Chromatography Theory II

Question 1

Plate number, N
(effective plate number. Neff)

Answer 1

The number of times a solute partitions between the two phases during its passage through the column

Each plate represents an equilibrium of the analyte between the two phases (MP and SP)

Question 2

Plate height

Answer 2

Used as a measure of column efficiency
- the smaller the plate height the more equilibrium steps can ‘fit’ in a column
- is constant when SP and column diameter are constant

Question 3

What do more efficient separations have?

Answer 3

• Smaller plate heights
• Small H values
• Larger number of theoretical plates
• Large N values

Question 4

Selectivity (a)

Answer 4

Relative retention of two solutes (where solute A elutes before solute B)

Question 5

Band broadening

Answer 5

A general term used to describe the overall dispersion (widening) of a sample peak as it passes through a separation system

In chromatography
- several causes (diffusion processes, transfer of solutes between MP/SP)
- goal is to relate width of analyte peak to chromatographic system parameters
- understanding how parameters affect band-broadening allows for better control of the separation

Question 6

What are some factors that effect band broadening?

Answer 6

Diffusion
Fluid flow
Equilibrium between SP/MP

Effects of these factors are additive
In CT theory the width of a peak is described in terms of theoretical plate height

Question 7

van Deemter equation

Answer 7

Conveys how the column and flow rate affect the plate height

Question 8

Eddy diffusion

Answer 8

‘A’ term in van Deemter equation

Some solute molecules move through column near the column walls where the density of packing material is relatively low
Other solute molecules pass through tightly packed centre of column with a more indirect path

Solutes with shorter paths elute before longer (more erratic) paths

Question 9

How to reduce Eddy diffusion

Answer 9

• Use exceedingly regular particles
• Use smaller particles
• Use a thinner bed
• If able, change to capillary column

Question 10

Longitudinal diffusion

Answer 10

‘B’ term in van Deemter equation

As the mobile phase moves through the column solute molecules diffuse in all directions (thin band first injected will broaden by diffusing outward while travelling through the column)

Question 11

How to reduce Longitudinal diffusion

Answer 11

• Use a thinner bed
• Use a capillary column
• Use higher flow rates

Only to a certain point, as there are other band broadening processes (Cm, Cs) we must be aware of

Question 12

Resistance to mass transfer

Answer 12

‘C’ term in van Deemter equation

Most important factor in GC and LC
Arises because the flow of MP disrupts the equilibrium distribution of analyte between the two phases

Sum of Cs (mass transfer in SP) and Cm (mass transfer in MP)

Question 13

What is Eddy diffusion proportional to?

Answer 13

Packing factor (tighter the better) and particle diameter (larger diameters = larger routes)

Question 14

What is Longitudinal diffusion proportional to?

Answer 14

Obstruction factor (accounts for influence of packing structure) and diffusion coefficient of solute in mobile phase (much larger for gases)

Question 15

What is Longitudinal diffusion inversely proportional to?

Answer 15

Mobile phase velocity
- most important contribution to band broadening (plate height) is longitudinal diffusion at low MP velocities

Question 16

How to reduce Cm contribution?

Answer 16

Reduce particle size (LC)
Reduce column diameter (GC)

Question 17

How to reduce Cs contribution?

Answer 17

Reduce thickness of SP
- reduces potential for band broadening by lowering sample capacity
- too high sample capacity = peak fronting

Question 18

Other important criteria in LC and GC

Answer 18

• minimum in curve represents highest efficiency and smallest plate height
• want to carry out separation at optimum velocity determined from min. of curve
• consideration should be given to working at slightly higher velocity to reduce analysis time depending on curve shape
• note for GC that for faster analysis time the main cause of band broadening is mass transfer in the mobile phase

Question 19

Why is LC more common than GC?
Why is GC chosen?

Answer 19

LC more common because most compounds are not sufficiently volatile for GC (too big or degrade with heat)

GC is less expensive, has better detectors, and generates less waste