Quant 8. Chromatography

chromatography is a widely used method for

separation, identification, and determination of chemical components in a mixture

stationary phase

fixed in place

mobile phase

moves through stationary phase carrying analyte mixture

in any general chromatography experiment, substances in the mixture interact with the

stationary phase

types of chromatography

1. adsorption chromatography (column, TLC, etc)

2. partition chromatography (gas, liquid-liquid, etc)

3. ion-exchange chromatography

4. size-exclusion chromatography

5. affinity chromatography

adsorption chromatography

solute adsorbs on surface of stationary phase

adsorption chromatography stationary phase

solid phase (silica, alumina)

adsorption chromatography mobile phase

liquid or gas (hexanes, water, chloroform, EtOH)

What is column chromatography?

A method of chromatography where substances are introduced onto the top of a column packed with an adsorbent.

What materials are commonly used as adsorbents in column chromatography?

Silica gel or alumina.

adsorption chromatography separation method

polarity

elution

the process in which solutes are washed through a stationary phase by the movement of a mobile phase

eluate

the mobile phase that exits the column

eluent

the solvent used to carry the components of a mixture through a stationary phase

T or F: the eluent comes through the column and comes out as an elaute

T

chromatogram

the record of chromatographic separation

chromatogram x vs y axis

x: time

y: detector response

retention time

amount of time between injection and the time it takes sample to reach the detector

substance peak - t0

dead time

the time it takes an unretained species to reach the detector

unretained species peak - t0

adjusted retention time

difference between the retention time and dead time

factors that affect how long it takes a substance to reach the detector

1. actual distance between injector and detector

2. flow rate

3. identity of mobile and stationary phase

4. temperature

5. identity of substance

if the experimental conditions are kept constant, the retention time is characteristic of the substance's ___________

identity

on a chromatogram, _____________ determines concentration

peak area

chromatographic calibration curve x vs y

x: concentration

y: peak area

chromatography pros

can detect multiple analytes in the same experiment

chromatography cons

- can take a long time

signs of a good chromatography

narrow peaks and no overlap

the goal of any chromatographic experiment is to ________________ the resolution or to _____________ zone spreading

maximize, minimize

sources of band broadening

1. multiple pathways

2. longitudinal diffusion

3. mass transfer

multiple pathways

some flow paths are longer/shorter

- molecules that enter at the same time can elute at different times

longitudinal diffusion

molecules move from areas of high concentration to low concentration as mobile phase moves throughstationary phase

mass transfer

as the mobile phase moves through the stationary phase, it takes time for molecules to interact with the stationary phase

- if interaction is slow compared to flow rate, then bands broaden

resolution formula

r= 2d/(W1 + W2)

r = resolution

d = peak height

w= peak width

for better resolution, __________ peak height and _________ peak width

increase, decrease

gradient method

change conditions to maximize R as the separation

gas chromatography mobile phase

gas -volatile substances

gas chromatography stationary phase

liquid phase immobilized on the surface of an inert solid packing or on the walls of a capillary tubing

mobile phase gases for GC

1. helium

2. argon

3. nitrogen

4. hydrogen

GC column types

1. packed columns

2. capillary columns

packed columns

stainless steel or glass tubes filled with particulate packing material (adsorbent or solid phase coated on a support material)

capillary columns

thin, fused silica glass tubes with a liquid phase, adsorbent material, or chemical bonding layer attached to the fused silica surface

capillary column pros

- better separation

- narrower peaks

capillary column cons

lower sample load compared to packed columns

conditions that affect elution in GC

- heat (primary method)

- flow rate (secondary method)

- column length and diameter

- stationary phase polarity

high-performance liquid chromatography (HPLC)

most versatile elution chromatography

HPLC pros

1. sensitivity

2. adaptive for quantitative measurements

3. easy to automate

4. suitable separation for nonvolatile

5. widely applicable

HPLC mobile phase

mobile: liquid solvent (analyte must be dissolved in mobile phase)

what's the most convenient way to change the separation of your mixture in HPLC

the mobile phase (liquid solvent)

factors to consider when choosing the correct mobile phase

1. polarity

2. viscosity

3. pH

4. boiling point (be careful cause BP affects density)

5. non-corrosive

6. toxicity

7. miscibility

8. transparency with signal

9. elution strength from the stationary phase

acetonitrile

used to clean HPLC after an experiment in case the analyte is stuck

normal phase

polar stationary (silica gel, alumina)

nonpolar mobile (hexane, DCE, DCM)

reverse phase

nonpolar stationary (C8, C18, C60)

polar mobile (water, MeOH, acetonitrile)

advantage of reverse phase

water is cheap, UV-transparent, and nontoxic

types of liquid chromatography

1. partition (liquid-liquid)

2. adsorption (liquid-solid, TLC)

3. ion-exchange

4. size exclusion

5. affinity

6. chiral

DEAE cellulose

anion- exchange

CM cellulose

cation exchange

ion exchange mobile phase

liquid solvent

how to elute molecules stuck on ion exchange chromatography

- change pH

- increase salt concentration (ionic strength)

if the mobile phase is too ______, band broadening occurs due to mass transfer

fast