2 Tosoh bioscience LLc. suite 100 king of Prussia, pa 19406, USA.

Transcription

1 TSKgel COLUMNS

2 Tosoh bioscience gmbh Im Leuschnerpark Griesheim Germany t + 49 (0) f + 49 (0) info.tbg@tosoh.com 2 Tosoh bioscience LLc 604 Horizon Drive, suite 00 king of Prussia, pa 9406, USA t f info.tbl@tosoh.com Tosoh corporation -8-2 Shiba, Minato-Ku tokyo japan t f info@tosoh.co.jp Tosoh bioscience SHANGHAI CO. LTD. room 0, plaza b, no. 289 yi shan road xu hui district shanghai, 2002, CHINA t f info@tosoh.com.cn Tosoh asia Pte. LTD. 6 market street #0-0 Bank of singapore centre singapore , singapore t f info.tsas@tosoh.com TOSOH HISTORY 9 Founding of Toyo Soda Manufacturing Co., Ltd. 96 Operation of Nanyo Manufacturing Complex begins 97 Scientific Instruments Division formed, First GPC column using TSKgel developed by Tosoh 974 High performance liquid chromatography column plant Is completed 979 Tosoh develops TOYOPEARL media 98 Tosoh develops Hydrophobic Interaction Media 987 TosoHaas US operations formed in Montgomeryville 989 TosoHaas GmbH operations formed in Stuttgart 99 Tosoh Nanyo gel facility receives ISO /200 all Tosoh affiliated scientific & diagnostic system related companies in Europe are unified under the name TOSOH BIOSCIENCE EcoSEC, THE 7TH GENERATION GPC SYSTEM IS INTRODUCED GLOBALLY 200 Tosoh celebrates its 7th year in business with the opening of five new plants, and continued rapid expansion in china 20 Tosoh Bioscience celebrates 40 Years of operation 202 Tosoh Releases first TOYOpearl mixed-mode resin toyopearl mx-trp-60m 20 Tosoh releases A high capacity Protein A Chromatography resin 204 Tosoh Bioscience GmbH celebrates its 2 th anniversary in Stuttgart 20 Tosoh bioscience successfully moves its sales & marketing offices to griesheim, darmstadt

3 Tosoh bioscience ANALYSIS HYDROP INTERACTION LIQUID CHROMATOGRAPHY Hydrophilic interaction liquid chromatography () is used primarily for the separation of polar and hydrophilic compounds. stationary phases are polar, similar to normal phase chromatography (NPC), but mobile phases are similar to reversed phase chromatography (RPC). Typical mobile phases are aqueous buffers with organic modifiers - primarily acetonitrile - applied in isocratic or gradient mode. In contrast to RPC, water has the highest elution power in mode. Therefore gradients usually start with a high percentage of acetonitrile. Typical stationary phases are silica or polymer particles carrying polar functional groups, e.g. hydroxyl, carbamoyl, amino or zwitterionic groups. Analysis of glycans, carbohydrates, peptides, polar drugs and metabolites, vitamins and other hydrophilic compounds are typical applications. is ideally suited for mass spectrometric analysis of water soluble polar compounds, because the high organic content in the mobile phase increases MS detection sensitivity. While using similar eluent systems and reversed phase can also be combined for two-dimensional liquid chromatography (2D-LC). Tosoh Corporation employs state-of-the-art manufacturing techniques that result in uniformly bonded packing materials with narrow pore size distributions and well-defined particle sizes to ensure high performance. Silica based TSK-GEL Amide-80 and NH2-00 columns enable the user to solve the most complex separation problems. HIGHLIGHTS offers orthogonal selectivity to reversed phase chromatography Covalently bonded carbamoyl and amino phases expand selectivity options Novel TSKgel NH2-00 columns show superior stability compared to conventional amino phases TSKgel Amide-80 columns provide unique retention mechanism for saccharide analysis Superior resolution and sensitivity with µm particle size

4 2 HOW IT WORKS It is commonly believed that in the aqueous content of the mobile phase creates a water rich layer on the surface of the stationary phase. This allows for partitioning of solutes between the more organic mobile phase and the aqueous layer. Hydrogen bonding and dipole-dipole interactions have been supposed to be the dominating retention mechanisms in mode (Figure ). The number of polar groups, as well as the conformation and solubility of the sample in the mobile phase determine the elution order. Since the retention is also related to the type of functional groups of the stationary phase, it varies between different phases. Compared to RPC the elution order in mode is inversed for most compounds. Figure 2 gives an example for the differences in selectivity of and RPC. Peptides were separated by C8 and columns of the same dimensions using the same eluents but almost inverse gradients. At low acetonitrile concentrations columns show a reversed phase mode of retention. The mode can only be executed when starting at high acetonitrile concentrations. offers unique advantages for mass spectrometric detection of very polar compounds when compared to reversed phase mode. The higher organic content of the eluent in mode supports efficient evaporation of the solvent thus enhancing sensitivity and altering ion suppression. In method development is an option as soon as polar compounds have to be analyzed and retention on reversed phase columns is too low. Since common RPC solvents can be used, TSK-GEL columns can be implemented in method development systems using automated column selection. A range of reversed phase columns differing in hydrophobicity or carrying polar embedded groups and one of the TSK-GEL column types should deliver an indication for the right direction of method development. TSK-GEL columns are available in various dimensions and particles sizes, functionalized with carbamoyl- or aminogroups. This enables the user to perfectly match selectivity to specific separation needs. figure principles figure 2 Peptides separated by RP chromatography and mv (A) (B) Columns: Sample: Elution: Flow rate: Detection: (A) TSKgel ODS-80TS, 4.6 mm ID x 2 cm L (B) TSKgel Amide-80, 4.6 mm ID x 2 cm L. PG; 2. LG;. FG; 4. EHP-NH2;. VGSQ; 6. GGYR; 7. WAGGDASGE; 8. DSDPR; (A) 0. % TFA/ACN, linear gradient of % - % ACN in 8. min (B) 0. % TFA/ACN, linear gradient of 97 % - % ACN in 70 min ml/min UV@2 nm

5 Tosoh bioscience ANALYSIS TSKgel Amide-80 TSKgel Amide-80 columns with small particle size ( µm) and a new high resolution type of TSKgel Amide-80 µm columns are the latest additions to the well-known TSKgel Amide-80 series. For years TSKgel Amide-80 columns are used successfully for separations of polar compounds, documented in more than 20 scientific publications. Packed with spherical silica particles that are covalently bonded with non-ionic carbamoyl groups (Figure ), they provide higher stability than conventional amino-phases and a unique selectivity. TSKgel Amide-80 µm columns reduce analysis time and improve peak capacity and sensitivity for both, HPLC and LC-MS analysis. An additional benefit of TSKgel Amide-80 for mass spectrometric as well as for evaporative light scattering detection is the virtual absence of column bleeding due to the covalently bonded functional groups. Separation of polar compounds Figure 4 shows the separation of sugar alcohols on a TSKgel Amide-80 µm column compared to a TSKgel Amide-80 µm column. Basically, the more hydroxyl groups in a compound the more polar it will be and the longer it will be retained on the column. figure Structure of TSKgel Amide-80 Comparison of the retention between mannitol and inositol, each with 6 hydroxyl groups, shows that inositol, which has a cyclic structure and lower solubility in the mobile phase is retained longer. Overall the µm column provides better resolution at reduced analysis time when compared to the µm TSKgel Amide-80 column. TSKgel Amide-80 long term stability The high stability of TSKgel Amide-80 columns is demonstrated in Figure showing the same analysis after 0, 660 and more than 000 runs compared to the first injection. Only % reduction of column performance (theoretical plates) is observed after more than 000 injections. figure 4 Separation of polyalcohols on TSKgel Amide-80 µm and µm figure Durability of TSKgel Amide-80 µm Mannitol TP=7884 AS=0.9 Mannitol TP=668 AS=.2 mv Inj. No. 08 Inj. No. 0 Inj. No. 660 TP =,264 (94,%) As f =.22 TP = 4,228 (0,%) As f =.8 TP = 4,024 (99,9%) As f = min Column: A) TSKgel Amide-80 µm (4.6 mm ID x cm L) B) TSKgel Amide-80 µm (4.6 mm ID x 2 cm L) Eluent: H 2 O/CH CN = 2/7 Flow rate:.0 ml/min Detection: Refractive index Temp.: 2 C Inj. volume : 0 µl Sample:. Ethyleneglycol 2. Glycerin. Erythritol 4. Xylitol. Mannitol 6. Inositol 00 0 Inj. No. TP = 4,04 As f = min Column: TSKgel Amide-80 µm (2.0 mm ID x cm L) Eluent : H 2 O/CH CN = /8 Flow rate : 0.2 ml/min Detection : UV@24 nm Temp. : 2 C Inj. volume : 2 µl Sample: Uracil (7 mg/l)

6 4 TSKgel NH2-00 TSKgel NH2-00 µm columns are the latest addition to the column family. They expand the selectivity range of TSK-GEL solutions by a new, robust amino-phase. In contrast to conventional silica-based amino phases the new column offers expanded stability under conditions. It is well suited for the analysis of all types of hydrophilic compounds like carbohydrates, peptides, vitamins, polar drugs or metabolites. figure 6 Structure of TSKgel NH2-00 The NH2-00 phase is based on a silica particle with µm particle and 00 Å pore size, treated with a special endcapping procedure. Amino groups are introduced step wisely after endcapping (Figure 6). The amino groups act as functional groups without any peak splits. Due to their high ligand density and large surface area TSKgel NH2-00 µm columns show high retention for very polar compounds. Separation of polar compounds Figure 7 shows the separation of a standard solution of water soluble vitamins on a TSKgel NH2-00 column compared to a TSKgel Amide-80 column. Dimension (4.6 mm ID x cm L), particle size ( µm), flow rate and mobile phase were identical for both columns. The elution order of the compounds changes when applying the same mobile phase to both columns: The TSKgel NH2-00 column shows stronger retention for nicotinic acid, vitamin C, and vitamin B2, while retention of vitamin B, B2, and pyridoxine is reduced. TSKgel NH2-00 long term stability The high stability of TSKgel NH2-00 columns is demonstrated in Figure 8 showing the change in retention time of inositol after more than 400 hours of flushing with mobile phase compared to the first injection. Only slight reduction of retention time is observed with the TSKgel NH2-00 column compared to a conventional amino-phase. figure 7 Separation of water soluble vitamins figure 8 Long term stability of TSKgel NH2-00 columns retention time [min] Columns: TSKgel Amide-80 µm, 4.6 mm ID x cm L TSKgel NH2-00 µm, 4.6 mm ID x cm L Eluent: 2 mm phosphate buffer (ph 2.)/ACN=0/70 Flow rate: ml/min Temp.: 40 C Detection: UV@24 nm Sample: Vitamin standard mixture: = Nicotinamide, 2 = Vitamin B2, = Pyridoxine, 4 = Nicotinic acid, = Vitamin C, 6 = Vitamin B, 7 = Vitamin B2 Injection: µl Column : TSKgel NH2-00 µm, 4.6 mm ID x cm L Conventional Amino column, 4.6 mm ID.x 2 cm L Eluent: H 2 O/ACN (2/7) Flow rate:.0 ml/min Detect: RI Temp.: 40 C Injection.: 0 µl Sample: Inositol

7 Tosoh bioscience ANALYSIS APPLICATIONS GLYCAN ANALYSIS Glycosilation is one of the most common post-translational modifications in eukaryotic cells. Complex N- and O-linked structures composed of repeating sugar moieties form the so called glycans. with fluorescence detection is the method of choice to effectively separate, identify and quantify glycans after exoglycosidase cleavage and fluorescent labelling. In order to normalize retention times of complex glycan structures a dextran ladder consisting of glucose oligomers is used as calibration reference. The calculated numbers of glucose units (GU) can be used in subsequent database queries (Glycobase, autogu) to predict the glycan structure. The selectivity of the new TSKgel NH2-00 series differs from TSKgel Amide-80 selectivity as shown in Figure 0. The type of column should be selected according to the sample type and separation need. If selectivity or regulatory requirements are not limiting the choice of columns we recommend selecting TSKgel Amide-80 columns instead of amino-phases because they show better long term stability. For years TSKgel Amide-80 μm columns have been used successfully in glycan analysis. Amide-80 chemistry is ideally suited for the separation of carbohydrate structures. With the new μm particles resolution and sensitivity can be further enhanced. Figure 9 shows the high-resolution separation of a 2-aminobenzamide (2AB) labeled dextran ladder within 0 minutes on a TSKgel Amide-80 µm column. figure 9 Separation of a 2AB-labeled Dextran Ladder on TSKgel Amide-80 figure 0 Separation of PA-Glycans on TSKgel NH (b) (a) retention time [min] Column: TSKgel Amide-80 ( μm, 2.0 mm ID cm L) Eluent: A) 0 mm Ammonium formate (ph 4.) B) Acetonitrile Gradient: 0- min - 7- % B Flow rate: 0.22 ml/min Detection: Fluorsecence Ex@60 nm, Em@42 nm Temperature: 0 C Injection vol.: µl Sample: CAB-GHP dextran ladder (Ludger; ~ 00 fmol for GU2) * Courtesy of K. Darsow & H. Lange, Institute of Bioprocessing, University of Nürnberg/Erlangen Column: (a): TSKgel NH2-00 µm, 4.6 mm ID x cm L (b): TSKgel Amide-80 µm, 4.6 mm ID x cm L Eluent: (a): (A): 0.2 M Triethylamine acetate (ph6.)/acn (0/70) (B): 0. M Triethylamine acetate (ph6.)/acn (60/40) (b): (A): 0.2 M Triethylamine acetate (ph6.)/acn (26/74) (B): 0.2 M Triethylamine acetate (ph6.)/acn (0/0) Gradient: 0% - 00% B in 0 min, hold at 00% B for min Flow rate:.0 ml/min Detect.: Fluorescence Ex@ nm, Em@80 nm Temp.: 40 C Inj. vol.: 0 µl

8 6 APPLICATION -MS High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has become a powerful tool when detection sensitivity is an issue. offers unique advantages for MS detection of very polar compounds when compared to reversed phase mode. The higher organic content of the eluent in mode supports efficient evaporation of the solvent thus enhancing sensitivity and altering ion suppression. separations are performed with gradients starting with high percentage of organic solvent and ending with a high portion of aqueous solvent - opposite to typical reversed phase gradients. The elution order of compounds is usually inversed as well. As a result polar compounds are very well separated according to increased polarity in mode. At the same time the portion of organic solvent in the mobile phase is relatively high. Figure shows the analysis of basic drug substances using a TSKgel Amide-80 µm column compared to the same analysis using a reversed phase TSKgel ODS-00V µm column. Ranitidine, a histamine H2 receptor antagonist, ondansetron, an antiemetic serotonin receptor antagonist, and labetalol, an alpha- and beta adrenergic blocker were selected to demonstrate the differences in selectivity and MS-signal response when applying different chromatographic modes. Ranitidine has the highest number of polar groups among these molecules and as a result shows the highest retention in and the lowest retention in RPC mode. Signal intensity is almost doubled for ranitidine in mode. For Labetalol a tenfold increase in signal height can be achieved by using instead of RPC. figure LC-MS/MS Analysis of basic drugs in and RPC mode Intensity, cps.0e Intensity, cps.00e.00e e e4 7.0 Intensity, cps.0e Intensity, cps.0e e 4.67.e4 7.2 Intensity, cps.0e Intensity, cps.0e Column: TSKgel Amide-80 µm (2.0 mm ID x cm L) Eluent : A: 0 mm Ammoniumformiate (ph.7) B: ACN Gradient : 0 min (B 90%) -> 0 min (B 40%) -> min (B 40%) Flow rate : 0.2 ml/min Inj. volume : µl (0 µg/l) Detection : QTrap LC-MS/MS (Applied Biosystems), ESI+ Column: : TSKgel ODS-00V µm (2.0 mm ID x cm L) Eluent : A: 0 mm Ammoniumformiate (ph.7) B: ACN Gradient : 0 min (B 0%) -> 0 min (B 80%) -> min (B 80%) Flow rate : 0.2 ml/min Inj. volume : µl (0 µg/l) Detection : QTrap LC-MS/MS (Applied Biosystems), ESI+

9 Tosoh bioscience ANALYSIS 7 APPLICATION DRUG METABOLITES The demand for separations in the analysis of drug substances is continuously increasing. Combined with tandem or hybrid mass spectrometric detection is a powerful separation mode for the analysis of polar metabolites in pharmacokinetics or metabolomics studies. Figure 2 shows the analysis of theophyline and its metabolites in serum after online deproteination, detected by UV absorption. Combining this separation with MS detection would further increase detection sensitivity and facilitate peak identification. figure 2 Separation of Theophyline and its Metabolites in Serum after online Deproteination DMU -MU retention time [min] Column: Analysis: TSKgel NH2-00 µm, 4.6 mm ID x cm L Deproteination: experimental BSA-ODS-00V precolumn 2.0 mm ID x cm L Eluent: Pretreatment; 0.2 M HCO 2 NH 4 (ph.6) 0-0. min A: ACN B: H 2 O/ACN=/8 C: 0.2 M HCO 2 NH 4 (ph.6)/acn=0/70 Step gradient: min A, min B, min C Flow rate:.0 ml/min, Detection: UV@24 nm, Temperature: 40 C Injection vol.: µl Sample: A: Standard. Caffeine (Cf), 2. Theobromine (Tb),.Theophyline (TP), 4. -Methylxanthine (-MX),. -Methylxanthine (-MX), 6.,-Dimethyluric acid (DMU), 7. -Methyluric acid (-MU) - 0 µg each B: Serum spiked with the standard samples

10 8 PRODUCT SPECIFICATION TSKgel Amide-80 TSKgel NH2-00 Base material Silica Silica Pore size 00 A 00 A Particle size µm, µm & 0 µm µm Functional group Carbamoyl Aminoethyl ORDERING INFORMATION Part # Description ID Length Particle Number Flow Rate (ml/min) Maximum (mm) (cm) Size (µm) Theoretical Range Max. Pressure Plates Drop (kg/cm 2 ) Stainless steel columns 2864 Amide , Amide , Amide , Amide , Amide Amide Amide , Amide , Amide , Amide , Amide , Amide , Amide , Amide , Amide , Amide , Amide , Amide , NH , NH , NH , NH ,000 0 Guard column products 2862 Amide-80 Guard cartridge, pk For 2.0 mm ID columns 286 Amide-80 Guard cartridge, pk.2. For 4.6 mm ID columns 294 Amide-80 Guard cartridge, pk For all 2.0 mm ID columns 902 Amide-80 Guard column For all 4.6 mm ID columns 900 Amide-80 Guard cartridge, pk.2. For all 4.6 mm ID columns 446 Amide-80 Guard column For 2. mm ID column 297 NH2-00 Guard cartridge, pk 2.0 For 2.0 mm ID columns 2972 NH2-00 Guard cartridge, pk.2 For 4.6 mm ID columns 908 Amide-80 Guard cartridge holder for 2.0 mm ID x.0 cm L guard cartridges 908 Amide-80 Guard cartridge holder for.2 mm ID x. cm L guard cartridges

11 TOSOH BIOSCIENCE ANALYSIS For detailed Toyopearl packing instructions, request our TOYOPEARL Instruction Manual. To get an overview about the whole range of TSKgel columns and small TOYOPEARL and TSKgel bulk media, please request our Chromatography catalog. For a deeper insight into applications and all questions related to the practical use of TSKgel and TOYOPEARL, check out the website and related catalogs or instruction manuals. Our technical experts are happy to discuss your specific separation needs by phone: +49 (0) or techsupport.tbg@tosoh.com

12 TOSOH BIOSCIENCE Im Leuschnerpark Griesheim, Germany Tel: Fax: info.tbg@tosoh.com BL44A