BioBasic Columns. Table 1: BioBasic Packing Characteristics Specifications

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1 TG 0- BioBasic Columns Introduction BioBasic columns are designed specifically for biochromatography of proteins, peptides, and nucleic acids The 00Å pore size, high purity silica, and stable bonding chemistry of BioBasic packings makes them ideal for life science applications. Improved HPLC performance for biomolecules Whether you re doing reversed phase or ion exchange, the BioBasic family of HPLC columns provides superior performance: improved resolution better reproducibility longer column lifetimes more efficient separations Table : BioBasic Packing Characteristics Specifications Phase Particle Size Carbon Load Pore Size Endcapping Silica Type BioBasic µm % 00Å yes high purity base deactivated BioBasic µm % 00Å yes high purity base deactivated BioBasic µm % 00Å yes high purity base deactivated BioBasic Phenyl µm - 00Å yes high purity base deactivated BioBasic CN µm - 00Å yes high purity base deactivated BioBasic AX µm - 00Å yes high purity base deactivated BioBasic SCX µm - 00Å yes high purity base deactivated Better Reproducibility BioBasic columns provide superior chromatography, run after run, column after column. The extra dense bonding chemistry used for BioBasic reversed phase packings gives a highly stable, reproducible surface for reliable results. Figure demonstrates the reproducible performance of batches of BioBasic packing materials. Longer column lifetimes BioBasic packings are designed to hold up to harsh mobile phase conditions for longer column lifetimes. Even when subjected to acid hydrolysis conditions at ph. and 0 C using 0.% TFA, BioBasic columns show superior results. Figure demonstrates a comparison of column lifetime under these difficult gradient conditions. A Range of Stationary Phases with Different Selectivities BioBasic reversed phase packings are available in several chemistries, including C, C, C, phenyl and cyano. BioBasic ion exchangers include BioBasic AX for anion exchange and SCX for cation exchange. Table provides a summary of characteristics for each BioBasic packing. Figure Reproducibility of BioBasic Figure Acid Hydrolysis Study BioBasic, µm, 0x.mm Gradient: A: 0.% TFA in H B: 0.% TFA in ACN %B to 00%B in 0 min Flow:.mL/min Detector: Theophylline. p-nitroaniline. Methyl Benzoate. Phenetole. o-xylene,0 plates/meter Average Efficiency Percent Capacity Factor -0 Number of Gradient Cycles 0 MIN /00

2 00Å Pore Size for Better Protein and Peptide Separations Pore size can exert a significant influence on the chromatography of biomolecules. Figure shows the higher resolution of the 00Å pore size BioBasic column for a tryptic digest separation, compared to a 0Å pore size C column. In particular, the peaks eluting near and minutes show much higher resolution on the BioBasic column. Figure shows a comparison of three proteins separated on a 00Å BioBasic column versus a 0Å C column. The BioBasic column provides taller, sharper peaks with less tailing. Figure demonstrates the changes in selectivity that occur due to differences in bonded phase chain length. The BioBasic and BioBasic columns show a reversal of peak order for the two myoglobin peaks, which are not separated on the BioBasic column. Figure Figure Effect of Pore Size on Tryptic Digest Separation 0Å, BetaBasic µm, 0x.mm Tryptic Digest of Bovine Serum Albumin 0 0 MIN Gradient: A: 0.% TFA in H B: 0.% TFA in ACN 0% to 0% B in 0 min Flow:.0 ml/min Detector: UV@ 0 Temp: Ambient -0 00Å, BioBasic µm, 0x.mm -00 Figure Effect of Bonded Phase Chain Length on Protein Separation 0 0 MIN B A A, B BioBasic µm, 0x.mm Gradient: A: 0.% TFA in H B: 0.% TFA in ACN to 00% B in 0 min Flow:. ml/min Detector: UV@ Temp: Ambient Proteins MW pi. Ribonuclease A KD.. Insulin KD.. Lysozyme KD.0. Myoglobin KD.0. valbumin KD. -00 BioBasic µm, 0x.mm Effect of Pore Size on Protein Separation 00Å, BioBasic, µm, 0x.mm 0Å, BetaBasic, µm, 0x.mm Proteins. Bovine Serum Albumin, kd. ß-Amylase, 00kD. Apoferritin, kd MIN A BioBasic µm, 0x.mm MIN Gradient:A: 0.% TFA in H B: 0.% TFA in ACN % to 00% B in 0 min Flow:. ml/min Detector: UV@ Temp: Ambient 0 0 MIN B MIN

3 NEW! BioBasic AX Anion Exchanger The BioBasic family now includes BioBasic AX columns for anion exchange. BioBasic AX columns give you superior performance for proteins, peptides and nucleic acids (Figure ), using proteinfriendly ion exchange conditions. BioBasic AX columns give you: better reproducibility longer column lifetimes more efficient separations multiple modes of interaction What is BioBasic AX packing material? BioBasic AX columns are made from polyethyleneimine (PEI) covalently bonded to a highly base deactivated 00Å µm silica (see Table ). PEI forms a polymeric structure on the silica surface which is protein-friendly. The covalent bond to the silica surface provides better stability and longer column lifetime than typical PEI-coated particles. BioBasic AX columns can also be used in non-buffered conditions for Hydrophilic Interaction Liquid Chromatograpy (HILIC). HILIC is closely related to normal phase chromatography, although HILIC uses a high aqueous component in the mobile phase, as shown in Figure. Table Specifications: Figure Nucleotides MIN BioBasic AX, µm, 0x.mm Gradient: A: mm KH P, ph. B: 0.M KH P, ph. 0-00%B in 0 min. Flow:.0 ml/min Detector: CMP. UMP. AMP. GMP. CDP. ADP. UDP. GDP. CTP 0. ATP. UTP. GTP 0-00 Phase Particle size Pore size Ion exchange ligand Ion exchange capacity BioBasic AX µm 00Å PEI 0. mequivalents/gram BioBasic SCX µm 00Å Sulfonic Acid 0.0 mequivalents/gram NEW! Versatile BioBasic SCX Cation Exchange Columns NEW! Versatile cation exchanger 00Å pore size for better protein and peptide separations Superb stability under demanding ph conditions Exceptional efficiency from µm silica particles NEW! BioBasic SCX Cation Exchanger BioBasic SCX columns are a versatile new cation exchange column providing you with superior performance for proteins, peptides and small molecules. BioBasic SCX columns are particularly useful for protein fractionations for proteomics analyses by LC-MS, including -dimensional fractionation when coupled to a BioBasic capillary column. Better Ion Exchange Performance BioBasic SCX columns are designed to give superior reproducibility, both columnto-column and batch-to-batch. The µm 00Å silica provides significantly higher efficiency than typical polymer-based ion exchangers. Every BioBasic SCX column packed and tested shows over 0,000 plates per meter as tested under normal phase conditions. Figure Phospholipids in HILIC Mode. L-erythro-Sphingosine. L-α-Phosphatidyl choline, egg. Phosphatidyl ethanolamine, bovine. lyso-lecithin, egg MIN BioBasic AX, µm, 0x.mm Gradient: A: % ACN / % mm NH Formate, ph. B: 0% ACN / 0% mm NH Formate, ph. 0%B 0- min., 0-00%B - min. Flow:.0 ml/min Detector: ELSD Figure. Trypsinogen. Ribonuclease A. Chymotrypsinogen A. Cytochrome C. Lysozyme BioBasic SCX, µm, 0x.mm Gradient: A: 0.0M Tris, ph B: A +.0M Na Acetate, ph 0-00%B in 0 min. Flow:.0 ml/min Detector: 0 Proteins MIN -00

4 Tunable Ion Exchange Both the BioBasic AX and BioBasic SCX packing materials can be used across a broad range of both ph and ionic strength. By manipulating buffer concentration, separations can be optimized for maximum retention, high efficiency, or rapid throughput. Figures and 0 demonstrate the effect of buffer concentration on retention for a series of organic acids using the BioBasic AX column. As buffer concentration decreases, retention increases. Figure shows the same effect of buffer strength on the BioBasic SCX column for a series of basic analytes. In Figure, the change in retention with change in ph is demonstrated on the BioBasic SCX column, showing longer retention and improved separations with decreased ph for a series of peptides. Figure Effect of Buffer Concentration on Ion Exchange Retention Figure 0 Effect of Buffer Concentration on BioBasic AX Figure Figure Effect of Buffer Concentration on BioBasic SCX Effect of ph on BioBasic SCX. Uracil. Shikimic Acid. Ascorbic Acid. Phenylacetic Acid 0mM 0mM 0mM 0mM 0 0 MIN BioBasic AX, µm, 0x.mm Eluent: A: NH Acetate, ph. B: Acetonitrile %A / %B Flow:.0 ml/min Detector: Sorbic Acid. Benzoic Acid. Vanillic Acid. p-coumeric Acid -00. Caffeine. Dextromethorphan. Pyridine. Pseudoephedrine. Diphenhydramine. Nicotine BioBasic SCX, µm, 0x.mm Eluent: 0% NH Acetate, ph. / 0% ACN Flow:.0 ml/min Detector: 0 mm 0 mm 0 mm MIN. Gly-Tyr. Val-Tyr-Val. Methionine Enkephalin. Leucine Enkephalin ph.0 ph. ph.0 ph. 0 MIN BioBasic SCX, µm, 0x.mm Eluent: A: mm NH Formate B: A + M NaCl % A / % B Flow:.0 ml/min Detector: 0-00

5 Better Performance from a Better Ion Exchange Column BioBasic AX columns are designed to give superior reproducibility, both column-tocolumn and batch-to-batch. Stringent quality control tests using ionic analytes and buffered mobile phase conditions ensure a reproducible ion exchange surface and consistent results. The exceptional ion exchange reproducibility of the BioBasic AX packing is demonstrated in Figure. BioBasic AX columns provide superb stability at both low and high ph ranges, unlike many other silica-based ion exchangers. Figures and show column lifetime studies done with,000,000 column volumes at ph. and ph. At these ph extremes, many silica-based columns will show permanent degradation of the bonded phase (ph.) or the underlying silica (ph ). At either ph, the BioBasic AX columns are easily regenerated with a salt gradient to remove impurity buildup caused by the buffers, and return to their original performance. Ion exchange columns often show poor efficiency, especially those that are based on polymeric materials. The BioBasic AX packing is manufactured on a basedeactivated silica, which provides exceptional efficiency in an ion exchange column. In addition, the surface characteristics of the PEI-bonded BioBasic AX stationary phase provide rapid mass transfer for more efficient separations. Figure shows a standard quality control test for the BioBasic AX packing run under normal phase conditions, demonstrating a minimum efficiency of 0,000 plates per meter for each column packed and tested. Figure Stability of BioBasic AX at ph. Figure BioBasic AX Batch-to-Batch Reproducibility PK MIN BioBasic AX, µm, 0x.mm Eluent: 0.0M KH P, ph. Flow:.0 ml/min Detector: Uracil. UMP. AMP. GMP Lot# PK0 RJ Column retention restored Figure Individual Column Test. Toluene. Nitrobenzene. o-nitroaniline. m-nitroaniline. p-nitroaniline Figure Stability of BioBasic AX at ph >0,000 plates per meter Column retention restored -0 0 MIN BioBasic AX, µm, 0x.mm Eluent: % Isoctane / % EtH (0.% HH) Flow:. ml/min Detector:

6 Better Performance from Better Biochromatography Columns BioBasic AX columns provide better separations than many other choices for ion exchange of biological molecules. Figures and show comparisons of superior performance for both nucleic acids and proteins versus other choices for ion exchangers. Figure demonstrates the excellent selectivity and resolution of the BioBasic AX column for a complex mixture of nucleotides compared to a popular 0Å pore size anion exchange column. Figure shows three proteins chromatographed with good separation and efficiency on the BioBasic AX column versus a popular 00Å anion exchanger. BioBasic columns also demonstrate superior performance when compared to other wide-pore reversed phase HPLC columns. Compare the difference in resolution for a BioBasic column and another 00Å C column. Figure (shown on page ) demonstrates the higher resolving power of the BioBasic column for a complex protein mixture, showing an additional peak separated with baseline resolution. Not Just for Large Molecules BioBasic columns are designed to give superior results for the chromatography of proteins, peptides and biomolecules. However, BioBasic columns also give outstanding results for small molecule separations, whether by reversed phase or ion exchange. Please refer to pages - for chromatograms that demonstrate the usefulness of BioBasic columns for proteins, peptides, and oligonucleotides, as well as a number of small molecule applications. Figure BioBasic AX Comparative Nucleotide Separation * Conditions were optimized for the BioBasic AX, then alternative column was run under identical conditions. BioBasic AX 0x.mm MIN Gradient: A: mm KH P, ph. B: 0.M KHP, ph. 0-00%B in 0 min Flow:.0 ml/min Detector: Figure BioBasic AX Comparative Protein Separation BioBasic AX 0x.mm, Alternative column µm, 0Å 00x.mm 0,. Human Transferrin. β-lactoglobulin B. β-lactoglobulin A CompetC * Conditions were optimized for the BioBasic AX, then alternative column was run under identical conditions.. CMP. UDP. UMP. GDP. AMP. CTP. GMP 0. ATP. CDP. UTP. ADP. GTP MIN Alternative column µm, 00Å 00x.mm CompetD MIN MIN Gradient: A: 0.0M TRIS ph B: A +.M NaAcetate, ph 0-00%B in 0 min Flow:.0 ml/min Detector: 0

7 Figure BioBasic Comparative Protein Separation Tryptic Digest of BSA BioBasic µm, 0x.mm Competitor C µm, 0x.mm Tryptic digest of bovine serum albumin MIN Gradient: A: 0.% TFA in H B: 0.% TFA in ACN %B to 00%B in 0 min Flow:.mL/min Detector: Protein Mix compet 0 MIN BioBasic, µm, 0x.mm Gradient: A: 0.% TFA in H B: 0.% TFA in ACN % B - 0% B in 0 min. Flow:.0 ml/min Detector: MIN Peptide Fragments Peptide fragments from BSA treated with Lysylendopeptidase β-lactoglobulins Tryptic Digest of β-lactoglobulin A Sample concentration: ug/0ul of 0mM tris buffer Data courtesy of Miyako Kawakatsu, M&S Instruments Trading, Inc., saka, Japan. β-lactoglobulin B. β-lactoglobulin A β-lactoglobulin A tryptic digest 00ng 00ng 0ng MIN BioBasic, µm, 0xmm Gradient: A: 0.% TFA in H B: 0.% TFA in %ACN -00% to B in 0 min. Hold for min. Flow: 0 µl/min Detector: UV@ 0 System: Gilson small bore HPLC MIN BioBasic AX, µm, 0x.mm Gradient: A: 0.0M TRIS, ph B: A + M NaAcetate, ph 0-00%B in 0 min. Flow:.0 ml/min Detector: MIN BioBasic AX, µm, 0x.mm Gradient: A: 0.0M TRIS, ph B: A + M NaAcetate, ph 0-00%B in 0 min. Flow:.0 ml/min Detector: 0

8 Tryptic Digest of valbumin ligonucleotides Food Additives Tryptic Digest of valbumin ligothymidylic Acid. 0 mer. mer. mer. mer. mer. mer. mer. mer. Uracil. Acesulfame. Benzoic Acid. Sorbic Acid. Aspartame MIN BioBasic AX, µm, 0x.mm Gradient: A: 0.0M TRIS, ph B: A + M NaAcetate, ph 0-00%B in 0 min. Flow:.0 ml/min Detector: MIN BioBasic AX, µm, 0x.mm Gradient: A: mm KH P, ph. B: 0.M KH P, ph. -00%B in min. Flow: 0. ml/min Detector: MIN BioBasic, µm, 0x.mm Gradient: A: 0.0M KH P, ph B: MeH % B to 0% B in 0 min. Flow: ml/min Detector: UV@ 00-0 Aromatic Acids on BioBasic Aromatic Acids on BioBasic AX 0. Uracil. Protocatachuic Acid. Caffeic Acid. p-hydroxybenzoic Acid. Phthalic Acid. Vanillic Acid. Syringic Acid. Phenylacetic Acid. Benzoic Acid 0. Salicylic Acid. p-coumeric Acid. Ferulic Acid. trans-cinnamic Acid. Uracil. Shikimic Acid. Ascorbic Acid. Phenylacetic Acid. Sorbic Acid. Benzoic Acid. Vanillic Acid. p-coumeric Acid MIN BioBasic, µm, 0x.mm Gradient: A: 0.M KH P, ph B: MeH 0%B to 0%B in 0 min. Flow:.0 ml/min Detector: MIN BioBasic AX, µm, 0x.mm Eluent: A: 0mM NH Acetate, ph. B: Acetonitrile %A / %B Flow:.0 ml/min Detector:

9 Small rganic Acids - Variation with ph Sugars in HILIC Mode ph.. Benzoic Acid. Sorbic Acid ph. Glucose. Maltose. Maltotriose. Maltotetraose. Maltopentaose MIN BioBasic, µm, 0x.mm Eluent: A: 0% 0.0M KH P, ph. B: 0% MeH Flow:.0 ml/min Detector: -00 BioBasic, µm, 0x.mm Eluent: A: 0% KH P in H, ph B: 0% MeH Flow:.0 ml/min Detector: MIN MIN BioBasic AX, µm, 0x.mm Eluent: % ACN / % H Flow:.0 ml/min Detector: ELSD Antibiotics. Cephaloridine. Amoxicillin. Ampicillin. Cephalosporin C. N-Acetylpenicillamine. Penicillin G For more information on protein and peptide chromatography, request our publication Guide to Reversed Phase HPLC of Peptides and Proteins MIN BioBasic AX, µm, 0x.mm Eluent: A: 0mM NH Acetate B: Acetonitrile 0%A / 0%B Flow:.0 ml/min Detector: 0 Temp. C BetaBasic and BioBasic are registered Trademarks of Thermo Hypersil-Keystone. 00 Thermo Hypersil-Keystone. All Rights Reserved.