OChem Class 2 Flashcards
2 phases in chromatography
Stationary Phase - substance that supports mixture & allows compounds to be retained
Mobile phase - fluid that carries mixture of compounds to be separated
What is chromatography?
Pass mobile phase along stationary phase which allows compounds to be distributed between them
The greater the affinity/interaction a compound has with stationary phase, the longer it’s retained
Size-exclusion chromatography
Separates based on molecular size
Mobile phase - is the aqueous phase that helps dissolve proteins (aqueous buffer)
Stationary Phase - inert, porous beads
Large compounds don’t go through beads so elute first, small compounds take longer path by going through beads so elute last
Thin-Layer Chromatography (TLC)
Analytical technique (tests small amounts)
- separates small amounts of solids or high boiling point liquids
Stationary Phase - silica gel which is polar and forms H-Bond to compounds
Mobile Phase - shallow solvent bath (usually contains ethyl acetate : heptane ration)
Non-polar compounds have weaker interactions because less affinity to gel so migrate faster. Polar compounds have stronger interactions because of H-bonds with gel so migrate slower
Rf Value
Rf = migration distance of the spot / migration distance to solvent front
Rf is never negative or greater than 1
Larger Rf = non-polar compounds
Non-polar compounds consist of
alkenes, aromatics
Polar compounds
H-bond acceptors
ketones, ester, ether, alkyl halides
Highly polar compounds
H-bond donors
alcohol, carboxylic acid, amines
Column Chromatography
- similar to TLC except separates larges amounts of solids or high boiling point liquids (based on polarity)
High performance liquid chromatography
-Same principles as TLC & Column chromatography (separates based on difference in polarity)
Normal HPLC:
Stationary phase - polar (Silica Gel)
Mobile phase - non-polar (ethyl acetate : heptane)
- non polar elutes first
Reverse HPLC:
Stationary phase - nonpolar (Silica Gel capped with large hydrocarbon)
Mobile phase - water:methanol
- polar elutes first
A pump will push the solvent through the column at a much higher pressure which provides quality separation (better purity)
Ion exchange chromatography
Separates based on differences in charge (+,-, or neutral)
- Separates proteins, nucleotides, amino acids
Stationary phase- resin containing anionic/cationic groups with counter-ions
Mobile phase- buffered solution (helps maintain pH)
Anion-exchange resin: retain anions (resin itself is cationic)
Cation-exchange resin: retain cations (resin itself is anionic)
Eg. Cation-exchange resin
Anions flush out first while cations attach to the resin, then to flush out cations you put in excess of cations to displace & elute it
Affinity Chromatography
- Separates based on highly specific lock & key interactions
- used to separate proteins from blood serum or cell lysate
Stationary phase - small particles of resin linked to ab-binding protein
- Add antibody to serum that’s against protein of interest added
- Stationary phase (G°) added, binds to ab and now have trimeric compound; protein of interest is in solid phase now
- Bead complexes collected by centrifugation; supernatant and solid particles form pellet
- supernatant decanted
- To elute target protein, add competitive ab-binding protein to have greater affinity so protein of interest can elute alone
Metal Ion Affinity Chromatography
Stationary Phase - nickel based resin inside the column
Mobile phase - cell lysate which includes multiple proteins
- The protein of interest is tagged with histadine amino acids
- Histidine has amidizole rings which has a high affinity for Nickel cation
- To elute the protein of interest, lower the pH bc amidizole if weakly basic
- Then add large volume of amidizole which will outcompete the amidizole component
Gas Chromatography
Mobile Phase - gas stream
Stationary phase - liquid absorbant that lines the column
- Separates based on differences in volatility/bp
- Used to separate small amounts of low bp compounds
- Compound mixture is heated to vaporize before entry into column
- less volatile (high bp) will stick to column while lower BP exit first
Gas Chromatograph
Provides information about:
- # of compounds in mixture equal to # of peaks
- relative quantity of each compound from peak area
- volatility/bp of compounds
Distillation
- separates differences in bp
- separates large amounts of low bp compounds
Volatility
Tendency of molecule to convert to gas
Boiling Point
Measure of intermolecular forces between liquid molecules
Factors that affect BP
- IMF (the more IMF = the higher the BP)
- Branching (the more branches = lower the BP)
- Molecular weight (the heavier the molecule = the higher the BP)
Simple vs Fractional distillation
Simple
- component BP differences are > 30°C
- remove impurities from a relatively pure liquid
Fractional
- component BP differences are <30°C
- useful for separating diastereomers
Simple distillation
- Boil the liquid and it boils through tube
2. Goes through vaporization, liquid with lower bp will condense back to liquid phase & collect in other flask
Fractional distillation
- Compound mixture is boiled and the tube has packing material to increase SA. High SA allows for vaporization, condensation, revap, condense, etc
- More volatile will move up the glass and vaporize and condense back to liquid in second flask
- Meanwhile, the less volatile portion will make contact with solid and go back to liquid in original flask
Solvent Extraction
Separates compounds based on differences in solubility in polar/non-polar solvents
Solubility Rules:
- Like dissolves like
- Compounds with 5 or less carbons and a polar group are water soluble
- Charged functional groups are more soluble in water than organic solvents
Acidic functional groups
carbonyls (pKa 20) < alkyl alcohols (pKa 15) < phenol (pKa 10) < carboxylic acid (pKa 5) < amine (basic functional group - pKa 10)
Extraction Conditions
- Aqueous NaHCO3 (weak base); ONLY deprotonates carboxylic acid
- Aqueous NaOH (strong base); ONLY deprotonates carboxylic acid and phenols (*Alkyl alcohols are not acidic enough to be useful in extractions because you need approx 10 M NaOH vs 1.0 or 0.1 M)
- Aqueous HCl (strong acid); ONLY protonates amines
Layers in Extraction
Organic mixture - contains compounds you want to separate
Then add aqueous solvent
Neutral compounds are usually less dense so found on top layer. Aqueous layer now contains salts of compounds being separated (so what you’ve protonated and deprotonated)
Why can you not use any other method except resolution to separate enantiomers?
Helps separate enantiomers because they share the same physical & chemical properties so cannot by directly separated by any physical process
Resolution
- Convert enantiomers into diastereomeric salts with a chiral resolving agent
- Separate salts using conventional means (now have different BP, polarity, etc)
- Revert salts to original enantiomers by treating with an acid or base (eg. NaOH to relieve tartaric acid)
How do we see colours?
an object absorbs a certain wavelength of visible light and the remainder of wavelengths are reflected to us
R G
O B
Y V
When you see a certain colour, all the other colours are absorbed but the one directly connected to x colour is most STRONGLY absorbed
Energy spectrum
Goes from highest energy, frequency (left to right)
Goes from lowest to high wavelength (left to right)
Gamma ray / X Ray / UV Ray / Visible Light / Infrared / Microwave / Radiowaves
Mass Spectroscopy
- Used to determine the molecular weight of a compound
- Determines the elemental & isotopic composition of molecule
Infrared Spectroscopy (IR)
- Light in IR range causes different bonds to vibrate at distinct frequencies
- Indicates which function group is present (but not how many or where)
- Useful for distinguishing constitutional isomers not stereoisomers
- Peaks expressed as wavenumber (cm-1) in spectrum 1/λ)
Important Wavenumbers: O-H C=O C=C C≡N/ C≡C
O-H: 3200 - 3600
C=O: 1700
C=C: 1650
C≡N/ C≡C: 2100-2260
UV/Visible spectroscopy
- Indicates presence of conjugated pi system (alternating double and single bonds)
- more alternating bonds = longer the wavelength of light absorbed)
H-NMR (NMRI Spectroscopy)
Radiowaves determine information about H atoms in molecules
What are the 4 key features of H-NMR Spectrum?
- # of signals (how many non-equivalent hydrogens)
- Splitting pattern n+1 rule (# of non-equivalent neighbouring hydrogens)
- Area under signal/ integration (#of hydrogens represented by each signal)
- Chemical shift (ppm) (chemical environment of that hydrogen)
N+1 (splitting pattern) refers to…
# of peaks for a signal 1 - singlet 2 - doublet 3 - triplet 4 - quarter 5+ - multiplet
Chemical shift (δ)
δ indicates location of signal on H-NMR spectrum (expressed in ppm)
Downfield shift (high ppm)
- carbon attached to e- withdrawing group
- proton is deshielded
Upfield shift (low ppm)
- proton is shielded
- carbon attached to e- donating group
10 - 9: aldehyde 8-7: aromatic phenol 6-5: vinyl 5-2: alcohol 4-2: H-C-Y 2-0: alkyl
