Chromatography

Question 1

What do the terms Stationary Phase and Mobile Phase refer to?

Answer 1

Stationary Phase: Where the separation happens.
Mobile Phase: move the sample through the stationary phase - may also interact with the sample.

Question 2

What is a chromatogram?

Answer 2

The graph of detection versus the retention time - peak area.

Question 3

Define Eluent, Elution, Eluate and Elution Order

Answer 3

Eluent: The mobile phase
Elution: The analyte coming off the column
Eluate: The fluid in the column.
Elution Order: The order in which things come off the column.

Question 4

what is the relative polarity of the stationary phase compared to the mobile phase in reverse phase?

Answer 4

Stationary: non-polar or weakly polar
Mobile: more polar

Question 5

What is the relative polarity of the stationary phase compared to the mobile phase in normal phase?

Answer 5

Stationary: polar - solvent less polar
Mobile: a more polar solvent

Question 6

what is the basis for normal phase and reverse phase separations?

Answer 6

Normal: compounds are attracted to, or not, the stationary phase or mobile phase based on the polarity.
Stationary: rely on differences in the hydrophobicity / polarity for separations - most common mode of HPLC.

Question 7

What is ion exchange chromatography?

Answer 7

• Analytes are removed by flushing with mobile phase OR by changing the composition of the mobile phase.
• Stationary phase: anions or cations are covalently attached to the stationary phase - usually resin.
• Charge of the resin-bound ions balanced by weakly bound counterions such as H+ (Cl-)
• Mobile phase: liquid
• Ions are separated based on differences in the strength with which they bind with resin-bound.

Question 8

What is size exclusion chromatography?

Answer 8

stationary phase: porous gel
mobile phase: liquid or gas
separates molecules by size, with larger solutes passing through most quickly.
- too large molecules: enter pores stream past the porous gel coating or particles and exit the column rapidly
- small molecules: enter the pores, sheltered from mobile phase flowing around gel particles (elute more slowly)

Question 9

how does size exclusion chromatography differ from gel permeation chromatography?

Answer 9

Size exclusion: uses water traditionally.
Gel permeation: uses organic solvents traditionally.

Question 10

what is the simple schematic of an HPLC

Answer 10

solvent (mobile phase) –> pump –> injector (autosampler) –> HPLC column (packing material) –> detector –> chromatogram

Question 11

what is the difference between binary and quarternary pumping?

Answer 11

Binary: two pumps - can only mix 2 solvents at a time.
Quarternary: one pump - can mix up to 4 solvents at a time (proportioning valve)

Question 12

what is an isocratic method? gradient?

Answer 12

Isocratic: can stay unchanged during entire run - simpler
Gradient: can change composition with time - can help to analyze a sample with very different compounds.

Question 13

what are the two types of injectors for an HPLC

Answer 13

Manual Injectors: introduce the sample to the instrument.
Autosampler: can accurately partially fill a loop (more volume options)

Question 14

what are the criteria for a carrier gas?

Answer 14

it must be pure and non reactive - to eliminate ghost peaks/contamination.

Question 15

what are common carrier gases?

Answer 15

He, H2, N2, Ar

Question 16

what is the role of a septa in the injector?
what happens when it fails?

Answer 16

Septa: acts as a barrier between the GC and the outside world.
Fails: loss of analyte and contamination.

Question 17

what temperature range relative to the analyte do we aim for? why?

Answer 17

heated to >50 C above the bp of the least volatile component of the sample (if known) - to ensure quick vaporization.

Question 18

What phases can we inject into GC?

Answer 18

Gas

Question 19

why do we want fast injection?

Answer 19

means the sample will enter the column as a tight band.
if too slow or too big of a sample, it will result in band spread and decreased resolution.

Question 20

what are the two types of columns available?
compare them.

Answer 20

Packed column: glass or stainless steel tubes packed with a solid material that has the liquid adsorbed to the surface.
Capillary column: open tubes - fused silica with an adsorbed liquid layer (stationary) on the inner surface (outer surface coated in polymer to reduce fragility)

Question 21

What must the analyte have or do in relation to the stationary phase for retention?

Answer 21

analyte must have some compatibility / solubility with the stationary phase.
like dissolves like (polarity)

Question 22

What is an FID? what does it detect? what are the advantages?

Answer 22

“Flame Ionization Detector”
The current is proportional to the amount of carbon entering the flame, which is proportional to the total analyte amount.
Advantages: high sensitivity
low noise
rugged and easy to use

Question 23

what other detectors exist?

Answer 23

Mass Spectrometry
Thermal Conductivity