Final Exam Test Review Notes

UV-Vis Spectrometers

• Slitwidth is programmed to change as a function of wavelength to maintain constant spectral resolution across the UV-Vis range.

• At longer wavelengths (lower energy), resolution decreases, so a narrower slit compensates.

• Advantages of increased slitwidth:

  • Higher signal intensity (better sensitivity)

  • Better signal-to-noise ratio

• Disadvantages:

  • Lower spectral resolution (peaks may broaden or overlap)

  • Potential loss of fine spectral details

Light Separation

• A prism separates UV or visible light by Refraction.

Gas Chromatography Detectors: FID vs TCD

• Flame Ionization Detection (FID)

  • Advantages: High sensitivity (especially to hydrocarbons), wide dynamic range

  • Disadvantages: Destroys sample, not universal (poor response to non-organics)

• Thermal Conductivity Detection (TCD)

  • Advantages: Universal (detects all compounds), non-destructive

  • Disadvantages: Lower sensitivity than FID

Fluorescence Quenching

• A quencher decreases fluorescence intensity in the emission spectrum.

Beer’s Law in Emission Spectroscopy

• Beer’s Law applies when emission intensity is proportional to analyte concentration.

• This holds at low concentrations where self-absorption and inner filter effects are negligible, and the excitation source is stable.

Spectroscopy Sources

• IR spectroscopy source: Globar (silicon carbide), Nernst glower, or heated filament

• Raman spectroscopy source: Monochromatic laser

HPLC Plate Height and Particle Size

• Plate height decreases as particle size decreases.

• Smaller particles improve efficiency (more theoretical plates) but increase backpressure.

Chromatography R Groups

• (a) Normal Phase: Polar groups like –SiOH, –NH₂, –CN

• (b) Reverse Phase: Nonpolar/hydrophobic groups like –C₈H₁₇ (C8), –C₁₈H₃₇ (C18)

Mass Spectrometry Methods for Molecular Weight Determination

• Methods to determine the molecular weight of a large biomacromolecule or polymer:

  • MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight)

  • ESI (Electrospray Ionization)

  • FT-ICR (Fourier Transform Ion Cyclotron Resonance) for high resolution

Chemical Ionization (CI) in Mass Spectrometry

• Chemical Ionization (CI) produces [M + H]+ (or [M + other adduct]+).

• The molecular ion weight is 1 amu higher than M, due to protonation.

n to sigma* Absorption

• Molecules that can have an n to sigma* absorption: NH₃ and H₂O

NMR Signals

• The aldehyde (CH₃)₃CCH₂CHO has three 1H NMR signals.

NMR Multiplicities

• For butanone, Hs on C1, singlet; Hs on C3, quartet; Hs on C4, triplet.

• The proton signal splits into n + 1 peaks when the adjacent carbon has n equivalent hydrogens.

• C1 is the isolated methyl, and C3 is the methylene linked to C4 (methyl) in butanone.

NMR Experiment

• NOT true about the NMR experiment: The energy required to flip the spin of a proton is in the infrared region of the electromagnetic spectrum.

Spin 1/2 Nucleus

• For a spin 1/2 nucleus in a magnetic field, there are 2 energy levels.

FT NMR Signal Acquisition

• In FT NMR signal acquisition takes place using a sequence described by

• wait time - a 90° pulse – acquisition.

Mass Analyzers

• Not a scanning mass analyzer: TOF

FT Instrument Resolution

• Resolution of an FT instrument depends on the Time over which the interferogram is acquired.

Wavelength Shift

• An unknown compound has an absorption at 600 nm in water, 620 nm in methanol (CH₃OH), and 640 nm in decane. There is a hypsochromic shift in the wavelength on going to more polar solvents.

Vibrational Modes of CO₂

• The symmetric and asymmetric stretch vibrations and the bending mode vibration of CO₂ are Raman active, IR active and IR active.

NMR Components

• Main components:

  • Magnet: Provides a strong, stable magnetic field

  • RF transmitter/receiver coil: Sends/receives radiofrequency pulses

  • Sample probe: Holds NMR tube in the field

  • Shims: Adjust field homogeneity

  • Computer/console: Controls pulses, acquires, and processes data

• The Probe: The coil emits the RF pulse and reads the signal from the sample

Relaxation

• Spin-lattice relaxation (T1) converts the excess energy into translational, rotational, and vibrational energy of the surrounding atoms and molecules (the lattice).

• Spin-spin relaxation (T2) transfers the excess energy to other magnetic nuclei in the sample.

Equivalent Protons

• 2 sets of protons are equivalent in meta-xylene (1,3-dimethylbenzene).

Protons in a Magnetic Field

• When an external magnetic field is applied, some protons align with the field and some align opposite to it.

Deshielded Protons

• CH₃Cl has the MOST deshielded protons.

NMR Spectrum Assignment

• Match the 1H NMR spectrum below to its corresponding compound, and assign all of the signals: 2-pentanone

• There is no signals at about 9 ppm for the aldehyde hydrogens in the spectra, so the spectrum is not for compound butaldehyde.

Van Deemter Equation

• A: eddy diffusion (multiple paths)

• B: Longitudinal diffusion

• C: Mass transfer resistance

• Does not contribute: A & B

Chromatography Phases

• (a): Polar (Normal)

• (b): Non-Polar (Reverse)

Bulk Property Detector

• Refractive index detector

HPLC Separation

• Gel permeation chromatography

Van Deemter Plot

• Optimum mobile phase flow rate

Chromatography Retention

• Polar and non-polar

Theoretical Plates

• Estimate the efficiency of a column

Reverse Chromatography

• The stationary phase is made non-polar.

Gel Permeation Chromatography

• Small Molecules elute last

• Large Molecules elute first

Gas Chromatography

• Major contribution to brand broadening: Longitudinal Diffusion

The efficiency of the column can be increased

• Plate number becomes greater; plate height becomes smaller

Detectors

• Flame ionization detector [FID]: Mass Flow-dependent signals

• Thermal Conductivity Detector [TCD]: Concentration

Carrier Gas

• Inert Gas: no interaction with the sample

• Thermal conductivity

• Cheap

• Example: He

EI and CI Analysis

• Molecules with boiling points less than 500 °C and weighing less than 1,000 Da

Michelson Interferometer

• If the reflected and transmitted beams are in phase at the beam splitter, then maximum intensity will reach the detector.

• True

Zone Broadening

• A decrease in the retention factor

• A decrease in the selectivity factor

• A decrease in resolution between any two peaks

• NOT TRUE: An increase in retention time

WCOT column

• WCOT columns may be constructed of metal, glass, plastic, or fused silica.

• True

GC Detection

• FID

  • High sensitivity and not water sensitive

  • Destroys sample, not universal (poor response to non-organics)

• TCD

  • Universal (detects all compounds), non-destructive

  • Lower sensitivity than FID

Flame Ionization Detector

•  A flame ionization detector easily detects organics with alcohol, carbonyls, halogens, and amine functional groups → False (Burns organics)

Raman

• Undergo a change in polarizability upon vibration

FTIR Instrument

• Reference for acquisition, acquiring digital data

Ion Source in MS

• Hard Ion source: More Fragmentation

• Soft Ion source: Less Fragmentation

MS Analysis

• Scanning: Magnetic sector & quadrupole

• Non-Scanning: Time of flight [TOF]

van Deemter

• van Deemter: Relationship between the height equivalent to a theoretical plate

• Longitudinal Diffusion (B/u)

  • more significant in GC [coffee example]

  • In HPLC, it is small or negligible due to the low diffusion coefficient

• Plot

  • GC: more pronounced curve at low velocities

  • HPLC: the curve is flatter

Carrier gas in GC

• Helium

Normal Phase HPLC

• The mobile phase is nonpolar

• The stationary phase is polar.

Ion-exchange chromatography

• A type of chromatography in which the analyte ions in the mobile phase are attracted to the counter ions in the stationary phase.

• This separates polar molecules.

 liquid Chromatography

• depends on interactions between solute, mobile phase, and immobilized liquid stationary phase: Partition

retention factor, k'

• Relates to time spent in the stationary vs mobile phase

Detector response

• not determine the column efficiency in liquid chromatography

Gel-Permeation Chromatography

• Small molecules elute first

Size exclusion chromatography

• work by the same principles of Size exclusion

  • Gel filtration or gel permeation chromatography

EI & CI analyze

• Molecules with boiling points less than 500°C and weighing less than 1000 Da

MS Ions detect

• MS uses an electron multiplier for detection

State of matter for mass spectroscopy

• Perform in the Gaseous state

GC-MS was developed in what system? 

• Packed column

Disadvantages of reciprocating pump (Liquid Chromatography)

• Produces pulsed flow

Not used in liquid or high-performance liquid chromatography 

• Capillary column

Hard and soft Ion Source

• Soft; (M+1)⁺, (M-1)⁺

• ESI: Soft; Multiple Charged Ions, Mn⁺

• MALDI: Soft; M⁺, MH⁺,(M+Na⁺)

• Which MS methods may be used to determine the molecular weight of a large biomacromolecule or polymer? 

  • MALDI-TOF MS

Longitudinal diffusion affects the chromatograph

• Brand Broadening

Two types of elutions are used in HPLC  

• Isocratic Elution: Constant solvent composition.

• Gradient Elution: Changes in solvent polarity to improve separation

Energy and Wavelength

• High energy → Lower Wavelength

• Low energy → Higher Wavelength

Monochromator

• A component of a spectrophotometer that isolates radiant energy of a specific wavelength and excludes that of other wavelengths 

UV-Vis

• Methods requiring the measurement of absorbance in the UV region of the electromagnetic spectrum require that the sample cuvette be made of Quartz.

Atomic Absorption Spectroscopy

Radiation is absorbed by non-excited atoms in the vapour state and excites them to higher states

Probes are used for the introduction of the sample 

Silica

process of 'sputtering' that occurs in the Hollow Cathode Lamp

Positive ions collide with the cathode surface, and metal atoms from the cathode are ejected

Optically thin conditions

Less than 0.05 absorbance

Quenching

• Deactivation due to “losing” energy to the solvent.

• Visible effect: Decrease in intensity of the spectrum

near infrared spectrometer wavenumber

12500 – 4000 cm-1

UV-Vis 

A conventional bench-top UV-Vis spectrometer uses a H₂ lamp as a UV source and a Xe or Tungsten lamp as a source in the visible region. 

Decrease the reciprocal linear dispersion of a monochromator 

Increase in focal length

Michelson Interferometer [False]

•  If the reflected and transmitted beams are in phase at the beam splitter, then maximum intensity will reach the detector. FALSE

Atoms in the gas phase produce

Discrete line spectra

Atomic Absorption Spectroscopy

• generally used radiation

  • Hollow cathode lamp

PMT turns radiant energy into an electrical current 

True

The type of radiation has the highest energy 

X-ray

T = P/Po

• Transmittance is given as T = P/Po.

• If Po is the power incident on the sample

• What does P represent?

  • Radiant power transmitted by the sample

Chopper

Not a component of the emission system in the Flame photometer 

fluorescence

• A photon is absorbed by a molecule, which then emits a photon of $λ=630 nm$

Raman

A molecule absorbs a photon at 2175 cm^-1, producing a v = 0 to v = 1 transition in the symmetric stretch normal mode of CO2; then the photon is re-emitted with no change in the dipole moment.

IR

• The absorption band is at $2130 cm^{- 1}$

Collision increases

• As the Temperature increases

Stokes shift

Due to the relaxation from the vibrational excited state down to the ground vibrational state being so much faster than the electronic state lifetime (10^{- 8} s), emission almost always takes place from the ground vibrational state. Hence, the emitted photon is usually of lower energy (longer wavelength) than the exciting photon. This shift to longer wavelengths is called the Stokes shift.

ICP-AES over Flame AE

• Increasing the sensitivity of the older flame methods.

• Good emission results from nearly all elements under a single set of conditions

• Spectra for a wide range of elements can be recorded simultaneously

• High temperature limits chemical and spectral interference and permits the determination of nonmetals such as chlorine, bromine, and sulfur

• Lower limits of detection are usually in the ppb region compared to the ppm region for flame AES

Boltzmann distribution.

• Energy levels in UV/Visible spectroscopy are far apart in energy, and, according to the Boltzmann distribution,

  • The population resides almost entirely in the lowest energy state.

  • As a result, UV/Visible spectroscopy is an extremely sensitive technique and is commonly used in analytical chemistry to deal with very small amounts of sample.

• In NMR, the energy separation of the spin states is comparatively very small and while NMR is very informative, quantum mechanically it is considered to be an insensitive technique.

internal conversion

When two singlet states overlap, energy is usually passed to the lowest vibrational state of the lowest electronic state by a process called internal conversion.

Intersystem crossing

Intersystem crossing is a process in which the spin state of an electron is reversed and energy from an excited singlet state is passed on to an excited triplet state.

Collision quenching

• Deactivation of an excited electronic state may involve transfer of energy to the solvent. In this case, no photon is emitted. The process is called external conversion or collision quenching.

Decrease the reciprocal linear dispersion of a monochromator

Decrease distance (d) and increase the focal length (F)

Fluorescence

Fluorescence: from an excited singlet electronic state

Phosphorescence

Phosphorescence: from an excited triplet state

Singlet state

Singlet state: contains no unpaired electrons

Triplet state

Triplet state: contains two electrons with the same spin

{-20}J$$

UV-Vis

• slit width is programmed to change as a function of wavelength. 

• What are the advantages and disadvantages of increased slit width

  • Advantage: power increase

  • Disadvantage: wider bandwidth

• The prism separates UV or visible light by 

  • Refraction 

• The sample cuvette should be made of

  • Quartz 

• UV-Vis absorption spectra result from the Excitation of electrons 

Raman Spectroscopy

Source: Laser

wavelength of light fluoresced by molecules

• The wavelength of light fluoresced by molecules is longer than that absorbed because 

  • Some of the energy absorbed by the molecules is lost to molecular vibrations before the fluorescence is produced,

  • which results in a longer wavelength.