Table 1 identifies several common lasers used for pharmaceutical applications. UV lasers have also been used for specialized applications but have various drawbacks that severely limit their utility for general analytical measurements.

Table 1. Typical Lasers Used in Pharmaceutical Applications

A wide variety of sampling arrangements are possible, including direct optical interfaces, microscopes, fiber optic-based probes (either noncontact or immersion optics), and sample chambers (including specialty sample holders and automated sample changers). The sampling optics may also be designed to obtain the polarization-dependent Raman spectrum, which often contains additional information. Selection of the sampling device will often be dictated by the analyte. However, considerations such as sampling volume, speed of the measurement, laser safety, and reproducibility of sample presentation should be evaluated to optimize the sampling device for any given application.

Scattered light at the laser wavelength (Rayleigh) is many orders of magnitude greater than the Raman signal and must be rejected prior to the detector. Notch filters are almost universally used for this purpose and provide excellent rejection and stability combined with small size. The traditional use of multistage monochromators for this purpose, although still viable, is now rare. In addition, various filters or physical barriers to shield the sample from external radiation sources (e.g., room lights, laser plasma lines) may be required depending on the collection geometry of the instrument.

The wavelength may be processed by either dispersion or interferometry (Fourier transform). The specific benefits and drawbacks of each of the dispersive designs compared to the FT instrument are beyond the scope of this chapter. Any properly qualified instruments should be suitable for qualitative measurements. However, care must be taken when selecting an instrument for quantitative measurements, as dispersion and response linearity may not be uniform across the full spectral range (for example, when using an echelle spectrograph).

The silicon-based charge-coupled device (CCD) is the most common detector for dispersive instruments. The cooled array detector allows fast, full-spectrum measurements with low noise. It also has peak wavelength responsivity when matched to the commonly used 785-nm diode laser. Fourier transform instruments typically use single-channel germanium or indium–gallium–arsenide (InGaAs) detectors responsive in the NIR to match neodymium:yttrium–aluminum–garnet (Nd:YAG) 1064-nm excitation.

In the case of FT–Raman instruments, primary wavelength-axis calibration is maintained with an internal He–Ne laser. Most dispersive instruments utilize atomic emission lamps for primary wavelength-axis calibration. In all Raman systems suitable for analytical Raman measurements, the vendor will offer a procedure of x-axis calibration that can be performed by the user. For dispersive Raman instruments, a calibration based on multiple atomic emission lines is preferred. The validity of this calibration approach can be verified subsequent to laser wavelength calibration using a suitable Raman shift standard. For scanning dispersive instruments, calibration may need to be performed more frequently, and precision in both a scanning and static operation mode may need to be verified.1

Laser wavelength variation can impact both the wavelength precision and the photometric (intensity) precision of a given instrument. Even the most stable current lasers can vary slightly in their measured wavelength output. The laser wavelength must therefore be confirmed to ensure that the Raman shift positions are accurate for both FT–Raman or dispersive Raman instruments. A reference Raman shift standard material such as those outlined in ASTM E1840-96(2002)1 or other suitably verified materials can be utilized for this purpose. [NOTE—Reliable Raman shift standard values for frequently used liquid and solid reagents, required for wavenumber calibration of Raman spectrometers, are provided in the ASTM Standard Guide cited. These values can be used in addition to the highly accurate and precise low-pressure arc lamp emission lines that are also available for use in Raman instrument calibration.] Spectrophotometric grade material can be purchased from appropriate suppliers for this use. Certain instruments may use an internal Raman standard separate from the primary optical path. External calibration devices exactly reproduce the optical path taken by the scattered radiation. [Note—When chemical standards are used, care must be taken to avoid standard contamination and to confirm standard stability.]

Unless the instrument is of a continuous calibration type, the primary wavelength axis calibration should be performed, as per vendor procedures, just prior to measuring the laser wavelength. For external calibration, the Raman shift standard should be placed at the sample location and measured using appropriate acquisition parameters. The peak center of a strong, well-resolved band in the spectral region of interest should be evaluated. The position can be assessed manually or with a suitable, valid peak-picking algorithm. The software provided by the vendor may measure the laser wavelength and adjust the laser wavelength appropriately so that this peak is at the proper position. If the vendor does not provide this functionality, the laser wavelength should be adjusted manually.

Calibration of the photometric axis can be critical for successful quantitation using certain analytical methods (chemometrics) and method transfer between instruments. Both FT–Raman units and dispersive Raman units should undergo similar calibration procedures. The tolerance of photometric precision acceptable for a given measurement should be assessed during the method development stage.

To calibrate the photometric response of a Raman instrument, a broad-band emission source should be used. There are two accepted methods: Method A, which utilizes an NIST-traceable tungsten white light source2 (and is applicable to all common laser excitation wavelengths listed in Table 1) and Method B, which utilizes NIST SRM 2241,3 a doped-glass fluorescence source that is currently available only for systems with 785-nm nominal excitation.

Detailed functional validation employing external reference standards is recommended to demonstrate instrumental suitability for laboratory instruments, even for instruments possessing an internal calibration approach. The use of external reference standards does not negate the need for internal quality control procedures; rather, it provides independent documentation of the fitness of the instrument to perform the specific analysis/purpose. For instruments installed in a process location or in a reactor where positioning of an external standard routinely is not possible, including those instruments possessing an internal calibration approach, periodic checking of the relative performance of an internal vs. external calibration approach should be made. The purpose of this test is to check for change in components that may not be included in the internal calibration method (process lens, fiber-optic probe, etc.), e.g., photometric calibration of the optical system.

It is important to ensure the precision of the wavelength axis via calibration to maintain the integrity of Raman peak positions. Wavelength calibration of a Raman spectrometer consists of two parts: primary wavelength axis and laser wavelength calibration. After calibrating both the primary wavelength axis and the laser wavelength, instrument wavelength uncertainty can be determined. This can be accomplished using a Raman shift standard such as the ASTM shift standards or other suitably verified material. Selection of a standard with bands present across the full Raman spectral range is recommended so that instrument wavelength uncertainty can be evaluated at multiple locations within the spectrum. The tolerance of wavelength precision that is required for a given measurement should be assessed during the method-development stage. [NOTE—For scanning dispersive instruments, calibration may need to be performed more frequently, and precision in both a scanning and static operation mode may need to be verified.]

Laser variation in terms of the total emitted photons occurring between two measurements can give rise to changes in the photometric precision of the instrument. Unfortunately, it is very difficult to deconvolute changes in the photometric response associated with variations in the total emitted laser photons from the sample and sampling-induced perturbations. This is one of the reasons why absolute Raman measurements are strongly discouraged and why the photometric precision specification is set relatively loosely. The tolerance of photometric precision required for a given measurement should be assessed during the method-development stage.

The wavelength precision should be measured by collecting data for a single spectrum of the selected Raman shift standard for a period equal to that used in the photometric consistency test. Peak positions across the spectral range of interest are used to calculate precision. Performance is verified by matching the current peak position to those collected during the previous instrument qualification and should not vary by more than ±0.3 cm–1, although this specification may be adjusted according to the required accuracy of the measurement.

The photometric consistency should be measured by collecting data for a single spectrum of a suitably verified reference standard material for a specified time. The areas of a number of bands across the spectral range of interest should be calculated using an appropriate algorithm. The most intense band area is set to an intensity of 1, and all other envelopes are normalized to this band. Performance is verified by matching the current band areas to their respective areas collected during the previous instrument qualification. The areas should vary by no more than 10%, although this specification may be adjusted according to the required accuracy of the measurement.

This test is applicable only to Raman instruments with automatic, internal laser power meters. Instruments without laser power measurement should utilize a calibrated laser power meter from a reputable supplier. The laser output should be set on a representative output, dictated by the requirements of the analytical measurement and the laser power measured. The output should be measured and checked against the output measured at instrument qualification. The power (in milliwatts or watts) should vary by no more than 25% compared to the qualified level. If the power varies by more than this amount, the instrument should be serviced (as this variation may indicate, among other things, a gross misalignment of the system or the onset of failure of the laser).

For instruments with an automatic, internal laser power meter, the accuracy of the values generated from the internal power meter should be compared to a calibrated external laser power meter at an interval of not more than 12 months. The internally calculated value should be compared to that generated by the external power meter. Performance is verified by matching the current value to that generated during the previous instrument qualification. The manufacturer may provide software to facilitate this analysis. If the instrument design prevents the use of an external power meter, then the supplier should produce documentation to ensure the quality of the instrument and provide a recommended procedure for the above analysis to be accomplished during a scheduled service visit.