1H T1ρ Edited 13C CPMAS Spectra - Pharmaceutical Analysis

Active pharmaceutical ingredients (API's) are often mixed with other compounds (excipients) used to dilute, stabilize, sweeten, color, flavour, bind, coat (etc...) the medication.  Often the API is a crystalline compound while the excipients are noncrystalline or amorphous.  When analyzing pharmaceutical pills by solid-state ¹³C CPMAS NMR, one often wants to observe the active crystalline API and not the amorphous non-active excipients.  This can sometimes be accomplished by collecting spectra with differing recycle delays, as the excipients often have shorter ¹H T₁ relaxation times than the crystalline API.  An example of this is given here.  Crystalline API compounds often have much longer ¹H T_1ρ's than the amorphous excipient compounds.  One can modify the simple CPMAS pulse sequence to discriminate against the excipients with short ¹H T_1ρ's by introducing an additional ¹H spin locking pulse before the cross polarization during which the transverse ¹H magnetization of the excpient decays to zero while that of the API decays to a much smaller extent.  After the ¹H spin locking pulse, cross polarization is applied, transferring polarization from the remaining API ¹H magnetization to the API ¹³C.  This modification to the CPMAS experiment is shown in the figure below.

The figure below illustrates this technique applied to generic acetylsalicylic acid (ASA) tablets.

The spectrum in the bottom panel is a ¹³C CPMAS spectrum of a crushed generic ASA tablet with a 60 second recycle time and a 2 msec contact time.  One can see the nine ¹³C resonances of the ASA plus the broad excipient signals between 50 ppm and 110 ppm (highlighted in yellow).  The spectrum in the middle panel was acquired under identical conditions with an additional 10 watt, 12 msec ¹H spin locking pulse applied prior to cross polarization.  Clearly, the broader signals of the excipients (with short ¹H T_1ρ) are suppressed leaving only the resonances of the ASA.  The top panel is a weighted difference of the two spectra showing only the signals of the excipients.  Although a pharmaceutical example is used here, the technique is applicable generally to any mixture of solids with different ¹H T_1ρ's.

Cross Polarization Based Mixture Resolution in Solids

One of the most common techniques used to collect solid-state NMR data for spin I = ½ nuclides is a combination of cross polarization (CP) and magic angle spinning (MAS).  CPMAS provides high sensitivity from the CP and high chemical shift resolution from the MAS.  Furthermore, the scan repetition rate depends on the shorter relaxation time of the protons rather than the longer relaxation time of the spin I = ½ nuclide therefore, more scans can be collected per unit time.  It must be remembered however, that the success of the CP technique depends on the dipolar coupling interaction between proximate protons and the nucleus being observed.  In the absence of dipolar coupled protons, CP signals are not observed.  For this reason, it is sometimes necessary to use a conventional one-pulse method (Bloch decay) which can be used to observe the spin I = ½ nuclide, albeit with lower sensitivity, whether protons are present or not.  When a sample consists of a mixture with some protonated components and some components without protons, then it may be advantageous to collect both a CPMAS and a Block decay spectrum.  When both methods are used, the data can be used to resolve the spectrum of the mixture into subspectra; the protonated components in one spectrum and those components without protons in another.  An example of this is shown with the ¹³C NMR data for a common antacid tablet in the figure below.

The two most abundant carbon containing components of an antacid tablet are calcium carbonate and sucrose.  Spectrum (a) is the CPMAS spectrum.  It consists only of the resonances of sucrose (color coded in yellow) since calcium carbonate (color coded in pink) contains no protons.  Spectrum (b) is the Bloch decay spectrum with high power proton decoupling.  It consists of both the resonances of sucrose and calcium carbonate.  Spectrum (c) is a linear combination of (a) and (b) and represents, primarily, the spectrum of calcium carbonate.  Another interesting example of CP based mixture analysis is given here using Christmas shortbread cookies as an example.

Mixture Resolution in 13C CPMAS NMR

The recycle delay necessary to get the highest signal-to-noise ratio in a multi-scan ¹³C CPMAS NMR spectrum depends on the relaxation properties of the protons in the sample.  The protons in pure solid samples normally belong to a single homogeneous dipolar coupled network.  As a result, all of the protons in the coupled network have a common T₁ relaxation time.  One would expect the same behavior for a mixture of compounds only if the components were mixed at the molecular level.  If the compounds are not mixed at the molecular level, the sample consists of domains of pure materials, each of which has a common proton T₁.  If the proton T₁'s of the domains are significantly different, then one has a means of discriminating between the domains and hence the compounds of the mixture with ¹³C CPMAS NMR data.  The figure below illustrates this principle for a tablet of vitamin C ground into a powder.  The vitamin C tablet consists primarily of ascorbic acid for which the structure is shown in the figure.  The other major solid organic additives are hypromellose (hydroxypropyl methylcellulose), stearic acid (n-C₁₇H₃₅COOH), magnesium stearate and carnauba wax (a complex mixture of C₂₆ to C₃₀ acids, esters and alcohols).  When the tablet is ground up, the powder consists of ascorbic acid domains, stearic acid domains, magnesium stearate domains and carnauba wax domains.

¹³C CPMAS NMR spectra were acquired with a 30 second and a 2 second recycle delay and are shown in (a) and (b), respectively.  One can see that relative intensity of the components in the mixture depends on the recycle delay.  The proton T₁ of the ascorbic acid is obviously longer than that of the other components of the mixture.  The spectra in (c) - (e) are linear combinations of (a) and (b).  The linear combination in spectrum (c) was created such that the ascorbic acid resonances were nulled.  The resulting spectrum is that of only the organic additives. The hypromellose resonances are in the 50 ppm to 110 ppm range.  The aliphatic resonances of the stearic acid, magnesium stearate and carnauba wax overlap in the 10 ppm to 50 ppm range and appear to have similar proton T₁'s.  The linear combination in spectrum (d) was created such that the aliphatic stearic and wax resonances were nulled.  The resulting spectrum is that of ascorbic acid and the inverted spectrum of the hypromellose.  The linear combination in spectrum (e) was created such that the hypromellose resonances were nulled. The resulting spectrum is that of the ascorbic acid with the stearic acid, magnesium stearate and carnauba wax additives.  This combination allows observation of the ascorbic acid with no overlapping resonances from the additives.

1D TOCSY for Mixture Analysis

Many two dimensional techniques such as the 2D TOCSY, have similar 1D analogs where selective pulses are used to get effectively only one slice of the comparable 2D experiment. If you are only interested in a few correlations, then the 1D experiments can represent a tremendous time saving ( i.e. money saving). Below is a series of 1D TOCSY spectra run on a mixture of toluene, p-dichlorobenzene and pyridine. In this case, the technique can be used to separate out the components of the mixture. In the figure, the lower trace is the standard ¹H spectrum. In the other traces red arrows indicate the resonance selectively irradiated with the shaped pulse.