Application Note #42

Using TLC to Accurately Predict FLASH Purification Results

J. Robert Bickler May 5, 2003

Introduction
Thin-layer chromatography (TLC) is a commonly used method development tool for flash purification. Product mixtures separated using TLC generally can be purified by flash chromatography using the identical solvent system. The method transfer accuracy of TLC to flash is based on the TLC silica and flash silica properties. When identical, the equation 1/Rf = CV holds, where Rf is the retention factor of a compound separated by TLC, and CV is the number of column volumes required to elute a compound. Cartridge loading capacity is based on the difference in CV (D CV) between two adjacent compounds. Although many TLC and flash-grade silicas have the same physical specifications (surface area, porosity, etc.) differences in their qualities exist. These differences manifest themselves as gross errors in the calculated CV, variable selectivity, and unreliable loading capacity calculations. Because of these real method transfer problems it is very important that the TLC silica and the flash silica be the identical and from the same vendor.

Procedure
Biotage, the leader in flash purification technology, now offers glass-backed TLC plates and prepacked flash cartridges made from the same silica. As an example of why matching TLC and flash silica is important, a 5-component dye mixture (Camag dye mix 2) was separated using a Biotage FLASH TLC plate and a competitive TLC plate using toluene as the solvent. Both manufactures TLC silica have virtually identical physi2 cal properties; surface area = 500 m /g and pore diameter = 60 . TLC Rf to Flash CV Correlation
Biotage TLC Rf Red Orange Black Yellow Blue 0.84 0.34 0.30 0.18 0.10 Calc. CV 1.19 2.94 3.33 5.56 10.00 D CV Biotage % FLASH Relative CV Error 1.25 4.76 3.00 1.96 3.25 -2.56 5.50 -1.01 9.25 -8.11 Comp. TLC Rf 0.82 0.33 0.28 0.25 0.09 Calc. CV 1.22 3.03 3.57 4.00 11.11 D CV Biotage % FLASH Relative CV Error 1.25 2.44 3.00 -1.01 3.25 -9.89 5.50 27.27 9.25 -20.12

1.75 0.39 2.23 4.44

1.81 0.54 0.43 7.11

Table 1. Shows improved flash purification predictability when the TLC silica and flash silica are the same. Much greater reliability in calculated elution volumes (solvent consumption) is possible. In this example, the yellow dye has an Rf of 0.18 (5.56 CV) on the Biotage FLASH TLC plate but an Rf of 0.25 (4.00 CV) on the competitive plate. When the dye mix was separated on a Biotage Flash 12+ cartridge the yellow dye eluted in 5.5 CV. The Biotage FLASH TLC Plate correlates to the Biotage FLASH 12+ CV data with more accuracy than the competitive plate. More accurate CV calculations will result in better throughput, higher purity, greater yield, and reduced solvent cost.

Results
Although Rf values are very similar for most compounds (red, orange, black, and blue), the yellow dye shows a remarkably different Rf value, 0.18 (5.56 CV) on Biotage, 0.25 (4.00 CV) on the competitors plate, Table 1. When this dye mix was purified on a Biotage FLASH 12+M cartridge, the yellow dye eluted in 5.50 CV, as predicted by the Biotage TLC plate, Fgure 1.

Figure 1. TLC separation on Biotage FLASH TLC plate and FLASH chromatogram of Camag dye mix #2 separated on a Biotage FLASH 12+M cartridge using toluene as the eluent. The black dye has poor UV absorption @ 254 nm and is only partially resolved from the orange dye, as predicted by TLC.

Conclusion
TLC to flash method transfer accuracy is improved when TLC plates and flash cartridges made with identical silica from Biotage are used. In the example cited above, the Biotage FLASH TLC plate Rf data correlate to FLASH CV with greater accuracy than a competitive plate with the same silica specifications. For synthetic chemists, the benefits of matched TLC plates and flash cartridges are better purification throughput, increased compound purity and yield, and reduced solvent cost.

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