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How to Evaluate a Benchtop NMR Instrument’s Technical Performance Part 4: 13C Sensitivity

July 17th, 2019, by

After 1H, 13C is easily the next most important nuclide in the NMR periodic table; 13C measurements can provide a wealth of valuable structural info. Unfortunately, with a receptivity that is around 5,500 smaller than that of 1H, 13C is a much less sensitive nuclide. This lower sensitivity demands the maximum performance from the NMR spectrometer to keep the measurement times and sample concentration within practical limits. Since 13C NMR has the reputation to be challenging even for high field spectrometers, people tend to think that only overnight experiments can be performed on bench top systems. In the first example below we want to show you that even at frequencies like 43, 60 or 80 MHz high quality 13C spectra can be acquired in a single scan. If your goal is to teach the principles of 13C NMR to students, it is worth knowing that good 13C NMR spectra can be acquired on concentrated organic liquid samples in just under a minute. Moreover students can collect a whole set of powerful multidimensional heteronuclear experiments in well under an hour. The spectrum below of neat propylbenzoate could serve as a useful example for teaching 13C NMR in an educational environment.

Figure1: 1D 13C NMR spectra of neat Propylbenzoate acquired with a single scan (blue), 4 scans (green) and 16 (red) scans totalling 5, 20 and 80 seconds of acquisition time respectively.

As the concentration of the sample decreases the experiment will take more time depending on the concentration and 13C sensitivity of your instrument. As described in the previous post, the effect of lower sensitivity can cause the experiment time to increase dramatically because of the square root relationship with the number of scans. Because 13C sensitivity is such a critical parameter we want to provide some standards that can be used as reference to understand and evaluate 13C measurements when considering a benchtop NMR instrument.

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Multiple Peak Solvent Suppression

June 25th, 2019, by

The Spinsolve ULTRA is able to suppress multiple solvent peaks (up to 3 separate peaks) which allows you to resolve compounds dissolved at sub-millimolar concentrations in protonated organic solvents, such as ethanol.  While single peak solvent suppression is useful for samples where water is the solvent, many organic solvents have multiple strong NMR peaks.  If you want to resolve or quantify compounds dissolved in an organic solvent, the ability to suppress all the solvent peaks is very useful as shown in Figure 1 below.

Figure 1: The image above shows a zoom of 1D proton spectrum of 170mM Paracetamol dissolved in normal protonated Ethanol acquired on a Spinsolve 60 ULTRA (top, red). The second spectrum (bottom, blue) shows the same sample acquired with multiple peak solvent supression at the frequencies indicated by the 3 red arrows.

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How to Evaluate a Benchtop NMR Instrument’s Technical Performance: Part 1

June 13th, 2019, by

When evaluating a benchtop NMR instrument there are several key performance characteristics that have a very significant impact on how the instrument will perform in your lab. These key performance characteristics are:

  • The spectral resolution, which is directly related to the magnet and determines the width or shape of the NMR lines (often called lineshape), and in turn the ability to separate signals in the spectrum
  • The sensitivity which determines the limits of detection (LOD) and quantitation (LOQ), and in turn how long sample measurements take
  • The stability of the magnet and instrument over time, which impacts the ability to make longer measurements, and the overall ease of use of the spectrometer

Before I examine these performance characteristics in more detail, it’s worth emphasising from the outset that the biggest aspect of a benchtop NMR system’s design that dictates how well the system performs is the “quality” of the magnetic field produced by the magnet. By “quality” we are referring to how uniform the magnetic field is over the sample volume, often referred to as the B0 homogeneity. To illustrate the importance of this key aspect of magnet design, Figure 1 shows a series of spectra collected under varying degrees of B0 homogeneity.

Figure 1. Effect of static magnetic field (B0) homogeneity on the NMR spectrum. As the homogeneity gets worse, both the resolution and sensitivity are negatively affected. The series of spectra on the left are shown at the same scale and show how the 2 peaks can no longer be resolved, and the signal intensity decreases. The series on the right is the same spectra shown with the peak intensities normalised which how the signal-to-noise ratio is decreasing (the noise is increasing) when the field is less homogeneous.

 

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Boron NMR Spectroscopy

November 29th, 2018, by

There are two naturally occurring NMR active nuclei of Boron, 11B (80.1%) and 10B (19.9%). Both nuclei are quadrupolar with spin of greater than ½. 11B has a spin of 3/2 and 10B is spin 3. In terms of sensitivity, 11B is the better nucleus to use as it has a higher natural abundance, a higher gyromagnetic ration, and a lower quadrupole moment. A Spinsolve benchtop NMR spectrometer with a proton frequency of 60 MHz can be configured to measure the 11B NMR signal which has a frequency of 19.2 MHz.

The 11B NMR spectrum of a 0.23 M solution Sodium tertraphenylborate in MeOH-d4 is shown below. The first spectrum shows the excellent sensitivity of Spinsolve using just 8 scans to acquire a spectrum in only 16 seconds.

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How to Evaluate a Benchtop NMR Instrument’s Technical Performance Part 3: 1H sensitivity

July 4th, 2019, by

In my recent posts on evaluating benchtop NMR system performance, I discussed the fundamental role the static (B0) magnetic field homogeneity plays in defining the lineshape and with it the resolution performance of the instrument. However, the quality of the magnetic field affects much more than just the instrument’s lineshape and resolution: since broadening of the lines due to B0 inhomogeneity causes them to be lower in amplitude, the quality of the field also directly affects the instrument’s sensitivity. In this post I explore the concept of instrument sensitivity in more detail and look at how to measure  1H sensitivity.

 

What is Meant by Sensitivity?

A formal definition of sensitivity is the ability of an instrument to detect a target analyte. This is usually expressed in NMR as the signal-to-noise ratio (SNR) for a defined concentration of reference substance. Simply put, the more sensitive the NMR spectrometer, the less sample you need to get the same SNR in your spectrum. The two principal enemies of any analytical measurement are higher noise levels and a lower intensity of the signal measured by the instrument’s detector for a sample of given concentration. With modern electronics the noise levels are consistent and should not vary much between different instruments.  This means the sensitivity depends primarily on the signal amplitude, which in turn depends on the lineshape and resolution of the instrument. A poor lineshape results in spectra with broad lines that are lower in amplitude, which decreases the SNR, thereby degrading the sensitivity of the instrument and increasing the amount of sample and/or measurement time required to get the same SNR in the spectrum, as we will see below.

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Benchtop NMR characterisation of diethyl phthalate containing land leeches repellent

June 18th, 2019, by

In Vietnam, for the observation of animals in the jungle of the national park of Cat Tien (and in other parts of the country and in Asia), the rangers give the tourists leech socks and a repellent cream for land leeches to put on the socks. Land leeches are terrestrial blood-sucking worm-like parasites. Reading the cream container, I noticed that it contains diethyl phthalate (DEP). Out of curiosity, I dissolved some of the cream in CDCl3 and acquired a NMR spectrum with the Spinsolve 80 MHz benchtop NMR spectrometer.

The 1D 1H spectrum confirms that the cream is mainly composed of diethyl phthalate (Fig. 1, a). A zoom of the spectrum (Fig. 1, b) shows the presence of some additional compounds overlapping with the 13C satellite peaks of DEP (0.55% of the main peaks). To simplify the identification of the additional compounds present in the cream I acquired a 1D 1H spectrum using the carbon decoupling protocol available in the Spinsolve software (Fig. 1, c). This method removes the satellites from the spectra making it possible to detect compounds dissolved at concentration smaller than 1% with respect to DEP.

Typical excipients used in such creams are fatty acid mixtures from butter and/or oils, glycerol/glycine, alcohol (multiplet ~ 3.5 ppm, CH2-OH) and PEG based compounds (peak ~ 3.6 ppm) and even perfume(s).

In our case, the fatty acid peaks are easily recognized. The terminal methyl of fatty acids is observed in region F around 0.8 ppm, the aliphatic chain in region E and probably under the CH3 of DEP, and the olefinic protons of saturated fatty acids around 5.2 ppm in the region A. As no signal is observed around 2.8 ppm, the saturated fatty acids present in the cream are mono unsaturated. The singlet at 2.47 ppm (singlet C) could be a residual solvent like DMSO or 1,3-dioxan, common solvents contaminating cosmetic cream. To check this hypothesis, ~ 2 µL of solvent was added. If the cream contains the solvent, the integral of peak C would increase, but in our case new peaks were observed (data not show). Region B correspond to a CH3 group next to a (mono or di) substituted aliphatic. The area D could be a triplet with a J coupling of 7 Hz. These peaks probably belong to a perfume, where the additional peaks of the perfume molecule overlap with peaks of DEP.

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Spinsolve 80 Phosphorus – Limit of Detection

May 29th, 2019, by

The best-selling Magritek 80 MHz Spinsolve benchtop NMR is also available with the X-channel set to 31-Phosphorus.  31P NMR spectroscopy is routinely used by chemists to determine structure and measure impurities.  When looking for impurities it is important to know the lower limit of detection (LOD). The LOD is the lowest concentration of a molecule that can be distinguished from the absence  of that molecule.

In NMR it is the sensitivity that determines the  LOD for a particular substance, and the higher magnetic field of an 80 MHz magnet brings a number of advantages including increased sensitivity.  We thought it would be interesting to determine the LOD for tetramethylphosphonium chloride with different acquisition times.  We defined the LOD as an NMR peak with signal height that was 3 times the noise level, i.e. an SNR of 3.


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Multiple peak solvent suppression with 13C decoupling

June 25th, 2019, by

In a previous post we demonstrated the use of multiple peak solvent suppression on the Spinsolve ULTRA benchtop NMR spectrometer.  While the sequence effectively suppressed 3 NMR solvent peaks, it did not reduce the carbon satellites of the solvent which are at a different frequency, and had a similar intensity to our compound of interest and therefore might interfere with our measurements. Fortunately on a Spinsolve ULTRA Carbon spectrometer we can use the carbon channel to do 13C decoupling at the same time as suppressing the solvent peaks.  An example of multiple peak solvent suppression with carbon decoupling is shown below in Figure 1. Notice how the peak labelled with the purple dot is revealed once the carbon satellites are suppressed.

Figure 1: The image above shows a 1D proton spectrum of 170 mM Paracetamol dissolved in normal protonated Ethanol acquired with multiple peak solvent suppression at the frequencies indicated by the 3 red arrows on a Spinsolve 60 ULTRA Carbon (top, red). The green dots identify the carbon satellites of the solvent peaks in the spectrum. The second spectrum (bottom, blue) shows the same sample acquired with multiple peak solvent suppression but now with carbon decoupling. This causes the carbon satellites of all peaks to be removed, including those from the solvent. The peaks which remain belong to the paracetamol and are identified with the the blue, orange, purple and yellow dots.

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How to Evaluate a Benchtop NMR Instrument’s Technical Performance Part 2: 1H Lineshape and Resolution

June 17th, 2019, by

In this post, I’m going to discuss a specific test for evaluating the resolution or lineshape of a benchtop NMR system. This test is measured on proton (1H) as the NMR spectrum is sensitive to the spectrometer resolution and we can make the measurement with a single scan. Resolution and lineshape refer to the width of a particular NMR spectral line, measured at 50% and 0.55% of the height of the line, as explained below.  The smaller the linewidth value, the better the resolution.

 

1H lineshape and resolution

The information content of any NMR spectrum depends on the ability to observe and resolve different signals, or peaks, in the spectrum. It is easier to distinguish two sharp (narrow) peaks close together, than two broad peaks. The key technical factor defining the sharpness of the lines in the spectrum (the lineshape) is the homogeneity of the magnetic field generated by the magnet.  Although NMR systems utilize an array of coils (“shims”) to further improve the B0 homogeneity, the achievable lineshape and resolution are strongly influenced by the inhomogeneity of the magnet itself. The process of calibrating the field and optimizing the B0 homogeneity is usually referred to as shimming. The homogeneity and resolution will gradually degrade over time, so you should carry out the shimming procedure whenever you want to ensure you have the best resolution your spectrometer is capable of.  You should also do the shimming procedure immediately before making the linewidth test described here.

 

Measuring 1H lineshape and resolution

The way to measure instrument resolution is to collect a spectrum containing a naturally sharp line. The standard approach for is to use a sample containing chloroform in acetone-d6 – the chloroform signal from this sample has a very narrow natural linewidth.1 Figure 1 shows a 1H NMR spectrum sample of a 20% chloroform in acetone-d6 NMR reference sample collected on an 80 MHz benchtop NMR spectrometer. (https://www.sigmaaldrich.com/catalog/product/sial/611859)

Figure 1. 80 MHz 1H NMR spectrum of 20% chloroform in acetone-d6 (“lineshape”) sample

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