Anna Horgan Lectures

Define chromatography

physical method of seperation in which the components are distributed between TWO phases

what’s the mobile phase

a liquid which percolates through the

what’s the stationary phase?

column packing material

what’s the Rf value

retardation factor

distance travlled by component/distance travelled by solvent

describe the solvent set up in HPLC

• degassing

• dust removal

• several reservoirs

  • gradient/isocratic

descrive gradient elution

change in solvent composition

• may be contanstly changing or changing in a stepwise manner

why is degassing needed

• avoids band spreading

  • avoids the detector detecting gas bubbesw

why is dust removal needed

• protects the column from clogging

• protects the pump

• detection interference

  • band spreading

what are the solvents used for reversed phase lc

water

methanol

polar stuff

describe the pump in hplc

• constant rate

  • resistant to corrosion

describe the pressures in pumps

• normally 400-600bar

• 6000psi-9000psi

in ultra high pressure systems = 1300bar

describe two types of injectors

1. autosampler (multiple samples)

2. manual

describe the physical needs of an injector

must be resistaant to high pressures of the liquid

describe the column

• small particles inside cause backpressure

  • can be made of stainless steel/heavy wall glasss/peek tubing

how is the column protected

guard column remives particular matter

when can heavy wall glass be used

pressure below 600psi

whata is column packing made up of

alumina/silica/divinylbenzene synthetic/ ion echange resin

what does a detector do

quantative

qualatative analysis

whats the most used detetctor

uv/ visable light detector

what is the most senstive lc detetctor

fluorescence detectorwh

what’s the issue w/ fluorescence detectors

Fluorescent derivatives must be synthesized

what detector is used for lipids/proteins/polymers

refractive index

why is refractive index used for polymers

non uv absorbing

what’s the partitioning co-eff

the partitioning relationship a solute has between teh stat and mobile phase expressed as a ratio

K= Cs/Cm (the solid Cork floats on the liquid)

why do solutes have a different eluting time

each solute has a different solubility due to the stationary phase

whats dead time

time between injection point and dead point

what’s dead volume

vol of mobile phase passed through the column between injection point and dead point

what’s the retention time

time between injection point and peak MAXIMUM

what’s corrected retention volume

vol. of mobile phase passed through the column between dead point and peak maximum

(as opposed to the injection point)4444

whats the capacity factor

k’= (tr-t0) /t0

what’s t0

dead time

what happens to k’ if a compoment spends longer in the column

increased capacity factor

what’s the selectivity (a)

relative retention of two compouds

a= k’2/k’1

the larger k’ will be on the top (gay fanfic laws)

in gc what is the mobile phase

a carrier gas

what is the stat phase in GC

the liquid film that coats the column filling (packed tubular)

or

the liquid film that coats the column walls (open/ capillary columns)

what happens at the GC inlet

injection point

• the sample solution is vapourised amd taken to the column by the carrier gas

what are the sepating forces in GC

1. temperature (boiling points)

   1. stationary phase interactions

how does seperation based on temp happen in GC

the capillary column is in an oven

oven temp increases (gradually! also called ramped)

lower b.p. components elute quicker

what happens at the base line of a chromatogram

that is the part of the chromatogram where only the mobile phase is emerging from the column (analytes stuck in the column due to interactions)

how to calculate flow rate

dead volume= flow rate * dead time

retention volume= flow rate* retention time

what do sharp, symmetrical peaks represent

a good seperation

what’s column efficiency

a measure of how good the stat phase is able to seperate components

what are theoretical plates

a measure of how efficient the column is

what’s plate height

HEPT (height eq. to one plate) = lenght of column/ no. of theoretical plates

(how wide the plate is, smaller the plate the more specific seperation)

give the equation for Neff

5.54( tr/ FWHM)²

give the van deemter eq and describe what it does

height eq of one plate= A + B / u+ Cu

describes the factors that impact peak broadeining (what things will make the column less efficient)

what’s eddy diffusion

the column is packed therefore each molecule will take a different path towards the end of the column

different path will take different amounts of time

what’s longitudal diffusion

the conc. of the analyte is the highest in the centre of the column

diffusion to the edges takes time

how to decrease longitudal diff

increase the velocity of the mobile phase

→ decreasing the time spent in column decreases the longitudal diffusion

what’s resistance to mass transfer

the analyte with a strong affinity to the stationary face will stay behind while the analyte that is in the mobile phase will zoom past it

this broadens the band especially badly when the velocity of the mobile phase is high

what causes resistance to mass transfer

the analyte takes time to equilibrate between mobile and stat phase

draw a van deemter plot

the optimum velocity is the x variable of the min point

which term in the equasion is independent of velocity

eddy diffusion

random paths will occur at different speeds

describe what happens to longitudal diffusion as velocity increases

decreases (flaccid graph line thing )

describe what happens to resistance to mass transfer as velocity increases

increases

gets worse as the mobile phase zooooms past the analyte that is has a high affinity to the stat. phase

which type of gc column has a lower hept

open tubular as no eddy diffusion happens

list all the factors that impact gc seperations

1. volatility of a compound

2. polarity of compouns

3. polarity of column

4. column temperature

5. flow rate of the gas

6. the leght of the column

A has a b.p. of 55C

B has a b.p. of 200C

Which travels faster

A, the compounds with a lower b.p. will travel faster

A is polar

B is non polar

Which travels faster

the polar will travel more slowly , especially when the column is polar as well (like attarcts like)

column a is set to 50 C

column b is set to 500 C

which tarvels faster

The column with a higher temperature will have the components move faster

What gases are used as carrier gases

Argon

Helium

Nitrogen

Hydrogen

What affects the choice of the carrier gas

1. Sample type

2. sample matrix

3. detector used

4. safety

5. purity

6. price

7. availibity

What does a carrier gas must be

inert

pure

compatible with detector used

in GC, where is the inlet attached

column head

how is the analyte injected

microsyringe through a rubber septum

whta happens to the analyte when injected

flash vapourisation

how is the injector temperature chosen

needs to volatilize the sample

cant decompose it

50C above b.p, of the less volatile compound

what are the types of open tubular columns

wcot

plot scot

whats plot

capilarry column with a porous layer of stationary phase

whats scot

support coated ot column

whats wcot

wall coated column ( walls coated w/ liquid stat phase)

what are the types of packed gc columnns

gls—> more common

gsc

what are packed gc columns made of

outside—> stainless steel/ glass(when it needs to be an inert column)

inside—> inert support is tighly packed

the support is coated with a thin film of liquid (3%-10% of stat phase)

what is the support made up of in the packed column

diatomaceus earth

what are the disadv. of packed columns

limited resolution (N<80.000)

long lenghts are impractical PRESSURE DROP

eddy diffusion

What are OT capillary columns made up of

out—> fused silica (inert)

in—> open tube, low resistance to flow, can get loooong lenghts (L>100m)

how is the stationary phase arranged in OT

thin , uniform liquid film that coats the wall fo the tubing

which type of column in gc is

1. more common

2. higher res

1. open

2. open tubular

What is the res of the OT

open tubing has 100,000= N

what type of column shpuld be used to analyse polar compounds

polar columns

what is the stat. phase in a polar column

CArbowax 20M (composed of polyethylene glycol stat phase)

what column should be used for non poalr compounds

non polar stat phase

—> CP Sil 5CB

dimethylpolysiloxane

why can’t the temperature be too high

high temp= fast elution time= less interaction= less seperation= broad peaks

what does the temp @ injector influence

the vapourisation of the sample

what does the temp @ column influence

the retetention time

the resolution

how is the detector selected

"based on sample matrix= everything that is present in the typical sample except for the analytes of interest

the sensitivity

what are the perfect gc detector properties

• stability

• reproducibility

• linear response to many orders of magnitude

• similar response to all analytes

• temp. range from room temp to 400 C

• non destructive

• easy to use

  • short response independent of flow rate

how can we classify detectors

1. bulk property / solute property

2. destructive/ non destructive

3. conc. sensitive/ mass sensitive

4. specific / non-specific

describe bulk property / solute property

bulk= measures bulk phys property of eluent

solute= chem/phys properties unique to solute

give example of bulk property

dielectric constant

refractive index

give solute propertie example

heat of combustion

fluroresnence

describe specific detectors

higher sensitivities

only respond to selected substances

eg nitrogen phosphorous detector detects N or P

what are the advantages of GC

1. fast

2. small samples only needed (microliters/grams)high resolution

3. reliable

4. cheap

5. non-destructive

6. sensitive (ppm/ppb )

7. complex samples in trace amounts

8. organic materials caqn be anylised

disadv. of gc

olatile only (temp. max =380, analyte must have a bp below 500)

1. cant use w/ thermally labile samples

2. samples need prep sometumes

3. samples cant react w/ column

4. samples must be soluble

5. requires spectroscopy (MS)

6. samples must be stable below 400C

7. most common detectors are unselective→ less sensitiv

what causes the rate of the elution

the equilibrium constants of the components in the stationary and mobile phase

K= Cs/Cm (cork floats on water)

give the formula for selectivity

a= k1/k2 the larger on top

what causes peak broadening

• dispersion of analyte in dead volume, connection between injector and column and between the column and detector (need to min dead volume)

• column broadening - van demteer

  broadening doesnt occur onky in the column!

what temp. is used for LC and why is that an advantage

room temp.

heat sensitive compounds can be analysed

what is seperation optimasation and where can it be done

LC- seperation is based on the interaction of solutes with the mobile/stat phase so you can tweak either

GC seperation is set on bioling point- cant change that

what can you seperate using

1. gc

2. lc

1. volatile compounds and gaseous mixtures

2. soluble compounds (aa, proteins, drugs, polymers, nucleic acids)