NMR coupling constants are a valuable tool in the structure determination process. The

increasing resonance frequency of modern NMR spectrometers commonly allows

first-order multiplet analysis. While this can easily be done for the simplest patterns more

complex patterns can be extremely difficult to analyze. This is certainly an issue for

synthetic organic chemists accessing walk-up spectrometers and who have not received

the appropriate training in spectral analysis.

We previously reported on the development of J-coupler, a multiplet analysis software

tool [1]. The algorithm deduces the coupling constant values from first-order multiplets

using a general inverted splitting tree algorithm [2]. A peak intensity normalization

procedure utilizes multiplet symmetry and generates a set of possible first-order intensity

distribution patterns. With this procedure we have shown it is possible to find an intensity

pattern that allows deduction of a proper set of coupling constants. Furthermore the

software tool allowed to manually enter the number of protons associated with multiplets

or to calculate this number based on comparison of multiplet integral and the number of

protons in proposed structure.

This approach ultimately fell short of the fully automated multiplet analysis required to

provide a table of chemical shifts and coupling constants for a proton spectrum as a

result of a simple "click of a button". We report here our continued innovations in this

regard. The improved algorithm is part of the ACD/NMR Manager package and allows

multiplet analysis of routine 1H NMR spectra as follows:

⇒ Automatically produces a list of chemical shifts and coupling constants with a

single mouse-click.

⇒ Automatically determines the number of protons for each multiplet even if the

chemical structure is absent.

⇒ Provides convenient navigation through the multiplets with user-defined or

automated zooming.

The application of the J-coupler tool to a particular multiplet is straightforward. The next

logical step is to identify distinct multiplets whenever possible and allocate the correct

number of protons to them. The flow chart describing the multiplet analysis of 1H NMR

spectra is described in Figure 1. Prior baseline correction and peak-picking is assumed.

The strategy can benefit from prior knowledge regarding signals such as impurities,

solvent, and artifact signals. These signals can be masked using dark regions to ignore

these resonances. It is beneficial to set these dark regions prior to analysis. Solvent

resonances which are masked as dark regions can still contribute to the overall integral of

multiplets located nearby unless solvent suppression is utilized. In this case dark regions

can be used to mask the solvent suppression residual artifacts as shown in Figure 2. This

may be the preferred method relative to the established fitting of solvent signal using

analytical functions [3,4].

Figure 1 Flow chart of Multiplet Analysis.

Figure 2 A typical 1H NMR spectrum with three dark regions masking an impurity signal

(dinitromethane @ 5.8 ppm), the residual water resonance (@ 3.3 ppm), and an area

confined between the DMSO satellites.

Mask known impurities and artifacts with dark regions(optional).

Spectrum integration. All integrals should include at least98% of the total spectrum.

Separate the integral zones as follows:

Integral normalization. Delete all integrals < SIV.

Multiplet analysis by J-coupler.

Final adjustment of numbers of protons. Sum is equal to thenumber of protons in the structure (optional).

Unite all integrals smaller that SIV & separated by < Jmax.

When the impurities are masked, the spectrum is integrated. The integration threshold is

interactively adjusted so that the resulting integrals will account for 98-99% of the total

spectrum integral. This ensures that integral zones include all peaks present in the

spectrum. This procedure could fail in two cases: when the spectrum was not baseline

corrected or large inverted peaks exist. However, baseline correction is a required

preprocessing step and inverted peaks are commonly artifacts in routine 1D NMR spectra.

It is possible to also use in the analysis a user-supplied list of integrals.

In the next stage of the analysis the resulting integral zones are analyzed. The goal is to

ensure that no multiplets were separated into smaller parts during the integration process.

Two neighboring zones separated by less than Jmax, the closest distance between peaks

inside the zones, are associated if they are smaller than the Smallest Integral Value (SIV).

The SIV is defined as the relative integral value with all integrals summed to 100%. The

value of SIV is to retain only real signals and reject any small contributions from

impurities.

For a pure compound SIV would equal the integral of one proton and is calculated as the

inverse of the number of protons in the structure N. This would ensure that integrals < SIV

represent impurities only. In practice relaxation delays employed in routine 1D

experiments result in non-quantitative integrals. Based on our analyses the optimal value

for SIV is found to be 2/3 * (1/NS) * 100%. When the hypothetical structure is not avail-

able as a basis there are simple rules to follow. Usually an SIV value of 2-3% works for

structures of 20-40 protons while SIV = 1.5-2% for structures with 40-60 protons.

During the next stage the initial integral zones are separated into distinct multiplets. The

benefits of distinguishing individual multiplets are more correct measures of the chemical

shifts and the possibility of determining the values of the coupling constants and the

number of associated protons. J-coupler-II uses several criteria for separation of the

multiplets.

1) Maximum multiplet width: This parameter would be large enough to

accommodate two or three very closely overlapping multiplets (bottom of Figure 3)

or a multiplet with an unusually large coupling constant (top of Figure 3, the

multiplet has 2JHF > 50 Hz).

Figure 3

The maximum width should not be too large to combine several well resolved

multiplets. The recommended value is 80-100 Hz and 100 Hz has been used in the

examples above. If a multiplet is found to exceed the limiting value, it is

separated into smaller parts by repeated integration with increasing threshold.

2) Jmax : Maximum allowed coupling constant. The neighboring peaks with a

difference > Jmax are considered to belong to different multiplets. For 1H NMR

spectra the recommended value is 18-20 Hz unless couplings with magnetically

active nuclei such as 19F and 31P etc... are present.

3) Separate Symmetric Multiplets: This option utilizes a check of symmetry to

distinguish the presence of overlapping multiplets. For a multiplet to be separated

its individual parts have to be symmetric and have an integral >SIV.

The following figures show the algorithm at work.

Figure 4

Figure 4 displays two methyl singlets and one doublet combined into a single multiplet.

This result was obtained without multiplet separation checking.

Figure 5

When Jmax is selected two methyl singlets result since the difference between them,

19 Hz, is >Jmax. However, the upfield methyl singlet and doublet are still associated.

When the “Separate symmetric multiplet” option is selected all three multiplets are

separated and their chemical shifts and coupling constants are determined correctly.

Figure 6

Consider the 1H NMR 300 MHz spectrum of 3'-Deoxy-3'-fluorothymidine. The resonances

of H-9’ and H-9” protons are of interest since they have rather large 3JHF coupling

constants.

Figure 7

In the figure above is the result of the automated analysis obtained using Jmax =18 Hz

and a maximum multiplet width of 60 Hz. The multiplet separation options were not

used. During the analysis the upfield multiplets were separated into two parts because

the multiplet has a width greater than 60 Hz. The resultant resonances have an integral

value which is only half of the threshold value. They were therefore discarded. The

overall result is that the wide multiplet was not identified.

The low-field multiplet was successfully identified but the coupling pattern was not

recognized since it shows a coupling constant of 3JHF > Jmax.

O1

2

N3

NH

4

56

7O8

9

10

O11

12

13CH314

15

F16

OH17

Figure 8

When Jmax is increased to 60 Hz to accommodate the very large 2JHF with H-13, and the

maximum multiplet width is increased to 80 Hz, both multiplets are correctly identified

and the lowfield multiplet has the correct coupling pattern determined.

Prior to the normalization of integrals all those < SIV are discarded. When a molecular

structure is proposed all remaining integrals are normalized to the number of protons N.

When a structure is not available the following procedure is followed. The smallest

determined integral is set to 1. All integrals within a value of 1.0-1.5 are found and their

average value is set to a value of one proton. There are two cases when this approach

could give incorrect results. 1) When integral values cannot distinguish the actual number

of protons. For example consider the integral ratio of 2:1 corresponding to the (CH2)n-

(CH)n- fragment. Here n could be 1, 2 or even more. 2) When a structure does not

contain a methine proton and consists of fragments like CH3-CH2-. While this example

could be treated correctly by improving the normalization procedure, the first example is

impossible to resolve based only on the NMR spectrum.

Once the integral zones are identified the application of J-coupler is straightforward to

provide the chemical shifts, coupling constants (if the multiplet is first-order), and the

number of protons attributed to the multiplet. The number of protons is obtained simply

by rounding off the integral values. If the multiplet is not recognized as first order its

chemical shift is calculated as the average position of the peaks weighted according to

their heights.

If the proposed structure is available then it is possible to ensure that the number of

protons is equal to the sum of protons contained within the multiplets using the “Fixed

Number of Protons” option. The algorithm is interactive and proceeds as follows. Let

Int(i) and IN(i) be the real and integer values of integral, N be the sum of protons in the

molecule, and Nsum is the sum of IN(i). If Nsum > N then find the multiplet with the

maximum positive difference IN(i)-Int(i) and decrease its IN(i) by one. If Nsum < N, find the

multiplet with the maximum negative difference IN(i)-Int(i) and increase its IN(i) by one.

New tools to aid visual inspection of the results in J-coupler have been introduced. There

are now left- and right-arrow buttons to switch to the next multiplet in ascending or

descending order.

Figure 9

When Multiplet Zoom is “Auto” the multiplets are displayed to provide the most detailed

multiplet presentation as shown in the picture above. Alternatively the user-defined zoom

can be maintained during multiplet navigation.

ConclusionA simple and straightforward method and interface has been developed to provide, in an

automated manner, a list of multiplets with chemical shifts and coupling constants as well

as the number of assigned protons. The method works across an experimental spectrum

following baseline correction and peak-picking. An additional useful preprocessing step is

to mask additional signals using “dark regions” assuming prior knowledge. Effective

multiplet separation can be achieved using several criteria including symmetry, maximum

peak distance, and multiplet width. The method requires a good estimate regarding the

size of the molecule. The method works best when there is no significant overlap of the

signals

References1. S.Golotvin, E. Vodopianov, and A.J. Williams, Magn. Res. Chem., 2002 (paper in press).

2. T. R. Hoye, P.R Hanson, and J.R. Vyvyan, J. Org. Chem., 1994, 59, 4096-4103.

3. Bartels. P.Guntert and K. Wuthrich, J. Mag. Reson, 1995, A 117, 330-333.

4. Griffiths L., Magn. Reson. Chem., 2001, 39, 194-202.

Further Adventures in the FULLY Automated First Order Multiplet AnalysisSergey Golotvin* and Eugene Vodopianov

Advanced Chemistry Development, Russian Office, 6 Bakuleva Street,

Moscow 117513, Russia

Antony WilliamsAdvanced Chemistry Development,90 Adelaide Street West, Suite 600,

Toronto, Ontario, M5H 3V9

Advanced Chemistry Development90 Adelaide St. W., Suite 600

Toronto, Canada M5H 3V9Tel: (416) 368-3435 Fax: (416) 368-5596

Toll Free: 1-800-304-3988Email: info@acdlabs.com

www.acdlabs.com