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Stable performance It ismost desirable to be able to run the compatible normal, polar organic and, reversedmobile phases without irreversibly damage the stationary phase. Switching between compatible normal to polar organicmobile phases will not lead to any degradation in performance. By using Kromasil AmyCoat and CelluCoat there are no need for solvent dedicated columns. In order to test the stability of Kromasil AmyCoat, in this case, the chromatographic performance was evaluated before and after high flow rate conditions. As shown in figure 4, the column efficiency wasmaintained even after the harsh conditions of the test sequence.


Figure 6b. Overloaded injections. Solute: Ethylmandelate 140 mg/ml in mobile phase. Column: Kromasil CelluCoat 10µm, 4.6 x 250 mm. Flow rate: 0.7 ml/min. Mobile phase: heptane/2-propanol (90/10). Detection: UV @ 254 nm.


Figure 4. Maintained efficiency after extended use. Separation of trans-stilbene oxide in heptane/2-propanol (90/10), detection UV @ 229 nm, temp. 25°C, column Kromasil AmyCoat, 3 µm, 4.6 x 150 mm


Scalability Depending on the purpose of the chiral separation method the


optimum particle size of the CSP varies. Small particles (3 µm and 5 µm) should be applied in analytical scale work and larger particles used when going to preparative scale.


When particle sizes from 3 µm to 10 µm giving identical selectivity, Kromasil AmyCoat and CelluCoat make it easy to scale up while retaining excellent performance. Figure 5 shows the separation of trans-Stilbene oxide on Kromasil CelluCoat 3 µm, 5 µm, and 10 µm.


Additives in the mobile phase In preparative chromatography additives are undesirable since they complicate the solvent recovery process and they could also reduce the stability of the enantiomers in the mobile phase, particularly during evaporation. Screening without additives with analytical injections should however be made with careful considerations. A study performed on the separation of Metoprolol clearly indicated, using analytical injections, that 0.1% DEA was needed in the mobile phase. However, at overloaded conditions no additive was needed in the mobile phase or in the injection solvent, as can be seen very clearly from the fraction analysis presented in Figure 7a and 7b. This behaviour could be explained by the substance buffering the phase itself at overload conditions.


0


Figure 7a. Separation with additive, 0.1 % DEA


Figure 5. Consistent retention and selectivity independent of particle size. Separation of trans-stilbene oxide in heptane/2-propanol (90/10), detection UV @ 220 nm, flow rate 0.5 ml/min, column Kromasil CelluCoat 3, 5 and, 10 µm, 4.6 x 150 mm


Semi-preparative chiral separations Important aspects in preparative chiral separations are productivity, loadability, selectivity and, solubility. Figure 6a and 6b illustrate a semi- preparative application of Ethylmandelate on Kromasil 10-CelluCoat. The analytical chromatogramwas run on a 3 µmCelluCoat column and since an identicalmanufacturing technology is used for all particle sizes the results could easily be translated to a column packed with 10 µmparticles.


Figure 7b. Separation without additive


Conclusion Kromasil AmyCoat and CelluCoat are two fully back integrated chiral stationary phases from Kromasil. The specially designed silica offers a high mechanical stability, which allows columns to be operated at pressures up to 400 bar.


The amylose- and cellulose-selector are well known for their ability to resolve a broad range of racemates.


Figure 6a. Analytical injection. Solute: Ethylmandelate 9 mg/ml in mobile phase. Injection: 5 µl. Column: Kromasil CelluCoat 3 µm, 4.6 x 150 mm. Flow rate: 1.0 ml/min. Mobile phase: heptane/2-propanol (90/10). Detection: UV @ 230 nm.


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