NIR Fundamentals and “a little more…” Graduate Students – Yleana M. Colón Andres Román Daniel Mateo

Electromagnetic Spectrum 12,800 cm-1 (780 nm) 4,000 cm -1 (2500 nm) Frequency (cm-1) 108 107 106 105 104 103 102 101 1 10-1 10-2 10-3 -Ray X – Ray Ultraviolet v i s i b l e NIR MIR FIR ESR NMR Region Infrared Microwave Radio, TV Waves Nuclear Transitions Inner Shell Electronic Transitions Valance Electron Transitions Molecular Vibrations Molecular Rotations Spin Orientation in Magnetic Field Interaction Wavelength (nm) 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 1 11 Courtesy of Bruker Optics

Spectroscopy … is based on the interaction of electromagnetic waves and matter. Spectral Absorptions Microwave Rotation of molecules IR Fundamental molecular vibrations NIR Overtones and combinations of IR UV / Visible Electronic transitions X-Ray Core electronic transitions in the atom

Units of spectra- nm, m, cm-1 1cm = 1 x 107 nm 1 nm = 1 x 10-3 µm Where cm-1 = 1 x 107 # nm Sometimes see cm-1 : 10,000 cm-1 = (1/10,000) cm or 0.0001 cm = 1 m = 1000 nm 6,000 cm-1 = (1/6000) cm or 0.000167cm = 1.67 m = 1670 nm 5,000 cm-1 = (1/5000) cm or 0.0002 cm = 2 m = 2000 nm 4000 cm-1 = (1/4000) cm or 0.00025 cm = 2.5 m = 2500 nm.

What is Infrared Spectroscopy? Sir Isaac Newton set up an experiment in which a beam of sunlight passed through window shutters into a dark room. Reference software (Algodoo v1.8.5)

What is Infrared Spectroscopy? (cont) Much later, Frederic William Herschel, the discoverer of planets and many other celestial objects, imagined the existence of other components of white light, outside the visible region. The region after the red part is called Infrared Region. Herschel set up an experiment to measure this radiation under the red which is not visible to human eye, thus he used a thermometer. References!

What is Infrared Spectroscopy? (cont) In March of 1800 Herschel placed a sample of water in the path of the beam, and the difference of temperature was then associated with absorption.

Mid-IR Today, the mid-infrared region is normally defined as the frequency range of 4000 cm-l to 400 cm-1. The upper limit is more or less arbitrary, and was originally chosen as a practical limit based on the performance characteristics of early instruments. The lower limit, in many cases, is defined by a specific optical component, such as, a beamsplitter with a potassium bromide (KBr) substrate, which has a natural transmission cut-off just below 400 cm-1. Introducion de “sissler”. Modificar y add algo mas visual. Comparacion de los rangos en el mismo slide. Comparaciones H.W. Siesler, “Basic Principles of Near Infrared Spectroscopy”, In Handbook of Near Infrared Analysis Ed. D.A. Burns and E.W. Ciurczak, 3rd ed., CRC Press, Boca Raton, FLA. 104 103 102 101 Frequency (cm-1) NIR MIR FIR Infrared J. Coates, “Vibrational Spectroscopy: Instrumentation for Infrared and Raman Spectroscopy”, Applied Spectroscopy Reviews, 1998, 33(4), 267 – 425.

Far IR The region below 400 cm-1, is now generally classified as the far infrared, characterized by low frequency vibrations typically assigned to low energy deformation vibrations and the fundamental stretching modes of heavy atoms. There is only one IR-active fundamental vibration that extends beyond 4000 cm-1, and that is the H-F stretching mode of hydrogen fluoride. The original NIR work was with extended UV-Vis spectrometers. Indicates that mid and NIR should be considered the same field. NIR 104 103 102 101 Frequency (cm-1) NIR MIR FIR Infrared J. Coates, “Vibrational Spectroscopy: Instrumentation for Infrared and Raman Spectroscopy”, Applied Spectroscopy Reviews, 1998, 33(4), 267 – 425.

Spectroscopy Provides Information Presence of functional groups Variation of functional groups, or elements throughout a surface (chemical information) Differences in the crystal structure of compounds Qualitative and quantitative analysis

Mid-IR Spectroscopy widely used in: Identification of pharmaceutical raw materials and finished products. Combination with MS and NMR to determine structure of process impurities and degradation products. Characterization of natural products, use of GC/FT-IR. Forensic analysis, IR-Microscopy. Environmental analysis: GC/FT-IR. Surface analysis, diffuse reflectance, attenuated total reflectance, grazing angle. Studies of protein structure and dynamics.

NIR Spectroscopy used in: Identification of solid sample forms Physical characteristic analysis of solid samples such as particle size and packing density of a material. Provide information on moisture content Monitor process parameters such as flow rates, blending process end time and even by-products. Non-invasive remote monitoring of different processes. Medical uses such as measurement of the amount of oxygen content of hemoglobin.

Molecular Vibrational Spectroscopy The physical origin of molecular vibrations are due to: - absorption of radiation by a material (MIR and NIR techniques) - scattering of radiation by a material (Raman technique) Vibrational frequencies are very sensitive to the structure of the investigated compound - structure elucidation, finger print spectra

Hooke’s Law In order to understand the absorption phenomenon, let’s compare a molecule to the vibration of a spring, Vibrating bond Vibrating spring F = -k x F – restoring force exerted by the spring k – rate of spring constant x – displacement of the spring from equilibrium m2 m1

Simple Harmonic Oscillator For diatomic molecules it is possible to calculate: Potential energy k – force constant of the bond, r – inter nuclear distance during vibration, re – equilibrium inter nuclear distance, q – displacement coordinate Vibrational frequency -or- Wavenumber m – reduced mass c – speed of light Energy curve for vibrating spring where, V – potential energy E – total energy K – kinetic energy “as a function of position”

Quantized Vibration Theory In the harmonic oscillator model, the potential energy well is symmetric. where, h – Plank’s constant 0 - vibrational frequency n – quantum number Molecular vibrations have: Discrete energy values, Energy levels are equally spaced, Each energy level is defined by n quantum number whose integers values are 0, 1, 2,… Only effective for relatively small deformations in the “spring”.

Vibration Theory On the basis of the equation above it is possible to state the following: 1) The higher the force constant k, i.e., the bond strength, the higher the vibrational frequency (in wavenumbers). 3 absorption peaks for different force constants. Note that by convention, in infrared spectroscopy wavenumbers are plotted right-to-left; i.e., highest wavenumber to the left. Courtesy of Bruker Optics

Vibration Theory 2) The larger the vibrating atomic mass, the lower the vibrational frequency in wavenumbers. 3 absorption peaks for different atomic masses. Note that by convention, in infrared spectroscopy wavenumbers are plotted right-to-left; i.e., highest wavenumber to the left. Courtesy of Bruker Optics

A Molecule Absorbs Infrared Energy when: Change in dipole moment must occur. The dipole moment is a measure of the degree of polarity of molecule (magnitude of the separated charges times the distance between them). A measurement of degree of unequal distribution of charges in molecule. + - H Cl

Molecular Dipole HBr does have a dipole change as it stretches, the intensity of the absorption is related to the magnitude of the dipole change. This dipole aligns with the electric field of the beam of light, then the light is absorbed.

Band Intensity in IR and Spectrum Band intensity depends on the rate of change of dipole moment during absorption of IR light. Stronger bands occur when the change in dipole moment is greatest. A spectrum is a plot that shows the absorption or reflection of radiation as wavelength or frequency of the radiation is varied. 3rd overtone CH3-Sym 3rd overtone CH2- Sym A.S. Bonanno, J. M. Olinger, and P.R. Griffiths, in Near Infra-Red Spectroscopy, Bridging the Gap Between Data Analysis and NIR Applications, Ellis Horwood, 1992.

Molecules that absorb Infrared energy vibrate in two modes: Stretching is defined as a continuous change in the inter-atomic distance along the axis of the bond between two atoms. Bending is defined as a change in bond angle MOLECULAR VIBRATION There are three motions involved in vibration molecular, called normal modes. Bend Symetric stretch Antisymetric stretch All non linear molecule has 3N-6 normal modes (N is the number of atoms in the molecule)

Molecular Spectroscopy This situation is simplified considering every functional group in the molecule independently. Each functional group has a set of group frequencies which correspond to the normal modes for the group.

Degrees of Freedom Molecule Degrees of freedom Non linear Linear 3N -6 Example: The fundamental vibrations for water, H2O are given in below. Water which is nonlinear, has three fundamental vibrations. Symmetric stretching Anti-symmetric stretching Symmetric Bending

Molecular Vibration Hexane C6H14 has 20 atoms (3(20)-6 = 54) normal modes, it is very difficult to analyze each mode.

NIR bands O-H, N-H, C-H, S-H bonds etc., are NIR strong absorbers since they have the strongest overtones as the dipole moment is high R-H stretch or R-H stretch / bend form most NIR bands The overtone and combination bands are 10 – 100 X less intense than the fundamental bands in mid-IR. Differences in spectra are usually very subtle. Instruments have a high signal to noise ratio.

Combination Bands The frequency of a combination is approx. the sum of the frequencies of the individual bands. Combinations of fundamentals with overtones are possible as well as well as fundamentals involving two or more vibrations. The vibrations must involve the same functional group and have the same symmetry. C.E. Miller, “Chemical Principles of Near Infrared Technology”, Chapter 2 in Near Infrared Technology: In the Agricultural and Food Industry, P. Williams and K. Norris (Editors), Amer. Assn. of Cereal Chemists; 2nd Ed. (November 15, 2001) .

NIR & Anharmonicity A number of bands are observed that cannot be explained on the basis of the harmonic oscillator. A more accurate model of a molecule is given by the anharmonic oscillator. The allowed energy levels for an anharmonic oscillator have to be modified: Where χ is the anharmonicity constant. The potential energy curve is represented by an asymmetric Morse function. Ref siesler

Morse Potential – Simple Anharmonic Oscillator Transition Name Range n=0 n=1 Fundamental mid-IR n=0 n=2 1st Overtone mid-NIR n=0 n=3 2nd Overtone NIR Interaction of two Combination NIR or more different vibrations

Example 1st Overtone Fundamental Vibration 2nd Overtone 2876cm-1 -C-H2 sym 2930cm-1 -C-H2 asym 2962cm-1 -C-H3 asym 2885cm-1 -C-H3 sym 2nd Overtone Analytical lab examples

Calculations of overtones and anharmonicities The wave number position of the fundamental position v1 or an overtone vn of the anharmonic oscillator can be given by: v0 is not directly accessible from the absorption spectra only the wave number v1, v2 …. may be obtained. Reference book Thus, if the wave number position £˛ν1 of the fundamental vibration and the anharmonicity constant χ are known, the wave number positions of the overtones can be calculated by Equation (2.13). Alternatively, χ can be calculated if, for example, £˛ν1 and £˛ν2 are known. H.W. Siesler, “Basic Principles of Near Infrared Spectroscopy”, In Handbook of Near Infrared Analysis Ed. D.A. Burns and E.W. Ciurczak, 3rd ed., CRC Press, Boca Raton, FLA.

NIR gets complicated Fermi Resonance Is an interaction between transitions of the same symmetry that occur at approximately the same wavenumber as that of a fundamental vibration. Firstly, if the transitions do not absorb at exactly the same wavenumber, the wavenumber of the lower frequency band is decreased while that of the higher frequency band is increased by about the same amount. In addition, the intensity of the weaker band is increased while that of the stronger band is decreased ("intensity borrowing”). Mid IR spectrum magnesium stearate solid sample C.E. Miller, “Chemical Principles of Near Infrared Technology”, Chapter 2 in Near Infrared Technology: In the Agricultural and Food Industry, P. Williams and K. Norris (Editors), Amer. Assn. of Cereal Chemists; 2nd Ed. (November 15, 2001) .

NIR continues to complicate Local Mode Treats a molecule as if it was made up of a set of equivalent diatomic oscillators: As the stretching vibrations are excited to high energy levels, the anharmonicity term χν0 tends to overrule the effect of interbond coupling and the vibrations become uncoupled vibrations and occur as “local modes”. NR spectrum n-pentane liquid sample C.E. Miller, “Chemical Principles of Near Infrared Technology”, Chapter 2 in Near Infrared Technology: In the Agricultural and Food Industry, P. Williams and K. Norris (Editors), Amer. Assn. of Cereal Chemists; 2nd Ed. (November 15, 2001) .

NIR complicates even more Darling-Dennison Resonance May lead to the presence of two bands where only one would be expected. Resonance between higher order overtone modes and the more intense combination bands. Particularly evident for X-H vibrations since interacting energy levels are close together and vibrational anharmonicity is high. Provides a complicating effect in NIR spectra, different from the simplifying effect that would be expected from local modes. Add example C.E. Miller, “Chemical Principles of Near Infrared Technology”, Chapter 2 in Near Infrared Technology: In the Agricultural and Food Industry, P. Williams and K. Norris (Editors), Amer. Assn. of Cereal Chemists; 2nd Ed. (November 15, 2001) . And L. Bokovza in Chapter 2 of Near Infrared Spectroscopy, H. W. Siesler, Y. Ozaki, S. Kawata, H.M. Heise, Wiley, VCH.

Electronic NIR Spectroscopy Electronic NIR bands Involves the change in the electronic state of a molecule (movement of an electron between different energy levels) Electronic transitions are generally of higher energy than vibrational transitions higher-energy visible and ultraviolet regions of the spectrum Electronic NIR bands are affected by intermolecular interactions and sample state. Although most practical NIR applications exploit vibrational absorption bands, the presence of electronic NIR bands cannot be overlooked. When atoms are combined to make molecules, the electrons of the atoms are distributed between newly defined molecular orbitals, each of which corresponds to a different electronic energy level. C.E. Miller, “Chemical Principles of Near Infrared Technology”, Chapter 2 in Near Infrared Technology: In the Agricultural and Food Industry, P. Williams and K. Norris (Editors), Amer Assn of Cereal Chemists; 2nd Ed. (November 15, 2001) .

Electronic NIR Spectroscopy Add reference Verify if some minerals may contain electronic aspects highly conjugated organic molecules such as hemoglobin C.E. Miller, “Chemical Principles of Near Infrared Technology”, Chapter 2 in Near Infrared Technology: In the Agricultural and Food Industry, P. Williams and K. Norris (Editors), Amer Assn of Cereal Chemists; 2nd Ed. (November 15, 2001) .

The NIR Complicating Factor Multitude of overtone and combination bands produced from only a few vibrations Large number of NIR-active groups (e.g CH, NH, OH, and C=O), each of which contributes its own set of overtone and combination bands Possibility of resonances between vibrational modes. which results in bands that cannot be assigned to "pure vibrations” in the molecule Possibility of several molecular configurations, each of which could produce a slightly different spectrum. This complications are also an advantage: The complexity of NIR spectra help to identify every single difference (Chemical and Physical). Referencia a Miller (estara presente) C.E. Miller, “Chemical Principles of Near Infrared Technology”, Chapter 2 in Near Infrared Technology: In the Agricultural and Food Industry, P. Williams and K. Norris (Editors), Amer Assn of Cereal Chemists; 2nd Ed. (November 15, 2001) .

The NIR Complicating Factor (CHCl3) C.E. Miller, “Chemical Principles of Near Infrared Technology”, Chapter 2 in Near Infrared Technology: In the Agricultural and Food Industry, P. Williams and K. Norris (Editors), Amer Assn of Cereal Chemists; 2nd Ed. (November 15, 2001) .

Understanding Hydrogen Bonding on vibrational spectra Solid minerals Wavenumber (cm-1) Response donor H : acceptor H ----- : acceptor Lone pair of e- Hydrogen bond Free surface O-H Hydrogen bonded surface O-H 1st overtone region for O-H bond stretching and free surface water Miller, C.E. (2001). Chemical Principles of Near-Infrared Technology. In: Williams, P. and Norris, K. Near-Infrared Technology in the Agricultural and Food Industries. 2nd ed. Minnesota, USA: American Association of Cereal Chemists, Inc. St. Paul, p19-36.

MIR and NIR Absorption Bands Typical MID IR Spectra Typical NIR Spectra Characterized by Wide Bands

MIR and NIR Absorption Bands Absorbance Wavenumber cm-1 Oleic Acid NIR MIR Courtesy of Bruker Optics 41

IR Instrumentation Mid IR Near IR

Advantages of Near Infrared Spectroscopy over Mid-IR No sample preparation required leading to significant reductions in analysis time and waste and reagents.(non-destructive testing). Possibility of using it in a wide range of applications (physical and chemical), and viewing relationships difficult to observe by other means. In-line monitoring of process. Spectrum may be used to identify the formulation and to quantify drug in the formulation.* Pharmaceutical samples have both chemical and physical properties. The physical properties are usually of significant interest to industrial pharmacists and process engineers. However, as the majority of analytical samples require sample preparation and dissolving this information is lost or thrown away. NIRS offers an opportunity to use this information. *M. Blanco, J. Coello, A. Eustaquio, H Iturriaga, and S. Maspoch, Development and Validation of a Method for the Analysis of a Pharmaceutical Preparation by Near-Infrared Diffuse Reflectance Spectroscopy, Journal of Pharmaceutical Sciences, 1999, 88(5), 551 – 556.

Infrared Equipment Classical (Dispersive) Spectrum Sample Reference Thermocouple (Detector) Sample Diffraction Grating Reference Spectrum

Infrared Equipment Modern (Fourier Transform)

Visualizing the Interaction of Light & Particles No sample preparation in NIR spectroscopy. Light interactions with particles. Need to learn to visualize the particles and their interaction with light. J.L. Ramirez, M. Bellamy, R.J. Romañach, AAPS Pharmscitech, 2001, 2(3), article 11.

Common NIR Techniques Tramittance Light may be remitted, transmitted & absorbed Diffuse Reflectance Detector for transmission

Scattering = reflection + refraction + diffraction. No interaction undeviated ray Diffracted ray Reflected ray Transmitted after internal reflection Transmitted ray Refracted ray Contributes to remission Contributes to transmission Isc = Iin (θ, λ, d, n) The intensity of scattered light is a function of the scattering angle, the wavelength λ, particle size d, and the refractive index n. Scattering = reflection + refraction + diffraction. Dahm DJ, Dahm KD. 2001. The Physics of Near-Infrared Scattering. In Williams P, Norris K, editors. Near Infrared Technology in the Agricultural and Food Industries, 2nd ed., Saint Paul: American Association of Cereal Chemists, p 19-37.

Scattering and Diffuse Reflectance Light propagates by scattering. As light propagates, both scattering and absorption occur, and the intensity of the radiation is reduced. The radiation that comes back to the entry surface is called diffuse reflectance.

Visualizing light interaction Low Scattering High scattering Smaller particle sizes More remission, less transmission Larger particle sizes Less remission, more transmission *Multiple path lengths are possible Prepared by Martha Barajas Meneses, MS 2006.

Subtle Differences,Valuable Info. Cristallinity – high degree of molecular order (narrower bands) Amorphous – no molecular order (broader bands) Crystalline sugar Amorphous sugar

Changes in spectra due to physical properties of a material Particle size effect Jackeline I. Jerez, Sept. 2009

Tablet Packing density Changes in spectra due to physical properties of a material Tablet Packing density NIR spectra of pure lactose tablet at different packing density Ropero, J. et al. 2011. Near-Infrared Chemical Imaging Slope as a New Method to Study Tablet Compaction and Tablet Relaxatio. Appl. Spect. 65, 4.

Probe-sample distance Changes in spectra due to variation in analysis Probe-sample distance Response Wavelength (nm) 1.3 cm 0.3 cm 0.6 cm 0.8 cm 1.0 cm NIR spectra of pure lactose analyzed at different distances

Changes in sugar spectra due variation in temperature Wavenumber (cm-1)

NIR aspects as functions of wavelength

CDI Lab Scale NIRS system, www.controldevelopment.com NIR Applications CDI Lab Scale NIRS system, www.controldevelopment.com

Powder and Solids Probe – Courtesy Bruker Optics Diffuse Reflection Probe Schematic IR Source IR Energy Sample Delivery Fiber Bundle Collection Fiber Bundle Reflected IR Energy Detector Powder & Solids Probe with liquid attachment references Extra-long immersion depth: 12”

Diffuse Reflectance Examples

Diffuse Reflectance for Flowing Powder I detected = 1/c x Ireflected Adetected = - log (Rdetected) = - log (Idetected/I0) = log c + log (I0/Ireflected) = c’ + A J. Ropero, L. Beach, M. Alcalà, R. Rentas, R.N. Davé, R.J. Romañach, Journal of Pharmaceutical Innovation, J. Pharm. Innov. 2009, 4(4), 187-197.

Transflection I0 Itrans Analyte Mirror or Reflector mirror Source I0 Detector Itrans Analyte Mirror or Reflector mirror Fiber probe for solids Courtesy Bruker Optics Pathlength is twice the distance from probe to mirror.

Transflectance using gold plate reflector. M. Blanco, M.A. Romero, “Near infrared transflectance spectroscopy Determination of dexketoprofen in a hydrogel”, Journal of Pharmaceutical and Biomedical Analysis, 30 (2002) 467–472.

Transmittance Tablet Sample Prepared by: María A. Santos Detector Position IR Beam Johansson and co-workers used time resolved spectroscopy to estimate that the optical path through a 3.5 mm thick tablet is 20 – 25 mm, because of the numerous reflections of the radiation with the particles within the tablet. J. Johansson, S. Folestad, M. Josefson, a. Sparen, C. Abrahamsson, S. Andersson-Engels, and S. Svanberg, Time-Resolved NIR/Vis Spectroscopy for Analysis of Solids: Pharmaceutical Tablets, Applied Spectroscopy, 2002, 58(6), 725 – 731. Tablet Sample Prepared by: María A. Santos R.J. Romañach and M.A. Santos, “Content Uniformity Testing with Near Infrared Spectroscopy”, American Pharmaceutical Review, 2003, 6(2), 62 – 67.

Transmittance Transmittance mode preferred since radiation interacts with a greater sample volume. Very interesting and often complex interaction between radiation and particles. Depth penetration depends on particle size (scattering properties) of particles within the tablet (Iyer, Morris, Drennen, J. Near Infrared Spectrosc., 2002, 10, 233 – 245.). It is very important to visualize particle and radiation interactions.

Recommended reading J. Coates, “Vibrational Spectroscopy: Instrumentation for Infrared and Raman Spectroscopy”, Applied Spectroscopy Reviews, 1998, 33(4), 267 – 425. A.S. Bonanno, J. M. Olinger, and P.R. Griffiths, in Near Infra-Red Spectroscopy, Bridging the Gap Between Data Analysis and NIR Applications, Ellis Horwood, 1992. C.E. Miller, “Chemical Principles of Near Infrared Technology”, Chapter 2 in Near Infrared Technology: In the Agricultural and Food Industry, P. Williams and K. Norris (Editors), Amer. Assn. of Cereal Chemists; 2nd Ed. (November 15, 2001) . H.W. Siesler, “Basic Principles of Near Infrared Spectroscopy”, In Handbook of Near Infrared Analysis Ed. D.A. Burns and E.W. Ciurczak, 3rd ed., CRC Press, Boca Raton, FLA. M. Blanco, J. Coello, A. Eustaquio, H Iturriaga, and S. Maspoch, Development and Validation of a Method for the Analysis of a Pharmaceutical Preparation by Near-Infrared Diffuse Reflectance Spectroscopy, Journal of Pharmaceutical Sciences, 1999, 88(5), 551 – 556. Dahm DJ, Dahm KD. 2001. The Physics of Near-Infrared Scattering. In Williams P, Norris K, editors. Near Infrared Technology in the Agricultural and Food Industries, 2nd ed., Saint Paul: American Association of Cereal Chemists, p 19-37.