Lab Techniques: Separations & Spectroscopy Flashcards
(142 cards)
1
Q
How does solvent extraction generally occur?
A
Isolation of natural products from marine organism, plants, and other natural sources is facilitated be exploiting the particular solubilities of organic compoundes in various solvents
2
Q
Complex mixtures of organic compounds can be separated using….
A
careful choice of solvents based on the differential solubilities of the various compounds of the mixture
3
Q
What is the purpose for extraction?
A
It allows the chemist to separate one substance from a mixture of substanes by adding a solvent in which the compound of interest is hightly soluble
4
Q
What is liquid-liquid extraction?
A
Occurs when the solution containing a compound of interest is shaken with a second solvent (commetely immiscible with the first) and allowed tp separate into two distinct phases, the compound of interest will distribute itself between the two phases based upon its solubility in each of the individual solvents
5
Q
Define the distributio (or ‘partition’) coefficient
A
the ratio of the substance’s solubilities in the two solvents
6
Q
What are the two things that solubility rely on?
A
- The polarity of the solute
- The polarity of the solvents
7
Q
When it comes to solubility…
A
like dissolves like
8
Q
What are polar molecules soluble in?
A
Polar solvents
9
Q
What are nonpolar molecules solvent in?
A
Nonpolar solvents
10
Q
What is the simplest liquid-liquid extraction?
A
When an organic compound is extracted with water
11
Q
What does a simple water extraction remove?
A
It can remove substances that are highly polar or charged, including inorganic salts, strong acids and bases, and polar, low molecular weight compounds (less than five carbons), such as alcohols, amines, and carboxylic acids
12
Q
A second type of organic extractions involves the use of what?
A
acidic or basic water solutions
13
Q
How can basic organic compounds (like amines) be extracted from mixtures of organic compounds?
A
upon treatment with dilute acids (usually 5-10% HCl)
◦ As this treatment will protonate the basic functional group, forming a positively charged ion
◦ The resulting cationic salts of these basic compounds are usually freely soluble in aqueous solution and can be removed from the organic compounds that remain dissolved in the organic phase
14
Q
What occurs with extraction with a dilute weak base?
A
◦ typically 5 percent sodium bicarbonate (NaHCO3)
◦ results in converting carboxylic acids into their corresponding anionic salts
15
Q
What happens with the extraction of carboxylic acids?
A
◦ Anionic salts are generally soluble in aqueous solution and can be removed from the organic componds that remain dissolved in the organic phase
◦ Dilute sodium hydroxide could also be used for this kind of extraction, but it is basic enough to also convert phenols into their corresponding anionic salts
16
Q
When phenols are present in a mixture of organic compounds and need to be removed…
A
a dilute sodicum hydroxide solution (usually about 10%) will succeed in converting phenols into their corresponding anionic salts
◦ the anionic salts of the phenols are generally soluble in the aqueous phase and can therefore be removed from the organic phase
17
Q
What is a separatory funnel?
A
The apparatus in which extractions are typically carried out
18
Q
To perform a solvent-solvent extraction, the solution containing the mixture of organic compounds and the extraction solvent of choice are pooured into the …
A
separatory funnel, and the apparatus is fitted with a stopper
◦ After mixing, the two layers may be separated from one another by removing the stopper at the top and slowly collecting each phase into separaste receiving flasks by opening the stopcock at the bottom of the funnel
19
Q
What does an extraction that will separate four organic compounds (para-cresol, benzoic acid, aniline and naphthalene) from one another?
A
◦ All four solutions are dissolved in diethyl ether
◦ The mixture is first extracted with an euqal volume of aqeous sodium bicarbonate (which is a weak bicarbonate), which is sufficiently basic to deprotonate benzioc acid and convert it to an anionic salt, but not strong enough to deprotonate para-cresol
◦ The bicarbonatre extraction will not affect aniline or naphthalene, thus they will remain dissolved in the ether phase, while the benzoic acid, now in its anionic salt form, will be extracted into the aqueous layer
◦ The ether layer (now containing 3 components) is extracted with a social hydroxide solution. The strongly basic hydroxide ion is strong enough to deprotonate para-cresol and convert it to its anionic salt form. The basic conditions will not affect aniline or naphthalene, so para-cresol is the only compound that is extracted into the aqueous phase. The aniline and naphthalene will remain dissolved in the ether layer
◦ The remaining two components can be separated from one another by an acidic extraction with a 10% HCl solution. The solution is acidic enough to protonate the lone pair of electrons of aniline and to convert aniline to its cationic salt. Naphthalene will not be affected and will remain dissolced in the ether layer
◦ The final extraction of aniline into the aqueous phase completes the separation. Naphthalene can be isolated by evaporating off the diethyl ether
20
Q
What are the eight types of chromatography?
A
- Thin-layer chromatography (TLC)
- Column (flash) chromatography
- Column chromatography
- Ion exchange chromatography
- High performance liquid chromatography (HPLC)
- Size exclusion chromatography
- Affinity chromatography
- Gas chromatography
21
Q
What are the basic features that all types of chromatography have in common?
A
◦ They are all used to separate mixtures of compounds, though some are used mostly for identification purposes, while others are generally used as purification methods
22
Q
What is the purpose of thin-layer chromatography?
A
TLC is used to separate compounds based on differing polarities
◦ It is a solid-liquid partitioning technique in which the mobile liquid phase ascends a thin layer of absorbant (generally silica, SiO2) that is coated onto a supporting material such as a glass plate
◦ This thin layer of absorbant acts as a polar stationary phase for the sample to interact with
23
Q
Why is thin-layer chromatography frequently used in organic chemistry laboratories?
A
because the speed of separation and the small sample amounts that can be successfully analyzed
24
Q
What are the steps involved in TLC?
A
- A very small amount (~1 mL) of sample is spotted near the based of the plate (~1 cm from the bottom) before placing the plate upright in a sealed container with a shallow layer of solvent
- As the solvent slowly ascends the plate via capillary action, the components of the spotted sample are partitioned between the mobile phase and the stationary phase (each component of the sample experiences many equilibriums between the mobile and the stationary phases as the development proceeds)
- Separation of the compounds occurs b/c different components travel along the plate at different rates
- Once the solvent nearly reaches the top of the plate, the plate is removed and allowed to dry (if the compounds are colour, we’d see a vertical series of spots, otherwise a different method of visualization would be needed)
25
Define developing or running
◦ Occurs during TLC
◦ It occurs when the solvent slowly ascends the plate via capillary action, the components od the spotted sample are partitioned between the mobile phase and the stationary phase
26
How many points of equibilibrium does each component of the sample experience between the mobile and stationary phases as the development proceeds?
They each experience many equilibriums
27
Why does the separation of the compounds occur during TLC?
because the different components travel along the plate at different rates
28
How do polar and unpolar components travel differently during TLC?
◦ The more polar components of the mixture interact more with the polar stationary phase and travel at a slower rate
◦ The less polar components have a greater affinity for the solvent than the stationary phase and travel with the mobile solvent at a faster rate than the more polar components
29
What are the visualization methods for TLC?
◦ If the compounds are coloured, you'd see a vertical series of spots on the plate
◦ If the compounds are not-coloured, visualization methods like, shining ultraviolet light on the plate, placing the thin layer plate in the presence of iodine vapour, and a host of other chemical staining techniques, are used
30
What is the Rf value?
◦ the "ratio to front"
◦ Is the distance traveled by an individual component divided by the distance traveled by the solvent front
*Rf is always positive and never greater than 1
31
What chromatography method is good for separating very small amounts of material in order to assess how many compounds make up a mixture?
TLC
32
What chromatography method is good for isolating bulk compounds?
Column (flash) chromatography
33
How does column (flash) chromatography work?
1. The column is filled with silic get (predominantly SiO2)
2. The silica gel is saturated with a chosen organic solvent, and the mixture of compounds to be separated is then added to the top and allowed to travel down through the silica-packed column
3. Excess solvent is periodically added to the top of the column, and the flow of solvent (along with the separated compounds) is collected from the bottom
34
What happens to polar and nonpolar compounds in column (flash) chromatography?
◦ Polar compounds will spend more time adsorbed on the polar solic phase, and therefore travel more slowly down the column than nonpolar compounds
◦ Compounds can be expected to leave the column, and be collected, in order of polarity (least polar to most polar)
35
What is ion exchange chromatography primarily used for?
when the materials to be separated have varying charge states
◦ also in the separation of mixtures of proteins
36
How are mixtures of proteins separated via ion exchange chromatography?
◦ At any given pH, proteins within a mixture may exist in a variety of charge states
◦ If such a mixture has passed through a cation exchange resin (one functionalized with negatively charge groups and cationic counterions), those proteins with pI values greater than the pH of the mobile phase will be positively charged and elute slowly compared to those with pI values below the solution pH
◦ If the same mixture at the same pH were passed through an anion exchange resin, the opposite would be true, and proteins with pI values above the pH of the solution would elute first
◦ If the pI values of the proteins to be separated are known, the pH of the mobile phase may be buffer to a specific pH, thereby ensuring different charge states and good separation
36
How does ion exchange chromatography work?
◦ It involves passing a mobile liquid phase containing the analyte through a column packed with a solid stationary phase, utilizes a polymeric resin functionalized with either positive or negative negatively charged moieties on the polymer surface
◦ As the analyte passes through the resin, positively charge groups displace sodium ions and coordinate to the anionic functionalities tethered to the polymer surface
◦ while these groups are retained, and their progress through the column retarded, the negatively charge groups and neutral species quickly pass through the material and are eluted first
◦ Once all the negatively charged and neutral species have been eluded, the column can be treated with a concentrated sodium containing solution to displace all absorbed positively charged species
37
What is high performance liquid chromateography used for?
◦ It takes advantage of the different affinities of various compounds for either the stationary or mobile phase
◦ Because the mobile phase is forced through the stationary phase at very high pressures, both the speed and efficiency of the separatation is increased, making this technique an improvement over the column chromatography
38
How is high-performance liquid chromatography done?
◦ The pumping unit is where pressurized of the mobile phase first occurs
◦ The samples to be separated is solubilized and injected by syringe, then the mobile phase carries the sample to the column
◦ The sample is separated into its constituent components, which are detected and analyzed as they exit the column
◦ The eluent is collected after detection, and the components can be isolated after evaporation of the solvent, if desired
39
During HPLC, what does the elution time depend on?
The elution time of any compound is dependent upon the mobile and stationary phase is used
40
For most HPLC separations of organic compounds, what is the stationary phase made out of?
The stationary phase is a silica gel that has been bonded to a nonpolar group (ex, octadecylsilane), creating a relatively nonpolar stationary phase - this is called reverse phase HPLC
◦
41
During HPLC, the mobile phase is generally ______ ______ than the stationary phase
More polar
42
If the mobile phase is generally more polar than the stationary phase, what does this mean in regards to the order of elution?
This means the order of elution will be the reverse of what occurs on a TLC plate or in a simple column chromatography
◦ More polar compounds allude first in HPLC as they have a high affinity for the mobile phase
◦ Less polar compounds are slowed by their interactions with non-polar stationary phase, and therefore last
43
Do polar or non-polar compounds elute first or last in an HPLC?
◦ More polar compounds elute first in HPLC as they have a high affinity for the mobile phase
◦ The less polar compounds are slowed by their interactions with a nonpolar, stationary phase, and therefore elute last
44
For the analysis of charge compounds, such as amino acid, what is the stationary phase and what is the mobile phase?
◦ The stationary phase is often an ion exchange column, usually cation exchange
◦ The mobile phase is a polar, protic (ex. CH3OH or H2O) or acidic solvent that ensures solubility and suppresses the dissociation of the COOH group on the amino acid
45
What is the difference in affinity to the column in HPLC attributed to?
It is attributed to the effects of a various R groups of the amino acids
46
What is the elusion order in HPLC?
It can be predicted based on the analysis of the intermolecular forces of the side chains
47
What is size exclusion chromatography?
A technique used to separate bulk materials based on molecular size
◦ It is frequently used for the separation of large polymers from small oligomeric fragments, or the separation of full proteins from smaller peptide chains
48
How does size exclusion chromatography work?
The materials to be separated are dissolved in solvent, loaded onto a column packed with a stationary phase, and allowed to travel to the bottom of the column where they are collected
◦ The stationary phase employed most often consists of chemically inert, porous polymer beads
◦ The size of the pores in the beads are carefully controlled to allow permeation of small molecules in the eluent, which excluding larger ones
◦ The exclusion of large molecules from the pore volume creates a more direct path down the column for large species then the more complicated, intraparticle pathway taken by compounds small enough to permeate the beads - the overall result is the quick elution of large molecules and longer retention of smaller species
49
What kind of molecules are eluded first in size exclusion chromatography?
Quick elusion of large molecules and longer retention of smaller species
50
What is the downside of size exclusion chromatography?
The materials of different sizes are easily separated, the technique is not particularly effective at separating different compounds of similar sizes
51
What is affinity chromatography used for?
It is used to purify proteins or nucleic acids from the complex biochemical mixtures, like cells lysates, growth media, or blood, rather than a reaction mixture
52
How does affinity chromatography work?
◦ It is based on highly specific interactions between macromolecules
◦ Due to the specific binding, the target molecule is trapped on the stationary phase, which is then washed to remove the unwanted components of the mixture
◦ The target protein is then released (or eluded) off the solid phase in a highly purified state
53
What is the stationary phase in a column impact with in large scale work for affinity chromatography?
The stationary phase is a column packed with a solid resin, and the sample is poured through the column
54
What can affinity chromatography look like in smaller scale experiments?
The solid phase can be mixed in a small tube with a sample to allow interaction with the components of the mixture
◦ The sample is in centrifuged, so that heavy solid rise and settles to the bottom of the tube
◦ Since the protein of interest is bound to the solid, resin, the liquid (or supernatant) is simply decanted, leaving the desired compound behind
55
How can you isolate a protein of interest in affinity chromatography?
The highly specific interactions of antibiotics can be used
◦ A commercially available antibody specific for the protein is added to the sample
◦ To isolate the antigen antibody complex, one of the three common microbe, derived proteins (protein A, protein G, or protein, L) is covalently linked to a solid support
◦ These proteins are useful because they bind meal antibodies so upon mixing, complex is made of protein of interest – antibody – protein A/G/L – solid support bead, form in solution
◦ the target is then isolated after centrifuge the sample and canteen the supernatant
56
How can you purify a protein of interest using an antibody and protein A linked beads?
1. Cells are collected and contain many proteins
2. And antibody against the protein of interest is added
3. Protein A linked beads are added, and bind antibodies
4. Beads and complexes are collected by centrifugation
5. Complexes are purified
57
Instead of centrifugation, what can be used as the solid phase for affinity chromatography?
Magnetic beads
58
How can magnetic beads be used as a solid phase in affinity chromatography?
The beads are isolated from solution by using a magnet to hold them (bound to the protein of interest) against the sides of the tube, while the solution containing any undesired compound is decanted
◦ Then the desired compound can be released from the beads in a pure state
59
What are the steps for using magnetic beads as a solid phase in infinity chromatography?
1. Addition of magnetic beads
2. Specific binding
3. Supernatant removal
4. Wash and elution
60
Do all proteins of interest have a commercially available antibody?
No - so researchers can use an affinity tag
61
What is an affinity tag?
Short peptide sequences or protein domain that is genetically fused to a protein of interest to facilitate the purification for affinity chromatography
◦ Using recombinant technology, a small molecular tag is added to the N-terminus or the C-terminus of the protein
◦ Affinity tagged proteins can be produced in large amounts in laboratory bacteria, and the cell lysate collected is rich in tagged protein
◦ Affinity tags are generally small enough that they don't interfere with protein, folding or function
62
Explain what His tags are
◦ A commonly used affinity tag
◦ Made of 6 to 10 Histine amino acids, which bind ions, such as nickel
◦ When a cell life is applied to a column, packed with nickel based resin, the tag proteins bind to the resin
◦ This is done under high pH conditions, and the his-tagged protein can be eluded off the solid phase using lower pH conditions
63
What is gas chromatography used for?
It is a form of column chromatography in which the partitioning of the components to be separated, takes place between a mobile gas phase and a stationary liquid phase
◦ This separation between mixtures of compound occurs based on their different volatility
64
How does gas chromatography work?
◦ Typically a sample is loaded into a syringe and injected into the device through a rubber septum
◦ The sample is then vaporized by a heater in the injection port, and carried along by a stream of inert gas (typically helium)
◦ The vaporized sample is quickly moved by the inert gas stream into a column composed of particles that are coded with a liquid absorbent
◦ As the compounds of the sample pass through the column, they interact differently with the absorbent based on their relative volatilities
◦ Each component of the sample is subjected to many gas-liquid partitioning processes which separates the individual components
◦ As each component exits the column, it is burned, and the resulting ions are detected by an electrical detector that generates a signal that is recorded by a chart recorder - the chart recorder print out, enables us to determine the number of components and their relative amounts
65
Detail the separation process by examining a typical GC column
With a mixture of two individual components, as a mixture enters the column, it begins to interact with the stationary phase, which is composed of support material coded with a liquid absorbent
◦ The liquid absorbent can range from hydrocarbon mixtures that are very nonpolar to polyesters that are polar
◦ as the mixture passes through the column, the components equilibrate between the carrier gas and the liquid phase
◦ The less volatile components will spend more time dissolved in the liquid stationary phase than the more volatile components that will be carried along by the carrier gas at a faster rate
◦ It is this equilibrium between the components (the absorbed liquid phase and the carrier, gas mobile phase) the results in the separation of the mixture
◦ If the interactions of the substrates with the column are similar (this is usually the case with most GC columns), the more volatile components emerged from the column first, while the less volatile components emerged from the column later
66
What two physical properties of a compound is most important to consider when conducting a gas chromatography experiment?
1. It's volatility
2. It's boiling point
67
What are melting points and boiling points indicators of?
They are indicators of how well identical molecules interact with (attract) each other
68
What attractive force causes non-polar molecules to interact because of?
They interact primarily because of an attractive force known as the London dispersion force, one of the intermolecular (Van der Waals) forces
69
What is the function of the London dispersion force?
It exists between temporary dipoles formed in non-polar molecules as a result of a temporary asymmetric electron distribution
70
What has to happen to melt a nonpolar compound (solid to liquid) or to boil a nonpolar compound (liquid to gas)?
Intermolecular forces like London dispersion force have to be overcome
71
The greater the attractive forces between the molecules, the ____ ______ will be required to get the compound to melt or boil
More energy
72
The weaker the attractive forces between molecules, the ____ melting or boiling point
Lower
73
For hydrocarbons, what is the most significant factor in determining the degree to which molecules of a given compound will interact?
Branching
74
Explain what branching is
Branching tends to inhibit Van der waal forces by reducing the surface area available for intermolecular interactions
◦ Thus, branching tends to reduce attractive forces between molecules and to lower both melting point and boiling point
75
Would a branched isomer or an unbranched isomer have a higher or lower melting or boiling point?
◦ Unbranched isomer require much more energy to melt or boil, because unbranched, it experiences greater Van der waal forces than does the branched isomer
76
What are the two main factors that influences melting point in bowling point for hydrocarbons?
1. Branching
2. Molecular weight
77
The greater than molecular weight of a compound, the _____ ____ ______ there is to interact, the ______ the number of van der waal interactions, and the _____ the melting point in boiling point
1. More surface area
2. Greater
3. Higher
78
Which would have a higher melting point in boiling point, hexane or propane?
Hexane - because it is a six carbon alkane, whereas propane is a three carbon alkane
79
What are the trends for hyper carbons regarding,
◦ Small hydrocarbons (1 to 4 carbons)
◦ Intermediate hydrocarbons (5 to 16 carbons)
◦ Large hydrocarbons (More than 16 carbons)
◦ Small hydrocarbons tend to be gases at room temperature
◦ Intermediate hydrocarbons tend to be liquids at room temperature
◦ Large hydrocarbons tend to be (waxy) solids at room temperature
80
What is responsible for the difference in boiling points between n-butanol and diethyl ether?
Because in butanol has the ability to form intermolecular hydrogen bonds while diethyl ether cannot
81
What does hydrogen bonding occur between?
A hydrogen bond donor (a hydrogen covalent bonded to a nitrogen, oxygen, or fluorine atom) and the hydrogen bond acceptor (a loan pair of electrons on a nitrogen, oxygen, or fluorine in another molecule, or part of the first molecule)
82
Explain how alcohols for an intermolecular hydrogen bonds
Because they have hydroxy (-OH) groups
◦ This results from a strong dipole in which the hydroxide groups hydrogen acquires a substantial partial positive charge (s+) and oxygen acquire a substantial partial negative charge (s-)
◦ The partial positive hydrogen can interact electrostatically with a non-bonding pair of electrons on nearby oxygen, resulting in a hydrogen bond
83
Is a hydrogen bond a covalent bond?
No - it's an intermolecular interaction
84
Does hydrogen bonding interactions that occur intermolecularly, increase or decrease boiling and melting points?
They increase their boiling and melting points
85
Does hydrogen bonding interactions that occur, intramolecularly, increase or decrease boiling or melting points
The intramolecular hydrogen bonds interactions, decrease the amount of intermolecular hydrogen bonding interactions that can occur between the molecules, thereby decreasing the melting and boiling points
86
What does the process of distillation entail?
It is the process of raising the temperature of a liquid until it can overcome the intermolecular forces that hold it together in the liquid phase
◦ The vapour then condensed back to the liquid phase and subsequently collected in another container
86
The more hydrogen bond donors and hydrogen bond acceptor there are in a molecule...
The higher, the boiling and melting points will be
◦ This is because the hydrogen bonds, like dispersion forces, act to hold the molecules together, resisting the change to become either a liquid or a gas
86
What are the two types of distillations?
1. Simple distillation
2. Fractional destination
87
Describe what a simple distillation is
◦ A simple distillation is performed when trace impurities need to be removed from relatively pure compound, or when a mixture of compounds with significantly different boiling points need to be separated
◦ ex. An appropriate use of a simple distillation would be to purify fresh drinking water away from a salt water solution. The more volatile water can be boiled away, then condensed and collected, leaving behind the non-volatile salts.
88
Describe what a fractional distillation is
A fractional distillation is a type of distillation process that is used when the difference in boiling points of the compounds in the liquid mixture is not large
◦ A fractional distillation column is packed with an appropriate material, such as glass beads or a stainless steel sponge
◦ The packing of the column results in the liquid mixture of being subjected to many vaporization–condensation cycles as it moves up the column toward the condenser
◦ As the cycles progress, the composition of the vapour gradually becomes enriched in the lower boiling component
◦ Near the top of the column, nearly pure vapour reaches the condenser and condenses back to the liquid phase where to subsequently collected in receiving flask
89
What is a molecules ground state?
Molecules that normally exist in their lowest energy form
90
What is a molecule that is in an excited state?
It occurs when a molecule is exposed to light, and it may absorb a photon (provided the energy of this photon matches the energy between the two fixed electronic energy levels of the molecule)
91
Do molecules prefer to be in a ground state or an excited state?
Molecules tend to prefer to be in their ground state to an excited state, but in order for them to return to their ground state, they must lose the energy they have gained
92
How do molecules lose energy they have gained?
The loss of energy can occur by the omission of heat, or less commonly, light
93
Explain absorption spectroscopy
◦ Scientists induce the absorption of energy by a sample of molecules by exposing the sample to various forms of light, thereby exciting molecules to a higher energy state
◦ They can measure the energy released as the molecules relaxed back to their ground state
◦ This measured energy can reveal structural features of the molecules in the sample
94
What are the four main types of spectroscopy?
1. Mass spectrometry
2. Ultraviolet/visible (UV/Vis) spectroscopy
3. Infrared (IR) spectroscopy
4. 1H nuclear magnetic residence (NMR) spectroscopy
95
What is mass spectrometry used for?
It allows researchers to determine the mass of compounds in a sample
96
How does mass spectrometry work?
Within the mass spectrometer, molecules are ionized and a high vacuum, usually by bombarding them with high energy electrons
◦ Once ionized, compounds enter a region of the spectrometer where they are acted on by a magnetic field
◦ This field causes the flight path of the charge species to alter, and the degree to which the path is changed is determined by the mass of the ion
◦ This difference is detected and translated into a mass read out in the detector
97
What does the M/e label on the X axis of a readout from a mass spectrometer represent?
◦ It represents the ratio of maths (M) to charge (e)
◦ In most cases, e = +1, so peaks can simply be viewed as molecular mass
98
What are the two variables present on a readout from a mass spectrometer?
◦ On the X axis is the M/e label (representing the ratio of mass to charge)
◦ On the Y axis is abundance (representing the relative abundance of each species of a particular mass detected in the sample). Masses are measured in amu
99
What does the abundance variable on the readout from a mass spectrometer represent?
The relative abundance of each species of a particular mass detected in the sample
◦ Masses are measured in amu
100
If the molecular weight of a compound is 128 g/mol, why are there peaks greater than this value, as well as lower than this value?
◦ Because Adams can come in a number of different isotopes
◦ Small peaks with mass as large larger than the main peak represent molecules that have one or more of these less abundance isotopes
◦ The mass is lower than the 128 represent the masses of molecular fragments - the high energy beam of electrons used to ionized molecules in the mass spectrometer can cause the molecule to break into smaller parts
101
What are the two most important particular atoms that can give characteristic peaks in their mass spectra due to isotopic ratios?
1. Bromine
2. Chlorine
102
Why do bromine and chlorine have the ability to give characteristic peaks in their mass spectra things to isotopic ratio?
◦ Growing naturally occurs into isotopes (79 and 81) of nearly identical, natural abundance. This means that any mass spectrum involving a dominated compound will have two major peaks, nearly equal height, two amu apart
◦ Chlorine also occurs as two main isotopes; 35 (75% natural abundance) and 37 (25% natural abundance). Mass spectra for chlorinated molecules will have a peak, two amu heavier than the main peak, and about 1/3 its height.
103
What is ultraviolet/visible spectroscopy used for?
It's a type of absorption spectroscopy
◦ It's similar to IR, but instead focusses on the slightly shorter, more energetic wavelength of radiation in the ultraviolet invisible area of the spectrum
◦ The wavelength in the UV invisible ranges of the electromagnetic spectrum are strong enough to induce, electronic excitation, promoting ground state, valence electrons into excited states
104
In general, what two kinds of molecules is UV/Vis spectroscopy used for?
1. Useful in monitoring complexes of transition metals
2. It is used to study highly conjugated organic systems
105
Why is UV/Vis spectroscopy, useful and monitoring complexes of transition metals?
Because the easy promotion of electrons from ground to excited states in the closely spaced d-orbitals of many transition metals gives them their bright colour (by absorbing wavelengths in the visible region), and since many of these promotions involve energies in the UV range, these promotions allow study of these species
106
Why is UV/Vis spectroscopy used to study highly conjugated organic systems?
◦ Molecular orbital theory tells us when molecules have conjugated pi-systems, orbitals form many bonding, non-bonding, and anti-bonding orbitals
◦ These orbitals can be reasonably close together and energies, and in fact, close enough to allow promotion of electrons between electronic states through absorption of ultraviolet, or even visible photons
◦ The wavelength of maximum absorption for any compound is directly related to the extent of conjugation in the molecule. The more extensive the conjugated system is, the longer the wavelength of maximum absorption will be.
◦ With the absorption of each aromatic ring, the conjugated system grows longer in the wavelength of maximum absorption increases. Since each 'λmax' corresponds to a particular colour of light, a simple colour wheel can be used to predict the colour othe compound will appear.
◦ Generally, speaking, the colour compound maximally absorbs is complementary to the colour it will appear to her eyes
◦ For a compound that only absorbs ultraviolet radiation, all of the visible wavelength will be reflected in thus the compound will appear white or colourless. However, a compound that absorbs blue light will appear to us as orange since blue and orange are complementary colours on opposite sides of the colour wheel.
107
What is infrared spectroscopy used for?
It is the measurement of the interaction of infrared radiation with matter by absorption, emission, or reflection.
◦ It is used to study and identify chemical substances or functional groups in solid, liquid, or gaseous forms
108
How does infrared spectroscopy work?
Electric magnetic radiation in the infrared (IR) range λ = 2.5 to 20 um is the proper energy to cause bonds in organic molecules to become vibrationally excited
◦ With a sample of organic compound is radiated with infrared radiation in the region between 2.5 and 20 um, it's covalent bonds will begin to vibrate at distinct energy levels (wavelength frequencies) within this region
◦ These wavelength correspond to frequencies in the range of 1.5×10^13 Hz to 1.2x10^14 Hz
◦ In IR spectroscopy, vibrational frequencies are more commonly given in terms of the wavenumber
◦ When a bond absorbs IR radiation of a specific frequency, that frequency is not recorded by the detector and is that seen as a peak in the IR spectrum [since low transmitting corresponds, naturally, to absorbance)
109
Explain what a wavenumber is
◦ Used to represent vibrational frequencies in IR spectroscopy
◦ Wavenumber (v) Is simply the reciprocal of wavelength:
v^- = (1/ λ) = (1/c) v
◦ Wave number is directly proportional to both the frequency (since λv = c = 3x10^10 cm/sec) and the energy of radiation (E = hv)
◦ Therefore, the higher the wavenumber, the higher, the frequency, and the greater the energy
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The higher the wavenumber...
The higher, the frequency and the greater the energy
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How are wavenumbers usually expressed?
In reciprocal centimetres, cm^-1
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What range will the Mcat IR spec typically cover?
From 4000 to 1000 cm^-1
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What are the important stretching frequencies needed to know for the MCAT?
◦ The double bond stretcesh
◦ The triple bond stretch
◦ The O-H stretches
◦ The C-H stretches
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What do we need to know about the double bond stretches regarding IR spectroscopy?
◦ They're two types of double bond stretches, the carbonyls and the alkenes
◦ The carbon stretch is centred around 1700 cm^-1, and is very strong and very intense
◦ In any spectrum, always look for the stretch first, as if it is not present, you can eliminate a wide range compounds that contain a carbon group, including aldehydes, ketones, carboxylic, acids, acid chloride, esters, amides, and anhydrides (on the other hand, if the carbonation stretches present, you know that one of the carbon containing functional groups is present)
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When reading an IR spectroscopy graph, what is the first stretch that one should look for?
The carbonate stretch, located at 1700 cm^-1, because it is very strong and very intense
◦ If it is not present, you can eliminate a wide range compounds a container, carbon group, including aldehydes, ketones car, carbonic acids, acid chloride, esters, amides, and anhydrides
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When looking at an IR spectroscopy graph, what do the words strength and intensity indicate?
◦ Strength is reflected in the precent absorbance (or transmittance)
◦ Intensity reflected in the sharpness or distinctiveness ("V" shape) of the spike appearing on the spectrum
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At what locations are carbonyls and alkenes centered around in IR spectroscopy?
◦ Carbons are centred around 1700 cm^-1
◦ Alkeenes are centred around 1650 cm^-1
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At what location is a triple Bond stretch present?
Between 2260-2100 cm^-1
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If a triple bond stretches present, what would the triple bond be between?
Either:
◦ C and C
◦ C and N
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What do we need to know regarding the O-H stretch?
◦ The hydroxy stretch (for alcohols) is strong and very broad (the broadness is due to hydrogen bonding)
◦ The OH stretch can occur between 3600-3200 cm^-1
* a means also have stretches in this region, although they vary in intensity
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For IR spectroscopy, what do the words strength and broadness indicate?
◦ Strength is reflected as the degree of absorption of peak displays on the spectrum
◦ Broadness is reflected as a wide "U"-shaped appear appearance on the absorption, spectrum, as opposed to a "V" shape
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What do we need to know about the C-H stretches?
The C-H stretching region is 3300 to 2850 cm^-1
◦ Since the vast majority of organic compounds contain C-H bonds, you will almost always see absorbances in this region
◦ Note that aliphatic C-H bonds stretch out wavelengths a little less than 3000 cm^-1, and aromatic C-H bonds stretched wavelength slightly greater than 3000 cm^-1
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◦ What is the location for a sp3 C-H carbon?
◦ What is the location for a sp2 C-H carbon?
◦ What is the location for a sp C-H carbon?
◦ sp3 C-H carbon = 3000-2850 cm^-1
◦ sp2 C-H carbon = 3150-3000 cm^-1
◦ sp C-H carbon = 3300 cm^-1
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List of summary of relevant IR stretching frequencies and their intensity, for the following:
◦ C=O
◦ C=C
◦ C tb C
◦ C tb N
◦ C-H
◦ N-H
◦ O-H
◦ C=O, 1735-1680 cm^-1 (intensity = strong)
◦ C=C, 1680-1620 cm^-1 (intensity = variable)
◦ C tb C, 2260-2100 cm^-1 (intensity = variable)
◦ C tb N, 2260-2220 cm^-1 (intensity = variable)
◦ C-H, 3300-2700 cm^-1 (intensity = variable)
◦ N-H, 3150-2500 cm^-1 (intensity = moderate)
◦ O-H, 3650-3200 cm^-1 (intensity = broad)
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What is 1H an MR spectroscopy used for?
◦ Also commonly called proton MNR
◦ Light from the radio frequency range of the electromagnetic spectrum is used to induce energy absorptions
◦ It is primarily used for determining the structure of organic compounds. It provides detailed information about the hydrogen atoms within a molecule, including their number, chemical environment, and how they are bonded to other atoms. This information helps scientists identify and confirm the structure of unknown molecules or verify the structure of known compounds.
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What are the four essential features of a molecule that can be deduced from its 1H NMR spectrum?
1. The number of sets of peaks in the spectrum tells one the number of chemically non-equivalent sets of protons in the molecule
2. The splitting pattern of each set of peaks tells how many protons are interacting with the protons in that set
3. The mathematical integration of the sets of peaks indicates the relative numbers of protons in each set
4. The chemical shift values of those sets of peaks gives information about the environment of the protons in that set
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Explain how determining chemically equivalent, hydrogen impacts NMR spectroscopy
Determine which hydrogen, or protons, or equivalent in organic molecule is a first skill needed for NMR spectroscopy
◦ Equivalent hydrogen in a molecule are those that have identical, electronic environments, and such hydrogens have identical locations in the 1H NMR spectrum and are therefore represented by the same signal, or resonance
◦ Non-equivalent hydrogen will have different locations in the 1H NMR spectrum and maybe be represented by different signals
◦ Being able to determine which hydrogens (or groups of hydrogen) are equivalent to which other groups, so that you can predict how many distinct NMR signals there will be in any 1H NMR spectrum
◦ Hydrogen are considered equivalent if they can be interchange by a free rotation or a symmetry operation (mirror plane or rotational access)
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How are hydrogens considered equivalent for NMR spectroscopy?
If they can be interchange by a free rotation or a symmetry operation (rotational plane or rotational axis)
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What equation is used to compute the degrees of unsaturation?
d = [2(#C) + 2 - (#H)] / 2
◦ If d = 0, there are no double bonds or rings present
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What is the spin-spin splitting in terms of NMR spectroscopy?
This occurs when non-equivalent hydrogen interact with each other
◦ This interaction exists because a magnetic field felt by a proton is influenced by surrounding protons
◦ This effect tends to fall off with distance, but it can often extend over to adjacent carbons
◦ Nearby protons that are non-equivalent to the proton in question will cause splitting in the observed 1H NMR signal
◦ The degree of splitting depend depends on the number of adjacent hydrogens, and a signal will be split into n + 1 lines, where n is the number of non-equivalent, neighbouring (interacting) protons
◦ The important information one must determine is how a proton or a group of chemically equivalent protons will be split by their hydrogen neighbours
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What is the n + 1 rule?
(from 0 to 5)
◦ The n plus one rule states that the number of neighbouring nonequivalent hydrogens is n +1 (if two hydrogens neighbor a different hydrogen, said different hydrogen would signal split into three peaks)
If;
◦ 0, splitting is 1 (singlet)
◦ 1, splitting is 2 (doublet)
◦ 2, splitting is 3 (triplet)
◦ 3, splitting is 4 (quartet)
◦ 4, splitting is 5 (quintet or multiplet)
◦ 5, splitting is 6 (sextet or multiplet)
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What role does integration play in NMR spectrometry?
Is the NMR instrument obtains the spectrum of the sample, it performs a mathematical calculation, called an integration, thereby measuring the area under each absorption peak (resonance)
◦ The calculated area under each peak is proportional to the relative number of protons, giving rise to each peak - thus, the integration indicates the relative number of protons in each side of molecule
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What role does chemical shift play in NMR spectroscopy?
Chemical shift indicates the location of residence (set of peaks) in the 1H MMR spectrum
◦ Differences in the chemical shift values for different sets of protons in a molecule are the result of differing electronic environments that different sets of protons experience
◦ The magnetic field created by electrons nearer, proton, will shield the nucleus from the applied magnetic field created by the instrument, shifting the residence upfield
◦ The more proton is dish shielded (the more distorted away from the atom the electron cloud is), the further down field (to the left) in an NMR spectrum it will appear
◦ ex. Aceta proton is near an electronegative group is said to be shielded and will appear downfield (to the left) in the 1H NMR spectrum, relative to asset of protons that are further away from the electronegative group, which is more shielded and appear more upfield (to the right) in the 1H NMR spectrum
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If something is said to be either more downfield or more upfield. What does that mean?
◦ If something is downfield, it's considered more deshielded
◦ If something is upfield, it's considered less deshielded
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What are the three factor factors involved in proton deshielding?
1. The electronegativity of the neighbouring atoms
2. Hybridization
3. Acidity and hydrogen bonding
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How does electronegativity effect chemical shift values?
If an electronegative atom is in close proximity to a proton, it will decrease the electron density near the proton in thereby deshield it
◦ This will result in a downfield shift in the chemical shift value
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How does hybridization effect chemical shift values?
The hybridization effect occurs as a result of the varying bond characteristics of carbon atoms connected to the hydrogens
◦ The greater the s-orbital character of a C-H bond, the less electron density on the hydrogen
◦ When considering the hybridization effect alone, the greater the s-orbital character, the more deshielded the set of protons is, which will result in a downfield shift for the peak corresponding to that set of protons
◦ Hybridization affects alone, would indicate that an alkene proton would be more shielded than the alkene, proton, however, due to more complicated physical phenomenon that turns out not to be the case
◦ Two other very characteristic chemical shifts, you should be familiar with are that of the aromatic protons (s = 6.5-8 ppm) and alkene protons (s = 5-6 ppm)
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How does a acidity and hydrogen bonding effect chemical shift values?
Protons that are attached to heteroatoms (oxygen and nitrogen) are quite shielded
◦ Acidic protons on carbonic acid are an extreme example of a large downfield shift
◦ Additionally, hydrogen bonding can cause a wide variation of chemical shift - ex. The resonance of the alcohol proton in methanol varies with both solvent and temperature (different degrees of H bonding).
◦ You should be aware that the chemical shift of alcohol protons are quite variable, depending on the particular compound, but are in the range of s = 2-5 ppm
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List the following values for 1H NMR chemical shift:
◦ alkyl (RxCH(4-x)
◦ alkyne (RC tb CH)
◦ alcohol (R-OH)
◦ X = NR2, OR, halogen (RCH2X)
◦ benzylic (RCH2C6H5)
◦ vinyl (r-r-C=C-R-H)
◦ aromatic (RC6H5)
◦ aldehyde (RCHO)
◦ carboxylic acid (R-CO2H)
◦ alkyl (RxCH(4-x) = 0-2 ppm
◦ alkyne (RC tb CH) = 2 ppm
◦ alcohol (R-OH) = 2-5 ppm
◦ X = NR2, OR, halogen (RCH2X) = 2.5-4 ppm
◦ benzylic (RCH2C6H5) = 2.5-4.5 ppm
◦ vinyl (r-r-C=C-R-H) = 5-6 ppm
◦ aromatic (RC6H5) = 6.5-8 ppm
◦ aldehyde (RCHO) = 9-10 ppm
◦ carboxylic acid (R-CO2H) = 10-13 ppm
