Resolution Modeling

for HPLC:
It’s Really Quite Simple
John W. Dolan
LC Resources Inc.

paper 230-1
presented at
Pittcon 2007, Chicago, IL

1
 Effect of Solvent Strength on Retention
and Band Spacing

tR-t0
k= t
0

“Rule of Three”

2
 Effect of %B on Retention and Selectivity

log k

%B

log(k) = log (kW) - Sφ

φ
3
 Resolution, Rs

tR2 - tR1
RS =
0.5 (wb1 + wb2 )

Rs = (3.15 - 2.95) / (0.12) = 1.7

4
 Fundamental Resolution Equation

log(k) = log (kW) - Sφ

φ α = k2/k1

RS ≈ ¼ [k/(k+1)] (α-1) N ½

150 mm, 5 µm
or N ≈ 10,000
100 mm, 3 µm

5
 Effect of %B on Retention and Selectivity

log k

%B

α - 1)(N)1/2
Rs = ¼ [k / (k + 1)] (α
6
 Window Diagram (Rs)

Resolution

1,2 2,1

40 50 60 70
%B

7
 Window Diagram (Rs)

2,3
Resolution

1,2 1,4 2,1

5,6
3,4

40 50 60 70
1 234 %B
56

8
 Window Diagram (Rs)

9
 (Minimum) Resolution Map

10
 Resolution Map

Rs

0
0 %B 100

11
 DryLab

A computer program
for optimization
of ISOCRATIC LC runs.

copyright (c) 1985 LC Resources Inc., all rights reserved

Press any key to continue

12
 N-calc
C:>?

13
 1.73
* *
* * * *
* * * *
1.30 * *
* *
Rs(10K) * *
* *
* *
0.87 * * * *
* * * * *
* * * * *
* * * * *
0.43 * * * * *
* * * * *
* * * * *
* * ** *
* * *
40 45 50 55 60 65 70
% Organic
Press any key to continue

14
15
 Initial Experiments
Isocratic

90%B, 80%B, 70% B…

(1 < k < 20)

DryLab

Isocratic Predictions

16
 Predicting Resolution
Isocratic
Retention log(k) = log (kW) - Sφ

RS ≈ ¼ [k/(k+1)] (α-1) N½
Gradient tG F
k* =
Retention Vm S ∆φ

RS ≈ ¼ [k*/(k*+1)] (α∗-1) N* ½

17
 Initial Experiments
Gradient

10 min, 20 min, 30 min…

(2 < k* < 10)

DryLab

Gradient or Isocratic
Predictions

18
 HPLC Resolution Modeling Modes

2 Input Runs 3 Input Runs

• Isocratic • Isocratic
• Gradient • Normal phase
• Temperature • pH
• (GC temperature) • Ionic strength
• Ion pairing
• ...

19
 Two-Dimensional Modeling

2 or 3 Runs per Dimension

• Isocratic / Temperature
• Gradient / Temperature
• Isocratic or Gradient / pH
• Gasoline Price / Phase of the Moon
• ...

20
 3-D Resolution Mapping
Isocratic %B – Ion Pair
30%B, 0 mM 30%B, 10 mM 30%B, 50 mM

45%B, 0 mM 45%B, 10 mM 45%B, 50 mM

21
 Isocratic %B – Ion Pair
*
30%B, 0 mM 30%B, 10 mM 30%B, 50 mM

0 2 4 6 8 10 12 14 0 2 4 6 8 10 0 2 4 6 8 10

1 overlap

3 bases 5 neutrals
2 overlaps

**

0 2 0 2 0 2
45%B, 0 mM 45%B, 10 mM 45%B, 50 mM
22
 Rs
50

40

30

20

10

mM
10 20 30 40 50 60 %B
17%, 7 mM 22%, 17 mM 33%, 25 mM
Rs = 2.46 Rs = 2.43 Rs = 2.03

0 20 40 0 10 20 0 2 4 6
23
 Summary (1)
• Simple model
– 2-point linear fit
– multipoint curve fit (up to 16 points)
• Accuracy (GIGO)
– Retention, α: ± 1-5%
– Resolution: <10%
• Flexibility
– Isocratic, gradient, ºC, pH, etc.
– 3-D mapping: 4 – 16 runs
– Design your own model
reprints: www.lcresources.com
24
 Summary (2)
• Scientific support
– Published algorithms (>20 yr)
– Dozens of articles
• Proven track record
– 20+ years commercially available
– 3 commercial products
• And…
– It’s easy & fun
– It encourages experimental discipline
– It gives you confidence in your results
reprints: www.lcresources.com
25