5 Columns and bulk packings for BIO-MACRO Molecular Separations

Transcription

1 YMC GENERAL CATALOG Columns and bulk packings for BIO-MACRO Molecular Separations Types and characteristics of columns and bulk packing for BIO-MACRO molecular separations YMC-BioPro Ion Exchange columns YMC-BioPro QA-F YMC-BioPro SP-F YMC-BioPro QA YMC-BioPro SP YMC-BioPro Ion Exchange media YMC-Pack Diol Reversed-phase columns for bio molecules YMC-Pack PROTEIN-RP YMC-Pack Diol-NP YMC-Pack Polyamine 6 7 8,9,,, 第 章.indd..8 :: AM

2 Columns and bulk packings for BIO-MACRO molecular separations It is increasingly more important to separate bio molecules such as peptides, proteins and nucleic acids, due to progress of bio pharmaceuticals like monoclonal antibodies. To meet such demands, YMC offers abundant columns and bulk packings for ion exchange, size exclusion, reversed-phase and normal-phase chromatography. Depending on target compounds or scale of the separation, choose appropriate columns and packings. Columns and bulk packings for BIO-MACRO separations (See P, for choosing appropriate column for target compounds.) Separation mode Product name Base material Functional group Purpose compounds Page YMC-BioPro QA-F YMC-BioPro SP-F non-porous CHN + (CH) CHCHCHSO Ion exchange YMC-BioPro QA YMC-BioPro SP YMC-BioPro Q YMC-BioPro Q7 YMC-BioPro S YMC-BioPro S7 Polymer porous porous CHN + (CH) CHCHCHSO CHN + (CH) CHCHCHCHSO Proteins Peptides Nucleic acids Oligonucleotides 6 Size exclusion YMC-Pack Diol Silica gel 6,,, Dihydroxypropyl Reversed-phase Normal phase Triart C8 Hybrid silica YMC-Pack Pro C8 Hydrosphere C8 YMC-Pack Pro C8 RS 8 YMC-Pack ODS-A,, YMC-Pack ODS-AQ, C8 YMC-Pack C8 Silica gel,, C8 YMC-Pack C,, C YMC-Pack Ph Phenyl YMC-Pack CN, Cyanopropyl YMCbasic C8 Proteins Peptides Nucleic acids Oligonucleotides Carbohydrates Proteins Peptides Nucleic acids Oligonucleotides Nucleotides Nucleosides Nucleobases Carbohydrates, YMC-Pack PROTEIN-RP, YMC-Pack PolymerC8 Polymer C8 YMC-Pack Diol-NP 6, Dihydroxypropyl Peptides Nucleotides Silica gel Nucleosides YMC-Pack Polyamine Ⅱ Polyamine Nucleobases Carbohydrates 第 章.indd..8 :: AM

3 Separation of proteins Comparison of separation in different mode Human serum Ion exchange IgG Albumin Size exclusion Albumin Transferrin Transferrin IgG N8E min N866A 6 min Eluent : A) mm Tris-HCl (ph 8.6) B) mm Tris-HCl (ph 8.6) containing. M NaCl -% B (- min), -% B (- min) Flow rate :. ml/min Temperature : Detection :UV at 8 nm Injection : µl ( µl/ml) Eluent :. M KHPO-KHPO (ph 7.) containing. M NaCl Flow rate :. ml/min Temperature :ambient ( ) Detection :UV at 8 nm Injection : µl ( µl/ml) Proteins in human serum are separated by the difference in the surface charge on ion exchange chromatography (IEC) and by the difference in the molecular weight on size exclusion chromatography (SEC). Mouse monoclonal IgG anti-human IgG (Purified by DEAE chromatography, containing NaN) Ion exchange NaN Mouse monoclonal IgG (Anti-human IgG) P8A Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl -% B (-8 min) Flow rate :. ml/min Temperature : Detection :UV at nm Injection : µl (. mg/ml) min P8A Mouse monoclonal IgG (Anti-human IgG) NaN min Eluent :. M KHPO-KHPO (ph 7.) Flow rate :.7 ml/min Temperature :ambient ( ) Detection :UV at nm Injection : µl (. mg/ml) Mouse monoclonal antibody against human IgG is analyzed on ion exchange chromatography (IEC) and size exclusion chromatography (SEC). Several peaks possibly derived from isoform of antibody are observed in ion exchange mode, while a single peak is detected in size exclusion mode. Mouse IgG Fc fragment (Prepared from normal serum) Size exclusion R86D. Rabbit IgG. Mouse IgG Fc fragment min Size exclusion Reversed-phase R869B Mouse IgG Fc fragment min Eluent :. M KHPO-KHPO (ph 6.9) containing. M NaCl Flow rate :. ml/min Temperature :ambient (7 ) Detection :UV at nm Injection : µl (. mg/ml) Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -% B (- min) Flow rate :. ml/min Temperature :7 Detection :UV at nm Injection : µl (. mg/ml) Size exclusion chromatography (SEC) is useful for separation of substances which have distinct differences in molecular weight, like between IgG and its fragments. On the other hand, reversed-phase chromatography (RPC) is suitable for a precise analysis of peptides and proteins with a molecular weight of less than kda such as IgG Fc fragment. 第 章.indd..8 ::8 AM

4 Columns and bulk packings for BIO-MACRO molecular separations Separation of proteins Comparison of separation in different mode Tryptic digests of BSA Ion exchange N8B 6 min * Eluent : A) mm Tris-HCl (ph 8.6) B) mm Tris-HCl (ph 8.6) containing. M NaCl -% B (- min), -6% B (-6 min) Flow rate :. ml/min Temperature : Detection :UV at nm Injection : µl Size exclusion 6 Reversed-phase N86L * MW 6 min N88B 6 min * undigested BSA * Calibration curve of peptides and proteins. Myoglobin (MW 7,). Insulin (Bovine) (MW,7). Neurotensin (MW,67). Tetraglycine (MW 6). Glycine (MW 7) Eluent :. M KHPO-KHPO (ph 7.) containing. M NaCl/acetonitrile (7/) Flow rate :.7 ml/min Temperature :ambient ( ) Detection :UV at nm Injection : µl Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -% B (- min), -% B (- min), % B (-6 min) Flow rate :. ml/min Temperature :7 Detection :UV at nm Injection : µl These chromatograms show separation of tryptic digests of BSA (MW: 66,) in ion exchange chromatography (IEC), size exclusion chromatography (SEC) and reversed-phase chromatography (RPC). The molecular weight of the digests is estimated to be approximately from to, by SEC chromatogram. IEC and RPC chromatograms show many peaks of fragments which are separated by the difference in structure, charge and hydrophobicity. Separation of sugar chains Comparison of separation in different mode Pyridylamino (PA) -Sugar chains Reversed-phase Normal-phase G967A min G96A min Eluent :methanol/ mm NHHPO (/9) Flow rate :. ml/min Temperature :7 Detection :FLS at Ex. nm, Em. nm Injection : µl (. pmol/ml) Sample : PA-Sugar Chain Series, manufactured by TAKARA SHUZO CO., LTD. Eluent :methanol/ mm NHHPO (8/) Flow rate :. ml/min Temperature :7 Detection :FLS at Ex. nm, Em. nm Injection : µl (. pmol/ml) Sample : PA-Sugar Chain Series, manufactured by TAKARA SHUZO CO., LTD. Pyridylamino (PA) sugar chains are often analyzed for structural determination of sugar chain in glycoproteins and glycolipids. Separations of PA sugar chains in reversed-phase (RP) mode and normal-phase (NP) mode are shown. Two dimensional HPLC combining two different modes, such as RP mode and NP mode, is useful tool for structural determination of sugar chain. 第 章.indd..8 :: AM

5 Separation of nucleic acids Comparison of separation in different mode DNA fragments Kb DNA ladder (7 -,6 bp) Ion exchange N8G min Eluent : A) mm Tris-HCl (ph 8.) containing.7 M NaCl B) mm Tris-HCl (ph 8.) containing. M NaCl -% B (- min) Flow rate :. ml/min Temperature : Detection :UV at 6 nm Injection : µl DNA fragments are analyzed with YMC-BioPro QA-F ion exchange column. mm length column of YMC-BioPro QA-F is ideal for high-resolution analysis of nucleic acids. Plasmid pbr restriction fragments Ion exchange Plasmid pbr Hae III digests (8-87 bp) Size exclusion N86F Plasmid pbr (,6 bp) min P867A Plasmid pbr (,6 bp) Plasmid pbr Hae III digests (8-87 bp) min Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl 7-8% B (- min), 8% B (- min) Flow rate :. ml/min Temperature : Detection :UV at 6 nm Injection : µl Eluent :. M KHPO-KHPO (ph 7.) containing. M NaCl Flow rate :.7 ml/min Temperature :ambient ( ) Detection :UV at 6 nm Injection : µl The separation of plasmid pbr restriction fragments (8-87 bp) is compared between in ion exchange mode and size exclusion mode. Ion exchange chromatography (IEC) is applicable to identification of each fragment requiring high resolution and size exclusion chromatography (SEC) is usable for characterization of molecular weight distribution. Primer of DNA sequencing ( mer) Reversed-phase '-CCGCTCGAGCTAAAAAAAGCCTGTGTTACC-' ( mer) mer Eluent : A) mm DBAA* (ph 6.) B) mm DBAA* (ph 6.)/acetonitrile (/) 8-6% B (- min), 6% B (- min) Flow rate :. ml/min Temperature : Detection :UV at 69 nm and ESI negative-mode Injection : µl ( pmol/component) * di-n-butylammonium acetate F6C- min UV TIC This figure shows LC/MS analysis of a mixture of synthetic 7- mer oligonucleotides in reversed-phase mode. Hydrosphere C8 columns have been designed for enhanced retention and selectivity of highly polar compounds. They can achieve excellent separation by one-nucleotide difference and sufficient intensity in UV and ESI-MS. 第 章.indd..8 :: AM

6 Ion Exchange Columns YMC-BioPro Ion Exchange Columns YMC-BioPro ion exchange columns are specially designed for separation of proteins, peptides, and nucleic acids. YMC-BioPro ion exchange columns are available in QA and SP chemistries and are based on µm porous and nonporous hydrophilic polymer beads with low nonspecific adsorption. Ion exchange columns ideal for analysis of proteins, peptides, and nucleic acids Features Ion exchange columns designed for analytical and laboratory-scale purification of proteins, peptides, and nucleic acids Newly developed hydrophilic polymer beads with low nonspecific adsorption Effective surface structure designed for maximum interaction with biomolecules Available in a strong anion exchanger (QA, quaternary ammonium) and a strong cation exchanger (SP, sulfopropyl) Non-porous type for increasing resolution and throughput Porous type for higher binding capacity and recovery Specifications SEM images of polymer beads of YMC-BioPro ion exchange columns Non-porous polymer beads Porous polymer beads YMC-BioPro QA-F YMC-BioPro SP-F YMC-BioPro QA YMC-BioPro SP Matrix Hydrophilic non-porous polymer Hydrophilic porous polymer Particle size µm Charged group CHN(CH) + CHCHCHSO CHN(CH) + CHCHCHSO Counter ion Cl Na + Cl Na + Ion exchange capacity (meq/ml-resin) Dynamic binding capacity (mg/ml-resin) >(BSA) >(human-igg) >(BSA) >7(human-IgG) Usable temperature - 6 Usable ph range. -. Column material PEEK 6 第 章.indd 6..8 ::6 AM

7 Ion Exchange Columns YMC-BioPro QA-F / YMC-BioPro SP-F Ion exchange column based on non-porous polymer beads High efficiency with low operating pressure mm length column for ultra high-throughput analysis mm length column for high-resolution analysis Matrix:Hydrophilic non-porous polymer beads Usable ph range:.. Ion exchange columns for high-throughput and high-resolution analysis of proteins, peptides, and nucleic acids YMC-BioPro QA-F/SP-F columns are ion exchange columns based on non-porous hydrophilic polymer beads with high chemical and mechanical stability, and low nonspecific adsorption of biomolecules. The short columns ( mm, mm) are useful for the fast analysis at a higher flow rate, and the mm length columns are best choice for the quality control assessment of biopharmaceuticals requiring a high-resolution. Ultra high-throughput analysis of proteins AU... N7K min. Ribonuclease A. Cytochrome c. Lysozyme High-resolution analysis of proteins NaN High-resolution analysis of nucleic acids P8A MAb min Eluent : A) mm KHPO-KHPO (ph 6.8) B) mm KHPO-KHPO (ph 6.8) containing. M NaCl -% B (- min) Flow rate :. ml/min ( cm/hr) Temperature : Detection :UV at nm Injection : µl Pressure :.8-. MPa The high mechanical stability of non-porous polymer beads and the short column length enable faster elution of proteins at a higher flow rate. Monoclonal antibody (MAb) against human IgG Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl -% B (-6 min) Flow rate :. ml/min (6 cm/hr) Temperature : Detection :UV at nm Injection : µl (. mg/ml) Sample : Mouse monoclonal IgG anti-human IgG (Purified by DEAE chromatography, containing NaN) min NaN MAb Two different lots of commercially available MAb purified by DEAE chromatography, are analyzed with mm length column of YMC-BioPro QA-F. The MAb is resolved into several peaks, and the lot-to-lot variability is observed. mm length column of YMC-BioPro QA-F/SP-F, which has high efficiency, is ideal for characterization of glycoproteins such as monoclonal antibodies P8B and for quality control assessment of biopharmaceuticals. DNA fragments Kb DNA ladder (7 -,6 bp) Eluent : A) mm Tris-HCl (ph 8.) containing.7 M NaCl B) mm Tris-HCl (ph 8.) containing. M NaCl -% B (- min) Flow rate :. ml/min (8 cm/hr) Temperature : Detection :UV at 6 nm Injection : µl (. mg/ml) N8G min The separation of DNA fragments is shown. YMC-BioPro QA-F of mm length column is good choice for high-resolution analysis of nucleic acids. Ordering information Packing material Particle size (µm) QA-F S- nonporous.6 QFS-6WP.6 QFS-6WP.6 QFS-6WP Packing material Particle size (µm) SP-F S- nonporous.6 SFS-6WP.6 SFS-6WP.6 SFS-6WP 7 第 章.indd 7..8 ::9 AM

8 Ion Exchange Columns YMC-BioPro QA / YMC-BioPro SP Ion exchange column based on porous polymer beads Excellent resolution High binding capacity and high recovery of biomolecules Suitable for laboratory-scale purification Matrix:Hydrophilic porous polymer beads Usable ph range:.. Ion exchange columns for analysis and laboratory-scale purification of proteins, peptides, and nucleic acids YMC-BioPro QA/SP columns are ion exchange columns based on porous hydrophilic polymer beads with low nonspecific adsorption of biomolecules. YMC-BioPro QA/SP columns have superior resolution, high binding capacity and high recovery of various biomolecules, and they allow highly effective analysis and laboratoryscale purification of biopharmaceutical proteins such as antibodies. Excellent resolutions AU. N76E The separation of standard protein mixture is compared between YMC-BioPro SP and a commercial porous polymer cation exchange column. Many impurities are resolved from the main peaks of proteins on YMC-BioPro SP with superior peak shapes. 6 min AU. 6 min N8E IgG IgG Transferrin Transferrin N78F. Ribonuclease A (. mg/ml). Cytochrome c (. mg/ml). Lysozyme (. mg/ml) Separation of proteins in human serum Comparison of separation on YMC-BioPro QA and commercial porous Q type columns Albumin min N8A IgG Transferrin Albumin min Albumin Eluent : A) mm KHPO-KHPO (ph 6.8) B) mm KHPO-KHPO (ph 6.8) containing. M NaCl -% B (-6 min) Flow rate :. ml/min (8 cm/hr) Temperature : Detection :UV at nm Injection : µl Peptide mapping Comparison of separation on YMC-BioPro QA and commercial porous Q type columns 6 min 6 min N8B N87C peaks = peaks = 8 peaks = N8G min N87G 6 min Eluent : A) mm Tris-HCl (ph 8.6) B) mm Tris-HCl (ph 8.6) containing. M NaCl -% B (- min), -% B (- min) Flow rate :. ml/min (8 cm/hr for.6 mmi.d., cm/hr for. mmi.d.) Temperature : Detection :UV at 8 nm Injection : µl Sample :Human serum ( µl/ml) Eluent : A) mm Tris-HCl (ph 8.6) B) mm Tris-HCl (ph 8.6) containing. M NaCl -% B (- min), -6% B (-6 min) Flow rate :. ml/min (8 cm/hr for.6 mmi.d., cm/hr for. mmi.d.) Temperature : Detection :UV at nm Injection : µl Sample :Tryptic digest of BSA 8 第 章.indd 8..8 ::6 AM

9 High binding capacity and recovery Comparison of dynamic binding capacity (DBC) and recovery for BSA Dynamic binding capacity (mg/ml-resin, % breakthrough) Eluted amount (mg/ml-resin) Recovery* (%) YMC-BioPro QA 6 9 Brand T (porous Q type) Brand G (porous Q type) *Recovery:(Eluted amount/dynamic binding capacity) AU.. % breakthrough BSA amount loaded (mg/ml-resin) Column : YMC-BioPro QA.6 mmi.d. Brand T (porous Q type).6 mmi.d. Brand G (porous Q type). mmi.d. Linear velocity :8 cm/hr Equilibration buffer : mm Tris-HCl (ph 8.6) Elution buffer : mm Tris-HCl (ph 8.6) containing. M NaCl Sample : mg/ml Bovine serum albumin (BSA) in equilibration buffer Detection :UV at 8 nm YMC-BioPro QA gives the superior DBC and recovery compared with conventional porous polymer anion exchange columns. The surface structure of YMC-BioPro which is designed for maximum interaction with proteins provides high binding capacity, and the hydrophilic property of polymer beads remarkably reduces nonspecific adsorption of proteins High loadability Comparison of the effect of sample load on YMC-BioPro QA and commercial Q type column N86B N87C N87B N86A Loading amount µg µg µg µg min N86E N88C N88B N86D min µg µg µg µg. Ovalbumin. Trypsin inhibitor Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl -8% B (- min) Flow rate :. ml/min (8 cm/hr for.6 mmi.d., cm/hr for. mmi.d.) Temperature : Detection :UV at 8 nm Injection : µl YMC-BioPro QA shows the excellent resolution and peak shapes even when the loading amount increases. The porous type YMC-BioPro columns are suitable for laboratory-scale purification of proteins. Ordering information Analytical columns Packing material QA S- Particle size (µm) porous.6 QAAS-6WP.6 QAAS-6WP.6 QAAS-6WP Packing material SP S- Particle size (µm) porous.6 SPAS-6WP.6 SPAS-6WP.6 SPAS-6WP 9 第 章.indd 9..8 ::9 AM

10 Ion Exchange Media YMC-BioPro Ion Exchange Media Hydrophilic polymer beads with low nonspecific adsorption Strong anion exchanger (Q type) and strong cation exchanger (S type) Effective surface structure designed for maximum interaction with biomolecules High binding capacity and recovery Matrix:Hydrophilic porous polymer beads Usable ph range:.. Ion exchange media for capture and intermediate purification of biomolecules YMC-BioPro ion exchange media are available in Q and S chemistries on 7 µm and µm of hydrophilic porous polymer beads with low nonspecific adsorption and high binding capacity. YMC-BioPro ion exchange media have similar retention selectivity to YMC-BioPro QA/SP columns, and it allows predictable scale up from analytical to preparative separation. Specifications YMC-BioPro Q YMC-BioPro Q7 YMC-BioPro S YMC-BioPro S7 Matrix Hydrophilic porous polymer beads Particle size µm 7 µm µm 7 µm Charged group CHN(CH) + CHCHCHCHSO Usable ph range. -. High dynamic binding capacity (DBC) for proteins YMC-BioPro ion exchange media have. to.8 times higher DBC of protein than commercial ion exchange media. YMC-BioPro ion exchange media are effective in protein purification from capture step requiring high capacity to intermediate step requiring high efficiency. Anion exchanger Particle size Ion exchange capacity DBC * (µm)(meq/ml-resin) (mg/ml-resin) YMC-BioPro Q Brand G(porous Q type) 9.9 Cation exchanger Particle size Ion exchange capacity DBC * (µm)(meq/ml-resin) (mg/ml-resin) YMC-BioPro S Brand G(porous S type) 9. 8 Dependency of DBC to linear velocity YMC-BioPro ion exchange media show high DBC over a wide range of linear velocity, and the difference of DBC is less than % between cm/hr and cm/hr. YMC-BioPro ion exchange media give increased productivity and reduced cost in biopharmaceutical production. 6 8 * Dynamic binding capacities were determined at % breakthrough under following conditions: Column :.6 mmi.d. Linear velocity :8 cm/hr for anion-exchange resin Equilibration buffer : mm Tris-HCl (ph 8.6) Elution buffer :. M NaCl in equilibration buffer Sample :. mg/ml BSA in equilibration buffer Detection :UV at 8 nm for cation-exchange resin Equilibration buffer : mm Glycine-NaOH (ph 9.) Elution buffer :. M NaCl in equilibration buffer Sample :. mg/ml Lysozyme in equilibration buffer Detection :UV at nm Column :. mmi.d. Equilibration buffer : mm Glycine-NaOH (ph 9.) Elution buffer :. M NaCl in equilibration buffer Sample :. mg/ml Lysozyme in equilibration buffer Detection :UV at nm 第 章.indd..8 :: AM

11 Excellent durability under CIP condition with M NaOH Separation of standard proteins Determination of DBC and recovery CIP with M NaOH ( column volumes at cm/hr) Cleaning in place (CIP) is an important procedure for cleaning and sterilization of columns used for protein purification. The DBC and the selectivity of proteins are unaffected following cycles of CIP with M NaOH. The high chemical stability of BioPro ion exchange media allows effective cleaning with alkaline solution. DBC Conditions of DBC measurement Column :YMC-BioPro S7. mmi.d. Linear velocity :8 cm/hr Equilibration buffer : mm Glycine-NaOH (ph 9.) Elution buffer :. M NaCl in equilibration buffer Sample :. mg/ml Lysozyme in equilibration buffer Detection :UV at nm DBC is determined at % breakthrough 8 6. Ribonuclease A. Cytochrome c. Lysozyme min Purification of IgY from egg yolk extract mv 6 min Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl % B (- min), % B (- min), 9% B (- min) Flow rate :. ml/min (8 cm/hr) Temperature :ambient Detection :UV at 8 nm Injection : ml (ca. mg Protein) *Courtesy of Pharma Foods International Co., Ltd. min Egg yolk antibody (IgY) can be isolated with high purity more than 99% by two chromatographic purification steps, which consist of a capture step by ion exchange chromatography on YMC-BioPro Q7 and a polishing step by size exclusion chromatography on YMC-Pack Diol-. mv 7 SEC Fraction from polishing step by SEC 7. IgY.. purity >99%. min Eluent :. M KHPO-KHPO (ph 6.9) containing. M NaCl Flow rate :.7 ml/min Temperature :ambient Detection :UV at 8 nm Injection : ml (ca.. mg IgY) 97. kda 66. kda. kda 9. kda. kda. kda Non-reduced SDS-PAGE IEC Marker Crude extract fraction SEC fraction Standard IgY IgY Ordering Information Packing material Particle size (µm) Product number Volume ml ml L L L YMC-BioPro Q QAAS * * * * * YMC-BioPro S SPAS * * * * * YMC-BioPro Q7 7 QAAS7 * * * * * YMC-BioPro S7 7 SPAS7 * * * * * 第 章.indd..8 ::6 AM

12 Silica gel SEC YMC-Pack Diol µm silica-based column with high mechanical stability Low-cost size exclusion chromatography (SEC) column Useful for molecular weight determination of proteins and sugars Particle size: µm :6,,, Å Usable ph range:. 7. S i l i c a - b a s e d s i z e e x c l u s i o n c h r o m a t o g r a p h y ( S E C ) c o l u m n YMC-Pack Diol is a size exclusion chromatography column based dihydroxypropyl-bonded silica, and available in four different pore sizes. Diol-,, and are suitable for separation or molecular weight determination of proteins with molecular weights of, to several hundred thousand. Diol-6 is the most suitable for separation of peptides or oligosaccharides whose molecular weights are, or less. Specifications Column Base Functional group Diol-6 Pore size 6 Particle size (µm) Usable ph range Characteristics For molecular weight below, Diol- For molecular weight, to, Silica gel Dihydroxypropyl 7. Diol- For molecular weight, to ca., Diol- For molecular weight ca., to,, Calibration curves of various proteins for three different pore sizes MW 6 Diol- 6 Diol- 7 9 Elution volume (ml) Diol- Column : YMC-Pack Diol 8. mmi.d. Eluent :. M KHPO-KHPO (ph 7.) containing. M NaCl Flow rate :. ml/min Temperature : Detection :UV at 8 nm Separation for standard protein markers MW. IgM 9,. Thyroglobulin 67,. IgA 9,. Fibrinogen,. γ-globulin 8, 6. IgG, 7. Transferrin 7, 8. HSA (human serum albumin) 66, 9. α-antitrypsin,. Ovalbumin,. Carbonic anhydrase,. Trypsin inhibitor,. Myoglobin 7,. α-lactalbumin,. Ribonuclease A,7 6. Cytochrome c, Diol-, Diol- and Diol- are suitable for the separation or molecular weight determination of proteins with molecular weights of, to several hundred thousand. MW. Glutamate dehydrogenase 9,. Lactate dehydrogenase,. Enolase 67,. Adenylate kinase,. Cytochrome c, Column : YMC-Pack Diol 8. mmi.d. Eluent :. M KHPO-KHPO (ph 7.) containing. M NaCl Flow rate :.7 ml/min Temperature :ambient Detection :UV at 8 nm min min min For molecular weight, to, compounds, Diol- is suitable for the separation. 第 章.indd..8 ::9 AM

13 Separation for molecular weight below, peptides min MW. Insulin (Bovine),7. Neurotensin,67. Angiotensin II,. Glycine 7 Column : YMC-Pack Diol-6 8. mmi.d. Eluent :. M KHPO-KHPO (ph 7.) containing. M NaCl/ acetonitrile (7/) Flow rate :.7 ml/min Temperature :ambient Detection :UV at nm MW MW. Myoglobin 7,. Ribonuclease A,7. Cytochrome c,. Insulin (Bovine),7. Insulin B chain,96 6. α-mating factor,68 7. Neurotensin,67 8. Angiotensin I,96 9. CCK-Octapeptide,. Bradykinin,6. Angiotensin II,. Oxytocin,7. Angiotensin III 9. Met-Enkephalin 7. Leu-Enkephalin 6. Tetraglycine 6 7. Glycine 7 For molecular weight below, peptides, Diol-6 is suitable for the separation. Elution volume (ml) Separation of oligo- and polysaccharide min MW. P-8 8,. P- 8,. P-,7. P-,8 Column :YMC-Pack Diol, 8. mmi.d. Eluent :water Flow rate :. ml/min Temperature :ambient Detection :RI Ordering information Analytical columns(stainless columns) Packing material MW Diol-6 Diol- Diol- 9 Diol- Elution volume (ml) MW. Pullulan (P-8) 8,. Pullulan (P-) 8,. Pullulan (P-) 86,. Pullulan (P-),. Pullulan (P-) 8, 6. Pullulan (P-),7 7. Pullulan (P-), 8. Pullulan (P-),8 9. Maltopentadecaose (G),8. Maltoundecaose (G),8. Maltoheptaose (G7),. Maltopentaose (G) 8. Maltotriose (G). Maltose (G). Glucose (G) 8 For separation or molecular weight determination of water-soluble oligo- and polysaccharides, Diol-6, Diol-, Diol-, and Diol- are useful individually or in combination. Diol DL6S-6WT 8. DL6S-8WT 8. DL6S-8WT Diol-.6 DLS-6WT 8. DLS-8WT 8. DLS-8WT Diol-.6 DLS-6WT 8. DLS-8WT 8. DLS-8WT Diol-.6 DLS-6WT 8. DLS-8WT 8. DLS-8WT For preparative columns with an inner diameter of mm or more, see page. Guard columns(stainless columns) Packing material Diol DL6S-8WTG Diol- 8. DLS-8WTG Diol- 8. DLS-8WTG Diol- 8. DLS-8WTG Analytical columns(glass columns) Diol DL6S-8FG 8. DL6S-8FG Diol- 8. DLS-8FG 8. DLS-8FG Diol- 8. DLS-8FG 8. DLS-8FG Diol- 8. DLS-8FG 8. DLS-8FG 第 章.indd..8 :: AM

14 Reversed-phase columns Reversed-phase columns for BIO-MACRO molecular separations Abundant gel types Excellent peak shapes High resolution R e v e r s e d - p h a s e c o l u m n s f o r p e p t i d e s, p r o t e i n s a n d n u c l e i c a c i d s For BIO-MACRO molecular separations, YMC has many choices for reversed-phase HPLC. A listing of the most popular column is found below. Specifications For products other than below listed, see the "Reversed-phase C8 columns (ODS)" and "Reversed-phase columns (other than ODS)" sections. Separation for molecular weight below, (pore size 8 Å, Å) Column Base Functional group Triart C8 Pro series YMC-Pack series Pro C8 Hydrosphere C8 Pro C8 RS Hybrid silica Silica gel Particle size (µm) Carbon Usable content (% ) ph range C8,,.. C8 *,,, 6 *,,. 8. 8,.. Characteristics Excellent chemical durability Superior peak shapes in every condition Processed with YMC's advanced endcapping technology Superior separation for basic compounds Superior separation for hydrophilic compounds Can be used with % water mobile phase Highly durable ODS Superior separation for basic compounds and hydrophobic compounds Currently in use worldwide Available from analytical to preparative ODS-A,,,, 7 ODS-AQ,. 7. Superior separation for hydrophilic compounds Ph Phenyl,,,, 9 Reversed-phase packing material with π electrons PolymerC8 Polymer C8 6,.. Polymer based ODS * Materials for µm is YMC-UltraHT products. Separation for molecular weight, -, (pore size Å, Å) Wide pore column Column Base Functional group Particle size (µm) Carbon Usable content (% ) ph range Characteristics,, ODS with wide pore size ODS-A C8, 7 For separation of peptides and proteins ODS-AQ C8,, Low carbon ODS,, 7 C8 with wide pore size C8 C8, For separation of relatively highly hydrophobic compounds. 7. Silica,, C with wide pore size C gel C, Superior separation for proteins CN cyano propyl YMCbasic C8, 7 PROTEIN-RP. 7. How to select reversed-phase columns To separate proteins or peptides, select columns based on the molecular weight of the compounds to be separated is important. As shown in the table on the right, the C8 column with Å pore size is generally suitable for small peptides up to MW,. In the case of large peptides or small proteins up to MW,, the C8 column with Å pore size often gives the best column efficiency. Furthermore, most of proteins are eluted effectively by the C column with Å. Separation may also be influenced by the hydrophobicity of the analyte and the type of the functional group as well as molecular weight. If the sufficient separation is not achieved with columns marked with a double circle, perform optimization as indicated by the arrows shown in the table. In addition to columns C8, C8, and C shown in the table, PROTEIN-RP and CN type columns with different selectivity are also useful. CN with wide pore size Unique selectivity due to cyano group Superior separation for proteins and peptides, especially for insulin Specialized column with excellent acid resistance for separation of proteins and peptides Molecular weight of sample,,, Functional group C8 C8 C Å Å Å 第 章.indd..8 :: AM

15 Separation of peptides (MW 7 -,6) Excellent peak shapes for basic peptides 6 min 7 A8B 6 min 7 AD. BAM-P (MW,). [D-Ala,Met ]-Enkephalinamide (MW 87). α-endorphin (MW,76). Met-Enkephalin (MW 7). [D-Ala,Met ]-Enkephalin (MW 88) 6. γ-endorphin (MW,899) 7. β-endorphin (MW,6) Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -% B (- min), % B (- min) Flow rate :. ml/min Temperature :7 Detection :UV at nm Generally, the conventional C8 column with Å pore size is suitable for analysis of small peptides up to, in molecular weight. Especially Pro series ODS columns, which are processed with advanced endcapping technology, are ideal for separation of basic peptides. As shown in the above, Hydrosphere C8, a Pro series column, exhibits excellent separations and superior peak shapes of basic peptides (peak and 7), in contrast to the commercial ODS column for hydrophilic compounds, Brand E. Separation of peptides and proteins (MW, - 7,) Comparison of separation on columns with different pore size and functional group C8, Å C8, Å D 8.D min C8, Å C, Å C8, Å 6 8.D, min D 7.D min. Cytochrome c (MW,). Insulin (Bovine) (MW,7). Amyloid β-protein (MW,). Lysozyme (MW,). α-lactalbumin (MW,) 6. Myoglobin (MW 7,) Column : µm,.6 mmi.d. Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -6% B (- min) Flow rate :. ml/min Temperature :7 Detection :UV at nm For proteins and peptides with molecular weight of, to 7,, separation characteristics are compared using columns with different pore size and functional group. In accordance with the table on the previous page, the suitable column is C8, Å for groups of compounds with a molecular weight within this range. If either pore size or functional group of the packing material is not optimized, peak broadening and poor resolution are observed. By using the most suitable column (C8, Å) for the target compounds, sharp peak shapes and excellent separation are achieved. 第 章.indd..8 ::6 AM

16 Reversed-phase columns Reversed-phase columns for BIO-MACRO molecular separations Separation of proteins (MW 66, - 96,) Optimization of eluent conditions (C, Å). BSA (MW 66,). Conalbumin (MW 77,). Lipoxidase (MW 96,).D min 97.D min Column : YMC-Pack C ( µm, Å).6 mmi.d. Eluent : -7% B (- min), 7% B (- min) Flow rate :. ml/min Temperature :7 Detection :UV at nm Gradient elution of water and acetonitrile containing TFA are often employed in an analysis of proteins and peptides. In some cases, addition of a third solvent is effective for change in selectivity and separation. The above example shows the resolution between highmolecular weight proteins (peak and ) is improved by adding -propanol into the standard mobile phase of acetonitrile/water/tfa. Comparison of separation on columns with different pore size and functional group C, Å 7.D C, Å 8 C, Å 97.D 6 min. BSA (MW 66,). Conalbumin (MW 77,). Lipoxidase (MW 96,) Column : µm,.6 mmi.d. Eluent : A) water/tfa (/.) B) acetonitrile/-propanol/tfa(//.) -7% B (- min), 7% B (- min) Flow rate :. ml/min Temperature :7 Detection :UV at nm C8, Å 78.D C8, Å 7.D 8 6 min 8.D 8 6 min Separation characteristics of proteins with molecular weight of 66, to 96, are compared using columns with different pore size and functional group. The columns with smaller pore size, which have the same C functional groups, provide broader peak shapes and poor separations. In comparison among the Å pore columns with different functional groups, the longer alkyl chain such as C8 and C8 results in poor resolution. It is important to choose optimal pore size and functional group depending on molecular weight of proteins for better peak shapes and resolutions. Proteins with molecular weight of, to, are separated effectively by the C column with Å pore size. 6 第 章.indd 6..8 :: AM

17 Separation of nucleic acid bases and nucleotides Nucleic acid bases Nucleotides. '-UTP. '-UDP. '-CMP. '-UMP. '-dcmp 6. '-GMP 7. '-ATP 8. '-IMP 9. '-ADP. '-TMP. '-AMP min S999A min A9A Column : Hydrosphere C8 ( µm, Å).6 mmi.d. Eluent : mm KHPO-KHPO (ph 6.9) Flow rate :. ml/min Temperature :7 Detection :UV at nm Column : Hydrosphere C8 ( µm, Å).6 mmi.d. Eluent : mm KHPO-KHPO (ph.) Flow rate :. ml/min Temperature : Detection :UV at 6 nm Hydrosphere C8 is designed for separation of hydrophilic compounds and can be used with % aqueous mobile phase. Hydrosphere C8 is favorable to the analyses of highly polar compounds such as nucleic acid bases and nucleotides. Separation of oligonucleotides T T T T- T T min Purification of highly polar compounds -oligonucleotide- Crude synthetic mer oligonucleotide Impurities T T mer T J9A JE J9B T JA Recovery 6% purity>99% Oligonucleotides d(pt)- Column : µm,.6 mmi.d. Eluent : A) mm or mm TEAA* (ph 6.) B) mm or mm TEAA* (ph 6.)/acetonitrile (8/) -6% B (- min) Flow rate :. ml/min Temperature : Detection :UV at 69 nm * triethylammonium acetate Hydrosphere C8 shows strong retention and excellent resolution of oligonucleotides even at a low concentration of triethylamine compared to ordinary C8 columns. '-CCGCTCGAGCTAAAA AAAGCCTGTGTTACC-' Eluent : A) mm DBAA*(pH 6.)/methanol(6/) B) mm DBAA*(pH 6.)/methanol(/8) -% B(- min) Temperature :ambient Detection :UV at 69 nm Sample :synthetic oligonucleotide( µm) * di-n-butylammonium acetate min. 7. min Impurities mer min 7.. min Recovery % purity>99% In analytical scale, many impurities could be separated from the target compound by onenucleotide difference on Hydrosphere C8. Even in purification scale, YMC-Actus gave superior separation and recovery. YMC-Actus Hydrosphere C8 is useful for purification of hydrophilic compounds such as oligonucleotides, organic acids, oligosaccharides and glycosides. See P8- for details of YMC-Actus series. 7 第 章.indd 7..8 :: AM

18 Reversed-phase columns Reversed-phase columns for BIO-MACRO molecular separations Ordering information YMC-Pack ODS-A Analytical columns Particle size (µm) S-.6 AAS-6WT.6 AAS-6WT. AAS-WT. AAS-WT. AAS-PWT. AAS-PWT. 7 AAS-LWT. AAS-WT. AAS-WT.6 7 AAS-L6WT.6 AAS-6WT.6 AAS-6WT.6 AAS-6WT.6 AAS-6WT 6. AAS-6WT 6. AAS-6WT 6. AAS-6WT 6. AAS-6WT S-.6 AAS-6WT.6 AAS-6WT.6 AAS-6WT.6 AAS-6WT 6. AAS-6WT 6. AAS-6WT 6. AAS-6WT 6. AAS-6WT Semi-preparative columns S- AAS-WT AAS-WT AAS-WT AAS-WT AAS-WT AAS-WT AAS-WT S- AAS-WT AAS-WT AAS-WT AAS-WT AAS-WT AAS-WT AAS-WT See P for preparative columns other than those listed above. Columns shown in above are only wide pore size columns. Details of pore sizes for 8 Å and Å columns, see the pages of reversed-phase columns. Guard cartridge columns (with inner diameter.,., or. mm: /pack,. mm: /pack) Particle size (µm) S-. AAS-GCC. AAS-CC. AAS-PCC. AAS-CC. AAS-GCC A guard cartridge holder will need to be purchased separately before using this product for the first time. Cartridge holder Semi-micro guard cartridge holder (inner diameter:.,., or. mm) Cartridge holder (inner diameter:. mm) XPCHSMW XPCHW YMC guard cartridge pack S-. AAS-GCCPK. AAS-GCCPK A guard cartridge pack includes a cartridge holder ( set), a guard cartridge column, and a column coupler (produced by PEEK). Guard columns S-. AAS-WFG AAS-WTG. AAS-WFG. AAS-WFG AAS-WTG AAS-WTG S- AAS-WTG. AAS-WFG. AAS-WFG AAS-WTG AAS-WTG 8 第 章.indd 8..8 :: AM

19 Ordering information YMC-Pack ODS-AQ YMC-Pack CN Analytical columns Analytical columns Particle size (µm) Particle size (µm) S-.6 AQS-6WT.6 AQS-6WT Semi-preparative columns S- AQS-WT AQS-WT S- AQS-WT AQS-WT See P6 for preparative columns other than those listed above. Guard cartridge columns (three-pack) S-. CNS-WT. CNS-WT. CNS-PWT. CNS-PWT. CNS-WT. CNS-WT.6 7 CNS-L6WT.6 CNS-6WT.6 CNS-6WT.6 CNS-6WT.6 CNS-6WT 6. CNS-6WT 6. CNS-6WT 6. CNS-6WT 6. CNS-6WT S-. AQS-GCC A cartridge holder will need to be purchased separately before using this product for the first time. Cartridge holder Cartridge holder (inner diameter:. mm) YMC guard cartridge pack XPCHW S-. AQS-GCCPK A guard cartridge pack includes a cartridge holder ( set), a guard cartridge column, and a column coupler (produced by PEEK). Guard columns S-. AQS-WFG AQS-WTG S- AQS-WTG Columns shown in above are only wide pore size columns. Details of pore sizes for 8 Å and Å columns, see the pages of reversed-phase columns. Semi-preparative columns S- CNS-WT CNS-WT See P for preparative columns other than those listed above. Guard cartridge columns (three-pack) S-. CNS-GCC A cartridge holder will need to be purchased separately before using this product for the first time. Cartridge holder Cartridge holder (inner diameter:. mm) XPCHW Guard columns S-. CNS-WFG. CNS-WFG CNS-WTG CNS-WTG 9 第 章.indd 9..8 ::6 AM

20 Reversed-phase columns Reversed-phase columns for BIO-MACRO molecular separations Ordering information YMC-Pack C8 Analytical columns Particle size (µm) S-.6 OCS-6WT.6 OCS-6WT. OCS-WT. OCS-WT. OCS-PWT. OCS-PWT. OCS-WT. OCS-WT.6 7 OCS-L6WT.6 OCS-6WT.6 OCS-6WT.6 OCS-6WT.6 OCS-6WT 6. OCS-6WT 6. OCS-6WT 6. OCS-6WT 6. OCS-6WT S-.6 OCS-6WT.6 OCS-6WT.6 OCS-6WT.6 OCS-6WT 6. OCS-6WT 6. OCS-6WT 6. OCS-6WT 6. OCS-6WT Semi-preparative columns S- OCS-WT OCS-WT OCS-WT OCS-WT OCS-WT OCS-WT OCS-WT S- OCS-WT OCS-WT OCS-WT OCS-WT OCS-WT OCS-WT OCS-WT See P8 for preparative columns other than those listed above. Columns shown in above are only wide pore size columns. Details of pore sizes for 8 Å and Å columns, see the pages of reversed-phase columns. Guard cartridge columns (with inner diameter.,., or. mm: /pack,. mm: /pack) Particle size (µm) S-. OCS-GCC. OCS-CC. OCS-PCC. OCS-CC. OCS-GCC A cartridge holder will need to be purchased separately before using this product for the first time. Cartridge holder Semi-micro guard cartridge holder (inner diameter:.,., or. mm) Cartridge holder (inner diameter:. mm) XPCHSMW XPCHW Guard columns S-. OCS-WFG OCS-WTG. OCS-WFG. OCS-WFG OCS-WTG OCS-WTG S- OCS-WTG. OCS-WFG. OCS-WFG OCS-WTG OCS-WTG 第 章.indd..8 ::7 AM

21 Ordering information YMC-Pack C Analytical columns Guard cartridge columns (with inner diameter.,., or. mm: /pack,. mm: /pack) Particle size (µm) S-.6 BUS-6WT.6 BUS-6WT. BUS-WT. BUS-WT. BUS-PWT. BUS-PWT. BUS-WT. BUS-WT.6 7 BUS-L6WT.6 BUS-6WT.6 BUS-6WT.6 BUS-6WT.6 BUS-6WT 6. BUS-6WT 6. BUS-6WT 6. BUS-6WT 6. BUS-6WT S-.6 BUS-6WT.6 BUS-6WT.6 BUS-6WT.6 BUS-6WT 6. BUS-6WT 6. BUS-6WT 6. BUS-6WT 6. BUS-6WT Semi-preparative columns S- BUS-WT BUS-WT BUS-WT BUS-WT BUS-WT BUS-WT BUS-WT S- BUS-WT BUS-WT BUS-WT BUS-WT BUS-WT BUS-WT BUS-WT See P9 for preparative columns other than those listed above. Columns shown in above are only wide pore size columns. Details of pore sizes for 8 Å and Å columns, see the pages of reversed-phase columns. Particle size (µm) S-. BUS-GCC. BUS-CC. BUS-PCC. BUS-CC. BUS-GCC A cartridge holder will need to be purchased separately before using this product for the first time. Cartridge holder Semi-micro guard cartridge holder (inner diameter:.,., or. mm) Cartridge holder (inner diameter:. mm) XPCHSMW XPCHW Guard columns S-. BUS-WFG BUS-WTG. BUS-WFG. BUS-WFG BUS-WTG BUS-WTG S- BUS-WTG. BUS-WFG. BUS-WFG BUS-WTG BUS-WTG See P76 for YMCbasic. See P, for YMC-Pack PROTEIN-RP. 第 章.indd..8 ::8 AM

22 Reversed-phase columns YMC-Pack PROTEIN-RP Improved recovery of proteins or peptides Improved durability when used with TFA solution Enables elution of high molecular weight proteins Carbon content:% Usable ph range:. 7. R e v e r s e d - p h a s e c o l u m n f o r s e p a r a t i o n o f p r o t e i n s o r p e p t i d e s YMC-Pack PROTEIN-RP is a reversed-phase column utilizing a silica gel base. It contains a stationary phase, specifically designed for separation of proteins or peptides. Problems that are associated with conventional reversed-phase columns with short alkyl chain lengths are minimized. Robust column lifetime and excellent recovery of hydrophobic proteins are typically possible on this phase. Improved recovery of proteins or peptides Recovery of proteins or peptides Sample YMC-Pack PROTEIN-RP Recovery(%) C column, company A C column, company B Ribonuclease A Cytochrome c Lysozyme Myoglobin BSA Ovalbumin Transferrin Insulin(bovine) Insulin B chain α-mating factor Leu-Enkephalin Gly-Gly-Gly-Gly Improved durability when used with TFA solution Recovery of various proteins and peptides is shown. YMC-Pack PROTEIN-RP gives greater recovery than competitive C columns. left graph shows the test result of the stability of the stationary phase with.% aqueous TFA. YMC-Pack PROTEIN-RP The Retention of diphenyl on C columns manufactured by other companies greatly decreases as time passes. This is caused by detachment of butyl groups from the packing material due to acid hydrolysis. Retention of diphenyl on YMC-Pack PROTEIN-RP is shown to be stable after hours of mobile phase flow.. Uracil. Benzene. Naphthalene. Diphenyl Flow conditions Eluent :water/tfa (/.) Flow rate :. ml/min Temperature :ambient min min Measurement conditions Column :YMC-Pack PROTEIN-RP.6 mmi.d. Eluent :acetonitrile/water (/6) Flow rate :. ml/min Temperature : Detection :UV at nm,. AUFS 第 章.indd..8 :: AM

23 A p p l i c a t i o n Proteins. Cytochrome c (MW,). BSA (MW 66,). Ferritin (MW,) min Column :YMC-Pack PROTEIN-RP.6 mmi.d. Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -9% B (- min, linear) Flow rate :. ml/min Temperature :ambient Detection :UV at 8 nm,. AUFS A p p l i c a t i o n min Ordering information Analytical columns Particle size (µm) S-. PR99S-WT. PR99S-WT.6 PR99S-6WT.6 PR99S-6WT Semi-preparative columns S- PR99S-WT PR99S-WT PR99S-WT Proteins and peptides. Met-Enkephalin (MW 7). Leu-Enkephalin (MW ). Oxytocin (MW,7). Bradykinin (MW,6). Angiotensin I (MW,96) 6. Ribonuclease A (MW,7) 7. α-mating factor (MW,68) 8. Insulin(bovine)(MW,7) 9. Cytochrome c (MW,). Lysozyme (MW,). BSA (MW 66,). β-lactoglobulin (MW 8,). Ovalbumin (MW,) Column :YMC-Pack PROTEIN-RP.6 mmi.d. Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -9% B (-6 min, linear) Flow rate :. ml/min Temperature :ambient Detection :UV at nm,. AUFS Cartridge holder Cartridge holder (inner diameter:. mm) XPCHW Guard columns S-. PR99S-WFG PR99S-WTG PR99S-WTG Guard cartridge columns (three-pack) S-. PR99S-GCC A cartridge holder will need to be purchased separately before using this product for the first time. 第 章.indd..8 :: AM

24 Normal-phase columns YMC-Pack Diol-NP Silica gel bonded with dihydroxypropyl group Two pore sizes of Å and 6 Å are available Normal-phase separation using non-polar solvents Useful for hydrophilic interaction chromatography (HILIC) Different separation characteristics from bare silica gel : Å, 6 Å Usable ph range:. 7. Diol column for non-aqueous Normal-Phase (NP) and Hydrophilic Interaction Chromatography (HILIC) YMC-Pack Diol-NP shows retention behavior of normal-phase chromatography when it is used with low-polarity mobile phases. Hydroxy groups on the surface of the stationary phase act as polar groups. YMC- Pack Diol-NP is as widely applicable to normal-phase separation as silica gel. It is also useful in cases where separation optimization is difficult to achieve using bare silica gel. In addition, YMC-Pack Diol-NP is usuable as a HILIC mode column when it is used with a mobile phase consisted of a high concentration of organic solvent in water or an aqueous buffer. Separation of highly hydrophilic compounds in HILIC mode Nucleic acid bases and Nucleosides HN O O N H HN O HOHC O O N N NH N N N HOHC O OH OH OH OH OH OH OH OH. Uracil. Uridine. Adenosine. Cytosine. Cytidine 6. Guanosine O N NH N H N O HOHC O NH N HN HN HOHC O N O N N min R96P 6 Column : YMC-Pack Diol-NP ( µm, Å). mmi.d. Eluent :water/acetonitrile (/9) containing mm CHCOONH Flow rate :. ml/min Temperature : Detection :UV at nm YMC-Pack Diol-NP shows superior performance as a HILIC mode column when it is used with a mobile phase consisted of a mixture of organic solvent/aqueous solution. Separation of hydrophilic compounds which are hardly retained on reversed-phase column A99E 6 min Eluent : A) acetonitrile/tfa/tea (/./.) B) water/tfa/tea (/./.) -% B (- min), % B (- min) Flow rate :. ml/min Temperature :ambient Detection :UV at nm, Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -% B (- min), % B (- min) Flow rate :. ml/min Temperature :ambient Detection :UV at nm A997I 6 8 min Peptides. TRP-GLY (MW 6). GLY-GLY-PHE (MW 79). Angiotensin I (MW,96). Substance P (MW,8). Insulin (bovine) (MW,7) Comparison of peptide separation on YMC-Pack Diol-NP and YMC- Pack ODS-A is shown. YMC-Pack Diol-NP offers favorable resolution of between peak and peak, highly hydrophilic peptides which cannot be separated in reversedphase mode with ODS column. An interaction between hydroxyl groups on the Diol-NP stationary phase and highly hydrophilic compounds provides enhanced retention and superior resolution. Shipping solvent for Diol-NP is hexane/-propanol (99./.). In case of using mobile phase including water, take care of miscibility. For details and ordering information, see P86, 87. 第 章.indd..8 ::6 AM

25 Normal-phase columns YMC-Pack Polyamine Ⅱ Silica gel chemically bonded with polyamine The most suitable column for separation of sugars Solves durability problem of conventional silica-based amino columns Useful for separation of hydrophilic compounds including vitamins Useful for separation of fat-soluble compounds using nonaqueous mobile phase : Å Usable ph range:. 7. A m i n o c o l u m n w i t h i m p r o v e d d u r a b i l i t y YMC-Pack Polyamine Ⅱ is a silica-based packing bonded with polyamine. It is particularly useful for separation of sugars. The column lifetime of YMC-Pack Polyamine Ⅱ in aqueous mobile phase is longer than conventional silica-based amino columns, and thus is applicable to separation of oligosaccharides using mobile phase with relatively higher water content. In addition, YMC-Pack Polyamine Ⅱ can be used to separate ionic compounds with a combination of normal-phase mode and weak anion exchange mode. Suitable column for separation of sugars Elution volume of sugars and sugar alcohols. Fructose. Glucose. Sucrose. Maltose. Lactose 6 6 Lactitol Palatinit Lactose Maltose min F68H Column : YMC-Pack Polyamine Ⅱ.6 mmi.d. Eluent :acetonitrile/water (7/) Flow rate :. ml/min Temperature : Detection :RI, -6 RIU/FS Excellent durability Durability in 7% acetonitrile mobile phase condition Rate of initial retention of Lactose (%) 8 6 Elution volume (ml) Raffinose Maltitol Inositol Palatinose Sucrose Galactose Mannose Glucose Mannitol Sorbitol Fructose Arabitol Ribitol Xylitol Xylose Fucose Erythritol Rhamnose Glycerol Sucralose Column volumes 7% 8% 8% YMC-Pack Polyamine Ⅱ shows little change in retention time of lactose after passing, column volumes of 7% acetonitrile mobile phase. On the other hand, retention time on conventional silica-based amino column decreases to 8% of the initial value continuously from the beginning. Column : YMC-Pack Polyamine Ⅱ.6 mmi.d. Temperature : Acetonitrile concentration (%) Not detected with 8% acetonitrile For details and ordering information, see P88, 89. 第 章.indd..8 ::9 AM

26 第 章.indd 6..8 ::9 AM