Download Method Development Guide v4 December2005 FEW

TABLE OF CONTENTS
1
Introduction................................................................................................. 5
2
General information .................................................................................... 10
3
2.1
Explanation of Figures ............................................................................ 10
2.2
Techniques ........................................................................................... 10
2.2.1
1D Off-line analysis .......................................................................... 10
2.2.2
2D Analysis ..................................................................................... 11
2.2.3
Bidirectional analysis ........................................................................ 13
2.2.4
Gradient elution (step and continuous) ................................................ 14
2.2.5
Infusion OPLC.................................................................................. 15
2.2.6
Over-running .................................................................................. 16
2.2.7
Multiple Development ....................................................................... 17
2.2.8
Preparative Isolation/Flash Chromatography ......................................... 18
Getting started........................................................................................... 22
3.1
4
The stationary phase.............................................................................. 22
3.1.1
Choosing the stationary phase............................................................ 22
3.1.2
Capacity Guidelines .......................................................................... 26
3.1.3
Handling......................................................................................... 27
3.1.4
Activation ....................................................................................... 28
3.1.5
Prewashing ..................................................................................... 29
3.1.6
Preconditioning ................................................................................ 29
3.1.7
Presaturation .................................................................................. 29
3.2
Sample Application ................................................................................ 30
3.3
Choosing a starting mobile phase ............................................................. 32
3.3.1
A TLC method exists for the samples ................................................... 34
3.3.2
No TLC method is available, but HPLC methods are known ...................... 35
3.3.3
No chromatographic method is available .............................................. 35
Optimizing off-line OPLC separations .............................................................. 36
4.1
Pure, 1-component solvent systems .......................................................... 36
4.1.1
Components near the origin ............................................................... 37
4.1.2
Components migrate to the end of the column ...................................... 37
4.2
Binary solvent mixtures .......................................................................... 38
4.2.1
Case a : My products are behind all demixing fronts............................... 39
4.2.2
Case b : The peaks remain at the head of the column ............................. 40
4.2.3
Case c : The peaks are found at the end of the column ........................... 41
4.2.4
case e : Products are close to or in the demixing front ............................ 42
4.3
Ternary solvent mixtures and beyond........................................................ 43
2/51