10/11/ ENGINEERING RESEARCH CENTER FOR S TRUCTURED O RGANIC P ARTICULATE S YSTEMS RUTGERS UNIVERSITY PURDUE UNIVERSITY NEW JERSEY INSTITUTE OF TECHNOLOGY UNIVERSITY OF PUERTO RICO AT MAYAGÜEZ Vibrational Spectroscopy for Pharmaceutical Analysis Part V. Methods for Obtaining Mid-IR spectra of Samples. Rodolfo J. Romañach, Ph.D.

2 Working with Liquids  Spectra of Solute are not as simple as in Ultraviolet region because solvents absorb IR radiation.  Some solvents have spectral windows, CCl 4 from cm -1 and CS 2 from cm -1.  Hexane and iso-octane may also be used but with narrower spectral windows.

3 Working with Liquids  Capillary film method (like closing a door).  Adjustable pathlength cells are available.  Fixed pathlength cells.  For quantitative work, thickness should such that the transmission is between % (absorbance is ).

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5 Sealed (Fixed Pathlength) Cells  Sealed cells should not be opened, should be left together.  If cell thicker than 0.1 mm, hold the cell at a slightly inclined angle(pencil underneath) and press gently on the syringe to fill cell.

6 Flow through cell to be used with luer lock syringe. Sealed Transmission Cell

7 Sealed Cells(thicker than 0.1 mm)  Hold at an Inclined Angle.  Place empty syringe at upper port, and the syringe with sample at lower port.  Fill the cell by withdrawing liquid with the empty syringe.  Excess liquid should be removed from the filling ports.  The plugs should be placed in the ports and rotated to aid in producing a tight seal between the plug and cell.

8 Working with Solids (Mulls)  Powdered crystalline solids transmit very little IR radiation, for the light will be scattered by multiple reflections of the particles.  The technique of dispersing a solid in a liquid can be used to reduce the amount of light scattered from the solid.

9 Working with Solids (Mulls) (2)  Scattering will be reduced by reducing particle size and sorrounding particles with a medium whose refractive index closely matches theirs.  Want to reduce the particle size of the solids to less than 2 m, when preparing mulls.

10 Mulls (Technique)  Use a large mortar and pestle to put enough pressure on sample to grind it.  Place about 25 mg of material in mortar.  After crushing, grind with a circular motion until it cakes on side of mortar and forms a glossy surface reflecting visible light.  Add a small drop of mulling fluid to mortar, mix to form a paste. With rubber policeman to place as thin film between cells.

11 Mulls (Technique) (2)  Use mineral oil ( cm -1 ) or fluorolube ( cm -1).  The compound will not be reduced after addition of mulling agent, so it is important to grind well previously.

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13 KBr disk-Advantages  Spectra are obtained which are free from interfering bands.  Less light is scattered.  Pellets can be stored for some time.

14 KBr disk-disadvantages  Some water always remains as an impurity.  Spectra of some samples do not agree with those measured in other media.  Physical or chemical changes can occur in the sample during the grinding or pressing process.  For ionic samples, exchanges of the halide with sample ions can occur.

15 KBr disk-Procedure  First grind the sample to about m particle size.  About 3 mg of sample is mixed gently with mg of spectral quality KBr.  KBr should be placed in drying oven at C about once a week and dried overnight. A vacuum oven is even better.

16 Making a KBr Pellet

17 KBr pellet holder

18 Modern ATR Accesories MIRacle TM Single Reflection ATR (left), HATR Multi- Reflection ATR (right). Available from Pike Techonology, (

19 Attenuated Total Reflectance (ATR) An ATR crystal (crystal with a high refractive index) is in contact with sample. The radiation is focused on the crystal and total internal reflection occurs at the crystal, but part of the electric field (evanescent wave) penetrates into sample at the point of reflection. The sample will absorb some of the radiation, and thus a transmission spectrum is obtained.

20 ATR Crystal The ATR Crystal (also called internal reflection element) is made with materials that have a high refractive index, such as ZnSe, Si, Ge, and diamond. Some of these accessories are configured to allow the radiation to bounce several times against crystal (increase sensitivity as radiation penetrates sample several times).

21 ATR - Advantages Non-destructive method of analysis. May be used for samples that are too thick and are not suitable for transmission analysis (the radiation cannot pass through a sample that is too thick). Allows analysis of fibers, pastes, powders, viscous liquids, papers, rubber, coatings, adhesives. May be used to obtain spectra in aqueous solutions (short pathlength limits the absorbance of O-H bands). Good method for analysis of surfaces as the radiation penetrates only a few micrometers into sample.

22 ATR Spectra vs. Transmission Spectra ATR and transmission spectra resemble each other, but there will be some differences because the pathlength is not uniform in ATR.

23 Gas Cells The molecules are dispersed, but the radiation is reflected several times through the cell. A 20 m cell path-length may be obtained with a cell cavity of about 0.5 m in length. Pike Technologies J.M. Chalmers and G. Dent, Industrial Analysis with Vibrational Spectroscopy, The Royal Society of Chemistry 1997, page 141 – 142.

24 Diffuse Reflectance (DRIFTS) - Reflectance is termed diffuse where the angle of reflected light is independent of the incident angle Spectra Affected by:  Particle size of sample.  Packing density of sample, and pressure on sample.  Refractive index of sample.  Crystalline form of sample.  Absorption coefficients of sample.  Characteristics of the sample’s surface. J.M. Chalmers and G. Dent, “Industrial Analysis with Vibrational Spectroscopy”, Royal Society of Chemistry, 1997, pages

25 Diffuse Reflectance For mid-IR spectroscopy diffuse reflectance spectra normally require grinding of the sample and KCl powder. The analyte is usually diluted to about 10% (wt/wt) in KCl. Dilution is not necessary in NIR spectroscopy. Pike Technologies J.M. Chalmers and G. Dent, “Industrial Analysis with Vibrational Spectroscopy”, Royal Society of Chemistry, 1997, pages

26 Infrared Microscopy Infrared Microscopy may be used to focus beam on a small sample area. A spectrum may be obtained for a sample as small as 10 μm. Slide Courtesy Smiths Detection

27 Optics ATR objective Slide Courtesy Smiths Detection

28 Spectrum obtained may be compared to spectra in library. Slide Courtesy Smiths Detection

29 Validation of an IR Method  Validated a method to identify desiccant raw material as an alternate method to XRF spectroscopy (USP method).  Selected several lots of the desiccant which had been previously identified as desiccant according USP procedure.

30 Validation of an IR Method  Obtained 3 spectra from each batch of the desiccant.  Removed desiccant from capsule, milled with mortar and pestle. Prepared KBr pellet.  Checked interbatch variation of spectra.  Prepared procedure for KBr pellet sample preparation.  Evaluated analyst to analyst variation by having two other analysts prepare samples.  Checked method with other batches (validation batches).

31 Caution with KBr  Very hygroscopic.  If you do not protect the pellet, the OH stretch band will grow.  OH band could become the strongest band in the spectrum.

32 Desiccant spectrum – IR method developed.

33 Assay Validation Requirements Studies IDImpurities Final Product Dissolution, CU Accuracy -++ Repeatability +++ Interm. Precision +++ Specificity (2) +++ Detection Limit -+- Quantitation Limit -+- Linearity -++ Range -++