PChem Exam 3

what are the separation methods

solvent extraction

chromatography

spectroscopy

types of chromatography

HPLC

gas chromatography

mass spectrometry

thin layer chromatography

how to calculate the partition coefficient

K=C1/C2

K=g compounds per mL of organic solvent/water

how to calculate the fraction remaining in water

final/initial = v2/(v2+v1K)^n

v1= vol of organic solvent

v2= og vol of water

how to make solvent extraction more efficent

do more extractions (bigger n)

more solvent comes out with each extraction

what molecules go into the aqueous layer

charged species, anything ionized

ex: water

what molecules go into the organic layer

neutral species, not ionized

ex: ethyl

to extract an acid into water, pH must be

higher than pKa

to extract a base into water, pH must be

lower than pKa

pH

becomes ionized (charged)

gains H

pH>pKa

does not become ionized

loses H

what does NaOH do

deprotonates

takes away H

usually from OH

this makes it charged so it goes into the aqueous layer

what does HCl do

protonates

adds an H

usually on NH2s (amines)

this makes it charged so it goes into the aqueous layer

what can HCl not protonate

amides (NH)

extraction with a metal chelator (aqueous and organic layers)

metal chelators are weak acids

aqueous phase: metal ion (M^n+)

organic phase: metal ligand (MLn)

extraction with a metal chelator what can occur

modifying pH can bering a metal into either phase

if the pH is high, it will be extracted into the organic

if pH is low, it will remain in aqueous

what is chromatographic separation based on

polarity

functional group

size

boiling point

what is a stationary phase and common examples

immobile phase that compound and mobile phase moves over

SiO2, C8, C18

what is a mobile phase and common examples

a solvent or gas that moves over the stationary phase carrying the compound of interest

water/methanol, hexane/isopropanol

if the stationary phase is SiO2, how do molecules move

SiO2 is very polar

polar molecules interact with SiO2 so those will moves slower

parameters of chromatography that can be manipulated

stationary/mobile phase composition

flow rate of mobile phase

size of stationary particle

level of packing and amount of stationary phase

height and width of column

what is band broadening and why do we want to avoid it

when peaks become thick and wide

more broad = less efficient column

things that can cause band broadening (refers to what occurs on the graph)

longitudinal diffusion (refers to the movement)

long retention time (too long on column)

particle size (bigger particle makes it hard to move through the column)

flow rate (too faster causes less separation)

injection time

column length (longer column inc retention time)

eddy diffusion (different paths taken through column, some may be longer than others, effects retention time)

amount of compound (more compound more broadening)

poor set up

ways to reduce longitudinal diffusion

faster flow rate

shorter column

smaller particles

open tubular columns

column is lined with the stationary phase which results in

high resolution

increased sensitivity

short analysis times

small sample capacity

packed columns

column is packed with tiny particles lined with stationary phase which leads to

low resistance

high flow rate

can make column longer to give more plates which increases efficiency

ideal resolution and what can effect it

>1.5

can be effected by

column length (longer column better resolution but not too long are can cause too much band broadening)

stationary phase

mobile phase

temperature GC

ideal asymmetry factor

AF=b/a=1

causes for fronting (AF less than 1)

overloading (too much compound, interactions are limited)

poor trapping (molecule doesn't interact well with solid phase)

injection solvent too strong (HPLC)

causes for tailing (AF greater than 1)

absorption to column (binds too well, hard to get off)

too much dead volume

ion exchange chromatography

separates based on the charge

stationary phase can be cationic/anionic

if made up of cations (+), anions (-) will be attracted and + ions pass through

anion exchange resin in ion exchange chromatography

only anions are attracted to it

molecular/size exclusion chromatography

separates based on size

small molecules go through pores (slow)

large molecules are excluded and go around (fast)

affinity chromatography

separates based on affinity

stationary phase is made up of things that attract other things (ligand)

attracts what has an affinity to it (ligand of interest)

if not attracted, then molecule will just wash through

area under the curve (AUC) is

proportional to the concentration

peaks require both

AUC and calibration curve

** peak must go back down to baseline for an accurate AUC

thin layer chromatography

separates by polarity to identify compounds by comparing to standards

stationary phase: SiO2 (polar)

mobile phase: hexane (nonpolar) /ethyl acetate (polar)

how do molecules travel on thin layer chromatography

stationary phase is POLAR

NONpolar molecules travel farther (top)

POLAR molecules do not travel as much (bottom)

silica holds polar closer, nonpolar doesnt interact and goes up

what happens to molecule if there is more hexane (NP) than ethyl acetate in the solvent (mobile phase)

hexane is nonpolar

more nonpolar, less interactions with silica, moves uo

ex: 30% hexane, 50% hexane, 70% hexane

70% hexane will have spots that are furthest down compared to 30% hexane which is closer to silica

what would you do if you want to increase the separation of a TLC plate

increase NONpolar solvent (hexanes) only when using SiO2 as a stationary

nonpolar will separate more bc it doesn't interact with SIO2

what is HPLC

High Performance Liquid Chromatography

form of column chromatography

pumps a sample mixture or analyte in a solvent (mobile phase) at high pressure through a column with chromatographic packing material (stationary phase)

parameters that can be manipulated in HPLC

stationary/mobile phase composition

flow rate of mobile phase

size of stationary particle

level of packing and amount of stationary phase

height and width of column

what limits HPLC

pressure and time

if you decrease particle size, what happens

efficiency increases

column/plate height decreases

plate number increases

pressure increases

why do you want a smaller particle size

allows for more interaction

it gives the sample more opportunity to bump into the particles to go through the stationary or mobile phase

what are common polar phases

amino, cyano, diol

what are common nonpolar phases

C18, C8, C4, phenyl

what are some stationary phase compositions

C18, C8, SiO2

what do you need for a mobile phase

2 solvents with differing polarities

(nonpolar and polar)

reverse phase (stationary phase)

C18

reverse phase (mobile phase)

polar: water

nonpolar: methanol, acetonitrile

normal phase (mobile phase)

polar: methanol, isopropanol

nonpolar: hexane

normal phase (stationary phase)

SiO2

isocratic elution does what to concentration

holds concentration constant (same throughout)

what is isocratic elution used for

used to separate early OR late peaks

cons of isocratic elution

easy separations can cause long elution times

gradient elution does what to concentration

varies the concentration by changing the mobile phase solvents/gradients

what is gradient elution used for

used to separate BOTH early AND late peaks

what is the relationship between pressure and solvent

the more viscous (thick) the solvent is the higher the pressure on the system

water vs MeCN viscosity

water is more viscous than MeCN so it causes more pressure

MeCN is less viscous than water so it causes less pressure

compound on a C18 column (reverse or normal phase)

reverse phase

how would compounds elute on a C18 column with water/methanol as a mobile phase

mobile phase is water/methanol which is nonpolar

most POLAR molecule will elute FIRST because it has the most interactions with the NONpolar mobile phase

how would you increase the resolution of separation on a C18 column with water/methanol as a mobile phase

normal phase

decrease particle size to increase efficiency

increase water concentration

(C18 is nonpolar, water is polar, more separation of polar compounds)

how would you increase the resolution of separation on a SiO2 column with hexane/isopropanol as a mobile phase

reverse phase

decrease particle size to increase efficiency

increase hexane concentration

(SiO2 is polar, hexane is nonpolar, more separation of nonpolar compounds)

why would you increase the water concentration to increase resolution of separation on a C18 column with water/methanol as a mobile phase

bad resolution or separation occurs due to fast retention time

if you decrease the retention time, the compounds would have more time to interact with the mobile phase

mobile phase is water/methanol which is nonpolar

if you increase water, you decrease polarity which allows the compounds to interact more with the nonpolar molecules since there isn't as many of them

this causes a longer retention time which results in better separation

what would happen if you increase the methanol concentration instead

too much nonpolar compounds to react which make the retention time super fast which results in bad resolution and low separation

compound on a SiO2 column (reverse or normal phase)

normal phase

how would compounds elute on a SiO2 column with hexane/isopropanol as a mobile phase

mobile phase is hexane/isopropanol which is polar

most NONpolar molecule will elute FIRST because it has the most interactions with the POLAR mobile phase

what moves faster in a reverse phase

polar moves faster

what moves faster in a normal phase

nonpolar moves faster

how does pH effect the eluent

effects retention time, separation, and resolution

how can you manipulate the pH of the eluent

used in reverse phase

uses buffers (salt mixtures) at various pH to change composition

when pH is near the pKa of the compound what happens to resolution/separation

compounds do not have bad resolution because they do not move or elute as much

when pH is not close to the pKa of the compound what happens to resolution/separation

compounds have better separation because they move/elute more

how does temperature effect HPLC

if you increase temperature

capacity factor decreases

retention time decrease

peak efficiency increase

elution time decreases

selectivity decreases

what is a molecular ion

M+ or M+H

doesn't always have to be the largest peak, sometimes does not show up

components of an ideal detector

sensitive and selective

universal

provide linear response

not broaden peaks

application of HPLC with an external standard

1. run chromatograms with varying concentrations of the analyte (crush a pill and run it on HPLC)

2. generate a calibration curve to determine concentration and amount (use pure standards at different concentrations)

3. run the sample to determine the concentration and compare to standard

**essentially comparing the standard to your unknown to determine what you have and how much you have

application of HPLC with an internal standard

choose an internal standard that is

close in structure to the elute (creates a similar detector response)

stable and pure

make sure you have a known amount

then you run it through a chromatograph and use the area under the curve and compare concentrations

**inject a similar compound (betamethasone) to the compound (hydrocortisone) to determine how much you have

requirements of gas chromatography

molecules must be small and volatile

column needs to be long BUT

flow rate needs to be high

mobile phase of gas chromatography

carrier gases such as He, N2, H2

stationary phase of gas chromatography

SiO2

how does gas chromatography work

analyte is injected into a heart port where it evaporates and then moves through the column by a carrier gas (He, N2, H2)

parameters of capillary gas chromatography

carrier gas flow (needs to be high)

carrier gas type (He, H2, N2)

column temp (lower temp for better resolution)

column length (longer length for lower temp for better resolution)

column diameter (smaller is better)

types of gas chromatography detectors

flame ionization

mass spectrometry

types of mass spectrometry

magnetic sector

single/triple quadrupole

time of flight (TOF)

ion trap

magnetic sector

ions accelerated by magnet where only certain ions can get through (depends on voltage)

small goes up (top)

heavy goes down (bot)

single quadruple

ions that resonate with the frequency (radio wave) applied can get through

**certain freq give certain ion

triple quadruple

uses 2 quadrupoles that can select for mass (thus 2 mass separations)

hits ions with collision gas which causes FRAGMENTATION

time of flight (TOF)

ions get accelerated

small ions travel faster but HIGH ENERGY travels first

reflectron opposes direction of high energy ion delaying the time (why small ions reach detector first even though high energy travels first)

VERY SENSITIVE

ion trap

ions injected into a trap and start oscillating at a certain frequency

VERY SENSITIVE

what happens when you combine TOF and ion trap

since both are very sensitive, you can get up to 4 decimal places which helps determine molecular formula

ionization methods

electron impact

chemical ionization

electron spray ionization

atomic pressure chemical ionization

MALDI

DART?

electron impact

molecule is hit with very high energy

CAUSES FRAGMENTATION

chemical ionization

ionizes molecule with methane (a chemical instead of energy like electron impact)

similar to electron impact just not as high energy

lets you see parent ions molecular weight

electronspray ionization (ESI)

+ current

- ions attracted

+ions pass through

atomic pressure chemical ionization

used for nonpolar molecules

eluent passes through heated tube which forms an aerosol in which voltage is applied to generate ions

CAUSES FRAGMENTATION

MALDI (matrix associated laser deposition ionization)

used for large molecules (proteins or polymers)

sample gets blasted with laser where ion forms

DART (direct analysis in real time)

plasma contains either + or - and neutral atoms which water with water and become charged

types of HPLC detectors

ultraviolet

refractive index

evaporative light-scattering

fluorescence

mass spectrometry

fourier transform infrared