tag:blogger.com,1999:blog-24083813751331141862024-03-25T15:37:33.097-07:00UCSD SSPPS NMR FacilityBlog for the NMR Facility at the UC San Diego Skaggs School of Pharmacy - with an emphasis on practical NMR.Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.comBlogger103125tag:blogger.com,1999:blog-2408381375133114186.post-31132256026321969912024-03-05T09:40:00.000-08:002024-03-05T09:40:11.631-08:00The impact of poor tuning and matching<p>
Tuning and matching is the process of optimising the frequency and resistance
of the circuit that includes the detection coil. Every sample has a slightly
different ionic content and so the probe should be tuned and matched for each
sample. Modern NMR probes have automatic tuning and matching devices, but the
Skaggs NMR Facility probes do not. Here, I look at the impact of poor tuning
and matching on a 1D <sup>1</sup>H spectrum.
</p>
<span><a name='more'></a></span>
<p>
The NMR spectra were collected using a sample of cholesteryl acetate in
chloroform-<i>d</i> using a room temperature 5 mm broad band inverse detection
probe. The spectra were collected using a single scan, a one minute relaxation
delay, and the <i>zg</i> pulse sequence that implements a single 90<sup
>o</sup> pulse followed by data acquisition. Initially, the probe was tuned for a
methanol-<i>d<sub>4</sub></i> sample, a 1D <sup>1</sup>H spectrum was
collected, and the 90<sup>o</sup> pulse with this tuning was measured. The
probe was then tuned and matched for chloroform-<i>d</i>, another 1D
<sup>1</sup>H spectrum collected with identical parameters, and the 90<sup
>o</sup> pulse redetermined.
</p>
<p>
A stackplot of the two spectra plotted with the same scaling is shown below.
The lower spectrum in blue was collected with poor tuning and matching, while
the upper red spectrum was collected with good tuning and matching.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a
href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhpFAqU1oY-YqdvH0u5N8_Lxv64Zqv6qjwBSJ74XLDSr4PIZVCn1z2stM6Djkymxhqc8xP1TjBH3NzSknlocdlBdhxS4CKjd7yaID4otezoHr6zipDL4uOogyNXMo9LgrPHO7XYnYSk3gFlXtOVoXa1Vrjnl6g5Zl_yGTUAtqNzRMnKZZDNtLHgIIvZLEDL/s765/stackplot.png"
style="margin-left: 1em; margin-right: 1em;"
><img
border="0"
data-original-height="357"
data-original-width="765"
height="186"
src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhpFAqU1oY-YqdvH0u5N8_Lxv64Zqv6qjwBSJ74XLDSr4PIZVCn1z2stM6Djkymxhqc8xP1TjBH3NzSknlocdlBdhxS4CKjd7yaID4otezoHr6zipDL4uOogyNXMo9LgrPHO7XYnYSk3gFlXtOVoXa1Vrjnl6g5Zl_yGTUAtqNzRMnKZZDNtLHgIIvZLEDL/w400-h186/stackplot.png"
width="400"
/></a>
</div>
<p>
Clearly, improving the tuning and matching has improved the sensitivity.
Measuring the signal-to-noise on the peak at 5.40 ppm gives 777.13 for the
poorly tuned spectrum and 1607.32 for the well tuned one.
</p>
<p>
The poorly tuned spectrum is less sensitive because the 90<sup>o</sup> pulse
is not calibrated correctly. Poor tuning and matching results in a longer
pulse being required to rotate the magnetisation 90<sup>o</sup> from
equilibrium along the <i>z</i>-axis, to the <i>x-y</i> plane where the signal
can be detected. For the well tuned spectrum the 90<sup>o</sup> pulse length
was 7.70 µs. For the poorly tuned spectrum the 90<sup>o</sup> pulse was
24.25 µs. Both spectra were recorded with a 7.45 µs pulse. Thus, the
poorly-tuned spectrum used a 28<sup>o</sup> pulse which would give 46.4% of
the signal, while the well tuned spectrum was recorded with an 87<sup>o</sup>
pulse which would give 99.9% of the maximum signal. The signal-to-noise
measurements gave values with a very similar ratio.
</p>
<table border="1" cellpadding="3" cellspacing="0" style="margin: auto;">
<tbody>
<tr>
<th style="text-align: right;">tuning</th>
<td style="text-align: center;">poor</td>
<td style="text-align: center;">good</td>
</tr>
<tr>
<th style="text-align: right;">90<sup>o</sup> pulse</th>
<td style="text-align: center;">24.25 µs</td>
<td style="text-align: center;">7.70 µs</td>
</tr>
<tr>
<th style="text-align: right;">effective pulse</th>
<td style="text-align: center;">28<sup>o</sup></td>
<td style="text-align: center;">87<sup>o</sup></td>
</tr>
<tr>
<th style="text-align: right;">signal available</th>
<td style="text-align: center;">46.4%</td>
<td style="text-align: center;">99.9%</td>
</tr>
<tr>
<th style="text-align: right;">signal-to-noise</th>
<td style="text-align: center;">777.13<br /></td>
<td style="text-align: center;">1607.32</td>
</tr>
</tbody>
</table>
<p>
The 5 mm probes are much more sensitive to tuning and matching than the 1.7 mm
probe because the volume of solvent is so much larger. If you want to maximise
your sensitivity make sure the probe is tuned and matched for your solvent.
</p>
Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-65936726613487836062024-02-06T10:22:00.000-08:002024-02-06T10:22:49.519-08:00Altmetrics and citations<p>
Publications arising from work at the SSPPS NMR Facility are tracked on the
Facility's
<a href="http://sopnmr.ucsd.edu/sopnmr-publications.htm">Publications webpage</a>. Along with breakdowns of the list by year, lab and journal, this page also reports the Altmetric score for each publication. This is a
measure of the attention the article has received, predominantly on social
media. To see how the Altmetric score compares with citations I prepared a few
graphs.
</p>
<span><a name='more'></a></span>
<p>
The Altmetric score is calculated from the number of times an article is
mentioned in traditional media, on blogs, on
<a href="http://twitter.com">Twitter</a>,
<a href="http://LinkedIn.com">LinkedIn</a>,
<a href="http://www.reddit.com">Reddit</a>, and various other sources. The
score is provided by a company called
<a href="http://www.digital-science.com">Digital Science</a>. For citations I
turned to <a href="http://www.webofscience.com/wos">Web of Science</a> and
used their core database citation count. Of the 218 papers on the Facility
publication list, 207 were indexed in Web of Science, and 155 had Altmetric
scores. The graph below shows Altmetric scores plotted against year with three
of the higher scoring papers labelled. As might be expected, more recent
papers tend to have higher Altmetric scores as social media is increasingly
used to promote publications. The Jang et al 2013 paper is unusual it that it
was mentioned by news organisations more than social media.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhs5KGIYse6eC8Akv3YRHM19f4pvAcCgbg3zdp6nUTPvhD2vfJDhRa72sGu9jWSrJyql3emA-c16OtDFdDP28b4YBH0EW28zVaLqpxyjhvrxt2ESQpSzbmGRG3i72P03oNOyGJn1y8TA6XNZajbg6lwN8Z55lGsmPMfn96FfK2ll1PZtgfeJABhuMR7Qlkx/s640/altmetric.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="480" data-original-width="640" height="300" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhs5KGIYse6eC8Akv3YRHM19f4pvAcCgbg3zdp6nUTPvhD2vfJDhRa72sGu9jWSrJyql3emA-c16OtDFdDP28b4YBH0EW28zVaLqpxyjhvrxt2ESQpSzbmGRG3i72P03oNOyGJn1y8TA6XNZajbg6lwN8Z55lGsmPMfn96FfK2ll1PZtgfeJABhuMR7Qlkx/w400-h300/altmetric.png" width="400" /></a>
</div>
<p>
The graph of citations versus publication year appears below and shows a
different trend, the more recent papers have less citations. Again, this is to
be expected as it takes time for a paper to accumulate citations. To show the
data more clearly this graph excludes two papers with very high citation
counts, Wang et al 2014 with 2,237 citations, and Yamanaka et al 2014 with
318. Three of the more highly cited papers are identified and none of these,
or the two that were excluded, include the three with high Altmetric scores
labelled on the first graph.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhBR5ritXPQSZJfp6g5506vBekNgJqMQi5G2KWSWBb-IKcBghK1-rDZI2OZa-dzDF5K6dmucJtsbe1bVC-ecUoxg0a7ag_Ai-oV6Do-NBwJoM1vhBhQQmlW0dW-R809l2xSzym1PXy7BcSdSqIuQ073upRH4_u-l5NsqZXP19xtu_Gsz9ZUWrswVHydLk2i/s640/citation.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="480" data-original-width="640" height="300" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhBR5ritXPQSZJfp6g5506vBekNgJqMQi5G2KWSWBb-IKcBghK1-rDZI2OZa-dzDF5K6dmucJtsbe1bVC-ecUoxg0a7ag_Ai-oV6Do-NBwJoM1vhBhQQmlW0dW-R809l2xSzym1PXy7BcSdSqIuQ073upRH4_u-l5NsqZXP19xtu_Gsz9ZUWrswVHydLk2i/w400-h300/citation.png" width="400" /></a>
</div>
<p>
Finally, the graph of Altmetric score against citations is shown below with
selected papers labelled. The straight line shows an attempt to fit the points
to a linear correlation (R<sup>2</sup>=0.095), which is obviously not there.
The inset shows an expansion of the lower left corner of the graph.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjLesXQ_YgsL52BJKn2B-eYUYaHo9cMk5sgf4PA9LNjGbZdVvq2ObaJVdGXYpLSVsq17aO-h0wwwl0Y4-SiJ-cQ0u0dux5WapAs0PhwauRP3B8nJMkcmV33glu-qQRct5_RkwggJfCdD72fJsGyVZBjKU8WppozCjCFLaabyHtW-BOYOydaIFht_8aYkEJQ/s640/citationValtmetric.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="480" data-original-width="640" height="300" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjLesXQ_YgsL52BJKn2B-eYUYaHo9cMk5sgf4PA9LNjGbZdVvq2ObaJVdGXYpLSVsq17aO-h0wwwl0Y4-SiJ-cQ0u0dux5WapAs0PhwauRP3B8nJMkcmV33glu-qQRct5_RkwggJfCdD72fJsGyVZBjKU8WppozCjCFLaabyHtW-BOYOydaIFht_8aYkEJQ/w400-h300/citationValtmetric.png" width="400" /></a>
</div>
<p>
Digital Science developed the Altmetric score to measure the early impact of a
publication as citation counts may take years to reflect a paper's impact.
These graphs suggest that it does report on something different from citation
counts and that the Altmetric score really is an alternative metric.<br />
</p>
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MS/MS networking guided analysis of molecule and gene cluster families. </a><br />
Proc Natl Acad Sci U S A. 2013 Jul 9;110(28):E2611-20
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Moore BS<br />
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Nat Chem Biol. 2014 Aug;10(8):640-7
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<p>
7. Yamanaka K, Reynolds KA, Kersten RD, Ryan KS, Gonzalez DJ, Nizet V,
Dorrestein PC, Moore BS<br />
<a href="http://dx.doi.org/10.1073/pnas.1319584111">
Direct cloning and refactoring of a silent lipopeptide biosynthetic gene
cluster yields the antibiotic taromycin A. </a><br />
Proc Natl Acad Sci U S A. 2014 Feb 4; 111(5), pp 1957-62
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Watrous J, Kapono CA, Luzzatto-Knaan T, Porto C, Bouslimani A, Melnik AV,
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Kersten RD, Pace LA, Quinn RA, Duncan KR, Hsu CC, Floros DJ, Gavilan RG,
Kleigrewe K, Northen T, Dutton RJ, Parrot D, Carlson EE, Aigle B, Michelsen
CF, Jelsbak L, Sohlenkamp C, Pevzner P, Edlund A, McLean J, Piel J, Murphy BT,
Gerwick L, Liaw CC, Yang YL, Humpf HU, Maansson M, Keyzers RA, Sims AC,
Johnson AR, Sidebottom AM, Sedio BE, Klitgaard A, Larson CB, Boya P CA,
Torres-Mendoza D, Gonzalez DJ, Silva DB, Marques LM, Demarque DP, Pociute E,
O'Neill EC, Briand E, Helfrich EJ, Granatosky EA, Glukhov E, Ryffel F, Houson
H, Mohimani H, Kharbush JJ, Zeng Y, Vorholt JA, Kurita KL, Charusanti P,
McPhail KL, Nielsen KF, Vuong L, Elfeki M, Traxler MF, Engene N, Koyama N,
Vining OB, Baric R, Silva RR, Mascuch SJ, Tomasi S, Jenkins S, Macherla V,
Hoffman T, Agarwal V, Williams PG, Dai J, Neupane R, Gurr J, Rodríguez AM,
Lamsa A, Zhang C, Dorrestein K, Duggan BM, Almaliti J, Allard PM, Phapale P,
Nothias LF, Alexandrov T, Litaudon M, Wolfender JL, Kyle JE, Metz TO, Peryea
T, Nguyen DT, VanLeer D, Shinn P, Jadhav A, Müller R, Waters KM, Shi W, Liu X,
Zhang L, Knight R, Jensen PR, Palsson BØ, Pogliano K, Linington RG, Gutiérrez
M, Lopes NP, Gerwick WH, Moore BS, Dorrestein PC, Bandeira N<br />
<a href="http://dx.doi.org/10.1038/nbt.3597">
Sharing and community curation of mass spectrometry data with Global Natural
Products Social Molecular Networking. </a><br />
Nat Biotechnol. 2016 Aug 9;34(8):828-837
</p>
<p>
9. Nguyen DD, Melnik AV, Koyama N, Lu X, Schorn M, Fang J, Aguinaldo K,
Lincecum TL Jr, Ghequire MG, Carrion VJ, Cheng TL, Duggan BM, Malone JG,
Mauchline TH, Sanchez LM, Kilpatrick AM, Raaijmakers JM, Mot R, Moore BS,
Medema MH, Dorrestein PC<br />
<a href="http://dx.doi.org/10.1038/nmicrobiol.2016.197">
Indexing the Pseudomonas specialized metabolome enabled the discovery of
poaeamide B and the bananamides. </a><br />
Nat Microbiol. 2016 Oct 31;2:16197
</p>
<p>
10. Brunson JK, McKinnie SMK, Chekan JR, McCrow JP, Miles ZD, Bertrand EM,
Bielinski VA, Luhavaya H, Oborník M, Smith GJ, Hutchins DA, Allen AE, Moore
BS<br />
<a href="http://dx.doi.org/10.1126/science.aau0382">
Biosynthesis of the neurotoxin domoic acid in a bloom-forming diatom. </a><br />
Science. 2018 Sep 28;361(6409):1356-1358
</p>
<p>
11. Wirth DM, Jaquez A, Gandarilla S, Hochberg JD, Church DC, Pokorski JK<br />
<a href="http://dx.doi.org/10.1021/acsami.0c02683">
Highly Expandable Foam for Lithographic 3D Printing. </a><br />
ACS Appl Mater Interfaces. 2020 Apr 22;12(16):19033-19043
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12. Reher R, Kim HW, Zhang C, Mao HH, Wang M, Nothias LF, Caraballo-Rodriguez
AM, Glukhov E, Teke B, Leao T, Alexander KL, Duggan BM, Van Everbroeck EL,
Dorrestein PC, Cottrell GW, Gerwick WH<br />
<a href="http://dx.doi.org/10.1021/jacs.9b13786">
A Convolutional Neural Network-Based Approach for the Rapid Annotation of
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<p>
13. Liang Z, Soriano-Castell D, Kepchia D, Duggan BM, Currais A, Schubert D,
Maher P<br />
<a href="http://dx.doi.org/10.1016/j.freeradbiomed.2022.01.001">
Cannabinol inhibits oxytosis/ferroptosis by directly targeting mitochondria
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</p>
Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-85131863673821589072024-01-03T10:49:00.000-08:002024-01-03T10:49:33.832-08:00The acquisition time<p>
In an NMR experiment the acquisition time is the period used to record the
signal. Changing this value can affect the quality and appearance of your
spectrum. Typically, larger acquisition times are used for one-dimensional
spectra, with shorter values used in multidimensional spectra. The impact of
using different acquisition times on 1D <sup>1</sup>H spectra are shown in
this post.
</p>
<span><a name='more'></a></span>
<p>
To record the spectra a halogenated cholestane in CDCl<sub>3</sub> was used.
Five spectra were collected with the acquisition time initially set to 0.143
seconds and then doubled for each successive spectrum. The spectra were
processed identically with exponential line broadening of 0.3 Hz and
zero-filling to 64K points. A stackplot of the spectra showing the upfield
region with the methyl peaks is shown below.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj88goTAR2UAbm_dOMdHm64V78lBZZVbSJaPmm4kdXd8nkflPOoZFQyArM8w2wyaqX2a5AzE3UUNJuKHW-rUChLE0ObRUMxBzo3-OGpth8JuCcmuuccd-kE2EEyelslAExwTLw4H0pKFt4SpUGlaT_kF8t0XOGjygb8THkzkbGIN8DC_1zZ5lfhBQXl_Zoi/s789/stackplotLabelled.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="644" data-original-width="789" height="326" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj88goTAR2UAbm_dOMdHm64V78lBZZVbSJaPmm4kdXd8nkflPOoZFQyArM8w2wyaqX2a5AzE3UUNJuKHW-rUChLE0ObRUMxBzo3-OGpth8JuCcmuuccd-kE2EEyelslAExwTLw4H0pKFt4SpUGlaT_kF8t0XOGjygb8THkzkbGIN8DC_1zZ5lfhBQXl_Zoi/w400-h326/stackplotLabelled.png" width="400" /></a>
</div>
<p>
The first thing to notice is that at shorter acquisition times the peaks are
broader and resolution is reduced. The doublets at 0.88 ppm are only resolved
when the acquisition time is at least 0.570 s. Other multiplets (0.75, 1.30,
1.54 ppm) also show reduced resolution at the shorter acquisition times. When
longer acquisition times are used the signal has time to decay to near zero,
but with shorter acquisition times the signal is still significant at the end.
During processing, a window function is used to scale the data so that it is
close to zero at the end of the acquisition time. This reduces truncation
artifacts, but it makes the signals appear to decay more rapidly than they
actually do. The increased apparent decay rate translates into broader peaks
and a loss of resolution.
</p>
<p>
The other effect of the reduced acquisition time is the artifacts in the
baseline, particularly obvious above 1.00 ppm. These "sinc wiggles" are due to
the presence of significant signal at the end of the acquisition time. The
fourier transform converts the intensity difference between the signal and the
zeros used for zero filling into a sinc function in the baseline.
</p>
<p>
Based on these examples one might assume that the longer the acquisition time
the better, but in fact this is not true. The figure below shows the
signal-to-noise ratio, measured using the peak at 0.68 ppm, plotted against
the acquisition time. The signal-to-noise rapidly increases to a maximum and
then slowly decreases.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj2S90D6q7QxL0VIPFj2uTqN3prnpvfsWJWpC0l3o8yp3mLLScEGoCmuZJAxyNDaxEsJ_Z4GhzbPqccmWrTJh1wFzhwGowo1ljFBDARlL2XdCye7jY_zEQIFw5UkfExqHOgx01QnCT0iU0Ky-P2Rf0T_8DpwUoiDHp7saqix90O9FDfKiAqSn8gyqdqcOR6/s640/snr.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="480" data-original-width="640" height="300" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj2S90D6q7QxL0VIPFj2uTqN3prnpvfsWJWpC0l3o8yp3mLLScEGoCmuZJAxyNDaxEsJ_Z4GhzbPqccmWrTJh1wFzhwGowo1ljFBDARlL2XdCye7jY_zEQIFw5UkfExqHOgx01QnCT0iU0Ky-P2Rf0T_8DpwUoiDHp7saqix90O9FDfKiAqSn8gyqdqcOR6/w400-h300/snr.png" width="400" /></a>
</div>
<p>
Signal-to-noise in NMR experiments has long been known to be maximal near
1.26T<sub>2</sub><sup>1</sup>. This is because the noise is constant, and will
continue to increase if recorded for longer, but the signal decays. After a
certain point in the acquisition time, 1.26T<sub>2</sub>, the signal has
decayed sufficiently that the noise is greater. Increasing the acquisition
time beyond 1.26T<sub>2</sub> will provide increased resolution but at the
expense of sensitivity and the cost of increased experimental time. </p><p>For quick
1D experiments, where resolution of multiplets is important, longer acquisition
times are typically used. For multi-dimensional experiments where crosspeak
fine structure is not important, but total experiment time is, acquisition
times are kept short. This also has the benefit of reducing the size of the
processed multidimensional data. At the Skaggs NMR Facility the acquisition
time is typically set to 1.140s for 1D <sup>1</sup>H spectra, 0.865s for 1D <sup>13</sup>C spectra, and 0.285s for 2D
and 3D spectra.
</p>
<p>
<u>References</u><br />
1. Rovnyak D.<br />
<a href="https://doi.org/10.1002/cmr.a.000.21473">The past, present, and future of 1.26T<sub>2</sub>.</a><br />
Concepts Magn Reson Part A. 2019; 47A:e21473</p><p><u>Acknowledgments</u><br />Thanks to Prof Ted Molinski for providing the cholestane sample.<br /></p>
Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-91669738232748982332023-12-04T10:25:00.000-08:002023-12-04T10:25:47.642-08:00The relaxation delay<p>
The relaxation delay is one of the basic NMR parameters. Optimising the
relaxation delay can help improve the appearance of your spectra and increase
the accuracy of your integrals. In this post its impact on 1D <sup>1</sup>H
spectra is shown.
</p>
<span><a name='more'></a></span>
<p>
The relaxation delay is the time between recording the NMR signal from one
scan and the first pulse of the next scan. Its purpose is to allow the
magnetisation to return to equilibrium before starting the next scan. This
provides a uniform starting point for each scan. To obtain complete
equilibrium, however, the relaxation delay needs to be much longer than the
time taken to generate and record the signal. In most cases a reduced
relaxation delay, that gives reduced signal, is used and more scans are
recorded to compensate.
</p>
<p>
To demonstrate the impact of the relaxation delay a series of 1D <sup>1</sup>H
spectra were recorded on a sample of 1% ethylbenzene in deuterated chloroform.
A 90<sup>o</sup> pulse and eight scans were used. The figure below shows a
stackplot of the spectra with the relaxation delay initially set to 0.25s and
then doubled in each successive spectrum. Clearly, the longer the relaxation
delay the more intense the signals.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi62uQ222ic_AYhzmJtmqYzyiytgPqhFM2ONKj16c_KXDMSc548DIY1SBlocJURx4FG94heWNUjYqLfuRVuO1OJJYfpN4pgKbYPepOaQ9o7uq3w4KVzDYk5X4ynNMChRknhUM4J5GSbDDirEIcQIkXhIVXwHhcMvRWzedLV42UW2GtM6Qx56ZS2UEEWrteG/s789/stackplot.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="644" data-original-width="789" height="326" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi62uQ222ic_AYhzmJtmqYzyiytgPqhFM2ONKj16c_KXDMSc548DIY1SBlocJURx4FG94heWNUjYqLfuRVuO1OJJYfpN4pgKbYPepOaQ9o7uq3w4KVzDYk5X4ynNMChRknhUM4J5GSbDDirEIcQIkXhIVXwHhcMvRWzedLV42UW2GtM6Qx56ZS2UEEWrteG/w400-h326/stackplot.png" width="400" /></a>
</div>
<p>
This suggests that long relaxation delays should be used to maximise signal
intensity but, as usual, its not quite that simple. If the integrals of the
signals are plotted against the relaxation delay it can be seen that the
intensity gains are not linear. In the figure below it can be seen that the
relationship is more like a logarithmic one. The curves indicate that
increasing the relaxation delay would provide diminishing returns.<br />
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhB8pbePdkOIgS07HF5IB_fx7ecwL_SkthDQYrJ8fGjL9bxsQ4uOg049PzhFXg25PU2ff4fTtlQZIKeCkNYl-_TEhYdXFvNv47bW57XvEKUrUYKF7GiS8Bx-sGW4qNT7fps6YO6px72BsisVyqznqm7MumUTLNTR2gEk_A9tmKAuUxhERojjdyq8Lnd62av/s640/relaxationDelay.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="480" data-original-width="640" height="300" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhB8pbePdkOIgS07HF5IB_fx7ecwL_SkthDQYrJ8fGjL9bxsQ4uOg049PzhFXg25PU2ff4fTtlQZIKeCkNYl-_TEhYdXFvNv47bW57XvEKUrUYKF7GiS8Bx-sGW4qNT7fps6YO6px72BsisVyqznqm7MumUTLNTR2gEk_A9tmKAuUxhERojjdyq8Lnd62av/w400-h300/relaxationDelay.png" width="400" /></a>
</div>
<p>
For the figure above the integral of the methyl resonance in the spectrum with
the longest relaxation delay (16s) was set to three and used to calibrate all
the other integrals. Looking at the curves its obvious that the integrals
would increase further if the relaxation delay was increased. Slightly less
obvious though, is that the integrals of the aromatic peaks are continuing to
increase while those of the methylene and methyl are starting to flatten out.
From the curves fitted to the integrals we can obtain rates for the curves.
The rates for the two aliphatic peaks are similar, while the aromatic rates
are all similar and slightly slower, and the chloroform rate is the slowest of
all. These rates are a mixture of the T<sub>1</sub> and T<sub>2</sub>
<a href="http://sopnmr.blogspot.com/2017/01/t-1-and-t-2-relaxation.html">relaxation times</a>
of the signals and each signal has a different relaxation time that depends on
its atom's magnetic environment.
</p>
<p>
For maximum intensity and the most accurate integrals it is generally
recommended that the relaxation delay be set to five times the T<sub>1</sub>
relaxation time. The T<sub>1</sub> relaxation time of the ethylbenzene methyl
group has been reported as 17.4s<a href="#ref1"><sup>1</sup></a>, requiring a relaxation delay of 87s. This is the fastest relaxing proton in
ethylbenzene so theoretically the relaxation delay should be even longer to
get accurate integrals of the other signals. A relaxation delay of more than
90s is not very practical, however. The usual solution to this problem is to
reduce the relaxation delay and the pulse width. The shortened relaxation
delay will reduce the accuracy of the integrals but still provide reasonable
estimates. Reducing the pulse width will reduce the signal intensity, but will
also reduce the time needed to return to equilibrium. The standard 1D
<sup>1</sup>H parameters at the SSPPS NMR Facility use a relaxation delay of
0.5s and a 30<sup>o</sup> pulse. For more accurate integrals the relaxation
delay can be increased.
</p>
<p>
<u>References</u><br />
<a name="ref1">1.</a> "NMR relaxation",
<a href="https://chem.ch.huji.ac.il/nmr/techniques/other/t1t2/t1t2.html">
https://chem.ch.huji.ac.il/nmr/techniques/other/t1t2/t1t2.html</a>
</p>
Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-34307276475695297282023-11-03T10:18:00.002-07:002023-11-03T10:18:58.614-07:00Fill volume for 1.7mm NMR tubes<p>
The SSPPS NMR Facility routinely uses a 1.7mm micro-cryoprobe that takes
capillary NMR tubes with a diameter of 1.7mm instead of the standard 5mm NMR
tubes. These capillary tubes require much less sample volume than a standard
tube, but exactly how much should be used? And what happens if there is too
little? Or too much?
</p>
<span><a name='more'></a></span>
<p>
To determine the optimum volume for the 1.7mm NMR tubes a series of 1D
<sup>1</sup>H NMR spectra were recorded with different volumes of
methanol-d<sub>4</sub>. Initially 20μl of methanol-d<sub>4</sub> was added and
a spectrum recorded, then solvent was added in 5μl aliquots up to a total
volume of 65μl. Spectra were recorded after each addition using a 90<sup
>o</sup
>
pulse, 2.0s relaxation delay and a 2.28s acquisition time.
</p>
<p>
The figure below shows expansions from a stackplot of the ten spectra. The
hydroxyl resonance is on the left and the methyl resonance on the right. At
the lower volumes both peaks are broad, misshapen and small. Gradient shimming
was not possible for the 20μl and 25μl samples. As the volume was increased
the peaks sharpened, lineshape improved, and the intensity increased until
there was no further improvement with further additions.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a
href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiY268qJto6QgeZMiopR8GU6HsXswlyvGZjkO3iJj9XRQBg9U5lSxpoqILQOyjzMfIgBlQtHvYyl3aclS0uc-lje_ZoSZ6Fcxc17cKDSkm_pnnaGiy8Y6HvfDgd60hQ0lUAyMdbCzpODm64mYb9e0qrG5_y4ibtXQGQqb1v9sjDGmRw_ciiRz6gQH4kHMA_/s1578/methanolStackplot.png"
style="margin-left: 1em; margin-right: 1em;"
><img
border="0"
data-original-height="644"
data-original-width="1578"
height="164"
src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiY268qJto6QgeZMiopR8GU6HsXswlyvGZjkO3iJj9XRQBg9U5lSxpoqILQOyjzMfIgBlQtHvYyl3aclS0uc-lje_ZoSZ6Fcxc17cKDSkm_pnnaGiy8Y6HvfDgd60hQ0lUAyMdbCzpODm64mYb9e0qrG5_y4ibtXQGQqb1v9sjDGmRw_ciiRz6gQH4kHMA_/w400-h164/methanolStackplot.png"
width="400"
/></a>
</div>
<p>
At the lower volumes there is insufficient sample to fill the entire volume
inside the detection coils in the probe. This results in poor shimming, and
thus broad and distorted peaks. It also reduces the intensity of the signal
because the number of atoms inside the detection volume is less than the
maximum. Once the sample volume is large enough to fill the detection coils
then the shimming improves and the signal intensity reaches a maximum.
Increasing the sample volume beyond this simply adds solution above the coils,
so the shimming does not improve and the signal no longer increases because
the extra atoms are outside the detection volume.
</p>
<p>
One way to determine the minimum required volume is to identify when the
shimming no longer improves. Linewidth, the width of a peak at half its
maximum height, is a good measure of shimming. The narrower a peak and the
smaller its linewidth, the better the shimming. Plotting the linewidth against
volume in the figure below we can see that linewidth decreases until 35μl has
been added, after which it remains fairly constant.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a
href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiRH20yUh0aB3nzWvh7gl4DDyR6eBNg7ADZexxYPxRh4xGaqLsoO-ilrIcwtJbAxi3RGY3P-cHBQAZzjQJLvs-__e-RV4wmSEMdahY-ugCW-hZbmw_bQacuZZRUL6-hJaeJRN_JPUPAPEuSOBOJ46ici070T_9tQUUZg8wh4YXyBD89P4PXQFovgAn9yAWe/s640/linewidth.png"
style="margin-left: 1em; margin-right: 1em;"
><img
border="0"
data-original-height="480"
data-original-width="640"
height="300"
src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiRH20yUh0aB3nzWvh7gl4DDyR6eBNg7ADZexxYPxRh4xGaqLsoO-ilrIcwtJbAxi3RGY3P-cHBQAZzjQJLvs-__e-RV4wmSEMdahY-ugCW-hZbmw_bQacuZZRUL6-hJaeJRN_JPUPAPEuSOBOJ46ici070T_9tQUUZg8wh4YXyBD89P4PXQFovgAn9yAWe/w400-h300/linewidth.png"
width="400"
/></a>
</div>
<p>
Another way to determine the minimum required volume is to identify when the
signal reaches a maximum. When the signal is maximised the detection volume
must be full. Adding further sample will not be useful as it will be outside
the detection coils and not contribute to the signal. Peak integrals are a
good measure of signal as, unlike peak intensity, the lineshape does not
matter. Plotting integrals against volume in the figure below we find that the
integrals increase up until the sample volume reaches 35ul, consistent with
the value obtained from linewidths.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a
href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhqqhop6X5YH0plQrfmne8SEgkVUJROjjEDGr_2BLitWXm5e0VPKWFYZ_xzCSBTY_L9UXNa-8djbzAFfUu6HzS2NS_-g26kOIpKUoi3M3DESZbpheg5ilW1HHHj3sD5OrQS4UHpCCbIgEOkz9OLY7oheDicrrNGXte1jNrVqB8ZL5IguxA67cqdUawPsTsX/s640/integrals.png"
style="margin-left: 1em; margin-right: 1em;"
><img
border="0"
data-original-height="480"
data-original-width="640"
height="300"
src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhqqhop6X5YH0plQrfmne8SEgkVUJROjjEDGr_2BLitWXm5e0VPKWFYZ_xzCSBTY_L9UXNa-8djbzAFfUu6HzS2NS_-g26kOIpKUoi3M3DESZbpheg5ilW1HHHj3sD5OrQS4UHpCCbIgEOkz9OLY7oheDicrrNGXte1jNrVqB8ZL5IguxA67cqdUawPsTsX/w400-h300/integrals.png"
width="400"
/></a>
</div>
<p>
The Bruker marketing literature and manuals state that the 1.7mm micro
cryoprobe needs a sample volume of 30ul which corresponds to a fill height of
23mm. I measured fill heights for all the volumes used and found 30μl
corresponds to a fill height of 17mm and a fill height of 23mm corresponds to
a volume of 42μl (figure below). My measurements may be a little inaccurate
but I don't think they are that far off. It could also be that the Bruker
numbers were obtained for aqueous buffer solutions, instead of an organic
solvent, and surface tension differences could affect the volume delivered.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a
href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEit_OCiPh_cQuUw9Sm7AaYhVzwdqIZWAPo_ZobeEHhRSfymzHOZv6pcQCpz93wZXbWLUKHXt1VxG0iR8qgLn-wctBeVMko7e_R6ouafHIvOYREegq85hmxDUMqDdYDpOHzYVql1x718IIROQF437RX2Y9CN-vdY76_5VMIzSU2dLk68fatKl2uNyfzkoOmI/s640/fillHeight.png"
style="margin-left: 1em; margin-right: 1em;"
><img
border="0"
data-original-height="480"
data-original-width="640"
height="300"
src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEit_OCiPh_cQuUw9Sm7AaYhVzwdqIZWAPo_ZobeEHhRSfymzHOZv6pcQCpz93wZXbWLUKHXt1VxG0iR8qgLn-wctBeVMko7e_R6ouafHIvOYREegq85hmxDUMqDdYDpOHzYVql1x718IIROQF437RX2Y9CN-vdY76_5VMIzSU2dLk68fatKl2uNyfzkoOmI/w400-h300/fillHeight.png"
width="400"
/></a>
</div>
<p>
The measurements here suggest that 35μl is the minimum required volume for
methanol-d<sub>4</sub>. I would recommend adding a little more, 40μl, to
compensate for sample loss during transfer. Anything more than 50μl is not
necessary, and if material is limited, will only dilute your sample.<br />
</p>
Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-69501835477474955582023-10-03T09:36:00.000-07:002023-10-03T09:36:33.108-07:00Fluorine and phosphorus heteronuclear coupling<p>
The most commonly encountered coupling in NMR spectra is due to <sup>1</sup>H, but other nuclei
can induce splitting of signals as well. The <a href="http://sopnmr.blogspot.com/2023/09/13c-satellites.html">previous post</a> discussed how
coupling to <sup>13</sup>C at natural abundance produces the small
<sup>13</sup>C satellite peaks. The other heteronuclear couplings that
are most likely to be observed are due to <sup>19</sup>F and <sup>31</sup>P.
Examples of these in 1D <sup>1</sup>H and <sup>13</sup>C spectra are
shown below.<br />
</p>
<a name='more'></a>
<p>
Trimethyl phosphate has three magnetically-equivalent methoxy groups attached to a
central phosphate atom. The 1D <sup>1</sup>H spectrum taken in D<sub>2</sub>O (shown below) shows what
at first looks like a singlet, as might be expected if considering just the
hydrogens. However, closer examination shows that the peak is a doublet with a
coupling of 13.3 Hz, due to the <sup>3</sup><i>J</i><sub>HP</sub> coupling.<br />
</p>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiev4XYnQUHGM1P5j8YBu1w8ieenp9UIa9oqyif_wAMHlaBG46nriN__KHcQU6ruAAXE0SQN69Xy9JNilxF6CvkaHdcY543_HsSDTtRm7nhUeT8UBkHH3qYNgiiWsxCwfzCIapHOExpGn4078F8h2INk48oPLHUbdu2w40VlwKzFr2-ywW3kdKJWDzFANtO/s761/trimethylPhosphate1H.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="638" data-original-width="761" height="335" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiev4XYnQUHGM1P5j8YBu1w8ieenp9UIa9oqyif_wAMHlaBG46nriN__KHcQU6ruAAXE0SQN69Xy9JNilxF6CvkaHdcY543_HsSDTtRm7nhUeT8UBkHH3qYNgiiWsxCwfzCIapHOExpGn4078F8h2INk48oPLHUbdu2w40VlwKzFr2-ywW3kdKJWDzFANtO/w400-h335/trimethylPhosphate1H.png" width="400" /></a>
</div>
<p>
The 1D <sup>13</sup>C spectrum (shown below) shows a similar situation. At
first glance the spectrum appears to consist of a single peak, as one might
expect considering only the magnetically-equivalent methoxy carbons. However,
the peak shows a splitting of 6.0 Hz, which can be assigned to the <sup>2</sup><i>J</i><sub>CP</sub> coupling.
</p>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgstOVeHNR5vtwu-EkFJjk5X_l_R2UYg65RBV5JlebwiqSLSXhL0zGtjMwmzA0WcqjJfbXPDSR8Bv1VYPZNC00DPC3CY0agpTiCo4z4lVHJmJCi6xZ7KMlh5DmgbBuujMXJw6tZD1GRv-QCAJNPelPYXStmSrEewCD1ZC3HXeld2vqq2Fo3tiWTqSAlGGgI/s761/trimethylPhosphate13C.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="638" data-original-width="761" height="335" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgstOVeHNR5vtwu-EkFJjk5X_l_R2UYg65RBV5JlebwiqSLSXhL0zGtjMwmzA0WcqjJfbXPDSR8Bv1VYPZNC00DPC3CY0agpTiCo4z4lVHJmJCi6xZ7KMlh5DmgbBuujMXJw6tZD1GRv-QCAJNPelPYXStmSrEewCD1ZC3HXeld2vqq2Fo3tiWTqSAlGGgI/w400-h335/trimethylPhosphate13C.png" width="400" /></a>
</div>
<p>
The compound <i>p</i>-fluorobenzoic acid contains a single fluorine atom attached to an aromatic ring. Its 1D <sup>1</sup>H spectrum acquired in D<sub>2</sub>O
(shown below) shows the expected two aromatic resonances, but the couplings of
these peaks are complicated by the presence of the fluorine atom. The
downfield peak at 7.88 ppm appears as a doublet of doublets with couplings of
5.6 and 8.8 Hz. The 8.8 Hz splitting is likely a three bond coupling to the
neighbouring <sup>1</sup>H, but the 5.6 Hz coupling is too large for a four
bond coupling to the symmetry related <sup>1</sup>H, and must be due to the
<sup>19</sup>F.<br />
</p>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhhZo5n5cC_Iifvm2cPXR9YUg5d61Sab1JYjtRJX0MM8EijzGdZuY95TaGEWjZ9m76Cyl5_vvX8zElzmMdTXdeeGe84OiQV71sxjeRaBlKjozikqkfP-FpQoWT661P4N6nodADknuXPW_X1zGwaiCiiLKRxIyyD_Ktm6ciY7Ycsnnanigrz1kPqHA1qzCpK/s761/fluoroBenzoicAcid1H.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="638" data-original-width="761" height="335" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhhZo5n5cC_Iifvm2cPXR9YUg5d61Sab1JYjtRJX0MM8EijzGdZuY95TaGEWjZ9m76Cyl5_vvX8zElzmMdTXdeeGe84OiQV71sxjeRaBlKjozikqkfP-FpQoWT661P4N6nodADknuXPW_X1zGwaiCiiLKRxIyyD_Ktm6ciY7Ycsnnanigrz1kPqHA1qzCpK/w400-h335/fluoroBenzoicAcid1H.png" width="400" /></a>
</div>
<p>
The upfield resonance at 7.16 ppm appears as a triplet with a coupling of 8.9
Hz. This matches the larger coupling on the downfield resonance, consistent
with <sup>3</sup><i>J</i><sub>HH</sub> coupling to the neighbouring aromatic <sup>1</sup>H. The
second coupling of 8.9 Hz, again, is unlikely to be a long range coupling to
the symmetry related <sup>1</sup>H and must be a <sup>3</sup><i>J</i><sub>HF</sub> coupling from the fluorine.
Since the upfield peak shows a larger <sup>19</sup>F coupling than the other, the 7.16 ppm peak is likely due to the hydrogens ortho to the <sup>19</sup>F and the 7.88 ppm peak to the meta hydrogens.
</p>
<p>
Turning to the 1D <sup>13</sup>C spectrum of <i>p</i>-fluorobenzoic acid
(shown below) we see six peaks, when there are only five magnetically distinct
carbons in the molecule. The two largest peaks can be assigned to the
symmetrical protonated carbons. The expansions show that both of these peaks
are in fact doublets. The more upfield of these two peaks at 117.7 ppm has the
larger coupling (21.9 Hz), suggesting this peak is from the carbons ortho to
the fluorine, while the peak at 134.0 ppm with the 9.1 Hz coupling is from the
carbons meta to the fluorine.
</p>
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</div>
<p>
The remaining peaks must all be due to quaternary carbons. The small peak at 135.1 ppm shows a coupling of 2.8
Hz, likely a fluorine coupling, and the small magnitude suggests this carbon is further from the
fluorine than the four protonated carbons. Assigning this peak to the carbon
para to the fluorine, leaves three peaks, all singlets, to assign to two
quaternary carbons. The peak at 177.5 ppm is typical for a carboxylic acid, thus the last two
peaks at 168.2 and 166.2 ppm must be a doublet from the fluorinated carbon,
with a <sup>1</sup><i>J</i><sub>CF</sub> of 248 Hz.
</p>
<p>
Heteronuclei such as <sup>19</sup>F and <sup>31</sup>P can produce confusing
spectra if one is not familiar with their effects. The large size of these
couplings, compared to <sup>1</sup>H coupling, needs to be kept in mind.
Unlike any other elements, the NMR active isotopes of fluorine and phosphorous
are 100% abundant and have spin ½, making them easy to detect by NMR. After <sup>1</sup>H, <sup>13</sup>C and
<sup>15</sup>N, these two nuclei are the ones most likely to be encountered by
organic chemists.
</p>
<p><u>Acknowledgements</u><br />
Spectra were obtained from the BioMagResBank entries <a href="http://dx.doi.org/10.13018/BMSE000774">bmse000774</a> for trimethyl phosphate and <a href="http://dx.doi.org/10.13018/BMSE000739">bmse00739</a> for <i>p</i>-fluorobenzoic acid.</p>
Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-19232210486031438352023-09-06T14:12:00.000-07:002023-09-06T14:12:44.305-07:0013C satellites<p>
Recently I was asked about the small peaks appearing symmetrically around a
large peak in a 1D <sup>1</sup>H spectrum. These peaks are called
<sup>13</sup>C satellites and arise from heteronuclear coupling to the small
amount of <sup>13</sup>C naturally present.
</p>
<span><a name='more'></a></span>
<p>
The figure below shows a 1D <sup>1</sup>H spectrum of
<a
href="http://sopnmr.blogspot.com/2017/02/assigning-simple-compound-odcb.html"
><i>o</i>-dichlorobenzene</a
>
in acetone-d<sub>6</sub>. The aromatic ODCB signals appear at 7.57 and 7.35
ppm, the residual undeuterated acetone signal at 2.05 ppm, and the chemical
shift reference compound tetramethylsilane (TMS) at 0.00 ppm. I'm not sure
what causes the peak at 3.59 ppm. Note the small peaks either side of the TMS
signal, these are the <sup>13</sup>C satellites. The insets show expansions of
the acetone and TMS peaks, including their <sup>13</sup>C satellite peaks.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a
href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhpGylpQajFlnmY_kur9GVL4MmdNE_XA6M98f5JfCvCwXwCq6i09HGatacGuhOpCzY3f-IWuUPRp4__R4eBwP-tYUcL1E94TJvw5Tx5gtIQ4sHEq94POHg5EhEs_qdBkVDd33rheuf1O-S2RYMOQGNd9TMMCe3QzuqnrcBEu_IOQ08IXogqwDq2ZPErSmwQ/s761/c13satellites.png"
style="margin-left: 1em; margin-right: 1em;"
><img
border="0"
data-original-height="641"
data-original-width="761"
height="338"
src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhpGylpQajFlnmY_kur9GVL4MmdNE_XA6M98f5JfCvCwXwCq6i09HGatacGuhOpCzY3f-IWuUPRp4__R4eBwP-tYUcL1E94TJvw5Tx5gtIQ4sHEq94POHg5EhEs_qdBkVDd33rheuf1O-S2RYMOQGNd9TMMCe3QzuqnrcBEu_IOQ08IXogqwDq2ZPErSmwQ/w400-h338/c13satellites.png"
width="400"
/></a>
</div>
<p>
Typically we think of the peak splitting in NMR spectra as being due
exclusively to nearby hydrogen atoms because this is the most common case, but
coupling from other elements is possible as well. In fact coupling to carbon
is present in all 1D <sup>1</sup>H spectra of organic compounds. However,
since the natural abundance of the NMR active carbon isotope, <sup>13</sup>C,
is only 1.1% the multiplets due to carbon coupling account for just 1.1% of
the total intensity. In the TMS inset above the two small peaks either side of
the large central peak are due to the 1.1% of the TMS methyl groups where the
carbon atom is <sup>13</sup>C. These two peaks form a doublet where the
<sup>1</sup><i>J</i><sub>CH</sub> coupling is 118.1 Hz. The large central peak
is due to the remaining 98.9% of the TMS methyl groups where the carbon atom
is <sup>12</sup>C, which cannot be detected by NMR. Since the <sup>13</sup>C
satellites combined account for 1.1% of the signal, each peak in the doublet
is 0.55% of the intensity of the original signal.
</p>
<p>
The acetone inset shows the peak from the undeuterated acetone. This is a
-CHD<sub>2</sub> peak split into a 1:2:3:2:1 pentet due to <sup>2</sup>H
coupling, like those of methanol and DMSO
<a
href="http://sopnmr.blogspot.com/2023/07/methanol-and-dmso-solvent-peaks.html"
>discussed previously</a
>. The <sup>13</sup>C satellites of this peak can be observed showing a
<sup>1</sup><i>J</i><sub>CH</sub> coupling of 126.0 Hz. Interestingly, these
peaks show the same splitting pattern as the central peak due to the
<sup>2</sup>H coupling, so these multiplets arise from <sup>1</sup><i>J</i
><sub>CH</sub> coupling and <sup>2</sup><i>J</i><sub>DH</sub> coupling.
</p>
<p>
Note that the <sup>1</sup><i>J</i><sub>CH</sub> coupling is different for the
acetone and TMS peaks. <sup>1</sup><i>J</i><sub>CH</sub> couplings cover a
range of 120-250 Hz and it is possible to use the size of <sup>1</sup><i>J</i
><sub>CH</sub> coupling to assign peaks and stereochemistry<a href="#ref1"
><sup>1</sup></a
>. It is also possible to use the intensity of the <sup>13</sup>C satellites
to determine sample concentration<a href="#ref2"><sup>2</sup></a
>.
</p>
<p><u>References</u></p>
<p>
<a name="ref1">1.</a> R.H. Contreras, J.E. Peralta<br /><a
href="https://doi.org/10.1016/S0079-6565(00)00027-3"
>Angular dependence of spin–spin coupling constants</a
><br />Prog. NMR Spec 2000 37(4) 321-425
</p>
<p>
<a name="ref2">2.</a>D.S. Dalisay, T.F. Molinski<br /><a
href="https://doi.org/10.1021/np900009b"
>NMR Quantitation of Natural Products at the Nanomole Scale</a
><br />J. Nat. Prod. 2009 72(4) 739–744
</p>
Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-59508237633413897622023-08-01T12:40:00.000-07:002023-08-01T12:40:31.286-07:00Receiver gain<p>
The receiver gain is one of the parameters that should be optimised before
recording an NMR spectrum. Here I'll explain what the receiver gain is and why
it needs to be optimised.
</p>
<span><a name='more'></a></span>
<p>
When recording an NMR spectrum the coils in the probe detect a continuously
varying voltage. This analog signal needs to be digitised, converted to a list
of numbers, for processing by the computer. This is done by an analog to
digital converter (ADC). The ADC has a limited range so if the analog signal
is too large, part of that signal will be lost and fourier transforming such a
truncated signal will produce artifacts. On the other hand, if the analog
signal is too small then the digitised data will be small as well, and smaller
peaks may not be detectable. To get the best possible spectrum the analog
signal fed to the ADC should be as large as possible without exceeding its
capacity. The receiver gain is a scaling factor applied to the analog signal
before digitisation.
</p>
<p>
In the figure below, a 1D <sup>1</sup>H spectrum was acquired with different
values for the receiver gain. All the remaining parameters were identical and
the spectra are plotted with the same scaling. As the receiver gain is
increased the intensity of the peaks increases. At the highest value, however,
the baseline shows a lot of noise due to the signal being truncated during
digitsation.<br />
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjLSyeDHdlNs-bItpB6FKU7dbOafYKaDk9pXAv5mqqpVzNWHwGjuedaC5Nj2HcJ3lybqgxXr7clQJW2WHkmCwgNqC8KYNd8GwpjIlEEQlPm1_PE1IArrXzXE-EO8Qsowc12mgfCmbM3sEyoI_0ez3_YR2OHVrzZ7CIvALbQuqMIakCGCCeRi9D_559f-iQg/s1610/rgStackplot.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="819" data-original-width="1610" height="204" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjLSyeDHdlNs-bItpB6FKU7dbOafYKaDk9pXAv5mqqpVzNWHwGjuedaC5Nj2HcJ3lybqgxXr7clQJW2WHkmCwgNqC8KYNd8GwpjIlEEQlPm1_PE1IArrXzXE-EO8Qsowc12mgfCmbM3sEyoI_0ez3_YR2OHVrzZ7CIvALbQuqMIakCGCCeRi9D_559f-iQg/w400-h204/rgStackplot.png" width="400" /></a>
</div>
<p>
The standard parameters used at the SSPPS NMR facility automatically set the
receiver gain before starting each experiment. This is done by running test
scans with different gain values until one is found at which the signal is
not truncated. Sometimes, however, the spectrometer still produces a warning
that the receiver was exceeded, like that shown below. In most cases the
spectrum will still be fine and the warning can be ignored. If the spectrum
does show artifacts then it is possible to use <a href="http://sopnmr.blogspot.com/2016/03/processing-linear-prediction.html">linear prediction</a> to replace
the points that were truncated using the information in the logfile reported
by the error message, but if the spectrum is a quick 1D <sup>1</sup>H it is
quicker and easier to run the spectrum again with a reduced receiver gain
(start by halving it) and the automatic receiver gain setting disabled.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiAU6PeMxIp2_RLeOEckjX6OBSYtCgLenfRj40F1E22-Ort4am8ajfcO7k4qaMHboOSZS96aazJXrEgHOKTvrk4cMffGOsJzooq5jHvu9ivFet2MCl7cupWnlmzba9-OK2AlEnACybZ3NZxctkUsOFamt3T9T_iuzyYwS2rgyE1RzIlPCFh93EmS6CDpxyJ/s498/errorDRU.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="219" data-original-width="498" height="141" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiAU6PeMxIp2_RLeOEckjX6OBSYtCgLenfRj40F1E22-Ort4am8ajfcO7k4qaMHboOSZS96aazJXrEgHOKTvrk4cMffGOsJzooq5jHvu9ivFet2MCl7cupWnlmzba9-OK2AlEnACybZ3NZxctkUsOFamt3T9T_iuzyYwS2rgyE1RzIlPCFh93EmS6CDpxyJ/s320/errorDRU.png" width="320" /></a>
</div>
<p>
For the spectra shown in the first figure, warnings were generated for the
three largest receiver gain values, but only the spectrum with the highest
value shows artifacts. For this sample the receiver gain optimisation selected
a value of 228. One might think a slightly higher value would be better, since no artifacts can be seen, but
this is not necessarily the case. The graph below plots signal to noise vs
receiver gain for the spectra in the first figure. As receiver gain is
increased signal to noise rapidly increases until it plateaus. At very high
receiver gains (2050 here) artifacts are produced and signal to noise is
greatly reduced. The selected, optimal value of 228 is where the curve starts
to flatten out and increases are no longer worthwhile because scaling the
signal further just increases the noise along with the signal.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg1Tn6cxUxrtWDHMN_txuaXC1U5swxa1d4x6kgJpRKGt8hEL494SaJL6k6QouAsgRWWq14wZ6yAzAvOAsid7tzltPqXyBonI3hZCkRNzm5lk4drFSJIjxkgKIggoxSY_0fCyQp9Gb7vdrtYgD8HiNPJMIY1WMTB6RwWZwZTDGRSWm2pgPF8xnzr_rAxk2SH/s640/snVrg.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="480" data-original-width="640" height="300" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg1Tn6cxUxrtWDHMN_txuaXC1U5swxa1d4x6kgJpRKGt8hEL494SaJL6k6QouAsgRWWq14wZ6yAzAvOAsid7tzltPqXyBonI3hZCkRNzm5lk4drFSJIjxkgKIggoxSY_0fCyQp9Gb7vdrtYgD8HiNPJMIY1WMTB6RwWZwZTDGRSWm2pgPF8xnzr_rAxk2SH/w400-h300/snVrg.png" width="400" /></a>
</div>
<p>
In most cases the automated receiver gain optimisation selects the best value.
If warnings are obtained they can usually be ignored. Only if the spectra look
bad should something different be tried.
</p>
Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-32608953194399307252023-07-05T12:55:00.002-07:002023-08-29T12:02:21.598-07:00Methanol and DMSO solvent peaks<p>
Two common deuterated NMR solvents, methanol and dimethyl sulfoxide, produce
very similar, characteristic peaks with an unusual splitting pattern. What is
the cause of these peaks?
</p>
<a name='more'></a>
<p>
The figure below shows the characteristic peaks of methanol-d<sub>4</sub> and
dimethyl sulfoxide-d<sub>6</sub> observed in <sup>1</sup>H NMR spectra. Both
peaks show five lines with intensities in the ratio 1:2:3:2:1. Both peaks are
from methyl groups, with no other nearby protons, so what causes these
characteristic peaks?
</p>
<div class="separator" style="clear: both; text-align: center;">
<a
href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEifPNCCeYowoDOE9fAjJcu10GlLnQA7iFlvISCW7iEyEpoF3cqz7ThQ9FZiWb3febglDXO_kQHTD-US2bCb7M5fvlmRXgvK3YeGqRD-bsVeDsuh9dwydELg9jspK_mrD9gQW7nkWp0NvRtyoB111KPiPSDHIoYRiRR7XqEIh7YD-tBScw0Gy1m2SMMJs0Oq/s1536/meod+dmso.png"
style="margin-left: 1em; margin-right: 1em;"
><img
border="0"
data-original-height="637"
data-original-width="1536"
height="166"
src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEifPNCCeYowoDOE9fAjJcu10GlLnQA7iFlvISCW7iEyEpoF3cqz7ThQ9FZiWb3febglDXO_kQHTD-US2bCb7M5fvlmRXgvK3YeGqRD-bsVeDsuh9dwydELg9jspK_mrD9gQW7nkWp0NvRtyoB111KPiPSDHIoYRiRR7XqEIh7YD-tBScw0Gy1m2SMMJs0Oq/w400-h166/meod+dmso.png"
width="400"
/></a>
</div>
<p>
The first thing to remember is that these signals are observed in a
<sup>1</sup>H spectrum so must be due to <sup>1</sup>H. Secondly, deuterated
solvents are never completely deuterated. A small percentage (0.1 or 0.2%) of
the hydrogens are <sup>1</sup>H instead of <sup>2</sup>H. In methanol and DMSO
this means that 0.1 or 0.2% of the methyl groups contain one <sup>1</sup>H
atom and two <sup>2</sup>H atoms, ie -CHD<sub>2</sub>. There is an even tinier amount
(0.1x0.1=0.01% or 0.2x0.2=0.04%) of methyl groups where two of the hydrogens
are <sup>1</sup>H and one is <sup>2</sup>H, but this is so small that it is
not detectable. The pentets then are due to -CHD<sub>2</sub> groups, but what
causes the unusual splitting pattern?<br />
</p>
<p>
The splitting of the peaks is due to <sup>2</sup>H coupling. Unlike
<sup>1</sup>H, which is a spin ½ nucleus, <sup>2</sup>H is a spin 1 nucleus.
Coupling to a spin 1 nucleus produces three states, rather than the two
produced by a spin ½ nucleus. The <i>n+1</i> rule for determining the
splitting of a resonance is a specific case for spin ½ nuclei. The more
general rule is <i>2nI+1</i>, where <i>I</i> is the spin state. With the
general rule we can calculate that coupling to two spin 1 nuclei, like the two
deteurons on -CHD<sub>2</sub>, will produce a pentet, as observed.
</p>
<p>
The relative intensities of the lines in a multiplet are typically explained
by
<a href="https://en.wikipedia.org/wiki/Pascal%27s_triangle"
>Pascal's triangle</a
>, but Pascal's triangle does not produce the 1:2:3:2:1 ratio observed for the
methanol-d<sub>4</sub> and dimethyl sulfoxide-d<sub>6</sub>
peaks. Using Pascal's triangle to predict multiplet intensity patterns assumes
that the splitting is due to a spin ½ nucleus. In the case of a spin 1 nucleus
each state must be split into three instead of two. The figure below shows how
two levels of splitting into three produces the intensity ratio 1:2:3:2:1.
</p>
<p></p>
<div class="separator" style="clear: both; text-align: center;">
<a
href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhcm5cgdW3SHWQiC1GzC3tmMbffMM3Feyl3mJtHyIA4okCorJ8VvLsx4AEaQgVzS9BqNvkPmpuDMCHT150UnQ8Ioo-u82S6hGK8iV3AqvSJOmLamw66b4-eIf7gp8EiK51Hggs1yqmWEbfi1Xa9jH4fwrS0G4Z_M4J7jge6COaYvW1w3Bp_S5sRZ663ZfVM/s160/splittingPattern.png"
style="margin-left: 1em; margin-right: 1em;"
><img
border="0"
data-original-height="121"
data-original-width="160"
height="121"
src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhcm5cgdW3SHWQiC1GzC3tmMbffMM3Feyl3mJtHyIA4okCorJ8VvLsx4AEaQgVzS9BqNvkPmpuDMCHT150UnQ8Ioo-u82S6hGK8iV3AqvSJOmLamw66b4-eIf7gp8EiK51Hggs1yqmWEbfi1Xa9jH4fwrS0G4Z_M4J7jge6COaYvW1w3Bp_S5sRZ663ZfVM/s1600/splittingPattern.png"
width="160"
/></a>
</div>
<p>
Thus, the characteristic pentets of methanol-d<sub>4</sub> and dimethyl
sulfoxide-d<sub>6</sub> are due to residual, partially deuterated solvent.
<sup>2</sup>H coupling creates a characteristic splitting pattern that cannot
be produced by spin ½ nuclei.
</p>
Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com2tag:blogger.com,1999:blog-2408381375133114186.post-33483272024203665062023-06-08T15:41:00.002-07:002023-06-08T15:41:18.680-07:00Determining stoichiometry from NMR titrations<p>
NMR titrations are often used to demonstrate binding, locate active sites, and
measure affinities. By measuring changes in chemical shifts or linewidths as
the concentration of one of the binding partners is changed, quantitative
data, such as dissociation constants and dissociation rates, may be obtained.
In most cases the systems studied involve 1:1 binding. Systems with multiple
binding sites are more difficult to analyse but, with some simplifying
assumptions, quantitative information, such as stoichiometry, may still be
obtained.
</p>
<span><a name='more'></a></span>
<p>
For a 1:1 interaction between a macromolecule, M, and a ligand,
L, the equilibrium can be written as
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjCUt65UkD3xGjEwruZvRdXcRQNv0cv0qDk0WI-XHywri173xp48-XGpiVMMQTCvHInP9hbpMq5s96_DJBFKmZMBCE__RG6CS4CQJncPyFY_ZT5ZGgF_RnQNSlrxblNGPyH6DQ-x2JC9KO-lgXXbt3UOwPSLv2fc92nDYfGTrVy52vGGqPQQRA-tjpWhw/s242/equilibrium1.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="37" data-original-width="242" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjCUt65UkD3xGjEwruZvRdXcRQNv0cv0qDk0WI-XHywri173xp48-XGpiVMMQTCvHInP9hbpMq5s96_DJBFKmZMBCE__RG6CS4CQJncPyFY_ZT5ZGgF_RnQNSlrxblNGPyH6DQ-x2JC9KO-lgXXbt3UOwPSLv2fc92nDYfGTrVy52vGGqPQQRA-tjpWhw/s16000/equilibrium1.png" /></a>
</div>
<p>
with the dissociation constant, <i>K<sub>D</sub></i>, the concentration of products over reactants for the dissociation reaction.
In the case of two ligands binding to a single macromolecule the two sites may
have different properties and the equilibrium now involves four different
dissociation constants.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhiq59XjQxbry8ollovJojiN8j2nfyFSAxhUfNl42xm-MiO2SsFKXylS4gOkJEOk3EJedLKEQFLDxrmtBcuO5KktCkHhLFOxdAG33zKrz9i-BWB5qPu58GhOCR5fGjl_zn833EK-SHmmlSrT-IFlUqH-Qlp-dAbgUCvQQxpo689kKEPFVNkj3cppGyGwQ/s301/equilibrium2.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="183" data-original-width="301" height="183" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhiq59XjQxbry8ollovJojiN8j2nfyFSAxhUfNl42xm-MiO2SsFKXylS4gOkJEOk3EJedLKEQFLDxrmtBcuO5KktCkHhLFOxdAG33zKrz9i-BWB5qPu58GhOCR5fGjl_zn833EK-SHmmlSrT-IFlUqH-Qlp-dAbgUCvQQxpo689kKEPFVNkj3cppGyGwQ/s1600/equilibrium2.png" width="301" /></a>
</div>
<p>
Expressing the binding as a series of steps in which one ligand binds at a
time creates a dissociation constant for each step. If we wanted to describe a
system with even higher stoichiometry the situation would be more complex
still. To handle these situations some simplifying assumptions are required. Binding
of an indeterminate number of ligands to a single macromolecule could be
written as
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgK4TzooICRH3R5C36ghyg7iKBIR0XAXVtYRatfARwTx9rDw6ryLF4d1-LLp1r5Zbdn2SZShz-e3sHrpfqGbyLhZqvrFXsLzwZKJdG4iPa8tQdPaIXqYwLv7PEmInv_u0aLyc8DSavQE5z8unRQSXosHSppILK3mH3gIAti8FRpVQm0ChVLbOAQeevWqg/s248/equilibriumN.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="40" data-original-width="248" height="40" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgK4TzooICRH3R5C36ghyg7iKBIR0XAXVtYRatfARwTx9rDw6ryLF4d1-LLp1r5Zbdn2SZShz-e3sHrpfqGbyLhZqvrFXsLzwZKJdG4iPa8tQdPaIXqYwLv7PEmInv_u0aLyc8DSavQE5z8unRQSXosHSppILK3mH3gIAti8FRpVQm0ChVLbOAQeevWqg/s1600/equilibriumN.png" width="248" /></a>
</div>
<p>
In this formulation, all the ligand molecules simultaneously bind to, or
dissociate from, the macromolecule, a highly unlikely event. Furthermore, this simplified
formulation assumes; (1) that the affinity of each site is identical; (2) that
the binding of one ligand does not affect the binding of another, i.e. the
site and order of binding is unimportant; and (3) that all the individual
events can be encompassed by a single, general, dissociation constant. While
these are rather broad assumptions, they do provide a framework to start to
understand and analyse the system. Under the simplified formulation the bound population of the ligand,
<i>P<sub>b</sub></i>, is given by<sup><a href="#ref1">1</a></sup>
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjXS5N3pGEXNjEmenk7YAmtADBaNtX8Qu4Hd9hocxo8s9fYDkskCmBs7ztcoZpn3FqONRwPCyMhab1iCIeeRQTKtndj2Rc3zKiP3_pdW_sFrUswuGtEuPj419YYuIzyP77Ihp0KGrdjyjbFF8RM8KCHNzHNPuEh862pODYuyo1aG-INygcqzJsVRgo-AQ/s392/equationsN.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="39" data-original-width="392" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjXS5N3pGEXNjEmenk7YAmtADBaNtX8Qu4Hd9hocxo8s9fYDkskCmBs7ztcoZpn3FqONRwPCyMhab1iCIeeRQTKtndj2Rc3zKiP3_pdW_sFrUswuGtEuPj419YYuIzyP77Ihp0KGrdjyjbFF8RM8KCHNzHNPuEh862pODYuyo1aG-INygcqzJsVRgo-AQ/s16000/equationsN.png" /></a>
</div>
<p>
These equations give us a way to relate <i>K<sub>D</sub></i> to
<i>P<sub>b</sub></i>. For a system in fast exchange the chemical shift is the average of the free and bound chemical shifts weighted by <i>P<sub>b</sub></i>. By
recording the chemical shift at different known ligand or macromolecule concentrations, <i>K<sub>D</sub></i> can be determined.</p>
<p>The equations also allow us to simulate the behaviour of peaks in systems with a variety of affinities and stoichiometries. The figure below shows the impact of <i>K<sub>D</sub></i> and <i>n</i> on chemical shift. The graph plots the change in chemical shift in Hz, Δν, against the ratio of ligand to macromolecule concentrations. Titrating a macromolecule with a ligand moves to the right along the horizontal axis with each addition of ligand. The vertical axis is the difference between the observed chemical shift and the chemical shift of the free ligand, so when [L]:[M] is near zero the curve approaches the bound chemical shift, and when [L]:[M] is large the observed chemical shift approaches the free chemical shift. The curves were calculated using a macromolecule concentration of 100 μM and a difference between free and bound chemical shifts of 50 Hz.
</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhe2sApbTZWUnaAwwTjQCdqrPTZ1_MBuCpl1D-4TwtxxRwgbxA0UJURPEBgKcqWOIxar0Abr2vIgY88RFLGERRZt1z4qRNQsL9KR7S-XF-gMZuAGMlwXV7AEDz0Av32TwwA3gLg148eAChBN6tUirEGYyd_8zuUioEDg9P6lZmAFa_oa3rSBxTDLveFVg/s569/stoichiometrySimulations.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="314" data-original-width="569" height="221" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhe2sApbTZWUnaAwwTjQCdqrPTZ1_MBuCpl1D-4TwtxxRwgbxA0UJURPEBgKcqWOIxar0Abr2vIgY88RFLGERRZt1z4qRNQsL9KR7S-XF-gMZuAGMlwXV7AEDz0Av32TwwA3gLg148eAChBN6tUirEGYyd_8zuUioEDg9P6lZmAFa_oa3rSBxTDLveFVg/w400-h221/stoichiometrySimulations.png" width="400" /></a>
</div>
<p>
Looking first at the solid lines that were all calculated using <i>K<sub>D</sub></i>=10<sup>-8</sup> M, we see that the observed chemical shift remains at the bound chemical shift until [L]:[M] reaches the stoichiometry of the system. In these tight binding systems the observed chemical shift does not change until all the binding sites are occupied because nearly all the ligand is in the bound state. Once there is more ligand than binding sites, then the chemical shift starts to move towards the free chemical shift. The higher the stoichiometry of the system, the shallower the curve and the slower the movement.</p><p>The dashed lines were calculated using increased <i>K<sub>D </sub></i>values, 10<sup>-6</sup> M for n=1 (blue), 10<sup>-5</sup> M for n=2 (red), and 10<sup>-4</sup> M for n=5 (green). As <i>K<sub>D</sub></i> increases (blue to red to green), the observed chemical shift moves away from the bound chemical shift, even at sub-stoichiometric levels. This is because the binding is not tight enough to keep all the ligand in the bound state.</p><p>Fitting these curves to experimental data can provide the stoichiometry and affinity of a system, however, it is important to sample as wide a range of [L]:[M] as possible. At high values of [L]:[M] there is little difference in the simulated curves. Only when data is available at the stoichiometric point, and lower [L]:[M] values, can the data be fitted properly.<br /></p>
<p></p>
<p><u>References</u>
</p><p><a name="ref1">1.</a> Fielding L.<br /><a href="https://doi.org/10.1016/j.pnmrs.2007.04.001">NMR methods for the determination of protein–ligand dissociation constants</a><br />Prog NMR Spec. 2007;51(4):219-242</p><p></p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com2tag:blogger.com,1999:blog-2408381375133114186.post-17110886784834038862023-05-02T11:44:00.000-07:002023-05-02T11:44:19.008-07:00i-HMBC : Identifying two bond correlations<p>
The HMBC experiment provides <sup>1</sup>H-<sup>13</sup>C correlations over multiple bonds and is
essential for assigning small molecules. The main
drawback to the HMBC is that it does not identify the length of the
correlation and in some cases may be ambiguous. A recent publication describes
a method, called i-HMBC, for distinguishing two bond HMBC correlations from
longer ones.
</p>
<span><a name='more'></a></span>
<p>
The i-HMBC is essentially a standard HMBC experiment recorded with vastly
increased resolution in the <sup>1</sup>H dimension. The increased resolution allows
small differences in the chemical shifts of peaks to be measured. Wang et al
report that peaks from two bond correlations are shifted upfield by 0.3-1.6 ppb
relative to three bond or longer correlations.
</p>
<p>
The Facility's standard HMBC parameters acquire 4096 points over 12 ppm at 600
MHz. During processing zero filling is not used for this dimension, so the
processed data contains 2048 points in the <sup>1</sup>H dimension with a digital
resolution of 3.5 Hz/point. For the i-HMBC the number of acquired points was
increased to 32768 and zero filling was applied to give 65536 points in the <sup>1</sup>H dimension and a digital resolution of 0.11 Hz/point. This increased number of points does not greatly increase the experiment time as the relaxation delay can be reduced to keep the time taken for a single scan the same as typically used. It does, however, produce a much larger processed data matrix and this can be slow to work with.<br /></p>
<p>
With this greatly increased resolution, distortions in the peak shape become
much more apparent and make it difficult to measure chemical shifts. Wang et al report that distortions can be reduced by turning the
lock off and on during the pulse sequence and by reducing the lock power. I
modified the pulse sequence to turn the lock off and on at the appropriate
times and found this improved the line shape. I haven't tried reducing the
lock power yet.
</p>
<p>
The test sample was a 100 mg/ml solution of cholesteryl acetate in
chloroform-d. The structure and the atom numbering is shown below.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiMCeGxZXWuTJDDmS2vkTss_1JnPP2D0DgnJUOiSZQjNa1ZkRu_7xLs5yp8ZiIeXKgCSZuhI80Qw57LdPVcAShWzW61vHAw6fd-1Z8jOryAi-6_ixkCfqQUDdFdmLo6JlNrbyEyWn1bWO1o2g-xYC1nxOxGf_AFmEXCGXI_IuO5y3zXmWhI5QuOBMQgpQ/s683/cholAc.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="438" data-original-width="683" height="256" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiMCeGxZXWuTJDDmS2vkTss_1JnPP2D0DgnJUOiSZQjNa1ZkRu_7xLs5yp8ZiIeXKgCSZuhI80Qw57LdPVcAShWzW61vHAw6fd-1Z8jOryAi-6_ixkCfqQUDdFdmLo6JlNrbyEyWn1bWO1o2g-xYC1nxOxGf_AFmEXCGXI_IuO5y3zXmWhI5QuOBMQgpQ/w400-h256/cholAc.png" width="400" /></a>
</div>
<p>
The left panels in the figures below show expansions of selected peaks from the 2D i-HMBC. The vertical line indicates the chemical shift of the <sup>1</sup>H
involved in these peaks. Close inspection of the peaks shows that though most
line up well, some are shifted upfield (to the right). For H18, the C13 peak
is offset. For H21 the C20 peak is shifted. The shifted peaks correspond to
the two bond correlations. Extracting 1D slices through these peaks allows the
shifts to perhaps be seen more clearly, and to be measured. In both cases the two-bond correlations are shifted upfield by about 0.5ppb.
</p>
<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi_7YpJH15acF-i7kp3Ipc2dE9VKd5U5hdI-zx-JOIC_BNfHpakRWNiSiNVyjRQYzpOj8uLjVzUY-vLbwv0CMC3W_mCTivNqh739hYDG6VdXy6PAH1cYif8kNCjY0tACFlBPOwqy90yOvigCaGB4ddljgj1d6G05b38fOM15V2Rcf39_RRXlhWibkTAYw/s1200/iHMBC-H18.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="643" data-original-width="1200" height="214" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi_7YpJH15acF-i7kp3Ipc2dE9VKd5U5hdI-zx-JOIC_BNfHpakRWNiSiNVyjRQYzpOj8uLjVzUY-vLbwv0CMC3W_mCTivNqh739hYDG6VdXy6PAH1cYif8kNCjY0tACFlBPOwqy90yOvigCaGB4ddljgj1d6G05b38fOM15V2Rcf39_RRXlhWibkTAYw/w400-h214/iHMBC-H18.png" width="400" /></a>
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiA4mkb7SLB-enh4Wn2VgqqfX8zImcb0xkguYr0-JnWB2xJ1dpkWDlmxkSN54Pb451iBKoAG7SBMU1HGvnUuyWXbi1_5tMxg4TXoWhVQYF2ULld2SyOAFb-tEytwpXi3_X1PUYDaURfjbpeFSZqTLCx-tCGnXinY6LFnGonqCO_vG73Pr9-VK1z-b_LMw/s1186/iHMBC-H21.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="643" data-original-width="1186" height="216" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiA4mkb7SLB-enh4Wn2VgqqfX8zImcb0xkguYr0-JnWB2xJ1dpkWDlmxkSN54Pb451iBKoAG7SBMU1HGvnUuyWXbi1_5tMxg4TXoWhVQYF2ULld2SyOAFb-tEytwpXi3_X1PUYDaURfjbpeFSZqTLCx-tCGnXinY6LFnGonqCO_vG73Pr9-VK1z-b_LMw/w400-h216/iHMBC-H21.png" width="400" /></a>
</div>
<p>
These two examples were the clearest in the i-HMBC I recorded. Other sets of
correlations involved more couplings and more complex peak shapes making
measuring the chemical shift differences difficult. Perhaps reducing the lock
power during acquisition would have helped. Happily, the i-HMBC gives better
and simpler lineshapes in proton deficient systems, which is perhaps where it
is most needed. Standard parameters for the i-HMBC are available at the Facility.<br /></p>
<p><u>References</u></p>
<p>
<a href="dx.doi.org/10.1038/s41467-023-37289-z">Unequivocal identification of two-bond heteronuclear correlations in
natural products at nanomole scale by i-HMBC</a><br />Yunyi Wang, Aili Fan, Ryan D Cohen, Guilherme Dal Poggetto, Zheng
Huang, Haifeng Yang, Gary E Martin, Edward C Sherer, Mikhail Reibarkh, Xiao
Wang<br />Nat Commun 2023 Apr 3;14(1):1842.
</p>
Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com1tag:blogger.com,1999:blog-2408381375133114186.post-49302450128011670712023-04-03T12:47:00.000-07:002023-04-03T12:47:54.172-07:00Cleaning a cryoprobe<p>
One of the Skaggs NMRs is normally fitted with a 1.7mm cryoprobe. This piece
of equipment is essential for our natural products and metabolomics users who
have limited amounts of their samples. The capillary sample tubes for the
1.7mm cryoprobe use special shuttles that do not grip the tube. If the probe
gets dirty then the tube can fail to enter the top of the probe and instead
gets pushed up out of the shuttle. This becomes apparent when the sample fails
to produce a lock signal. Repeatedly ejecting and inserting the sample tube
can sometimes get the tube to drop into place by luck, but this is not really
a viable solution. If the 1.7mm tubes are not entering the probe reliably then
the probe needs to be cleaned.
</p>
<a name='more'></a>
<p>
The Bruker cryoprobe user manual provides the following instructions on
cleaning cryoprobes.
</p>
<span style="font-family: arial; font-size: x-small;">
<p>
<i><b>Cleaning the sample cavity</b></i>
</p>
<p>
The CryoProbe sample cavity is extremely fragile. Even a tiny scratch inside
can spoil the CryoProbe performance and entail a major repair action.
Preventive cleaning is not recommended - clean only in case or problems.
</p>
<b
>CAUTION: Do not put any objects or cleaning devices into the sample cavity!
in particular, soft cotton buds must not be introduced under any
circumstances - the CryoProbe cavity would almost certainly be damaged!</b
>
<p>
If dirt or liquid must be removed from the sample cavity, follow the
procedure given in Table 6.4 below.
</p>
<p>Table 6.4. Clean the CryoProbe sample cavity</p>
<table
border="1"
cellpadding="3"
cellspacing="0"
style="font-family: arial; font-size: x-small;"
>
<thead>
<tr style="text-align: left;">
<th style="width: 7%;">step</th>
<th>action</th>
</tr>
</thead>
<tbody>
<tr>
<td>k.1</td>
<td>
<b>Remove</b> the cryoprobe from the magnet and observe all handling
precautions.
</td>
</tr>
<tr>
<td>k.2</td>
<td>
Put the Cryoprobe <b>upside down</b> onto the edge of a level surface,
e.g. a table, such that it cannot fall down. Its tube must point down
but without touching anything.
</td>
</tr>
<tr>
<td>k.3</td>
<td>Protect your eyes with <b>goggles</b>.</td>
</tr>
<tr>
<td>k.4</td>
<td>
Connect the <b>VT gas</b> to its regular input at the Cryoprobe bottom
and select a flow rate ≥ <b>1000 l/h</b> in edte.
</td>
</tr>
<tr>
<td>k.5</td>
<td>
If some debris or liquid is trapped inside the sample cavity,
<b>flush</b> it out with jets of (1st) <b>water</b> and (2nd)
<b>alcohol</b>. Use a syringe or a wash-bottle which you direct from
below in to the sample cavity. <b>CAUTION</b>: Do not immerse in
alcohol for an extended period of time. Do not use solvents other than
those listed above! Take extreme care not to touch the inside of the
cavity. Do not flush anything but VT gas through the VT gas channel
inside the CryoProbe. Do not reverse the direction of VT gas flow.
</td>
</tr>
<tr>
<td>k.6</td>
<td>Wait until the VT gas stream has <b>dried</b> the entire cavity</td>
</tr>
<tr>
<td>k.7</td>
<td>
Set the <b>VT gas flow</b> rate back to its previous value and detach
the VT gas hose from the CryoProbe.
</td>
</tr>
</tbody>
</table>
<p>If this procedure does not solve the problem contact BRUKER.</p>
</span>
<p>
The protocol above is useful if a tube has broken in the probe, but in our
case the cryoprobe normally has a buildup of dirt and oil transferred from
fingers when handling the tubes. To remove this buildup a Bruker engineer
recommended the following protocol.</p>
<ol style="text-align: left;">
<li>Warm up the probe and remove it from the magnet.</li>
<li>
Place the probe on a flat level surface with the opening for the sample
tubes facing downwards, e.g. the cryoplatform.
</li>
<li>
Beneath the cryoprobe tube place a 600ml beaker on a stir plate and a lab
jack.
</li>
<li>Add 200ml of methanol and a magnetic stir bar to the beaker.</li>
<li>
Using the lab jack lift the beaker up so that the first 5cm of the cryoprobe
tube is covered with methanol.
</li>
<li>
Using the stir bar agitate the methanol to wash the probe for 15 minutes.
</li>
<li>
Remove the probe from the methanol and place in a stable position where it
can be connected to the VT gas, e.g. cryoprobe case.
</li>
<li>
Connect the VT gas, set the flow rate to 1000 l/h using edte and dry for an
hour.
</li>
<li>Reinstall the probe and cool down.</li>
</ol>
<p></p>
<div class="separator" style="clear: both; text-align: center;">
<a
href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjEnihpMgY1TOFDihvac5p4zsKZWLsqiPiIR7A_bsODLJlVGS-Tag166ka2cNi2r2GeSAOPLnknpkHviWUqZ9t3Oxg3C9mik1rBcmQf4eThWpSmMIkev4_c1cE9nyKC1TK3wgh2vgyfXHqsArhlj8acTFwxwmOBOcu5TE4pmxpqkOJZl6foKXdvVZYQZQ/s4368/cleaningPhotos.jpg"
imageanchor="1"
style="margin-left: 1em; margin-right: 1em;"
><img
border="0"
data-original-height="2880"
data-original-width="4368"
height="264"
src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjEnihpMgY1TOFDihvac5p4zsKZWLsqiPiIR7A_bsODLJlVGS-Tag166ka2cNi2r2GeSAOPLnknpkHviWUqZ9t3Oxg3C9mik1rBcmQf4eThWpSmMIkev4_c1cE9nyKC1TK3wgh2vgyfXHqsArhlj8acTFwxwmOBOcu5TE4pmxpqkOJZl6foKXdvVZYQZQ/w400-h264/cleaningPhotos.jpg"
width="400"
/></a>
</div>
<p></p>
<p>
I have successfully used this protocol several times to restore the ability to
get a lock signal. Ultimately though, the best solution is to prevent the
probe from getting dirty. All sample tubes should be cleaned with Kimwipes
before being placed in the magnet.
</p>
<p><u>Acknowledgments</u></p>
<p>
Thanks to Joshua Schweer from the Siegel lab for providing all the lab
equipment.
</p>
Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-90395759548973496372023-03-03T10:49:00.001-08:002023-03-03T10:51:32.781-08:00NMR assignment exercises<p>Interpreting NMR spectra is a skill that every chemist needs, but it requires practice and experience. A recent paper describes a new interactive website that offers many NMR assignment exercises based on experimental one- and two-dimensional spectra. Working through this website allows a user to practice and improve their NMR assignment skills. In addition, there are several other websites available that provide NMR spectra, along with MS and IR data, for users to try their hand at. This post lists some of the currently available resources.</p>
<a name='more'></a>
<p><a href="https://nmr-challenge.uochb.cas.cz">NMR Challenge</a> is a Czech website described in a recent paper. It provides over 160 problems in a modern interactive interface where users draw a proposed structure. Submitting the structure provides the user with an immediate assessment of its appropriateness. Basic problems use only 1D <sup>1</sup>H, <sup>13</sup>C and <sup>19</sup>F NMR data. Advanced problems introduce 2D spectra, such as <sup>1</sup>H,<sup>13</sup>C-HSQC and <sup>1</sup>H,<sup>13</sup>C-HMBC, and in some cases COSY and <sup>1</sup>H,<sup>15</sup>N-HMBC.</p>
<p><a href="https://nmr.tips">NMR Tips</a> is a German website available in German and English. The 75 problems are presented as PDF slideshows with multiple steps shown for solving the problems. Exercises are broken up into introductory 1D spectra, coupling constants, 2D spectra, and non-categorized.</p>
<p><a href="https://webspectra.chem.ucla.edu/">WebSpectra</a> is one of the oldest NMR assignment problem websites. It was developed at UCLA and provides 74 problems based on 1D <sup>1</sup>H and <sup>13</sup>C spectra. Some problems include <sup>13</sup>C DEPT or COSY spectra, and some include IR data. Expansions of the peaks in the NMR spectra allow the user to examine the splitting patterns. Spectra and the correct structure are provided as separate links.</p>
<p><a href="https://structureworkbook.nd.edu/">Organic Structure Elucidation Workbook</a> offers 64 problems using 1D <sup>1</sup>H and <sup>13</sup>C NMR spectra as well as IR and mass spectrometry data. The user can toggle between the various spectra. Answers are not provided but can be requested by emailing the authors at the University of Notre Dame.</p>
<p><u>References</u><br>
<a href="https://doi.org/10.1021/acs.jchemed.2c01067">NMR-Challenge.com: An Interactive Website with Exercises in Solving Structures from NMR Spectra</a><br />
Ondřej Socha, Zuzana Osifová, and Martin Dračínský<br />
J. Chem. Educ. 2023, 100, 2, 962–968<br />
</p>
<p><u>Acknowledgments</u><br>
This post was suggested by Jim La Clair</p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-33376866205154992332023-02-08T15:45:00.000-08:002023-02-08T15:45:28.848-08:00Assigning stereochemistry with NOEs<p>Relative stereochemistry can often be determined by analysis of nuclear Overhauser effects or NOEs. I have seen many papers where the stereochemistry was defined based on the observation of one particular peak, but this may not be sufficient and some care must be taken when interpreting NOEs. Here I offer a few pointers on how best to interpret the crosspeaks in NOESY and ROESY spectra.</p>
<a name='more'></a>
<p>The nuclear Overhauser effect (NOE) is a through-space interaction over short distances, typically assumed to be less than 5 Å. NOESY and ROESY experiments correlate atoms via NOEs, so a crosspeak in a NOESY or ROESY spectrum indicates that the atoms involved are within 5 Å of each other. This seems simple enough, but there are some complications.</p>
<p>In addition to through-space interactions, NOESY and ROESY spectra often show zero-quantum artifacts for strongly coupled atoms, typically those with a significant <i>J<sub>HH</sub></i> coupling. This normally appears as DQF-COSY-like correlations. If the spectrum is processed in magnitude mode, then these zero-quantum artifacts will look identical to through-space interactions. It is possible to suppress zero-quantum artifacts through the combination of an adiabatic pulse and a weak gradient, but it doesn't eliminate them completely. The figure below shows the structure and atom numbering of 2β-chloro-3α bromocholestane, the H12-H11 crosspeaks of cholesterol acetate in a standard NOESY, and the H12-H11 crosspeaks of 2β-chloro-3α bromocholestane in a NOESY with zero quantum suppression. H12 and H11 in both molecules are in the same chemical environment and should produce the same NOEs. Obviously the NOESY with zero quantum suppression is a lot cleaner.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiTrBKjjIPFUFCUBu2HKIaHuCRb2YtBECfc5PPOEuKGPH1KGA4gkWCy_-HWJ_JyHiHbHXJK-Iz6yE0DdpnD3QuyZ-7BEggMM-c46V-9cojt5aBo5zXR91ODelu68xpNT4VZYQbEpAD9QDDAimbVKyF2MfBxTrdw-IFbY2EBJ8B00oOCzSsJyqM7VZEnww/s2382/noesyZeroQuantumArtifacts.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="644" data-original-width="2382" height="108" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiTrBKjjIPFUFCUBu2HKIaHuCRb2YtBECfc5PPOEuKGPH1KGA4gkWCy_-HWJ_JyHiHbHXJK-Iz6yE0DdpnD3QuyZ-7BEggMM-c46V-9cojt5aBo5zXR91ODelu68xpNT4VZYQbEpAD9QDDAimbVKyF2MfBxTrdw-IFbY2EBJ8B00oOCzSsJyqM7VZEnww/w400-h108/noesyZeroQuantumArtifacts.png" width="400" /></a></div>
<p>I recommend to always use phase sensitive processing, use pulse
sequences with zero-quantum suppression whenever possible, and be
particularly wary of peaks that could be due to atoms separated by two
or three bonds.</p>
<p>Observation of an NOE indicates that the atoms involved are likely closer than 5 Å, but without careful calibration from multiple spectra it is difficult to say exactly how close. If one is looking at a single crosspeak does that mean the atoms are 2 Å apart or 5 Å? For this reason NOEs should be interpreted in pairs whenever possible. For example, the intensity of an NOE between atom A and atom B should be compared to the intensity of an NOE between atom A and atom C. Only if the NOEs differ in intensity can conclusions about the relative positions of the atoms be made. For example, in the expansion below on the left the cholestane shows NOEs from H2 to the adjacent diastereotopic methylene protons H1+ and H1- ("+" signifies the downfield peak and "-" the upfield one). However, the peaks have similar intensities so from these NOEs no conclusions can be drawn about the stereochemistry.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjUom8RRjBXAb14Cuf9MFgwgh53bT8MIdInRybmeVAszxhOH2Qfng2jGCZWQKArKB92javptiAN3W-AhZSq1pwHXkw011vDqQW1LlbwQ0nvxEnZS5gTzcUaT57IsHsjV8lsE9ccsaNCWw16OxtopBiLm4NQ3VBoiLfD9F6EifoDpfJbdnaLKXsimdRKGg/s1633/labelledNoesy.jpg" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="677" data-original-width="1633" height="166" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjUom8RRjBXAb14Cuf9MFgwgh53bT8MIdInRybmeVAszxhOH2Qfng2jGCZWQKArKB92javptiAN3W-AhZSq1pwHXkw011vDqQW1LlbwQ0nvxEnZS5gTzcUaT57IsHsjV8lsE9ccsaNCWw16OxtopBiLm4NQ3VBoiLfD9F6EifoDpfJbdnaLKXsimdRKGg/w400-h166/labelledNoesy.jpg" width="400" /></a></div>
<p>In the expansion on the right H1+ shows a sizeable NOE to H19 whereas the H1- NOE is much smaller. The difference in intensity allows H1+ to be identified as closer to H19 than H1-. Similarly, H12+ shows an NOE to H18 but H12- shows none at this contour level, allowing H12+ to be assigned as closer to H18 than H12-. Other NOEs show H12+ closer to H21 than H12-, while H12- is closer to H9 than H12+.</p>
<p>This information must be verified by comparison with a three dimensional model of the structure. Models can be physical, built from kits, or virtual ones constructed with chemical modelling software. Be careful with virtual structures, though, as not all packages generate reasonable conformations. Finally <b>all</b> of the NOEs must be consistent with the proposed stereochemistry. If some of the observations cannot be explained then the proposal needs to be revised. The figure below shows how the NOEs in the expansions above are consistent with the expected three dimensional structure of 2β-chloro-3α bromocholestane.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjzwSQLEqHFmFOCx50rpyIwg3nPMbx5oAbvdH3loaHhNwc12pn8WYyloF1V1QvoasNJFIcZgksp0fdIz3DNsRTsNB9XF8ZiJ-UyNc20kBPEsjLzaNJw-x4NABQEIUDyOn2AFqmDLB7iu7qt3qHfGza7lGHiAA-0tvYNRROzcEdl9U4I_nTE7ThErUwbTw/s1461/cholestane3D.jpg" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="483" data-original-width="1461" height="133" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjzwSQLEqHFmFOCx50rpyIwg3nPMbx5oAbvdH3loaHhNwc12pn8WYyloF1V1QvoasNJFIcZgksp0fdIz3DNsRTsNB9XF8ZiJ-UyNc20kBPEsjLzaNJw-x4NABQEIUDyOn2AFqmDLB7iu7qt3qHfGza7lGHiAA-0tvYNRROzcEdl9U4I_nTE7ThErUwbTw/w400-h133/cholestane3D.jpg" width="400" /></a></div>
<p><u>References</u></p>
<p>Thrippleton MJ, Keeler J.<br />
<a href="http://dxdoi.org/10.1002/anie.200351947">Elimination of zero-quantum interference in two-dimensional NMR spectra.</a><br />
Angew Chem Int Ed Engl. 2003 Aug 25;42(33):3938-41
</p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-42877550742281874582023-01-03T09:26:00.000-08:002023-01-03T09:26:31.663-08:00GEMSTONE-NOESY<p><a href="https://sopnmr.blogspot.com/2022/12/gemstone-ultra-selective-excitation.html">GEMSTONE</a> is a new highly selective NMR technique. Combining it with traditional homonuclear 2D experiments creates exceptionally selective 1D versions of these experiments, which can be useful when trying to characterize highly overlapped spectra. In this post I compare the GEMSTONE-NOESY experiment with the standard 1D selective NOESY.</p>
<span><a name='more'></a></span>
<p>To compare GEMSTONE-NOESY with a standard 1D selective NOESY I used the halogenated steroid dissolved in chloroform-d that I have used in <a href="https://sopnmr.blogspot.com/2022/04/nus-for-hmbc-and-lr-hsqmbc-experiments.html">many</a> <a href="https://sopnmr.blogspot.com/2022/05/nus-for-cosy-experiments.html">recent</a> <a href="https://sopnmr.blogspot.com/2022/06/nus-for-asap-hsqc-experiments.html">posts</a>. The structure of the compound and the numbering of its carbon atoms is shown below.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjM6Zl9dnGc9w35bH-lLwrubRChs5NPPflbKfTjjfDyMWM9gDLy_8ZWlfBWXLCYb3FAQoAimF6DcIjLgcpCCbu8pkhsYlwIWBkWtToP_I9H-EI4avczJ7LQgbARRsU3oMTdVO1YeXorgLuG8pf6N5-ZRhOLUozdEllpAO2XwaSugtxCtIOvMUEF6k63YA/s577/ClBrCholestane.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="430" data-original-width="577" height="238" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjM6Zl9dnGc9w35bH-lLwrubRChs5NPPflbKfTjjfDyMWM9gDLy_8ZWlfBWXLCYb3FAQoAimF6DcIjLgcpCCbu8pkhsYlwIWBkWtToP_I9H-EI4avczJ7LQgbARRsU3oMTdVO1YeXorgLuG8pf6N5-ZRhOLUozdEllpAO2XwaSugtxCtIOvMUEF6k63YA/w320-h238/ClBrCholestane.png" width="320" /></a></div>
<p>Spectra were collected with selective excitation at 1.803 and 1.841 ppm, which correspond to the H5α and H16α resonances, respectively. An Rsnob shaped pulse with a selective excitation bandwidth of 40 Hz was used. Despite partial overlap in the 1D <sup>1</sup>H spectrum, GEMSTONE can separate these resonances, as seen in <a href="https://sopnmr.blogspot.com/2022/12/gemstone-ultra-selective-excitation.html">the previous post</a>. A NOESY mixing time of 500 ms was used. </p><p>The spectra are shown in the stackplot below. The GEMSTONE-NOESY spectra are the top two traces shown in yellow and purple.
For reference, a standard 1D <sup>1</sup>H spectrum is shown in green in the middle
with several of the resonances labelled. The lower two traces in red and blue are the standard selective NOESY spectra. The large negative peaks in the selective NOESY spectra are the excitation sites.<br /></p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiTUGafJucRQb1U85zIVorqrQZPnKbB-pUEPtmybOLFi-kN-a595NuRdKkJoorgfTykqir4j9SEPv2WZSy7EZkv8bNKRMzFaHsYwgBeVSibeJNzAHIiP3ERo5Yo094h50BjmNwqCxqLcSKmbu4jip3Ep-cpgA-9biU6FhPgARgBMmon0keG75XgJsGd9Q/s850/stackplotLabelled.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="701" data-original-width="850" height="330" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiTUGafJucRQb1U85zIVorqrQZPnKbB-pUEPtmybOLFi-kN-a595NuRdKkJoorgfTykqir4j9SEPv2WZSy7EZkv8bNKRMzFaHsYwgBeVSibeJNzAHIiP3ERo5Yo094h50BjmNwqCxqLcSKmbu4jip3Ep-cpgA-9biU6FhPgARgBMmon0keG75XgJsGd9Q/w400-h330/stackplotLabelled.png" width="400" /></a></div>
<p>One of the first observations is that the selective NOESY spectra both show NOEs to H2α and H3β, whereas only the H5α GEMSTONE-NOESY spectrum does. The H16α spectra should not show NOEs to H2α or H3β. Observation of these signals in the H16α selective NOESY experiment indicates that the selective excitation was not selective enough.</p><p>Signals from protons on the opposite face of the molecule (H1β, H12β and H7β) from the selected protons (H5α and H16α) are not observed in any of the selective NOESY spectra, as expected. A signal from the degenerate H4αβ signals is seen in both selective NOESY spectra, but only in the H5α GEMSTONE-NOESY. Again, this demonstrates that the selective NOESY was not selective enough.</p><p>The H1α and H15α resonances are separated in the GEMSTONE-NOESY spectra, but the H16α selective NOESY shows them both, when it shouldn't. Finally, the H7α and H9α signals appear in the H5α GEMSTONE-NOESY spectrum but not the H16α, as they should. These two peaks are present in both selective NOESY spectra.</p><p>The GEMSTONE-NOESY experiment clearly performs better than the standard selective NOESY experiment. There is no loss in sensitivity associated with the extra selectivity, and setup is no more difficult. There appears to be no reason not to use the GEMSTONE technique when recording 1D selective spectra.</p><p>The GEMSTONE selection can also be applied to ROESY, TOCSY and COSY experiments. The COSY variant may not seem particularly useful at first, but when trying to identify highly overlapped multiplets being able to identify the adjacent protons may prove very useful.<br /></p>
<p>
<u>Acknowledgments</u><br />Thanks again to Prof. Ted Molinski for preparing and providing the sample.
</p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-8330070655994542522022-12-01T10:21:00.002-08:002022-12-16T12:05:57.683-08:00GEMSTONE - Ultra selective excitation<p>Interpretation of NMR spectra is often hampered by the overlap of signals. Particularly for compounds with numerous complex multiplets, assigning individual signals can be tricky. However, a recent improvement in selective excitation techniques, GEMSTONE, offers a method to tease apart the overlapping signals.</p>
<span><a name='more'></a></span>
<p>The GEMSTONE experiment is a 1D pulse sequence that produces a spectrum of a single, targeted multiplet. To test the sequence I used a halogenated steroid with numerous overlapping multiplets. The figure below shows a stackplot with the normal 1D <sup>1</sup>H spectrum at the top, and a series of GEMSTONE spectra arranged below. Compare how the GEMSTONE spectra separate overlapped resonances like those at 1.841 and 1.803 ppm, and the group between 1.40 and 1.25 ppm, with the mess of signals in the standard 1D <sup>1</sup>H spectrum at the top of the figure.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh5FDglqQ0rvcEaAxl0vFrES5pfuHIO7botkjovinK8K_ymBfsE7OvyctC3SMkIxD5cnZ3EDxmwE2Q4ROsCRTukpog6a6veJLYa-w5_wBvHwGMZAl3SP3ITwKz3WFRPqCQOZnGs5Urcxew-oexJd100HVzFA30nUfDy8GB1hjWPKuyzyDQzrLyG1WQtlw/s1012/gemstoneStackplot.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="649" data-original-width="1012" height="256" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh5FDglqQ0rvcEaAxl0vFrES5pfuHIO7botkjovinK8K_ymBfsE7OvyctC3SMkIxD5cnZ3EDxmwE2Q4ROsCRTukpog6a6veJLYa-w5_wBvHwGMZAl3SP3ITwKz3WFRPqCQOZnGs5Urcxew-oexJd100HVzFA30nUfDy8GB1hjWPKuyzyDQzrLyG1WQtlw/w400-h256/gemstoneStackplot.png" width="400" /></a></div>
<p>Peak positions for selective excitation in the GEMSTONE spectra were determined by recording a pure shift PSYCHE spectrum. Each GEMSTONE spectrum takes about the same amount of time as a standard 1D <sup>1</sup>H spectrum, and the selective excitation does not cause a loss in sensitivity. Setup is easy. For the spectra above the only parameter I changed was the position of the peak to excite. Some broad multiplets, such as 2.246 and 1.803 ppm, required a wider excitation range (40 Hz vs 25 Hz) to prevent reduced intensity of the edges of the multiplet.<br /></p>
<p>The figure below compares the pulse sequence of a standard selective experiment with the GEMSTONE sequence. Selective excitation in these sequences is produced by the shaped 180<sup>o</sup> pulse flanked by gradient pulses. The enhanced specificity of the GEMSTONE sequence is achieved by the addition of a pair of adiabatic pulses applied simultaneously with low power gradient pulses. Adiabatic pulses sweep through a range of frequencies exciting different frequencies at different times. In the GEMSTONE sequence the first adiabatic pulse sweeps from low frequency to high while the second goes in the opposite direction.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh6aYMEdY0bix5QoeeMZ_IKfmu8fFn27gQJy2JHF9MAVth14nHSz9Y3T4w2E3qLg5tYhPW8tuLqPqmwJGKgsUynwJdXEmuCvObHJmf3zglPQemKzmZfTASIum7AUhCqfqy08o-yOwIhip1ft9MGvGC4gNVGMHIWJzxpUEJXL-_zic3Qe5taQfzY1gvi0w/s659/gemstonePulseSequence.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="213" data-original-width="659" height="103" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh6aYMEdY0bix5QoeeMZ_IKfmu8fFn27gQJy2JHF9MAVth14nHSz9Y3T4w2E3qLg5tYhPW8tuLqPqmwJGKgsUynwJdXEmuCvObHJmf3zglPQemKzmZfTASIum7AUhCqfqy08o-yOwIhip1ft9MGvGC4gNVGMHIWJzxpUEJXL-_zic3Qe5taQfzY1gvi0w/s320/gemstonePulseSequence.png" width="320" /></a></div>
<p>The net effect of the adiabatic pulse and the simultaneous gradient pulse is a phase difference that depends on both chemical shift and position. Signals on resonance at the center of the spectrum remain in phase, but those further away do not. The phase difference also depends on the position of the molecule in the NMR sample tube. As indicated by the figure below, off resonance peaks from sample near the top of the tube (yellow and purple) acquire a different phase from those due to sample near the bottom (red and blue). However, each scan collects signal from the entire sample column at once so that the off resonance signals, with a mix of phases, are averaged out and do not produce a signal.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh91kvmew8GdDq-U3wFIVrCXnMQVEEfm7c4i8IZuABk7KpsxV-lN-prLSGuPsbnJLyDE2WjWpguIFRyprI4debCVIT2iVXIrY_GgR0nnTO90iTibnvn6Hj3CgTyRTdyoDqhUucG8iHKq7UjTKn0983UlVM4TNbc0WybNzJd7A_Ky6qtpPaq-AKZ9yg9YQ/s1822/tubeStackplot.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="739" data-original-width="1822" height="163" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh91kvmew8GdDq-U3wFIVrCXnMQVEEfm7c4i8IZuABk7KpsxV-lN-prLSGuPsbnJLyDE2WjWpguIFRyprI4debCVIT2iVXIrY_GgR0nnTO90iTibnvn6Hj3CgTyRTdyoDqhUucG8iHKq7UjTKn0983UlVM4TNbc0WybNzJd7A_Ky6qtpPaq-AKZ9yg9YQ/w400-h163/tubeStackplot.png" width="400" /></a></div>
<p>The GEMSTONE sequence provides a quick and easy way to tease apart overlapping resonances. It can also be combined with TOCSY or NOESY experiments to produce 1D selective versions of these 2D experiments, and I will cover these in the next post.</p>
<p><u>References</u><br />
<a href="https://doi.org/10.1002/anie.202011642">Single-Scan Selective Excitation of Individual NMR Signals in Overlapping Multiplets.</a><br />
Kiraly P, Kern N, Plesniak MP, Nilsson M, Procter DJ, Morris GA, Adams RW.<br />
Angew Chem Int Ed Engl. 2021 Jan 11;60(2):666-669. doi: 10.1002/anie.202011642.</p>
<p><u>Acknowledgments</u><br />
Thanks again to Prof. Ted Molinski for preparing and providing the sample.</p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-32467782734808986992022-11-02T10:25:00.000-07:002022-11-03T11:02:04.742-07:00Assigning a 19F spectrum<p>Recently I was asked to help assign a <sup>19</sup>F spectrum. The Skaggs NMRs are not capable of recording <sup>19</sup>F spectra so I have not had much experience interpreting them. The extent of my <sup>19</sup>F knowledge was an awareness of a large chemical shift range and large scalar couplings. Nevertheless, what follows is our rationalisation of the spectrum. Please let me know if you have a better explanation!</p>
<span><a name='more'></a></span>
<p>The figure below shows the full <sup>19</sup>F spectrum recorded on the Department of Chemistry's JEOL 400. The large peaks near -65 ppm are a compound added for chemical shift referencing. The two peaks of interest appear near -107 ppm and in the lower inset an expansion is shown where it can be seen they appear as triplets. Integrations of the two triplets are nearly equal. Both triplets show the same coupling of 6.6 Hz and are separated by 238.3 Hz. The structure of the compound is shown in the upper inset.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEie2Am1K6t3yK1uE8Cm49MY49OHRpwrqzfZ1sUL20firUheTgso8GsDpc8FX-TDSNBo4lBeCo50FRmkHPiwqtVa9q5H77AHEcKOkI31lY3MdEhETCgLyA-S6um2ZPVHWkjcGo1MPYvkoRG5ijVZeVQC0-CVsiMCRHT72WA4HJbtv590H7nXYbAW1UEMag/s713/full&zoom&structure.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="538" data-original-width="713" height="301" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEie2Am1K6t3yK1uE8Cm49MY49OHRpwrqzfZ1sUL20firUheTgso8GsDpc8FX-TDSNBo4lBeCo50FRmkHPiwqtVa9q5H77AHEcKOkI31lY3MdEhETCgLyA-S6um2ZPVHWkjcGo1MPYvkoRG5ijVZeVQC0-CVsiMCRHT72WA4HJbtv590H7nXYbAW1UEMag/w400-h301/full&zoom&structure.png" width="400" /></a></div>
<p>Looking at the structure we first assumed that the fluorine atoms would be in magnetically equivalent environments due to symmetry. The triazopyrimidine substituent is likely perpendicular to the fluorinated ring and, if sufficiently planar, would leave the aromatic ring symmetrical. The two <sup>19</sup>F peaks could then be the result of long range <sup>19</sup>F-<sup>19</sup>F coupling. Coupling to the protons ortho and para to each fluorine might then create the observed triplets. In this explanation the <i><sup>4</sup>J<sub>FF</sub></i> coupling would be 238.3 Hz and the <i><sup>3</sup>J<sub>HF</sub></i> and <i><sup>5</sup>J<sub>HF</sub></i> couplings would both be 6.6 Hz.</p><p></p>
<p>I have collected from the literature some <a href="http://sopnmr.ucsd.edu/coupling.htm#fluorine">typical <sup>19</sup>F coupling values</a> on the Facility website but none of these were helpful in assigning this example. The best source of small molecule NMR data I have found on the web is <a href="https://organicchemistrydata.org/hansreich/">The Reich Collection</a>. On <a href="https://organicchemistrydata.org/hansreich/resources/nmr/?index=nmr_index%2F19F_coupling">this page</a> of The Reich Collection we found reported values for <i><sup>4</sup>J<sub>FF</sub></i> on an aromatic ring of 6.6 Hz, 8.9 Hz for <i><sup>3</sup>J<sub>HF</sub></i> and 0.22 Hz for <i><sup>5</sup>J<sub>HF</sub></i> - none of which supported our first rationalisation.</p>
<p>Assuming that the aromatic ring is not symmetrical and each triplet is due to one fluorine atom, the splitting pattern could be explained as the result of <i><sup>4</sup>J<sub>FF</sub></i> coupling and <i><sup>3</sup>J<sub>HF</sub></i> coupling, both of 6.6 Hz. The fact that the two couplings are coincidentally the same makes the peaks appear as triplets. In this rationalisation, the <i><sup>5</sup>J<sub>HF</sub></i> coupling from the protons para to the fluorines would be too small to be detected. This explanation is more consistent with the reported <i>J</i> couplings found on The Reich Collection. It is also supported by the <sup>13</sup>C spectrum which shows a signal for each carbon atom in the fluorinated aromatic ring, indicating that it is not magnetically symmetric.
</p><p><u>Acknowledgments</u></p>
<p>Thanks to Thibault Alle in the Ballatore group for allowing use of his spectrum.</p><p></p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com6tag:blogger.com,1999:blog-2408381375133114186.post-8030382153541244052022-10-03T10:00:00.002-07:002022-10-04T08:54:21.770-07:00Comparing processing in Mnova and TopSpin<p>Occasionally users have told me that spectra they have collected on the Skaggs NMRs do not look as good when they take them back to their own computers. This could be because they did not process their data with optimised parameters. Or, since most users use Mnova, it may be due to real processing differences between Mnova and TopSpin. Here I examine a few spectra processed with both packages to see if there is a real difference between processing with Mnova and TopSpin.</p>
<span><a name='more'></a></span>
<p>The most obvious difference I have noted between data processed with Mnova and TopSpin is the noise in 1D spectra. The figure below shows an expansion of a <sup>13</sup>C DEPTQ spectrum. The TopSpin processed data is the upper spectrum, and the Mnova processed the lower one.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgX8Nj1Ddapkbd3x7TaWfR6jxrYdOuTok3zJwf8D6yfmr8MS4HMMR7_vgyPqImvOthcTCVBDUDe-0334jD-6rDNIexLpF4Fwy84nylTqWiDqQ1vrFANHpA9Es6qEACjgzvhaiKPrd7oUTv_yIRNpBWKcn3MkZLzFBgvCBBSmgE5ayxcBszesX2BRgrvMw/s1611/comparisonProton.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="1611" data-original-width="1088" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgX8Nj1Ddapkbd3x7TaWfR6jxrYdOuTok3zJwf8D6yfmr8MS4HMMR7_vgyPqImvOthcTCVBDUDe-0334jD-6rDNIexLpF4Fwy84nylTqWiDqQ1vrFANHpA9Es6qEACjgzvhaiKPrd7oUTv_yIRNpBWKcn3MkZLzFBgvCBBSmgE5ayxcBszesX2BRgrvMw/w270-h400/comparisonProton.png" width="270" /></a></div>
<p>There is little difference between the peaks, but the noise does appear different. To my eye, the Mnova noise does not look truly random. It looks as though it was generated to fill a specified range.</p>
<p>More concerning than differences in noise, though, are reports of peaks seen in TopSpin processed data but not in Mnova spectra. The figure below shows an expansion of the aromatic region of a HSQC spectrum. The TopSpin processed data is at the top and the Mnova below. Identical window functions and amounts of linear prediction in the indirect dimension were used in both packages. Contour intervals are the same in both plots. The threshold is roughly the same but could not be matched exactly as the packages scale the data differently. The TopSpin data appears to have slightly better resolution in the <sup>1</sup>H
dimension and slightly better signal-to-noise. </p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgs9shvYDnTIejLh_clGOPUbxmqX5czyFAEzicMSwdOwU27N_ZOG8K2rq8RuaACd32GMSR0abplEf9NSavz08gdibZbh2b3PZ38bcoy8RuAXNFngtbZvgcZ7AO600Gkrg0JofVCnJ7ni733aFdHQR9ahDSo_McNrJzH95Rr8OIIvjju-MoUxMvkD9rrQg/s1449/comparisonHsqc.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="1449" data-original-width="849" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgs9shvYDnTIejLh_clGOPUbxmqX5czyFAEzicMSwdOwU27N_ZOG8K2rq8RuaACd32GMSR0abplEf9NSavz08gdibZbh2b3PZ38bcoy8RuAXNFngtbZvgcZ7AO600Gkrg0JofVCnJ7ni733aFdHQR9ahDSo_McNrJzH95Rr8OIIvjju-MoUxMvkD9rrQg/w234-h400/comparisonHsqc.png" width="234" /></a></div>
<p>When I analysed this data using TopSpin I picked six peaks in the expansion shown above. In addition to the four obvious peaks at 6.23-141.9, 6.21-133.5, 6.07-139.5 and 5.81-129.8, I picked another at 6.06-129.4 and a sixth at 5.93-143.1. The sixth peak is very weak and I used other spectra to help convince me that there was a peak at this location. However, in the Mnova processed spectrum this peak is even less convincing.</p>
<p>While the Mnova data did not look as good as the TopSpin data the difference was not that great. I reprocessed the spectrum with Mnova's default parameters to see if this could cause the disappearance of peaks. The result is shown below. Here the resolution in the <sup>13</sup>C dimension is not as good as the spectra above. The weak sixth peak is not visible, and even the fifth peak at 6.06-129.4 is dubious.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgGdVMbGx6a0tnPNXnUbFuchqm2qFhHxzkJK5B56xmh995FgSaZmRYXyUymYqQ7BEFhY7B2sz7xTlLKq-S2uaeZrWKcVDdLyOXiXFsuwWk6pe1Hmvafmpz13AQqcdDbvV64QB07IXDGlrjL8RxocG_nNnfr8jbtz12KDFBTW8J-yufwHR2N8dCq_CFM5Q/s854/hsqcMnovaDefault.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="704" data-original-width="854" height="264" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgGdVMbGx6a0tnPNXnUbFuchqm2qFhHxzkJK5B56xmh995FgSaZmRYXyUymYqQ7BEFhY7B2sz7xTlLKq-S2uaeZrWKcVDdLyOXiXFsuwWk6pe1Hmvafmpz13AQqcdDbvV64QB07IXDGlrjL8RxocG_nNnfr8jbtz12KDFBTW8J-yufwHR2N8dCq_CFM5Q/s320/hsqcMnovaDefault.png" width="320" /></a></div>
<p>The answer seems to be that Mnova's default processing parameters do not always give the best results. This is probably true of any data processing package. It is worth taking the time to learn how <a href="https://sopnmr.blogspot.com/2016/01/processing-window-functions.html">window functions</a>, <a href="https://sopnmr.blogspot.com/2016/02/processing-zero-filling.html">zero filling</a>, and <a href="https://sopnmr.blogspot.com/2016/03/processing-linear-prediction.html">linear prediction</a> can alter the appearance of your data. You can probably get more information out of your data than you realise.<br /></p>
<p><u>Acknowledgements</u><br />Thanks to Gisela Camacho-Hernandez and Jim La Clair for the use of their data.</p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com2tag:blogger.com,1999:blog-2408381375133114186.post-59508373119136074922022-09-02T16:17:00.007-07:002022-09-06T11:04:02.532-07:00Extracting 1H-1H coupling constants<p>When publishing NMR data <sup>1</sup>H-<sup>1</sup>H couplings are often reported. These are most easily obtained from a 1D <sup>1</sup>H spectrum. Measuring the couplings can be made easier, and more accurate, if the spectrum is processed to enhance resolution.</p>
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<p>The figure below shows two expansions of a doublet of doublets in a 1D <sup>1</sup>H spectrum. The upper red spectrum was processed with the standard parameters, an <a href="https://sopnmr.blogspot.com/2016/01/processing-window-functions.html#em">exponential window</a> with 0.3 Hz line broadening. The lower blue spectrum is the same data reprocessed with a resolution enhancing <a href="https://sopnmr.blogspot.com/2016/01/processing-window-functions.html#gm">gaussian window</a> using gaussian broadening of 0.7 and -3.0 Hz line broadening. The measured couplings in Hz are shown above the peaks. The gaussian window greatly reduces the signal intensity so the lower spectrum is scaled up by a factor of 16.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEggN8pEB7ieBoisHySbrCW06PSUMjcF_7gq3ZhSCCntg7M75vVGSo3oAk02HIqAmcIlTseF1TUuaMKnsGU7LSeUoBAZizbIlj1yiTYci4Uxc4X3bFlpXCjaqegT0tOWSWEX0_dH9JBCYJrqH6JCKJ5kqaYVmOVBwZg-uJWEYt88BRXf2jVaukbRnbKuSw/s850/spectraLabelled.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="701" data-original-width="850" height="330" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEggN8pEB7ieBoisHySbrCW06PSUMjcF_7gq3ZhSCCntg7M75vVGSo3oAk02HIqAmcIlTseF1TUuaMKnsGU7LSeUoBAZizbIlj1yiTYci4Uxc4X3bFlpXCjaqegT0tOWSWEX0_dH9JBCYJrqH6JCKJ5kqaYVmOVBwZg-uJWEYt88BRXf2jVaukbRnbKuSw/w400-h330/spectraLabelled.png" width="400" /></a></div>
<p>The benefits of the resolution enhancing processing are immediately obvious. While the large coupling in the doublet of doublets is easily identified in both spectra, the smaller coupling could be missed in the upper spectrum. More importantly, the values of the measured couplings are different. In the upper spectrum the broader peaks overlap more and shift their maxima closer to each other, resulting in reduced values. While overlap may not be completely removed in the lower, resolution-enhanced spectrum, it is greatly reduced, and the measured couplings will be closer to their true values.</p><p>Resolution enhancement may also allow measurement of couplings in peaks that would normally be labelled as "multiplets". The peak at 3.425 ppm shows no measurable splitting in the upper spectrum, but in the lower spectrum some of the lines are resolved. <br /></p>
<p>When measuring <sup>1</sup>H-<sup>1</sup>H couplings from 1D <sup>1</sup>H spectra I recommend recording a good quality spectrum with high signal-to-noise. The spectrum should then be processed at least twice; firstly, with a sensitivity enhancing window such as the exponential multiplication; and then with one or more resolution enhancing windows to obtain more accurate coupling constants.</p><p>Another approach is lineshape fitting of the experimental spectra. This gives the most accurate values for peak positions and linewidths, but is the most involved and likely only carried out for peaks of particular interest in a spectrum.<br /></p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com1tag:blogger.com,1999:blog-2408381375133114186.post-20660145449613154892022-08-03T15:46:00.002-07:002022-08-04T10:35:12.916-07:00Care and cleaning of NMR tubes<p>Every NMR user ends up with a collection of used NMR tubes. These tubes are often cleaned and used over and over, however, some care needs to be taken when cleaning tubes to prevent them from being damaged. Damaged tubes give poorer quality spectra, and may even break the spectrometer probe. This post provides some guidelines on best practices for cleaning NMR tubes.</p>
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<p>The purpose of an NMR tube is to hold the sample in a perfect cylinder within the magnetic field. Any deviation will result in broadening of the peaks and a loss of resolution. For this reason any cleaning procedure that will affect the <a href="https://sopnmr.blogspot.com/2022/07/nmr-tube-specifications.html#concentricity">concentricity</a> or <a href="https://sopnmr.blogspot.com/2022/07/nmr-tube-specifications.html#camber">camber</a> of the NMR tube needs to avoided.</p>
<p>Several NMR tube manufacturers provide guides to cleaning<sup><a href="#ref1">1</a></sup>, and most users will have their own cleaning protocols that incorporate many of these steps. To some extent the steps taken depend on what was in the tubes and what the cleaned tubes will be used for. In general, however, cleaning requires the following four steps:</p>
<h4>1. Remove old solution</h4>
<p>The old solution should be removed from the tube before the solvent has evaporated. This prevents the solute from sticking to the surface of the tube, making cleaning more difficult. Extra long Pasteur pipettes that will reach to the bottom of an NMR tube can be purchased to help with this<sup><a href="#ref2">2</a></sup>. It is also possible to use a flame to melt a standard glass Pasteur pipette and draw out an extended pipette.</p>
<h4>2. Soak</h4>
<p>If the old solution was not removed before the solvent evaporated it may be necessary to soak off any deposits. If the original solvent does not remove the material, <a href="https://en.wikipedia.org/wiki/Aqua_regia">aqua regia</a> or <a href="https://en.wikipedia.org/wiki/Piranha_solution">piranha solution</a> may be used. Do not use chromic acid to clean NMR tubes. Chromium is paramagnetic and residual ions in your NMR tube will lead to peak broadening. Do not use base to clean tubes as it will etch the glass. Brushes are best avoided as they may scratch the internal surface of the tube.</p>
<h4>3. Rinse</h4>
<p>After removing the old sample, the tube needs to be rinsed. Using a series of solvents of differing polarity will ensure that everything is removed. The last solvent used should be one that evaporates easily. A final rinse with the deuterated solvent that will be used for the next sample will leave a smaller residual solvent signal.</p>
<p>Devices for rinsing 3 mm and larger tubes are commercially available<sup><a href="#ref3">3</a></sup>. These use a vacuum to move solvent through the NMR tube. Several papers describing how to do this with standard lab equipment have appeared recently<sup><a href="#ref4">4</a></sup>. For 1.7 mm NMR tubes, one of the facility's users, Brenda Andrade, used a length of teflon tubing projecting from a vacuum flask to remove solvent from the capillary tubes, like the commercial device from Wilmad<sup><a href="#ref3b">3b</a></sup>.</p>
<h4>4. Dry</h4>
<p>Following rinsing, the tubes need to be dried. Drying under vacuum is the best option. Using an oven may cause the tubes to bend. While the increased camber may not be visually detectable, a distortion of 0.3 mm is large enough to exceed the tolerances of modern NMR probes and may damage the spectrometer.</p>
<p> </p>
<p>These guidelines are written with 5 mm tubes in mind. For 1.7 mm tubes I generally recommend they be treated as disposable. The quality of 1.7 mm tubes is comparable to economy 5 mm tubes, and at less than $5.00 each the cost of 1.7 mm tubes is probably less than the cost of the solvents and time spent cleaning them.</p>
<p><u>References</u></p>
<p><a name="ref1">1.</a> (a) <a href="https://sp-wilmadlabglass.com/wp-content/uploads/2019/12/NMR_Cleaning.pdf">Wilmad - Proper Cleaning Procedures for NMR Tubes</a></p>
<p> (b) <a href="https://newera-spectro.com/nmr-sample-tube-washers-2">New Era - Cleaning NMR sample tubes</a> </p>
<p> </p>
<p><a name="ref2">2.</a> (a) <a href="https://www.nmrtubes.com/accessories/pasteur-pipettes-for-nmr-tubes">Norell - Pasteur Pipettes for NMR Tubes</a></p>
<p> (b) <a href="https://chemglass.com/glass-pasteur-pipets-nmr-tubes">ChemGlass- Glass Pasteur Pipettes, NMR Tubes</a></p>
<p> (c) <a href="https://www.sigmaaldrich.com/US/en/product/aldrich/z255688">Sigma-Aldrich - NMR Pipette</a></p>
<p> </p>
<p><a name="ref3">3.</a> (a) <a href="https://sp-wilmadlabglass.com/product/economy-tube-washers-cleaners-for-nmr-and-epr-sample-tubes">Wilmad - Economy Tube Washers/Cleaners for NMR and EPR Sample Tubes</a></p>
<p> <a name="ref3b">(b)</a> <a href="https://sp-wilmadlabglass.com/product/universal-solvent-jet-tube-cleaner-for-2-5-mm-5-mm">Wilmad - Universal Solvent Jet Tube Cleaner</a></p>
<p> (c) <a href="https://www.nmrtubes.com/accessories/5-position-nmr-tube-cleaner">Norell - 5 Position NMR Tube Cleaner</a></p>
<p> </p>
<p><a name="ref4">4.</a> (a) <a href="https://doi.org/10.1021/acs.oprd.6b00001">Vacuum Desiccator as a Simple, Robust, and Inexpensive NMR Tube Cleaner</a><br />Thanh Binh Nguyen<br />Org. Process Res. Dev. 2016, 20, 2, 319</p>
<p> (b) <a href="https://doi.org/10.1021/acs.jchemed.1c00337">Easy-to-Assemble NMR Tube Cleaner Made from Common Laboratory Equipment</a><br />Sean C. Butler<br />J. Chem. Educ. 2021, 98, 10, 3405–3408</p>
<p></p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-20828865556558227772022-07-05T09:21:00.003-07:002022-08-03T15:45:24.337-07:00NMR tube specifications<p>NMR sample tubes come in a variety of grades. Good quality tubes will not only give you better quality spectra, but are less likely to damage the instrument. In this post the specifications used to distinguish a good NMR tube from a bad one are explained and how these parameters affect your spectra are discussed.</p>
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<p>NMR tube manufacturers produce tubes in disposable, economy and performance grades. The highest grade tubes are the straightest and have the most uniform wall thickness. The specifications used by manufacturers to assess tube quality are outer tube diameter, inner tube diameter, concentricity, and camber. These specifications are usually given in fractions of a millimeter or in microns.</p>
<p>Outer tube diameter is the distance across the NMR tube from the outer surface on one side to the outer surface on the other side. Inner tube diameter is measured in the same way but between the inner surfaces. These measurements are made at many points along the length of the tube and reported as a mean and standard deviation. If the outer tube diameter is too large, then the tube may contact the inner surface of the probe and perhaps damage it. If the outer diameter is too small, then the tube may slip through the spinner and again come into contact with the probe. In modern probes the space between the sample tube and the probe is around 0.3 mm, so there is not a lot of room to accommodate misshapen tubes.</p>
<p><a name="concentricity">Concentricity</a> measures how well the cylinders defined by the inner and outer walls of the tube overlap and is reported as a deviation. A perfect tube will have zero concentricity. Variations in concentricity could lead to some areas of the sample being outside the cylindrical space where the magnetic field is uniform, which will lead to peak broadening.</p>
<p><a name="camber">Camber</a> is a measure of the straightness of a tube and like concentricity is reported as a deviation. Tubes with high camber will not spin well and may come into contact with the probe coils, perhaps damaging them. They are also likely to place the sample outside the volume of the uniform magnetic field.</p>
<p>The table below shows the specifications of some different NMR tubes to give an idea of the range of values. Not all specifications are reported by all suppliers, so some cells are empty</p>
<p>
</p><table border="1" cellpadding="3" cellspacing="0" style="font-size: x-small;">
<thead style="background-color: black; color: white;">
<tr>
<th>Manufacturer</th>
<th>ID</th>
<th>Type</th>
<th>Outer diameter (mm)<br /></th>
<th>Inner diameter (mm)<br /></th>
<th>Concentricity (mm)<br /></th>
<th>Camber (mm)<br /></th>
<th>Recommended<br /> field (MHz)<br /></th>
</tr>
</thead>
<tbody>
<tr><td>Wilmad</td><td>WG-1241-7-5</td><td>5mm Economy</td><td>4.946 ± 0.019</td><td>4.516 ± 0.019</td><td>0.0038</td><td>0.0038</td><td>600</td></tr>
<tr><td>Wilmad</td><td>535-PP-7-5</td><td>5mm Precision</td><td>4.9635 ± 0.0065</td><td>4.2065 ± 0.0065</td><td>0.0013</td><td>0.0006</td><td>600</td></tr>
<tr><td>Norell</td><td>502-7</td><td>5mm Economy</td><td>4.97 ± 0.05</td><td>4.20 ± 0.05</td><td>0.020</td><td>0.070</td><td><br /></td></tr>
<tr><td>Norell</td><td>509-UP-7</td><td>5mm Precision</td><td>4.97 ± 0.006</td><td>4.20 ± 0.012</td><td>0.004</td><td>0.006</td><td>600</td></tr>
<tr><td>New Era</td><td>NE-UL5-7</td><td>5mm Precision</td><td>4.960 ± 0.006</td><td>4.560 ± 0.006</td><td>0.003</td><td>0.003</td><td>500-700</td></tr>
<tr><td>Bruker</td><td>Z112273</td><td>5mm SampleJet</td><td>5.00</td><td>4.62</td><td><br /></td><td>0.06</td><td><br /></td></tr>
<tr><td>Bruker</td><td>Z106462</td><td>1.7mm SampleJet</td><td>1.70</td><td>1.50</td><td><br /></td><td>0.06</td><td><br /></td></tr>
</tbody>
</table>
<p>As expected the economy tubes have much greater variation in outer and inner diameter and have larger concentricity and camber. The Wilmad tubes seem to have the best specs, consistent with their reputation. Many of the specifications for the Bruker SampleJet tubes were not available, but the camber numbers suggest these tubes are comparable to the economy tubes of the other manufacturers.</p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com2tag:blogger.com,1999:blog-2408381375133114186.post-48316628625423250342022-06-06T12:02:00.002-07:002022-12-16T12:06:16.828-08:00NUS for ASAP-HSQC experiments<p><a href="https://sopnmr.blogspot.com/2016/05/non-uniform-sampling.html">Non Uniform Sampling</a> (NUS) speeds the collection of multidimensional NMR spectra by measuring only a fraction of the data and predicting what was omitted. The quality of the reconstructed data depends on many factors and what works well for one experiment many not work well for others. The last two posts examined how NUS affects <a href="https://sopnmr.blogspot.com/2022/05/nus-for-cosy-experiments.html">COSY</a> and <a href="https://sopnmr.blogspot.com/2022/04/nus-for-hmbc-and-lr-hsqmbc-experiments.html">HMBC</a> experiments. This post examines if NUS impacts the ASAP-HSQC experiment more than the traditional HSQC.</p>
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<p><a href="https://sopnmr.blogspot.com/2016/12/asap-acceleration-by-sharing-adjacent.html">ASAP</a> (Acceleration by Sharing of Adjacent Polarisation) is an innovation that reduces NMR data collection time by allowing the time between scans to be dramatically reduced. For any NMR experiment more complicated than a simple 90<sup>o</sup> pulse and record, reducing the time between scans generates many artifacts as the magnetisation does not have time to return to equilibrium and the carefully planned manipulation of the magnetisation is disrupted. ASAP actively returns magnetisation to equilibrium using a short TOCSY mixing period in the relaxation delay to transfer magnetisation from protons attached to spin inactive nuclei, such as <sup>12</sup>C in a <sup>13</sup>C experiment. Thus, ASAP is useful in heteronuclear experiments on unlabelled samples where the majority of the sample does not produce a signal.</p>
<p>As well as reducing the time between scans, the ASAP-HSQC parameters implemented at the SSPPS Facility reduce the time spent recording the signal. This speeds the experiment further, and also reduces the fraction of time spent decoupling. If decoupling is active for too high a fraction of the experimental time then the probe can be damaged. The ASAP-HSQC parameters in use at the Facility do not cause excessive probe heating and appear to be safe. They also reduce the experimental time by a factor of six.</p>
<p>To see if NUS impacts the ASAP-HSQC more than the standard HSQC the halogenated steroid sample from the previous posts was used. Spectra were collected identically except for the amount of NUS sampling and the pulse sequence used. Processing with TopSpin 4.0 used cosine squared apodisation in both dimensions and four-fold linear prediction in the indirect dimension. In the ASAP-HSQC spectra four-fold linear prediction was also used in the directly detected dimension. Spectra were collected at four different levels of NUS and are shown below plotted with the same threshold and contour intervals.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiIkumhLWziV5iE9bvIdi4Dgbmk-FelWU3Jx7V0bAu1ZwNyWluCwSlRwxxi1LjLDaahsGZ-V5pbVxZQTqco0v19yZGoEn683Mji_mvdCAtIRIL0FM-8twUVsPiXkEwHHzHlv4cR3-8yqtVwyIe10tTKWIseli9jkJ_cuPRjQSY83vHO5APibaR6M2j4LQ/s1840/nusAsapHsqc.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="1840" data-original-width="1200" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiIkumhLWziV5iE9bvIdi4Dgbmk-FelWU3Jx7V0bAu1ZwNyWluCwSlRwxxi1LjLDaahsGZ-V5pbVxZQTqco0v19yZGoEn683Mji_mvdCAtIRIL0FM-8twUVsPiXkEwHHzHlv4cR3-8yqtVwyIe10tTKWIseli9jkJ_cuPRjQSY83vHO5APibaR6M2j4LQ/w261-h400/nusAsapHsqc.png" width="261" /></a></div>
<p>Both sets of spectra show a loss of signal intensity as the amount of sampling is reduced, but this is to be expected as the measurement time is reduced. (The 12.5% ASAP-HSQC spectrum was recorded in less than a minute.) The ASAP-HSQC spectra are less intense than the standard HSQC, so the loss of intensity with reduced sampling is more noticeable. Noise does not appear to increase with reduced sampling as dramatically as with the HMBC and COSY experiments.</p>
<p>Examining slices along the <sup>13</sup>C dimension shows that the experiments are remarkably robust. Slices from the HSQC spectra taken at 1.1 ppm in the <sup>1</sup>H dimension are shown below.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjMliu1BT6Bp8o2jtSUj2WJJQlnE5Yp64gSc51REIpVdVb8xiPu0Hxu79dluDMM6eeZ1td0QcaRmG8821HASHoO4Z4WqzfpN9cJRV9WCF29k1iY6gPMjpb1eK6_GVKocaqBDK3z-FElBfi0nSrCkk-J6KoKfFHhs6YtBmI2Y_4B43fQXYx2IBij6Iv-wA/s852/slicesHsqc.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="688" data-original-width="852" height="258" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjMliu1BT6Bp8o2jtSUj2WJJQlnE5Yp64gSc51REIpVdVb8xiPu0Hxu79dluDMM6eeZ1td0QcaRmG8821HASHoO4Z4WqzfpN9cJRV9WCF29k1iY6gPMjpb1eK6_GVKocaqBDK3z-FElBfi0nSrCkk-J6KoKfFHhs6YtBmI2Y_4B43fQXYx2IBij6Iv-wA/s320/slicesHsqc.png" width="320" /></a></div>
<p>The peaks at 15, 24, 40 and 57 ppm are all true signals. It is only when the sampling is reduced to 12.5% that the smaller true signals become indistinguishable from noise and significant artifacts appear (74, 45 and 27 ppm). Matching slices from the ASAP-HSQC spectra are shown below.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgKykaz3zfXiSXMXJPcW-DirmRGR0Lrd4JoDsjT0-KypZFrW81AgmdGAPTuKOToEsjCAMUe3U5Bi1-mbBcjQcJVSRtd9AYuYXGE9WWRfC-c4k_JQfgaPUcq430c5rjKDWmM3lga_bYrLVgr8P2zz67utm50nLo_ky92cLTrNH9GYqGwgpZBLABrPEr6Kg/s852/slicesAsapHsqc.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="688" data-original-width="852" height="258" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgKykaz3zfXiSXMXJPcW-DirmRGR0Lrd4JoDsjT0-KypZFrW81AgmdGAPTuKOToEsjCAMUe3U5Bi1-mbBcjQcJVSRtd9AYuYXGE9WWRfC-c4k_JQfgaPUcq430c5rjKDWmM3lga_bYrLVgr8P2zz67utm50nLo_ky92cLTrNH9GYqGwgpZBLABrPEr6Kg/s320/slicesAsapHsqc.png" width="320" /></a></div>
<p>The ASAP-HSQC slices show more baseline noise and artifacts appear earlier than in the standard HSQC, at 25% sampling, but all the true signals are present. For many users the six fold reduction in acquisition time may be worth accepting the increased artifacts, and most users are now opting for the ASAP-HSQC, but for those that prefer the traditional HSQC those parameters are still available. The default parameters have the ASAP-HSQC using 50% NUS, while the standard HSQC uses 25%. These values can easily be changed in the IconNMR interface.</p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-4501554338869338592022-05-02T12:47:00.004-07:002022-12-16T12:06:38.713-08:00NUS for COSY experiments<p>Non Uniform Sampling (NUS) reduces the time taken to acquire multi-dimensional NMR spectra by predicting a fraction of the normal data instead of measuring it. The most commonly used algorithm for reconstructing the missing data requires the collected data to be properly phased in the indirect dimension. For this reason I have not recommended using NUS with HMBC and gCOSY experiments. However, <a href="https://sopnmr.blogspot.com/2022/04/nus-for-hmbc-and-lr-hsqmbc-experiments.html">last month's post</a> showed that unphaseable HMBC experiments cope with NUS just as well as the phaseable LR-HSQMBC. In this post I compare the unphaseable gCOSY experiment with the phaseable CLIP-COSY to see how they are impacted by NUS</p>
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<p>The sample for the experiments was the same halogenated steroid in chloroform-d that was <a href="https://sopnmr.blogspot.com/2022/04/nus-for-hmbc-and-lr-hsqmbc-experiments.html">used previously</a>. This compound only shows signals between 0.0 and 5.0 ppm, so reduced sweep widths were used. The standard Bruker pulse sequences, <i>cosygpqf</i> and <i>clipcosy</i>, were used. Four spectra were collected for each experiment, the first without NUS, then 50% NUS, 25% and 12.5%. The number of points in the indirect dimension was 256 for the fully sampled experiment. The 100% spectra were recorded in about 60 minutes, while the 12.5% took 8 minutes. All spectra were processed with TopSpin 4.0.8 using the Bruker implementation of the Iterative Soft Thresholding (IST) algorithm.</p>
<p>The figure below shows the eight spectra. For ease of comparison the gCOSY spectra had to be plotted with a contour threshold a factor of 4 lower than the CLIP-COSY spectra. The intervals between the contours are the same in all spectra. Click on the figure for a larger version.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgnyJmVH_hyliDy6BZiMgw11m8-s-_9g9IbuJRm9ldVVFX3d7PoFpqQ3G3pTGAXVFzXh9CY1hBY2b-CX7C0Bi6-Ebb0kf4ZA8nJ_qbZEaTuo5qyUl6V9M5Psd8B-cNxNYCCtFahWt-kzgbayYjrvU6-W5ihLFmGcrhta9MZOW0_NbrgdaxZ9lTHR-B9qg/s2821/gCosyVclipCosy.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="2821" data-original-width="1732" height="640" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgnyJmVH_hyliDy6BZiMgw11m8-s-_9g9IbuJRm9ldVVFX3d7PoFpqQ3G3pTGAXVFzXh9CY1hBY2b-CX7C0Bi6-Ebb0kf4ZA8nJ_qbZEaTuo5qyUl6V9M5Psd8B-cNxNYCCtFahWt-kzgbayYjrvU6-W5ihLFmGcrhta9MZOW0_NbrgdaxZ9lTHR-B9qg/w392-h640/gCosyVclipCosy.png" width="392" /></a></div>
<p>For both experiments the quality decreases as the sampling is reduced; peaks disappear, artifacts become numerous, and <i>t<sub>1</sub></i> noise increases. The majority of peaks are present in both experiments but there are some peaks that are present only in the gCOSY spectra and some only in the CLIP-COSY.</p>
<p>For a more detailed examination of the quality of the spectra, columns were extracted from all experiments at 1.294 ppm. The gCOSY columns are shown below. Only the region between 3.0 and 0.0 ppm is shown as there were no signals downfield of 3.0 ppm. The peak with the shoulder near 1.3 ppm is the diagonal peak, while the peak at 1.97 ppm is an expected three bond correlation. Other expected correlations are a geminal coupling at 1.44 ppm, and vicinal couplings at 0.75, and 1.10 ppm, but these show only weak signals. As the amount of sampling is reduced the 1.97 ppm signal decreases dramatically, some of the weak correlations disappear completely, and artifacts appear. The 50% NUS column, though, is little different from the 100% column, indicating that the gCOSY experiment could be run with 50% NUS without a significant loss of quality.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjOKyQXGcbgZHTgDA6C1TQwCGu_tTkC2A-RYnzTY134fyTmbNV2qFPlpOK3OMARXW-3-XSTgxsC91HxEcnqM4-tX3HonsXS-PdJjp3k8EAnmeXt-FNP45TPXEZ6qcEFKA9L4Sci5Onnvyv70pNJGU8xwhYJyRGQCsccZPk0-cL-SO4gcOdZk7a-rf9zPQ/s853/gCosyColumns.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="688" data-original-width="853" height="258" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjOKyQXGcbgZHTgDA6C1TQwCGu_tTkC2A-RYnzTY134fyTmbNV2qFPlpOK3OMARXW-3-XSTgxsC91HxEcnqM4-tX3HonsXS-PdJjp3k8EAnmeXt-FNP45TPXEZ6qcEFKA9L4Sci5Onnvyv70pNJGU8xwhYJyRGQCsccZPk0-cL-SO4gcOdZk7a-rf9zPQ/s320/gCosyColumns.png" width="320" /></a></div>
<p>Columns extracted from the CLIP-COSY spectra appear in the figure below. These were extracted at the same frequency as the gCOSY columns above and should show the same signals. The most intense peaks correspond to the expected signals; the diagonal (1.29 ppm), a geminal correlation (1.44 ppm), and three vicinal correlations (1.97, 1.10, and 0.75 ppm). The medium intensity signals (1.76 and 0.98 ppm) appear to be five-bond correlations. Like the gCOSY columns, the CLIP-COSY columns show a significant loss of quality as the sampling is reduced below 50%; peak intensities are reduced, some correlations disappear entirely, and artifacts begin to appear.</p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjRfGSkrlRwL7KBAXFxvO52M5a3EtjBqDHa3jPRVKCKpDjbsW6Zn3SxMK-wedObVJACOQqHng6ckATbtfdy5jaBqezfJPQTU6-5IrGVar8QWBduFdBhAVx-_iIga3i-qCmIbEeRyHxtCjD2ihScOoZHEp5nqrPZnidi9pVbdAW4KoXwYPx1akXmhKkE-g/s853/clipCosyColumns.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="688" data-original-width="853" height="258" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjRfGSkrlRwL7KBAXFxvO52M5a3EtjBqDHa3jPRVKCKpDjbsW6Zn3SxMK-wedObVJACOQqHng6ckATbtfdy5jaBqezfJPQTU6-5IrGVar8QWBduFdBhAVx-_iIga3i-qCmIbEeRyHxtCjD2ihScOoZHEp5nqrPZnidi9pVbdAW4KoXwYPx1akXmhKkE-g/s320/clipCosyColumns.png" width="320" /></a></div>
<p>The columns from the two sets of spectra show that both experiments cope with reduced sampling similarly; 50% sampling shows little decrease in quality, but at 25% and 12.5% there is significant signal loss. Like the <a href="https://sopnmr.blogspot.com/2022/04/nus-for-hmbc-and-lr-hsqmbc-experiments.html">previous comparison</a> of the HMBC and LR-HSQMBC experiments, I am surprised that the unphaseable experiment (gCOSY) copes with NUS as well as the phaseable experiment (CLIP-COSY). Perhaps the Bruker reconstruction algorithm has been modified to cope with unphaseable data. With these COSY experiments, though, 50% sampling seems to be the limit. This may be because the homonuclear COSY experiments contain more signals in each column than the heteronuclear HMBC and LR-HSQMBC. Successful NUS reconstruction requires sparse data. In the Facility's standard parameter sets the NUS sampling will be set to 50% for homonuclear experiments and 25% for heteronuclear.<br /></p>
<p><u>Acknowledgments</u><br />Once again, Prof. Ted Molinski is acknowledged for preparing and providing the sample used to record the data presented here.<br /></p><p> </p><p><br /></p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-50362924468964823352022-04-06T13:12:00.001-07:002022-12-16T12:06:52.421-08:00NUS for HMBC and LR-HSQMBC experiments<p>Non Uniform Sampling (NUS) of multidimensional NMR data can greatly reduce the time taken to record a spectrum by recording only a subset of the normal data. A variety of algorithms are available to reconstruct the omitted data based on the data that was recorded. The most commonly used algorithm is Iterative Soft Thresholding (IST). Most implementations of the IST algorithm rely on the peaks in the detected dimension being phased correctly and positive. For most modern experiments this is not a problem, but in the HMBC experiment it is not possible to phase the peaks. For this reason I have not recommended using NUS with HMBCs. The LR-HSQMBC experiment, however, can be phased and I recommend using NUS with it. In this post, spectra recorded with different levels of NUS were recorded to determine how NUS affects HMBC and LR-HSQMBC experiments.</p>
<span><a name='more'></a></span>
<p>The sample used to record the spectra was a halogenated steroid dissolved in chloroform-d. All of its <sup>1</sup>H peaks appear between 0 and 5 ppm, and its <sup>13</sup>C peaks between 0 and 70 ppm, so the spectral widths were reduced. The experiments were optimised to detect long range couplings of 8 Hz. ASAP versions of the pulse sequences were used with a relaxation delay of 30 ms, a DISPI-2 mixing period of 25 ms, and an acquisition time of 513 ms. <sup>1</sup>H decoupling was not used. All experiments were run with 16 scans and nominally 256 <i>t<sub>1</sub></i> increments. The fully sampled (100%) spectra took 50 minutes, while the 12.5% sampled spectra took 6.5 minutes. Spectra were processed identically with Topspin 4.0.8 using the IST algorithm. The figure below shows the spectra. All spectra are plotted with the same contour threshold and level spacing. Click on the figure to see a larger version.</p>
<p></p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjgfB3AwgYUHPUFm3jNhmCGhqe2lbLWU7IPJPXAevIdgNRCNVgQzVFmRD6atZqqaX5Q-oTMlcHTZrT4F-7Wsdb15f8fbdr89XmQt-2QY7vuL2v_XkEj8bWbxEKQSdhw61TyoykcKVziI7nakD4LC0Hh-Jsa7ZbHmif0ecyWfQVaZXPlIwHw2mKqUr49ZA/s2398/2Ds.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="2398" data-original-width="1522" height="640" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjgfB3AwgYUHPUFm3jNhmCGhqe2lbLWU7IPJPXAevIdgNRCNVgQzVFmRD6atZqqaX5Q-oTMlcHTZrT4F-7Wsdb15f8fbdr89XmQt-2QY7vuL2v_XkEj8bWbxEKQSdhw61TyoykcKVziI7nakD4LC0Hh-Jsa7ZbHmif0ecyWfQVaZXPlIwHw2mKqUr49ZA/w406-h640/2Ds.png" width="406" /></a></div>
<p>The spectra clearly show increased noise as the amount of sampling is reduced from 100% to 12.5%. In particular the tallest, sharpest peaks show increasing streaks of truncation artifacts as the sampling is reduced. The HMBC spectra appear to show more noise than the LR-HSQMBC spectra, but the HMBC signals are more intense and the noise is more obvious. Close inspection reveals the two experiments show little difference in the noise as sampling is decreased.<br /></p><p></p><p>To make comparison easier, <sup>13</sup>C traces running through the large signals near 1.1 ppm in the <sup>1</sup>H dimension were extracted and are shown below.<br /></p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiNRufS47_k_95vTSAH0_9Fn5SlMBwtoPjq9ZjghqSMr4Yl67-DHynXhx2xV4O60zI82YpRk0jZM4MIeRfdKkb3-oaPaL6vse2k-5ShrJqig8WkO3_e1CmGBE_M5qqwMxpvpMp2VDArV81GtbaC0AVD42tb4o5FVUihdj2tubKXFGH4tChrtEzmpBEKag/s853/columnsHmbc.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="688" data-original-width="853" height="258" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiNRufS47_k_95vTSAH0_9Fn5SlMBwtoPjq9ZjghqSMr4Yl67-DHynXhx2xV4O60zI82YpRk0jZM4MIeRfdKkb3-oaPaL6vse2k-5ShrJqig8WkO3_e1CmGBE_M5qqwMxpvpMp2VDArV81GtbaC0AVD42tb4o5FVUihdj2tubKXFGH4tChrtEzmpBEKag/s320/columnsHmbc.png" width="320" /></a></div>
<p></p>
<div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgVaPC9EfSvHK76MXSFvudRyUL5Qnx9xafZiPBBXUbBoXb_HXIiwbvGgT5-POAK8e3lppwVGbseZ4wBG2u7WDAhRQkmFIeYxX4DCUOWYoobBnNzO_Wa5aUHsUw_WqsS3C1m5w7_A4ClPkl89Mb0VNEpJA_Dnrq3I6trNmo9bysrW3JTiEdhIfxhloadvA/s853/columnsLrhsqmbc.png" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="688" data-original-width="853" height="258" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgVaPC9EfSvHK76MXSFvudRyUL5Qnx9xafZiPBBXUbBoXb_HXIiwbvGgT5-POAK8e3lppwVGbseZ4wBG2u7WDAhRQkmFIeYxX4DCUOWYoobBnNzO_Wa5aUHsUw_WqsS3C1m5w7_A4ClPkl89Mb0VNEpJA_Dnrq3I6trNmo9bysrW3JTiEdhIfxhloadvA/s320/columnsLrhsqmbc.png" width="320" /></a></div>
<p>The extracted <sup>13</sup>C traces show the appearance of artifacts as the sampling is reduced. At 12.5%, in particular, the artifacts become numerous enough to be problematic. Surprisingly, the HMBC experiment does not appear to have any more artifacts than the LR-HSQMBC. I was expecting the HMBC to be much worse. </p><p>These spectra indicate that HMBC and LR-HSQMBC experiments perform equally well with NUS. The spectra also indicate that reducing sampling to 12.5% produces lots of artifacts. The default sampling level on Skaggs Facility NMR experiments is 25%, except for the ASAP-HSQC where it is 50% because of the reduced acquisition time. I will update the HMBC parameters to include NUS with 25% sampling, but this value can easily be changed in the IconNMR interface when setting up experiments.<br /></p>
<p><u>Acknowledgements</u></p>
<p>The sample for this work was kindly prepared and provided by Prof. Ted Molinski.</p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0tag:blogger.com,1999:blog-2408381375133114186.post-8514035842156643542022-03-01T13:41:00.003-08:002022-03-02T09:34:09.463-08:00Ξ values for indirect referencing<p>In <a href="https://sopnmr.blogspot.com/2022/02/locating-hydroxyls-by-deuterium-exchange.html">the previous post</a> I mentioned that <a href="https://sopnmr.blogspot.com/2020/03/indirect-referencing.html">indirect referencing</a> was required to compare spectra collected in methanol-d<sub>4</sub> and methanol-d<sub>3</sub>. Indirect referencing relies on precisely determined ratios of the <sup>1</sup>H gyromagnetic ratio and the gyromagnetic ratio of the nucleus being referenced. Unfortunately, many different values of this ratio, known as Xi (Ξ), have been reported. Here I test several of these Ξ values to see how closely they match referencing to an internal standard.</p>
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<p>Most readers of this blog will be more concerned with referencing <sup>13</sup>C than any other nucleus, so I examined <sup>13</sup>C chemical shift referencing. A quick internet search found several different values for the <sup>13</sup>C/<sup>1</sup>H Ξ value. I used each of these values to reference the <sup>13</sup>C dimension of a HSQC spectrum of cholesteryl acetate in CDCl<sub>3</sub> containing TMS. I then read off the <sup>13</sup>C chemical shifts of TMS and CDCl<sub>3</sub>. The values should be 0.00 ppm for TMS and 77.16 ppm for CDCl<sub>3</sub><a href="#ref1"><sup>1</sup></a>. Results are in the table below.</p>
<p></p>
<table border="1" cellpadding="3" cellspacing="0" style="font-size: x-small;">
<thead style="background-color: black; color: white;">
<tr>
<th>Sample</th>
<th><sup>13</sup>C/<sup>1</sup>H Ξ</th>
<th>Source</th>
<th>δ<sub>C</sub> TMS</th>
<th>δ<sub>C</sub> CDCl<sub>3</sub></th>
</tr>
</thead>
<tbody>
<tr><td>10mM DSS in 99.8% D<sub>2</sub>O, 25<sup>o</sup>C</td><td>0.251449528</td><td><a href="#ref2">2</a><br /></td><td>2.615</td><td>80.002</td></tr>
<tr><td>10mM DSS in 99.8% D<sub>2</sub>O, 25<sup>o</sup>C</td><td>0.251449537</td><td><a href="#ref2">2</a><br /></td><td>2.570</td><td>79.972</td></tr>
<tr><td>DSS in 1M NH<sub>4</sub>NO<sub>3</sub> and 1M HNO<sub>3</sub>, 25<sup>o</sup>C</td><td>0.251449519</td><td><a href="#ref2">2</a><br /></td><td>2.659</td><td>80.042</td></tr>
<tr><td>5mM DSS in NH<sub>3</sub>, 25<sup>o</sup>C</td><td>0.251449531</td><td><a href="#ref2">2</a><br /></td><td>2.609</td><td>79.999</td></tr>
<tr><td>10mM DSS in 99.8% D<sub>2</sub>O, 25<sup>o</sup>C</td><td>0.251449530</td><td><a href="#ref2">2</a>,<a href="#ref3">3</a>,<a href="#ref4">4</a></td><td>2.603</td><td>79.999</td></tr>
<tr><td>5mM TSP in D<sub>2</sub>O, 22<sup>o</sup>C</td><td>0.25144954</td><td><a href="#ref5">5</a></td><td>2.563</td><td>79.959</td></tr>
<tr><td>1% TMS in CDCl<sub>3</sub>, 25<sup>o</sup>C</td><td>0.25145020</td><td><a href="#ref3">3</a>,<a href="#ref4">4</a></td><td>-0.078</td><td>77.333</td></tr>
<tr><td>1% TMS in CDCl<sub>3</sub>, 25<sup>o</sup>C</td><td>0.25145022</td><td><a href="#ref6">6</a></td><td>-0.134</td><td>77.255</td></tr>
</tbody>
</table>
<p></p>
<p>Clearly, accurate indirect referencing depends on using the correct Ξ value. Using values of Ξ obtained for DSS or TSP gives poor results. The most accurate result is obtained using the TMS derived value, Ξ=0.25145020. This is the value recommended by IUPAC.<sup><a href="#ref3">3</a>,<a href="#ref4">4</a></sup></p>
<p>Interestingly, IUPAC recommends two different values of Ξ, depending on the solvent.<sup><a href="#ref3">3</a>,<a href="#ref4">4</a></sup> For organic solvents, Ξ=0.25145020 is recommended. For aqueous systems, they recommend using Ξ=0.251449530 and noting that the chemical shifts are referenced to DSS. This is because the methyl groups of DSS in D<sub>2</sub>O do not resonate at the same frequency as the methyls of TMS in CDCl<sub>3</sub>. In other words, the chemical shift of DSS, which is normally set to 0.00 ppm, is not the same as the chemical shift of TMS, which is also set to 0.00 ppm.</p>
<p>Even comparing the chemical shift of TMS in different solvents is fraught because in solvents other than CDCl<sub>3</sub> the TMS signal does not resonate at 0.00 ppm. Tables of correction factors are available,<sup><a href="#ref4">4</a>,<a href="#ref7">7</a>,<a href="#ref8">8</a>,<a href="#ref9">9</a></sup> but are not entirely consistent, and it is not recommended to use them unless one has to compare chemical shifts in different solvents.</p>
<p>My recommendations for referencing spectra are;</p>
<ul style="text-align: left;">
<li>Whenever possible use TMS or DSS as an internal reference.</li>
<li>Without an internal reference, calibrate <sup>1</sup>H spectra using residual solvent peaks and use indirect referencing with the IUPAC recommended Ξ value to calibrate heteronuclear spectra.</li><li>Use the values in Gottlieb et al<a href="#ref1"><sup>1</sup></a> to calibrate <sup>1</sup>H spectra. Even though they may not be accurate, they are the most generally accepted values.</li><li>When publishing, describe how you referenced the spectra, including what values you used.<br /></li>
</ul>
<p><u>References</u></p>
<p><a name="ref1">1.</a> Gottlieb, H. E., Kotlyar, V., and Nudelman, A.<br />
<a href="https://doi.org/10.1021/jo971176v">
NMR Chemical Shifts of Common Laboratory Solvents as Trace Impurities</a><br />
J Org Chem. 1997;62(21):7512–7515<br />
</p>
<p><a name="ref2">2.</a> Wishart, D. S., Bigam, C. G., Yao, J., Abildgaard, F., Dyson, H. J., Oldfield, E., Markley, J. L., and Sykes, B. D.<br />
<a href="https://doi.org/10.1007/bf00211777">
<sup>1</sup>H, <sup>13</sup>C and <sup>15</sup>N Chemical Shift Referencing in Biomolecular NMR</a><br />
J Biomol NMR. 1995;6:135-140<br />
</p>
<p><a name="ref3">3.</a> Harris, Robin K., Becker, Edwin D., Cabral de Menezes, Sonia M., Goodfellow, Robin and Granger, Pierre<br />
<a href="https://doi.org/10.1351/pac200173111795">
NMR nomenclature. Nuclear spin properties and conventions for chemical shifts (IUPAC Recommendations 2001)</a><br />
Pure Appl Chem. 2001;73(11):1795-1818<br />
</p>
<p><a name="ref4">4.</a> Harris, Robin K., Becker, Edwin D., Cabral de Menezes, Sonia M., Granger, Pierre, Hoffman, Roy E. and Zilm, Kurt W.<br />
<a href="https://doi.org/10.1351/pac200880010059">
Further conventions for NMR shielding and chemical shifts (IUPAC Recommendations 2008)</a><br />
Pure Appl Chem. 2008;80(1);59-84<br />
</p>
<p><a name="ref5">5.</a> Bax, A. and Subramanian, J.<br />
<a href="https://doi.org/10.1016/0022-2364(86)90395-1">
Sensitivity-Enhanced Two-Dimensional Heteronuclear Shift Correlation NMR Spectroscopy</a><br />
J Magn Reson. 1986;67:565-570 (1986)<br />
</p>
<p><a name="ref6">6.</a>
<a href="https://www.cif.iastate.edu/nmr/nmr-tutorials/indirectref">
Indirect Referencing</a><br />
Iowa State Unversity, Chemical instrumentation Facility<br />
</p>
<p><a name="ref7">7.</a>
<a href="http://chem.ch.huji.ac.il/nmr/whatisnmr/chemshift.html">
Chemical Shift Referencing. Table 2. TMS chemical shifts and solvent susceptibilities in commonly used pure NMR solvents at 25°C</a><br />
The Hebrew University of Jerusalem, Institute of Chemistry, NMR Lab
</p>
<p><a name="ref8">8.</a> Hoffman, R.E.<br />
<a href="https://doi.org/10.1016/S1090-7807(03)00142-3">
Variations on the chemical shift of TMS</a><br />
J Magn Reson. 2003;163:325-331
</p>
<p><a name="ref9">9.</a> Hoffman, R.E.<br />
<a href="https://doi.org/10.1002/mrc.1801">
Standardization of chemical shifts of TMS and solvent signals in NMR solvents</a><br />
Magn Reson Chem. 2006;44:606-616
</p>Brendan Dugganhttp://www.blogger.com/profile/12187129197580998981noreply@blogger.com0