YMC-Actus. Columns for Semi-preparative HPLC. Selectivity Durability Loadability

Transcription

1 YMC-Actus Columns for Semi-preparative HPLC Selectivity Durability Loadability

2 Fast semi-preparative chromatography Semi-preparative chromatography is the link between analytical HPLC and preparative LC. Even though the chromatographic systems used for semi-preparative LC are not as large as preparative LC systems, the objectives remain the same: Purification and isolation of maximum sample quantity Savings in time and costs. With YMC-Actus, time is on your side! 2 YMC Europe GmbH Phone: +49 (0) Fax: +49 (0) info@ymc.de Website:

3 Stability Rapid pressure changes under high-speed gradient conditions may lead to column degradation and loss of column performance. With YMC-Actus, a new hardware and packing technology has been applied to these semipreparative columns to provide a uniform packing density which results in a longer lifetime than conventional semi-preparative columns. In order to determine the quality of the packing, the column performance has been evaluated every 100 runs with a sequential high-throughput gradient. YMC-Actus columns offer outstanding efficiency without compromising resolution. Furthermore, YMC-Actus columns provide reliable results, even after exposure to severe, rapid gradient conditions and multiple injections. Increase column lifetime = Decrease of cost Conventional Semi-preparative Column Sequential gradient test Colum: 50 x 20 mm ID, 5 µm or 50 x 19 mm ID, 5 µm Eluent: Gradient: A) water B) methanol 5% B (0-0.5 min), 5-95% B ( min), 95% B ( min), 5% B ( min) Flow rate: 50 ml/min Temp.: ambient Column performance test Column: 50 x 20 mm ID, 5 µm or 50 x 19 mm ID, 5 µm Eluent: methanol / water (70/30) Flow rate: 10 ml/min Sample: Naphthalene Application data by courtesy of YMC Co., Ltd. 3

4 YMC-Actus ION EXCHANGE MEDIA Axial Compression Technology for Ultimate Separation YMC-Actus series columns are semi-preparative HPLC columns that have excellent column stability and efficiency as a result of applying axial compression technology. YMC-Actus series columns show high stability under high flow rate or steep gradient conditions which are desirable for milligram scale preparative HPLC of various compounds. 10% increase in bed density Compressing Flow Test conditions (high-speed and high-pressure) Column: 5 µm, 50 x 20 mm ID Eluent: A) water B) methanol Gradient: 5%-95% B Flow rate: 50 ml/min Pressure: ~ 17 MPa Long Lasting Columns! after 700 runs initial Conventional Column Packing without compression Flow Void and crack Degradation of performance after 300 runs initial Uniformly high density packing is necessary for highly efficient and stable HPLC columns. DAC (Dynamic Axial Compression) columns are widely used for preparative separation in pilot or production scale. This allows uniformly high density packing and prevents formation of voids. YMC-Actus series columns have been developed by applying this Axial Compression Technology to semi-prep column production. The column bed is compressed appropriately when attaching the inlet end assembly of the newly designed YMC-Actus hardware. It provides increased bed density (10% higher than conventional columns) and bed uniformity. YMC Europe GmbH Phone: +49 (0) Fax: +49 (0) info@ymc.de Website: 4

5 YMC-Actus ION EXCHANGE MEDIA Excellent stability and efficiency under fast gradient conditions at high flow rate Glycyrrhizic acid in herb medicine Analysis Purification YMC-Pack ODS-AQ, 50 x 4.6 mm ID, 5 µm 3 ml/min, 10 µl injection YMC-Actus ODS-AQ, 50 x 20 mm ID, 5 µm 60 ml/min, 500 µl injection After purification 99.5% Glycyrrhizic acid Glycyrrhizic acid Available for high-throughput purification: Injection in DMSO 1. Ethenzamide 2. Clemastine fumarate 3. Ibuprofen Column: Eluent: Gradient: Flow rate: Temperature: Detection: Injection: YMC-Actus Pro C18 RS (5 µm, 8 nm), 50 x 20 mm ID A) water / TFA (100/0.1) B) acetonitrile / TFA (100/0.1) 10-90% B (0-3 min), 90% B (3-5 min), 10% B (5-10 min) 20 ml/min ambient UV at 270 nm 100 µl (0.2-2 mg/ml in DMSO) As shown in overlay chromatograms, YMCActus columns provide outstanding stability and reproducibility in the separation of pharmaceuticals dissolved in 100% DMSO, even after 1,000 injections under the test conditions. This makes YMC-Actus columns ideal for high-throughput purification in drug discovery. Application data by courtesy of YMC Co., Ltd. 5

6 Excellent separation of compounds with similar structure Capsaicinoids in red pepper Analysis 50 x 4.6 mm ID, 5 µm 2.0 ml/min, 20 µl injection Phenomenex Luna C18, 5 µm, 10 nm Waters XBridge C18, 5 µm YMC-Pack Pro C18 RS Purification YMC-Actus Pro C18 RS, 50 x 20 mm ID 5 µm Nordihydrocapsaicin Capsaicin Dihydrocapsaicin 40 ml/min, 400 µl injection Purity >99.5% Eluent: A) methanol / water / TFA (50/50/0.1) B) methanol / TFA (100/0.1) 0-30% B (0-5 min, 30% B (5-10 min) Temperature: 25 C for 50 x 4.6 mm ID ambient for 50 x 20 mm ID Detection: UV at 280 Sample: methanol extract of a commercial red pepper (1 g / 3 ml) Outstanding separation of highly polar compounds Crude synthetic 30mer oligonucleotide Analysis Hydrosphere C ml/min, 5 µl injection Purification 19 ml/min, 100 µl injection 50 x 4.6 mm ID, 5 µm 50 x 20 mm ID, 5 µm Impurities 30mer YMC-Actus Hydrosphere C18 Recovery 56% purity>99% 6 YMC Europe GmbH Phone: +49 (0) Fax: +49 (0) info@ymc.de Website:

7 YMC-Actus Preparative Column Selection Finder Optimisation of preparative chromatography! The main task for a preparative chromatographer is to find the suitable system. In order to simplify the considerations YMC developed a Preparative Column Selection Guide. R&D Scale-up Kit: Analytical and semipreparative columns packed with a stationary phase from the same lot. The Preparative Column Selection Guide will help to select:1.the most suitable column ID for the required sample loading 2.the appropriate particle size for optimum efficiency 3.the required column length for the necessary resolution R&D Kits To minimise the problems that the process of scale-up may introduce, YMC offers YMC R&D kits, which consist of one analytical column and one YMC-Actus column packed with exactly the same packing material from the same production lot. Therefore no further method development is needed, just a simple linear scale-up calculation. Application data by courtesy of YMC Co., Ltd. 7

8 Linear Scale-Up In most cases it is beneficial to develop a semi-preparative method on an analytical scale column. The analytical separation carried out on a 150 x 4.6 mm ID column has to be scaled up to 150 x 20 mm ID Therefore the chromatographic parameters such as flow rate and column load have to be adjusted according to the following equation: Linear Scale-Up SF = P 2 ID, prep P 2 ID, anal. = m prep m anal. SF = r 2 ID, prep / r 2 ID, analytical SF = 20 2 ID, prep / ID, analytical SF = g 4.6 mm ID 20 mm ID 37.8 g Inner diameter loadability / compound Guideline for Sample Load according to column ID Column ID (mm) Scale-Up factor Loadability (mg) YMC Europe GmbH Phone: +49 (0) Fax: +49 (0) info@ymc.de Website:

9 Scale-Up YMC linear scale-up is achieved in 4 steps: 1. Analytical scale method development. Determine the optimum separation conditions by using analytical columns packed with different stationary phases and various conditions. 2. Study the preparative scale. Select the particle size of the packing material and the inner diameter of column appropriate for the sample volume. 3. Optimise the separation conditions and perform loadability studies using analytical columns with inner diameter of 4.6 mm or 6.0 mm packed with the packing material selected for the preparative separation (scout column). If the particle size of the packing material is the same as in the Step 1, this process can be omitted. If the preparative column is more than 100 mm ID, it is advisable to insert another step with a scout column of 20 mm ID in order to accurately predict loadability and calculate the running costs. 4. Proceed with the preparative separation with scale-up of chromatographic parameters such as flow rate/ column ID/ sample load as necessary. From all the given steps above the most demanding step will be the scale-up of the chromatographic parameters in order to meet the preparative demands. There are a number of scalable parameters: flow rate, column ID, sample load, tubing ID, sample injection concentration, volume of sample loop, consumption of solvent, dead volume,fraction mass, size of the detector cell. How to adjust the scalable factors? In order to simplify your scale-up the three most important scale-up factors are summarised. Scalable factor SF Internal Diameter Linear Scale-Up Column length Time Column length and ID Volume SF = r 2 ID, prep / r 2 ID, anal. SF = l ID, prep / l ID, anal. SF = (r 2 ID, prep / r 2 ID, anal.) / (l ID, prep / l ID, anal. ) Impact Flow rate Eluent composition Retention time Cycle time Plate number Amount of adsorbent Application data by courtesy of YMC Co., Ltd. 9

10 Ordering Information High durability semi-preparative columns Packing material Particle size Pore size Column size [µm] [nm] length x ID [mm] Product Code 50 x 20 TA12S WX 100 x 20 TA12S WX 150 x 20 TA12S WX Triart C18 S x 20 TA12S WX 50 x 30 TA12S WX 75 x 30 TA12S05-L530WX 100 x 30 TA12S WX 50 x 20 AS12S WX 100 x 20 AS12S WX 150 x 20 AS12S WX Pro C18 S x 20 AS12S WX 50 x 30 AS12S WX 75 x 30 AS12S05-L530WX 100 x 30 AS12S WX 50 x 20 HS12S WX 100 x 20 HS12S WX 150 x 20 HS12S WX Hydrosphere C18 S x 20 HS12S WX 50 x 30 HS12S WX 75 x 30 HS12S05-L530WX 100 x 30 HS12S WX 50 x 20 RS08S WX 100 x 20 RS08S WX 150 x 20 RS12S WX Pro C18 RS S x 20 RS12S WX 50 x 30 RS08S WX 75 x 30 RS08S05-L530WX 100 x 30 RS08S WX 50 x 20 OS12S WX 100 x 20 OS12S WX 150 x 20 OS12S WX Pro C8 S x 20 OS12S WX 50 x 30 OS12S WX 75 x 30 OS12S05-L530WX 100 x 30 OS12S WX 50 x 20 AA12S WX 100 x 20 AA12S WX ODS-A S x 20 AA12S WX 50 x 30 AA12S WX 75 x 30 AA12S05-L530WX 100 x 30 AA12S WX 50 x 20 AQ12S WX 100 x 20 AQ12S WX ODS-AQ S x 20 AQ12S WX 50 x 30 AQ12S WX 75 x 30 AQ12S05-L530WX 100 x 30 AQ12S WX YMCbasic S x 20 BA99S WX Other phases are available on request. Columns with 50 mm ID are available on request. 10 YMC Europe GmbH Phone: +49 (0) Fax: +49 (0) info@ymc.de Website:

11 Ordering Information Guard cartridges Packing material Particle size [µm] Pore size [nm] Triart C18 S-5 12 Pro C18 S-5 12 Hydrosphere C18 S-5 12 Pro C18 RS S-5 8 Pro C8 S-5 12 ODS-A S-5 12 ODS-AQ S-5 12 Column size length x ID [mm] Product Code 10 x 20 TA12S GC 20 x 30 TA12S GC 10 x 20 AS12S GC 20 x 30 AS12S GC 10 x 20 HS12S GC 20 x 30 HS12S GC 10 x 20 RS08S GC 20 x 30 RS08S GC 10 x 20 OS12S GC 20 x 30 OS12S GC 10 x 20 AA12S GC 20 x 30 AA12S GC 10 x 20 AQ12S GC 20 x 30 AQ12S GC YMCbasic S x 20 BA99S GC Guard Cartridge Column Holder: Product Code XPCHSPW2 Application data by courtesy of YMC Co., Ltd. 11

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