Lecture 6 Molecular Absorption Spectrometry - Infrared Spectrometry Flipbook PDF

Learning Objectives At the end of this module, you should be able to: 1. Identify the sub-regions of the infrared spectr

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De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Molecular Absorption Spectrometry:

Infrared Spectrometry

A Learning Module

READING MATERIAL 

Chapter 5: Infrared Spectrophotometry in Pharmaceutical Analysis: A Textbook for Pharmacy Students and Pharmaceutical Chemists, 5th edition by David G. Watson

EXPECTED ACTIVITIES AND OUTPUTS     

Read Chapter 5 of Pharmaceutical Analysis: A Textbook for Pharmacy Students and Pharmaceutical Chemists, 5th edition by David G. Watson Watch the supplemental video by the Royal Society of Chemistry in this link: https://youtu.be/DDTIJgIh86E. Study and familiarize yourself with the Simplified IR Correlation Chart. Answer the formative assessments at the end of each section of the module. Write well-thought out answers in the integrative guide questions at the end of the module.

LEARNING OBJECTIVES At the end of this module, you should be able to:  Identify the sub-regions of the infrared spectrum and their analytical applications  Demonstrate an understanding of the principle of infrared spectrometry  Correlate the infrared spectra of molecular substances to their structure  Identify the components of the infrared spectrometer  Describe the different methods of sample preparation in infrared spectrometry and their applications in pharmaceutical analysis  Recognize the importance of infrared spectrometry in pharmaceutical analysis

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

INTRODUCTION Infrared (IR) Spectrometry is the measurement of the absorption of radiant energy in the infrared (IR) region that is associated with vibrational transitions of atoms within molecules. 1. The Infrared Region Infrared region refers broadly to that part of the electromagnetic spectrum between the visible and microwave regions (Figure 1).

Figure 1. Infrared Region of the Electromagnetic Spectrum Table 1. Divisions of the Infrared Region Region Wavelength (λ) Range, µm Near 0.78 to 2.5 Middle 2.5 to 50 Far 50 to 1,000

Wavenumber Range, cm-1 Frequency (ν) Range, Hz 12, 800 to 4,000 3.8 x 1014 to 1.2 x 1014 4,000 to 200 1.2 x 1014 to 6.0 x 1012 200 to 10 6.0 x 1012 to 2.0 x 1013

1.1. Near-IR corresponds to energies in the range 37 to 10 kcal mole -1. There are few absorptions of organic molecules in this range, but it has become a most important tool for the routine quantitative determination of constituents in finely ground solids. The most widespread use of this technique has been for the determination of protein, moisture, starch, oil, lipids, and cellulose in agricultural products such as grains and oilseeds. For example, the method can be used in place of the tedious Kjeldahl protein determination. Diffuse reflectance occurs in which radiation penetrates the surface layer of the particles, excites vibrational modes of the analyte molecule, and is then scattered in all directions. A reflectance spectrum is thus produced that is dependent upon the composition of the sample. 1.2. Middle-IR has energy of 10 to 1 kcal mole-1. The energy of this radiation corresponds to the differences commonly encountered between vibrational states. The most used region is one that is between 2.5 to 15 μm.

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PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

1.3. Far-IR occurs at energy ranging from 1.0 to 0.1 kcal mole -1. This is particularly useful for inorganic studies because absorption due to stretching and bending vibrations of bonds between metal atoms and both inorganic and organic ligands generally occur at frequencies lower than 650 cm -1 (>15μm). Molecules composed only of light atoms absorb in the far-infrared if they have skeletal bending modes that involve more than two atoms other than hydrogens. Pure rotational absorption by gases is observed in the far-infrared region, provided the molecules have permanent dipole moments. Examples include H2O2, O3, HCl, and AsH3. Absorption frequencies for metal-organic bonds are ordinarily dependent upon both the metal atom and the organic portion of the species. Far-IR studies of inorganic solids have also provided useful information about lattice energies of crystals and transition energies of semiconducting materials.

Figure 2. Divisions of the Infrared Region

Formative Assessment No. 1: The Infrared Region 1. Fill in the blank. Absorption of infrared radiation results in _____________ (electronic, vibrational) transitions of atoms within molecules. 2. Choose which among these is in correct order of increasing frequency of radiation. A. Radiowave < Infrared < Visible < Microwave B. Microwave < Visible < Infrared < Radiowave C. Microwave < Radiowave < Infrared < Visible D. Radiowave < Microwave < Infrared < Visible 3. Match the division of the infrared region (Column A) with its application in pharmaceutical analysis (Column B) Column A Column B 1. Near IR A. Analysis of finely ground solids 2. Mid IR B. Inorganic compound analysis 3. Far IR C. Organic structure analysis

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

2. Theory of Infrared Spectrometry Absorption of IR radiation is confined largely to molecular species that have small energy differences between various vibrational and rotational states. In order to absorb IR radiation, a molecule must undergo a net change in dipole moment as a consequence of its vibrational and rotational motion. The dipole moment is determined by the magnitude of the charge difference and the distance between the two centers of charge. If the frequency of the radiation exactly matches a natural vibrational frequency of the molecule, a net transfer of energy takes place that results in a change in the amplitude of the molecular vibration; absorption of the radiation is a consequence (Figure 3). Similarly, the rotation of asymmetric molecules around their centers of mass results in a periodic dipole fluctuation that can interact with radiation. No net change in dipole moment occurs during the vibration or rotation of homonuclear species such as O2, N2, or Cl2; consequently such compounds cannot absorb in the IR.

Figure 3. The frequency of radiation absorbed by a bond depends on the natural vibrational frequency of the molecule. 2.1. The Intensity of Absorption The intensity of absorption is determined by the dipole moment (bond dipole). Thus, the order of intensity of absorption for the following C–X bonds is: C–O > C–Cl > C–N > C–C–OH > C–C–H Similarly: OH > NH > CH The intensity depends on the relative electronegativity of the atoms involved in the bond. The intensity of the stretching of carbon-carbon double bonds is increased when they are conjugated to a polar double bond. The order of intensity is as follows: C=C–C=O > C=C–C=C > C=C–C– 2.2. Energy Level of Absorption The energy of absorption is determined by the mass of atoms. The natural vibrational frequency of a bond in a molecule is governed by Hooke's Law. The vibrational motion of a bond can be visualized as a simple harmonic motion of an object on a spring (Figure 3). That is, for purposes of explaining infrared spectroscopy, a molecule is viewed as being joined by bonds which behave like springs.

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Figure 4. The frequency of simple harmonic motion like a mass on a spring is determined by the mass m and the stiffness of the spring expressed in terms of a spring constant k. A mass on a spring has a single resonant frequency determined by its spring constant k and the mass m. The frequency f is directly proportional to the resonant vibrational frequency ω (Figure 4). The energy level of vibration of a bond, Evib, is shown to be proportional to similar factors: √ where k is a constant related to the strength of the bond, e.g., double bonds are stronger than single bonds and therefore absorb at a higher energy than single bonds; μ is related to the ratio of the masses of the atoms joined by the bond. This is given by the following equation: where m1 and m2 are the masses of the atoms involved in the bond. According to the μ term, the highest energy bonds are the X–H (OH, NH, CH). The order of energy absorption for some common bonds is as follows, which reflects μ and the strength of the bonds: O–H > N–H > C–H > C≡N > C≡C > C=O > C=C > C–O > C–C > C–F > C–Cl Therefore, strong bonds and light atoms vibrate at relatively high stretching frequencies (or wavenumbers, which are reciprocal wavelengths). Conversely, weak bonds and heavy atoms absorb at lower wavenumbers, as would be expected from Hooke's Law. Similar functional groups are made up of similar atoms. Thus, molecules with similar functional groups will absorb photons of similar energies. Watch the video by clicking this link to learn more about the principles, basic instrumentation, and analytical method of Infrared Spectroscopy.

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Formative Assessment No. 2: Theory of Infrared Spectrometry 1. ___________ is a physical property of a bond that determines the intensity of infrared absorption. 2. The frequency of IR absorption depends on the ___________ of the atoms. 3. True or False. A net change in dipole moment as a consequence of its vibrational or rotational motion is required for a bond to absorb in the infrared region. 4. True or False. Homonulcear species in the atmosphere, such as O2 and N2, present difficulties in infrared spectroscopy by absorbing significant radiation in the group frequency range. 5. True or False. Strong bonds and light atoms vibrate at lower frequencies than weak bonds and heavy atoms. 3. Vibrational Modes Transitions between the vibrational energy levels of molecules occur in the infrared region of the electromagnetic spectrum. Absorption of infrared radiation results in vibrational modes known as stretching and bending. 3.1. Stretching Stretching is a rhythmical movement along the bond axis such that the interatomic distance is increasing or decreasing. There is change in bond lengths. There are two types of stretching modes:  

In asymmetric stretching, as one atom moves toward the center, the other moves away. In symmetric stretching, both outside atoms move away from or toward the center.

Figure 5. Stretching vibrational modes 3.2. Bending Bending may consist of a change in bond angle between bonds with a common atom or the movement of a group of atoms with respect to the remainder of the molecule without movement of the atoms in the group with respect to one another. Below are the types of bending modes:    

Scissoring involves symmetric bending vibration in a plane Rocking involves asymmetric bending vibration in a plane Twisting involves symmetric bending vibration out of plane Wagging involves asymmetric bending vibration out of a plane

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In-plane bending modes

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Out-of-plane bending modes

Figure 6. Bending vibrational modes

Formative Assessment No. 3: Vibrational Modes 1. ___________ may consist of a change in bond angle between bonds with a common atom, or the movement of a group of atoms with respect to the remainder of the molecule without movement of the atoms in the group with respect to one another. 2. ___________ is a rhythmical movement along the bond axis such that the interatomic distance is increasing or decreasing. 3. Match Column A with Column B: Column A Column B 1. Symmetric out-of-plane bending vibration A. Scissoring 2. Symmetric in-plane bending vibration B. Rocking 3. Asymmetric out-of-plane bending vibration C. Twisting 4. Asymmetric in-plane bending vibration D. Wagging

INFRARED SPECTROMETRY 1. Interpretation of Infrared Spectra Similar functional groups are made up of similar atoms. Thus, molecules with similar functional groups will absorb photons of similar energies. Determining the absorption frequencies in the infrared (IR) region, therefore, can be helpful in the identification of molecules. IR absorption bands are described by either the wavelength, λ, of the absorbed light in micrometers or its reciprocal value, called wavenumber. A linear wavenumber scale is usually preferred in IR spectroscopy because of the direct proportionality between this quantity and both energy and frequency. The frequency of the absorbed radiation is, in turn, the molecular vibrational frequency actually responsible for the absorption process. For many groups containing only two atoms, the approximate frequency of the fundamental vibration can be calculated from a simple harmonic oscillator model. Calculations show that for most groups of interest, characteristic frequencies of stretching vibrations should lie in the region between 4000 cm -1 and 1000 cm-1. For most practical purposes, the group frequency region, which encompasses radiation from about 3600 cm-1 to approximately 1200 cm-1 determines what functional groups are most likely present in a molecule.

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

The fingerprint region, from 1200 cm-1 to 600 cm-1 is particularly useful because small differences in the structure and constitution of a molecule result in significant changes in the appearance and distribution of peaks in this region. The absorption in the fingerprint region is very complex and it is difficult to be confident in the assignment of absorptions to particular functional groups. The large number of bonds in polyatomic molecules means that the data obtained by IR analysis is extremely complex. Quite a lot of information can be obtained from an IR spectrum, but even with modern instrumentation, it is not possible to completely "unscramble" the complex absorbance patterns present in IR spectra. In order for the structure of an unknown molecule to be determined, the functional groups are first identified in the group frequency region, and the spectrum (particularly the fingerprint region) of the unknown is thoroughly compared with the spectra of pure compounds that contain all the functional groups found in the first step. The complexity of the absorption pattern in the fingerprint region, however, presents a problem when reading the IR spectrum without a database and computerized matching software.

Figure 7. Regions in the IR Spectrum 1.1. The Group Frequency Region In order to simplify the interpretation of IR spectra and to familiarize with the different absorption frequencies (or wavenumbers) of functional groups, the group frequecy region can be divided into five zones (Table 2):

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Table 2. Zones in Group Frequency Region Zones Wavenumbers Functional Groups Zone 1 3700 cm-1 – 3200 cm-1 Alcohol O–H, amide or amine N–H, terminal alkyne C–H Alkyl C–H, aryl or vinyl C–H, aldehyde C–H, carboxylic Zone 2 3200 cm-1 – 2700 cm-1 acid O–H -1 -1 Zone 3 2300 cm – 2100 cm Alkyne and nitrile triple bonds Zone 4 1950 cm-1 – 1650 cm-1 Carbonyl functional groups Zone 5 1680 cm-1 – 1450 cm-1 Alkene (–C=C–), benzene ring This table only incudes the most common functional groups found in compounds. A more detailed correlation chart may prove to be useful in complete interpretation of IR spectra. 1.2. Sample IR Spectra and Interpretation 1.2.1. Alcohols and Phenols Let us consider the spectra of similar alcohols including phenol.

Cyclohexylmethanol

Benzyl alcohol

Phenol

Figure 8 shows the IR spectrum of cyclohexylmethanol. Zonal analysis of the IR spectrum of cyclohexylmethanol shows that there are peaks in zone 1 (3500 cm-1 to 3100 cm-1) and zone 2 (2900 cm-1 to 2800 cm-1). The peak in zone 1 is due to absorption by the O–H bond. The O–H absorption band is characterized as broad and strong (intense). The broadness of this peak is due to hydrogen bonding to other alcohol molecules or to water. This band is one of the most prominent absorption peaks that can be observed in the IR spectra of alcohols. Hydrogen bonding of alcohols also results in absorption at lower wavenumbers than that observed in monomeric alcohols. Dry alcohols in dilute solution exhibit sharper bands in a narrower range (3620 cm-1 to 3650 cm-1).

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PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Figure 8. IR Spectrum of Cyclohexylmethanol The peaks in zone 2 are due to absorption by aliphatic C–H bonds. Compared to O–H peaks, C–H peaks are sharper and usually occur as multiplets. Splitting of peaks in this region is due to slightly different natural vibration frequencies of various types of C–H bonds in a molecule. The alcohol C–O stretch which can be observed at 1050 cm-1 is usually characterized as a strong absorption peak. The absence of C–H stretching bands at 3200 cm-1–3000 cm-1 and C–C stretching bands in zone 5 (1680 cm-1–1450 cm-1) confirms that the molecule is an aliphatic alcohol. The peaks that are observed at 1450 cm -1 and 1100 cm-1 are due to bending vibrations. Absorption peaks due to bending in the fingerprint region are most often difficult to unequivocally assign to a bond, so these are most often not included in spectral analyses. Now, let’s compare this with the IR spectrum of benzyl alcohol:

Figure 9. IR Spectrum of Benzyl Alcohol

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

The most prominent differences in the IR spectra of cyclohexylmethanol and benzyl alcohol are as follows: 





Presence of multiplet peaks in 3150 cm-1 – 3000 cm-1 which are due to aromatic C–H stretch. The aromatic C–H bonds absorb at higher wavenumbers than aliphatic C–H bonds because the latter have less energy than aromatic C–H bonds. Presence of weak to medium absorption peaks at about 1600 cm-1 and about 1500 cm-1 primarily corresponds to absorption by aromatic C=C bonds. The aromatic C=C stretch may be weak unless the aromatic ring is substituted with polar substituents, e.g., a phenol, aromatic ether or aromatic amine free base. Note that unconjugated C=C bonds absorb at 1680 cm-1 to 1610 cm-1; whereas, conjugated/aromatic C=C absorb at lower wavenumbers (1600 cm-1 to 1500 cm-1). Weak absorption peaks (multiplets) from 2000 cm-1 to 1600 cm-1 correspond to aromatic overtones, which are caused by overtones (harmonics) of the benzene ring vibrational modes having stretching frequencies in the IR spectrum's fingerprint region. Overtones occur when a vibrational mode is excited from v = 0 to v = 2, which is called the first overtone, or v = 0 to v = 3, the second overtone. The fundamental transitions, v = ±1, are the most commonly occurring, and the probability of overtones rapid decreases as the number of quanta (Δv = ±n) increases.

Coupling of stretching and bending vibrations at wavenumbers below 1500 cm -1 results in similar patterns with cyclohexylmethanol. This time, let us examine the IR spectrum of phenol:

Figure 10. IR Spectrum of Phenol The IR spectra of benzyl alcohol and phenol have quite a few number of differences as follows:  

Absence of aliphatic C–H stretch at less than 3000 cm-1 to 2800 cm-1 in phenol Strong absorption by aromatic C=C bond at 1600 cm-1 due to the substitution of the aromatic ring with a polar OH group

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PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Presence of strong band at 1250 cm-1 which is due to aromatic (phenolic) C–O absorption. Similar to aromatic C–H bonds, aromatic C–O bonds absorb at higher wavenumbers than aliphatic C–O bonds.

In addition, a medium intensity peak at 1350 cm-1 is more prominently observed in phenol than in cyclohexylmethanol and benzyl alcohol. This is due to O–H bending, which becomes more prominent because of the aromatic ring. 1.2.2. Amines and Amides Primary amines are characterized by a doublet peak, which is commonly medium intensity, between 3500 cm-1 and 3300 cm-1 (Figure 11). Secondary amines only show a single weak band at this region (Figure 12). These bands are due to N–H stretching vibrations in amines. In addition to N–H stretching bands, another characteristic feature of the IR spectra of amines is the C–N stretching bands that can be found between 1250 cm-1 and 1020 cm-1.

Figure 11. IR Spectrum of Benzylamine

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PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Figure 12. IR Spectrum of N,N-Diethylamine Amides are characterized by two distinct absorption peaks: those due to amide N–H stretch (3500 cm-1 to 3100 cm-1) and the amide C=O stretch (approximately 1680 cm-1) (Figure 1314).

Figure 13. IR Spectrum of Acetamide

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PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Figure 14. IR Spectrum of N-Methylacetamide Restricted rotation about N–CO bond produces diastereomers, giving two bands in the case of secondary amides (Figure 14). 1.2.3. Aldehydes and Ketones The common structural feature of aldehydes and ketones is the carbonyl (C=O) group (Figure 15-16). The C=O stretching frequency is unusually strong and typically appears in a relatively narrow range (1750 cm-1 – 1690 cm-1) in zone 4. Conjugation results in absorption peaks observed at lower wavenumbers (approximately 1650 cm-1).

Figure 15. IR Spectrum of Acetone (Propanone)

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PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Figure 16. IR Spectrum of Acetaldehyde (Ethanal) In aldehydes, an additional peak due to C–H stretch can be observed at 2830 cm-1–2695 cm-1. This is usually a medium intensity and doublet peak (Figure 16). 1.2.4. Carboxylic Acids and Esters

Benzoic Acid

Sodium Benzoate

Figure 17. IR Spectrum of Benzoic Acid

Methyl Benzoate

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PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

The carboxy group consists of a carbonyl group and an attached hydroxy substituent. Consequently, both characteristic stretching frequencies are seen in the infrared spectrum (Figure 17). The O–H bond gives rise to a broad band in zone 2 or in lower wavenumber (3300 cm-1 – 2500 cm-1) than is observed for alcohols, because of strong hydrogen bonding. Note that the carboxy C–O stretch (1300 cm-1) can also be found at higher wavenumber than aliphatic alcohols. This is due to the resonance effect of the carboxy group. A salt may not show the carboxylic acid O–H stretch (Figure 18). The IR spectrum of an ester, methyl benzoate, shows similar absorption patterns (Figure 19). In addition, the carboxy C=O stretch may also be observed at slightly higher wavenumber (1728 cm-1) because of the absence of intramolecular hydrogen bonding.

Figure 18. IR Spectrum of Sodium Benzoate

Figure 19. IR Spectrum of Methyl Benzoate

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

For a closer study of infrared spectra, check out the book Spectrometric Identification of Organic Compounds by R.M. Silverstein, G.C., Bassler, and T.C. Morrill, and the online Spectral Database for Organic Compounds SDBS by National Institute of Advanced Industrial Science and Technology (AIST), Japan available in this link: https://bit.ly/3bi2zHi.

Formative Assessment No. 5: Interpretation of Infrared Spectra True or False 1. Conjugation results in absorption at lower wavenumber as the pi (π) electrons are delocalized. 2. Intramolecular hydrogen bonding results in absorption at higher wavenumber as this strengthens the bond. 3. Aromatic substitution with polar groups results in a hyperchromic shift in C=C absorption peak because of the ease of changing the bond dipole upon vibration. 4. Narrowing of the O–H absorption band between 3500 cm-1 and 3100 cm-1 of an alcohol is a result of the intermolecular hydrogen bonding with other molecules of the alcohol or water. 5. But-2-yne (dimethylacetylene) shows a sharp peak in zone 3 of its infrared spectrum. H3C–C≡C–CH3 Multiple Choice 6. A bottle of aromatic liquid has a label of C5H11N. The infrared spectrum showed a single medium intensity peak in zone 1. Which among these statements is a correct assumption? A. The DBE is equal to zero so the compound neither has unsaturation nor ring. B. The DBE is equal to one so the compound is definitely unsaturated. C. The compound is a saturated cyclic secondary amine. D. The compound is an unsaturated aliphatic tertiary amine. Refer to the FTIR spectrum (in KBr disc) of C12H16O below:

7. What is the unsaturation number (double bond equivalent) of the compound? A. 3 C. 5 B. 4 D. 6

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Take note of the peaks and refer to the Summary of Group Frequencies for Organic Compounds. 8. The prominent peak at 1683 cm-1 is due to: A. sp2 C–H stretch B. C=O stretch

C. D.

alkene C=C stretch aromatic C=C stretch

9. The sharp peak at 1607 cm-1 is due to: A. sp2 C–H stretch B. C=O stretch

C. D.

alkene C=C stretch aromatic C=C stretch

Summary of the Group Frequencies for Organic Groups Bond C–H

Alkanes

C–H

Alkenes

C–H C–H

Alkynes Aromatic rings

O–H N–H C=C C=C C≡C C–N C≡N C–O C=O NO2

Type of Compound

Monomeric alcohols, phenols Hydrogen-bonded alcohols, phenols Monomeric carboxylic acids Hydrogen-bonded carboxylic acids Amines, amides Alkenes Aromatic rings Alkynes Amines, amides Nitriles Alcohols, ethers, carboxylic acids, esters Aldehydes, ketones, carboxylic acids, esters Nitro compounds

Frequency Range, cm-1 2850 – 2970 1340 – 1470 3010 – 3095 675 – 995 3300 3010 – 3100 690 – 900 3590 – 3650 3200 – 3600 3500 – 3650 2500 – 2700 3300 – 3500 1610 – 1680 1500 – 1600 2100 – 2260 1180 – 1360 2210 – 2280 1050 – 1300 1690 - 1760 1500 – 1570 1300 - 1370

Intensity Strong Strong Medium Strong Strong Medium Strong Variable Variable, sometimes broad Medium Broad Medium Variable Variable Variable Strong Strong Strong Strong Strong Strong

2. Instrumentation in Infrared Spectrometry 2.1. Types of Infrared Spectrometer 2.1.1. Dispersive Spectrometers Dispersive spectrometers were introduced in the mid-1940s and used since. It provided the rebust instrumentation required for extensive application of this technique. These instruments use a monochromator to select each wavenumber in turn in order to monitor its intensity after the radiation has passed through the sample. In other words, there is sequential scanning of each wavenumber. The spectral scan is complete in 2 to 3 minutes. There are two types of dispersive IR spectrometer:  Single-Beam IR Spectrometer  Double-Beam IR Spectrometer

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PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Single-Beam IR Spectrometer In single-beam IR spectrometer, the radiation source are allowed to fall on the sample cell. The sample cell absorbs the radiation and transmits some of it via the entrance slit onto the mirror. The radiation from the mirror are directed towards the prism and then, to the Littrow mirror (Figure 20). The Littrow mirror reflects the incident beam back to the mirror and through the exit slit onto the detector.

Figure 20. Littrow mirror/prism is a 30-degree prism with mirrored back which permits more compact monochromator designs. Some of the disadvantages of single-beam IR spectrometer are as follows:  The emission intensity of radiation source is not uniform  The IR spectrum of the sample solution shows extra bands due to the solvent Double-Beam IR Spectrometer In double-beam IR spectrometer (Figure 21), the source is separated into two separate beams: the sample and background are collected simultaneously. The monochromator scans through the wavelength region. Double-beam operation allows a stable 100% Transmittance baseline in the spectra. It also compensates for atmospheric absorption for the wavelength dependence of the source spectra radiance, the optical efficiency of the mirrors and grating, and the detector instability, which are serious in the IR region. Dispersive IR spectrophotometrs are generally double-beam recording instruments, which use reflection grating for dispersing radiation. Generally, these incorporate a low frequency chopper (5 to 30 cycles per second) that permits the detector to discriminate between the signal from the source and signals from extraneous radiation, such as IR emission from various bodies surrounding the transducer. Sample and reference are placed before the monochromator has the advantage that most scattered radiation and IR emission (generated within cell compartment) is effectively removed by the monochromator. In turn, this stray light does not reach the transducer.

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PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Figure 21. A double-beam dispersive IR spectrometer Some of the disadvantages of dispersive IR spectrometers, in general, are as follows:  Slow scan speed make dispersive instruments too slow for monitoring systems undergoing rapid changes (for example, in monitoring chromatography effluents)  Less resolution and sensitivity  Less accuracy compared to Fourier Transform Infrared (FTIR) instruments  Narrow range of wavelengths  Involvement of stray light  Atmospheric absorptions by carbon dioxide and water also take place  Mechanical slippage due to many moving parts 2.1.2. Fourier Transform Infrared (FTIR) Spectrometer FTIR instruments (Figure 22) use an interferometer, which generates a radiation source in which individual wavenumbers can be monitored within a ~1-second pulse of radiation without dispersion being required. These simultaneously collect all wavelengths and scan them at once. Spectral scan is complete in about 1 second. These are widely applied and quite popular in the far-IR and mid-IR spectrometry. FTIR instruments work based on Michelson interferometer (Figure 23), which has a beam splitter, a fixed mirror, and a movable mirror that enable the instrument to transmit waves with slightly different phases/amplitudes. These waves combine based on superposition principle and produce an interferogram. This complex beat pattern is processed using a common algorithm called Fourier transform. The Fourier transform converts one domain (in this case displacement of the mirror in cm) into its inverse domain (wavenumbers in cm −1). This results in a spectrum that can be interpreted easily.

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PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Figure 22. A Fourier transform infrared (FTIR) spectrometer

Figure 23. Michelson interferometer Some of the advantages of FTIR spectrometers include:        

Fast scanning time Sensitivity Simultaneous modulation of all frequencies Simple mechanical design with only one moving part Absence of stray light No need for external calibration (when using He-Ne laser as internal standard) Availability of easy sampling accessories Air pollutants like CO, ethylene oxide, etc. can be analyzed

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

2.2. Components of Infrared Spectrometers 2.2.1. Source The common infrared sources are inert solids heated to 1500 to 2000 K, a temperature at which the maximum radiant output occurs at 1.5 to 1.9 μm. Two common infrared sources are the Nernst glower and the Globar.  The Nernst glower usually operates from about 2 to 15 μm. This is a rod consisting of a mixture of rare earth oxides, such as zirconium oxide (ZrO2), yttrium oxide (Y2O3), and erbium oxide (Er2O3) at ratio of 90:7:3 by weight. It has a negative temperature coefficient of resistance and is nonconducting at room temperature. Therefore, it must be heated to excite the element to emit radiation, but once in operation it becomes conducting and furnishes maximum radiation at about 1.4 μm or 7100 cm-1 (1500–20000C).  The Globar ("glow bar") is a rod of sintered silicon carbide (SiC) heated to about 1300– 17000C. Its maximum radiation occurs at about 1.9 μm (5200 cm-1), and it must be watercooled. The Globar is a less intense source than the Nernst glower, but it is more satisfactory for wavelengths longer than 5 µm, because its intensity decreases less rapidly. 2.2.2. Sample Cells Solids The most common is a cell of sodium chloride prisms. Sodium chloride, however, becomes opaque at wavelengths longer than about 15 μm (660 cm -1). This spectral region became known as the rock-salt region. Fixed-thickness cells are available for these purposes and are the most commonly used. Sodium chloride cells must be protected from atmospheric moisture (stored in desiccators) and moist solvents. They also require periodic polishing to remove "fogging" due to moisture contamination. Potassium bromide prisms to extend the range to 25 μm (400 cm−1) and cesium iodide to 50 μm (200 cm−1). Silver chloride windows are often used for wet samples or aqueous solutions. These are soft and will gradually darken due to exposure to visible light/ Generally, the solid sample must be ground until its particle size is less than the wavelength of the radiation in order to avoid the effects of scattered radiation. Liquids (or Solutions) When samples exist as pure liquids, they are usually run without dilution (called "neat" samples) in the infrared region. For this purpose, the cell length must be short (generally within a range of 0.01–0.05 mm) in order to keep the absorbance within the optimum region. If a solution of the sample is to be prepared, a fairly high concentration is usually run, because no solvent is completely transparent in the IR region, and this will keep the solvent absorbance

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

minimal. Common solvents used are CCl4 and CS2. Short path lengths are required, generally 0.1 mm or less. In this case, the liquid sample is placed in between NaCl or KBr discs. Volatile liquids are placed in sealed cells. Gases Gases may be analyzed using a long path length cell (usually 10 cm in length). Cells as long as 20 m and up have been used in special applications. Gases are placed in cells that consist of a glass or metal body, two IR-transparent end windows, and valves for filling gas from external sources. 2.2.3. Thermal Transducers Thermal transducers are widely employed for the detection of infrared radiation. This responds to the average power of the incident radiation. Phototransducers are generally not applicable in the IR region because photons in this region lack the energy to cause photoemission of electrons. The principle of thermal transducers involves the measurement of the resultant temperature rise when a small black body absorbs the infrared radiation that impinges (or "shines") on it. The radiant power level from a typical infrared beam is minute (10-7 to 10-9 W) so that the heat capacity of the absorbing element must be as small as possible if a detectable temperature change is to be produced. In order for thermal transducers to measure small changes in temperature, the absorbing element (i.e., the black body) should have a minimum size and thickness, and the IR beam must be concentrated on its surface. Some of the common thermal transducers are (a) thermocouple, (b) bolometer and thermistor, and (c) pyroelectric transducers. 

Thermocouple consists of a pair of junctions formed when two pieces of a metal such as copper are fused to each end of a dissimilar metal (Figure 24). When a temperature difference exists between the two points, a potential difference is developed, which can be measured. A well-designed thermocouple transducer is capable of responding to temperature differences of 10-6 K. This difference corresponds to a potential difference of about 6 to 8 µV/µW.

Figure 24. Thermocouple

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry



Bolometer is a type of resistance thermometer constructed of strips of metals such as platinum or nicker; whereas, a thermistor is made from a semiconductor. These exhibit a relatively large change in resistance as a function of temperature. The responsive element is kept small and blackened to absorb radiant heat. Compared to a thermocouple, these resistance thermometers have more rapid response time (4 milliseconds compared to thermocouple's 60 milliseconds), so these provide improved resolution and faster scanning rates. These, however, are not so extensively used as other infrared transducers for the mid-IR region.



Pyroelectric transducers are constructed from single crystalline wafers of pyroelectric materials, which are insulators (dielectric materials) with very special thermal and electrical properties (Figure 25). When the temperature is changed by irradiation with IR radiation, the charge distribution across the crystal is altered creating a measureable current in an external circuit that connects the two sides of the capacitor. The magnitude of this current is proportional to the surface are of the crystal and to its rate of change of polarization with temperature. These transducers exhibit response times that are fast enough to allow them to track changes in the time-domain signal from an interferometer.

Figure 25. A pyroelectric transducer 3. Sample Preparation 3.1. KBr (or KCl) Pellet/Disc The sample, ~200 mg (usually 1% of the weight of KBr/KCl used) is ground to a powder (≤2 μm) with KBr or KCl and pressed at a pressure of 800 kPa. The KBr or KCl should be free from moisture. Discs should be discarded if they do not appear uniform. Any disc having a transmittance < 75% at 2000 cm-1 in the absence of a specific absorption band should be discarded. This method is not suitable for identifying polymorphs (different crystalline forms) of a drug because the pressures used to prepare the discs can cause polymorph interconversion.

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

3.2. Mull A

B

C

Figure 26. Differences in the IR spectra of acetanilide prepared by KBr disc (A) and Nujol mull (B). Additional peaks can be seen in the Nujol mull preparation of acetanilide: 2855 cm -1 and 2924–2955 cm-1, and 1323 cm-1 and 1436 cm-1, which correspond to the aliphatic C–H stretching and C–H bending vibrations of Nujol (mineral oil), respectively. The IR spectrum of Nujol film (C) shows that these peaks are due to absorption by the solvent, not acetanilide.

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Powders may be run as a suspension or thick slurry (mull) in a viscous liquid having about the same index of refraction in order to reduce light scattering. The sample is ground in the liquid, which is often Nujol, a mineral oil (thus, the sample preparation is called a Nujol mull). Alternatively, chlorofluorocarbon greases (i.e., Fluorolube) or hexachlorobutadiene is useful when the Nujol masks any C-H bands present (Figure 26). Then, the sample is run as a film sandwiched between two NaCl or KCl discs. The mull technique is useful for qualitative analysis, but it is still difficult to reproduce for quantitative work. Until recently, the standard method of sample preparation for characterizing polymorphs by IR spectrometry was by using a Nujol mull. However, the DRIFT (diffuse relectance infrared Fourier transform) technique has an advantage since it does not introduce the interefering peaks which are present in Nujol and which may obscure the areas of interest in the fingerprint region of the spectrum. In addition, low polarity samples may be soluble in Nujol, thus causing their polymorphs to break down. 3.3. Diffuse Reflectance A more recent development in sample preparation is the use of diffuse reflectance. Diffuse reflectance is a readily observed phenomenon. When light is reflected off a matt surface, the light observed is of the same intensity no matter what the angle of observation (Figure 27). Samples for diffuse reflectance are treated in the same way as those prepared for KBr disc method, but, instead of being compressed, the fine powder is loaded into a small cup, which is placed in the path of the sample beam. The incident radiation is reflected from the base of the cup and, during its passage through the powdered sample and back, absorption of radiation takes place, yielding an infrared spectrum which is very similar to that obtained from the KBr disc method.

Figure 27. In specular reflection (regular reflection), each incident ray is reflected at the same angle to the surface normal as the incident ray, but on the opposing side of the surface normal in the plane formed by incident and reflected ray. On the other hand, in diffuse reflectance, the light observed has the same intensity in all angles of observation.

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

The diffuse reflectance technique mainly used for acquiring spectra of powders and rough surface solids. It is widely used in near infrared spectrometry and it can also be used to examine films and coatings if they are put onto a reflective background. It is also a useful technique for examining polymorphs since the sample can be prepared for analysis with minimal grinding and compression, which can cause interconversion of polymorphs. 3.4. Attenuated Total Reflectance ATR occurs when a beam of radiation enters from a more-dense (with a higher refractive index) into a less-dense medium (with a lower refractive index). The evanescent wave (with a thickness of 0.5–2 μm) penetrates the thin film of the sample and its intensity is decreased when the sample absorbs the wavelength (Figure 28).

Figure 28. Attenuated total reflectance ATR is another recent development in sample handling. In this case, the sample may be run in a gel or cream, and this method may be used to characterize both formulation matrices and their interactions with the drugs present in them. If the active ingredient is relatively concentrated and if a blank of the matrix is run using the same technique it may be subtracted from the sample to yield a spectrum of the active ingredient. ATR also provides another technique which can be used for the characterization of polymorphs.

Formative Assessment No. 6: Instrumentation in Infrared Spectrometry 1. Differentiate a dispersive infrared spectrometer from a Fourier transform spectrometer in terms of: Dispersive IR FTIR Spectrometer Spectrometer a. Scan Speed (Fast/Slow) b. Resolution (High/Low) c. Sensitivity (High/Low) d. Accuracy (High/Low) e. Wavelength range (Wide/Narrow) f. Stray Light (Present/Absent) g. Mechanical Slippage (Present/Absent) (continued on the next page)

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

2. Match Column A with Column B: Column A 1. Sample preparation where the comminuted solid is mixed with dry KBr and compressed at a pressure of 800 kPa 2. Liquid samples run without dilution 3. Sample preparation where the comminuted solid is mixed with dry KBr and placed in a sample cup 4. A technique where the liquid or semisolid film is placed onto high refractive index crystal 5. A suspension or thick slurry of solid samples in mineral oil or chlorofluorocarbon solvent

A. B. C. D. E.

Column B Mull Disc or pellet DRIFT ATR Neat samples

3. True or False. In diffuse reflectance, the reflected light bounces off a smooth surface at an angle equal to the incident angle measured from the surface normal. 4. Which of the following techniques may be used in the identification and differentiation of polymorphic solids? I. Neat II. Disc/pellet III. Mull IV. ATR V. DRIFT A. B.

I and III only I, II, and III only

C. D.

I, III, and V only I, III, IV, and V only

5. Match Column A with Column B. Column A 1. An electrical device consisting of a metal that varies resistance with temperature 2. An electrical device consisting of two dissimilar electrical conductors forming an electrical junction and producing a temperature-dependent voltage as a result of the thermoelectric effect 3. An IR detector made of dielectric materials with very special thermal and electrical properties 4. A resistance thermometer that consists of a semiconductor with temperature-dependent electrical resistance

A. B. C. D.

Column B Thermocouple Bolometer Thermistor Pyroelectric transducer

REFERENCES

Silverstein, R.M., Bassler, G.C., and Morrill, T.C. (1981). Spectrometric Identification of Organic Compounds (4th ed.). New York: John Wiley & Sons. Watson, D.G. (2012). Pharmaceutical Analysis: A Textbook for Pharmacy Students and Pharmaceutical Chemists (3rd ed.). London: Elsevier Churchill Livingstone.

De La Salle Medical and Health Sciences Institute College of Pharmacy

PH-PHR 221 (Pharmaceutical Analysis II) Lecture 6: Infrared Spectrometry

Integration: Infrared Spectrometry Now that you have completed the Online Module in Infrared Spectrometry, I expect you to be able to answer the following questions. Contemplate on these and then, concisely answer each of the following in complete sentences. 1. What do you think will you observe in the infrared spectrum of a mixture of substances? In other words, how will you interpret the absorption peaks observed in a mixture? 2. How will you use infrared spectrometry in the quantitative determination of pharmaceutical mixtures or pharmaceutical products containing more than one component (e.g., combination of drugs and herbal medicines)?