the first four cases are relatively simple, keep in mind that the integration values provide ratios, not absolute numbers. In two cases additional information from infrared spectroscopy is provided. When you have made an assignment you may check your answer by clicking on the spectrum itself. In the sixth example, a similar constitutional isomer cannot be ruled out by the data given.

3. Carbon NMR SpectroscopyThe power and usefulness of 1H nmr spectroscopy as a tool for structural analysis should be evident from the past discussion. Unfortunately, when significant portions of a molecule lack C-H bonds, no information is forthcoming. Examples include polychlorinated compounds such as chlordane, polycarbonyl compounds such as croconic acid, and compounds incorporating triple bonds (structures below, orange colored carbons).

The most important operational technique that has led to successful and routine 13C nmr spectroscopy is the use of high-field pulse technology coupled with broad-band heteronuclear decoupling of all protons. The results of repeated pulse sequences are accumulated to provide improved signal strength. Also, for reasons that go beyond the present treatment, the decoupling irradiation enhances the sensitivity of carbon nuclei bonded to hydrogen. When acquired in this manner, the carbon nmr spectrum of a compound displays a single sharp signal for each structurally distinct carbon atom in a molecule (remember, the proton couplings have been removed). The spectrum of camphor, shown on the left below, is typical. Furthermore, a comparison with the 1H nmr spectrum on the right illustrates some of the advantageous characteristics of carbon nmr. The dispersion of 13C chemical shifts is nearly twenty times greater than that for protons, and this together with the lack of signal splitting makes it more likely that every structurally distinct carbon atom will produce a separate signal. The only clearly identifiable signals in the proton spectrum are those from the methyl groups. The remaining protons have resonance signals between 1.0 and 2.8 ppm from TMS, and they overlap badly thanks to spin-spin splitting.

Unlike proton nmr spectroscopy, the relative strength of carbon nmr signals are not normally proportional to the number of atoms generating each one. Because of this, the number of discrete signals and their chemical shifts are the most important pieces of evidence delivered by a carbon spectrum. The general distribution of carbon chemical shifts associated with different functional groups is summarized in the following chart. Bear in mind that these ranges are approximate, and may not encompass all compounds of a given class. Note also that the over 200 ppm range of chemical shifts shown here is much greater than that observed for proton chemical shifts.

The following problems focus on concepts and facts associated with nmr spectroscopy. The first two questions ask you to identify structurally equivalent groups of hydrogen and carbon atoms. The third question concerns both 1H & 13C nmr, and the fourth examines spin-splitting in proton nmr. The fifth & sixth questions require interpretation of a proton nmr spectrum. Questions 7, 8 & 9 present an assortment of unknowns for which a variety of spectroscopic data is given. Question 10 combines chemical and spectroscopic evidence for an unknown compound.

The study of carbon nuclei by magnetic resonance spectroscopy (NMR) is an important technique for determining the structures of organic molecules. Using it in conjunction with proton H NMR as well as infrared spectroscopy enables organic chemists to determine the complete structure of an unknown compound without contaminating their hands in the laboratory. New Fourier transforms NMR (FT-NMR) devices They make it easy to bring in carbon spectra. 041b061a72


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