Monday, May 8, 2017

Variants of the 1D 13C experiment

1D 13C spectra are often used to confirm compound identity. The simplicity of such spectra enable the number and type of carbon atoms present to be quickly evaluated. 2D 1H-13C correlation spectra, while inherently more sensitive and informative, require more work to interpret and may not show signals from all carbon atoms. Consequently, 1D 13C spectra still have a place in compound characterization. To increase the sensitivity and information content of 1D 13C spectra several variants of the original pulse sequence have been developed. Several of these are discussed below.

The figure below shows 1D 13C spectra recorded using 100mg/ml of cholesteryl acetate in CDCl3, processed in identical fashion, and plotted at the same vertical scale. All but the bottom spectrum were recorded with the same parameters - 128 scans, 2.0 s relaxation delay - and took 396 seconds. The bottom spectrum (zgpg30 in blue) was recorded with a 0.5 s relaxation delay and took 194 seconds.

The blue spectrum (zgpg30) was recorded using the standard 1D 13C parameters on the Facility spectrometers. It uses the zgpg30 pulse sequence which applies 1H decoupling during the relaxation delay to increase signal intensity via the nuclear Overhauser enhancement (NOE) and a 30o pulse to allow use of a short relaxation delay and more rapid pulsing. The red spectrum (zgpg) uses a 90o pulse and a longer relaxation delay, making the experiment longer, but the peaks are larger. The green APT spectrum uses 1H decoupling during the relaxation delay to increase signal intensity via the NOE, and adds multiplicity editing to discriminate CH3 and CH peaks from CH2 and quaternary carbons. You can also see that the baseline is much flatter due to the introduction of a delay between the final pulse and acquisition. The maroon DEPT spectrum was recorded with a 135o pulse to discriminate CH3 and CH carbons from CH2s. The DEPT sequence uses polarization transfer to increase signal intensity, but it cannot detect quaternary carbons. The brown INEPT spectrum also uses polarization transfer to increase signal intensity and adds shaped 13C pulses to give more uniform excitation and increased signal intensity. The orange DEPTQ spectrum uses 1H decoupling during the relaxation delay to increase signal intensity via the NOE, polarization transfer to improve sensitivity, shaped 13C pulses for uniform excitation, multiplicity editing, and can detect quaternary carbons.

To quantify the differences in the various experiments the signal to noise ratio was measured for five different peaks. The same noise region of 10,000 Hz was used for each peak, and the results were normalized to the values obtained in the zgpg30 experiment. The table below summarizes the results.

Experiment C18
mean S/N
zgpg30 1.0 1.0 1.0 1.0 1.0 1.0
zgpg 0.6 0.6 0.6 0.6 0.4 0.6
APT 1.3 2.1 2.3 3.8 2.4 2.4
DEPT 5.0 5.1 3.4 4.7 - 4.6
INEPT 2.1 3.4 4.0 3.8 - 2.7
DEPTQ 3.8 3.8 2.8 3.7 2.3 3.3

Surprisingly, the S/N in the zgpg experiment was less than in the zgpgp30 experiment, despite the peaks appearing larger. The noise must have increased as well as the signal. The APT gives increased S/N for all five peaks examined, but not as great an increase for the methyl resonance. The DEPT shows the greatest increase in S/N of all the experiments, but unfortunately does not detect quaternary carbons. The INEPT is similar but does not increase the S/N as much as the DEPT. While the DEPTQ gives the second largest increase in S/N and detects quaternary carbons.

I would recommend using the DEPTQ as the routine 1D 13C method. It gives a three fold increase in sensitivity, allows multiplicity editing, and detects quaternary carbons. Standard parameters for the DEPTQ are available on both Facility spectrometers as "UCSD_DEPTQ".

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