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Nanogram-scale preparation and NMR analysis for mass-limited small volatile compounds.

Nojima S, Kiemle DJ, Webster FX, Apperson CS, Schal C - PLoS ONE (2011)

Bottom Line: Semiochemicals are often produced in infinitesimally small quantities, so their isolation requires large amounts of starting material, not only requiring significant effort in sample preparation, but also resulting in a complex mixture of compounds from which the bioactive compound needs to be purified and identified.With this technique, (1)H-NMR spectra were obtained on 50 ng of geranyl acetate, which served as a model compound, and reasonable H-H COSY NMR spectra were obtained from 250 ng of geranyl acetate.This simple off-line integration of preparative GC and NMR will facilitate the purification and chemical identification of novel volatile compounds, such as insect pheromones and other semiochemicals, which occur in minute (sub-nanogram), and often limited, quantities.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology and WM Keck Center for Behavioral Biology, North Carolina State University, Raleigh, North Carolina, United States of America.

ABSTRACT
Semiochemicals are often produced in infinitesimally small quantities, so their isolation requires large amounts of starting material, not only requiring significant effort in sample preparation, but also resulting in a complex mixture of compounds from which the bioactive compound needs to be purified and identified. Often, compounds cannot be unambiguously identified by their mass spectra alone, and NMR analysis is required for absolute chemical identification, further exacerbating the situation because NMR is relatively insensitive and requires large amounts of pure analyte, generally more than several micrograms. We developed an integrated approach for purification and NMR analysis of <1 µg of material. Collections from high performance preparative gas-chromatography are directly eluted with minimal NMR solvent into capillary NMR tubes. With this technique, (1)H-NMR spectra were obtained on 50 ng of geranyl acetate, which served as a model compound, and reasonable H-H COSY NMR spectra were obtained from 250 ng of geranyl acetate. This simple off-line integration of preparative GC and NMR will facilitate the purification and chemical identification of novel volatile compounds, such as insect pheromones and other semiochemicals, which occur in minute (sub-nanogram), and often limited, quantities.

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Background signals of blank and control NMR samples.
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pone-0018178-g002: Background signals of blank and control NMR samples.

Mentions: All spectra were acquired at 25°C with a Bruker AVANCE 600 MHz spectrometer equipped with a 1-mm triple resonance z-gradient microprobe, with spectral widths of 4800 Hz (8.0 ppm). 1-D Proton spectra (Figs. 2 and 3) were acquired with a 4.18 µsec 90° pulse, with a 1.5 sec recycle delay and 1.5 sec acquisition time. Depending on the concentration of the sample, spectra were acquired with 4000 to 8000 scans. Data were processed with exponential decay equivalent to 1 Hz line broadening. The 2-D gradient magnitude calculated COSY spectrum (Fig. 4) was acquired with 2048 data points in F2 by 142 data points in F1, a 4.18 µsec 90° pulse, with a 1.0 sec recycle delay and 0.21 sec acquisition time with a total time of 50 hrs to acquire data. The COSY was processed as a 2k ×256 matrix with a shifted (p) qsin weighting function. Data were symmetrized. The signal of trace amounts of benzene, which is an unavoidable contaminant in NMR samples, was used as a reference.


Nanogram-scale preparation and NMR analysis for mass-limited small volatile compounds.

Nojima S, Kiemle DJ, Webster FX, Apperson CS, Schal C - PLoS ONE (2011)

Background signals of blank and control NMR samples.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3065492&req=5

pone-0018178-g002: Background signals of blank and control NMR samples.
Mentions: All spectra were acquired at 25°C with a Bruker AVANCE 600 MHz spectrometer equipped with a 1-mm triple resonance z-gradient microprobe, with spectral widths of 4800 Hz (8.0 ppm). 1-D Proton spectra (Figs. 2 and 3) were acquired with a 4.18 µsec 90° pulse, with a 1.5 sec recycle delay and 1.5 sec acquisition time. Depending on the concentration of the sample, spectra were acquired with 4000 to 8000 scans. Data were processed with exponential decay equivalent to 1 Hz line broadening. The 2-D gradient magnitude calculated COSY spectrum (Fig. 4) was acquired with 2048 data points in F2 by 142 data points in F1, a 4.18 µsec 90° pulse, with a 1.0 sec recycle delay and 0.21 sec acquisition time with a total time of 50 hrs to acquire data. The COSY was processed as a 2k ×256 matrix with a shifted (p) qsin weighting function. Data were symmetrized. The signal of trace amounts of benzene, which is an unavoidable contaminant in NMR samples, was used as a reference.

Bottom Line: Semiochemicals are often produced in infinitesimally small quantities, so their isolation requires large amounts of starting material, not only requiring significant effort in sample preparation, but also resulting in a complex mixture of compounds from which the bioactive compound needs to be purified and identified.With this technique, (1)H-NMR spectra were obtained on 50 ng of geranyl acetate, which served as a model compound, and reasonable H-H COSY NMR spectra were obtained from 250 ng of geranyl acetate.This simple off-line integration of preparative GC and NMR will facilitate the purification and chemical identification of novel volatile compounds, such as insect pheromones and other semiochemicals, which occur in minute (sub-nanogram), and often limited, quantities.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology and WM Keck Center for Behavioral Biology, North Carolina State University, Raleigh, North Carolina, United States of America.

ABSTRACT
Semiochemicals are often produced in infinitesimally small quantities, so their isolation requires large amounts of starting material, not only requiring significant effort in sample preparation, but also resulting in a complex mixture of compounds from which the bioactive compound needs to be purified and identified. Often, compounds cannot be unambiguously identified by their mass spectra alone, and NMR analysis is required for absolute chemical identification, further exacerbating the situation because NMR is relatively insensitive and requires large amounts of pure analyte, generally more than several micrograms. We developed an integrated approach for purification and NMR analysis of <1 µg of material. Collections from high performance preparative gas-chromatography are directly eluted with minimal NMR solvent into capillary NMR tubes. With this technique, (1)H-NMR spectra were obtained on 50 ng of geranyl acetate, which served as a model compound, and reasonable H-H COSY NMR spectra were obtained from 250 ng of geranyl acetate. This simple off-line integration of preparative GC and NMR will facilitate the purification and chemical identification of novel volatile compounds, such as insect pheromones and other semiochemicals, which occur in minute (sub-nanogram), and often limited, quantities.

Show MeSH