Sampling methods 77

Several different infrared sampling methodologies are commonly used; these are transmission, diffuse reflectance and attenuated total reflection. Diffuse reflectance based infrared spectroscopy uses diffusely scattered infrared light, and is often used when a sample scatters strongly but absorbs only weakly. It is often used for studying solid particulate samples as little preparation is required, however, solid samples require grinding and diluting with materials such as KBr which are non-absorbing. As the signal collected using this sampling approach is usually very low, long acquisition times are needed to produce an acceptable SNR. To compare the data between two samples reliably the particles in the sample need to be of similar size distribution.

Transmission mode is the most well known form of infrared spectroscopy. A beam of infrared light passes through a sample as shown in Figure 4-6. Examination of the resulting radiation can determine which frequencies of infrared light have been absorbed. The advantages of this methodology are that the SNR is good and the accessories necessary are relatively cheap. It is also the most straightforward approach for quantitative analysis, as the path length of the beam in the sample is the same as the effective path length used in quantitative analysis (see Section 4.2.8). A disadvantage of this approach is that the sample must be thin enough to allow the radiation to pass through, but it must also be thick enough such that a reasonable amount of absorbance occurs. This can often require very careful sample preparation. A suitable thickness is usually between 5 and 20 μm for studies in the mid-infrared region. The form of the chosen sample is also an important factor, fluids are simple to prepare as cells with fixed path lengths can be used, whereas, working with solid samples is often non-trivial. Powders cause random reflections of the light as it passes through the sample, thus the beam can become too scattered or they will be too opaque for sufficient signal transmission. Solid samples will often require mixing with other non-absorbing

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materials such as KBr to a concentration of around 0.2-0.5 wt% of the sample. The KBr and the sample are ground together and pressed into a disc.

Figure 4-6. Schematic diagram of transmission sampling.

Attenuated Total Reflection (ATR) is a significantly more versatile sampling methodology which is described in detail by Harrick (Harrick, 1967). This technique relies on the principle of total internal reflection. A material with a high refractive index such as diamond which has a refractive index of 2.4 or zinc selenide, which has a similar value of refractive index, is often used in the form of an inverted prism. The sample is placed on the top surface of the crystal; the infrared radiation enters the crystal and approaches the top surface of the crystal at an angle greater than the critical angle. Although total internal reflection occurs at the interface between the crystal and the sample, the radiation does penetrate a few micrometers into the sample, in the form of an evanescent wave, where absorption of by sample attenuates the beam. This is illustrated in Figure 4-7.

Figure 4-7. Diagram illustrating refraction below the critical angle and internal reflection above the critical angle, where n1 and n2 are the refractive indices of media 1 and 2, n1 > n2 and θc is the critical angle.

Source Refracted beams θc Evanescent wave Internally reflected beam n1– refractive index medium 1 n2- refractive index medium 1 Source Sample Detector Beam

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Figure 4-8. Exponential decay of evanescent field into the medium with lower refractive index. dp is the depth at which the field amplitude has decayed to 1/e of its initial value at

the interface.

The penetration depth of the evanescent wave into the next medium dp, which is defined as the distance in which the amplitude of the electric field falls to 1/e of its initial value at the surface, can be calculated using Equation 4-6, where θ is the angle of incidence, λ is the wavelength of light and n1 and n2 are the refractive indices of media 1 and 2.

⁄ ⁄

Equation 4-6

The depth of penetration in Equation 4-6 is dependent on several factors listed above. Longer wavelengths have greater depth of penetration, the consequences of this will be discussed further in Chapter 7. The expected depth of penetration using common polymeric materials with refractive indices of around 1.5, is usually in the range 1 to 5 μm when working in mid-IR (Gupper et al., 2002).

To measure a sample in ATR mode, it must be placed on the surface of the crystal, therefore sample preparation can be very simple. The sample must be pressed onto the crystal with sufficient force to ensure comprehensive contact between the two media because of the shallow penetration depth of the evanescent wave. The crystal must be transparent to infrared radiation and have a high refractive index. Zinc selenide crystals are brittle and easily scratched but a large, optically consistent crystal can be obtained

Evanescent wave dp Low refractive index medium High refractive index medium

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relatively cheaply. Diamond is another option however; it is expensive and absorbs certain frequencies of infrared radiation, especially at high temperatures. The main advantages of diamond are that it is able to endure high pressures, its chemical resistance and it is exceptionally hard making it scratch resistant.

A general perception about ATR spectroscopy is that a high level of force is required to achieve a good contact between sample and ATR crystal, which is often not the case. Adequate contact can be achieved with liquid samples simply by covering the top of the crystal with the sample, although, when working with polymeric materials, some compaction force is required to achieve sufficient contact. The required force can be minimised as flat surfaces help facilitate the contact, while malleable samples require minimum force in compaction. When working with some pharmaceutical polymeric materials in dissolution experiments, the formation of a gel upon contact of the polymeric sample with water greatly improves the contact of the polymer (van der Weerd and Kazarian, 2004b). Although the compaction force used for compacting tablets of the ATR crystal is significantly lower than in industrial tablet presses, the image data obtained using these in situ compaction methods show good contact for pharmaceutical formulations. This demonstrates that the force used to compact tablets in situ is sufficient to achieve good contact. Nevertheless, while compaction with a diamond as an ATR crystal is not an issue, it can be harder to achieve a satisfactory compaction on some other crystals, such as zinc selenide, without incurring damage to the crystal.