Optimization of HPLC separations:

(bring your laptop)

1.
2.

Explore a virtual HPLC and optimize separations

Stationary-phase design

III. Band broadening in LC

1. Create your own virtual chromatograms

Use of Excel 2010 is recommended, allow macro
The program incorporates effects of:
Van Deemter coefficients of packed beds in isocratic mode
Molecular diffusion (Dm depends on temperature)
Viscosity of the mobile phase ( depends on temperature)
Assumptions: ideal situation
No peak tailing
No bad columns or column-to-column variation
All components display the same influence of temperature on retention factors

Starting conditions:
Isocratic separation
Maximum pressure is 400 bar
Column temperature is 30C
250 mm x 4.6 mm column packed with 3.5 m reversed-phase (C18) silica particles
Sample mixture contains 3 pairs of closely-eluting compounds (critical pairs)

III. Band broadening in LC

1.1. Reference separation

Critical pair: 2 closest eluting compounds (in terms of resolution)

III. Band broadening in LC

1.2. Output parameters

Resolution (Rs)
Measure that defines how well two closely retained peaks are separated
Baseline resolution obtain if Rs > 1.5
Rs

t R,2 t R,1
(w b,1 w b,2 ) / 2

Plate height (H)

In analogy to a distillation process
H = column length (L) / Number of plates (N)
The lower H the better

t
N R
t

t
16 R
WB

III. Band broadening in LC

1.3. Improved separation

III. Band broadening in LC

1.4. Exercise 1: effect of flow rate on efficiency

Study the effect of flow rate (pressure) on the separation
Reference system: column packed with 3.5 um particles
Install a stronger pump and increase the pressure up to 1000 bar
Keep all other parameter the same
Determine the following parameters at 400 and 1000 bar:
The mobile-phase velocity (u0)
The retention factor (k) for the first critical pair
The selectivity () for the first critical pair
The plate number (N) for the first critical pair
The plate height (H) for the first critical pair
The resolution (Rs) for the first critical pair
Are we improving the separation or not?

Explain the effects of u0 on H and Rs

III. Band broadening in LC

1.5. Exercise 2: Effects of column length and particle size on efficiency

Speed-up the reference separation while keeping the critical-pair baseline separated by
adjusting:
The pressure drop
The column length
The particle size
How do these parameters influence the separation?

III. Band broadening in LC

1.6. Exercise 3: Effects of column temperature

Use the reference system and investigate the effect of column temperature on retention
and efficiency
Explain which parameters can be changed to achieve:
The highest separation efficiency?
The fastest possible critical-pair separation?