Pchem Exam 3 (pKa and chromatography)

what is pka

the propensity of a molecule to be protonated; an equilibrium

Equilibrium when pH=pKa

equal amounts of HA and A^-

Equilibrium when pH > pKa

solution is more basic so acid loses H more (towards right side); more A^- than HA

equilibrium when pH<pKa

solution is more acidic so acid loses H less (towards left side); more HA than A^-

pKa of COOH

4.75

pKa of imidazole

7

pKa of amine

11

pKa of phenol

10

pKa of pyridine

5

pKa of pyrrolidine

10

if pH > pKa

solution more basic so compound deprotonated

if pH < pKa

solution more acidic so compound protonated

Partition coefficient (K)

g of compound per mL of organic solvent/ g compound per mL water

higher K means what?

more solvent in organic solvent than water

define each variable

neutral species are more soluble in which layer

organic

charged species are more soluble in which layer

water (aq)

to extract an acid into water pH needs to be higher or lower than pKa?

higher (bc then solution wld act as base and steal the H from the acid making it negative so it wld be charged and go into water)

to extract a base into water pH needs to be higher or lower than pKa?

lower (bc then solution wld act as acid and donate H to the base making it positive so it wld be charged and go into water)

pKa of HCl and when wld u use it for seperations

-2; wld use to extract a base

pKa of H₂CO₃ and when wld u use it for separations

6.5; wld use to extract a stronger acid if you 2 acids want the weaker acid to stay in organic phase to j get the stronger one in water

pKa of NaOH and when wld u use it for separations

14; wld use to extract a weaker acid / acid in general

Chromatography; separation of a molecule is based on chemical structure so what properties?

polarity, functional grp, size, boiling point

4 types of chromatography we go over

thin liquid chromatography (tlc), liquid chromatography, gas chromatography, high performance liquid chromatography (hplc)

TLC stationary phase?

SiO₂

SiO₂ polarity?

polar

TLC mobile phases?

ethyl acetate (polar) and hexanes (nonpolar) mixture

TLC which wld elute first

nonpolar

TLC which wld have highest retention time

polar

if you increase mobile phase % polar solvent what happens?

lessen interactions with polar stationary phase so move FURTHER/FASTER and LESS separation between molecules

if you increase mobile phase % nonpolar solvent what happens?

molecules interact more with polar stationary phase, move LESS but BETTER separation between molecules

TLC parameters we can manipulate (7)

stationary phase composition

mobile phase composition

flow rate of mobile phase

size of stationary particle

level of packing of stationary phase

amount of stationary particle

height and width

how to get a sharper peak (don’t want broad bc can overlap and can’t tell proper diff between molecules and can’t find AUC)

smaller particles + good packing + optimized flow rate = sharper peaks

calculation of column efficiency

how good is the separation? good is sharp/narrow peaks

theoretical plates (N)

a plate is one “mixing and separating” cycle

if N is large what type of separation

better separation

broader peaks (band broadening) why bad?

the broader the peaks the less efficient the column

sources of band broadening?

Longitudinal diffusion, eddy diffusion, poor set up

Longitudinal diffusion

as it spends more time on column (plate) bands get wider (think the mobile phase pulling compound forward and stationary phase pulling compound the stay in place so getting more stretched with time)

ways to reduce longitudinal diffusion (4)

faster flow rates (less time on plate so less time to spread)

shorter column (less time on plate)

smaller particles (less time on plate bc move faster)

wider column (push mobile phase faster)

eddy diffusion

particles in the column (plate) not all perfectly uniform so different paths exist so molecules take different routes —> peaks widen (since same molecules in compound will elute at diff times)

what resolution is desirable?

> 1.5; want peaks to be as narrow as possible so we can see separation so want 2 signals that meet the baseline

factors that affect resolution? (4)

column (plate) length

stationary phase

mobile phase in LC

temperature in GC

Asymmetry factor formula

AF = b/a (right side area/ left side area)

AF ideal range

0.95-1.15

fronted asymmetry

A peak that rises slowly and then drops sharply, unlike a symmetrical peak.

causes for fronted

overloading (too much), poor trapping (lack of interactions with stationary phase), injection solvent too strong (HPLC); so think the mobile phase like a wave and washing over and taking everything in the beginning instead of throughout whole time

tailing asymmetry

The peak's front is steep and sharp, while the back gradually slopes down towards the baseline; trailing (back) half appearing broader than its leading (front) half

causes for tailing

adsorption to column stationary phase (sticking to stationary), too much dead volume (mobile phase isn’t sweeping it), common for polar molecules

ion exchange steps

1) deprot (ex the cooh to make neg) or prot (rnh2 —> rnh3+ if not alr positive)

2) bind ionically

3) any other molecules come out

4) change solvent/H2O in excess to wash way the one your trying to purify

1. Sample Loading: The sample (e.g., hard water or a protein solution) flows through the column, and target ions (positive or negative) bind to the oppositely charged sites on the resin, displacing the original counter-ions (like Na⁺ or OH⁻).

2. Washing: A buffer (often the same as the loading buffer) is passed through to wash away any non-target molecules or impurities that aren't strongly bound.

3. Elution: The bound target molecules are released by changing the buffer's salt concentration (e.g., adding NaCl) or pH, which disrupts the electrostatic attraction.

reason for ion exchange

to purify compounds (bc only the charged stuff you want can bind to the stationary phase (ion exchanger) so anything else in the compound will wash away and then u can release j the bound stuff you wanted)

what is cation exchange good for

purifying amines

what is the stationary phase in ion exchange (anion exchange)

mobile anions held near cations that are covalently attached to stationary phase

anion exchange resin, what can bind?

only anions can be attracted to it (resin has + ends)

size(molecular) exclusion chromatography - what elutes first and why?

small molecules penetrate pores of particles where large molecules are excluded (can’t go thru pores) so go around

large will elute first, and smaller take longer to elute

affinity chromatography

one kind of molecule in complex mixture becomes attached to molecule that is covalently bound to stationary phase; all other molecules wash through

what can affinity chromatography be used for

way to purify proteins

how does affinity chromatography purify proteins (what molecules bind to each other)

a protein that has histidine (his6)

the resin you use in stationary phase has nickel covalently bound

1) his6 binds to Ni to all protein you want bound to resin

2) then you wash away all other particles (ex a buffer)

3) then you elute the protein out to get the purified protein (ex use excess histidine/ elution buffer)

HPLC

shorter columns, higher pressure system —> fast flow rate; good to avoid band broadening

HPLC parameters you can manipulate (7)

stationary phase composition (C18)

mobile phase composition

flow rate of mobile phase

size of stationary particle

level of packing of stationary phase

amount of stationary particle

height and width

HPLC parameter limits

pressure and time

HPLC stationary phases

C18 (mostly this), C8, SiO₂

how does C18 have compounds stick to it as a stationary phase

hydrophobic interactions, hydrogen bonds, ionic bonds

how does particle size change column efficiency

Efficiency increases with decreasing particle size of the stationary phase

lower particle size —> lower column height —> greater theoretical plates

what does a smaller particle size mean for pressure/ peak

greater pressure/thinner peak

particles that elute at a later will hav what type of peak

broader, and the ones that elute first thinner peak

reverse chromatography stationary and mobile phases

sp: c18

polar mobile: water

nonpolar mobile: methanol, acetonitrile

normal chromatography

sp: sio2

p mp: isopropanol, methanol, ethyl acetate

np mp: hexanes

isocratic and why good and bad

same mp concentration whole time; good for easy separations but can result in longer elution times; able to separate early peaks or late peaks but not both

gradient and y good

concentration varies overtime (you wld slowly add more of the mp that the plate is so if the plate it polar wld slowly add more and more ethyl acetate conc in ur mobile phase)

better separation and can still see compounds that elute later

polarity ranking low to high of normal phase chromatography

hexane, dichloromethane, isopropanol, methanol

polarity ranking low to high of reverse phase chromatography

tetrahydrofuran, acetonitrile, methanol, water

for reverse phase if you increase polar mobile what happens/?

the np molecules will stick more to the np plate so spend more time on column so acc worse off cuz more longitudinal diffusion

hplc relationship between pressure and solvent

the more viscous the solvent the higher the pressure on the system

water—> very viscous (higher p)

MeCN —> less viscous (lower p)

effect of pH

can modulate pH to change separation; pH can affect the separation and resolution of compounds when the compounds pKa is close to that of the eluent (mobile phase); mostly used in reverse phase

effect of temp

retention time (capacity factor) decreases with increased temp, greater peak efficiency with higher temp

increase temp decreases elution time and selectivity/retention

HPLC detection methods

UV, refractive index, evaporative light - scattering, fluorescence, mass spec, fourier transform infrared

AUC is proportional to what

concentration (peak needs to go to baseline to find AUC and therefore conc)

AUC (y axis) vs concentration is called what

calibration curve

peak height whats used for?

nothing it don’t matter

hplc applications: external standard

use the pure compound and make calibration curve to find amount of it in your compound

1) run formulation HPLC: crush pill, take small amount, dissolve in eluent, run on HPLC

2) generate calibration curve by using pure standard at diff concs

3) use calibration curve to determine conc and amount of acc pure standard your looking for in tablet

hplc applications: internal standard how shld u choose it

closely related in structure (same func. grp) and therefore elute close to analyte and result in similar detector response

stable

Chromatographipally resolved from analyte and impurities

hplc applications: internal standard

1) add known amount of internal standard

2) use AUC to compare concentrations (compare ratio of peaks)

DONT need to make a calibration curve both internal standard and compound you investigating on same HPLC

GC mobile phase name and examples

carrier gas: H₂ (more common), He₂(more common), N₂

GC stationary phase

SiO₂

how GC works

analyte is injected through a septum into a heated port and evaporates (vaporize)

analyte then moves thru column by carrier gas

GC requirements

molecules must be sufficiently small/volatile (so can evaporate, < 250 g)

column is very long but flow rate is very high

parameters to manipulate in GC

column temp; as you heat molecule interacts less w stationary phase bc moving faster

lower temp for GC means what for resolution

better resolution

column length in GC

generally very long, longer columns allow lower temp

column diameter in GC

smaller the better (small pore so wtv goes thru can interact w stationary phase)

flow rate in GC

high flow rates

GC detectors

flame ionization detecter (most common), mass spec

flame ionization detector

since the molecules are coming off as a gas if you introduce a flame the gas —> ions and detected by ion detector

whats a benefit of the GC detectors

don’t need UV active molecules (FID, MS) and can do with any small molecule