OChem: Lab Techniques: Separations and Spectroscopy Flashcards
Chromatography employs two phases:
Stationary
Mobile
Stationary -> substance that supports and allows compounds to be retained
Mobile -> fluid that carries mixture of compounds to be separated
Size exclusion chromatography:
Property of separation
Used to separate
Elution order (what comes out first/last)
Property of separation -> molecular size
Used to separate -> large proteins (polymers) from smaller peptides (oligomers)
Elution order (what comes out first/last) -> largest polymer first, smaller oligomers later
Column packed with inert, porous beads as stationary phase - larger compounds are excluded from beads and go around so elute first
TLC Property of separation Used to separate Elution order (what comes out first/last) Equation for Rf value
Are the following compounds nonpolar, polar, or highly polar?
Ketone, alkene, alkyl halides, alcohols, amines, aromatics, esters carboxylic acids
Property of separation -> polarity
Used to separate -> SMALL amounts of high molecular weight compounds
not good for bulk compounds
Elution order (what comes out first/last) -> Stationary phase = plate coated in polar silica gel, silica can H-bond to compounds Mobile phase = Shallow solvent bath within a (sealable) development chamber Non polar compounds have weaker interactions with silica gel (migrate faster up the plate) Polar compounds have stronger interactions with silica gel (migrate slowly up the plate) Rf = migration distance of spot/ migration distance of solvent front
High Rf values/ nonpolar compounds -> alkenes, aromatics
Polar -> ketones, esters, alkyl halides
Low Rf values/Highly polar compounds -> alcohols, amines, carboxylic acids
polar is slower
Column Chromatography
Property of separation
Used to separate
Elution order (what comes out first/last)
looks like column with solvent running through
Property of separation -> polarity (same as TLC)
Used to separate -> LARGE amounts of high molecular weight compounds
Elution order (what comes out first/last) -> most nonpolar elute first, most polar last
High Performance Liquid Chromatography - HPLC
Normal phase HPLC
Why would you choose this methods over others like column chromatography?
Property of separation
Used to separate
Elution order (what comes out first/last)
Only difference for this next technique is that it is used at much higher pressures! Use if can’t purify by column chrom which doesn’t use much pressure, here get much better separation bc used with high pressure, use for hard separations (for things with similar Rf values/similar polarity)
Property of separation -> polarity (same as TLC)
Used to separate -> large amounts of high molecular weight compounds
Elution order (what comes out first/last) stationary phase = polar silica gel beads Mobile phase = nonpolar solvent most nonpolar elutes first
Reverse phase HPLC
= More common form of HPLC is reverse phase HPLC (if MCAT doesn’t specify which one it is using, it is this one)
Property of separation
Used to separate
Elution order (what comes out first/last)
Used to separate based on polarity
BUT stationary phase = nonpolar (hydrocarbon chains on silica gel beads)
Mobile phase = polar (solvent)
Elution order (what comes out first/last)
Most polar elutes first now!
Most nonpolar elutes last
Ion Exchange Chromatography:
Property of separation
Used to separate
Elution order (what comes out first/last)
What do anionic resins vs cationic resins elute first?
For a cation exchange, will the proteins (aa) with pI values greater than the pH of the mobile phase elute faster or slower compared to those with pI values below the solution pH
Property of separation -> charge states (+,-, or neutral)
Used to separate -> mixtures of charges amino acids, proteins, or nucleotides
Elution order (what comes out first/last) -> stationary phase = resin containing anionic/cationic groups with counterions Mobile phase: buffered solution
Anionic (-) resins elute anions first (retain cations), cationic (+) resins elute cations first (retain anions)
If there is a cation exchange resin, those proteins with pI values greater then 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 were passed through an anion exchange, the opposite would be true.
If the pI values of proteins to be separated are known, the pH of the mobile phase may be buffered to a specific pH, thereby ensuring the different charge states and hence good separation
Affinity Chromatography
Property of separation
Used to separate
Elution order (what comes out first/last)
What happens in immunoaffinity chromatography?
Property of separation -> lock-and-key interactions
Used to separate -> proteins from a biochemical mixture (e.g. blood serum)
ex. Immunoaffinity chromatography
a) have blood serum with many proteins
b) add Ab against protein of interested and they bind
c) add stationary phase which binds Abs bonded to those proteins
d) centrifuge and bead complexes are collected at the bottom of tube bc heavier
Elution order (what comes out first/last) Stationary phase = small particles or resin linked to Ab-binding protein To elute the target protein, add competitive Ab binding protein
*Not all proteins of interest have a commercial Ab available. In this case, researchers can use an affinity tag. Using recombinant tech, a small molecular tag is added to N-terminus or the C-terminus of the protein. DNA sequences coding for affinity-tagged proteins can be produced in large amounts in lab bacteria, and the cell lysate collected is rich in tagged protein. One class of commonly used affinity tags are the His tags (made of 6-10 histidine aa's), which bind ions such as nickel. When a cell lysate is applied to a column packed with nickel-based resin, the His-tagged proteins bind to the resin. This is done under high pH conditions, and the His-tagged protein can be eluted off the solid phase using lower pH conditions.
Lower yield/he skipped
Metal Ion Affinity Chromatography
How does it work?
Uses recombinant proteins: proteins grafted with a tag at its C or N terminus
SEE TEXTBOOK FOR REST
Gas Chromatography
Property of separation
Used to separate
Elution order (what comes out first/last)
Describe the phases
What can a gas chromatogram tell you
Property of separation -> volatility/boiling point BP
Used to separate -> small amounts of low BP compounds
mobile gas phase and liquid stationary phase
Elution order (what comes out first/last) -> lowest BP compounds exit first, higher BP compounds exit later (makes logical sense)
Mobile phase = gas stream
Stationary phase = column with liquid adsorbant retains high BP compounds
A gas chromatogram can tell you:
- The number of compounds in a mixture equals the number of peaks
- The relative quantity of each compound from peak area
- The volatility/BP of the compound can be read from the time axis -> The highest BP/lowest volatility is the later peak
BP is a measure of 3 intermolecular forces between liquid molecules: name them
*What factors affect BP?
Hydrogen bonds
Dpole-dipole forces
London dispersion
Factors that affect BP:
Molecular weight -> heavier molecules have higher BPs (bc have more SA to interact and have van der Waals interactions and this is true for all molecule types, not just hydrocarbons)
Branching -> more branches = lower BP
*MW factors are more important than branching
Another factor is hydrogen bonding
- Intramolecular hydrogen bonding can occur b/w the hydrogen of the hydroxyl group and a lone pair of e- on ex. the nitro group on the same molecule and these H-bonding interactions decrease the amount of intermolecular hydrogen bonding interactions that can occur b/w molecules thereby decrease the melt and boiling point.
Distillation
Property of separation
Used to separate
Elution order (what comes out first/last)
Property of separation -> volatility/boiling point (BP)
Used to separate -> LARGE amounts of low BP compounds (does same as gas chrom, but for larger amounts of material)
Elution order (what comes out first/last) -> Nonvolatile solutes remain after distillation of the lower BP compound
Distillation is the process of raising the temperature of a liquid until it can overcome the IMF that hold it together in the liquid phase. The vapor is then condensed back to liquid phase and subsequently collected in another container
Skipped and lower yield
Simple vs fractional distillation
Prob need to read book
Fractional distillation -> applicable when component BP differences are less than <30º
Useful for separating diastereomers
simple distillation is performed when trace impurities need to be removed from a relatively pure compound, or when a mixture of compounds with significantly different boiling pts needs to be separated
ex. distillation to purify drinking water away from salt water solution, the more volatile water can be boiled away, then condensed and collected, leaving behind nonvolatile salts
Fractional distillation used when the difference in bp of the components in the liquid mixture is not large
Solvent extraction
Property of separation
Solubility rules in terms what to consider polar vs nonpolar for like dissolved like, etc
Property of separation -> solubility in polar/nonpolar solvents
Solubility rules:
1. “like dissolves”
- Polar compounds are soluble in polar solvents
- Nonpolar compounds are soluble in nonpolar (organic) solvents
2. Compounds with < or equal to 5C atoms and a polar group are water soluble (unless there are MANY functional groups like in glucose which is polar)
3. Charged functional groups dissolve readily in a polar solvent
Important functional groups for extractions
Which FG is definitely not acidic enough for extraction (MCAT will try to trick you)?
Deprotonating acids or protonating acids renders them charged and more water soluble
Acidic functional groups
Pka in multiples of 5
carbonyl compounds (alpha protons -> H’s right next to carbonyl) pKa = 20
Alkyl alcohols pKa=15
Do not use for extraction!
Phenols (OH on end) -pKa 10
GOOD FOR EXTRACTION
Carboxylic acids = great! Pka = 5
GREAT FOR EXTRACTION
Basic FG:
Amines =
IMPORTANT FOR EXTRACTIONS
**Alkyl alcohols are not acidic enough to do an extraction pKa is 15
