A New Era in SFC Column Technology

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2 A ew Era in SFC Column Technology Combining state-of-the-art media manufacturing with the industry leading column technology, Viridis Supercritical Fluid Chromatography (SFC) columns bring a new level of reproducibility to the world of laboratory scale purification. Viridis SFC columns are now available for analytical and preparative chromatography as: Viridis SFC -Ethylpyridine columns Viridis SFC Silica columns TM A

3 Viridis SFC columns, manufactured for reliability and consistency Based upon over 50 years of experience in the separation science industry, Waters chromatographic media sets the standard for quality and batch-tobatch reproducibility. As a primary manufacturer of silica and hybrid chromatographic particles, all stages of product synthesis are meticulously monitored and controlled, ensuring complete characterization of the finished product. This level of manufacturing process understanding delivers chromatographic media with guaranteed consistent performance year after year. Wate rs Fine C hemi cal M an u fa ctu ring Plant in Taunton, Massachusetts, USA. Waters is committed to support the purification scientists who use SFC technology. With the acquisition of TharSFC and the development of Viridis SFC columns, Waters Corporation now offers a complete SFC solution that encompasses system, service, software and new columns. SFC-MS Prep 100

4 Viridis SFC Columns Manufactured with BD Technology Column Performance Identifying the Problem Laboratory-scale SFC purification presents many challenges to the chromatographer. Inconsistency in column-to-column performance and lifetimes often result in lost samples, repeat purification runs and poor scalability from small to larger volume columns. Common SFC prep column failures are due to mechanical instabilities, especially voids in the packed bed. The patented BD technology 1 combines optimized packing processes with a unique set of column designs ensuring increased lifetime and better reproducibility. When a column is used in a SFC system, it must remain stable during operations. The bed must be packed sufficiently dense to withstand the compressive fluid forces encountered during use. In the case of analytical column dimensions, the necessary packed bed density can be generally achieved using traditional slurry packing methods. As the diameter of the column increases, it becomes increasingly difficult to reach the bed density required for stable, long term performance, especially when using small particles. The optimum bed density is dependent upon the specific properties of the chromatographic particles and column dimensions. BD ptimized Bed Density High-pressure Slurry Packing Localized Bed Expansion ear Inlet BD Process ptimum Bed Density Throughout Axial-ram Slurry Packing Localized Compression ear Inlet ptimized Bed Density is achieved by applying pre-determined axial force only at the column inlet. 1 US Patent # 7,99,410; UK Patent # GB BD Column Design Improved Mechanical Stability Waters combines high-pressure slurry packing with a carefully calculated axial compression element localized at the less-dense inlet end of the bed. With careful tuning of the packing process for each particle type and column geometry, the Prep BD design and process results in predictable, uniform density profiles throughout the column. Established procedures ensure that the column bed is not over-compressed or disrupted during the final column packing process. Excess axial compression applied at the inlet can break particles, build bridges, and lower local bed permeability. Exploded Prep BD Column Hardware For purification, the Viridis SFC columns incorporate chemically inert seals made to prevent leaks and a pair of specially designed filter housings with frits and distributors built to withstand high pressures.

5 Viridis SFC -Ethylpyridine Columns -Ethylpyridine chemistry is successfully used for a wide range of achiral SFC applications due to its retention, peak shape and selectivity properties. Viridis SFC -Ethylpyridine columns also provide high reliability and reproducibility and can be used for analytical and preparative applications. Si -Ethylpyridine Ligand Excellent Peak Shape Viridis SFC -Ethylpyridine columns exhibit good peak shape whether separations are performed with or without additives such as diethylamine or ammonium acetate. High Mass Loading Viridis SFC -Ethylpyridine columns provide excellent mass loading to ensure maximum throughput. Conditions Conditions Viridis SFC -Ethylpyridine Column, 4.6 x 150 mm, 5 µm Part umber: Gradient Separation: 10 min Flow Rate:.5 ml/min at 40 C Wavelength: 54 nm System Back Pressure: 100 bar Injection Volume: µl Compounds 1 Caffeine Amitriptyline Thymine 4 Cytosine 5 Sulfamethoxazole 6 Sulfamethizole Viridis SFC -Ethylpyridine BD Column, 19 x 150 mm, 5 µm Part umber: Gradient Separation: 4. min Mobile Phase: 5-15% methanol with no additive Flow Rate: 85 g/min at 40 C Wavelength: 0 nm System Back Pressure: 10 bar Compounds 1 Coumarin Flavone Ibuprofen 4 Carbamazepine Mobile Phase: 5-40% methanol with no additive Injection Volume: 100 µl Sample Concentration: 80 mg/ml Total Mass Load: 8 mg Absorbance (mau) Absorbance (mau) Mobile Phase: 5-40% methanol with 0.% diethylamine (DEA) Injection Volume: 50 µl Sample Concentration: 80 mg/ml Total Mass Load: 8 mg -50 Absorbance (mau) Mobile Phase: 5-40% methanol with 0 mm ammonium acetate min Injection Volume: 750 µl Sample Concentration: 80 mg/ml Total Mass Load: 60 mg min 4

6 Viridis SFC Silica Columns Based on Waters long history of chromatographic silica production, the Viridis SFC Silica columns are designed to be highly reproducible and predictable based on tight product specifications and very low metal content. Selectivity Viridis SFC Silica columns offer added selectivity due to the highly polar, unbonded surface and will have greater interaction with polar functional groups when compared to Viridis SFC -Ethylpyridine columns. Viridis SFC Silica column versus Viridis SFC -Ethylpyridine column Conditions Gradient Separation: 4. min Mobile Phase: 5-15% methanol with no additive Flow Rate: 85 g/min at 40 C Wavelength: 0 nm System Back Pressure: 10 bar Compounds 1 Flavone Ibuprofen Carbamazepine 1 6.0e-1 4.0e-1.0e-1 Viridis SFC Silica BD Column, 19 x 150 mm, 5 µm Part umber: e-1 1.0e-1 6.0e e-1.0e-1 Viridis SFC -Ethylpyridine BD Column, 19 x 150 mm, 5 µm Part umber: e-1 1.0e min Using the same SFC gradient conditions, different selectivity can be achieved by using a Viridis SFC -Ethylpyridine column or Viridis SFC silica column. The retention time for ibuprofen was reduced and the carbamazepine retention time was increased by switching SFC column chemistries, with little change in the retention of flavone. 5

7 RDERIG IFRMATI Chemistry Particle Shape Particle Size (µm) Pore Size (Å) Surface Area (m /g) Pore Volume (cc/g) Carbon Load (%) Endcapped -Ethylpyridine Spherical no Silica Spherical n/a n/a Description Part umber Viridis SFC -Ethylpyridine columns Viridis SFC -Ethylpyridine Column, 4.6 x 50 mm, 5 µm Viridis SFC -Ethylpyridine Column, 4.6 x 100 mm, 5 µm Viridis SFC -Ethylpyridine Column, 4.6 x 150 mm, 5 µm Viridis SFC -Ethylpyridine Column, 4.6 x 50 mm, 5 µm Viridis SFC -Ethylpyridine Column, 10 x 50 mm, 5 µm Viridis SFC -Ethylpyridine Column, 10 x 100 mm, 5 µm Viridis SFC -Ethylpyridine Column, 10 x 150 mm, 5 µm Viridis SFC -Ethylpyridine Column, 10 x 50 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 19 x 50 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 19 x 100 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 19 x 150 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 19 x 50 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 0 x 50 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 0 x 75 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 0 x 100 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 0 x 150 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 0 x 50 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 50 x 50 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 50 x 100 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 50 x 150 mm, 5 µm Viridis SFC -Ethylpyridine BD Column, 50 x 50 mm, 5 µm Description Part umber Viridis SFC Silica columns Viridis SFC Silica Column, 4.6 x 50 mm, 5 µm Viridis SFC Silica Column, 4.6 x 100 mm, 5 µm Viridis SFC Silica Column, 4.6 x 150 mm, 5 µm Viridis SFC Silica Column, 4.6 x 50 mm, 5 µm Viridis SFC Silica Column, 10 x 50 mm, 5 µm Viridis SFC Silica Column, 10 x 100 mm, 5 µm Viridis SFC Silica Column, 10 x 150 mm, 5 µm Viridis SFC Silica Column, 10 x 50 mm, 5 µm Viridis SFC Silica BD Column, 19 x 50 mm, 5 µm Viridis SFC Silica BD Column, 19 x 100 mm, 5 µm Viridis SFC Silica BD Column, 19 x 150 mm, 5 µm Viridis SFC Silica BD Column, 19 x 50 mm, 5 µm Viridis SFC Silica BD Column, 0 x 50 mm, 5 µm Viridis SFC Silica BD Column, 0 x 75 mm, 5 µm Viridis SFC Silica BD Column, 0 x 100 mm, 5 µm Viridis SFC Silica BD Column, 0 x 150 mm, 5 µm Viridis SFC Silica BD Column, 0 x 50 mm, 5 µm Viridis SFC Silica BD Column, 50 x 50 mm, 5 µm Viridis SFC Silica BD Column, 50 x 100 mm, 5 µm Viridis SFC Silica BD Column, 50 x 150 mm, 5 µm Viridis SFC Silica BD Column, 50 x 50 mm, 5 µm H H H Carbamazepine Caffeine H Cytosine Flavone Ibuprofen H S H S S H Coumarin Amitriptyline H Thymine H Sulfamethizole H Sulfamethoxazole 6

8 Sales ffices Austria and European Export (Central South Eastern Europe, CIS and Middle East) Australia Belgium Brazil Canada x05 China CIS/Russia / Czech Republic Denmark Finland France Germany Hong Kong The etherlands orway Poland Puerto Rico Singapore Spain Sweden Switzerland Taiwan United Kingdom All other countries: Waters Corporation U.S.A Hungary India and India Subcontinent Ireland Italy Japan Korea Mexico The quality management system of Waters manufacturing facilities in Taunton, Massachusetts and Wexford, Ireland complies with the International Standard IS 9001:000 Quality Management and Quality Assurance Standards. Waters quality management system is periodically audited by the registering body to ensure compliance. A 010 Waters Corporation. Waters, The Science of What s Possible, Viridis, BD and SFC-MS Prep 100 are trademarks of Waters Corporation E April 010 KK-FP

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