TsK-geL reversed phase columns

Transcription

1 TsK-geL reversed phase columns

2 Tosoh bioscience gmbh Zettachring 707 Stuttgart Germany Tosoh bioscience LLc keystone drive montgomeryville, pa 89-97, USA Tosoh corporation Tosoh SHANGHAI. LTD -8- Shiba, Minato-Ku RM 8A, INTERN. TRADE ENTER, TKY 0-8 N. 0 YAN-AN WEST RAD SHANGHAI, 00 japan HINA t + 9 (0) f + 9 (0) info.sep.eu@tosoh.com t f info.tbl@tosoh.com t f info@tosoh.co.jp t f PAN@TSH.M.N TSH HISTRY 9 Founding of Toyo Soda Manufacturing o., Ltd. 9 peration of Nanyo Manufacturing omplex begins 97 Scientific Instruments Division formed, First GP column using TSK-GEL developed by Tosoh 97 High performance liquid chromatography column plant Is completed 979 Tosoh develops TYPEARL 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 IS In November former TosoHaas US operations becmes TSH BISEP LL, a 00% subsidiary of Tosoh orporation 00 In January former TosoHaas GmbH European perations becmes TSH BISEP GmbH, a 00% subsidiary of Tosoh orporation 00/ Tosoh orporation announces that all Tosoh affiliated scientific and diagnostic system 00 related companies in Europe, will be unified under the new name TSH BISIENE. 008 ESE, THE 7TH GENERATIN GP SYSTEM IS INTRDUED GLBALLY 009 0th anniversary of tosoh bioscience gmbh, stuttgart

3 Tosoh bioscience ANALYSIS REVERSED PHASE HRMATGRAPHY Reversed Phase (RP) hromatography is one of the most frequently used chromatographic modes for analytical separations. Starting in the mid 970s has become the standard technique to analyze small molecular weight compounds in industrial, academic and governmental laboratories. Applications range from neutral polar and non-polar solutes to acidic, basic, and amphoteric compounds and from small molecular weight compounds to biomolecules. is also an efficient technique for the analysis of derivatized amino acids, peptides, and proteins, although protein structure is not always maintained due to the high concentration of organic solvent required for their elution. Tosoh Bioscience offers analytical and semi preparative reversed phase (RP) HPL columns packed with silica or polymer based porous or nonporous beads. They are well suited for a broad range of applications in R&D, quality control or reaction monitoring. TSK-GEL DS-80, DS-00 and DS-0 silica based columns offer high resolution power for various applications. For high-speed separations we recommend the porous, silica-based TSK-GEL Super and DS-0HTP series or the nonporous, polymeric NPR columns. For better stability at high ph, or to benefit from alternative selectivity and a large pore size, we recommend polymer-based TSK- GEL columns. For very polar solutes, which are difficult to retain in RP mode, we offer a selection of silica based HILI columns, which are described in a separate brochure. Tosoh orporation employs state-of-the-art manu-facturing techniques that result in uniformly bonded packing materials with narrow pore size distributions and well-defined particle sizes to ensure high performance at high speed. TSK-GEL reversed phase columns enable the chromatographer to solve the most complex separation problems.

4 HW IT WRKS Reversed phase (RP) chromatography retains molecules based on their hydrophobic character on a non-polar stationary phase. In an aqueous, moderately polar solvent the hydrophobic patches of the analyte molecule bind to an immobilized hydrophobic ligand. A mobile phase of increasing hydrophobicity is used to release the bound molecule at a point at which the interaction between the exposed patches and the matrix is less favorable than the interaction between the molecule and the solvent. The molecule releases from the matrix and elutes. Retention can be decreased by adding less polar solvents (methanol, acetonitrile) to the mobile phase. Elution can be performed either in isocratic or gradient mode. Isocratic elution is easy to realize, less expensive and allows solvent recycling. Gradient elution the continuous reduction of polarity of the aqueous mobile phase by increasing percentage of organic solvent - delivers sharper peaks and faster separation. The binding of the analyte to the stationary phase is proportional to its hydrophobic surface area. Structural properties of the analyte therefore play an important role for reversed phase retention. Large hydrophobic surface areas increase retention whereas polar groups reduce retention. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. Very large molecules can show incomplete interaction between the large analyte surface and the alkyl chains of the stationary phase and might have problems entering the pores of the stationary phase. RP separation of peptides and proteins is usually performed by adding the volatile ionic modifier trifluoroacetic acid (TFA) to the mobile phase for ion pairing. Addition of TFA overcomes peak broadening and asymmetry (tailing) that are believed to result from interactions of peptides and proteins having a variety of polar, ionic, and hydrophobic sites with residual polar silica surfaces. For RP L/MS analysis formic acid or ammonium formate are the most common modifiers. For polar compounds, which are not retained on standard RP columns, HILI could be the solution. A separate HILI brochure is covering the features of TSK-GEL HILI columns. figure Reversed Phase hromatography Sample application H low salt/low organic Elution H/organic modifier Isocratic or gradient with increasing solvent concentration

5 Tosoh bioscience ANALYSIS STATINARY PHASES lica Based Stationary Phases lica particles functionalized with straight chain alkyl groups such as 8, 8 or or with aromatic groups such as phenyl are the most widely used RP stationary phases. Their high mechanical stability, mono disperse particles, high surface area, and easily tailored pore size distributions are the advantages of silica phases. lica bonding chemistry also allows for a variety of stationary phases with different selectivities. Surface functionalization of silica can be performed in a monomeric or a polymeric reaction. Nowadays, residual silanol groups of the matrix are usually endcapped with covalently-bound small organic silanes. Polymer Based Stationary Phases Polymer based RP columns are compatible with a wide range of mobile phase conditions. nce these columns are chemically stable from ph -, they allow robust, reproducible operation at basic ph where silica-based columns have limited chemical stability. Reversed Phase Selectivity Subtle differences in the surface chemistries of different RP stationary phases lead to changes in selectivity. A broad range of RP phases is available to meet specific separation needs. TABLE gives a quick overview of some of the features and benefits of silica- and polymer-based TSK-GEL columns. TABLe Properties of lica- and Polymer based TSK-GEL olumns TSK-GEL SILIA Bonding phase Functional group Endcapping Particle size (µm) arbon load Pore size (Å) Excl. limit (kda) Application/ Features DS-00V monomeric 8 complete % 00 8 DS-00Z monomeric 8 complete 0% 00 8 DS-0HTP polymeric 8 complete. % 0 0 Super-DS polymeric 8 complete 8% 0 0 Super-ctyl polymeric 8 complete % 0 0 Super-Phenyl polymeric H complete % 0 0 ligodna RP monomeric 8 none 0% 0 Higher surface polarity, compatible to 00% aqueous eluents, higher retention of polar compounds More hydrophobic than DS-00V; stronger retention and higher selectivitiy for non-polar compounds; higher steric selectivity High-throughput analyses of hydrophilic or hydrophobic peptides, tryptic digests/ peptide mapping, low MW pharmaceuticals, purines and pyrimidines, nucleosides, nucleotides TMS-0 monomeric complete 0 % 0 00 Specialty column for protein separations PLYMER Specialty column for analysis and preparative purification of oligonucleotides, RNA and DNAfragments ctadecyl- PW ctadecyl- PW Phenyl-PW RP ctadecyl- NPR monomeric monomeric monomeric H - 0 -, monomeric Nonporous >.000 Low MW peptides and pharmaceuticals unstable at low ph Medium and high MW peptides and proteins especially if unstable at low ph High MW peptides and proteins.phenyl group modifies selectivity Rapid separation of high MW peptides and proteins

6 TSK-GEL REVERSED PHASE LUMNS Tosoh Bioscience offers distinct reversed phase column types, either based on silica or methacrylate particles. For the development of new methods we recommend TSK-GEL DS-00 or fast L columns such as TSKgel DS-0HTP. Traditional, silica based TSK-GEL DS-80 and DS-0 columns are not described in detail in this brochure. Particle Design Tosoh manufactures porous and nonporous spherical packing materials for liquid chromatography covering silica as well as polymer-based particles from as small as µm to as large as 00 µm. The nomenclature for TSK-GEL columns is based on the bonded phase characteristics or on the application the column was designed for. Bonded Phase hemistry DS stands for octadecylsilyl groups attached to silica particles. D refers to an octadecyl carbon chain attached to a polymer-based resin. TMS indicates a primary bonding with trimethylsilyl groups. Phenyl, ctyl, N indicate stationary phases containing phenyl, octyl, or cyano groups. Specialty Phases ligodna RP is designed for analysis of oligo-nucleotides and DNA fragments. The Super and the DS-0HTP series of small particle size columns are designed for fast analysis and high throughput screening. NPR columns are packed with. µm nonporous resin (NPR) suitable for high speed analysis of biopolymers. TABLe olumn Selection for TSK-GEL Reversed Phase olumns Sample Solubility Sample type Example olumn omment rganic Soluble Water Soluble Lipophilic Steroids, fat soluble vitamins Polyaromatic hydrocarbons N-80TS, DS-00V DS-0A EPA method 0 Low MW Nonionic Water soluble vitamins DS-80TS, DS-00V/Z Ionic, ph > Sulfonic acids DS-80TM, DS-00V Purines, pyrimidines nucleosides, nucleotides DS-80TS, DS-00V Ionic, ph < 9 Basic drugs DS-80TS, DS-00V Ionic, ph > 9 Pharmaceuticals ctadecyl-pw Polymer based Medium MW ligomers ligosaccharides DS-80TS, DS-00V Aqueous mobile phase Peptides DS-80TM, DS-00V 00,000 Da ctadecyl-pw DS-0T Super-DS/ctyl/Phenyl, DS-0HTP High MW Proteins ctadecyl-npr TMS-0 ctadecyl-pw Phenyl-PW RP 00 8,000 Da 00 0,000 Da 00 0,000 Da,000,000,000 Da 00 00,000 Da,000 00,000 Da 0,000,000,000 Da ligonucleotides ligodna RP Up to,000 Da

7 Tosoh bioscience ANALYSIS TSK-GEL DS-00 UNIVERSAL RP LUMNS TSKgel DS-00V and TSKgel DS-00Z columns incorporate best-in-class surface properties to limit secondary interactions of basic, acidic and chelating compounds. They offer high efficiency and symmetrical peak shapes. The ultra high purity Type B base silica combined with monomeric bonding chemistry makes the best general purpose RP columns. TSK-GEL DS-00 columns are suitable for demanding separations in quality control as well as in R & D. TSKgel DS-00V TSKgel DS-00V columns are general purpose columns providing strong retention and high selectivity for polar solutes. Based on unique, highly efficient bonding and endcapping procedures, secondary interactions of basic, acidic, and chelating compounds are limited. Monomeric bonded phase chemistry provides complete wetting and retention stability in 00% aqueous mobile phase. The bonded phase is prepared by an incomplete first reaction with a difunctional octadecylsilane reagent, followed by endcapping with a mixture of two difunctional dialkylsilane reagents (Figure ). TSKgel DS-00Z TSKgel DS-00Z RP columns are a great choice when a change of selectivity from TSKgel DS-00V columns is needed. They contain a high density monomeric 8 bonded phase (Figure ) for maximum retention and selectivity of small molecular weight compounds. Exhaustive endcapping prevents secondary interaction with residual silanol groups. The TSKgel DS-00Z phase is prepared by a first reaction with a difunctional octadecylsilane reagent, followed by repeated endcapping with monofunctional trimethylsilane reagent. ontaining a high carbon content of 0 %, TSKgel DS-00Z columns exhibit a high stability at low ph. They provide longer retention for non-polar compounds than TSKgel DS-00V columns. Steric selectivity is higher as well for DS-00Z. TSKgel DS-00 columns are available in µm and µm particle size. µm columns, providing fast and highly efficient separation, are well suited for L/MS applications. figure Structure of TSKgel DS-00V figure Structure of TSKgel DS-00Z H H Difunctional endcapping reagents R R H R R H Residual silanol H Monofunctional endcapping reagent H H Residual silanol H H H H H H H H H H H

8 TSK-GEL DS-00 V/Z FEATURES Bonded Phase haracterization Standard Reference Material SRM 870 was developed by NIST (National Institute of Standards and Technology) as a means to classify reversed phase columns into closely related groups. Amitriptyline, a tertiary amine, and quinizarin, a strong chelating compound, are in SRM 870, together with other compounds. As shown in Figure, symmetrical peaks are obtained on both DS-00 columns for all compounds of SRM 870. Note the good peak shape for quinizarine (peak ) and for the basic amitriptyline (peak ). These results indicate the very low amount of non specific interactions of TSK-GEL DS-00 columns with chelating compounds and organic bases, respectively. Lot-to-Lot Reproducibility Figure shows the separation of SRM870 test mixture using bonding lots of TSKgel DS-00Z columns prepared from different base silica lots. The results show no marked differences among the chromatograms, confirming minimal lot-to-lot variability and high consistency of the manufactured packing material. TSKgel DS-00V TSKgel DS-00Z Matrix ultra-pure silica ultra-pure silica Particle size µm, µm µm, µm Pore size 00 Å 00 Å Specific surface area 0 m /g 0 m /g Functional group 8 8 arbon content % 0% Bonding phase monomeric monomeric Endcapping yes yes Sample type polar hydrophobic MW limit 0,000 Da 0,000 Da ph stability Temperature range Preferred sample type Polar, basic, acidic and chelating Non-polar figure Separation of SRM 870 figure TSKgel DS-00Z Lot-to-lot Variability mv A) B) Retention Time (min) olumns: (A) TSKgel DS-00V µm (. mm ID x cm L) (B) TSKgel DS-00Z µm (. mm ID x cm L) Eluent: 0 mmol/l Phosphate buffer (ph 7.0) /MeH (0/80) Flow rate :.0 ml/min Detection: nm Temp: 0 Inj. volume: 0 µl Sample:. TSKgel DS-00V.. Uracil,. Toluene,. Ethyl benzene,. Quinizarin,. Amitriptyline. AF=0.99. AF=.0 TSKgel DS-00Z Retention time (minutes) TSKgel DS-00Z, µm,. mm ID x cm L Eluent: 0 mmol/l phosphate buffer (ph 7.0)/MeH = 0/80 Flow rate:.0 ml/min Detection: UV@ nm Temperature: 0 Injection vol.: 0 µl Samples:. uracil. toluene. ethyl benzene. quinizarin. amitriptyline DS Lot F, Base silica Lot DS Lot E, Base silica Lot B DS Lot D, Base silica Lot A DS Lot, Base silica Lot A DS Lot B, Base silica Lot A DS Lot A, Base silica Lot A

9 Tosoh bioscience ANALYSIS 7 TSK-GEL DS-00V APPLIATIN DATA Separation of rganic Acids rganic acids play an important role in many metabolic processes, fermentation, and food products. TSKgel DS- 00V columns can be operated at low ph conditions in 00% aqueous mobile phases. These conditions are ideal for the separation of a broad range of organic acids. Figure shows a baseline separation (UV@ 0 nm) of organic acids on TSKgel DS-00V in less than minutes using a simple 0.% phosphoric acid mobile phase. Separation of Vitamins Water and lipid soluble vitamins were separated in a single run on a TSKgel DS-00V column as demonstrated in Figure 7. The sample is a mixture of vitamins ranging from the very polar water-soluble vitamin ascorbic acid to the very hydrophobic tocopherol derivatives. Polar vitamins elute in the beginning of the chromatogram under aqueous or low organic mobile phase conditions. A steep gradient from 0% AN to 00% AN is initiated from 0 to minutes to elute retinol and the tocopherols. The TSKgel DS-00V column provides high resolution for polar compounds, while at the same time delivers short analysis time for late eluting nonpolar compounds. figure Separation of rganic Acids figure 7 Separation of Vitamins 0 00 Intensity (mv) Intensity (mv) Retention time (minutes) TSKgel DS-00V, µm,. mm ID cm L Eluent: 0.% H P, ph. Flow rate:.0 ml/min Temperature: 0 Injection vol.: 0 µl Detection: 0 nm Samples:. oxalic acid (0. mg/ml). L-tartaric acid (0. mg/ml). formic acid (.0 mg/ml). L-malic acid (.0 mg/ml). L-ascorbic acid (0. mg/ml). lactic acid (.0 mg/ml) 7. acetic acid (.0 mg/ml) 8. maleic acid (0.0 mg/ml) 9. citric acid (.0 mg/ml) 0. succinic acid (.0 mg/ml). fumaric acid (0.0 mg/ml). acrylic acid (0. mg/ml). propionic acid (.0 mg/ml). glutaric acid (.0 mg/ml). itaconic acid (0.0 mg/ml) Retention time (minutes) TSKgel DS-00V, µm,. mm ID x cm L Eluent: A) 0.% TFA in H B) 0.% TFA in AN Flow rate:.0 ml/min Detection: 80 nm Temperature: 0 Injection vol.: µl Gradient: 0 min (0%B), 0 min (0%B), min (00%B), 0 min (00%B) Samples:. L-ascorbic acid. nicotinic acid. thiamine. pyridoxal. pyridoxine. caffeine 7. riboflavin 8. retinol 9. δ-tocopherol 0. α-tocopherol. α-tocopherol acetate

10 8 TSK-GEL DS-00V APPLIATIN DATA Separation of Nucleotides The separation of mono-, di-, and triphosphorylated nucleotides on a TSKgel DS-00V column is shown in Figure 8. The separation is accomplished by adding a short chain ion pairing agent, t-butylamine, and adjusting the mobile phase ph to ph.8. Separation of Tryptic Peptides The rapid identification of 0 peptides using a TSKgel DS-00V column is detailed in Figure 9. The high speed analysis and symmetrical peaks of basic compounds in low concentration ammonium formate buffer make this column an excellent choice for L/MS work. figure 8 Separation of Nucleotides figure 9 Separation of Peptides by L/MS TSKgel DS-00V (. mm ID cm L) Mobile phases: A) 0 mmol/l t-butylamine + H P (ph.8) B) A/MeH (90/0) Gradient: 0 min (B: 0%) -- min (B: 00%) Flow rate:.0 ml/min Temp: Detection: 0 nm Inj. Volume: µl oncentration: 0. g/l each Samples: Retention time (min). MP,. UMP,. DP,. dump,. GMP,. IMP, 7. UDP, 8. TP, 9. TMP, 0. GDP,. IDP,. AMP,. UTP,. dgmp,. TDP,. GTP, 7. ITP, 8. ADP, 9. TTP, 0. damp,. ATP Intensity (cps).x0.0x0.0x m/z :.0 : 09.0 : 7. : 87.8 : 9.9 : 7. 7:. 8: 7. 9: 9.0 0: 88.9 : 7.0 : : 0.0 : 9.7 : 8.0 : : 0. 8: 8.0 9: 9.0 0: Time (minutes) TSKgel DS-00V, µm,.0 mm ID x cm L Eluent: A: 0.% TFA in H, B: 0.% TFA in AN Flow rate: 0. ml/min Injection vol.: µl Gradient: 0 min (0%B), min (70%B), 7 min (70%B) Sample: ß-lactoglobulin tryptic digest Instrument: Q TRAP, ESI+ 9 0

11 Tosoh bioscience ANALYSIS 9 TSK-GEL DS-00Z APPLIATIN DATA Separation of Polyphenols atechins, which are found in large quantities in tea, are polyphenols. atechins have been extensively studied for their antioxidant properties. Figure 0 demonstrates the baseline separation of six catechins in the presence of caffeine on a cm TSKgel DS-00Z column. Separation of Tetracycline Antibiotics A cm TSKgel DS-00Z column was evaluated for its selectivity for a mixture of tetracycline-like chemical structures. Tetracycline is an impurity in oxytetracycline formulations. The two compounds have very similar structures and separation is difficult. As demonstrated in Figure, a TSKgel DS-00Z column provides superior resolution for oxytetracycline (peak ) and tetracycline (peak ) within the mixture. figure 0 Separation of atechins figure Separation of Tetracycline Retention time (minutes) Retention time (minutes) TSKgel DS-00Z, µm,. mm ID x cm L TSKgel DS-00Z, µm,. mm ID cm L Eluent: A: 0 mmol/l KH P (ph.) B: H H Flow rate:.0 ml/min Detection: 70 nm Temperature: 0 Gradient: 0 min (8%B), min (0%B) Injection vol.: µl Samples: : (-)-epigallocatechin (7 mg/l) : (-)-catechin (87 mg/l) : (-)-epigallocatechin gallate ( mg/l) : caffeine (7 mg/l) : (+)-epicatechin (87 mg/l) : (-)-epicatechin gallate ( mg/l) 7: (-)-catechin gallate ( mg/l) Eluent: Flow rate: Detection: Temperature: Injection vol.: Samples: 0 mmol/l formic acid/acetonitrile=8/7.0 ml/min nm 0 0 µl. tetracycline derivative. oxytetracycline (0 mg/l). tetracycline (0 mg/l). doxycycline derivative. chlortetracycline derivative. chlortetracycline (0 mg/l) 7. doxycycline (0 mg/l)

12 0 SUB- µm, HIGH THRUGHPUT RP LUMNS TSKgel DS-0HTP and TSKgel Super Series reversed phase columns are based on small. µm silica particles. They provide high resolution and short analysis times at moderately high pressures. TSKgel DS-0HTP TSKgel DS-0HTP columns were designed for use with either UHPL or conventional HPL systems. The backpressure of a TSKgel DS-0HTP column is less than half of the pressure of a.7 µm particle size column of the same dimensions. The polylayer bonding chemistry of TSKgel DS-0HTP columns results in highly efficient and physically stable columns when operated at high flow rates under high pressure. High efficiency and shorter retention make these columns an optimal fit for high throughput separations including drug discovery, pharmacokinetics and peptide digest separations. TSK-GEL Super Series TSKgel Super-DS, Super-ctyl and Super-Phenyl RP columns are based on monodisperse spherical. µm silica particles bonded with, respectively, 8, 8, and phenyl functional groups. The bonded phases have a polymeric structure. An exhaustive endcapping minimizes the presence of residual silanol groups. TSK-GEL Super series RP columns are recommended for small molecular weight compounds (<0 kda) such as peptides, amino acids, nucleotides, and small organic molecules. In order to fully exploit all benefits of small particle size stationary phases and to achieve optimum resolution it is highly recommended to optimize the HPL system with respect to low dead volume, fast detector response and high sampling rates. TABLe Properties of TSK-GEL sub- µm Reversed Phase olumns TSKgel DS-0HTP TSKgel Super DS TSKgel Superctyl TSKgel SuperPhenyl Matrix silica silica silica silica Particle size. µm. µm. µm. µm Pore size 0 Å 0 Å 0 Å 0 Å Endcapping yes yes yes yes Functional group Phenyl % arbon 8 ph stability Max. pressure 00 kg/cm 0/00 kg/cm 0/00 kg/cm 0/00 kg/cm Temperature range

13 Tosoh bioscience ANALYSIS TSK-GEL DS-0HTP APPLIATIN DATA System Requirements Sub- µm columns can be used on a regular HPL system if the dead volume is minimized. The following recommendations help the user to achieve optimum results with sub- µm columns: Use guard filters to reduce particulate contaminations. Injection volume should be as low as possible (<0 µl). To ensure minimal extra-column volume, keep tubing (< 0. mm ID) as short as possible. Use the smallest detector time constant and highest sampling rate. Use semi-micro or micro detector flow cell (< µl volume) Performance Data TSK-GEL DS-0HTP columns operate at lower pressure than competitive sub- μm columns (Figure ). The pressure drop of a cm TSKgel DS-0HTP column at cm/min is less than half of the pressure of smaller particle size competitive columns. Not surprisingly, the pressure drop over a 0 cm TSKgel DS-0HTP column was still lower than any of the competitive sub- µm cm columns. Separation of Herbal Extracts In hinese traditional medicine, an extract of rinum latifolium L is used to invigorate blood circulation. It is thought to possess antiviral and immunostimulative properties and shows immunomodulatory properties in human peripheral blood mononuclear cells. The analysis of products derived from plant extracts is a challenging chromatographic task. Due to the high number of components, the column needs to provide high peak capacity. As shown in Figure, the TSKgel DS-0HTP column is an excellent choice for plant extract separations. figure omparison of Pressure Drop figure Separation of rinum Latifolium L DS-0HTP ( cm) DS-0HTP (0 cm) ompetitor column A ompetitor column B ompetitor column Pressure (MPa) Linear velocity (cm/min) TSKgel DS-0HTP,. µm,.0 mm ID x cm L TSKgel DS-0HTP,. µm,.0 mm ID x 0 cm L ompetitor column A,.7 µm,. mmid x cm L ompetitor column B,.8 µm,. mm ID x cm L ompetitor column,.9 µm,. mm ID x cm L Eluent: H /AN=0/0 Detection: nm Temperature: Injection vol.: µl Sample: naphthalene Retention time (minutes) TSKgel DS-0HTP,. µm,. mm ID x 0 cm L Instrument: Acquity UPL System with TUV detector Eluent: A: H B: AN Flow rate: 0. ml/min Detection: 0 nm Temperature: Injection vol.: µl Gradient: 0 min (% B), 0.08 min (% B), 7.7 min (0% B),. min (00% B),. min (00% B),. min (% B) Sampling rate: 80 Hz Sample: 0 g/l extract of rinum latifolium L by 9% ethanol

14 TSK-GEL SUPER-SERIES APPLIATIN DATA Separation of Amino Acids The baseline separation of 8 PT-derivatized amino acids in minutes on a TSKgel Super-DS column is shown in Figure. omparison of Selectivity The different selectivity of Super-DS, Super-Phenyl and Super-ctyl columns is illustrated in Figure for the analysis of a mixture of six neuropeptides in less than one minute. figure omparison of Selectivity [mv] A. TSKgel Super-DS [mv] Minutes B. TSKgel Super-ctyl figure Separation of PT Amino Acids 0 00 (mv) Minutes [mv] 0 0. TSKgel Super-Phenyl 0 Minutes olumn: TSKgel Super-DS (. mm ID x 0 cm L) Sample:. Asp,. Glu,. Ser,. Gly,. His,. Arg, 7. Thr, 8. Ala, 9. Pro, 0. PT-NH,. Try,. Val,. Met,. ys,. Ile,. Leu, 7. Phe, 8. Lys Elution: a. AN/0 mm acetate buffer (ph.0)=/97 b. AN/H =0/0 Flow Rate:. ml/min Detection: nm Injection: µl (0 pmol) Temperature: ambient olumn: Sample: 0..0 Minutes Each. mm ID x cm L. oxytocin;. α-endorphin;. bombesin;. leu-enkephalin;. γ-endorphin;. somatostatin Elution: Buffer A. mm Hl ; Buffer B. mm Hl /H N = 0/80; % B to 80% B in a min linear gradient Flow rate:.0 ml/min Detection: 0 nm

15 Tosoh bioscience ANALYSIS TSK-GEL RP LUMNS FR PRTEIN ANALYSIS lica Based TSKgel TMS-0 reversed phase columns are based on µm, 0 Å pore size silica particles functionalized with trimethylsilyl groups. It allows unhindered access by large biomolecules and is therefore ideally suited for the analysis of proteins. Due to the low hydrophobicity of the ligand, excellent recoveries are common even when used with large proteins. Proteins such as adolase (8 kda) exhibit sharp peaks relative to wide pore 8 or 8 columns (Figure ). Although standard nomenclature designates the bonded phase of TSKgel TMS-0 as, its degree of hydrophobicity and selectivity is similar to those reversed phase columns, frequently used for protein separations. Polymer Based TSKgel ctadecyl-pw is based on 7 or µm particle size, polymeric resin with 00 Å pores. The highly cross-linked polymethacrylate base material provides excellent stability in high ph buffers and can withstand rigorous cleaning with either acid or base. The 00 Å pore allows for the analysis of proteins up to 00 kda while the particle size offerings allow for analytical and semi-preparative scale separations. TSKgel Phenyl-PW is based on 0 µm particle size polymethacrylate resin with 000 Å pores. The 000 Å pore size accommodates globular protein samples up to,000,000 Da (Figure 7). figure High Resolution Protein Separation on TSKgel TMS-0 TSK-gel TMS-0 figure 7 Purification and Puritiy heck of Proteins A. Purification B. Purity check Retention times (minutes) Sample: Elution: Flow Rate: Detection: 0 0 Minutes TSKgel TMS-0,. mm ID x 7. cm L µg g each of:. ribonuclease A,. cytochrome,. lysozyme,. bovine serum albumin,. aldolase,. carbonic anhydrase, 7. ovalbumin 0 min (TMS-0) linear gradient from 0% to 9% H N in 0.0% TFA, ph. 0. ml/min 0 nm TSKgel Phenyl-PW RP, 0 µm,. mm ID x 7. cm L Flow rate:.0 ml/min Detection: 0 nm Elution: min linear gradient from % to 0% AN in 0.0% TFA, followed by (A - 8 min/b - min) linear gradient to (A - 80%/B - 0%) AN in 0.0% TFA Sample: lactate dehydrogenase (700 kda) A. 0 µg in 00 µl B. purity check of fraction collected in part A

16 TSK-GEL PLYMER BASED RP LUMNS Polymer-based reversed phase columns offer the best solution for high ph separations. They are chemically stable from ph to, allowing operation at basic ph, where silica-based columns have limited chemical stability. The wider ph range also allows many basic compounds to be analyzed in their uncharged form, thus reducing secondary adsorption and improving peak shape. TSK-GEL polymerbased columns deliver improved recovery for peptides and proteins due to reduced secondary interactions. TSKgel ctadecyl-pw The highly cross-linked polymethacrylate base material of TSKgel ctadecyl-pw columns provides excellent stability in high ph buffer systems (Figure 8). They can withstand rigorous cleaning with either acid or base. The Å pores allow for analysis of peptides up to 8,000 Da. TSKgel ctadecyl-pw TSKgel ctadecyl-pw is based on a similar polymethacrylate base material but offering larger pore size (00 Å), suitable for analysis of proteins up to 00 kda (Figure 9). TSKgel ctadecyl-npr TSKgel ctadecyl-npr is based on small particles size (. µm), non porous polmethacrylate resin. It offers high resolution reversed phase separation of biomolecules at short analysis times. TSKgel ligodna RP TSKgel ligodna RP contains a monomeric 8 bonded phase and was designed for the analysis of oligonucleotides, RNAs and DNA fragments (up to 00mer). figure 9 Separation of Peptides at Acidic, Neutral or Basic ph on TSKgel ctadecyl-pw B 7 figure 8 Separation of Tricyclic Antidepressant Drugs on TSKgel ctadecyl-pw A TSKgel ctadecyl-pw 0 0 Time, minutes 0 0 Time, minutes 7 olumn: Sample: Minutes TSKgel ctadecyl-pw,. mm ID x cm L. desipramine,. imipramine,. amitriptyline,. trimipramine Elution: 0mM phosphate buffer (ph.0)/ acetonitrile, 0/0 Flow rate: A. 0. ml/min, B..0 ml/min Detection: nm Temp.: Ambient 8 0 Time, minutes olumn: TSKgel ctadecyl-pw,. mm ID x cm L Sample: -0 g each of:. Met-Enkephalin,. Bradykinin,. Leu-Enkephalin,. Neurotensin,. Bombesin,. Angiotensin, 7. Somatostatin, 8. Insulin Solvent progr.: 0 min linear gradient from 0% to 80% acetonitrile in: A: 0.% TFA (ph.9) B: 0 mm sodium phosphate (ph 7.) : 00 mm ammonia (ph 0.8) Flow rate:.0 ml/min Detection: nm Temperature:

17 Tosoh bioscience ANALYSIS RDERING INFRMATIN RDERING INFRMATIN Part # Description ID Length Particle Number Flow Rate (ml/min) Maximum (mm) (cm) ze (µm) Theoretical Range Max. Pressure Drop Plates (kg/cm ) Stainless steel columns 88 DS-00V, 00 Å.0., DS-00V, 00 Å.0.0, DS-00V, 00 Å, pk * -NEW DS-00V, 00 Å -NEW-.0.0, DS-00V, 00 Å.0., DS-00V, 00 Å.0.0, DS-00V, 00 Å , DS-00V, 00 Å.0 0.0, DS-00V, 00 Å.0.0 7, DS-00V, 00 Å -NEW , DS-00V, 00 Å -NEW-.0.0, DS-00V, 00 Å -NEW-.0., DS-00V, 00 Å.0.0, DS-00V, 00 Å , DS-00V, 00 Å.0 0.0, DS-00V, 00 Å.0.0 8, DS-00V, 00 Å -NEW , DS-00V, 00 Å -NEW-..0, DS-00V, 00 Å -NEW-.., DS-00V, 00 Å..0, DS-00V, 00 Å. 7. 9, DS-00V, 00 Å. 0.0, DS-00V, 00 Å..0 9, DS-00V, 00 Å -NEW-..0 0, DS-00V, 00 Å.0.0, DS-00V, 00 Å -NEW-.0 7., DS-00V, 00 Å -NEW , DS-00V, 00 Å.0.0, DS-00V, 00 Å -NEW , DS-00V, 00 Å -NEW-.0.0, DS-00V, 00 Å -NEW-.0., DS-00V, 00 Å -NEW-.0.0, DS-00V, 00 Å -NEW-.0 7., DS-00V, 00 Å -NEW , DS-00V, 00 Å -NEW-.0.0, DS-00V, 00 Å -NEW-.0.0, DS-00V, 00 Å -NEW-..0, DS-00V, 00 Å -NEW-.., DS-00V, 00 Å -NEW-..0, DS-00V, 00 Å -NEW , DS-00V, 00 Å -NEW , DS-00V, 00 Å..0, DS-00V, 00 Å..0, DS-00Z, 00 Å, pk * -NEW DS-00Z, 00 Å -NEW-.0.0, DS-00Z, 00 Å -NEW-.0., DS-00Z, 00 Å -NEW-.0.0, DS-00Z, 00 Å -NEW , DS-00Z, 00 Å -NEW , DS-00Z, 00 Å -NEW , DS-00Z, 00 Å -NEW , DS-00Z, 00 Å -NEW-.0.0, DS-00Z, 00 Å -NEW-.0., DS-00Z, 00 Å -NEW-.0.0, DS-00Z, 00 Å -NEW , DS-00Z, 00 Å -NEW , DS-00Z, 00 Å -NEW , DS-00Z, 00 Å -NEW , DS-00Z, 00 Å -NEW-..0, DS-00Z, 00 Å -NEW-.., DS-00Z, 00 Å -NEW-..0, DS-00Z, 00 Å -NEW , DS-00Z, 00 Å -NEW-. 0.0,00 0 *needs cartridge holder

18 RDERING INFRMATIN Part # Description ID Length Particle Number Flow Rate (ml/min) Maximum (mm) (cm) ze (µm) Theoretical Range Max. Pressure Drop Plates (kg/cm ) 7 DS-00Z, 00 Å -NEW-..0 9, DS-00Z, 00 Å -NEW-..0 0, DS-00Z, 00 Å, pk * -NEW DS-00Z, 00 Å -NEW-.0.0, DS-00Z, 00 Å -NEW-.0., DS-00Z, 00 Å.0.0, DS-00Z, 00 Å -NEW-.0 7., DS-00Z, 00 Å -NEW , DS-00Z, 00 Å.0.0, DS-00Z, 00 Å -NEW , DS-00Z, 00 Å -NEW-.0.0, DS-00Z, 00 Å -NEW-.0., DS-00Z, 00 Å -NEW-.0.0, DS-00Z, 00 Å -NEW-.0 7., DS-00Z, 00 Å -NEW , DS-00Z, 00 Å -NEW-.0.0, DS-00Z, 00 Å -NEW-.0.0, DS-00Z, 00 Å -NEW-..0, DS-00Z, 00 Å -NEW-.., DS-00Z, 00 Å -NEW-..0, DS-00Z, 00 Å -NEW , DS-00Z, 00 Å -NEW , DS-00Z, 00 Å..0, DS-00Z, 00 Å..0, Guard column products 997 DS-00V, Guardgel artridge, pk.0.0 For all µm DS-00V mm ID columns DS-00V Guard artridge, pk.. For all DS-00V. mm ID columns 8 DS-00Z Guard artridge, pk.0.0 For all DS-00Z mm ID columns DS-00Z Guard artridge, pk.. For all DS-00Z. mm ID columns 99 DS-00Z, Guardgel artridge, pk DS-00Z, Guardgel artridge, pk.0.0 Stainless steel columns 97 TSKgel DS-0HTP..0. 7, TSKgel DS-0HTP. 0.0., DS-80TS, 80 Å.0.0, DS-80TS, 80 Å.0.0 8, DS-80TS, 80 Å. 7., DS-80TS, 80 Å..0, DS-80TS, 80 Å..0 8, DS-80TS, 80 Å , DS-80TM, 80 Å. 7., DS-80TM, 80 Å..0, DS-80TM, 80 Å..0 8, DS-80TM, 80 Å , ctyl-80ts, 80 Å..0, ctyl-80ts, 80 Å..0 8, N-80TS, 80 Å..0, N-80TS, 80 Å..0 8, Guard column products 90 DS-80TS Guard cartridge, pk.. For all. mm ID DS-80TS columns 90 ctyl-80ts Guard cartridge, pk.. For all. mm ID ctyl-80ts columns 78 DS-80TS Guard column For P/N DS-80TM Guard column For P/N DS-80TM Guard cartridge, pk.. For. mm ID DS-80TM columns 90 N-80TS Guard cartridge, pk.. For. mm ID N-80TS columns 908 artridge Holder.0.0 For mm ID cartridges 908 artridge Holder.. For. and. mm ID cartridges *needs cartridge holder

19 Tosoh bioscience ANALYSIS RDERING INFRMATIN Part # Description ID Length Particle Number Flow Rate (ml/min) Maximum (mm) (cm) ze (µm) Theoretical Range Max. Pressure Drop Plates (kg/cm ) Stainless steel columns 07 DS-0A, 0 Å..0 7, DS-0A, 0 Å..0 0, DS-0A, 0 Å , DS-0A, 0 Å , DS-0T, 0 Å.0.0, DS-0T, 0 Å.0.0 0, DS-0T, 0 Å..0 7, DS-0T, 0 Å..0 0, DS-0T, 0 Å , DS-0T, 0 Å , Guard column products 900 DS-0T Guard cartridge, pk.. For. mm ID DS-0T columns 900 DS-0A Guard cartridge, pk.. For. mm ID DS-0A column Stainless steel columns 00 Super-DS, 0Å.0.0, Super-DS, 0Å.0.0, Super-DS, 0 Å.0 0.0, Super-DS, 0 Å..0 8, Super-DS, 0 Å. 0.0, Super-ctyl, 0 Å.0.0, Super-ctyl, 0 Å.0 0.0, Super-ctyl, 0 Å..0 8, Super-ctyl, 0 Å. 0.0, Super-Phenyl, 0 Å.0.0, Super-Phenyl, 0 Å.0 0.0, Super-Phenyl, 0 Å..0 8, Super-Phenyl, 0 Å. 0.0, Guard column products 97 Guard cartridge, pk.0.0 For mm ID Super-DS columns 807 Guard cartridge, pk.0.0 For. mm ID columns (Super-DS, -ctyl, -Phenyl) 80 artridge holder For P/N 807 Stainless steel columns ligodna RP, 0 Å..0 7, ligodna RP, 0 Å , TMS-0, 0 Å. 7. 0, Glass columns 007 Phenyl-PW RP Glass, 000 Å Stainless steel columns 00 ctadecyl-npr nonporous..., ctadecyl-pw, (00-00 Å).0.0, ctadecyl-pw, (00-00 Å)..0, ctadecyl-pw, (00-00 Å).0.0, ctadecyl-pw, 00 Å.0.0 7, ctadecyl-pw, 00 Å..0 7, ctadecyl-pw, 00 Å..0, ctadecyl-pw, 00 Å , Phenyl-PW RP, 000 Å Phenyl-PW RP, 000 Å Phenyl-PW RP, 000 Å..0, Guard column products 9007 Phenyl-PW RP artridge, pk.. 0 For P/N ctadecyl-pw Guard column..0 For P/N ctadecyl-pw Guard column.0.0 For P/N ctadecyl-pw artridge, pk.. 7 For P/N 79 ctadecyl-pw Prep Guardgel Kit For P/N 7 9 ctadecyl-pw Guard column For P/N 8

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