In an earlier post on qNMR we described how benchtop NMR can be used to quantify the concentration of a sample or measure its purity. When such quantitative methods are validated, there are standard requirements for accuracy, precision, range, and linearity over that range that need to be met.
For example, the United States Pharmacopeia (USP) specifies the general requirements for a Category I NMR method when measuring a drug substance (there are other specifications for finished products and impurities). These specifications are listed in the table at the end of this post and are compared to the measured Spinsolve performance.
We have validated the Spinsolve benchtop qNMR performance by measuring the purity of one reference standard, methylsulfonylmethane (MSM), with another, maleic acid. Maleic acid is a common reference standard for qNMR, so this was used as the reference to measure the known purity of MSM (specified 99.5% pure). A spectrum of the mixture in D2O is shown in Figure 1.
Despite the proliferation of new 2D techniques over the past four decades, one of the most commonly used experiments is the very first one to have come into existence, COSY1. It’s easy to see why it’s withstood the test of time: firstly, it’s an extremely useful experiment, providing a direct and easy way of establishing “through bond” proton-proton connectivities (“this hydrogen is near or next to that hydrogen”); secondly, because it’s a homonuclear 2D experiment (that is to say, it correlates protons with protons) its’s a very sensitive one. The high sensitivity also means that it’s a quick experiment to run, particularly if the experiment uses gradients for coherence selection (more on that below and in a future blog post).
Example COSY spectra recorded on Spinsolve can be found elsewhere on the Magritek website, but a typical example is also shown in Figure 1 below, recorded on a sample of ethyl crotonate. The sequence used to collect this spectrum utilizes gradients, meaning that it was run using only a single scan per t1 increment, and with 512 increments it only took 15 minutes to run.
Fig. 1. COSY spectrum of ethyl crotonate, collected using gradients and 512 t1 increments.
Dr Jonathan Harburn is a Lecturer in Medicinal Chemistry in the Wolfson Research Institute located in the School of Medicine, Pharmacy and Health at Durham University. Working together with Drs Stuart Cockerill and Jonathan Sellars, Dr Harburn’s research goals are to create clinical drug candidates for the treatment of viruses, bacteria and cancer.
In their research, recent progress has focussed repurposing novel fluorinated drug fragments on known drug scaffolds to develop hit identification. 19F NMR using Spinsolve is one of the most useful tools in confirming fluorinated fragment incorporation with spectra run in 3 minutes. Also, 1H NMR is routinely carried out for identification before further spectral data is acquired on higher field NMR.
Alistair Paterson, a Level 2 MPharm student at Durham University, uses Spinsolve to evaluate his sulfathiazole sample
When natural rubber (NR) latex is epoxydized, it is converted to a new plastic (Epoxydized Natural Rubber or ENR) with many favorable properties. Damping and anti-vibration characteristics are improved. ENR is oil resistant and has a better air impermeability i.e. better sealing properties. ENR or Epoxyprene is a speciality polymer produced from natural rubber latex, cis–1,4–polyisoprene, with epoxide groups randomly dispersed along the polymer backbone. Two grades are available commercially, Epoxyprene 25 and Epoxyprene 50, indicating 25% and 50% epoxidation respectively.
In collaboration with Kruthwong & Sprenger Co.Ltd, our main distributor for Southeast Asia, we measured the ENR concentration using the Spinsolve60 benchtop NMR spectrometer. Typical 60 MHz benchtop NMR spectra of ENR samples are shown in Figure 1. The peak at 2.7 ppm is from epoxy (E), the peak at 5 ppm is natural rubber (NR). The ENR ratio can be calculated by E/(E+NR). However, the epoxy peak at 2.7 ppm suffers from significant overlap by the tails of the CH2 resonances at 2.0 ppm. This makes the ENR measurement using standard 1D spectra inaccurate.
Figure 1: Spinsolve60 Proton NMR spectra of natural rubber samples with different ENR.
In order to obtain a clean baseline for peak integration in the region of 2.5-3.0 ppm, a T1-filter was implemented to suppress the CH2 resonances at 2.0 ppm. With this implementation, the epoxy and rubber signals can be nicely separated and integrated accurately to determine the amount of epoxy present, as shown in Figure 2.
Magritek is really excited to be this year at ILMAC Conference in Basel, Switzerland from 20-23 September 2016. You can find us at Booth 1.1 E118 and we really look forward to meeting you. Our team will have a live, working Spinsolve Benchtop NMR and our fabulous applications and support team will be there to answer all your benchtop NMR spectroscopy questions.
HTBLA Wels is a higher technical vocational college of chemistry in Austria. Here, Dr Harald Baumgartner is responsible for the instrumental analytical laboratory. The lab’s main focus is to teach students the basics of NMR (interpretation of spectra).
Dr Baumgartner says “Compared to the old 60 MHz spectrometer, the Magritek Spinsolve benchtop spectrometer is so much easier to use. It is software-based so collecting and processing data is quite straightforward. As well as 1H spectra, our Spinsolve allows us to measure more complex spectra including 13C-spectra. Even 2-dimensional experiments are now available to the students.”
A college student learns about NMR with the Magritek Spinsolve Carbon at HTLBA Wels in Austria
In part 5 we introduced the PGSE experiment to measure self-diffusion coefficients. We saw that if the peak integrals are displayed as a Stejskal-Tanner plot we can immediately identify if there is a single self-diffusion coefficient or not. This works pretty well for neat liquids, or solutions with a single type of molecule, or even polymer molecules with a size distribution. However, in real life we are often dealing with mixtures of molecules, and it would be nice if we could somehow separate the spectra of the individual compounds.
Consider for example the spectrum of a mixture of procaine and paracetamol in D2O. This is shown in the middle scan of Figure 1, along with the spectra of the pure compounds above and below. If we had only the mixture available, but not the pure compounds, it would be hard to figure out how many and which compounds are present in the mixture.
These spectra, along with all the others shown in this post, were acquired on a Spinsolve benchtop NMR spectrometer with additional hardware to enable PFGs for measuring diffusion.
Figure 1: Spectra of procaine (top), paracetamol (bottom), and a 1:1 mixture of both (middle) in D2O.
Dr Alan Kenwright is Reader in Spectroscopy and Manager of the solution-state NMR facility in the Chemistry Department at Durham University. His personal research is focussed on developing and using NMR techniques to solve a range of chemical problems. In choosing to use Magritek’s Spinsolve, Dr Kenwright anticipates it will allow the extension of his work in various areas in ways that he could not otherwise. He plans to use the equipment initially in three areas:
Dr Nicola Rogers is a post doc in the Kenwright group using the Magritek Spinsolve to make relaxation measurements on lanthanide complexes at Durham University. For ease of use, it is mounted on a trolley making it easy to move from lab to lab.
“The first application is in looking at lanthanide complexes of the sort used as contrast agents for MRI” … “being able to do measurements in the relatively low magnetic field (43 MHz) used by Magritek’s Spinsolve is a big advantage for us, particularly as the field it uses it not very different to the field actually used in many hospital MRI scanners. These measurements using the Spinsolve are just starting to appear in the literature.” (reference given at the end of this blog post)
Cleveland State University in Ohio is developing into one of the best urban universities in the nation. Investment at the ground roots graduate level is illustrated by the recent purchase of benchtop NMR spectrometry to offer students hands-on experience of the latest in scientific instrumentation.
Dr Vania De Paoli is an associate college lecturer in the Department of Chemistry where she is leading a program to create a solid environment for teaching Organic Chemistry. Prior to investing in the Magritek benchtop NMR spectrometer, students‘ practical options were limited to the measurement of melting points and refractive index. Students were not experiencing anything close to life in a modern organic chemistry laboratory.
As Dr De Paoli says, the Spinsolve has changed this position immensely. “It is a small system, portable and lightweight. It is quickly ready to use allowing the students to have a real NMR analysis experience (they prepare the samples in standard NMR tubes, add the solvent and record the spectra in similar ways to a research grade NMR). The spectra are of good quality. The software is friendly and, overall, Spinsolve is readily affordable.”
Quantification using any analytical method requires calibrating an instrumental response with a known reference, and then calculating the concentration of an unknown sample from the measured instrument response. One advantage of NMR compared to other analytical methods is that the signal response is linear, resulting the NMR signal intensity being proportional to the number of nuclei.
Sample concentrations and purities can be easily measured from known peaks once the proportionality constant is calibrated using a reference of known concentration and purity. Such measurement methods are known as quantitative NMR, or qNMR for short.
We regularly post testimonials on our blog, where our customers describe how they are using their Spinsolve and comment on their experience using it. We have a number of these user stories now, so we have conveniently compiled them all on a new page ‘What Customers Say‘.
Take a look to see how Spinsolve users all over the world, in teaching and research, are using their Spinsolves.
Serving requests from our followers, we have created a list of selected peer-reviewed publications in which our Spinsolve benchtop NMR spectrometer is featured. Spinsolve is being used for research in topics such as online Reaction Monitoring, Hyperpolarisation, Ultrafast 2D NMR, Residual Dipolar Couplings and Process Control.
Part 3 discussed how a matched pair of positive/negative magnetic field gradient pulses can be used to encode spins for their displacement. Although this simple sequence has great value from an educational point of view, it is rarely used in practice due to several drawbacks.
The delay time between the two gradient pulses can be quite long (up to several hundreds of milliseconds or even seconds). During that time the spins acquire additional phase information due to chemical shift evolution. This will make it impossible to phase the spectrum if it contains more than one peak.
During the evolution the magnetisation suffers from T2* relaxation, which can lead to significant signal attenuation.
For this experiment to work, it is extremely important that the two gradient pulses are very well matched in length and amplitude. This is very difficult to achieve with a positive/negative pair.
In 1965, E.O. Stejskal and J.E. Tanner published a famous paper describing an experiment which avoids these issues. It is called the Pulsed Gradient Spin Echo (PGSE) experiment, and I love this experiment because it’s so simple, yet technically very challenging. It is still being used in its original form after being around for half a century. The basic idea is shown in Figure 1.
Figure 1: (a) Schematic diagram of the PGSE pulse sequence. (b) The phase evolution of the spins at different locations along the gradient direction. Note that the 180 degree pulse inverts the phase wrap imposed by the first gradient pulse. The second gradient pulse, which is now identical to the first one in amplitude and length, completely refocuses this phase wrap.
Part 3 discussed how a matched pair of positive/negative magnetic field gradient pulses can be used to encode spins for their displacement. We can distinguish between two basic types of displacement:
All spins are moving together with the same speed along the same direction. This leads to a phase shift in the signal.
All spins are jumping randomly between positions. This results in an attenuation of the signal amplitude.
In this post we will have a closer look at this second type of movement.
It is closely related to the spreading of for example a blob of ink in a beaker filled with water, which is shown schematically in Figure 1. Initially, the blob is located within a small volume (a) near the wall, but over time it will spread out and eventually cover the entire volume (b). The Latin word for “spreading out” is “diffundere”, and therefore this process is known as diffusion.
Figure 1: (a) Some particles are dissolved in a beaker of water. Initially, the particles are located in a small volume near the wall. Over time, the particles randomly move around and spread evenly over the entire volume (b).
This type of random jumps was first visually observed by the Scottish botanist Robert Brown when he watched pollen grains suspended in water. Nowadays we know that diffusion (another name is Brownian motion) governs biochemical processes, and there is a great interest to measure and understand such random movements.