Editors
     
03/17
 Topic
Improving performance and efficiency in flash chromatography with spherical silica gels



Classical flash chromatography using a glass apparatus has been automated in recent years. The use of flash chromatography machines significantly increased the sample throughput. A further improvement in performance is possible by the use of spherical instead of irregular silica gels.


  Automated flash chromatography
  Flash chromatography is a proven method for the preparative purification of compound mixtures, which is mainly used in the synthesis laboratory. Traditionally, glass columns with silica gels are still filled manually. In a glass apparatus, the sample is then pressed with the eluent flow (mobile phase) through the column bed (stationary phase) under slight overpressure in order to get the compounds separated.

For a high sample throughput, flash chromatography machines are standard tools meanwhile, especially in the chemical and pharmaceutical industry, currently using pre-packaged plastic cartridges. Isco Companion® and other Teledyne Isco CombiFlash® systems are widely used. For these, as well as for Yamazen SmartFlash, Interchim® puriFlash® and other machines, MACHEREY-NAGEL offers the CHROMABOND® Flash RS cartridges packed with a wide range of different silica gels.

In contrast to preparative HPLC, low-cost fractionated silica gels have been dominant in flash chromatography. However, the demand for better efficiency in automated flash chromatography is the main reason to start thinking about spherical silica gels.

  Resolution of irregular and spherical silica particles
  The performance of a chromatographic system is mainly determined by the resolution Rs, the measure for the separation capacity of two sample compounds on a stationary phase.
  The resolution is influenced by efficiency, selectivity and retention. The most important parameter for improving the resolution is the selectivity. It can be changed by using another type of the stationary phase or by changing the mobile phase. The efficiency can also be improved by choosing sorbents with smaller particle sizes. The particle size is inversely proportional to the theoretical number of plates.


  For example, the flash chromatographic separation of two parabens and the structurally similar phenone shows a significant improvement when downsizing the median particle size from 40-63 µm to 15-40 µm (see figures 1, (3) and (2)).

By using spherical instead of irregular silica gel, a further improvement in the resolution can be achieved under the same chromatographic conditions and a base line separation can be obtained, too (see figure 1, (1)). The reason for this is that spherical particles with a narrow particle size distribution can be packed more uniformly into the cartridge than irregularly shaped  particles. In this way the formation of cavities or channels in the package are avoided and finally the resolution is increased.

 


 
Conditions
 Cartridges:


(1) Flash RS cartridge, 40 g spherical silica gel, 25 µm
(2) Flash RS cartridge, 40 g irregular silica gel, 15-40 µm
(3) Flash RS cartridge, 40 g irregular silica gel, 40-63 µm
 Substances: 3,5-dibenzyloxyacetophenone, butylparaben, methylparaben
 Sample loading:
165 mg
 Eluent A:
cyclohexane
 Eluent B: ethyl acetate
 Gradient: 20–80 % B in 4 min
 Flow rate:
80 mL/min

Figure 1: Chromatograms and conditions of the separation of a phenone from parabens on silica gels with different resolution capacities

  Loading capacity of irregular and spherical silica gels
  In preparative chromatography, the highest possible loading capacity is also decisive for a high yield. Spherical silica gel particles with a higher specific surface area can achieve up to twice as much loading as irregular particles depending on the application, since they generate a higher resolution and more interactions.

For example, pyrazines are to be separated with 40 g of silica gel cartridges. The cartridge packed with spherical silica gel of particle size 15 µm achieves a base line separation up to 800 mg sample amount while the fractionated silica gel, 40-63 µm does not completely
separate 800 mg of the substance mixture.

 


 
Conditions
 Cartridges:

(1) Flash RS cartridge, 40 g spherical silica gel, 15 µm
(2) Flash RS cartridge, 40 g irregular silica gel, 40-63 µm
 Substances:
2,5-dimethylpyrazine, 2-acetylpyrazine, 2-ethylpyrazine, 2-methoxypyrazine
 Sample
    loading:
800 mg

 Eluent A:
cyclohexane
 Eluent B: ethyl acetate
 Gradient: 10–80 % B in 6 min, 90 % B for 5 min
 Flow rate:
80 mL/min

Figure 2: Chromatograms and conditions of pyrazine separation showing the loading capacity of irregular and spherical silica gels

  Performance of the spherical silica gel CHROMABOND® SPHERE SiOH
  The new CHROMABOND® Flash RS SPHERE SiOH cartridges are packed with high-purity spherical silica gel in particle sizes of 25 µm or 15 µm. They exhibit the advantages of a significantly higher resolution and higher loadability of spherical silica gels and can be economically used in automated flash systems.

The following example shows the method transfer for the separation of 1 g of a phthalate mixture from a CHROMABOND® Flash RS 40 cartridge (packed with 40 grams irregular 40-63 µm silica) to a CHROMABOND® SPHERE SiOH 15 µm cartridge (filled with the same amount of spherical silica gel).

 


 
Conditions
 Cartridges:

(1) CHROMABOND® Flash RS 40 SPHERE SiOH 15 µm (REF 732763)
(2) CHROMABOND® Flash RS 40 SiOH 40-63 µm (REF 732803)
 Substances: dibutyl phthalate, diethyl phthalate, dimethyl phthalate
 Sample loading:
1 g
 Eluent A:
cyclohexane
 Eluent B: ethyl acetate
 Gradient:

(1) 20–80 % B in 8 min
(2) 5–30 % B in 10 min
 Flow rate:

(1) 80 mL/min
(2) 40 mL/min
 Total solvent
    consumption:
(1) 200 mL
(2) 340 mL
 Run time:

(1) 2.5 min
(2) 8.5 min

  Figure 3: Chromatograms and conditions for the optimization of phthalate separation with CHROMABOND® SPHERE SiOH


  Figure 3 shows the shorter run time of 8.5 min (2) compared to 2.5 min (1) and the saving in  solvent consumption from 340 mL (2) to 200 mL (1) when using CHROMABOND® SPHERE SiOH.

The spherical particle shape and the narrow particle size distribution of the SPHERE SiOH particles, enables a space-filling, dense packing in the cartridge. Therefore less back pressure is generated and higher flow rates can be applied.

  Conclusion
  Flash chromatography with CHROMABOND® SPHERE SiOH is a fast and economical way to purify organic compounds and ideally suited to enhance efficiency in automated (flash) systems.

  Trademarks
  Isco Companion®, CombiFlash® – Teledyne Isco Inc., USA
Interchim®, puriFlash® – Interchim, France
CHROMABOND® – MACHEREY-NAGEL GmbH & Co. KG, Germany

 
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