Infrared Microspectroscopy Reflection Theory Microscope Training Course

Reflectance Methods in FT-IR Microscopy Fundamental Considerations of FT-IR Microscopy 1. Specular 2. Diffuse 3. Reflection-Absorption 4. Grazing Angle 5. Internal Reflection (Attenuated Total Reflection) Infrared reflection measurements are perceived to be desirable because of reduced sample preparation. If the sample’s spectrum can be measured via reflectance, then it is unnecessary to prepare thin films needed for transmission measurements. Thick samples, thin films on metallic substrates, intractable surface contaminants, or samples that require nondestructive reflectance testing are all candidates for reflectance FT-IR microscopy. However, there is a price to be paid for reduction in sample preparation. By nature, reflectance spectra often require extra data manipulation steps prior to interpretation or special objectives for spectral collection. There are five types of reflection FT-IR microscopy methods. Specular, diffuse and reflection-absorption measurements are made with the standard objective lens in the reflection operational mode. Both grazing incidence and internal reflection require special objectives. The details of each reflection method is discussed in detail.

Specular Reflection Specular Reflectance Specular reflectance is the front surface reflection from the exterior surface of a material; i.e., the beam does not penetrate the material. The angle of incidence is equal to the angle of the reflection. Specular Reflectance

Specular Reflection Wavenumbers Polymer by Specular Reflectance Polymer after Kramers-Kronig 0.0 0.5 1.0 Absorbance 2000 4000 Wavenumbers 3000 1000 Specular reflectance spectra are markedly different from transmission spectra. The reflectance spectrum of the polymer possesses the derivative shaped bands typically observed for specular reflectance measurements. The efficiency for specular reflection depends on its refractive index. For a material with a refractive index of 1.5, the reflectivity is only 4%. The reflectivity for the present polymer is approximately 4.5%, as indicated by the spectral “baseline”. Specular reflectance data can be resolved into two optical parameters; refractive index and extinction coefficient, via the Kramers-Kronig theory. The distinctive shape of specular reflection data is a consequence of variations in these parameters. Notice that, as absorption maxima are approached from the higher wavenumbers, the refractive index decreases initially, causing the reflectivity to decrease. The refractive index increases rapidly (as does the extinction coefficient), resulting in sharp maxima. The specular reflection spectrum of the polymer sample after conversion via the Kramers-Kronig theory compares favorably with transmission like data. Band position and intensity are also transmission like, so that electronic libraries may be routinely used to identify materials.

Reflection vs Transmission Data Ratio of 1374 / 1466 cm-1 Peaks 3 45% 40% k Spectrum Ratio 25% 30% 14% 9% Specular reflection is useful as a quantitative technique. The table above shows the results of analyses on a series of copolymers that were studied in transmission and in reflection. The reflection spectra were transformed via the Kramers-Kronig theory, and the ratio of the areas under the 1313 cm-1 and 1466 cm-1 bands was calculated. The same band ratios were calculated for the transmission data, and a linear relationship was determined to exist between the reflectance data and the transmission data. 3 Absorbance Spectrum Ratio

Diffuse Reflection Sample KBr I T I S Specular Transmission I T & S I Typically, in diffuse-reflectance measurements, several interactions occur simultaneously. The most useful interaction (upper left) occurs when the input radiation passes through an analyte particle, then reflects from the KBr particle; this directs the radiation back to the detector. Specular reflection (upper right) occurs when the input ray reflects from the outer surface of an analyte particle. When the input radiation passes through one analyte particle and reflects from the surface of another analyte particle in route to the detector (lower left), a combination of transmission and specular reflection data is obtained. Yet another path that the radiation can follow is one of total absorption (lower right), where (due to repeated reflection and absorption) the input radiation does not re-emerge. Diffuse reflectance experiments and apparatus are designed to maximize transmission interactions (upper left) while minimizing all other modes. Specular contributions can be reduced by placing a physical obstruction between the source and the sample. This technique has been most useful for diffuse reflectance devices in which the source, sample and detector have been placed in a straight line. An alternate method is to collect the diffusely reflected radiation at angles other than 180 degrees. Difficulties in eliminating the spectular reflectance interaction and the low collection efficiency of diffuse measurements (typically, 4% to 10%) limit its utility in FT-IR microscopy. Transmission & Specular Total Absorption

Diffuse Experiment (1-R) k’ f (R) = = 2R s 1.6 Diffuse 1.4 1.2 1.0 Absorbance 0.8 Absorbance 0.6 0.4 0.2 0.0 A sample of a display monitor chassis was analyzed using both diffuse reflection and transmission. The display monitor chassis was analyzed via diffuse reflectance by rubbing the surface of the display monitor chassis with a piece of silicon-carbide paper and then collecting the diffusely-reflected radiation from the paper. A gold mirror was scanned to serve as a background for the measurement. The Kubelka-Monk relation which corrects for the inherent scatter losses in any diffuse-reflectance measurements, was applied to the diffuse data; the top spectrum is the result. The spectrum of the display monitor chassis measured in the transmission mode is shown for comparison. 3500 3000 2500 2000 1500 1000 Wavenumbers (cm-1) R = the ratio of the diffuse reflectance of the sample at infinite depth. s = a scatter constant. k’ =2.303kc, where k is the extinction coefficient. c = concentration (1-R) 2 k’ f (R) = = 2R s

Reflection Absorption Film Reflecting Substrate Reflection absorption occurs when a thin, absorbing layer of material is on the surface of a more highly reflective substrate. The input ray passes through the thin film, reflects from the substrate, passes through the film a second time, and then passes on to the detector. The ideal reflection-absorption interaction occurs when a thin, absorbing layer is supported by a polished metallic surface. The signal-to-noise ratio is very high for films on metal substrates due to the high collection efficiency of the measurement (typically 80%).

Sample by Reflection-Absorption Contamination on Circuit Board 0.6 0.4 Absorbance 0.2 This is a spectrum obtained from a contaminated circuit board. There were problems with solder sticking to the gold contacts. For many infrared spectroscopists, this will be an all-too-familiar spectrum. The contamination is poly(dimethylsiloxane) or silicone lubricant. It seems that the metal belt that transported the boards through the soldering machine was squeaking so someone went to the hardware store, bought some WD-40 and sprayed it on the belt. 4000 3000 2000 1000 Wavenumbers

Transmission / Reflection Absorption Comparison Comparison of the infrared spectrum of very thin polystyrene film recorded by transmission with that of the same film recorded by reflection absorption method. Reflection absorption measurements at near normal incidence (0-45 degrees) have reduced utility for films less than 1um thick. The table above summarizes a comparison between transmission and reflection absorption measurements made on a thin film of polystyrene. The sample under analysis was prepared by casting a thin film of polystyrene on water and then lifting the film from the water (using a perforated sheet of aluminum). Transmission measurements were made through the holes. In a reflection absorption measurement the infrared radiation passes through the film twice. Therefore reflection absorption measurements should be twice as strong as the transmission bands. Experimental data do not support this. The ratio of the transmission to reflection absorption data (last row in the table) is > 1 for all of the bands measured, indicating a reduction in sensitivity of reflection absorption interaction for the sub-micron film studied here.

Grazing Incidence I Thin Film Reflecting Substrate Films that are too thin for near normal reflection absorption spectroscopy (RAS) may often be detected using grazing incidence spectroscopy. Grazing incidence is achieved when the angle of incidence is between 60 degrees and 90 degrees. Grazing incidence measurements have greater sensitivity than near-normal RAS because the electric field’s strength is enhanced at the surface of the metallic substrate. Grazing angle measurements are commonly used to measure ultra-thin films from 2.0 nanometers to a micrometer. Even monolayers can be analyzed with grazing angle when they are on reflective metal surfaces. Grazing angle can be used in qualitative analysis of these films, to measure thickness and study molecular orientation.

Grazing Angle Objective Survey Mode Grazing Mode The grazing angle objective has two modes of operation, viewing and grazing. When the objective is in the view mode, an aperture with a central opening is used to allow near normal illumination of the sample. With the objective in the view mode, the area of interest is positioned in the center of the field of view. The mode is then changed to grazing incidence by sliding an annular ring into position. This blocks the near normal rays, allowing only rays between 65 degrees to 85 degrees to pass. The sample image is then refocused so that spectral data can be collected.

Monolayers Analysis by Grazing Angle Reflectance Spectroscopy Polarization Effects Phase shift of the electric vectors of “S” polarized light at the incident plane results in an electric field intensity of Zero. Only “P” polarized light produces a phase shift to enhance the electric field acting on the molecule The use of the Grazing Angle Objective (GAO) is to analyze the “P” polarized light from an ultra thin coating on an metal substrate. The electric field created by the “S” polarized light effectively cancels itself, adding nothing to the electric field potential.

Polarization Effects Polarization: E| | or Ep Polarization: E^ or Es Here is an illustration that shows the orientation of the electric field that occurs at the surface of the substrate. Notice the polarization effect in both illustrations, the “P” polarization and the “S” polarization. increased electric field at surface zero electric field at surface

to 15X & 32X Reflachromat Objectives Grazing Angle Objective Comparison to 15X & 32X Reflachromat Objectives 0.009 GAO - Aflunox 606 32x obj. - Aflunox 606 0.008 15x obj. - Aflunox 606 0.007 0.006 0.005 Absorbance 0.004 0.003 0.002 A thin film of Aflunox 606 on gold was analyzed using the Grazing Angle objective (red), the near normal incidence of the 15X Reflactromat objective (green), and the near normal incidence of the 32X Reflactromat objective (violet). The increase in intensity of the bands in the grazing incidence measurements illustrates the greater sensitivity of this technique over near normal measurements. 0.001 0.000 1600 1400 1200 1000 Wavenumbers (cm-1)

Internal Reflection n2 n1 n1 > n2 Critical Angle Source point The ATR Objective initiated a new field of FT-IR microscopy. With the ATR objective, any surface making contact with the internal reflection element will produce an infrared ATR spectrum. The basis for ATR microscopy is the internal reflection of infrared radiation within a high index internal reflecting element (IRE). Light that travels from a medium of a given refractive index (n1) to a medium with a lower refractive index (n2) at an angle above the critical angle will be reflected back into the first medium. This internal reflection creates an electric field (evanescent wave) that extends beyond the IRE’s surface. When a material is brought in contact with the IRE, the evanescent wave extends into the material and may be absorbed. Critical Angle n1 Source point

Attenuated Total Reflection Internal Reflection Crystal Here is an illustration of the IR beam path in the ATR crystal. Depending on the crystal material, the evanescent wave can have a depth of penetration from 0.5um to 2.0 um. Attenuated Total Reflectance (ATR) technology has revolutionized FT-IR non-destructive infrared surface analysis on a microscopic scale. Evanescent Wave Penetration Depth (0.5-2um)

with available ATR Crystals ATR Objective with available ATR Crystals Ge Diamond The ATR objective has (4) crystal materials to choose from. The standard ATR objective uses a, Zinc Selenide (ZnSe) crystal. The other (3) options are Diamond, Silicon (Si), and Germanium (Ge). ZnSe Si

ATR Objective Survey Mode Contact Mode ATR Mode In addition to its infrared measuring mode, the ATR objective has two viewing modes which enable the user to see the area of interest prior to analysis. In the survey mode, light passes through a central aperture, several glass lenses, and the ATR crystal (ZnSe or Diamond). In this mode the sample can be seen and the area of interest may be positioned in the center of the field of view. In order to collect spectra of a sample, the sample must be in contact with the IRE. Such contact is established in the second (viewing) mode, called the contact mode. When the ATR objective is in the contact mode, the light path is followed enabling the user to view the surface of the IRE. When the sample is brought into contact with the surface of the IRE, it is seen as a wetting effect. After contact is established, and while still in the contact mode, the appropriate aperture size is selected. Finally, the ATR mode is selected, and the spectral data are collected. In the ATR mode, infrared radiation passes into the IRE, interacts with the sample and then returns to the detector.

Visual Modes of an ATR Objective Survey Mode Contact Mode The above images show the use of the visual mode of the ATR objective when using the Zinc Selenide (ZnSe) or the Diamond IRE. One can survey the area of interest, then monitor the IRE making contact to the desired area of interest. Adjusting the variable mask, allows selection of the desired area of interest for data collection. ATR MODE Aperture Vertical Blades Aperture Horizontal Blades

Transmission vs ATR Spectrum Transmission Spectra / NEC multisync 4FG display monitor 100 80 %Transmittance 60 40 20 ATR objective ZnSe crystal spectra / NEC mulisync 4FG display monitor 100 80 %Reflectance The above spectra are of the same NEC multisync 4FG display monitor chassis. The upper spectrum was collected by transmission of a sample prepared in a micro-compression cell. The lower spectrum was collected using the ATR objective and the ZnSe IRE. The intensity differences observed between the two spectra are due to the change in the depth of penetration at different wavelengths for the ATR measurements. The relatively shallow depth of penetration (=1 um) obtained with the ATR objective enables the user to study surfaces. Surface treatments, surface oxidation, and surface coatings may all be examined with the ATR objective. With a thin film of polyethylene being analyzed by both transmission FT-IR microscopy and ATR microscopy, the transmission results would indicate that the sample is mostly polyethylene with a small amount of fluorine. The ATR results show that the sample is polyethylene with a greater amount of fluorine than is seen a transmission spectrum. When considered together, the two spectra indicate that a film was fluorinated at the surface. 60 40 20 3500 3000 2500 2000 1500 1000 Wavenumbers (cm-1)

Depth of Penetration dp = l / 2 p no (sin2Q - n2/no2) Refractive indices of IRE and Sample Angle of Incidence Wavelength of the Light The depth of penetration of an internal reflectance measurement is dependent on the wavelength of radiation, IRE refractive index, sample refractive index, and angle of incidence of the radiation. dp = l / 2 p no (sin2Q - n2/no2)

Depth of Penetration 1.8 Refractive Index of sample = 1.4 1.6 1.4 ZnSe 1.2 1.0 Penetration Depth (mm) 0.8 0.6 Ge 0.4 As the wavelength of the radiation increases, so does the depth of penetration. The consequence of this variation in penetration depth is a relative decrease in sensitivity in the shorter wavelength regions. 0.2 4000 3000 2000 1000 Wavenumbers (cm-1)

Depth of Penetration ZnSe (45o) Hexane RI = 2.4 Si (45o) Hexane RI = 3.4 1.0 Ge (45o) Hexane RI = 4.0 0.8 0.6 Absorbance 0.4 Increasing the refractive index of the IRE (ATR crystal) will decrease the depth of penetration. Higher refractive index IREs are often used when the outer most surface of a material is the area of interest. Depth of penetration may also be varied altering the angle of incident radiation. Higher angles of incidence are used when shallow depths of penetration are required. 0.2 0.0 3200 3100 3000 2900 2800 2700 Wavenumbers (cm-1)

Benefits of Different IRE Materials 100 95 90 %Reflectance Survey Mode 85 80 Silk Fiber 75 70 4000 3500 3000 2500 2000 1500 1000 Contact Mode Above are the major benefits in using the Zinc Selenide (ZnSe) crystal with the ATR objective. The refractive index of ZnSe is 2.4. Wavenumbers (cm-1) ZnSe ATR Internal Reflection Element : - Allows the ability to survey the area of interest (Survey Mode) - Gives confidence that crystal is in contact with sample (Contact Mode) - The ZnSe crystal has a depth of penetration of 2 um.

Benefits of Different IRE Materials 100 98 96 94 92 %Reflectance 90 88 86 Black Carbon O’Ring 84 82 80 78 Above are the major benefits in using the Germanium (Ge) crystal with the ATR Objective. The refractive index of Ge is 4.0. 76 4000 3500 3000 2500 2000 1500 1000 Wavenumbers (cm-1) Ge ATR Internal Reflection Elements: - Ideal for highly absorbing carbon filled material - The Ge crystal has a depth of penetration of 0.66 um

Benefits of Different Crystals 76 78 80 82 84 86 88 90 92 94 96 98 100 %Reflectance 1000 1500 2000 2500 3000 3500 4000 Wavenumbers (cm-1) Very Hard Crystal Above are the major benefits in using the diamond crystal with the ATR objective. The refractive index of diamond is 2.4. Diamond ATR Internal Reflection Element: - Has the ability to perform survey and contact modes - Ideal for very hard samples - The Diamond crystal has a depth of penetration of 2 um.

Reflection Experiments Practical Hands-On Applications Using Special Objectives