NMR

Mass-Resolving NMR with the DOSY Experiment

Two Examples on How to Take Laboratory Techniques to the
Next Level
Innovation with Integrity

Abstract
1
H Diffusion Ordered Spectroscopy (DOSY) investigates the diffusion of solubilized molecules. Diffusion is proportional
to the molecular mass (g/mol). This enables mass-resolving experiments which can be performed with a 1H NMR
spectrometer and opens the door to incredibly powerful analytical strategies as mass data and NMR data are
combined.

Synthesis Control with NMR

1
H NMR spectroscopy is an invaluable technique for synthesis control in the chemical laboratory. The molecular
structure of the main product can be derived, while at the same time, the high sensitivity of 1H NMR allows for the
detection of diluted byproducts and residuals solvents.

Unknown small signals are encountered in the products of most organic syntheses. The assignment of all signals is
necessary, including small signals of unknown origin, to fully understand reaction conditions, the conversion of starting
materials, and the impurity profile. The assignment of small signals is thus of utmost importance when studying
reaction conditions.

The assignment of small, unknown signals in the presence of intense signals from the matrix is a challenging and
time-consuming task – to the point where conventional 2D NMR spectroscopy can become ineffective to deliver
the required information with reasonable effort. In most cases, heteronuclear 2D experiments increase the spectral
resolution at the expense of the signal-to-noise ratio (S/N) which often decreases the signals of diluted compounds
to a value below the detection threshold. If small signals are above the detection threshold, they can be overlain by
other signals or they can be confused with 2D artifacts, such as from T1-noise. Hence, the assignment of unknown
impurities in mixtures is challenging and hard to accomplish with standard 2D NMR methods.
Liquid chromatography coupled to mass spectrometry (LCMS) can contribute valuable information but is intrinsically
“blind” when dealing with isomeric byproducts that have the same molecular mass as the main compound. This
makes it especially difficult to answer the question if a reaction occurred at the right “place” in the molecular
structure. Secondly, unknown impurities might not be ionizable and could remain undetected in LCMS. In many cases,
byproducts must then be synthesized on larger scale to be separated by chromatography and analyzed by NMR and
LCMS. This procedure can require weeks of work and can often not be performed due to financial or time constraints.

In the following, we show two examples how the DOSY experiment can be used to overcome these challenges. The
DOSY typically takes less than five minutes and often helps to avoid more time-consuming 2D NMR. The DOSY can
be applied to any routine NMR sample with the perspective to obtain results as if column chromatography was applied.
In view of the great advantages of the DOSY experiment, NMR scientists coined the term “chromatographic” NMR
spectroscopy.

Example 1: Mixture Analysis with DOSY

The first example shows a DOSY experiment that was performed on a sample of approx. 5 mg caffeine and ethylene
glycol in D2O.

Figure 1: 1H NMR DOSY experiment recorded with the DOSY_DC parameter set in 2:55 minutes on a sample of approx. 5 mg caffeine and ethylene glycol
in D2O using an AvanceCore 400 MHz NMR spectrometer. Note that the y-axis is plotted along the exponent of the diffusion constant with respect to the
base 10 (the DOSY exponent γ). This is described in more detail at the end of the application note.

The resulting spectrum of the DOSY experiment shows the standard 1D 1H NMR spectrum on the x-axis and correlates
each peak with a diffusion constant on the y-axis. The diffusion constant is obtained by spatially encoding the location
of the molecules within the NMR sample tube using a linear magnetic field gradient along the z-axis (parallel to
the magnetic B0 field). After a diffusion time of typically 100 ms, the diffusion constant is derived from the spatial
displacement of the molecules.

The diffusion constant is proportional to the molecular weight in g/mol (this is described in more detail at the end of this
application note). Signals that originate from heavy molecular entities appear on the top of the indirect dimension where-
as signals from light weight entities appear at the bottom.
Signals that are observed on a horizontal line are thus likely to originate from the same molecular entity. This principle
can be used for a quick and robust assignment of the 1H signals:

� Often, the product and polymers are the heaviest components in a reaction mixture and are observed at the top of
the DOSY spectrum. In the above example, this is caffeine (Figure 1, green).
� The mass resolving capabilities of the DOSY are usually sufficient to distinguish the heavy components from
precursors and fragments, which are often of intermediate molecular weight and therefore found in the center of
the DOSY spectrum. To illustrate this center region in the example above, ethylene glycol was added to the mixture
(Figure 1, red).
� Small organic molecules such as residual solvents and water are usually well resolved at the bottom of the DOSY
spectrum (Figure 1, residual water, blue).

Assigning a molecule according to its DOSY signals can be as straightforward as drawing a horizontal line in the
spectral plane. This makes the DOSY one of the easiest, yet most powerful NMR experiments. In contrast to
heteronuclear 2D experiments, the DOSY is of comparable sensitivity as the 1H experiment. Hence, traces in the 10
to 100 ppm concentration range can be detected within a few minutes of measurement time if approx. 10 to 50 mg of
sample material are available.

Example 2: Assigning Related Compounds with the DOSY Experiment

In chemical processes, side reactions often lead to a variety of byproducts that are structurally similar to the main
product and are often referred to as related compounds. Especially isomeric byproducts can be challenging, as they
have the same mass as the main product. They can hence not be distinguished from each other and from the main
product using standard mass spectrometry.

NMR is capable of elucidating differences in similar chemical structures, including isomers. However, structural
similarity leads to spectral similarity. Analytical samples obtained from chemical reactions are usually a mixture of
products, byproducts, starting materials, fragments, and residual solvents. Signals from concentrated species, such as
the product, are likely to overlay and mask signals of similar and more diluted species such as byproducts. An example,
shown in Figure 2, is the mixture of ibuprofen with its related compound ibuprofen methyl ester.

Figure 2: 1H DOSY spectrum of 20 mg ibuprofen and approx. 0.9% ibuprofen methyl ester in DMSO. Ibuprofen methyl ester is a byproduct of ibuprofen
and causes weak signals which are largely overlain by the sample matrix, with the exception of the signal at 3.57 ppm. This single signal of ibuprofen
methyl ester is sufficient to clearly identify a diffusion constant D which is similar – but not perfectly equal – to the diffusion constant of ibuprofen, which
indicates that the substance is different from the main product but of similar molecular mass. Even though the multiplicity of the signal at 3.57 ppm does
not exhibit any further information, it can be concluded that it does not originate from residual solvents but is likely to originate from a byproduct. This
information can be used to adapt the gravimetric share of the sample accordingly.
In the 1H spectrum, the main product ibuprofen largely overlays the signals from ibuprofen methyl ester except for
the signal at 3.57 ppm which is visible as a small singlet (Figure 2). This signal originates from the methyl group of
ibuprofen methyl ester. In this example, the 1H spectrum is not sufficient to fully characterize the sample as it is
unclear if the singlet at 3.57 ppm originates from a residual solvent, a polymer, or a related compound. The singlet line
shape can be well resolved. However, being a singlet, it cannot be used to deduce coupling partners in its molecular
vicinity. Other experiments such as the sensitive 1H TOCSY are thus unlikely to reveal correlations to other signals that
may be hidden below other signals. Hence, the TOCSY is unlikely to resolve this challenge. The same difficulty likely
restricts heteronuclear 2D experiments as well, which are, on top, usually not sensitive enough to reliably detect diluted
impurities.

The DOSY experiment presents a robust and reliable experimental strategy if the signal under question is well resolved
in the 1H spectrum and not overlain by other signals.

The DOSY spectrum of a mixture containing ibuprofen and ibuprofen methyl ester can now be evaluated by drawing
horizontal lines in the 2D plane:

� The green horizontal line in Figure 2 is assigned to the main compound ibuprofen.
� The signal at 3.57 ppm is assigned to a species with similar molecular weight as ibuprofen and is therefore likely to
be a related compound (Figure 2, red). In the example above, it is thus assigned to ibuprofen methyl ester. (Note that
the remaining signals of ibuprofen methyl ester are largely overlain by the signals of ibuprofen and can therefore not
be used for the DOSY evaluation).
� The signal of ibuprofen methyl ester can be clearly discriminated from light-weight residual organic solvents such as
DMSO or water which appear at the bottom of the DOSY spectrum (Figure 2, blue and light green).

Unlike other 2D experiments which rely on the assignment of all individual peaks in the spectrum, the DOSY does
not require knowledge of the precise chemical structure. A single signal is sufficient to derive the diffusion constant.
The strength of the DOSY experiment lies in the complementarity of mass data and NMR data which enables the
identification of entirely unknown species with high reliability and small risk of incorrect assignments.

How to Acquire the DOSY in Manual Mode on an AvanceCore 400 MHz NMR Spectrometer

Structure elucidation by NMR is often based on the inspection of peaks and coupling patterns (see application note:
“Learn How to Read and Interpret NMR Spectra in 15 Minutes”). In that regard, the most important information is if a
peak is present at all.

In contrast, the DOSY experiment is based on quantitative properties, i.e. correct and undisturbed peak intensities
are exceedingly important. Unlike many other NMR experiments, it is thus more challenging to optimize the DOSY
experiment as it is not straightforward to establish causal relations between experimental artifacts and their quantitative
influence on the signal.

Bruker scientists therefore carried out a systematic examination of the experimental procedure with the goal to
establish an easy-to-use DOSY experiment. This DOSY experiment is available in the DOSY_DC parameter set and was
tested for small organic molecules and polymers up to 30 kDa. The experiment works “out-of-the-box” without further
adjustments:

Create a new experiment or copy any old experiment by typing “edc” in the TopSpin command line.

�
Read the DOSY parameter set by typing “rpar DOSY_DC” in the TopSpin command line.
�
Read in the pulse power by typing “getprosol” in the TopSpin command line.
ƒ Start the experiment by typing “zg” (or “xaua”) in the TopSpin command line or by clicking the play button.
ƒ After the acquisition, the automated processing is triggered by typing “xaup” in the TopSpin command line. “xaup”
launches the processing AU program (automation program). This triggers the DOSY fitting by calling the Dynamics
Center software (v. 2.8 or higher), which can be downloaded from the Bruker website and is free to use for the
DOSY functionality.
ƒ The Dynamics Center automatically writes back the DOSY spectrum in the experiment folder (processing number 1)
where it can be evaluated with TopSpin in the same way as any other NMR spectrum.
How to Acquire the DOSY in Automation with IconNMR

The productivity of NMR is closely related to the possibility to perform NMR in automation with a sample changer.
Using the IconNMR software, the NMR spectrometer can be turned into an easy-to-use open-access measurement
platform which makes it easy to measure a large number of samples in full automation.

Figure 3: IconNMR is used for the automated acquisition of NMR spectra, turning the NMR spectrometer into an open-access platform that can serve a
group of non-expert users. Recording the DOSY_DC experiment only takes a few clicks and less than five minutes of measurement time so that the experi-
ment can be recorded in conjunction with every standard 1D 1H experiment.

Using IconNMR, NMR experiments can be set up with only a few clicks as shown in Figure 3:

1. Put the sample in a holder of the sample changer and double click the holder position in IconNMR to set up an
experiment for that particular sample.
2. Choose the solvent from the drop-down menu.
3. Choose the parameter set DOSY_DC from the drop-down menu.
4. Click “Submit” to start the acquisition or add the experiment to the measurement queue.
5. In the “Preceding Experiments” section in IconNMR, double click the DOSY experiment to display the result in
TopSpin (or find the result being written to a network folder or sent via e-mail).

The acquisition of the DOSY_DC parameter set takes less than five minutes so that it can be recorded together with
every standard 1H experiment.

How to Derive the Molecular Weight from the Diffusion Constant

In many cases, diffusion constants obtained from the DOSY experiment are advantageously used in a non-quantitative,
relative fashion by visual inspection of the spectra and comparing the signal of interest with a reference signal from the
same sample as outlined in the examples one and two. This guarantees that all components are measured under the
same experimental conditions such as temperature, ionic strength, etc.

If diffusion data of different samples is compared, this can be done by directly overlaying the spectra in the “Multiple
Display” of the TopSpin software or by calculating relative diffusion constants for each species. Relative diffusion con-
stants can be calculated by dividing the diffusion constant of a species by the diffusion constant of a known standard
component that is present in the same sample.
Yet another way to use diffusion data is to calibrate it as described in the following. Calibration can be used to derive
the absolute molecular weight in g/mol of unknown substances. At a given temperature, the fundamental relationship is
that heavy molecules diffuse slower than lighter molecules. Secondary effects such as 3-dimensional folding, intermo-
lecular hydrogen bonds and van-der-Waals forces influence the diffusion constant as well, and many theories have been
discussed to establish a universally valid model. Even though a closed analytical solution is not available, good approxi-
mative results can be obtained with the following relationship:

D ~ M�
Equation 1: D: Diffusion constant; M: Molecular weight; β: Exponent

For a single point calibration and according to theory, β is -0.5. With a multi-point calibration, it is possible to refine β
further.

Molecular Weight Calibration for D2O

M log (M DOSY D [m^2 / log (D [m^2 / M DOSY
Substance M %deviation
[g/mol] [g/mol]) Exponent γ (s*1E9)] (s*1E9)]) [g/mol]
H2O 18 1.255 -8.785 1.642 0.215 16 -10

MeOH 32 1.505 -8.944 1.138 0.056 34 6

Ethylene glycol 62 1.792 -9.088 0.816 -0.088 66 6

DSS* 218 2.338 -9.316 0.483 -0.316 188 -14

PEG 400 g/mol 400 2.602 -9.485 0.327 -0.485 408 2

PEG 3.86 kDa 3860 3.587 -10.031 0.093 -1.031 5025 30

PEG 10.37 kDa 10370 4.016 -10.206 0.062 -1.206 11238 8

PEG 31.63 kDa 31630 4.500 -10.381 0.042 -1.381 25131 -21
*DSS: sodium trimethylsilylpropanesulfonate

Molecular Weight Calibration for DMSO

M log (M DOSY D [m^2 / log (D [m^2 / M DOSY
Substance M %deviation
[g/mol] [g/mol]) Exponent γ (s*1E9)] (s*1E9)]) [g/mol]
DMSO 32 1.505 -9.230 0.589 -0.230 36 13

Ethylene glycol 62 1.792 -9.340 0.457 -0.340 62 -1

PhEtOH 122 2.086 -9.431 0.371 -0.431 96 -21

Ibuprofen 206 2.314 -9.586 0.259 -0.586 204 -1

PEG 400 g/mol 400 2.602 -9.753 0.177 -0.753 459 15

Linear Fitting D2O: log(D) vs. log(M) Linear Fitting DMSO: log(D) vs. log(M)
Slope -0.501 Slope -0.474

y-intercept 0.822 y-intercept 0.508

*DSS: sodium trimethylsilylpropanesulfonate

Table 1: Multi-point calibration of molecular masses M vs. diffusion constants D for D2O and DMSO at 25° C.
For the multi-point calibration, the known molecular mass M [g/mol] is plotted on the x-axis against the diffusion constant
D on the y-axis in Figure 4. The diffusion constant is obtained by reading the DOSY exponent γ from the TopSpin software
and inserting it into equation 2:

Figure 4: Multi-point calibration of molecular masses M vs. diffusion constants D for D2O
and DMSO at 25° C.

Figure 5: Reading the DOSY exponent γ of the diffusion

constant from the cursor position in the TopSpin software
(γ = -9.604).

γ*1E9
� = 10
Equation 2: D : Diffusion constant [m2 s-1 *1E-9]

There are several ways to conduct a regression analysis, for example by applying a linear regression as shown in Table 1.
Alternatively, a power regression can be used. In either case, the calculated molecular mass (M DOSY in Table 1) is put
in relation to the known true molecular mass (M [g/mol] in Table 1) to derive the deviation in percent (M %deviation in
Table 1).

When using the DOSY experiment to determine an unknown molecular mass according to the calibration, typical errors
can be on the order of ± 30 % (column M %deviation, Table 1). Errors can be smaller when comparing structurally
similar compounds with similar polarity and hydrogen bonding properties. On the other hand, errors can be larger if
structures differ in functional groups that might induce strong changes in polarity and hydrogen bonding as it is the case
for ibuprofen and ibuprofen methyl ester (Fig. 1). If only the influence of the molecular weight on the diffusion constant
is considered, ibuprofen methyl ester should be heavier and therefore diffuse slower than ibuprofen, i.e. the signals for
ibuprofen methyl ester should appear above the signals of ibuprofen. Since the difference in weight is small (only 14 g/
mol) and the change in polarity is large – as ibuprofen exhibits a polar carboxylic acid group (-COOH) which is not present
in ibuprofen methyl ester which contains a less polar ester group (-COOMe) – the opposite is the case.

Using water (18 g/mol) and ibuprofen (206 g/mol) as an example, and assuming a 30% deviation in the calculated molec-
ular weight results in 23 g/mol vs. 268 g/mol, respectively, which is sufficiently different to discriminate the components.
Hence, a relative error of ± 30 % does usually not limit the practical usability of the DOSY data.

Obtaining High Quality DOSY Spectra

To obtain good quality DOSY spectra, solvent convection in the NMR sample tube needs to be avoided. The easiest
way to accomplish this is by using high viscosity solvents such as D2O or DMSO in 5 mm sample tubes while avoiding
elevated temperatures. If low viscosity solvents such as CDCl3 must be used, 3 mm sample tubes are effective to avoid
convection in most cases if kept at room temperature.

For routine purposes and to obtain good quality spectra that are comparable between samples, it is recommended to
leave the standard parameters of the DOSY experiment unchanged. Changing parameters can influence the quantitative
properties of the DOSY experiment in many unforeseen ways.

Bruker BioSpin is continually improving its products and reserves the right to change specifications without
The diffusion constant in the indirect dimension of the DOSY experiment is obtained from a quantitative fitting procedure
of peak amplitudes. Standard NMR experiments are typically robust with respect to the experimental setup and the
sample that is used. However, the opposite is true for the DOSY. It critically relies on good control of all experimental
parameters and the sample properties (such as temperature, viscosity, concentration, etc.) to yield accurate and precise
diffusion constants. Unlike other 2D experiments, the resolution in the indirect dimension is largely independent of the
number of increments in the indirect dimension. To obtain good DOSY data in routine spectroscopy, and to fit single
exponential decay curves from non-overlain signals, at least three (two plus one) F1 increments are necessary for line
fitting and to derive the error of line fitting. The DOSY_DC parameter set uses eight F1 increments, and this parameter is
recommended for all routine applications as oversampling is not necessary.

To optimize the performance of the DOSY experiment, the scientific literature sometimes recommends optimizing the
DOSY gradients for each sample so that the largest range of values can be used for the numerical reconstruction of
the indirect dimension. This might be indicated from a mathematical point of view when acquiring DOSY data of heavy

notice. Order No. T192898 © 06/2023 Bruker BioSpin.

polymers (> 50000 g/mol). For routine applications with the DOSY_DC parameter set, optimizing the DOSY gradient is
not deemed necessary and will not increase accuracy, precision, or resolution significantly. In contrast, changing the
gradient duration or the gradient strengths (towards smaller attenuations) typically leads to the refocusing of artefacts
that are caused by interference with the spoil gradients. Hence, the duration and power of the spoil gradients must
not be changed as well. If artifacts are created, they usually strongly falsify the peak amplitudes and thereby influence
accuracy, precision, and resolution of the DOSY experiment.

Even insufficient relaxation can compromise the spectral reproducibility which compromises the ability of the phase
cycle to compensate errors. Hence, if attempting to optimize experimental parameters, the outcome needs to be judged
on a quantitative basis. If such an assessment is not possible, it is recommended to not change parameters except the
number of scans and the spectral width. Additional details on how to obtain good quality data are given in the title of the
DOSY_DC parameter set.

Figure 2 The power of NMR in narcotics analysis

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