OChem Class 2

Question 1

2 phases in chromatography

Answer 1

Stationary Phase - substance that supports mixture & allows compounds to be retained
Mobile phase - fluid that carries mixture of compounds to be separated

Question 2

What is chromatography?

Answer 2

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

Question 3

Size-exclusion chromatography

Answer 3

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

Question 4

Thin-Layer Chromatography (TLC)

Answer 4

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

Question 5

Rf Value

Answer 5

Rf = migration distance of the spot / migration distance to solvent front

Rf is never negative or greater than 1
Larger Rf = non-polar compounds

Question 6

Non-polar compounds consist of

Answer 6

alkenes, aromatics

Question 7

Polar compounds

Answer 7

H-bond acceptors

ketones, ester, ether, alkyl halides

Question 8

Highly polar compounds

Answer 8

H-bond donors

alcohol, carboxylic acid, amines

Question 9

Column Chromatography

Answer 9

• similar to TLC except separates larges amounts of solids or high boiling point liquids (based on polarity)

Question 10

High performance liquid chromatography

Answer 10

-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)

Question 11

Ion exchange chromatography

Answer 11

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

Question 12

Affinity Chromatography

Answer 12

• 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

1. Add antibody to serum that’s against protein of interest added
2. Stationary phase (G°) added, binds to ab and now have trimeric compound; protein of interest is in solid phase now
3. Bead complexes collected by centrifugation; supernatant and solid particles form pellet
4. supernatant decanted
5. To elute target protein, add competitive ab-binding protein to have greater affinity so protein of interest can elute alone

Question 13

Metal Ion Affinity Chromatography

Answer 13

Stationary Phase - nickel based resin inside the column
Mobile phase - cell lysate which includes multiple proteins

1. The protein of interest is tagged with histadine amino acids
2. Histidine has amidizole rings which has a high affinity for Nickel cation
3. To elute the protein of interest, lower the pH bc amidizole if weakly basic
4. Then add large volume of amidizole which will outcompete the amidizole component

Question 14

Gas Chromatography

Answer 14

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

1. Compound mixture is heated to vaporize before entry into column
2. less volatile (high bp) will stick to column while lower BP exit first

Question 15

Gas Chromatograph

Answer 15

Provides information about:

1. # of compounds in mixture equal to # of peaks
2. relative quantity of each compound from peak area
3. volatility/bp of compounds

Question 16

Distillation

Answer 16

• separates differences in bp

- separates large amounts of low bp compounds

Question 17

Volatility

Answer 17

Tendency of molecule to convert to gas

Question 18

Boiling Point

Answer 18

Measure of intermolecular forces between liquid molecules

Question 19

Factors that affect BP

Answer 19

1. IMF (the more IMF = the higher the BP)
2. Branching (the more branches = lower the BP)
3. Molecular weight (the heavier the molecule = the higher the BP)

Question 20

Simple vs Fractional distillation

Answer 20

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

Question 21

Simple distillation

Answer 21

1. 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

Question 22

Fractional distillation

Answer 22

1. Compound mixture is boiled and the tube has packing material to increase SA. High SA allows for vaporization, condensation, revap, condense, etc
2. More volatile will move up the glass and vaporize and condense back to liquid in second flask
3. Meanwhile, the less volatile portion will make contact with solid and go back to liquid in original flask

Question 23

Solvent Extraction

Answer 23

Separates compounds based on differences in solubility in polar/non-polar solvents

Solubility Rules:

1. Like dissolves like
2. Compounds with 5 or less carbons and a polar group are water soluble
3. Charged functional groups are more soluble in water than organic solvents

Question 24

Acidic functional groups

Answer 24

carbonyls (pKa 20) < alkyl alcohols (pKa 15) < phenol (pKa 10) < carboxylic acid (pKa 5) < amine (basic functional group - pKa 10)

Question 25

Extraction Conditions

Answer 25

1. Aqueous NaHCO3 (weak base); ONLY deprotonates carboxylic acid 2. 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) 3. Aqueous HCl (strong acid); ONLY protonates amines

Question 26

Layers in Extraction

Answer 26

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)

Question 27

Why can you not use any other method except resolution to separate enantiomers?

Answer 27

Helps separate enantiomers because they share the same physical & chemical properties so cannot by directly separated by any physical process

Question 28

Resolution

Answer 28

1. Convert enantiomers into diastereomeric salts with a chiral resolving agent 2. Separate salts using conventional means (now have different BP, polarity, etc) 3. Revert salts to original enantiomers by treating with an acid or base (eg. NaOH to relieve tartaric acid)

Question 29

How do we see colours?

Answer 29

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

Question 30

Energy spectrum

Answer 30

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

Question 31

Mass Spectroscopy

Answer 31

- Used to determine the molecular weight of a compound | - Determines the elemental & isotopic composition of molecule

Question 32

Infrared Spectroscopy (IR)

Answer 32

- 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)

Question 33

``` Important Wavenumbers: O-H C=O C=C C≡N/ C≡C ```

Answer 33

O-H: 3200 - 3600 C=O: 1700 C=C: 1650 C≡N/ C≡C: 2100-2260

Question 34

UV/Visible spectroscopy

Answer 34

- Indicates presence of conjugated pi system (alternating double and single bonds) - more alternating bonds = longer the wavelength of light absorbed)

Question 35

H-NMR (NMRI Spectroscopy)

Answer 35

Radiowaves determine information about H atoms in molecules

Question 36

What are the 4 key features of H-NMR Spectrum?

Answer 36

1. # of signals (how many non-equivalent hydrogens) 2. Splitting pattern n+1 rule (# of non-equivalent neighbouring hydrogens) 3. Area under signal/ integration (#of hydrogens represented by each signal) 4. Chemical shift (ppm) (chemical environment of that hydrogen)

Question 37

N+1 (splitting pattern) refers to...

Answer 37

``` # of peaks for a signal 1 - singlet 2 - doublet 3 - triplet 4 - quarter 5+ - multiplet ```

Question 38

Chemical shift (δ)

Answer 38

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 ```