TSKgel SW TSKgel SW XL TSKgel SuperSW. TSKgel PW TSKgel PW XL -CP. TSKgel Alpha TSKgel SuperAW. TSKgel H XL TSKgel H HR TSKgel Super H TSKgel Super HZ

Transcription

1 8 size exclusion CHROMATOGRAPHY

2 Tosoh bioscience ANALYSIS sec size exclusion chromatography 9 size exclusion CHROMATOGRAPHY PRODUCTS TSK-GEL SW-type TSKgel SW TSKgel SW XL TSKgel SuperSW TSK-GEL PW-type TSKgel PW TSKgel PW XL TSKgel PW XL -CP TSK-GEL Alpha-type TSKgel Alpha TSKgel SuperAW TSK-GEL H-type TSKgel H XL TSKgel H HR TSKgel Super H TSKgel Super HZ TOSOH FACT Tosoh has a long history in size exclusion chromatography (). In 978 Tosoh first introduced porous silica-based SW columns for the isolation of proteins using LC. These first gels had particle sizes from 0 to µm and were quickly adopted and referred to as the standard for analytical on FPLC and HPLC systems. As new packing materials were discovered and new bonding chemistries developed, the product line has grown into four major classes of columns. The following pages will help you choose the best column for your application.

3 0 INTRODUCTION TO TSK-GEL SIZE EXCLUSION COLUMNS Gel Filtration Chromatography (GFC) GFC is popular among biochemists for the isolation of proteins, for the removal of aggregates, to desalt a protein sample, to separate nucleic acid fractions, or to characterize watersoluble polymers used in food products, paints, pharmaceutical preparations, etc. Available TSK-GEL products are classified by application area and particle composition. Application Area: Water- and organic-soluble polymers Base material: polyvinyl Alpha SuperAW These columns are ideal for industrial polymers soluble in water, buffers and many organic solvents. Each of the types below is described in detail in this chapter. Application Area: Proteins and other biopolymers Base material: silica SW SW x l SuperSW These columns are ideal for proteins and nucleic acids using an aqueous buffer as mobile phase. Application Area: Water-soluble polymers Base material: polymethacrylate PW PW x l PW x l -CP These columns are ideal for industrial polymers, oligosaccharides, nucleic acids and small viruses using aqueous buffer or salt solutions as mobile phase. The PW XL -CP columns are developed to facilitate separation of cationic polymer under low salt conditions. Gel Permeation Chromatography (GPC) GPC plays an important role in the characterization of organicsoluble polymers in the chemical and petrochemical industries. TSK-GEL GPC columns contain particles prepared from polystyrene crosslinked with divinylbenzene. Available products are grouped according to their relative lack of adsorptive properties and the speed of analysis. Each of the types below is described in detail in this chapter. Application Area: Organic-soluble polymers Base material: polystyrene Ultra-low adsorption columns with limited solvent range SuperHZ (high throughput) H x l (conventional) Low adsorption columns with expanded solvent range SuperH (high throughput) H h r (conventional) FEATURES Rigid hydrophilic and hydrophobic packings Four series of columns with diferent ranges of solvent compatibility Easy scale up BENEFITS Minimal swelling and excellent physical strength Low adsorption resulting in high mass recovery Suitable for both types of size exclusion, aqueous (GFC) and nonaqueous (GPC) Analytical and preparative pre-packed column

4 Tosoh bioscience sec ANALYSIS SUMMARY OF TSK-GEL SIZE EXCLUSION COLUMN LINES Characteristics of TSK-GEL Size Exclusion Column Lines Column Line TSK-GEL SW TSK-GEL PW TSK-GEL Alpha TSK-GEL SuperAW TSK-GEL H Particle Composition Silica Methacrylate Polyvinyl PS-DVB No. of Available Pore Sizes ph Stability Solvent Compatibility 00% polar 0% polar 00% polar and nonpolar 00% nonpolar, limited polar Max. Temperature 0 C 80 C* 80 C C (H xl and SuperHZ) 0 C (H h r and SuperH) Pressure** (kg/cm ) Application Focus proteins water-soluble polymers intermediate polar polymers organic-soluble polymers * Except for the TSKgel G-DNA-PW, which can be operated up to 0 C and the mm ID TSK-GEL PW-type columns, which can be operated up to 60 C. When operating below 0 C, reduce the flow rate to ensure that the maximum pressure is not exceeded. ** Depends on column dimensions and particle size. Note: The operating conditions and specifications for each column are listed on the Operating Conditions and Specifications sheet (OCS) shipped with the column and in the Ordering Information section at the end of each section.

5 Column Selection Guide for TSK-GEL Gel Filtration Columns Sample Column selection Selection criteria First choice Alternative Carbohydrates polysaccharides TSKgel GMPW XL G000PW XL and G000PW XL Nucleic acids Proteins large pore size, linear calibration curve, small particles, high resolving power oligosaccharides TSKgel G-Oligo-PW G00PW XL small particles, high resolving power DNA fragments large TSKgel G-DNA-PW or TSKgel G000PW XL medium and small TSKgel G000SW XL, TSKgel BioAssist GSW XL, TSKgel SuperSW000, or G000SW XL, TSKgel BioAssist GSW XL large pore size, small particles, high resolving power suitable pore sizes RNA TSKgel G000SW XL suitable pore sizes TSKgel BioAssist GSW XL TSKgel SuperSW000, or G000SW XL, TSKgel BioAssist GSW XL oligonucleotides TSKgel G00PW XL small pore size, ionic interaction normal size small-medium proteins large proteins low density lipoprotein TSKgel SuperSW000, G000SW XL, SKgel BioAssist GSW XL TSKgel G000SW XL, TSKgel BioAssist GSW XL TSKgel SuperSW000, or G000SW XL, TSKgel BioAssist GSW XL TSKgel G6000PW XL or TSKgel G000PW XL G000PW XL or G000PW XL gelatin TSKgel GMPW XL G000PW XL and G000PW XL Peptides large TSKgel SuperSW000, G000SW XL, TSKgel BioAssist GSW XL or G000SW XL, TSKgel BioAssist GSW XL SuperSW000 or G000PW XL small TSKgel G00PW XL SuperSW000 or G000SW XL small particles small to medium range pore sizes large pore sizes large pore size, linear calibration curve small to medium range pore size, versatile linear calibration curve, high resolving power Viruses TSKgel G6000PW XL or TSKgel G000PW XL large pore size, high resolving power Synthetic polymers TSKgel GMPW XL or TSKgel Alpha-M G000PW XL and G000PW XL or Alpha-000 and Alpha-000 large pore size, low adsorption, linear calibration curve cationic TSKgel G000PW XL -CP, TSKgel G000PW XL -CP TSKgel G6000PW XL -CP medium to large pore size, low adsorption, linear calibration curve Synthetic oligomers nonionic TSKgel G-Oligo-PW, TSKgel G00PW XL or TSKgel Alpha-00 G00PW or SuperAW00 small pore size, high resolving power anionic TSKgel G00PW XL or TSKgel Alpha-00 G00PW or SuperAW00 small pore size, ionic interaction

6 Tosoh bioscience sec ANALYSIS TSK-GEL SW, SW xl and SuperSW Gel Filtration columns HIGHLIGHTS TSK-GEL SW-type columns (SW, SW XL and SuperSW column lines) are all based on spherical silica particles with very high internal pore volumes. Silica particles in SW-type columns are chemically bonded with a stationary phase containing polar diol groups. SW-type columns feature low residual adsorption and very high pore volumes, which are essential characteristics of high performance gel filtration columns. SW and SW xl columns lines are available in three pore size ranges with nominal pore sizes of Å, 0 Å and 0 Å. SuperSW and QC-PAK column lines are available in Å and 0 Å pores. SW columns are packed with 0 micron (G000SW and G000SW) or micron (G000SW) particles. SW XL columns contain micron (G000SW XL and G000SW XL ) or 8 micron (G000SW XL ) particles. SuperSW columns contain micron particles (SuperSW000 and SuperSW000) All SW-type columns are available in stainless steel hardware. SW and QC-PAK columns are also available in glass, while SW XL columns are also available in PEEK hardware. Recommendations for TSK-GEL SW series selection Samples of unknown molecular weight TSKgel G000SW XL is the ideal scouting column. If the protein of interest elutes near the exclusion volume, then G000SW XL is the logical next step. Conversely, if the protein of interest elutes near the end of the chromatogram, try the G000SW XL. Proteins (general) Choose one of the TSK-GEL SW XL columns using the calibration curves on page to select the appropriate pore size based on knowledge or estimate of protein size. Monoclonal antibodies TSKgel G000SW XL is commonly used for quality control. TSKgel SuperSW000 is utilized when sample is limited or at very low concentration. Peptides TSKgel G000SW XL is the first selection for the analysis of peptides. TSKgel SuperSW000 is utilized when sample is limited or at very low concentration. Other The use of TSK-GEL SuperSW columns requires optimization of the HPLC system with respect to extra-column band broadening. Capillary tubing ID, injection volume, detector cell volume, and detector time constant all need to be reduced to fully benefit from the high column efficiency and small peak volumes of the SuperSW columns. Use SW columns when not sample limited or when larger amounts of sample need to be isolated. Properties and separation ranges for TSK-GEL SW-type packings Molecular weight of sample (Da) TSK-GEL packing Particle Pore Globular Dextrans Polyethylene Size (µm) Size (Å) proteins glycols and oxides SuperSW000 x 0. x 0 x 0 x 0 x 0 x 0 G000SW XL /BioAssist GSW XL x 0. x 0 x 0 x 0 x 0 x 0 QC-PAK TSK 00 x 0. x 0 x 0 x 0 x 0 x 0 G000SW 0,, 0 x 0 x 0 x 0 x 0 x 0 x 0 SuperSW000 0 x 0 x 0 x 0 7 x 0 x 0. x 0 G000SW XL /BioAssist GSW XL 0 x 0 x 0 x 0 7 x 0 x 0. x 0 QC-PAK TSK 00 0 x 0 x 0 x 0 7 x 0 x 0. x 0 G000SW 0,, 0 0 x 0 x 0 x 0 7 x 0 x 0. x 0 G000SW XL /BioAssist GSW XL 8 0 x 0 7 x 0 6 x 0 x 0 x 0. x 0 G000SW, 7 0 x 0 7 x 0 6 x 0 x 0 x 0. x 0 Data generated using the following conditions: Columns: Two µm,.6 mm ID x 0cm L TSK-GEL SuperSW columns in series; two µm, 7.8 mm ID x 0 cm L TSK-GEL SW x l columns in series; two 0 µm, 7. mm ID x 60 cm L TSK-GEL SW columns in series Elution: Globular proteins: 0. mol/l NaCl in 0. mol/l (0.0 mol/l for SW x l columns) phosphate buffer, ph 7.0 Dextrans and polyethylene glycols and oxides (PEOs): distilled water

7 Calibration curves for TSK-GEL SW-type Gel Filtration columns The best results are obtained when selecting a column with the sample s molecular weight in the linear portion of the calibration curve. Polyethylene oxide, dextran and protein calibration curves for TSK-GEL SW columns TSKgel G000SW TSKgel G000SW TSKgel G000SW Molecular weight (Da) Column: Elution: Flow Rate: Detection: Elution volume (ml) Elution volume (ml) 0 0 Elution volume (ml) TSK-GEL SW, two 7. mm ID x 60 cm L columns in series proteins, polyethylene oxides, dextrans dextrans and polyethylene oxides: distilled water; proteins: 0. mol/l NaCl in 0. mol/l phosphate buffer, ph ml/min 0 nm and RI 0 Protein calibration curves for TSK-GEL SW XL columns Molecular weight (Da) TSKgel G000SW XL TSKgel G000SW XL TSKgel G000SW XL 0 Calibration curves for TSK-GEL SuperSW and SW XL 0 6 Molecular weight (Da) SuperSW000 G000SW XL 6 SuperSW000 G000SW XL 7 Column: Elution: Detection: Elution volume (ml) TSK-GEL SW XL columns, or 8 µm, 7.8 mm ID x 0 cm L proteins:. thyroglobulin (660,000 Da);. thyroglobulin (660,000 Da );. IgG (60,000 Da );. γ-globulin (0,000 Da);. BSA (67,000 Da);. BSA (67,000 Da);. ovalbumin (,000 Da);. β-lactoglobulin (8,00 Da);. lysozyme (,00 Da);. peroxidase (0,00 Da); 6. β-lactoglobulin (8,00 Da); 6. cytochrome C (,00 Da); 7. triglycine (89 Da) 7. myoglobin (6,900 D a) ; 8. ribonuclease A (,600 Da); Elution: 0. mol/l phosphate buffer (ph 6.8) 9. cytochrome C (,00 Da); 0. glycine tetramer (6 Da) Flow Rate: 0. ml/min for SuperSW;.0 ml/min for SW 0. mol/l NaCl in 0. mol/l sodium phosphate buffer, ph 7.0 XL Temperature: C 0 nm Detection: 80 nm (0 nm for triglycine)

8 Tosoh bioscience sec ANALYSIS Comparing TSK-GEL SW, SW xl and SuperSW Gel Filtration Columns Figure shows the increased resolution and faster separation time with a protein standard mixture on the TSK-GEL SW XL compared to TSK-GEL SW. This is due to the smaller particle size ( vs. 0 µm) of the TSK-GEL SW XL packing material. Figure & Figure show the increased resolution and sensitivity of the TSK-GEL SuperSW columns compared to TSK-GEL SW XL columns. This is due to the smaller particle size ( vs. µm) coupled with a narrow column (.6 mm ID). figure Comparison of TSKgel SuperSW000 and TSKgel G000SW XL for the separation of proteins mv figure Column: Elution: A. G000SW XL B. SuperSW000 Flow Rate: Temp: Detection: RS: 8. RS:.0 RS: Rs:.0 Rs:. Rs: A.TSKgel G000SW XL, 7.8mm ID x 0cm; B. TSKgel SuperSW000,.6mm ID x 0cm µl of a mixture of. thyroglobulin, 0.mg/mL (660,000Da);. γ-globulin,.0mg/ml; (0,000 Da);. ovalbumin,.0mg/ml (,000Da);. ribonuclease A,.mg/mL (,600Da);. p-aminobenzoic acid, 0.0mg/mL (7Da) 0.mol/L NaSO in 0.mol/L in phosphate buffer with 0.0% NaN, ph 6.7.0mL/min for G000SW XL ; 0.mL/min for SuperSW000 C 0nm Higher resolution with µm TSK-GEL SW XLcompared with 0µm TSK-GEL SW columns TSKgel G000SW TSKgel G000SW XL figure Comparison of TSKgel SuperSW000 and TSKgel G000SW XL for the separation of proteins Column: Elution: Flow Rate: Detection: Left: TSK-GEL SW, two 0µm, 7.mm ID x 0cm columns in series Right: TSK-GEL SW XL, one µm, 7.8mm ID x 0cm column. glutamate dehydrogenase (80 kda). lactate dehydrogenase ( kda). enolase (67 kda). adenylate kinase ( kda). cytochrome C (, kda) 0. mol/l NaCl in 0.0 mol/l phosphate buffer ph 6.9.0mL/min 0nm mv Column: Inj. Volume: Elution: Flow Rate: Temp: Detection: 0 A. G000SW XL B. SuperSW000 Rs:.7 Rs:.6 Rs:.9 mv Rs:6. Rs:.8 Rs: A. TSKgel G000SW XL, 7.8mm ID x 0cm; B. TSKgel SuperSW000,.6mm ID x 0cm. thyroglobulin (0.mg/mL);. albumin (.0mg/mL);. ribonuclease A (.0mg/mL);. p-aminobenzoic acid (0.0mg/mL) µl 0.mol/L phosphate buffer + 0.mol/L Na SO + 0.0% NaN (ph 6.7) 0.mL/min for SuperSW000;.0mL/min for G000SW XL C 80nm

9 6 Applications of TSK-GEL SW-type Gel Filtration columns Proteins The effect of different concentrations of surfactant on the separation of membrane proteins is seen in Figure. As the concentration of octaethyleneglycol dodecylether increases to 0.0%, the main peak becomes sharper and recovery increases. The TSKgel SuperSW000 provides an excellent high resolution separation of IgG from mouse ascites fluid as can be seen in Figure. figure Separation of membrane protein by with different surfactant concentration in the eluent Enzymes Mobile phase conditions in GFC are optimized to ensure little or no interaction of the sample with the packing material. This gentle technique allows for high recovery of enzymatic activity. For example, crude samples of peroxidase and glutathione S-transferase were separated in only minutes on a TSKgel G000SW XL column and activity recovery was 98% and 89%, respectively. The elution profiles of the separations in Figure 6 show that all of the activity eluted in a narrow band of about. ml. () () () figure Column: TSKgel G000SW, 7.mm ID x 60cm Membrane protein from a crude extract from rat liver microsome Elution: (0.mol/L sodium chloride + 0% glycerol + octaethyleneglycol dodecylether) in 0mmol/L phosphate buffer, ph7.0. Note: concentration of surfactant: () 0.00%, () 0.0%, () 0.0%, () 0.0% Flow Rate: Detection:.0mL/min 80nm The separation of monoclonal antibody (IgG ) from mouse ascites fluid () figure 6 Separation of crude protein samples on TSKgel G000SW XL A. crude peroxidase 0 0 B. crude glutathione S-transferase IgG (mv) 7 9 Column: Elution: Flow Rate: Detection: TSKgel SuperSW000,.6mm ID x 0cm µm of IgG from mouse ascites 0.mol/L phosphate buffer, ph mL/min 80nm micro flow cell 0 0 Column: TSKgel G000SW XL, µm, 7.8mm ID x 0cm A. crude peroxidase from Japanese radish, 0.mg in 0.mL B. crude glutathione S-transferase from guinea pig liver extract, 0.7mg in 0.mL Elution: 0.mol/L NaCl in 0.0mol/L phosphate buffer, ph 7 Flow Rate:.0mL/min Detection: 0nm (solid line) and enzyme assay tests (dashed line) Recovery: enzymatic activity recovered was 98% in A and 89% in B

10 Tosoh bioscience sec ANALYSIS 7 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 ) Glass columns 6 QC-PAK GFC 00GL 8.0 0, QC-PAK GFC 00GL 8.0 0, G000SW, Glass , G000SW, Glass , G000SW, Glass , G000SW, Glass , Stainless steel columns 867 SuperSW , SuperSW000 -NEW , SuperSW000 -NEW-.0 0, SuperSW , G000SW XL , G000SW XL , G000SW XL , QC-PAK GFC , QC-PAK GFC , G000SW , G000SW , G000SW , G000SW , G000SW , G000SW , G000SW , G000SW , G000SW. 0 8, G000SW. 60 0, G000SW. 60 0, G000SW , G000SW G000SW.0 0 0, G000SW G000SW , PEEK Columns 007 BioAssist GSW XL , BioAssist GSW XL , BioAssist GSW XL ,

11 8 ORDERING INFORMATION Part # Description ID Length Particle (mm) (cm) Size (µm) Guard column products 0880 SW Guard column, Glass For all 8 mm ID SW glass columns 6 SW Guard column, Glass For P/N 6 SW glass columns 876 SuperSW Guard column.6. For.6 mm ID SuperSW columns (contains SuperSW000 packing) 08 SW XL Guard column For all SW XL columns and P/Ns 6 and 609 (contains 000SW XL packing) 8008 SW XL Guard column, PEEK For all BioAssist SW XL, PEEK columns 07 SW Guard column For all 7. mm ID SW columns (contains 000SW packing) 078 SW Guard column. 7. For all. mm ID SW columns 077 SW Guard column.0.0 For mm ID SW columns Bulk packing 08 SW XL Top-Off, g wet gel For SW XL and QC-PAK columns 0689 SW Top-Off, g wet gel 0 For all 7. mm ID SW columns

12 Tosoh bioscience sec ANALYSIS 9 TSK-GEL PW and TSK-GEL PW x l columns Gel Filtration Chromatography of water soluble polymers HIGHLIGHTS Hydrophilic, rigid, spherical, porous methacrylate beads Excellent chemical and mechanical stability ph range of to, with up to 0% organic solvent Temperatures up to 80 C (0 C for TSKgel G-DNA-PW) Wide separation range up to 8 x 0 6 Da for linear polymers PEEK column hardware available for G6000PW packings for ultra-low sample adsorption during virus analysis Available in analytical, semi-preparative and preparative stainless steel columns TSK-GEL New PW XL -CP columns designed for low salt separations of cationic polymers. Polymeric TSK-GEL PW and TSK-GEL PW XL columns are designed for GFC of water soluble organic polymers, polysaccharides, oligosaccharides, DNA and RNA. For the analysis of proteins and peptides SW type columns are recommended. A number of specialty columns include columns for samples with a broad molecular weight range, oligosaccharides, DNA, and RNA. For analytical purposes the TSK-GEL PW XL columns are preferred. For preparative work, or for other cases in which large amounts of sample must be used, the 60 cm TSK-GEL PW columns are recommended because of their increased loading capacity. TSK-GEL PW XL -CP columns are especially suited for the separation of cationic polymers. PROPERTIES AND SEPARATION RANGES FOR TEK-GEL PW-TYPE PACKINGS Molecular weight of sample (Da) TSK-GEL packing Particle Pore Polyethylene Dextrans** Globular Size* (µm) Size (Å) glycols and oxides proteins** G000PW 0 < x 0 < x 0 G00PW x l 7 < 00 < x 0 < 8 x 0 G00PW, 7, 0 < 00 < x 0 G000PW x l 7 00 < x 0 < 6 x 0 x 0-8 x 0 G000PW 0, 7, 0 00 < x 0 G000PW XL -CP 7 < 00 < x 0 G000PW x l 0 00 < x 0 x 0-7 x 0 x 0 -. x 0 6 G000PW 7, 0 00 < x 0 G000PW x l < x 0 6 x 0 -. x 0 6 < x 0 G000PW 7, < x 0 6 G000PW XL -CP 0 < 000 < x 0 G6000PW x l > 000 < 8 x 0 6 x 0 - x 0 7 < x0 8 G6000PW / BioAssist G6PW 7 > 000 < 8 x 0 6 G6000PW XL -CP > 000 < 8 x 0 6 GMPW x l < x 0-8 x 0 6 < x 0 7 < x 0 8 GMPW 7 < x 0-8 x 0 6 G-Oligo-PW 7 < x 0 < x 0 G-DNA-PW 0 > 000 < 8 x 0 6 < x 0 7 < x 0 8 Column: TSK-GEL PW columns, 7. mm ID x 60 cm L; TSKgel PW x l, TSKgel PW x l -CP, G-Oligo-PW & G-DNA-PW, 7.8 mm ID x 0 cm L Elution: Polyethylene glycols and oxides: distilled water; dextrans and proteins: 0. mol/l phosphate buffer, ph 6.8 Flow Rate:.0 ml/min Note: *Larger particle sizes of each group are for. mm ID x 60 cm L semi-preparative and mm or 08 mm ID x 60 cm L preparative columns. **Maximum separation range determined from estimated exclusion limits.

13 0 CALIBRATION CURVES FOR TSK-GEL PW-TYP COLUMNS The best results are obtained when selecting a column with the sample s molecular weight in the linear portion of the calibration curve. Polyethylene glycol and oxide calibration curves on TSK-GEL PW and TSK-GEL PW XL columns COMPARISON BETWEEN TSK-GEL PW AND TSK-GEL PWXL The smaller particle sizes of the TSK-GEL PW XL columns provide almost. times the resolution of their TSK-GEL PW counterparts. With shorter TSK-GEL PW XL columns, similar or higher resolution separations are possible in less than half the time. Molecular weight (Da) TSK-GEL PW Columns 0 6 G A D C B 0 0 Elution volume (ml) E F TSK-GEL PW XL Columns Column: TSK-GEL PW columns: A. G000PW, B. G00PW, C. G000PW D. G000PW, E. G000PW, F. G6000PW, G. GMPW, all 7.mm ID x 60 cm TSK-GEL PW XL columns: H. G00PW XL, J. G000PW XL, K. G000PW XL, L. G000PW XL, M. G6000PW XL, N. GMPW XL, all 7.8 mm ID x 0 cm Elution: distilled water Flow Rate:.0 m L/min Detection: RI Molecular weight (Da) a b c d e f g K H N J M L 0 Elution volume (ml) Protein calibration curves on TSK-GEL PW XL columns Faster analysis and higher resolution with TSK-GEL PW XL columns A. TSKgel G00PW C. TSKgel G000PW SE0 SE SE B. TSKgel G00PW XL D. TSKgel G000PW XL SE SE0 0 0 Column: A. TSKgel G00PW, two 0µm, 7.mm ID x 60cm columns in series B. TSKgel G00PW XL, two 6µm, 7.mm ID x 60cm columns in series C. TSKgel G000PW, 7µm, 7.mm ID x 60cm D. TSKgel G000PW XL, 0µm, 7.8mm ID x 0cm A. & B.: polyethylene glycol 00 C. & D.: polyethylene oxide standards: SE-0, SE- and SE- in 00µL Elution: A. & B.: distilled water; C. & D.: 0.mol/L NaCl Flow Rate:.0mL/min Temp.: A. & B.: C; C. & D.: 0 C Detection: RI SE Elution volume (ml) Column:. G000PW XL,. G000PW XL,. G000PW XL,. G6000PW XL,. GMPW XL a. thyroglobulin (660,000 Da), b. γ-globulin (0,000 Da), c. albumin (67,000 Da), d. ovalbumin (,000 Da), e. β-lactoglobulin (6,000 Da), f. myoglobin (6,900 Da), g. cytochrome C (,00 Da) Elution: 0. mol/l phosphate buffer (ph 6.8) Flow Rate:.0 ml/min Detection: 80 nm

14 Tosoh bioscience sec ANALYSIS Columns for Specific Applications TSK-GEL PWXL-CP The new TSK-GEL PW XL -CP columns are designed to facilitate the separation of cationic polymers by at low salt conditions. They are based on the well known PW-type of polymeric resins for aqueous. Cationic surface modification enables low salt elution of cationic polymers with high recoveries. The columns show high theoretical plate numbers, linear calibration curves and high durability. They are produced with three pore sizes for diffrent ranges (G000-, G000- and G6000PW XL -CP). FIgure 7 shows the analysis of various cationic polymers on a series of TSKgel PW XL -CP columns. figure 7 Separation of cationic polymers mv PAA (MW:8kDa) PAA (MW:kDa) PEI (MW:66kDa) P(DADMACI) (MW:0kDa) PAS (MW:7800Da) PAS (MW:87kDa) Cationic Dextran (MW:kDa) Chitosan (MW:.kDa) TSK-GEL G-Oligo-PW The specialty column TSKgel G-Oligo-PW is designed for high resolution separations of nonionic and cationic oligomers. Because of the presence of residual cationic groups, this column is not recommended for separating anionic materials. The polyethylene glycol and polythylene oxide calibration curves for TSKgel G-Oligo-PW (not shown) are identical to the calibration curve for TSKgel G00PW XL (shown on the previous page) Columns: Elution Time (minutes) Eluent: 0.mol/L NaNO Flow Rate: ml/min Detection: RI Temperature: C Sample Load: g/l, 00µL TSKgel G000PW XL -CP, 7µm (7.8mm ID x 0cm), TSKgel G000PW XL -CP, 0µm (7.8mm ID x 0cm), TSKgel G6000PW XL -CP, µm (7.8mm ID x 0cm) TSK-GEL G-DNA-PW The TSKgel G-DNA-PW column is dedicated to the separation of large polynucleotides, such as DNA and RNA fragments of 00 to,000 base pairs. The exclusion limits for double-stranded DNA fragments are lower than those for rrnas, indicating that double-stranded DNA fragments have a larger molecular size in solution than rrnas of the same molecular weight. The packing of the TSKgel G-DNA-PW column has very large pores (>000 Å) and a small particle size (0 µm). For the separation of large DNA fragments greater than,000 base pairs, a four-column system is typically required. Baseline resolution of DNA fragments up to 7,000 base pairs can be achieved, provided there is a two-fold difference in the chain length of the fragments. TSK-GEL GMPW and TSK-GEL GMPWXL When the molecular weight range of the sample is broad or unknown, Tosoh Bioscience offers two mixed-bed columns, TSKgel GMPW and TSKgel GMPW XL, for analysis. The TSKgel GMPW column and its high resolution counterpart, TSKgel GMPW XL, are packed with the G00, G000 and G6000 PW or corresponding PW XL resins. They offer a broad molecular weight separation range. As shown on the previous page, the calibration curve for polyethylene glycols and oxides on these mixed-bed columns is fairly shallow and is linear over the range of 00-,000,000 Da. The introduction of mixed-bed columns has made the problems of analyzing polydisperse samples much easier. Previously, many two-column systems such as TSKgel G000PW and TSKgel G6000PW, were required to achieve good resolution with wide MW-range samples. The substitution of a TSK-GEL GMPW series column can save both time and money compared with multi-column systems.

15 OPTIMIZING GEL FILTRATION WITH TSK-GEL PW AND TSK-GEL PW XL COLUMNS Selecting Mobile Phase Buffers separation is based on the difference of apparent molecular size with no additional interaction between the column matrix and the sample molecules. In practice, however, a small number of weakly charged groups on the surface of PW-type packings can cause changes in elution order from that of an ideal system. The eluent composition can vary greatly with TSK-GEL PW columns to be compatible with a wide range of neutral, polar, anionic, and cationic samples. The table below lists appropriate eluents for GFC of major polymer types. For some nonionic, nonpolar polymers, such as polyethylene glycols, ideal size exclusion behavior can be obtained by using distilled water. More polar ionic polymers may exhibit abnormal peak shapes or minor peaks near the void volume when eluted with distilled water, due to ionic interactions between the sample and residual charged groups on the resin surface. To eliminate ionic interactions, a neutral salt such as sodium nitrate or sodium sulfate should be added. Generally, a salt concentration of 0. to 0. mol/l is sufficient to overcome undesirable ionic interactions. Hydrophobic Samples TSK-GEL PW-type resins are more hydrophobic than polysaccharide gels such as cross-linked dextran. Depending on the sample, this can lead to hydrophobic interaction as a secondary retention mechanism. The extent of hydrophobic interaction increases as the salt concentration of the eluent increases, but it can be reduced by the addition of an organic modifier such as acetonitrile. Water-soluble organic solvents are frequently used as modifiers to suppress hydrophobic interactions between the sample and the resin surface. Modifiers are also used for optimizing the elution of both charged and neutral hydrophobic polymers. Typical examples for a variety of sample types are given in the table below. All TSK-GEL PW-type column packings are compatible with 0 % aqueous solutions of methanol, ethanol, propanol, acetonitrile, dimethyl formamide, dimethyl sulfoxide, formic acid, and acetic acid. In addition, these columns can be operated in 0 % aqueous acetone. RECOMMENDED ELUENTS FOR GFC OF WATER-SOLUBLE POLYMER ON TSK-GEL PW-TYPE COLUMNS Type of polymer Typical sample Suitable eluent Nonionic hydrophilic polyethylene glycol distilled water soluble starch, methyl cellulose, pullulan 0.0N NaOH dextran, hydroxyethyl cellulose, 0% DMSO polyvinyl alcohol, polyacrylamide Buffer or salt solution (e.g., mol/l NaNO ) Nonionic hydrophobic polyvinylpyrrolidone Buffer or salt solution with organic solvent (e.g., 0% ACN in 0.mol/L NaNO ) Anionic hydrophilic sodium chondroitin sulfate, sodium alginate, Buffer or salt solution (e.g., 0. mol/l NaNO ) carboxymethyl cellulose, sodium polyacrylate, sodium hyaluronate Anionic hydrophobic sulfonated lignin sodium salt, Buffer or salt solution with organic solvent sodium polystyrenesulfonate (e.g., 0% ACN in 0. mol/l NaNO ) Cationic hydrophilic glycol chitosan, DEAE-dextran, poly(ethyleneimine), 0. mol/l acetic acid with 0. mol/l Na SO, or poly(trimethylaminoethyl methacrylate) iodide salt 0.8 mol/l NaNO (0. mol/l NaNO for PW XL -CP type) Cationic hydrophobic poly(-vinylbenzyltrimethylammonium chloride), 0. mol/l acetic acid with 0. mol/l Na SO poly(n-methyl--vinylpyridinium) iodide salt Amphoteric hydrophilic peptides, proteins, poly-and oligosaccharides, DNA, RNA Buffer or salt solution (e.g., 0. mol/l NaNO ) Amphoteric hydrophobic blue dextran, collagen, gelatin, hydrophobic proteins Buffer or salt solution with organic solvent hydrophobic peptides (e.g., 0% ACN in 0. mol/l NaNO or % ACN in 0.% TFA)

16 Tosoh bioscience sec ANALYSIS Applications of TSK-GEL PW-type Gel Filtration columns Nucleic acids Desalting of nucleosides can be accomplished using TSKgel G00PW XL, as depicted in Figure 8. Clearly, adenosine elutes after the void volume in the unbuffered water mobile phase. Polysaccharides TSK-GEL PW columns are recommended for polysaccharide analysis due to their ability to separate a wide molecular weight distribution. Nonionic polysaccharides are the least complicated molecules to analyze by because they seldom exhibit secondary interactions with the solid support. TSKgel G000PW and TSKgel G000PW in series are effective for the characterization of clinical dextran. Cationic samples can be adsorbed on the resin by electrostatic interaction. If the polymer is strongly cationic, a fairly high salt concentration is required to prevent ionic interactions with conventional packings. A mobile phase of 0. mol/l acetic acid with 0. mol/l Na SO can also be used. The new TSKgel PW XL -CP series enables elution of water soluble, cationic polymers under low salt conditions (e.g. 0. mol/l NaNO ). An effective separation of the anionic hydrophilic glucosaminoglycan, hydraluronic acid, is shown in Figure 9 on a TSKgel G6000PW and TSKgel G000PW column in series with a 0. mol/l sodium chloride mobile phase. figure 8 Desalting of nucleosides A B figure 9 Separation of hyaluronic acid. Hyaluronic acid. Hyaluronic acid. Polyethylene oxide SE0 C LS Column: TSKgel G00PW XL, 7.8mm ID x 0cm A. 0.mol/L NaCl; B. uridine; C. adenosine Eluent: distilled water Flow Rate:.0mL/min Detection: 60nm 0 0 Column: Elution: Flow Rate: Te mp: 0 TSKgel G6000PW + G000PW, two 7.mm ID x 60cm columns in series hyaluronic acid 0.mol/L NaCl 0.9mL/min 0 C

17 figure 0 Faster analysis and higher resolution of chito-oligosaccharides on a TSKgel G-Oligo-PW column A. TSKgel G000PW B. TSKgel G-Oligo-PW Oligomers The TSKgel G-Oligo-PW column is designed for high resolution separations of nonionic and cationic oligomers. Figure 0 demonstrates excellent resolution of chito-oligosaccharides obtained by using the smaller, 6 µm particle size packing in TSKgel G-Oligo-PW columns as compared with the resolution obtained with a TSKgel G000PW column. The pore sizes in both TSKgel G-Oligo-PW and TSKgel G000PW columns are about Å and both resins bear approximately 0. µeq/ml of cationic groups. Because of the presence of cationic groups, neither column is recommended for separating anionic materials. However, for nonionic oligomers, TSKgel G-Oligo-PW columns provide higher resolution than TSKgel G00PW XL columns Column: A. TSKgel G000PW, two 0µm, 7.mm ID x 60cm columns in series B. TSKgel G-Oligo-PW, two 6µm, 7.8mm ID x 0cm columns in series. chitohexaose,. chitopentaose,. chitotetraose,. chitotriose,. chitobiose. Elution: distilled water Flow Rate:.0mL/min Detection: RI 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 ) Stainless steel columns 080 G-Oligo-PW , G-DNA-PW , G00PW XL , G000PW XL , G000PW XL , G000PW XL , G6000PW XL , GMPW XL , G000PW XL -CP , G000PW XL -CP , G6000PW XL -CP , G000PW 7. 0, G00PW 7. 0, G000PW 7. 0,

18 Tosoh bioscience sec ANALYSIS 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 ) 076 G000PW , G000PW , G6000PW , GMPW , G000PW , G00PW , G000PW , G000PW , G000PW , G6000PW , GMPW , G00PW , G000PW , G000PW , G000PW , G000PW ND G000PW , PEEK columns 00 BioAssist G6PW , Guard columns 080 Oligo Guard column For 7.8 mm ID G-Oligo-PW columns 080 PW XL Guard column For 7.8 mm ID PW XL and G-DNA-PW columns (contains TSKgel G000PW packing) 876 PW XL -CP Guard column For 7.8 mm ID PW XL -CP columns 0676 PW Guard column For 7. mm ID G000PW and G000PW columns (contains TSKgel G000PW packing) 0676 PW Guard column For 7. mm ID G00PW through GMPW columns 0678 PW Guard column For. mm ID G00PW through G000PW columns 079 PW Guard column For mm ID G000PW through G000PW columns Bulk packing 080 PW XL Top-Off, g wet resin 0 For all PW XL and G-DNA-PW columns

19 6 TSK-GEL ALPHA AND SUPERAW GEL FILTRATION COLUMNS Gel Filtration and Gel Permeation Chromatography of water-soluble and polar organic-soluble polymers HIGHLIGHTS A unique hydrophilic, polyvinyl resin is available in conventional column dimensions (Alpha) and high throughput column format (SuperAW). Exhibits strong mechanical stability and minimal swelling characteristics A wide range of solvent compatibility, from 00% water to 00% non-polar organic solvents The reduced particle size and shorter column length of TSK-GEL SuperAW columns provide equivalent resolution in one half the time for high throughput applications. Unlike polystyrene-divinylbenzene (PS-DVB) resins that may adsorb polymers due to hydrophobic interaction, both the TSK-GEL Alpha and SuperAW columns allow for the separation of polymers soluble in methanol. Provide accurate molecular weight determination of samples in dimethyl formamide and exhibit normal retention of polystyrene polymers System peaks from salts in the eluent elute away from the oligomer of interest, providing accurate MW determinations. Column Selection The TSK-GEL Alpha Series consists of six columns with three particle sizes: 7, 0, and µm. These columns span a wide MW separation range from 00 to more than x 0 6 Da when using polyethylene oxide (PEO) as a MW standard. Exclusion limits for the TSK-GEL Alpha columns for polyethylene oxide (PEO), polyethylene glycols (PEG) and polystyrenes (PS) are shown in the table below. Calibration curves for the TSK-GEL Alpha Series columns are shown on the next page for polyethylene oxide, polyethylene glycol and polystyrene standards. The TSK-GEL SuperAW series contains a similar chemistry as the TSK-GEL Alpha series but offers the benefit of smaller particle sizes ( µm to 9 µm) and smaller column dimensions. Reductions in analysis time and mobile phase consumption make SuperAW columns ideal for high throughput applications. TSK-GEL Alpha and SuperAW columns are offered in discrete exclusion ranges and mixed bed. Both column types can accommodate polymer standards up to several million Dalton molecular weight (see calibration curves on the next page). Exclusion limits for TSK-GEL Alpha Series and SuperAW Series columns Column Particle Size (µm) Exclusion limit (Da) for various standards and eluents PEO a /H O PS b /0mmol/L LiBr in DMF PEG c /0mmol/L LiBr in MeOH Alpha-00 7 x 0 x 0 x 0 Alpha x 0 x 0 6 x 0 Alpha x 0 x 0 6 x 0 6 Alpha x x 0 6 N.D. Alpha-6000 > x 0 7 > x 0 7 N.D. Alpha-M > x 0 7 > x 0 7 N.D. SuperAW00 x 0 8 x 0 x 0 SuperAW000 9 x 0 8 x 0 x 0 SuperAW000 6 x x 0 6 x 0 SuperAW000 7 x 0 6 * N.D. N.D. SuperAW x 0 7 * N.D. N.D. SuperAWM-H 9 x 0 7 * N.D. N.D. N.D. = not determined a Polyethylene oxide b Polystyrene divinyl benzene c Polyethylene glycol * Exclusion limit for SuperAW000, SuperAW6000, and SuperAWM-H are estimated, respectively

20 Tosoh bioscience sec ANALYSIS 7 Calibration curves for TSK-GEL ALPHA AND SUPERAW Gel Filtration columns The best results are obtained when selecting a column with the sample s molecular weight in the linear portion of the calibration curve. Polyethylene oxide (PEO), polyethylene glycol (PEG) and polystyrene (PS) calibration curves for TSK-GEL Alpha columns Molecular Weight (Da) A. 6 PEO. Alpha-00. Alpha-000. Alpha-000. Alpha-000. Alpha Alpha-M 6 Molecular Weight (Da) B. PEG 6. Alpha-00. Alpha-000. Alpha-000. Alpha-000. Alpha Alpha-M 6 Molecular Weight (Da) C. 6 PS. Alpha-00. Alpha-000. Alpha-000. Alpha-000. Alpha Alpha-M Elution Volume (ml) Elution Volume (ml) Elution Volume (ml) Column: Eluent: Flow Rate: Temperature: Detection: TSK-GEL Alpha Series, 7.8 mm ID x 0 cm L A. H O; B. 0 mmol/l LiBr in Methanol; C. 0 mmol/l LiBr in DMF.0 ml/min A. C; B. C; C. 0 C RI Calibration curves for TSK-GEL SuperAW Series in different solvents with different polarity MW 0 7 MW A. B. C. 0 7 MW SuperAWM-H 6 SuperAWM-H SuperAW6000 SuperAW6000 SuperAW000 SuperAW SuperAW000 SuperAW000 SuperAW000 SuperAW000 SuperAW00 SuperAW SuperAWM-H SuperAW6000 SuperAW000 SuperAW000 SuperAW000 SuperAW Elution volume (ml) 0... Elution volume (ml) 0... Elution volume (ml) Column: TSK-GEL SuperAW Series (6.0 mm ID x cm L) Eluent: A. Water; B. MeOH containing 0 mmol/l LiBr; C. DMF containing 0 mmol/l LiBr Flow rate: 0.6 ml/min Temperature: C Detection: Refractive index detector Samples: Standard polyethylene oxide, polyethylene glycol, ethylene glycol

21 8 Applications of TSK-GEL ALPHA AND SUPERAW Gel Filtration columns The versatility of using TSK-GEL Alpha columns with various polar solvents is illustrated in Figure for the analysis of cellulose derivatives. A TSKgel Alpha-M column was used to separate ethylcellulose with the polar solvent DMF and ethylhydroxyethyl cellulose with methanol. The separation of polyvinylalcohol with different degrees of saponification is shown in Figure. This separation was performed with a TSKgel Alpha-000 and a TSKgel Alpha-000 column in series using a hexafluoroisopropanol mobile phase. Figure shows that the column efficiency of TSK-GEL SuperAW series columns is maintained in a wide variety of polar organic solvents. figure TSKgel Alpha-M separation of cellulose derivatives Polyvinylalcohol characterization using TSK-GEL Alpha-000 and Alpha-000 columns in series A B C figure Column: Eluent: Flow Rate: Temperature: Detection: TSKgel Alpha-000 and Alpha-000, 7.8mm ID x 0cm in series degree of saponification of polyvinyl alcohol: A. 7%; B. 88%; C. 00% hexafluoroisopropanol (HFIP) 0.mL/min 0 C RI 0 A. mv 0 0 TP 000 figure Solvent Compatibility of TSK-GEL SuperAW Series B. 000 mv Column: Elution: Flow Rate: Temperature: Detection: 0 0 TSKgel Alpha-M, 7.8mm ID x 0cm A. 0µL ethylcellulose, 0.%; B. 0µL ethylhydroxyethylcellulose, 0.% A. 0mmol/L LiBr in DMF; B. 0mmol/L LiBr in methanol 0.mL/min 0 C RI Original (H O) MeOH/H O=/ MeOH ACN/H O=/ ACN DMF SuperAW00 SuperAW000 SuperAW000 SuperAW000 SuperAW6000 SuperAWM-H DMSO HFIP Column: TSK-GEL SuperAW Series (6.0 mm ID x cm L) Eluent: Water Flow rate: 0.6 ml/min Temperature: C Detection: Refractive index detector Ethylene glycol Inj. volume: µl (. g/l) THF

22 Tosoh bioscience sec ANALYSIS 9 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 ) Stainless steel columns 89 Alpha , Alpha , Alpha , Alpha , Alpha , Alpha-M (mixed bed) , Guard columns 8 Alpha Guard column 6 For all Alpha columns VMpak columns* 00 VMpak-.0 7, VMpak-.0 7, Stainless steel columns 9 SuperAW , SuperAW , SuperAW , SuperAW >0, SuperAW >7, SuperAWM-H >7, Guard columns 9 SuperAW-L Guard Column.6. 7 For SuperAW columns. 9 SuperAW-H Guard Column.6. For SuperAW000-AWM-H columns *TSK-GEL VMpak- series contains a similar packing as TSKgel Alpha-00. It can be used for multimodal LC/LC-MS separations.

23 0 TSK-GEL H x l, H h r, SUPERH AND SUPERHZ GEL PERMEATION COLUMNS Polymer-based columns for Gel Permeation Chromatography of organic-soluble polymers HIGHLIGHTS Porous, highly cross-linked, spherical polystyrene divinylbenzene (PS-DVB) resin. Four different TSK-GEL H-type columns are available. Each of these are packed with different particle sizes (see table below). H-type packings are available in eight pore sizes. Expanded molecular weight ranges with exclusion limits from,000 Da to an estimated x 0 8 Da Minimal shrinking and swelling of the column bed Chemically and thermally stable Use.6 mm ID SuperH and SuperHZ columns for reduced solvent consumption in high throughput analysis. Novel multi-pore distribution in the TSKgel MultiporeH XL -M column provides linear calibration curves over a wider MW range. Mixed bed columns with optimized particle and pore sizes to prevent polymer sheering. TSK-GEL H-type packings are available in eight pore sizes and span four different column chemistries. For polymer samples with a broad molecular range, packing of several pore sizes are provided in the mixed bed columns: TSK-GEL SuperHZM series, TSK-GEL SuperHM series, TSKgel GMH XL, TSKgel GMH HR, and selected high temperature versions provide linear calibration curves up to several million Daltons (see page ). Column Selection The Super prefix refers to the efficiency of the column. The Super series columns contain ultra efficient particles as small as µm, housed in cm length columns. The smaller particle allows for equivalent resolution to conventional H XL columns, with 0% less run time due to the shorter column length. The Super series columns are an excellent choice for high throughput polymer analysis. Semi-micro SuperHZ columns now available as multipore columns with linear calibration curves. Series Type SuperHZ H XL SuperH H HR Application focus Particle size High-throughput polymer analysis with ultra low polymer adsorption. Limited solvent compatibility range., and 0 µm, depending on pore size Conventional polymer analysis with ultra low polymer adsorption. Limited solvent compatibility range., 6 and 9 µm, depending on pore size High-throughput polymer analysis with expanded solvent compatibility. and µm, depending on pore size Conventional polymer analysis with expanded solvent compatibility range. Theoretical plates 6,000/ cm column 6,000/0 cm column 6,000/ cm column 6,000/0 cm column Maximum temperature G000 - G C G000 - G C 0 C 0 C G000 - mixed 80 C G000 - mixed 80 C Standard shipping solvent THF THF THF THF µm THF can be switched to Other shipping solvents available? Number of solvent substitutions Solvent exchange instructions benzene, chloroform, toluene, xylene, dichloromethane and dicholoroethane yes One time only Several Linear gradient with a %/min rate of change at a flow rate <0. ml/min. Linear gradient with a %/ min rate of change at a flow rate <0. ml/min. see our website for detailed information no Linear gradient with a %/min rate of change according to flow rates listed on our website. ) Theoretical plates listed are based on smallest particle size listed ) High-temperature columns (HT) are shipped with OCDB (Orthochlorodivinylbenzene) as standard shipping solvent. ) Switching from THF to dichloromethane and dichloroethane is not recommended for G000 pore size columns. ) See our website for available shipping solvents ) After switching to a very polar solvent such as acetone, switching back to a nonpolar solvent is not recommended.

24 Tosoh bioscience sec ANALYSIS CALIBRATION CURVES FOR TSK-GEL H-TYPE GELPERMEATION COLUMNS Molecular weight (Da) Molecular weight Calibration curves for TSK-GEL H HR columns with polystyrene standards f e d b c a Elution Volume (ml) h Column: TSKgel H HR series (7.8 mm ID x 0 cm L) polystyrene standards Elution: THF g a. G000H HR b. G000H HR c. G00H HR d. G000H HR e. G000H HR f. G000H HR g. G6000H HR h. G7000H HR Molecular weight (Da) N M L H H. GMH HR -H M. GMH HR -M N. GMH HR -N L. GMH HR -L Elution Volume (ml) Flow Rate:.0 ml/min Temperature: C Detection: nm Calibration curves for TSK-GEL SuperHZ columns with polystyrene standards SuperHZ000 SuperHZ000 SuperHZ00 SuperHZ000 SuperHZ000 Molecular weight SuperHZM-N SuperHZM-M SuperHZM-H Molecular weight (Da) The best results are obtained when selecting a column with the sample s molecular weight in the linear portion of the calibration curve. Calibration curves for TSK-GEL H XL columns with polystyrene standards GMH XL G000H XL G6000H XL G000H XL GMH -L XL G00H XL G000H XL G000H XL G000H XL G7000H XL Elution volume (ml) Elution volume (ml) Column: TSK-GEL SuperHZ series (.6 mm ID x cm L) Temperature: C Eluent: THF standard polystyrene Flow rate: 0. ml/min Inj. volume: µl Calibration curves for TSK-GEL SuperH columns with polystyrene standards Molecular weight SuperH SuperH SuperH000 SuperH000 SuperH000 SuperH00 SuperH000 SuperH000 Molecular weight SuperHM-H SuperHM-M SuperHM-N SuperHM-L 0 Column size: Eluent: Flow Rate: Temperature: Detection: 7 9 Elution Time (min) 7.8 mm ID x 0 cm L polystyrene standards THF.0 ml/min C nm Elution volume (ml) Elution volume (ml) Column: TSK-GEL SuperH series (6.0 mm ID x cm L) Temperature: C Eluent: THF Detection: UV@ nm Flow rate: 0.6 ml/min polystyrene standards

25 MULTI-PORE SIZE DISTRIBUTION IN A POLYSTERENE PACKING MATERIAL Novel approach to GPC of samples with a wide range of molecular weights The TSKgel MultiporeH XL -M column offers a unique packing material and a novel strategy for the precise analysis of polymers by Gel Permeation Chromatography (GPC). Until now, the GPC separation of a sample containing a wide range of molecular weight polymers was performed by one of two strategies. One strategy combines columns with different pore sizes of packing material in series. The other strategy employs a single column with a blend of different pore sizes of packing materials, commonly referred to as a mixed bed. Mixed bed columns do not always provide linear calibration curves, which may result in broad or split peaks. With the introduction of the TSKgel MultiporeH XL -M column, a novel strategy was introduced using a single column containing a novel polystyrene packing material with a multi-pore size distribution. Figure illustrates the three strategies. The TSKgel Multipore column has several different pore sizes with continuous distribution in every bead. This results figure figure 6 in sharper peaks without inflections that may be observed using mixed-bed columns. The pore size distributions of the TSKgel MultiporeH XL -M column and a mixed-bed column are shown in Figure. The mixed-bed column shows a sharp maximum for pores with a diameter of 0.08 µm, though the overall pore size distribution ranges from to 0.6 µm in diameter. In the case of the TSKgel MultiporeH XL -M column, the pore size distribution exhibits a wider maximum range from 0.0 to 0. µm in diameter. This difference in pore size distribution may explain the reason for the inflection phenomenon. A comparison of calibration curves for polystyrene standards on the TSKgel MultiporeH XL -M column, the TSKgel GMH HR -H and PLgel Mixed-C, are shown in Figure 6. Both the TSKgel GMH HR -H and PLgel Mixed-C columns are mixed-bed columns. Strategies for wide range separation using Size Exclusion Chromatography Conventional Strategy New Strategy Large Pore Medium Pore Small Pore Multiple Pore Size Calibration curves for Multipore and mixed-bed columns 0 7 TSKgel MultiporeH XL -M TSKgel GMH HR -H 0 6 PLgel Mixed-C Connect columns with different grades of packings (TSKgel G000H+G000H+G000H) Blend (mixed bed) packings of different grades (TSK-GEL GMH series) Pure packings with multi-pore size distribution (TSKgel MultiporeH XL column) Molecular weight 0 0 figure Pore size distribution of TSKgel MultiporeHXL-M column and a mixed-bed column 0 Pore volume dv (log r) TSKgel MultiporeH XL -M Mixed-bed column Column: Elution: Flow Rate: Temperature: Detection: Elution volume (ml) TSKgel Multipore H XL -M, 7.8 mm ID x 0 cm L; TSKgel GMH HR -H, 7.8 mm ID x 0 cm L; PLgel Mixed-C, 7. mm ID x 0cm L polystyrene standards THF.0 ml/min 0 C nm Pore size (µm)

26 Tosoh bioscience sec ANALYSIS Applications of TSK-GEL H-TYPE Gel PERMEAtion columns Phthalate esters Figure 7 demonstrates the high efficiency separation on a TSKgel G000H XL column for low molecular weight phthalate esters. Resolution was close to baseline, even though the molecular weights of the esters differed by less than 0 Da. Phenol resin The TSKgel GMH XL -L column has been designed to provide a complete profile for high molecular weight samples that contain low molecular weight additives. The calibration curve for this mixed-bed column is shallow in the low molecular weight range figure 7 High resolution of phthalate esters on TSKgel G000H XL 6 of oligomers. Sample adsorption is not observed. For example, the complete profile of a phenol resin, with high resolution of the low molecular weight components, is shown in Figure 8. Other applications for the TSKgel GMH XL -L column include analyses of paint materials, bond and adhesive components and synthetic polymer additives. Fatty acids In Figure 9, two TSKgel G000H XL columns in series separate a mixture of fatty acids ranging from C to C0. figure 8 Separation of phenol resin on TSKgel GMH XL -L Column: Elution: Flow Rate: Detection: 0 TSKgel GMH XL -L, 7.8mm ID x 0cm phenol resin THF.0mL/min nm figure 9 Separation of fatty acids C C 0 C 8 C 6 C C C 0 C 8 Column: Elution: Flow Rate: Detection: TSKgel G000H XL, 7.8mm ID x 0cm. polystyrene (0,00Da),. dioctylphthalate (9Da),. dibutylphthalate (78Da),.dipropylphthalate (0Da),. diethylphthalate (Da), 6. dimethylphthalate (9Da), 7. n-propylbenzene (0Da), 8. ethylbenzene (6Da), 9. toluene (9Da), 0. benzene (78Da) THF.0mL/min nm 0 Column: Elution: Flow Rate: Detection: C 6 C C 6 C TSKgel G000H XL, two 7.8mm ID x 0cm in series fatty acids THF.0mL/min RI

27 Applications of TSK-GEL H-type Gel PERMEAtion columns Acrylic polymer Figure 0 shows the separation of an acrylic polymer on the TSKgel MultiporeH XL -M column compared with two commercially available mixed-bed columns. The arrows illustrate the inflections seen in the chromatograms from mixed-bed columns and the improvement achieved when using the TSKgel MultiporeH XL -M column. Polymethylmethacrylate The effect of different pore size distributions in the mixed beds of TSKgel GMH HR -H and TSKgel GMH HR -M is illustrated in Figure. The TSKgel GMH HR -M produces better resolution in the 8 x 0 to x 0 Da range. Epoxy resin Figure demonstrates the excellent fingerprint obtained using small diameter SuperHZM-M mixed bed columns. figure Comparison of TSKgel GMH HR -H and -M columns with polymethylmethacrylate standards A. TSKgel GMH HR -H B. TSKgel GMH HR -M Column: A. TSKgel GMH HR -H, 7.8 mm ID x 0 cm L; B. TSKgel GMHHR-M, 7.8 mm ID x 0 cm L polymethylmethacrylate:. 80,000 Da,. 67,000 Da,. 0,00 Da,.,90 Da figure 0 figure Separation of acrylic resin by on TSKgel MultiporeH XL -M and mixed-bed type columns 0 Chromatogram of epoxy resin A 0 mv 0 B mv mmI.D C.6mmI.D. 0 0 Column: A. TSKgel MultiporeH XL -M, two 7.8 mm ID x 0 cm L columns in series; B. Competitor P, two 7. mm ID x 0 cm L columns in series, mixed-bed type; C. Competitor S, two 8.0 mm ID x 0 cm L columns in series, mixed-bed type acrylic polymer (0.%, 0 µl) Elution: THF Flow Rate:.0 ml/min Temperature: 0 C Detection: RI (min) Column: TSKgel Super HZM-M x Eluent: THF Flow rate: 0. ml/min (.6 mm ID) 0.6 ml/min (6.0 mm ID) Temperature: 0 C Detection: RI Epoxy resin (0 g/l) Inj. volume: µl (.6 mm ID) 9 µl (6.0 mm ID)

28 Tosoh bioscience sec ANALYSIS 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 ) Stainless steel columns 7 G000H HR , G000H HR , G00H HR , G000H HR , G000H HR , G000H HR , G6000H HR , G7000H HR , GMH HR -L mixed-bed , GMH HR -N mixed-bed , GMH HR -M mixed-bed , GMH HR -H mixed-bed , GMH HR -H(S)HT mixed-bed , G000H XL , G000H XL , G00H XL , G000H XL , G000H XL , G000H XL , G6000H XL , G7000H XL , GMH XL mixed-bed , GMH XL -HT 7.8 0, GMH XL -L mixed-bed , Multipore H XL -M , TSKgel SuperH , TSKgel SuperH , TSKgel SuperH , TSKgel SuperH , TSKgel SuperH , TSKgel SuperH , TSKgel SuperH , TSKgel SuperH , TSKgel SuperHM-L 6.0 6, TSKgel SuperHM-N 6.0 6, TSKgel SuperHM-M 6.0 6, TSKgel SuperHM-H 6.0 6,

29 6 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 ) Stainless steel columns 909 TSKgel SuperHZ , TSKgel SuperHZ , TSKgel SuperHZ , TSKgel SuperHZ , TSKgel SuperHZ00.6 6, TSKgel SuperHZ , TSKgel SuperHZ , TSKgel SuperHZ , TSKgel SuperHZ , TSKgel SuperHZ , TSKgel SuperHZM-N.6 6, TSKgel SuperHZM-N 6.0 6, TSKgel SuperHZM-M.6 and 6, TSKgel SuperHZM-M 6.0 and 6, TSKgel SuperHZM-H.6 0 9, TSKgel SuperHZM-H , SuperMultiporeHZ-M.6 6,000 8 SuperMultiporeHZ-N.6 0, SuperMultiporeHZ-H.6 6,000 0 Guard columns 80 MultiporeH XL -M Guard For P/N H XL Guard Column For G000H XL through G000H XL columns 77 H XL Guard Column For G000H XL through GMH XL -L mixed-bed columns 768 H HR Guard Column For G H HR and GMHhr-L columns 769 H HR Guard Column For G H HR and and GMH HR -M; -N; -H columns 800 SuperH Guard Column.6. For SuperH SuperH Guard Column.6. For SuperH and HM-L;-N;-M;-H columns 9 SuperHZ Guard Column.6.0 For.6 mm ID SuperHZ and HZM-N &-M columns 9668 SuperHZ Guard Column For.6 mm ID SuperHZM-H columns 9666 SuperHZ Guard Column.6. For 6.0 mm ID SuperHZ and HZM-N &-M columns 9667 SuperHZ Guard Column.6. 0 For 6.0 mm ID SuperHZM-H columns 89 SuperMP-M Guard.6.0 For SuperMultipore HZ-M P/N SuperMP-N Guard.6.0 For SuperMultipore HZ-N P/N SuperMP-H Guard For SuperMultipore HZ-H P/N 887

30 Tosoh bioscience sec ANALYSIS 7 ECO GPC SYSTEM - BASED ON YEARS OF EXPERIENCE IN GPC Eco is a compact, all-in-one GPC system for fast, high resolution, semi-micro GPC. Comprising a precision solvent delivery system, automatic injector, column oven and a high performance refractive index detector, the design of the system components, their configuration and the optimized flow line provides outstanding performance with minimized dead volume. This makes Eco the ideal instrument to be used in combination with the well respected TSK-GEL semi-micro GPC/ columns. In Europe, Eco is offered in cooperation with Polymer Standards Service (PSS), an acknowledged leader in the field of polymer analysis.