CODEINE

C₁₈H₂₁NO₃, MW= 299.4
Data acquired on a JEOL Eclipse⁺ 400 spectrometer
¹H spectrum

Peak Assignments for Codeine

	13C (ppm)		1H (ppm)
1	146.38	q
2	142.23	q
3	133.43	CH	5.71
4	131.13	q
5	128.30	CH	5.29
6	127.30	q
7	119.58	CH	6.57
8	113.03	CH	6.66
9	91.39	CH	4.89
10	66.43	CH	4.18
11	58.92	CH	3.35
12	56.40	CH3	3.84
13	46.47	CH2	2.59, 2.40
14	43.12	CH3	2.44
15	42.99	q
16	40.82	CH	2.67
17	35.85	CH2	2.06, 1.88
18	20.46	CH2	3.04, 2.30
		OH	2.99

¹³C spectra 
18 mg sample, 1.3 hrs acquisition time


3.3 mg sample, 5 hrs acquisition time

COSY spectrum of codeine

This is a 2D experiment that shows which Hs are coupled to each other.  For simple spectra, this information can often be extracted by measuring and matching couplings, or by selective decoupling, but unless this can be done very quickly, a COSY is more efficient, because all coupling information is obtained in one experiment.

The sample was 3.3 mg of codeine in .65 ml CDCl3 The spectrum was acquired in 5 minutes (not including set-up and processing)

One way to view a 2D spectrum is as a 3D, or "stacked", plot.   However, it is difficult to measure peak frequencies using this type of plot, and many peaks are obscured behind larger peaks.  The more useful representation is a contour plot, in which peaks are represented by a series of concentric rings (contour lines) drawn at vertical intervals, analogous to a topological map.  Taller peaks are drawn with more contour lines.  The 2 plots below illustrate these 2 view for the same region of the COSY spectrum.


    
For COSY data (as with all homonuclear 2D plots), the diagonal (indicated by green line in the figure below) consists of intense peaks that match the normal spectrum, as do projections onto each axis. 
The coupling information is contained in the off-diagonal peaks, called cross-peaks.   The coordinates of each cross-peak are the chemical shifts of the 2 Hs that are coupled.  The cross-peak shown in red "connects" protons at 2.7 ppm and 5.7 ppm (H-16 and H-3, referring to the numbering scheme shown above).
The spectrum is symmetrical about the diagonal - a peak located in the upper left triangle will have a corresponding peak in the lower right triangle of the plot.


Coupling "networks" can be traced out, as shown in the figure below.

The colored arrows trace out coupling networks, corresponding to:H-3 —> H-5 —> H-10 —> OH H-10 -> H-9 H-3 —> H-16 H-16 —> H-11

Table of COSY correlations

shift	shift	Assignments
6.6	6.7	7 - 8
5.7	5.3	3 - 5
5.7	2.7	3 - 16
5.7	4.9	3-9 weak
5.3	4.2	5 - 10
5.3	2.7	5 - 16
4.9	4.2	9 - 10
4.2	2.9	10 - OH
3.3	2.7	11- 16
3.3	2.4	11 - 14
3.3	2.3	11 - 18'
3.0	2.4	18 - 14
3.0	2.3	18 - 18'
2.6	2.4	13 - 13'
2.6	2.1	13 - 17
2.6	1.9	13 - 17'
2.4	2.1	13' - 17
2.4	1.9	13' - 17'
2.1	1.9	17 - 17'

Acquisition Parameters:

512 complex points in direct dimension
128 t₁ increments
2 scans
1 sec relaxation delay
Total acquisition time: 5 min

Including setup, processing and plotting, total time was 15-20 min. 
Processing:

sine squared window function was applied in both dimensions (with 0 degree phase shift)
2x zero-fill was used in the indirect dimension, for a final data size of 512 x 512
magnitude calculation (no phasing is required)
symmetrization

NOESY spectrum of codeine

  The sample is 3.3 mg of codeine in .65 ml CDCl₃




A contour plot of the NOESY spectrum is shown below.  As with all homonuclear 2D plots, the diagonal consists of intense peaks that match the normal spectrum, as do projections onto each axis.   The interesting information is contained in the "cross-peaks", which appear at the coordinates of 2 protons which have an NOE correlation.
For small molecules, the NOE is negative.  Exchange peaks have the opposite sign from NOE peaks, making them easy to identify.  The water peak at 1.5 ppm exchanges with the OH at 2.9 ppm, shown here in red.
The spectrum is phased with the large diagonal peaks inverted (shown in red here), so the NOE cross-peaks are positive.








Expansion of the upfield region:


Table of NOEs: ( ' indicates the more upfield of geminal CH₂ protons)

8 - 7, 12 7 - 18, 18' 3 - 5, 10 5 - 11, 16, 18' 9 - 10, 17, 17' 10 - 16 11 - 18, 16, 14, 18' 18 - 13, 18' 16 - 14, 17 13 - 14, 17, 17' 13' - 17, 17' 17 - 17'

In addition to confirming assignments, the NOESY spectrum allows stereospecific assignments of methylene Hs.  The 3 cross-peaks indicated in red on the plot below distinguish between the 3 CH₂ pairs:

5 -18' 16 - 17 18 - 13

Acquisition parameters:

512 complex points in the direct dimension
256 t₁ increments
mixing time: 0.8 sec
experiment is hypercomplex (method of States, et al) and phase sensitive
16 scans
2 sec relaxation delay
Total time: 5 hrs.

Processing parameters:

cosine squared window function (sine function with 90 degree phase shift) in both dimensions
phased so all peaks in first slice are inverted
2x zero-fill in the indirect dimension
final size 512 x 512

HMQC   (1-bond CH correlation) of codeine

This is a 2D experiment used to correlate, or connect, ¹H and ¹³C peaks for directly bonded C-H pairs.  The coordinates of each peak seen in the contour plot are the ¹H and ¹³C chemical shifts.  This is helpful in making assignments by comparing ¹H and ¹³C spectra.   

This experiment yields the same information as the older "HETCOR" experiment,

 but is more sensitive, so can be done in less time and/or with less material.  This is possible because in the HMQC experiment, 

the signal is detected by observing protons, rather than carbons, which is inherently more sensitive, and the relaxation time is shorter.  This so-called "inverse detection" experiment is technically more difficult 

and is possible only on newer model spectrometers.  

Acorn NMR's new JEOL Eclipse⁺ 400 is equipped to perform inverse experiments, 

and uses Z-gradients for improved spectral quality.

The time required for an HMQC depends on the amount of material, but can be done in 1/2 hour or less, compared to several hours for a HETCOR spectrum.  

Contour plot of the HMQC spectrum.  Because it is a heteronuclear experiment, the 2 axes are different, and the plot is not symmetrical.  Unlike a COSY spectrum, there are no diagonal peaks.

Normal 1D ¹H and ¹³C spectra are shown along the edges.  Peaks occur at coordinates in the 2 dimensions corresponding to the chemical shifts of a carbon and its directly bonded proton(s).  For example, the contour peak indicated in red shows that the ¹³C with peak at 91.5 ppm is bonded to the ¹H with peak at 4.9 ppm.

Non-equivalent methylene protons are easily identified as 2 peaks located at the same ¹³C position.  There are 3 CH₂s in the codeine HMQC spectrum.

1H 13C Assignment 6.6 113 8 6.5 120 7 5.7 133 3 5.3 128 5 4.8 91 9 4.2 66 10 3.8 56 12 3.3 59 11 3.0 & 2.3 20 18 2.6 40 16 2.6 & 2.4 46 13 2.4 43 14 2.0 & 1.8 36 17

The sample is 3.3 mg codeine in ~ .65 ml CDCl₃

Acquisition Parameters:

512 complex points in direct dimension
128 t₁ increments
2 scans
2 sec. relaxation delay
Total acquisition time: ~ 10 min.

Processing:

sine squared window function in both dimensions with 45 degree phase shift
2x zero-fill in the indirect dimension
magnitude calculation (no phasing is required)
final data size 512 x 512

There are variations on this experiment, including a version in which CH₂s have phase 

opposite of that of CH and CH₃ peaks, called an HSQC-DEPT spectrum.  

Negative peaks are shown in red in the plot below,

 easily identifying the 3 CH₂s in codeine. 

HMQC / HSQC 1-bond CH correlation

Comparison of phase-sensitive HSQC using echo-antiecho  (JEOL experiment name: hsqc_pfg_s_phase_pn.exp ) and magnitude mode HMQC (JEOL experiment name:  hmqc_irr_pfg_s.exp )   (see about HMQC processing)
Comparison of experiments run on 4 mg codeine sample using

2 scans
128 t₁ increments (note that because hsqc is phase sensitive, it takes twice as long to acquire the same number of t₁ increments)
512 complex points in direct dimension

signal-to-noise ratio (S/N) measured at N-Me for HMQC (with phase of sine squared window fcn of 45) was twice that of HSQC, but S/N of the smaller peaks is comparable for both spectra.
HMQC:  (total acquisition time ~9 min)


HSQC - note that peaks are sharper, so overlapping peaks are less of a problem.   (total acquisition time ~18 min)

Processing:
HMQC:

sine squared window function in both dimensions with 45 degree phase shift
2x zero-fill in the indirect dimension
magnitude calculation (no phasing is required)
final data size 512 x 512

HSQC:

this is an echo-antiecho experiment;
sine squared window function in both dimensions with 90 degree phase shift
2x zero-fill in the indirect dimension
final data size 512 x 512

Processing HMQC spectra of codeine

There are several variations on ¹H-detected one-bond C-H correlation experiments.   Magnitude mode experiments have the advantage of simplified processing, because no phase correction is required.  The last step in processing is a magnitude calculation.  Choice of window function dramatically affects the quality of the resulting spectrum. 
The sample used here was 3.3 mg codeine in CDCl₃, acquisition time 2.5 hrs.

A 90-degree shifted sine squared function was used above.  Note the broad "wings" on the peaks that result from the subsequent magnitude calculation.  This is most severe for the intense methyl peak in the center of this plot.


A 0-degree shifted sine squared function is commonly used to avoid the broad wings in magnitude spectra.  However, in this case, use of this function led to complete loss of one peak!  The missing peak is broad in the ¹H spectrum, and is severely attenuated by the sine function.


A 30-degree (above) or 45-degree (below) shifted sine squared function both seem to be good compromises.

HMBC   (multiple-bond CH correlation) of codeine

This is a 2D experiment used to correlate, or connect, ¹H and ¹³C peaks for atoms separated by multiple bonds (usually 2 or 3).  The coordinates of each peak seen in the contour plot are the ¹H and ¹³C chemical shifts.  This is extremely useful for making assignments and mapping out covalent structure.
The information obtained is an extension of that obtained from an HMQC spectrum, but is more complicated to analyze.  Like HMQC, this is an "inverse detection" experiment, and is possible only on newer model spectrometers.  Acorn NMR's new JEOL Eclipse⁺ 400 is equipped to perform inverse experiments, and uses Z-gradients for improved spectral quality.
The time required for an HMBC depends on the amount of material, but is much greater than for HMQC, and can take from an hour to overnight.
The contour plot shown below is of 3.3 mg codeine in ~ .65 ml CDCl_3.  See also comparison to the HMBC spectrum of an 18 mg sample.
Normal 1D ¹H and ¹³C spectra are shown along the edges.  Peaks occur at coordinates in the 2 dimensions corresponding to the chemical shifts of a carbon and protons separated by (usually) 2 or 3 bonds.  The experiment is optimized for couplings of ~8 Hz.  Smaller couplings are observed, but their intensities are reduced.  Compare to the spectrum obtained when the experiment is optimized for 4 Hz.
The experiment is designed to suppress 1-bond correlations, but a few are observed in most spectra.  In concentrated samples of conjugated systems, 4-bond correlations can be observed.  There is no way to know how many bonds separate an H and C when a peak is observed, so analysis is a process of attempting to assign all observed peaks, testing for consistency and checking to be sure none of the assignments would require implausible or impossible couplings.
Because of the large number of peaks observed, analysis requires several expanded plots.  In this case, the spectrum has been divided into 4 sections, each of which is discussed below.

The discussion below uses the numbering system shown at right.  The numbers were assigned to peaks in the 1D 13C spectrum, starting downfield, moving upfield, and numbering each sequentially.   This generates a unique identifier for each Carbon, even before knowing any assignments.

In aromatic rings, the most common correlations seen in HMBC spectra are 3-bond correlations because they are typically 7-8 Hz, which is the value for which the experiment is optimized.  The coupling constant is affected by substituents, so 2-bond correlations are also sometimes observed. 
The red lines in the plot above show correlations from aromatic proton H-8 to aromatic carbons C-1 and C-6 (both are 3-bond couplings) and a weak correlation to C-2, a 2-bond coupling.
The other aromatic proton, H-7, has correlations to C-2 and C-4, both of which are 3-bond couplings.
The green lines in the plot above show correlations from proton H-9 to carbons C-1, C-3 and C-4 (all are 3-bound couplings).  With the poor digital resolution of the spectrum in the carbon dimension (512 data points spread over 17kHz), the peaks for C-3 and C-4 run together because they are barely resolved.

The peaks indicated in red above are due to 1-bond coupling in CHCl₃ solvent.  Note that the pair of peaks don't line up with any H peaks, but are symmetrically located about the CHCl₃ peak, with a separation equal to the 1-bond C-H coupling constant.
The other 2 peaks in this plot are H-7 to C-18 and H-9 to C-17.


The region in the above plot shows correlations between aliphatic Hs and aromatic Carbons.  In the lower left corner is a peak showing the 3-bond coupling between the methoxy Hs and C-2, the aromatic carbon bearing the methoxy.
Two protons show correlations to the same 3 carbons.  These are the geminal H-18 protons, showing coupling to C-7 and C-4 (both 3-bond couplings) and C-6 (2-bond coupling).  The remaining peak is H-17 to C-4.  Note that the corresponding coupling from H-17' is not observed.  As with H-H couplings, the value of the 3-bond coupling constant is dependent on the dihedral angle.

The last quadrant is shown above, showing correlations between aliphatic Hs and Cs.  The peaks are identified below, for each H, starting from the left.  Carbons 14 and 15 are only 0.1 ppm apart, and are not resolved in the 2D spectrum.

H-11 to C-14 and/or C-15
H-18 to C-16, C-11
H-13 to C-17, C-14 and/or C-15, C-11
H-14 to C-13 and C-11
H-18' to C-11
H-17 to C14 and/or C-15, C-13

During the process of assigning HMBC peaks, it can be useful to indicate on the plot the positions of the 1-bond correlations.  NUTS has the ability to do this, using the compare command.  In the HMBC plot below, 1-bond HMQC peaks are indicated by X.

Acquisition Parameters:

512 complex points in direct dimension
128 t₁ increments
8 scans
2 sec. relaxation delay
Total acquisition time: 35 min

Processing:

sine squared window function in both dimensions with 0 degree phase shift in t₂ and 90 degree phase shift in t₁
2x zero-fill in the indirect dimension
magnitude calculation (no phasing is required)
final data size 512 x 512

MASS SPECTRUM

IR

 
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DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO

 
 amcrasto@gmail.com

http://newdrugapprovals.org/

DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO

HMBC (multiple-bond CH correlation) of codeine

HMBC   (multiple-bond CH correlation) of codeine

This is a 2D experiment used to correlate, or connect, ¹H and ¹³C peaks for atoms separated by multiple bonds (usually 2 or 3).  The coordinates of each peak seen in the contour plot are the ¹H and ¹³C chemical shifts.  This is extremely useful for making assignments and mapping out covalent structure.

The information obtained is an extension of that obtained from an HMQC spectrum, but is more complicated to analyze.  Like HMQC, this is an "inverse detection" experiment, and is possible only on newer model spectrometers.  Acorn NMR's new JEOL Eclipse⁺ 400 is equipped to perform inverse experiments, and uses Z-gradients for improved spectral quality.

The time required for an HMBC depends on the amount of material, but is much greater than for HMQC, and can take from an hour to overnight.

The contour plot shown below is of 3.3 mg codeine in ~ .65 ml CDCl_3.  See also comparison to the HMBC spectrum of an 18 mg sample.

Normal 1D ¹H and ¹³C spectra are shown along the edges.  Peaks occur at coordinates in the 2 dimensions corresponding to the chemical shifts of a carbon and protons separated by (usually) 2 or 3 bonds.  The experiment is optimized for couplings of ~8 Hz.  Smaller couplings are observed, but their intensities are reduced.  Compare to the spectrum obtained when the experiment is optimized for 4 Hz.

The experiment is designed to suppress 1-bond correlations, but a few are observed in most spectra.  In concentrated samples of conjugated systems, 4-bond correlations can be observed.  There is no way to know how many bonds separate an H and C when a peak is observed, so analysis is a process of attempting to assign all observed peaks, testing for consistency and checking to be sure none of the assignments would require implausible or impossible couplings.

Because of the large number of peaks observed, analysis requires several expanded plots.  In this case, the spectrum has been divided into 4 sections, each of which is discussed below.

The discussion below uses the numbering system shown at right.  The numbers were assigned to peaks in the 1D 13C spectrum, starting downfield, moving upfield, and numbering each sequentially.   This generates a unique identifier for each Carbon, even before knowing any assignments.

In aromatic rings, the most common correlations seen in HMBC spectra are 3-bond correlations because they are typically 7-8 Hz, which is the value for which the experiment is optimized.  The coupling constant is affected by substituents, so 2-bond correlations are also sometimes observed.  

The red lines in the plot above show correlations from aromatic proton H-8 to aromatic carbons C-1 and C-6 (both are 3-bond couplings) and a weak correlation to C-2, a 2-bond coupling.

The other aromatic proton, H-7, has correlations to C-2 and C-4, both of which are 3-bond couplings.

The green lines in the plot above show correlations from proton H-9 to carbons C-1, C-3 and C-4 (all are 3-bound couplings).  With the poor digital resolution of the spectrum in the carbon dimension (512 data points spread over 17kHz), the peaks for C-3 and C-4 run together because they are barely resolved.

The peaks indicated in red above are due to 1-bond coupling in CHCl₃ solvent.  Note that the pair of peaks don't line up with any H peaks, but are symmetrically located about the CHCl₃ peak, with a separation equal to the 1-bond C-H coupling constant.

The other 2 peaks in this plot are H-7 to C-18 and H-9 to C-17.

The region in the above plot shows correlations between aliphatic Hs and aromatic Carbons.  In the lower left corner is a peak showing the 3-bond coupling between the methoxy Hs and C-2, the aromatic carbon bearing the methoxy.

Two protons show correlations to the same 3 carbons.  These are the geminal H-18 protons, showing coupling to C-7 and C-4 (both 3-bond couplings) and C-6 (2-bond coupling).  The remaining peak is H-17 to C-4.  Note that the corresponding coupling from H-17' is not observed.  As with H-H couplings, the value of the 3-bond coupling constant is dependent on the dihedral angle.

The last quadrant is shown above, showing correlations between aliphatic Hs and Cs.  The peaks are identified below, for each H, starting from the left.  Carbons 14 and 15 are only 0.1 ppm apart, and are not resolved in the 2D spectrum.

H-11 to C-14 and/or C-15
H-18 to C-16, C-11
H-13 to C-17, C-14 and/or C-15, C-11
H-14 to C-13 and C-11
H-18' to C-11
H-17 to C14 and/or C-15, C-13

During the process of assigning HMBC peaks, it can be useful to indicate on the plot the positions of the 1-bond correlations.  NUTS has the ability to do this, using the compare command.  In the HMBC plot below, 1-bond HMQC peaks are indicated by X.

Acquisition Parameters:

512 complex points in direct dimension
128 t₁ increments
8 scans
2 sec. relaxation delay
Total acquisition time: 35 min

Processing:

sine squared window function in both dimensions with 0 degree phase shift in t₂ and 90 degree phase shift in t₁
2x zero-fill in the indirect dimension
magnitude calculation (no phasing is required)
final data size 512 x 512

HMBC  of codeine optimized for small couplings

See explanation of the HMBC experiment.

Long range C-H couplings cover a range of values, typically less than 10 Hz.  The experiment is optimized for a specific coupling, and selection of that value is a trade-off.  Correlations due to couplings of other values are reduced in intensity.  To optimize for a specific J value, a delay in the pulse sequence is set to 1/(2J).  For very small couplings, this delay becomes so long that much of the magnetization is lost to relaxation.  Typically, the delay is optimized for J of 7-8 Hz, the expected value for aromatic 3-bond couplings.  However, longer-range couplings can be observed if the delay is optimized for smaller couplings.

The contour plots shown below are of 8 mg codeine in ~ .65 ml CDCl₃, in which the experiment was optimized for couplings of 4 Hz.

Compare to the HMBC spectrum optimized for 8 Hz.

Because of the large number of peaks observed, analysis requires several expanded plots.  In this case, the spectrum has been divided into 4 sections, each of which is discussed below.

The discussion below uses the numbering system shown at right.  The numbers were assigned to peaks in the 1D 13C spectrum, starting downfield, moving upfield, and numbering each sequentially.   This generates a unique identifier for each Carbon, even before knowing any assignments.

Compare to the same quadrant in the HMBC optimized for 8 Hz.  Several additional 2-bond couplings are observed, as are 1-bond couplings for 7 and 8.  The peaks indicated by red circles are 4-bond couplings: H-8 to C-4 and H-7 to C-1.  

Compared to the 8 Hz-optimized spectrum, one additional peak is observed in this quadrant, indicated by the red circle.  This is a 4-bond coupling from H-7 to C-15.  

In this quadrant, three 4-bond peaks are indicated by red circles (H-18 to C-8 and C-1; H-18' to C-8).  In addition, two 5-bond couplings are indicated by blue circles.  These are H-18 and H-18' to C-2.

The only additional correlations in the quadrant not seen in the 8 Hz-optimized spectrum are from H-13' (just upfield of the intense N-Me singlet) to carbons 17, 14/15 and 11.

Acquisition Parameters:

512 complex points in direct dimension
128 t₁ increments
64 scans
2 sec. relaxation delay
Total acquisition time: 4.5 hrs

Processing:

sine squared window function in both dimensions with 0 degree phase shift in t₂ and 90 degree phase shift in t₁
2x zero-fill in the indirect dimension
magnitude calculation (no phasing is required)
final data size 512 x 512