OChem: Lab Techniques: Separations and Spectroscopy Flashcards

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1
Q

Chromatography employs two phases:
Stationary
Mobile

A

Stationary -> substance that supports and allows compounds to be retained
Mobile -> fluid that carries mixture of compounds to be separated

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2
Q

Size exclusion chromatography:
Property of separation
Used to separate
Elution order (what comes out first/last)

A

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

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3
Q
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

A

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

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4
Q

Column Chromatography
Property of separation
Used to separate
Elution order (what comes out first/last)

A

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

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5
Q

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)

A

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
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6
Q

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)

A

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

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7
Q

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

A

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

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8
Q

Affinity Chromatography
Property of separation
Used to separate
Elution order (what comes out first/last)

What happens in immunoaffinity chromatography?

A

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.
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9
Q

Lower yield/he skipped

Metal Ion Affinity Chromatography

How does it work?

A

Uses recombinant proteins: proteins grafted with a tag at its C or N terminus

SEE TEXTBOOK FOR REST

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10
Q

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

A

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:

  1. The number of compounds in a mixture equals the number of peaks
  2. The relative quantity of each compound from peak area
  3. The volatility/BP of the compound can be read from the time axis -> The highest BP/lowest volatility is the later peak
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11
Q

BP is a measure of 3 intermolecular forces between liquid molecules: name them

*What factors affect BP?

A

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.

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12
Q

Distillation

Property of separation
Used to separate
Elution order (what comes out first/last)

A

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

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13
Q

Skipped and lower yield

Simple vs fractional distillation

A

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

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14
Q

Solvent extraction

Property of separation
Solubility rules in terms what to consider polar vs nonpolar for like dissolved like, etc

A

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

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15
Q

Important functional groups for extractions

Which FG is definitely not acidic enough for extraction (MCAT will try to trick you)?

A

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

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16
Q

Extraction conditions:

  1. Aqueous NaHCO3 (weak base): ONLY deprotonates ________
  2. Aqueous NaOH (strong base): deprotonates ____ AND ______.
    NOTE: _____ are not acidic enough to be useful in extractions
  3. Aqueous HCl (acid): protonates amines
A
  1. Aqueous NaHCO3 (weak base): ONLY deprotonates carboxylic acids (bc carboxylic acids so acidic)
  2. Aqueous NaOH (strong base): deprotonates phenols AND carboxylic acids.

NOTE: alkyl alcohol are not acidic enough to be useful in extractions

17
Q

Organic and Aqueous Layers, generally how does it work with extractions?

A

Add aqueous solvent to glassware with an organic mixture which contains the compounds you want to separate

Then you get the aqueous layer on bottom which contains salts of the compounds being separated
and the organic layer is on top (Pretty sure this layer still contains other OG mixture elements)

*Read more in book

18
Q
If have glassware with organic mixture 
it includes 
A -> phenol FG
B -> carboxylic acid and alcohol FGs
C-> alcohol and amine FG 
D -> benzene ring 

Which compound will always remain in the organic layer ?
Which compound is best extracted first? How? (Using what aqueous solvent)

A

Key: want to add an aqueous solvent which will react with only one of the compounds in the the organic mixture in order to separate it from them

Which compound will always remain in the organic layer ? D
Which compound is best extracted first? C due to unqiue (basic) NH2 group which differs from the other more acidic FG of other organic mixture options
B could work too if add weak base then you only extract B (carboxylic acid is strong enough of a acid FG to react with a weak base like NaHCO3)

C requires an acid (HCl) but will not affect A,B, or D

To extract A, need NaOH (a strong base) BUT this extracts both A and B so not a good option/not efficient

19
Q
Wash out the alcohol + amine compound from:
A -> phenol FG
B -> carboxylic acid and alcohol FGs
C-> alcohol and amine FG 
D -> benzene ring 

describe process and what you would use as aqueous layer added

A

Amine is base so want to add aqueous solution with an acid (HCl), then only the amine salt version will be present in the aqueous layer on the bottom

20
Q

If had a mixture of only phenol FG compound and benzene ring compound, how would you extract the phenol compound?

pKa of amide?

A

The phenol group is mildly acid and requires a strong base
Add aqueous solution of base (ex. NaOH) and then phenol group will be in the bottom aqueous layer

Then you can evaporate organic layer to get D isolated

pKa of amide about 20 so basic

21
Q

Are enantiomers easy or difficult to separate?

Resolution of enantiomers

A

Enantiomers share all physical and chemical properties, so they cannot be directly separated by any physical process
enantiomers share identical solubility, BP, polarity, etc.

Diastereomers -> different physical properties so can now be separated

Resolution separates enantiomers of a racemic mixture by:

  1. converting the enantiomers into diastereomeric salts with a chiral resolving agent which is added to the compound so that you have 2 diastereomers now (commonly an acid or a base)
  2. Separating salts using conventional means (e.g. recrystalization)
  3. Reverting salts to the original enantiomers
22
Q

What is mass spectrometry used to determine?

*What is UV/Vis spectroscopy used for?

A

Mass spec used to determine the molecular weight of a compound, determines the elemental and isotopic composition of a molecule

UV/Vis spectroscopy
Indicates the presence of a conjugated pi system in a molecule
As conjugation in a molecule increases, the wavelength of light absorbed increases or redshifts; a blue shift is the opposite
(bc as conjugation increases, resonance increases, lower E, also red shift makes sense bc if you imagine the energies of light, red has a greater wavelength than blue)
The wavelength absorbed is complementary to the color perceived

23
Q

What does IR tell you?

A

IR tells you which FG present, often used to monitor reactions on the MCAT
Peaks are expressed as wavenumbers (cm^-1)

24
Q

What FG wavenumbers do you need to know?

A

O-H -> 3200 - 3600 cm^-1 and BROAD vc
C=O -> 1700 cm^-1 variable
C=C -> 1650 cm^-1
C≡N/C≡C 2100 - 2260 cm^-1

The appearance/disappearance of a C-H bond is never diagnostic on the MCAT

25
Q

H-NMR
What does it tell you?
What is the chemical shift of aromatic protons? and alkene protons? alkyl?

A

of signals = # of sets of non-equivalent Hs

Splitting pattern (n+1 rule) = # of non-equivalent neighboring Hs (Hs 3 sigma bond aways)

Area under signal (integration) = # of Hs represented by the signal

Chemical shift (ppm) = chemical environment of H

Chemical shift of aromatic protons = 6.5 - 8 ppm and alkene protons 5 - 6 ppm
0 - 2 is alkyl

26
Q

What makes proton environment equivalent?

A

Free rotation or symmetry makes H’s equivalent (same chemical enviornment)
If H is on the plane of symmetry, then counts as TWO DIFFERENT H’s
Can have multiple planes of symmetry which render almost all H’s in the ring same environment

For each of non-equivalent Hs, a spectrum will have on H-NMR signal

27
Q

How does chemical shift work?

What it shielding?

A

Don’t go into detail with chemical shift values
Know signal shifts based on e- donating groups or withdrawing groups

Right side of axis -> upfield shift (low ppm), proton is shielded, Carbon attached to EDG

Left side of axis -> Downfield shift (high ppm), proton is deshielded, carbon attached to EWG

E- donating groups (alkyl) show up upfield bc proton shielded

28
Q

If the exact mass of something was found as the sodium complex, what m/z value was experimentally determined? (just how would you go about solving it)

A

add up molar mass of compound and molar mass of sodium