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.

Glycine as a 13C CPMAS Setup Sample

Glycine is an excellent setup compound for ¹³C CPMAS NMR measurements.  Its utility in this regard has been described in detail.^1,2  It can easily be observed in one scan and has reasonably short ¹H T₁'s, allowing it to be used for ¹H 90° pulse calibration and to setup the Hartmann Hahn matching condition.  The width of the methylene carbon signal can be conveniently used to evaluate the proton decoupling efficiency.  The width and shape of the carbonyl signal are very sensitive to the angle at which the sample is spun and can be used to set the magic angle with a reasonably high degree of precision.  In addition the carbonyl resonance is sharp and can be used as a secondary standard for chemical shift calibration.  If used as a secondary chemical shift standard, one must be aware that glycine has three polymorphic forms, each with different carbonyl chemical shifts.  The polymorphic form is not generally displayed on the reagent bottle and different suppliers may provide different polymorphs or mixtures of polymorphs.  It is therefore important to know which polymorph is being used to calibrate the chemical shift scale.   The α and γ polymorphs are the most common and stable, while the β polymorph is less stable and easily converted over time to the α polymorph.  Furthermore, the β polymorph has a very short ¹H T_1ρ at room temperature and therefore difficult to observe with typical millisecond CP contact times.  The γ polymorph can be converted to the α polymorph at 165°C.  The chemical shifts for the carbonyl resonances for the α and γ polymorphs are 176.5 ppm and 174.6 ppm, respectively.¹ The chemical shift of the β polymorph is between that of the α and γ polymorphs however, it is not usually observed.  The figure below shows the carbonyl region of the ¹³C CPMAS spectrum of three samples of glycine: pure α, pure γ and a mixture of the α and γ polymorphs.

If a single carbonyl resonance is observed for a sample of glycine using typical millisecond CP contact times, one can determine if it is the α or γ polymorph by measuring its chemical shift with respect to another chemical shift standard.  Alternatively, since the ¹H T_1ρ characteristics for the α and γ polymorphs are quite different from one another at room temperature, the authors of reference 1 report that a CPMAS spectrum collected with a 20 msec contact time will show almost no signal for the carbonyl carbon of the γ polymorph.  The carbonyl signal of the α polymorph, on the other hand, will be only slightly attenuated compared to a CPMAS spectrum measured with a 1 msec contact time.

1.  M.J. Potrzebowski, P. Tekely, Y. Dusausoy. Solid State Nuclear Magnetic Resonance. 11, 253 (1998).
2. R. E. Taylor. Concepts in Magnetic Resonance. 22A, 1 (2004).

What is T1ρ and How is it Measured?

The time constant for the build up of magnetization along the direction of the main magnetic field, B_o, (the z axis) either after a pulse or upon initially exposing a sample to the magnetic field is called the T₁ relaxation time or spin-lattice relaxation time. It is this relaxation time which determines the rate at which a pulse sequence can be repeated. The time constant for the decay of magnetization in the x-y plane of the rotating frame of reference after a pulse is called the T₂ relaxation time, the spin-spin relaxation time or the transverse relaxation time. It is this relaxation time which determines the natural line width of a particular resonance. There is another relaxation time constant of interest to NMR spectroscopists - T₁ρ. T₁ρ is the time constant for the decay of magnetization along the radio frequency field, B₁, of an applied spin locking pulse in the rotating frame of reference. It is analogous to T₁ except it describes relaxation along the radio frequency field of the pulse (which is static in the rotating frame) rather than relaxation along B_o. T₁ρ's are of interest in ROESY, TOCSY and cross polarization experiments. The T₁ρ is measured by first applying a 90 degree pulse to an equilibrium magnetization vector. A spin locking pulse is then applied. The phase of this pulse is shifted 90 degrees with respect to the excitation pulse such that the field of the spin locking pulse is coincident with the spin vector in the rotating frame of reference. During the spin locking pulse, the large magnetization vector (which was initially polarized in B_o) decays to its equilibrium value in the much smaller field, B₁, with time constant, T₁ρ. The T₁ρ is measured by analysing the intensity of the NMR signal in spectra collected as a function of the duration of the spin locking pulse. This is illustrated in the figure below.