Chromatography final

What is SEC?

Size exclusion chromatography

Uses aqueous solvents

Often with biomolecules

What is GPC?

gel permeation chromatography

Uses organic solvents

Used with polymers

SEC and GPC separation mechanism

Stationary phase beads with pores, smaller molecules interact more in and out of pores = longer retention

Axis in SEC

y - MW

X - normalized retention volume (Vr/VC)

Axis in GPC

Y - MW

X - elution volume

Axis in reverse phase

Y - peak area

X - concentration

Used for quantification

What is a calibration curve in SEC/GPC used for

Determine relative molecular weight

4 types of ion chromatography

Ion exchange

Ion exclusion

Ion pair

Ion suppression

What is ion chromatography?

Uses a charged stationary phase to create separations

Ion Exchange Mechanism

Charged stationary phase, compounds are repelled or attracted

weak ion exchangers

Ionizable, 2 pH units below for Bases, 2 units above PKA for acid

Strong ion exchangers

Permanent charge, ex. Phosphate, quaternary amines

Cation exchanger charge

Negative, binds positive

Anion exchanger charge

Positive, binds negative

Weak Cation Exchanger example

carboxymethyl

Strong cation exchanger example

methyl sulphonate

Weak anion exchanger example

Diethylaminoethyl

Strong anion exchanger example

quaternary ammonium

How can pH impact ion exchange separations

Charge on analytes and stationary phase

Both must be charged

How do we cause elution in ion exchange separations?

Use high concentration of ions to displace analyte

Salt introduced as gradient/stepwise

Or changing pH, changing prolongation state of resin or analyte

ion exclusion chromatography

Use for small weak acids/bases and hydrophilic species

Does not require gradient

Strong anion or cation resin

Ion exclusions separation based on?

Size, shape and charge

How does ion exclusion chromatography work?

Same charges repelled, small neutral molecules can go into and out of pores in resin beads

Analytes analyzed by ion exclusion

Carboxylic acids, ammonia, carbohydrates

affinity chromatography

Separates based on proteins binding affinity to ligands on stationary phase

What is affinity chromatography used for?

Biological applications, don't break down proteins

Key characteristics of a carrier gas

Mobile phase

Very pure

Non-reactive/inert (He, H2, N2, Ar)

Little impact on selectivity

Does not interact with analytes(just carries them)

Interactions with the column define the separation

What is the impact of changing the carrier gas o.n one's chromatography?

Faster flow rate

Effect of carrier gas on flow rates

Each gas has optimum flow rate for particular column ID (N2

2 main types of GC columns

Capillary and packed

More efficient GC column

Capillary much longer

Capillary Column (GC)

Long narrow columns made of fused silica

GC column with higher capacity

Packed

Impact of inner diameter on separations

Decreasing = improved resolution because increase interactions between solute and stationary phase

Impact of column length on resolution

Increase column = increase resolution but longer time because more partitioning between stationary and mobile phase

Impact of stationary phase thickness on resolution?

Thicker = Longer retention = better resolution

Packed Column (GC)

Filled with fine particles of solid support coated with a nonvolatile liquid stationary phase, or the solid itself may be the stationary phase

Broader peaks = less resolution

Longer retention times

Packed columns used for?

Preparative separations

Separations of poorly retained gases

Desirable qualities for GC stationary phase

High boiling point

Thermally stable

Inert

How to choose GC stationary phase

Match polarity of sample with stationary phase

Two things that determine retention time on GC?

Volatility and polarity

Impact of volatility on GC

Lower boiling point=more molecules are a vapour=faster elution

If two compounds have the same boiling point what will determine their retention time?

Polarity

If two compounds have the same attraction to a column what will determine their retention time?

Volatility

What is isothermic separation?

Unchanging temperature from start to finish

Benefits of isothermic separation

Faster and simpler, better reproducibility, less stress on instrument, ideal for volatile compounds

What is temperature programming?

Gradually changing temperature

Why do we do temperature programming

All compounds are eluted and peak separation is fairly uniform

Isothermal method would be equivalent to what type of LC method

Isocratic