Mobile Phase

The separations can be started in saturated or un-saturated chromatographic tanks. However, on starting the separation in an unsaturated chamber the chromatographic tank becomes saturated during the development because of the long separation time (1-2 h). If saturated chromatographic chambers are used, the optimized analytical mobile phase may be transferred unchanged to PPC. Because the particle sizes and size distribution of sorbents for preparative purposes are larger, and the plates are overloaded with the compounds to be separated, inferior separation is invariably achieved on preparative plates. This means that a successful preparative separation will need an optimized mobile phase.

The 'PRISMA' mobile phase optimization system enables not only optimization of solvent strength and mobile phase selectivity, but also transfer of the optimized mobile phase between the different planar chromatographic techniques. The system is based on the solvent classification by Snyder, who classified more than 80 solvents into eight groups for normal phase (NP) chromatography according to their properties as proton acceptors (Xa), proton donors (Xd), and their dipole interactions (Xn). Because

Sample streak Preadsorbent Sample streak

Figure 3 Comparison of separations on different preparative plates. (A) Pre-coated chromatographic plate without concentrating zone, (B) pre-coated chromatographic plate with concentrating zone, (C) pre-coated chromatographic plate with layer-thickness gradient.

Sample streak Preadsorbent Sample streak

Figure 3 Comparison of separations on different preparative plates. (A) Pre-coated chromatographic plate without concentrating zone, (B) pre-coated chromatographic plate with concentrating zone, (C) pre-coated chromatographic plate with layer-thickness gradient.

Xa + Xd + Xn =1, the solvents can be characterized by two of them (e.g. Xa and Xd), and so a new definition was introduced - the individual selectivity value (Sv), which is the ratio of Xa to Xd. The individual solvent strengths (Si), selectivity values, and viscosities of the solvents most often used for normal phase PPC, are listed in Table 1.

For characterization of multi-component mobile phases the total solvent strength (ST) can be defined as the sum of the Si of the components, weighted by multiplication by their volume fraction. The total selectivity factor (SV) can be also calculated similarly to the ST value.

The volatility of the individual solvents must be also considered during the optimization process, otherwise several problems can arise in subsequent steps (elution of the compound from the stationary phase, evaporation of the solvent). It also precludes the use of, e.g. acetic acid as a component of the preparative mobile phase, because of the possibility of chemical degradation during concentration of the isolated compounds. Multicomponent mobile phases should not be used repeatedly, whereas single-solvent mobile phases can be used repeatedly until they become contaminated.

Figure 4 shows the transfer possibilities of the mobile phase, where thick lines indicate direct transfers. The thin lines indicate transfers which are also possible, but the solvent strength and selectivity must generally be changed. The dashed lines indicate direct transfer possibilities for fully online separation processes.

Table 1 Selected solvents for normal phase PPC separation

Selectivity

Solvents

Si

c e

Viscosity

group

* Xd

(cP)

n-Hexane

0

0.01

0.31

I

Methyl-t-butyl ether

2.7

3.50

0.27

Diethyl ether*

2.8

4.08

0.23

II

n-Butanol

3.9

3.11

2.98

Ethanol *

4.3

2.74

1.20

Methanol

5.1

2.18

0.52

III

Tetrahydrofuran*

4.0

1.90

0.47

Methoxyethanol

5.5

1.59

0.95

V

Dichloromethane*

3.1

1.61

0.44

1,1-Dichloromethane

3.5

1.43

0.79

VI

Ethyl acetate*

4.4

1.48

0.45

Methyl ethyl ketone

4.7

1.59

0.42

Dioxane*

4.8

1.50

1.20

Acetone

5.1

1.52

0.32

Acetonitrile

5.8

1.15

0.39

VII

Toluene*

2.4

0.89

0.59

Benzene

2.7

0.72

0.69

VIII

Chloroform*

4.1

0.61

0.57

Water

10.2

1.00

0.95

* Preferred solvent.

Figure 4 Mobile phase transfer possibilities between different planar chromatographic methods and HPLC, and fully online FFPC techniques.

* Preferred solvent.

Figure 4 Mobile phase transfer possibilities between different planar chromatographic methods and HPLC, and fully online FFPC techniques.

The optimized TLC mobile phase can be transferred from unsaturated chromatographic tanks to analytical OPLC and U-RPC without modification of the selectivity. If the tank is saturated, the optimized mobile phase can be transferred to M-RPC. Transfer of the corresponding preparative methods (OPLC, U-RPC, M-RPC) from analytical normal-phase FFPC can be performed directly with the same mobile phase. Modification of the mobile phase used in the unsaturated TLC tank is necessary for transfer of preparative N-RPC or S-RPC separations. Direct mobile-phase transfer is also valid from OPLC and U-RPC to C-RPC. With the characterization of the different saturation grade of chromatographic chambers, excellent mobile phase transfer between analytical and preparative planar chromatographic methods and analytical HPLC can be achieved.

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