Class 2: Lab Techniques: Separation and Spectroscopy
Solvent Extraction: Organic & Aqueous Layers
*see notes
Diagnostic IR techniques
- All organic compounds have a C—H stretch (2950), so it will never be diagnostic (eliminate answers that say something like this: The appearance of aliphatic C—H stretching vibrations)
Distillations and Boiling Points
- BP are a measure of IMFs between liquid molecules IMFs in liquids = van der Waals: - H bonds (strongest) - Dipole-dipole -LDFs Other factors that affect BP: 1. MW (heavier molecules have higher BP; more electrons = more polarizable making dispersion forces stronger) a. small hydrocarbons (1-4C) = gases at RT b. 5 - 16 C = liquids at RT c. large hydrocarbons > 16C = (waxy) solids at RT WHEN LOOKING AT A COMPOUND COUNT ITS CARBONS TO DETERMINE ITS STATE (S, L, OR GAS) 2. Branching (more branching = lower BP/lower melting point because less stacking and inhibits van der Waals forces by reducing the surface area available for intermolecular interaction) - (Z) = better stacking, more solid-like = higher BP - (E) = kinks, less solid-like = lower BP BUT cis unsaturated FA = kinks trans unsaturated FA = stack
Important Isotopes for Mass Spectrometry
- Bromine naturally occurs in 2 isotopes (79 and 81) of nearly identical natural abundance - this means that any mass spectrum involving a brominated compound will have 2 major peaks, nearly identical in height, 2 amu apart - Chlorine also occurs as 2 main isotopes; 35 (75% natural abundance) and 37 (25% natural abundance) - mass spectra for chlorinated molecules will have a peak 2 amu heavier than the main peak, and about 1/3 its height
Factors that influence gas chromatography retention time
- In gas chromatography, the compound with the highest boiling point will have the longest retention time. - Molecular weight, van der Waals forces, and hydrogen bonding are properties that can influence the boiling point of a compound. - a compound that can form more hydrogen bonds than all the other compounds will have the highest boiling point and the longest retention time
Gas Chromatography details
- Molecular weight, the amount of branching, and intermolecular interactions all influence boiling points. To increase the boiling point the molecule needs a high molecular weight, no branching, and stronger intermolecular interactions (ion-dipole, H-bonding, dipole-dipole, dipole-induced dipole, LDFs) (high BP, low volatility)
Toluene and water are two solvents that are slightly miscible and have boiling points of 111°C and 100°C, respectively. Which of the following techniques would best separate the solvents? A. Mass spectrometry B. Extraction C. Simple distillation D. Fractional distillation
- Since the solvents are only slightly miscible, they will separate into two layers during an extraction. However, they are not completely immiscible, therefore this is not the best technique to use because complete separation would not occur (choice B is incorrect). Simple distillation also would not afford complete separation as the two solvents have similar boiling points (choice C is incorrect). Fractional distillation is the best technique to separate two solvents that have similar boiling points (choice D is correct). solvent extraction = good for immiscible solutions because different layers will be obvious simple distillation = need >30 degrees difference in BP
2 Chromatography techniques used to separate small amounts:
- TLC - Gas Chromatography
Which of the following methods would be best for isolating the chemical components of a perfume? A. Thin layer chromatography B. High-performance liquid chromatography C. Affinity chromatography D. Gas chromatography
- The fragrance of a perfume is caused by volatile compounds, which must become air-borne to be detected by receptors in the nose. Gas chromatography separates analytes in the gas phase and is ideally suited for volatile compounds (choice D is correct). - TLC and HPLC are generally used on nonvolatile compounds so are less ideal methods of isolation (USE FOR POLARITY) - Affinity chromatography requires a specific interaction between the analyte and the stationary phase, and is typically used on large biomolecules like proteins and antibodies. Since the chemical components of the perfume are unknown, choice C can be eliminated. (Although the compounds in the perfume will interact with an olfactory receptor protein, there are hundreds of receptor proteins, making affinity chromatography highly impractical in this particular case.)
Ultraviolet/Visible Spectroscopy details
- a type of absorption spectroscopy used in o-chem, very similar to IR but instead focuses on the slightly shorter, more energetic wavelengths of radiation in the ultraviolet and visible area of the spectrum - the wavelengths in the UV and visible ranges of the EM spectrum are strong enough to induce electronic excitation, promoting ground state valence electrons into excited states Used to study 2 kinds of molecules: - monitoring complexes of transition metals because of easy promotion of electrons from ground to excited states in the closely spaced d-orbitals, giving them their bright color (by absorbing wavelengths in the visible region) - also used to study highly conjugated organic systems; when molecules have conjugated pi systems, orbitals from many bonding, non-bonding, and anti-bonding orbitals. These orbitals can be reasonably close together in energy; the wavelength of maximum absorption for any compound is directly related to the extent of conjugation in the molecule (the more conjugated = the longer the wavelength of maximum absorption) - with the addition of each aromatic ring, the conjugated system grows longer and the wavelength of maximum absorption increases (3 rings = max wavelength is 363 nm, absorbs UV and appear white VS 5 rings = max wavelength is 595 nm, absorbs yellow/orange and appears blue/violet) **the color a compound maximally absorbs is complementary to the color it will appear to our eyes ** for a compound that absorbs only UV radiation, ALL of the visible wavelengths will be reflected and thus the compound will appear white or colorless ex: a compound that absorbs blue light will appear to us as orange
Common polar stationary phases
- alumina = silica gel
Solvent Extraction summary
- extraction allows you to separate one substance from a mixture of substances by adding a solvent in which the compound of interest is highly soluble - compounds are washed from the organic to the aqueous layer by choosing a complementary reagent that will leave the desired species charged, and therefore water soluble - stronger carboxylic acid groups must be separated before the less acidic phenolic groups by using a weaker base (NaHCO3) - water can immediately separate charged compounds while non-charged compounds will move into organic layer WHOLE POINT IS TO SEPARATE CHARGED COMPOUND BECAUSE THEY BECOME WATER SOLUBLE
1. Non-Equivalent Protons (hydrogens)
- free rotation or symmetry makes Hs equivalent (same chemical environment) - for each set of non-equivalent Hs, a spectrum will have ONE (or the same) H-NMR signal, or resonance - non-equivalent Hs will have different locations in the H-NMR spectrum and be represented by different signals *can identify DOU equation to identify structure if given molecular formula Q: What is the number of signals expected in compound?
downfield (a, a, v)
- higher ppm - electron density if 'H" is deshielded because electron density is being pulled away from it due to EWGs - protons that are more deshielded (near electronegative groups) ex: aldehyde, aromatic (H-Ph), vinyl (H-C=C)
Affinity Chromatography book
- large scale. stationary phase is a column packed with a solid resin, and the sample is poured through column (small scale in test tube) - the solid phase can be mixed in a small tube with the sample to allow interaction with the components of the mixture - the sample is then centrifuged so the heavy solid resin 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 ex: - cell lysates are collected and contain many proteins - an antibody against the protein of interest is added - protein A-linked beads are added that bind the antibodies - beads and complexes are collected via centrifugation - complexes are purified *instead of centrifugation, magnetic beads can be used as the solid phase - the beads are isolated from solution by using a magnet to hold them (bound to protein of interest) against the sides of the tube * not all proteins of interest have a commercial antibody available, can use an affinity tag - recombinant technology = a small molecular tag is added to N-terminus or C-terminus of protein - DNA sequences coding for affinity tags are well known, these can be subcloned into a plasmid with the gene of interest - these tags are usually small enough so don't interfere with protein folding or function
upfield (a, H, alk)
- lower ppm - protons that are more shielded (near electron donating groups) - more p-character - R groups are EDGs and they are pushing e- density away from themselves = shielding - ex: alcohol (H-O-C), H-C-Y (halogen), alkyl (H-C-R)
Spectroscopy intro book
- molecules normally exist in their ground state - when a molecule is exposed to light it MAY absorb a photon, provided the energy of this photon matches the energy between two of the fixed electronic energy levels of the molecule - when this happens, molecule is in excited state - molecules prefer to be in their ground state, but in order for them to return, they must lose the energy they have gained - this loss of energy can occur by the emission of heat, or less commonly light absorption spectroscopy = induce the absorption of energy by a sample of molecules by exposing sample to various forms of light, exciting the molecules to a higher energy state - they then measure the energy released as the molecules relax back to their ground state - this measured energy reveals structural features of the molecules in the sample - the different forms of like in the EM spectrum induce different transitions in ground state molecules to different excited states of the molecules
Resolving agent for Resolution Separation Technique
- must be chiral - must be an acid or a base (this has to be compatible with pair of enantiomers) - if enantiomers are bases, use acidic resolving agent enantiomers on left = base; diastereomer salt =right
cation exchange resin
- one functionalized with negatively charged groups and cationic counterions
UV/Vis Spectroscopy (low yield)
- similar to IR but instead focuses on shorter, more energetic wavelengths of radiation in the UV and visible area of the spectrum - the wavelengths in the UV and visible ranges of EM spectrum ae strong enough to induce electronic excitation, promoting ground state valence electrons into excited states = indicates the presence of a conjugated pi system in a molecule (extensive resonance in a molecule) = if molecule is able to absorb energy in the UV-Visible light spectrum, it is due to having a pi system (chain of sp2 hybridized atoms) - think benzene (3+ pi bonds allow molecules to produce colors and absorb visible lights) - ex: beta-keratin (in colors) has tons of resonance and is what allows carrots to produce the colors - as conjugation (delocalization due to pi bonds) in a molecule increases, the wavelength of light absorbed increases, or redshifts - a blueshift is the opposite REDSHIFT: - if a substance has lots of resonance = absorbing red light = so you appear as green - redshifts have increased wavelengths BLUESHIFT: - if a substance has less resonance in a compound = absorbing blue/violet light = so you appear orange/yellow - blueshifts have decreased wavelengths - less resonance in the fall (blueshift), more resonance in the summer (think leaves) UV absorption: - very short range of sp2 hybridized atoms (very little resonance) will absorb in UV light and object won't be colored at all
Extractions - book
- simplest liquid-liquid extraction = when organic compound is extracted with water - a simple water extraction can remove substances that are highly polar or charged, like inorganic salts, strong acids and bases, and polar low MW weight compounds (<5C) - like amines, alcohols, and carboxylic acids
Important MCAT IR wavenumbers
- the higher the wavenumber, the higher the frequency and the greater the energy **(A wavenumber is the inverse of the wavelength, λ, in cm: It has units of 1/cm or cm-1. It is directly proportional to the frequency and the energy of the radiation: radiation with a high wavenumber has higher frequency and energy than radiation with a low wavenumber.) O-H peak is broad due to H bonding which is why it has large range (U or V shape) C=O, carbonyl, is variable because lots of functional groups contain the carbonyl (aldehydes, ketones, carboxylic acids, 4 acid derivatives) and will have slightly different wave numbers associated with them C=C, alkenes
Spectroscopy Reflect
- with spectroscopic information, we can "read" the structural elements of a molecule - techniques to identify and distinguish between different molecules: - IR (functional groups) (higher yield) - H-NMR (connectivity of H atoms; non-equivalent Hs) (higher yield) - UV/Vis (conjugated pi systems) - MS (mass spec) (MW)
When to use Distillation
- would be the most convenient laboratory technique for the separation of acetone from nonane. Due to the large difference in molecular size and weight between acetone (3-carbon chain) and nonane (9-carbon chain), they have very different boiling points (56.5°C and 150.8°C, respectively). In general, a large increase in molecular weight between two compounds results in a large increase in boiling point. Distillation can be conveniently used to exploit this difference.
Thin Layer Chromatography (TLC)
-= separates compounds based on polarity - used to separate small amounts of solids or high boiling point liquids (oils) - large molecules but not macro-molecules Stationary phase: - plate is coated with silica gel (POLAR) - silica gel can H-bond to compounds Mobile phase: - shallow solvent bath within a (sealable) development chamber - solvent carries compounds as it is drawn upward via capillary action - solvent is NP NONPOLAR = compounds have weaker interactions with silica gel (migrate faster up the plate; will have greater RF value, max value =1) POLAR = compounds have stronger interactions with silica gel (migrate slowly up the plate as they H-bond and stick to the gel, minimum =0) *can approximate Rf values as a percent
Separation of Compounds Techniques:
1. Chromatography - good for separating immiscible compounds (oil and water) - size-exclusion (size) - TLC (polarity, small amounts) - Column (polarity) - HPLC (polarity) - Ion Exchange (charge) - Affinity (lock-and-key interactions ) - Gas (volatility/boiling point, small amounts) 2. Distillation (BP, larger amounts) 3. Solvent Extraction (solubility) 4. Resolution of Enantiomers
Solvent Extraction Reactions (NaHCO3)
1. aqueous NaHCO3 (weak base) ONLY deprotonates carboxylic acids (making it water soluble, forms a anionic salt) O- (Na+ dissociates) and acts as a nucleophile and removes H from carboxylic acid, now negative O picks up Na+ and is positively charged due to Na
Gas Chromatogram
1. number of compounds in mixture = number of peaks (2) 2. relative quantity = greatest intensity because this compound has greater peak area (there is more A than B in this mixture) 3. time - A comes out first, B comes out after it = this means that B has the higher BP (and lower volatility)
Solvent Extraction Reactions (NaOH)
2. aqueous NaOH (strong base): deprotonates phenols AND carboxylic acids (forms an anoinic salt that is more water soluble bc charged) **alkyl alcohols are not acidic enough to be useful in extractions (cannot deprotonate them with anything; NaOH is not strong enough to pull off alcoholic hydrogen)
Solvent Extraction Reactions (HCl)
3. aqueous HCl (strong acid) protonates amines (forming a charged molecule, a cationic salt that is soluble in water) = sp3 - LP isn't localized through resonance = basic vs. sp2 - LP on N is delocalized through resonance with carbonyl so it is not basic and won't pick up H from HCl
3. Area under signal (integration)
= # of Hs represented by the signal - simply count Hs based on rule #1 (determining groups of non-equivalent Hs) - 6H is indicative of symmetry (anything greater than 3H)
Fractional Distillation (useful for separating which kind of stereoisomers?)
= a different type of distillation process that is used when the difference in boiling points of the components in the liquid mixture is not large (within <30C of each other) **useful for separating diastereomers (similar properties and MW because same molecular formula)** - a fractional distillation column is packed with an appropriate material, such as glass beads or a stainless steel sponge - contains additional glass column packed with beads to aid in further separation - the packing of the column results in the liquid mixture being subjected to many vaporization-condensation cycles as it moves up the column toward the condenser - as the cycle progresses the composition of the vapor gradually becomes enriched in the lower boiling component - near the top of the column, nearly pure vapor reaches the condenser and condenses back to the liquid phase where it is subsequently collected in a receiving flask
Distillation
= a larger scale version of Gas Chromatography (separates larger amounts) = 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 vapor is then condensed back to the liquid phase and subsequently collected in another container *separates large amounts (gas chromatography = small amounts) of compounds
What color of light do the pigments in carrots absorb the strongest?
= blue How about in eggplant? = yellow Thus, when a certain wavelength of visible light is absorbed, we can "read" a specific property of an object that we perceive as color Similarly, all spectroscopy allows us to "read" different information about molecules based on the type of light energy that is absorbed (i.e absorbing UV light, IR...etc) Questions commonly asked about spectroscopy methods: 1. Which method is best used to distinguish between any two molecules or identify a molecule? 2. What structural feature of the molecule can be deduced from the spectroscopic data given?
To separate molecules
= depends on the types of individual molecules in the mixture, as well as what property distinguishes the molecules
Simple Distillation
= performed when trace impurities need to be removed from a relatively pure compound, or when a mixture of compounds with significantly different boiling points needs to be separated - applicable when compound BP differences >30C - used to separate LARGE AMOUNTS of LOW BP compounds ex: purifying fresh drinking water away from a salt water solution - the more volatile water can be boiled away, then condensed and collected, leaving behind the nonvolatile salts
Column (flash)Chromatography
= same principle as TLC (separates based on differences in polarity) - separates large amounts of solids or high boiling point liquids (high mw compounds > 200g/mol = 15 + carbons) Stationary phase: - packed silica gel (POLAR) Mobile phase: - NP solvent NONPOLAR compounds will elute first and travel through column POLAR compounds retained in column elute last Elution/Retention time = time it takes compounds to come out of the column (NP take less time, P take more time)
High Performance Liquid Chromatography (HPLC)
= same principle as TLC/Column (separates based on differences in polarity) - more efficient that column Normal phase HPLC: - stationary phase = polar (silica gel packed beads) - mobile phase = nonpolar - the pumping of the technique only increases the speed and efficiency of the separation NONPOLAR = compounds will elute first Reverse phase HPLC (more common): - stationary phase = nonpolar - mobile phase = polar NONPOLAR compounds will now elute last (compounds LDF bond to the stationary phase) with the most POLAR compounds eluting first
Gas Chromatography (hi/low BP exit first?)
= separates based on differences in volatility/boiling point (BP) - used to separate small amounts of low boiling point compounds (these compounds will have a low mw < 200g/mol so less than 15 carbons) - volatility = the tendency of a molecule to convert to a gas (= a low BP) = high volatility = low BP Steps: - compound (liquid) mixture heated to vaporize components on entry - gas stream (mobile phase=inert gas like He) pushes components through column - gas stream rapidly carries low BP compounds through (these molecules have an easy time maintaining their gaseous state) - column is coated with liquid absorbant (stationary phase, sticky; could be NP hydrocarbon or polar polyester) so high BP compounds will be retained (these molecules condense, stick, and slow down through column - will eventually re-vaporize and come out of column) *compounds with the lowest BP exit first (highest volatility); higher BP compounds (lower volatility) exit last *time spent in column tells you about volatility/BP of compounds
Affinity Chromatography
= separates biochemical mixtures based on highly specific lock-and-key interactions between macromolecules (i.e. enzymes-substrate, ligand-receptor, antigen-antibody) - used to separate proteins from blood serum or a cell lysate Immunoaffinity chromatography: - separates proteins from blood - add antibody to bind to protein of interest - pass down column chromatography so antibody is recognized or could add stationary phase (small particles of resin linked to antibody-binding proteins, polymeric beads) - stationary phase binds to antibodies (now there are 2 lock-and-key interactions) - protein of interest is now much heavier - protein of interest gets centrifuged, so heavier parts of mixture get separated from light - remove supernatant (light proteins in mixture that were not of interest will be at top) - now to isolate/elute protein of interest from large bead complex, add competitive binding protein - competitive binding protein will outcompete protein of interest and bind to antibody-stationary phase complex - this allows the protein to be separated from the antibody and can be eluted and collected *technique relies on a series of lock-and-key interactions to isolate ONE protein in a complex mixture *other variations of affinity chromatography use magnetic beads as the stationary phase/solid phase. The beads are isolated from solution by using a magnet to hold them against the sides of the tube *mobile phase is dependent on the biochemical mixture (i.e blood serum would use water) *exploiting the binding properties of a single protein
Ion Exchange Chromatography
= separates compounds based on difference in charge states (+, -, or neutral) - used to separate mixtures of charged amino acids, proteins, or nucleotides Stationary phase: - resin containing anionic/cationic groups with counterions - pore is not size limited Mobile phase: - buffered solution - sets pH value for the column and does not change - this technique is pH dependent because these charges depend on the pH and we want that to be consistent (a negative charge may only be negative at a certain pH but then turn neutral at a different pH) column/pore-SO3(-)-Na(+) = cation exchange and (+) charges in mixture will replace Na(+) = cation-exchange resin (retains cations) vs. anion-exchange resin (retains anions, meaning it is a positively charged column); functional group attached to column/pore would then be positive *once mixture has run through column/pore and counterions have been replaced with the same charges from the mixture, the column must be flushed with the original counterion (i.e Na+) to displace all adsorbed positively charged species
Solvent Extraction
= separates compounds based on differences in solubility in polar/nonpolar solvents Solubility Rules: 1. Like Dissolves Like: - polar compounds are soluble in polar solvents ex: water, alcohols - nonpolar compounds are soluble in nonpolar (organic) solvents ex: hexane, diethyl-either 2. Compounds with less than or equal to 5 carbons and a polar group are water soluble (aka 10C group with 2 polar groups = water soluble) ratio = 5C : 1polar group to dissolve that amount of carbon 3. Charged functional groups are more soluble in water than organic solvents (charge gives you more solubility no matter the amount of carbon) ex: an 3C alcohol will dissolve in a polar (water) solvent ex: a 6C ring with a NO2 group will dissolve in an organic solvent (NP) ex: a 11C chain with two polar groups (one is charged) will dissolve in water (polar) solvent ex: a 6C chair conformation structure with 5OH groups and an O (C6H10O6) = will dissolve in a polar compound (5C:6 polar groups) **small polar (and charged) molecules are more soluble in the aqueous layer; larger, nonpolar molecules are more soluble in the organic layer
Size-exclusion Chromatography
= separates compounds based on molecular size - used to separate full proteins (large) from peptide fragments (small) or even amino acids (smallest) - cannot separate compounds that are similar in size - column is packed with inert, porous beads that serve as stationary phase - larger compounds are excluded from beads and go around - travel a shorter more direct path, so elute first - smaller compounds enter the beads - travel a longer, less direct path, so elute later
2. Splitting pattern (n+1 rule)
= the number of non-equivalent neighboring Hs - splitting pattern indicates # of Hs on adjacent carbons - (n+1) = # of peaks for a signal, where n = # of non-equivalent neighboring Hs (Hs within 3 sigma bonds) N = 0 = SINGLET
Mass Spectrometry (low yield)
= used to determine the molecular weight of a compound - It is specific enough to identify amino acids and proteins - determines the elemental and isotopic composition of a molecule (how many C, H's, O's are present) - can determine the difference between 2 compounds that have different weights - can also tell apart compounds that have identical weights
Mass Spectrometry details
= used to determine the molecular weight of a compound = determines the elemental and isotopic composition of a molecule - within the mass spectrometer, molecules are ionized in a high vacuum, usually bombarding them with high energy electrons - once ionized, compounds enter a region of the spectrometer where they are acted on by a magnetic field - the field causes the flight path of the charged species to alter, and the degree to which the path is changed is determined by the mass of the ion M/e labeled x-axis: - represents the ratio of mass (M) to charge (e) - in most cases e=+1, so peaks can be simply viewed as molecular mass - masses are usually measured in amu Y-axis = relative abundance of each species Interpreting the spectra: - small peaks with masses larger than the main peak represent molecules that have one or more of these less abundant isotopes (i.e hydrogen vs deuterium, 12C vs 13C) - the masses lower than the greatest peak (the 43) represent the masses of molecular fragments because the high energy beam of electrons used to ionize molecules in the mass-spectrometer can cause the molecule to break into smaller parts (a nonane group might have lost the terminal CH3 group making it 15 less than the peak of 43)
Chromatography (high yield)
All forms of chromatography employ 2 phases: 1. Stationary phase: will be usually packed into a column (solid, beads) = a substance that supports the mixture and allows compounds to be retained 2. Mobile phase: fluid that carries mixture of compounds to be separated All forms of chromatography employ 2 key ideas: 1. Passing the mobile phase along the stationary phase allows compounds to be distributed between them - compounds attracted to mobile phase elute quickly/first - compounds attracted to stationary phase elute last 2. The greater the affinity/interaction a compound has with the stationary phase, the longer it is retained
If a solution containing the compounds shown in Figure 4, is injected into a gas-liquid chromatograph, the first peak observed in the gc trace is attributable to which compound? A.2-Methyl-2-butanol B.2-Methyl-2-butene C.2-Chloro-2-methylbutane D.2-Bromo-2-methylbutane
B.2-Methyl-2-butene 2-methyl-2-butene will exhibit the lowest molecular weight and also the weakest intermolecular forces of attraction
Which one of the following solvent mixtures could be used to perform a solvent extraction? A. Methanol and water B. Formaldehyde and water C. Acetone and water D. Diethyl ether and water
D. Diethyl ether and water = NP and P Solvent extractions require two solvents that aren't soluble in one another. The only combination that qualifies is choice D.
The environment of the retinal binding site is most likely: A.hydrophilic. B.positively charged. C.negatively charged. D.hydrophobic.
D.hydrophobic. because retinal is composed of mainly carbon and hydrogen, making it largely hydrophobic. 1 polar group to HOW MANY MILLION CARBONS
Infrared (IR) Spectroscopy (high yield)
Details: - light in the IR range causes different bonds to vibrate at distinct frequencies - indicates which functional groups are present in a molecule - often used to monitor reactions on the MCAT - peaks are expressed as wavenumbers (cm^-1) in a spectrum (1/wavelength) = vibrational frequency IR Spec limitations: - have an OH group will be identified around 3200-3600 cm^-1 but this doesn't tell you WHERE the hydroxyl is located on the molecule - this technique does not tell you the structure or connectivity - how do you tell if there are 2 hydroxyl's in your compound? (they will both produce a wavenumber in the same territory); only one signal will be present but you don't actually know how many total are in the molecule - this technique is useful for distinguishing between constitutional isomers (same molecular formula, different bond connectivity THINK D-GLUCOSE VS D-FRUCTOSE SO ALDEHYDE VS KETONE), but not stereoisomers (same bond connectivity, different spatial arrangement of atoms)
Resolution of Enantiomers
Enantiomers share all physical and chemical properties (except optical activity) so they cannot be directly separated by any physical process - a pair of enantiomers = a racemic mixture (enantiomers have identical solubility, BP, polarity, etc) Resolution separates enantiomers of a racemic mixture by (3 steps): 1. Converting the enantiomers into diastereomeric salts with a chiral resolving agent (commonly an acid or a base) - if you have R-base and S-base as racemic mixture will use acidic resolving agent to do acid/base chemistry - mixture must be enantiomerically pure (cannot be a mixture of other stereoisomers) 2. Separating salts using conventional means (i.e recrystalization, simple distillation); now use base to remove acidic resolving agent 3. Reverting salts to the original enantiomers (R-base and S-base)
vinyl
H-C=C
A common column material used in size-exclusion chromatography is dextran, a polysaccharide of glucose. Which type of interaction most likely occurs between proteins and the dextran column material? A. Aromatic B. Hydrophobic C. Salt bridge D. Hydrogen bonding
Hydrogen bonding because a polysaccharide of glucose has numerous hydroxyl groups that can hydrogen bond to the polar side chains that are typically exposed on a protein surface.
Solvent Extraction: Acidic Functional Groups
Most acidic: - carboxylic acid (pka=5) - phenols (pka=10) - alkyl alcohols (pka=15) - carbonyl compounds has alpha protons (pka=20) *carboxylic acid is 10^5 times more acidic than phenol *removing H on these compounds makes them more water soluble because they become charged
tertiary amines
N-C3
secondary amines
N-R2 (N bonded to 2 Cs) ex: Arg
Rf values
Nonpolar compounds (alkenes, aromatics) = high Rf Polar compounds (ketones, esters, alkyl halides) Highly polar compounds (alcohols, amines, carboxylic acids) = low Rf *polar is lower and slower for molecules with polar groups * H-bonders will always be the lowest
Ion Exchange Chromatography
Property Exploited: - charge differences Used to Separate: - amino acids, proteins, nucleotides
Affinity Chromatography
Property Exploited: - lock-and-key-interaction Used to Separate: proteins, antibodies (enzymes-substrates ligands-receptors, antigens-antibodies) macromolecules
TLC/Column/HPLC Chromatography hi or low MW compounds?
Property Exploited: - polarity Used to Separate: - high MW compounds (solids or high BP liquids) with different functional groups
Size Exclusion Chromatography
Property Exploited: - size Used to Separate: - proteins, peptide fragments, amino acids
Extraction
Property Exploited: - solubility (in polar vs. organic solvent) Used to Separate: - high MW compounds with acidic and/or basic functional groups (carboxylic acids, phenols, amines)
Resolution
Property Exploited: - usually polarity of diasteromers (but we would never know this) Used to Separate: - enantiomers, racemic mixtures
Gas Chromatography/Distillation hi or low MW compounds
Property Exploited: - volatility, BP Used to Separate: - can only separate low BP compounds (low MW = <200g/mol <15C)
primary amines
R-NH2 (ex: Lysine)
4. Chemical shift (ppm)
REMEMBER WE ARE LOOKING AT THE H'S = H-NMR = chemical environment of that H - where signal appears on spectrum (10-0 ppm) - delta indicates the location of a signal on a HNMR spectrum (expressed in ppm) - a signal will be "shifted" according to the presence of nearby electrons, or the presence of EDGs or EWGs (in proximity to the H) 10ppm<-downfield (proton more deshielded = carbon attached to EWGs) ->upfield (proton is shielded = carbon attached to EDGs)->0ppm Factors involved in proton deshielding: 1. the electronegativity of the neighboring atoms (if EN atom is close to H = decrease electron density, deshield it, downfield shift) 2. hybridization (greater s-orbital character = more downfield shift) 3. acidity and hydrogen bonding (H attached to O and N = deshielded; acidic protons like on a carboxylic acid = downfield shift) aldehyde (H-C=O) = 9-10 ppm aromatic (H-Ph) = 8-7 vinyl (H-C=C) = 6-5 H-C-Y (halogen) = 4-2 alcohol (H-O-C) -- broad = 5-2 alkyl (H-C-R) = 2-0
Diagnostic Techniques; allow us to identify and distinguish differences between molecules:
Spectroscopy (identification of compounds) - Mass spec/UV-Vis - Infrared (IR) - H-NMR
A mixture of acetic acid and which of the following will be LEAST effectively separated via distillation? A. C2H6 Your Answer B. C15H31COOH C. CH3CHO D. CH2ClCH2OH
acetic acid = C2H3O2- = MW = 60 g/mol - Compounds with similar boiling points are difficult to sepatrate via distillation, which relies on a difference in boiling points to be effective. - Of the choices given, acetic acid will have a boiling point most similar to that of 2-chloroethanol (choice D), since they have similar molecular weights and both experience hydrogen bonding.
H-Nuclear Magnetic Resonance (NMR) Spectroscopy (high yield)
aka proton NMR = radiowaves are used to determine info about H atoms in a molecule - there are 4 key features to a H-NMR spectrum: 1. # of signals = # of non-equivalent Hs 2. splitting pattern (n+1 rule) = # of non-equivalent neighboring Hs 3. area under signal (integration) = #Hs represented by the signal (counting) 4. Chemical shift (ppm) = chemical environment of that H
Gas Chromatograms can be generated in:
all chromatography: - size-exclusion (size) - Column (polarity) - HPLC (polarity) - Ion Exchange (charge) - Affinity (lock-and-key interactions ) - Gas (volatility/boiling point) EXCEPT: - TLC (polarity) - doesn't have a column Properties: 1. the number of compounds in a mixture equals the number of peaks (still applies to all) 2. the relative quantity of each compound from peak area (still applies to all) 3. the volality/BP of the compounds can be read from the time axis (this VARIES depending on technique) ex: Reverse HPLC Chromatogram: 3. time axis would tell you polarity so stationary phase is NP so whatever elutes first (less time on time axis) would be more polar compound (A would be more polar compound and B would be more NP) ex: Size-exclusion Chromatogram - A peak would elute first so this means those compounds are the largest
Spectroscopy Preview
color: Orange what allows us to see this color? - colored objects absorb certain wavelengths of visible light, and the remainder of wavelengths NOT absorbed make it to our eyes (gets reflected) if the color we see is being reflected, what color(s) is/are being absorbed? - orange is being reflected so ALL of the other colors are being absorbed (red, yellow, green, blue, purple, pink etc) - BUT the complimentary color (blue) is absorbed the STRONGEST
What element is not visible in proton NMR spec?
deuterium is not visible in proton NMR spectroscopy
Solvent Extractions: Important Functional Groups
most important are - carboxylic acids (pka = 5) - phenols (pka = 10) - amines (basic) * deprotonating acids or protonating bases renders them charged and more water soluble **the solubility of a substance can be easily altered via acid/base reaction if an amine, phenol, or carboxylic acid is present in the compound
(H-NMR) spin spin coupling
n+1 subpeaks n= # of non-equivalent hydrogen neighbors Spin-spin coupling, or splitting, is an interaction between neighboring non-equivalent sets of hydrogens. CH3CHBr2 is the only choice with more than one set of hydrogens that are three bonds apart.
hexane
nonpolar solvent (organic solvent)
diethyl ether
nonpolar solvent (organic solvent) ether: R-O-R (not to be confused with an ester = RCOOR
Solvent Extraction: Basic Functional Group
only one: amines R3N (amine = bonded to non carbonyl carbons)
polarimetry
this measures the change in the degree of rotation of plane-polarized light caused by one of the components over time - must have chiral centers
Basic nitrogens
will have (+) charge from resonance - HCl protonates amines making a basic nitrogen with a + charge - think about basic amino acids - HCl can only protonate sp3 amines without resonance
