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Chapter 28. Spectroscopy

Recommended Article: 【Organic Chemistry】 Organic Chemistry Table of Contents

1. Overview

2. Mass Spectroscopy

3. Infrared Spectroscopy

4. Ultraviolet-Visible Spectroscopy

5. Nuclear Magnetic Resonance Spectroscopy

6. Raman Spectroscopy

7. Other Spectroscopy Methods

8. Design of Spectroscopy Instruments

a. Spectroscopy Examples

1. Overview

⑴ Photoelectron Spectroscopy: Observing electron transitions by X-rays

⑵ Raman Spectroscopy: Emitting visible light lasers and observing changes in wavelength

⑶ Infrared Spectroscopy: Observing molecular vibrations by infrared. Observing how much the same wavelength of infrared is observed after irradiating infrared

⑷ Microwave Spectroscopy: Observing molecular rotations by microwaves

⑸ Nuclear Magnetic Resonance Spectroscopy: Observing nuclear spin transitions by radio waves

⑹ Here are some tips for solving spectroscopy problems:

① Consider the Degree of Unsaturation First: If the degree of unsaturation is 4 or higher, the presence of a benzene ring is strongly suspected.

② Focus on ¹H NMR: It’s recommended to solve problems primarily using 1H NMR, as it can be approached like solving a math problem and provides a substantial amount of information.

③ Use the Chemical Formula to Narrow Down Possibilities: It’s advisable to deduce possible structures based on the given chemical formula.

④ Utilize Symmetry: Taking advantage of symmetry can make solving problems significantly easier.

⑤ Differentiate cis/trans Alkenes with 1H NMR: The cis/trans isomers of alkenes can be distinguished using ¹H NMR.

⑥ Chemical Shifts of Hydrogens on the Same Carbon: Hydrogens attached to the same carbon can have different chemical shift values (ppm) in ¹H NMR depending on their spatial arrangement.

2. Mass Spectroscopy (MS)

⑴ Principle

Figure 1. Diagram of Mass Analysis Method

① 1^st. entrance region

○ 1-1. Sample molecules are introduced: Molecules are mostly neutral

○ 1-2. Molecules pass through metal plates with positive potential

② 2^nd. ionization region

○ 2-1. In a mass spectrometer, electrons with an energy of 70 eV are emitted through an electron beam.

○ 2-2. Ionization stage: Organic molecules colliding with high-energy electrons lose one electron and form molecular ions

○ Mass of molecular ion = Molecular weight

○ Molecular ion takes radical form: Can be expressed as M^+ㆍ

○ 2-3. Fragmentation: Molecular ions are very unstable, breaking into radical parts and positively charged parts

○ Preferred fragmentation reactions exist depending on the functional groups

○ Fragmentation of polypeptides: b represents a fragment with NH2 at the end, and y represents a fragment with -COOH at the end

Figure 2. Example of Polypeptide Fragmentation

Figure 3. Forms of Fragmented Ions

○ When two peaks differ exactly by 28 Da, it is estimated to be an a-b pair or an x-y pair

○ The a ion appears less frequently than the b ion: C≡O⁺ is more unstable than C=N⁺

○ 2-4. Generation of 1+, 2+ ions, etc.: Generally, 1+ ions are formed

③ 3^rd. acceleration region

○ 3-1. The electric field is formed due to the metal plates, each with zero and negative potential, along with the metal plate in the front with positive potential.

○ 3-2. Positively charged molecules are accelerated according to the electric field

○ Only positively charged chemical species reach the recorder

④ 4^th. drift region

○ Positively charged molecules move in a straight line in the drift region where there is no electric field when passing through metal plates with negative potential

⑤ 5^th. detector region

○ Type 1: Reflectron mode: See Figure 1.

○ Principle: Lorentz Force

○ In other words, when ions pass through a magnetic field, they can reach the detector differently depending on their m/z ratios.

○ Type 2: Linear mode

○ A representative example is quadrupole mass spectrometry (QMS).

○ In QMS, an oscillating electric field is provided through RF (radio frequency), allowing ions to reach the detector differently depending on their m/z ratios.

○ Principle 1: Time-of-Flight (TOF) Mass Analyzer

○ Utilizes the principle that ions of different masses take different times to reach the detector based on m/z

○ In other words, ions with smaller masses reach the detector faster than ions with larger masses.

○ Principle 2: Liquid Chromatography (LC) Analyzer

○ Specific m/z can be selectively accepted, not the entire m/z (e.g., MS/MS)

○ A single substance can produce multiple ions, thus having multiple m/z values: Deconvolution algorithm creates a single peak

⑵ Interpretation of Mass Analysis

① The highest peak in mass analysis is called the base peak

② The intensity of the base peak is set as 100, and the others are measured as relative values.

③ Example of m/z: The more stable the positive chemical species, the larger the peak

Figure 4. Example of m/z

④ Nitrogen rule

○ Hydrocarbons composed of C, H, O elements always have even molecular weights, so molecular ions are even in m/z ratio.

○ If an odd-numbered molecular ion in m/z ratio is observed in organic molecules, it can be assumed to contain an odd number of nitrogen atoms.

○ Example: The odd m/z in the Figure 4 is due to not considering proton transfer. Refer to MALDI-TOF.

○ CH₄: 16

○ C_nH_2n+2: Even

○ C_nH_2n: Even

○ C_nH_2n-2: Even

○ When substituting -H with -OH, -F, -Cl, -Br, -I, -SH, -SiH₃ groups: Even

○ When substituting -H with -NH₂ group: Odd

⑶ Mass Analysis and Isotopes

① High-resolution spectrometers can distinguish isotopes

Table 1. Abundance of Isotopes Found in Organic Compounds (Natural Abundance)

② Cl exists in a ratio of 3:1 with masses 35 and 37

③ Br exists in a 1:1 ratio with masses 79 and 81

④ The height difference between M and M+2 indicates the presence of certain elements

⑷ Type 1: Matrix-Assisted Laser Desorption/Ionization (MALDI-TOF): Koichi Tanaka won the Nobel Prize related to this.

① 1^st. Sample (M) is mixed with a matrix, and when nitrogen laser light (337 nm) is applied to the matrix, heat is generated

② 2^nd. Due to the generated heat, the matrix vaporizes along with the sample

③ 3^rd. The matrix is vaporized and simultaneously protonated: M → MH⁺

④ 4^th. Proton transfer with A (anion): MH⁺ + A → M + AH⁺

Figure 5. Principle of MALDI-TOF

⑤ Targeted towards solid samples. Signal splitting occurs up to about 1+, 2+, and 3+ ions

Figure 6. MALDI-TOF Mass Analysis Result of IgG before Deconvolution

⑸ Type 2: Electron Spray Ionization (ESI): John B. Fenn received the Nobel Prize regarding this.

① Liquid samples after passing through High-Performance Liquid Chromatography (HPLC) are sprayed in a nebulizer gas-covered high-voltage tube, forming a spray

② Targeted towards liquid samples. Significant signal splitting occurs

Figure 7. ESI Mass Spectrum of Trypsinogen (Mw : 23983) before Deconvolution

⑹ Applications: Quantification of Mass Analysis (Focused on Protein Analysis)

① Background: Mass analyzers are not inherently quantitative, so various techniques have been developed

○ Reason 1. Laser power can vary depending on the measurement spot

○ Reason 2. The degree of ionization varies for different substances.

② Type 1: Relative Quantification

○ 1-1. Labeling

○ ICAT (Isotope-Coded Affinity Tag): Chemical

○ Structure: Affinity tag (biotin) + Linker region (light: 8 ¹H, heavy: 8 ²H) + Cysteine binding motif (iodoacetamide)

○ Assumption: Target protein contains Cys

○ Principle: Selectively label with a light linker according to the experimental conditions, and compare with the heavy linker labeled under different conditions.

○ 1^st. Attach light ICAT tag to one experimental group and heavy ICAT tag to another experimental group

○ 2^nd. Combine proteins obtained from each experimental group and perform avidin affinity chromatography

○ 3^rd. Selectively collect tagged proteins: The tag only binds to proteins containing Cys, thus many other proteins are removed.

○ 4^th. Perform LC/MS

○ Labeling stage: Protein

○ Disadvantage 1. Difficult to separate target protein and streptavidin

○ Disadvantage 2. Because the biotin tag is more prone to fragmentation than the protein, signal interference due to biotin can occur.

○ SILAC (Stable Isotope Labelling with Amino Acids in Cell Culture): Metabolic

○ Use: Protein expression study, unbiased analysis of post-translational modification

○ 1^st. Provide cells in the heavy side with ¹³C, ¹⁵N-Arg, ¹³C-Lys

○ 2^nd. Provide cells in the light side with ¹²C, ¹⁴N-Arg, ¹²C-Lys

○ 3^rd. Combine extracts from each source and perform LC/MS/MS

○ 4^th. Identify and relatively quantify through spectra

○ Labeling stage: Cell

○ Advantage 1. Allows reliable correction in data analysis as even the same protein shows slight variations in signal

○ Advantage 2. Labels occur naturally without separate labeling reactions

○ Advantage 3. Labeling of the cells themselves, i.e. analysis on a global scale, is possible.

○ Disadvantage 1. Multiplexing is Limited to 3.

○ Disadvantage 2. If signals overlap in the spectrum, it becomes difficult to distinguish between heavy-side and light-side signals.

○ BEMAD (β-Elimination Followed by Michael Addition of DTT): Chemical

○ DTT functions as a reducing agent, removing disulfide bonds (-S-S-) within proteins and making -SH.

○ Principle: Using ²H₆-DTT instead of DTT and relative comparison in the spectrum.

○ Labeling stage: Protein

○ ¹⁶O / ¹⁸O: Enzymatic

○ Principle: Analyzing the products generated when hydration occurs in water under trypsin conditions

○ Labeling stage: Protein

○ ¹⁵N: Metabolic

○ Labeling stage: Protein

○ 1-2. Label-Free

○ Peak intensity

○ Spectral counting

○ Spectral TIC

③ Type 2: Absolute Quantification

○ 2-1. Labeling

○ iTRAQ (Isobaric Tag for Relative and Absolute Quantitation): Chemical

○ iTRAQ reagent: Reporter group (cationic) + Balance group (neutral) + Amine binding motif (NHS)

○ Reporter group and balance group together are referred to as isobaric tag

○ Relative Quantification Principle: Vary reporter group and balance group to shift peak positions in the spectrum

○ Absolute Quantification Principle: Measure the charge amount of the reporter group in each extract

○ Labeling stage: Peptide

○ Advantages: Comparatively flexible multiplexing is possible. Both relative and absolute quantification are feasible

○ Disadvantages: Can only be used with substances containing amines. Unlike SILAC, comparison on a global scale is not possible

○ MRM (Multiple Reaction Monitoring)

○ TMT (Tandem Mass Tags)

○ 2-2. Label-Free

○ APEX

④ Relative quantification methods can also become absolute quantification

○ Method 1: Mix a labeled substance with a known quantity with a substance whose quantity is to be determined, and then perform relative quantification.

○ Method 2: Drawing calibration curves

⑺ Applications: MS/MS (MS², Tandem Mass Spectrometry)

① Definition: Combination of two mass spectrometers

○ Generally utilizes triple-quadrupole structure (linear detector mode)

○ Component 1: MS1 (quadrupole structure; Q1): Separates ion X with specific m/z after ionizing the given sample

○ Ion that passes through MS1 is referred to as precursor ion

○ Component 2: Fragmentation part (quadrupole structure; Q2): Breaks down ion X with specific m/z into smaller fragments

○ Methods like CID (collision-induced dissociation), ion-molecule reaction, photodissociation are used in Component 2

○ Component 3: MS2 (quadrupole structure; Q3): Separates fragments with specific m/z

○ Ion that passes through MS2 is referred to as product ion

○ The types of signals that can be obtained through MS/MS have become very diverse.

○ Recombining m/z signals of each fragment allows estimation of m/z value of ion X: Area of algorithms

② Type 1: Shotgun technique

○ Definition: Technique to detect spectra of all chemical species without filtering in MS1 and MS2

○ Used in shotgun proteomics, etc. to determine relative distribution of entire proteins.

○ Drawbacks: Time-consuming, low sensitivity, inability to investigate specific m/z values due to limited time

○ 1-1: DDA (data-dependent acquisition) method: Selects single peak from MS1 and performs MS2. Requires significant time

○ 1-2: DIA (data-independent acquisition) method: Selects peak range from MS1 and performs MS2

Figure 8. Comparison between DDA and DIA methods

③ Type 2: SRM (selected reaction monitoring)

○ Definition: Technique to detect spectra of fragments with specific m/z

○ Advantages: Automation, no need for antibody purchase, increased sensitivity (reduced background interference), improved selectivity, expanded dynamic range

○ Drawbacks: Limited to absolute quantification

④ Type 3: MRM (multiple reaction monitoring)

○ Definition: Technique to detect spectra of one or more product ions from one or more precursor ions

○ SRM and MRM are sometimes included under the broader meaning of SRM

⑻ Applications: McLafferty rearrangement

Figure 9. McLafferty rearrangement

① Characteristic 1: Movement of hydrogen radical

② Characteristic 2: Six-membered ring transition state

③ Characteristic 3: Decomposition into neutral alkene and radical cation

3. Infrared Spectroscopy (IR spectrum)

⑴ Definition

① Experimental method for analyzing functional groups of compounds by analyzing the absorption wavelengths of infrared radiation

② Used in pH sensors, etc.

③ Models for predicting IR spectra using AI are being developed

⑵ Vibrations: Oscillation of electrons, categorized into stretching vibrations and bending vibrations

① Infrared absorption wavelengths are in the mid-infrared range (2,500-8,000 nm)

② Mid-infrared light has a high absorption rate and weak signal strength, so near-infrared light is generally used instead.

③ Attempts are being made to utilize mid-infrared

⑶ Type 1: Stretching vibrations

① Definition: Temporary transfer of electrons from an atom with high electronegativity to an atom with lower electronegativity

② Types: Symmetric stretch, asymmetric stretch

③ Most IR spectroscopy is related to stretching vibrations

④ Atoms with dipole moments absorb infrared radiation and reduce the size of the dipole moment

○ Similarly, there must be a change in dipole moment for infrared absorption in IR spectroscopy.

⑤ Infrared absorption wavelengths are quantized

⑥ In IR, infrared absorption lines are represented as wavenumbers (cm^-1), the reciprocal of wavelength in centimeters, generally ranging from 4000-400 cm^-1

○ 521 cm^-1 refers to 521 waves per centimeter

⑦ Factor 1: Stronger bonds require more energy, leading to increased wavenumbers

○ E = hν (where ν is wavenumber) closely related

○ C≡C ＞ C=C ＞ C-C

○ C=O bond in 2-pentanone is a double bond, leading to observation of wavenumber 1720 cm^-1.

○ C=O bond in 2-cyclohexanone has lower degree than 2 due to resonance, leading to observation of wavenumber 1680 cm^-1, which is less than 1720 cm^-1.

⑧ Factor 2: Smaller atoms have less electron dispersion, leading to increased wavenumbers

⑨ Factor 3: Larger dipoles indicate stronger bonds, leading to increased wavenumbers

○ O-H ＞ N-H ＞ C-H

○ Here, dipole refers to dipole in a single bond, not the dipole of the entire molecule (ref)

Figure 10. A example comparison of C=O stretching vibration wavenumbers

○ The right structure has a higher wavenumber from C=O stretching vibration.

○ Reason 1: Due to hybridization effects, electrons move from the methyl group (sp³) to the ketone carbon (sp²) → bond energy increases → wavenumber (ν = E/h) increases.

○ Reason 2: When electrons are delocalized by the benzene ring, the resonance wavelength increases, and the wavenumber decreases (cf. particle in a box).

○ The relationship between wavenumber and dipole moment seems to be limited to single bonds because, in the left structure, 1) the dipole intermediate is stabilized, and 2) the distance between the dipoles is greater, resulting in a larger dipole moment for the left structure.

Figure 11. The reason the left structure has a larger dipole moment

⑩ Factor 4: When hydrogen bonding is present, because intermolecular bonds resemble intramolecular bonds and there are a variety of hydrogen bond forms, the absorption lines become very broad.

○ OH bond is observed as a broad spectrum due to hydrogen bonding

⑪ Stretching vibration absorption wavenumbers for major functional groups

Table 2. Stretching vibration absorption wavenumbers for major functional groups

⑷ Type 2: Bending vibrations (bending vibration, deformation vibration)

① Definition: Temporary change in bond angle

② Types

○ Symmetric in-plane bend (scissor)

○ Asymmetric in-plane bend (rock)

○ Symmetric out-of-plane bend (twist)

○ Asymmetric out-of-plane bend (wag)

③ Primarily found in carbon-hydrogen bonds

④ Bending vibrations require less energy than stretching vibrations, resulting in smaller wavenumbers

⑤ Bending vibration absorption wavenumbers for major functional groups

Table 3. Bending vibration absorption wavenumbers for major functional groups

⑸ Type 3: Rotational energy: Rotational energy subdivides vibration energy levels further

⑹ Examples: Sequence of examples follows organic chemistry index

① Example 1: Alcohol

Figure 12. IR spectroscopy of alcohol

○ 3400: -OH

○ 1060: C-O

② Example 2: 2-propyn-1-ol

Figure 13. IR spectroscopy of 2-propyn-1-ol

○ 3300: OH group

○ 2950: sp³ CH

○ 2100: Alkyne

③ Example 3: Diethyl ether

Figure 14. IR spectroscopy of diethyl ether

④ Example 4: Ketone

Figure 15. IR spectroscopy of ketone

○ 1720: C=O

○ Finger region

⑤ Example 5: Aldehyde

Figure 16. IR spectroscopy of aldehyde

○ 2700, 2800: C-H

○ 1720: C=O

⑥ Example 6: Benzaldehyde

Figure 17. IR spectroscopy of benzaldehyde

○ 3050: sp² CH

○ 2810, 2730: Unique twin peaks of aldehyde

○ 1700: Partial single-bond character carbonyl

○ 1600, 1460: Benzene ring

⑦ Example 7: Carboxylic acid

Figure 18. IR spectroscopy of carboxylic acid

○ 3200, 2600: -OH

○ 3000: C-H

○ 1700: C=O

○ 1200: C-O

⑧ Example 8: Ester

Figure 19. IR spectroscopy of ester

○ 1760: C=O

○ 1100, 1200: C-O

○ Finger region

⑨ Example 9: Amide

Figure 20. IR spectroscopy of amide

○ 1660: C=O

○ Finger region

⑩ Example 10: N-methylethanamide

Figure 21. IR spectroscopy of N-methylethanamide

○ 3300: N-H stretching vibration

○ 2950: sp³ CH

○ 1660: Amide carbonyl

○ 1560: N-H bending vibration

⑪ Example 11: Amine

Figure 22. IR spectroscopy of amine

○ 3400, 3300: N-H stretching vibration

○ 1600: N-H bending vibration

○ Finger region

⑺ Application 1: Vibrational-rotational spectrum

① Definition: Visualization of information on vibration and rotation obtained through IR or Raman spectroscopy

② R-branch: ΔJ = 1, Δv = 1

4. Ultraviolet-Visible Spectroscopy (UV-Vis spectrum)

⑴ Principle 1: Determination of absorption wavelengths

① π bonds can absorb UV-visible radiation, and longer conjugation lengths result in longer absorption wavelengths

② π bond electrons transition to π* orbitals upon absorbing UV-visible radiation

⑵ Principle 2: Beer-Lambert law

① Proof

○ I: Intensity of light

○ d: Path length of light penetration

○ a: Absorption coefficient depending on medium

○ T: Transmittance

○ A: Absorbance (unit: OD, optical density)

○ c: Molar concentration

○ ε: Molar absorptivity

② Conclusion: Absorbance is directly proportional to substance concentration

⑶ Absorption color and observed color based on absorption wavelength

table 4. Absorption color and observed color based on absorption wavelength

⑷ Examples of absorption wavelengths

① Chemical substances

○ Methyl vinyl ketone: 219 nm

○ 3,5-hexadien-2-one: 249 nm

○ anilinium ion: 254 nm

○ benzene: 255 nm

○ phenol: 270 nm

○ aniline: 280 nm

○ phenolate ion: 287 nm

② Biomolecules

○ Nitrogen bases of nucleic acids: 260 nm

○ Phenyl groups in Phe, Trp, Tyr: 280 nm

○ NADH: 340 nm

○ Ninhydrin: 405 nm. Ninhydrin reacts with amino acids to form a purple product with an absorption wavelength of 570 nm

○ β-carotene: 455 nm

○ Lycopene: 474 nm

○ Carotenoids: 500 nm

○ Hemoglobin: 560 nm

○ Chlorophyll: 680 nm, 700 nm

○ Pigment molecules of photosynthetic bacteria (e.g., cyanobacteria): 840 nm, 870 nm

5. Nuclear Magnetic Resonance (NMR) Spectroscopy

⑴ Principle

① Chemical species with nonzero spin quantum numbers like ¹H, ¹³C, ¹⁵N, ¹⁹F, ³¹P can be studied using NMR.

② Such chemical species exhibit quantized states when subjected to an external magnetic field.

③ The electromagnetic waves corresponding to energy differences between these states are radiofrequency waves.

④ Information obtained through NMR: Type and number of specific chemical species.

⑤ Voxel size in MRI is generally 1 × 1 × 5 mm³, while in MRS it is larger at 15 × 15 × 15 mm³.

⑵ ¹H NMR (Proton NMR)

① ¹H possesses two quantized states with respect to the magnetic field B₀: α-spin state and β-spin state.

Figure 23. Two quantized states with respect to the magnetic field

② Spin flip: When ¹H absorbs energy proportional to the magnetic field strength, it transitions from the α-spin state to the β-spin state.

③ Principle 1. Integral ratio

○ Integral ratio of NMR signals represents the ratio of equivalent 1H nuclei.

○ Reason: Signal is linearly proportional to the number of equivalent 1H nuclei. Other factors have minimal influence.

④ Principle 2. Chemical shift

○ Lenz’s Law: When there’s a change in the external magnetic field, electrons induce a magnetic field in the opposite direction to cancel it.

○ Chemical shielding: Refers to local variations in the magnetic field, which are proportional to the basic magnetic field B₀.

○ B_i: Local magnetic field

○ σ_i: Shielding factor reflecting the shielding effect on proton i

○ Shielding factors are generally small (10^-4 to 10^-6), so magnetic field differences are very small.

○ Using ratios instead of absolute values to reflect magnetic field differences

○ Calculation of index: The difference between local magnetic field and reference frequency divided by the reference frequency. Commonly expressed in ppm units.

○ This term representing the magnetic field difference is called chemical shift.

○ The value of ω_ref is arbitrary but often chosen as ω_TR of the transmitter.

○ Main advantage: Independent of B₀

○ Example: In a 300 MHz ¹H nuclear magnetic resonance (NMR) spectrum, acetone exhibits a single resonance line with a chemical shift δ of 2.0 ppm with reference to tetramethylsilane (TMS). How many Hz away from TMS is this acetone’s resonance line? 600 Hz

○ Protons in an electron-deficient environment experience a strong external magnetic field, resulting in higher resonance frequency and larger δ value.

○ Protons in an electron-deficient environment are called deshielded protons or downfield protons.

○ Associated with the deshielding effect

○ Protons in an electron-rich environment experience a weaker external magnetic field, leading to lower resonance frequency and smaller δ value.

○ Protons in an electron-rich environment are called shielded protons or upfield protons.

○ Associated with the shielding effect

○ Protons closer to atoms with high electronegativity experience more deshielding, resulting in larger δ values.

○ Example: In CH₃CH₂CH₂NO₂, δ for H is 1.04 ppm, δ for H is 2.07 ppm, δ for H is 4.37 ppm.

○ Example: Methyl proton corresponds to 0.85 ppm, methylene proton corresponds to 1.20 ppm, and methine proton corresponds to 1.55 ppm.

⑤ Principle 3. Spin-Spin Splitting: Also called coupling, multiplicity.

○ When there are n equivalent 1H atoms around a specific ¹H atom, the NMR signal of that ¹H atom splits into n+1 peaks and follows a binomial distribution.

○ Background 1. Each neighboring ¹H can adopt one of two spin states: α-spin state or β-spin state.

○ Background 2. The state of each neighboring ¹H affects the NMR signal of the specific ¹H.

○ ortho hydrogen > meta hydrogen > para hydrogen in terms of splitting influence: That is, the impact magnitude is increasing as they get closer.

Figure 24. Spin-Spin Splitting Constants according to Hydrogen Orientation

○ When only ortho hydrogens are present

○ NMR signal from that ¹H is split into a total of n+1 signals, and the relative size of the k-th signal is proportional to binomial coefficients _nC_k.

○ Depending on the number of splittings, signals are classified as singlet, doublet, triplet, quartet, quintet, sextet, septet, octet, nonet, etc.

⑥ Application 1: Multiple Bonds and ¹H NMR

○ For the following compounds, list them in order of increasing chemical shift (δ) values for the protons in each compound:

Figure 25. Multiple Bonds and ¹H NMR

○ Right (6.5-8 ppm) ＞ Center (5.3 ppm) ＞ Left (2.4 ppm)

○ Benzyl proton: The circulating electrons create a magnetic field in the same direction as the external magnetic field → Higher resonance frequency and δ value.

○ Alkene proton: Electron motion generates a magnetic field in the same direction as the external magnetic field → Higher resonance frequency and δ value.

○ Alkyne proton: Only induced magnetic field opposite to the external magnetic field according to Lenz’s Law → Lower resonance frequency and δ value.

Figure 26. ¹H-NMR δ values and their principles for the given compounds

⑦ Actual ¹H NMR

○ TMS (tetramethylsilane): Used as a reference substance, its signal is set to 0 ppm.

○ CDCl₃, acetone, methanol, DMSO, and benzene are also used as NMR solvents.

○ δ values of other ¹H NMR signals are expressed relative to TMS.

○ Reason 1. High frequency values

○ Reason 2. δ values are independent of the magnetic field (because frequency is proportional to the magnetic field)

○ Examples of chemical shifts: Benzene hydrogen = 6.5-8, aldehyde hydrogen = 9.0-10

Figure 27. Examples of ¹H NMR chemical shifts

⑧ ¹H NMR Examples (ref)

⑶ ¹³C NMR

① Principle 1. Integral ratio

② Principle 2. Chemical shift: After 220 ppm

③ Principle 3. Spin-Spin Splitting

④ Actual ¹³C NMR

○ TMS (tetramethylsilane): Used as a reference substance, its signal is set to 0 ppm.

○ CDCl₃, acetone, methanol, DMSO, and benzene are also used as NMR solvents.

○ ¹³C NMR values for various substances: R-CH₃ = 0-35, C-O = 50-90, benzene = 110-170, C=O = 205-220

Figure 28. Examples of ¹³C NMR chemical shifts

⑤ ¹³C NMR Examples (ref)

Figure 29. ¹³C NMR Examples

The answer is ①

○ A: There are a total of three types of carbon positions: Cl position (2 places), positions next to Cl (2 places), and positions next to the positions next to Cl (2 places).

○ B: There are a total of four carbon positions: Cl position (2 places), position to the right of Cl (1 place), positions to the left of Cl (2 places), and a position next to the positions next to Cl (1 place).

○ C: There are a total of two types of carbon positions: Cl position (2 places) and positions next to Cl (4 places).

6. Raman Spectroscopy

⑴ Definition: Observing wavelength changes by shining monochromatic light onto a target.

Figure 30. Difference between FTIR (above) and Raman spectroscopy (below)

⑵ Inelastic Scattering: Also known as Raman scattering

① Definition: Scattering with energy change. Incident and scattered wavelengths are different.

② Raman shift is used to denote how much the wavelength has shifted from Rayleigh scattering.

○ Unlike IR spectroscopy, it doesn’t directly measure vibrational energy.

○ Raman shift is measured in cm^-1.

③ Quantum mechanical understanding: Energy difference before and after Raman scattering corresponds to molecular vibrational energy.

○ When a molecule gains energy, it’s called Stokes scattering.

○ When a molecule loses energy, it’s called anti-Stokes scattering.

○ Stokes scattering is generally more prominent than anti-Stokes scattering because ground state molecules outnumber vibrationally excited molecules.

⑶ Raman Spectrum Fingerprint Region

Figure 31. Raman spectrum fingerprint region

⑷ Spectroscopic Process

① 1^st. Monochromatic light is directed onto the sample.

② 2^nd. Both Raman-scattered light and Rayleigh-scattered light pass through a notch filter, mostly containing Rayleigh scattering.

③ 3^rd. The notch filter selectively blocks Rayleigh-scattered light.

④ 4^th. Raman-scattered light that passes through the notch filter is separated by a grating based on its wavelength.

⑤ 5^th. The specific wavelength Raman-scattered light is read by a CCD.

⑸ Using visible light on human skin results in strong fluorescence, making it difficult to observe Raman scattering spectra.

① Strategy 1. Use longer wavelengths to reduce fluorescence interference.

○ Example 1. Near-IR FT Raman spectroscopy using Nd:YAG laser with a wavelength of 1064 nm.

○ Example 2. Dispersion Raman spectroscopy using CCD (charge coupled device) detector.

② Strategy 2. Use shorter wavelengths to separate the fluorescence region from the Raman spectrum region.

○ Example 1. Ultraviolet-resonance Raman spectroscopy (UVRR)

7. Other Spectroscopy Methods

⑴ Surface Plasmon Resonance Spectroscopy

① The principle of surface plasmon resonance(SPR)

⑵ Refractive Index Microscopy

8. Design of Spectroscopy Instruments

⑴ Definition: Device used to measure the reflection, transmission, absorption, scattering, and fluorescence of a given sample at different wavelengths, when the light is incident on the sample.

⑵ Component 1. Light Source

⑶ Component 2. Wavelength Selector: Prism, grating, optical filters, etc.

⑷ Grating (Diffraction Grating)

① Definition: Made by cutting sawtooth-shaped grooves into a smoothly polished metal surface.

○ Reflective diffraction gratings used in the ultraviolet and visible light spectrum have 300 to 6,000 grooves per millimeter.

○ Reflective diffraction gratings used in the infrared spectrum have 10 to 200 grooves per millimeter.

○ The grooves must all be of the same size, parallel, and equally spaced.

② Echellette Type Diffraction Grating

○ When parallel rays of light hit the reflecting surface, they follow the law of reflection at each surface.

○ Reflected rays interfere with each other.

○ Reinforcement interference occurs when the difference in path traveled by adjacent rays is an integer multiple of the wavelength.

Figure 32. Reinforcement Interference Conditions for Echellette Type Grating

⑸ Optical Filters

① Interference Filter

○ Consists of two partially transparent thin films separated by a transparent thin dielectric layer.

○ Bragg diffraction: As the thickness of the dielectric layer decreases and the angle of incidence increases, the wavelength of the radiation increases.

Figure 33. Bragg Diffraction

○ Forms the principle of X-ray diffraction (XRD).

○ Feature: Narrow effective wavelength width (FWHM, full width at half magnitude).

② Absorption Filter

○ A filter that overall weakens incident light.

○ Wide effective wavelength width (FWHM, full width at half magnitude).

○ Examples: UV cut-off filter, NIR absorption filter

⑹ Type 1. Single-Beam Spectrophotometer

○ Multi-color light → Monochromator → Monochromatic light

○ Measures transmittance P/P₀

○ Alternately measures sample and solvent: Error occurs due to changes in light source intensity and detector sensitivity.

⑺ Type 2. Double-Beam Spectrophotometer

○ The light alternately passes through the sample and reference containers.

○ Minimizes errors due to changes in light source intensity and detector sensitivity over time.

Input: 2019.05.02 09:32

Modified: 2020.03.04 15:29