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In General Chapter <781> Optical Rotation, “Specific Rotation” and “Angular Rotation” are two different procedures. Angular rotation requires reporting the observed value of rotation but not calculating the specific rotation. Angular rotation is used for pure materials where there is no dilution. When reporting the specific rotation, the equations in General Chapter <781> are used to calculate the result of specific rotation. Therefore, these two procedures are not interchangeable.
Some USP monographs require the test of “Specific Rotation” or “Angular Rotation”. However, if a USP monograph does not require these tests, an appropriate method that is fit for purpose may be used for the test material.
To measure specific rotation for a liquid, the equations in General Chapter <781> shall be used to calculate the result of the neat liquid specific rotation. To report a specific rotation for a pure liquid, a user would calculate the result based on the density of that liquid. Please refer to the following equation:
Density is only needed in the calculation as specified in General Chapter <781>.
No, the value will be reported without further calculations. However, USP monographs require measurements on solutions (i.e., diluted analytes) that always use specific rotation rather than angular rotation. Angular rotation is used for neat liquids.
Yes, the density value needs to be measured. To report a specific rotation for a solution when making a solution on a weight/weight basis (or sample solution, g/g), the density is important, and its value of the solution needs to be measured. Please refer to the following equation:
Density is only needed in the calculation as specified in General Chapter <781>.
No. Density value is not required when making a solution on a volume basis (or sample solution, g/mL) to report a specific rotation for a solution. Please refer to the following equation:
Density is only needed in the calculation as specified in General Chapter <781>.
The qualification of an instrument shall be designed to ensure that it is fit for purpose. If an instrument is used for identification testing of a raw material, the bandwidth and wavelength may not be as critical if the instrument is used for quantification of the concentration of an optically active analyte. Therefore, it is not appropriate to require every optical rotation instrument to have a specific requirement for the light source. It is important that the instrument parameters be documented as fit for purpose (including the details of wavelength and bandwidth) and these parameters can be used for re-qualification and for assay transfer exercises.
The wavelength and bandwidth may affect the accuracy of the specific rotation value. If an instrument is used for an identification measurement, it may still allow an instrument to be fit for purpose without strictly controlled bandwidth. If an instrument is used to assess the concentration of an optically active analyte, then it may not be fit for use without the appropriately controlled bandwidth.
It is the responsibility of the user to demonstrate that the instrument is fit for purpose. A user may consult their specific equipment manual or contact the manufacturer regarding accuracy of the wavelength and bandwidth.
Linearity is specifically listed as part of the OQ section in General Chapter <781>. Linearity is required if the optical rotation measurement is used for quantitation of an enantiomer. The user needs to determine if a test shall be part of the ongoing PQ based on the experimental purpose.
General Chapter <781> does not specify whether linearity needs to be determined with a dextrorotary or levorotatory compound. USP does not provide a specific recommendation for a dextrorotary or levorotary compound. A user may consult their specific equipment manual or contact the manufacturer regarding appropriate standards.
For a linearity test, an example is shown in the Table below. The Table shows preparation sucrose solutions at nominal values of 10%, 20%, 30%, 40%, and 50% w/v and evaluates the linearity of the optical rotation. The acceptance criterion is that the R2 value should be NLT 0.99.
Table. Reference Values for Optical Rotation of Sucrose Solutions at 589 nm (sodium D-line) at a Temperature of 20°C and 25°C
| Sucrose % w/v |
1-dm cell, 20 °C Optical Rotation (degrees) |
1-dm cell, 25 °C Optical Rotation (degrees) |
|---|---|---|
| 0.0 | 0.000 | 0.000 |
| 10.0 | 6.664 | 6.637 |
| 20.0 | 13.319 | 13.258 |
| 30.0 | 19.958 | 19.862 |
| 40.0 | 26.575 | 26.442 |
| 50.0 | 33.158 | 32.989 |
Linearity of the instrument and linearity of the assay on a particular compound should be considered two separate issues.
Linearity of the instrument is assessed during the IQ/OQ/PQ of the instrument and may use a well-characterized standard compound.
Verification of the method for a particular compound or sample is typically performed to assess the assay performance and the verification should be determined according to General Chapter <1226> (see also General Chapter <1225>).