Handbook of HiPep-Columns for Proteins & Peptides Catalog and Technical Notes HiPep Laboratories  URL：www.hipep.jp   info@hipep.jp Tel：075-813-2101  Fax：075-801-0280 from abroad voice +81-75-813-2101, FAX +81-75-801-0280
BACKGROUND The founder of HiPep Laboratories, Dr. Nokihaara has continued to research peptides and their derivatives since 1973. In the meantime, he anticipated HPLC which was introduced in bio-research field, and invested a lot of efforts to purification and analysis. HiPep mainly used a wide variety of C18 reversed-phase silica gel columns for synthetic peptides generated by the SPPS which have different molecular-weights up to 15KDa, also natural peptides isolated from tissue (organs). After foundation of HiPep Laboratories in 2002 the best packing for peptide analysis, separation, and preparative purification had been found and commercialized listed below. Purification tests had been carried out with varieties of packing materials with different derivatization and optimized by using specially designed synthetic peptides. A column sized 20 id X 150 mm can separate around 10 mg of crude peptides at one run. Purification recovery depends on crude materials and generally 30 to 50%. Important performances for HPLC columns: >  High resolution, reproducibility, endurance ▷ Easy migration to semi-preparative purification ▷  Higher recovery in preparative purification
All columns above have greater performances than the others in theoretical plate number, resolution, residual silanol, acid resistance and lower nonspecific adsorption. For designing, we gave priority to highly-efficient separation for peptide and related materials. All analytical columns are LC-MS-suitable, while HiPep Labs routinely use those columns for own R and D as well as contract research.
Precautions for column handling
Please avoid excessive mechanical shock. When you connect columns: There is a mark of FLOW on the column's label. Please connect the column in the direction of eluent flow. When you use a wrench to set tube-in-tube, please put the wrench at the hexagonal part of its end-fitting top. Do not put the wrench at a slit on column's pipe. Before use insoluble material must be removed from sample solution by centrifugation in advance, and have to be filtered with membrane-filters of 0.45 μm or 0.2 μm. Floating materials or precipitates may cause pressure increase trouble by clogged columns. Normal operation pressure: approximately 20 MPa (maximum). In consideration of solvent's viscosity, please set flow volume not to exceed maximum pressure. Appropriate range of column temperature is 15 - 65 ℃. Please set columns temperature, in consideration of peak shape, retention capacity and separation characteristic. Generally silica-based columns should be used between pH 2 - 7. Please set pH according to the purpose of your analysis. When use in alkaline-condition in the long term, column life time may shorten. Any case column life can be changed depending on the analyses conditions such as organic solvent concentration, pH adjuster composition, temperature and structure of analytes. In case of long-term storage of columns after use, please make sure to displace them in column storage solvent. Long-term storage in alkaline or acidic conditions must be avoided. Column-wash: please remove analytes completely by higher concentration of the organic solvent. After analyses, columns have to be stored by displacing in approximately 60% aqueous acetonitrile or approximately 70% aqueous methanol solution, tightly closed and store in room temperature. Next page show the resolution comparison. The key issue in  discovery from natural resources: resolution of peaks patteren HiPep Laboratories  URL：www.hipep.jp   info@hipep.jp Tel：075-813-2101  Fax：075-801-0280 from abroad voice +81-75-813-2101, FAX +81-75-801-0280 HiPep Laboratories Flow rate: 1.0 mL/min (1% = 1 min) HiPep-Cadenza-C18, 3.0 id X 150 mm Flow rate: 0.5 mL/min (1% = 1 min) Comparison of Efficiency in Separation/ Resolution: Analyte as an example: Solid- phase syntheses peptides before purification mol. weight: approximately 3200  (Linear Peptide with Arg x 4, Lys x 9 ) Column Length 150 mm,UV 210 nm, Eluent: A=0.01N HCl, B=0.01 N 90% Acetonitrile, A/B=95/15-65/35 in 30min. Efficient separation and purification for structured peptides with difficult sequences using HiPep-Intrada HiPep-Intrada: Wide-pore Reverse-phase Packing Materials Preparative purification and analysis with HiPep-Intrada Preparative purification with C18 column (20 id x 150 mm) Preparative separation by HiPep-Intrada 20 id x 250 mm (60℃): sharper peak ★ Analytical conditions of purification samples: HiPep-Intrada 3.0 id×150 mm, Flow Rate: 0.3 mL/min A= 0.1% TFA. B= 0.1% TFA in 90%ACN Gradient : B= 45-75% in 30 min  60 ℃ This packing material for HiPep-Intrada has been designed that the resolution in separation is the first priority, thus the column-life is relatively shorter than that of conventional reverse-phase columns. This column is very sensitive against strong acid or alkaline treatment. Following points should be considered in operation to keep longer life. HiPep Laboratories  URL：www.hipep.jp   info@hipep.jp Tel：075-813-2101  Fax：075-801-0280 from abroad voice +81-75-813-2101, FAX +81-75-801-0280
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1.Do not start gradient elution from 100% water. 2.Do not use 100% organic solvents at the end of elution and never washed with 100 % organic solvents. 3.Column Storage: in 30% aqueous methanol solution containing 0.1% acetic acid. Avoid to use 100% water or 70% aqueous methanol solution for storage. 4.HiPep-Intrada often discriminates conformational difference of peptides even the same primary structures (sequence). In fact, sensitivity in separation of conformational differences is often influenced by isomers or other components existing in analytes. Selection of solvent mixture and their ratio for injection for sample are important. Reproducibility of peak profiles is also dependent on the dead space between injector and column-inlet. These phenomena are often observed when analyte is structured peptide such as amyloid, Ab-peptides. Ab-peptides were dissolved in DMSO (100%) and injected to sample loop, the analyte was mixed with the initial eluent in the sample loop, many peaks could be observed, of which mass unit were the same. Those peak patterns obtained were split or broadening. HiPep-Intrada is filled with extremely sensitive derivatized silica.

Analyses and purificaqtionn of βAmyloid and Prion related peptides by HiPep-Intrada
In recent years, some diseases caused by peptides conformational changes are highlighted as intractable disease. In the research of peptides which have specific secondary/tertiary structures in solutions, single substance and their mixture may have different HPLC profiles due to the some interactions. These phenomena seems to be caused by sample preparation procedures not in the columns. In the case that multiple peptides which have specific structures are combined, interactions among peptides may caused, thus same peptides are eluted at different retention times due to their structural difference. Below indicated analyses of mixtures of Αβ(1-40), Αβ(1-42), Αβ(1-43), hence sample preparations is important. Hirata, A., Miyajima, M., and Nokihara, K. Separation of peptides having structures and derivatives of mimosine, a non- proteinogenic amino acid, by a novel reverse-phase HPLC column packed with wide-pore silica,  J. Liquid Chromatography & Related Technologies, 36, 2960-2967, 2013. Structured peptides by the conventional C18 colum gives broad peak and difficult to separate, although HiPep-Intrada Give a sharp peak and easyer to characterize (a)  HiPep-Cadenza C18 (b) HiPep-Intrada (c)  HiPep-Intrada for mixture of Aβ (d)  Enlarged (c) at 60℃ HiPep Laboratories  URL：www.hipep.jp   info@hipep.jp Tel：075-813-2101  Fax：075-801-0280 from abroad voice +81-75-813-2101, FAX +81-75-801-0280
Peptides which have structures, cause aggregation are/or poor solubility show often broad peak on HPLC elution profiles. In these cases purification is difficult and after purification not only purity but also recovery generally low due to broad peaks. To improve these purification, sample preparation methods have been studied. Column: HiPep-Intrada (20 x150 mm), A:0.1% TFA in H₂O; B:0.1% TFA in 90% Acetonitrile, flow rate: 9.9 mL/min, detector: UV at  210 nm, 60 °C) ☆ DMSO preparation Example 1. Peptide A ☆ Nonafluoropentanoic Acid preparation ☆ DMSO preparation ☆ Nonafluoropentanoic Acid preparation Example 3. Peptide C (peak was not detected in DMSO preparation) ☆HFIP preparation ☆ Nonafluoropentanoic Acid preparation Analysis of Αβ(1-43) by HiPep-Intrada 3.0 φ x 150 mm discriminate structure of peptides and proteins. Reference: Peptide Science 2007: S. Aimoto and S. Ono (Eds) The Japanese Peptide Society (2008) page 143-146. HiPep Laboratories  URL：www.hipep.jp   info@hipep.jp Tel：075-813-2101  Fax：075-801-0280 from abroad voice +81-75-813-2101, FAX +81-75-801-0280
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Prion protein related fragment peptides for research (a), (b); PrP(106-126),  (c), (d); PrP[(169-176)+180-195)] HiPep-Cadenza C18 gave single component, although HiPep-Intrada gave multiple components, which was been identifile by LC-MS as multiple MS-signals. Therefore HiPep-Intrada allows components which can not be separated by HiPep-Cadenza C18. Separation example of HiPep-Intrada  single peak doesn't mean a single component The series of this packing materials has been developed for difficult and /or aggregated structured peptides such as  βAmyloid Peptides or Prion related peptides Peptide Analyses: Elution Pattern + Resolution + Recovery → Improved Characterization & Preparative separation by HiPep-Intrada Peptide Science 2007, JPS, pp143, 2008 ; Peptide Science 2008, JPS, pp 533, 2009 ; Peptide Science 2009, JPS, pp 405, 2010; J. Liq. Chrom & Rel. Tech , 2013, 36, 2960. HiPep Laboratories  URL：www.hipep.jp   info@hipep.jp Tel：075-813-2101  Fax：075-801-0280 from abroad voice +81-75-813-2101, FAX +81-75-801-0280 Improved chiral analysis for non proteinogenic amino acid mimosine (a), (b)  GITC-D,L-Ala; lower (c), (d): GITC-D,L-Mimosine; Left (a), (c) = conventional ODS; Right (b), (d): HiPep-Intrada （HPLC condition ambient temp.）. Nokihara, K, et al., Amino Acids, 54, 27-36, 2012. Preparative scale isolation, purification and derivatization of mimosine, a non-proteinogenic amino acid
Evaluation of Packing Materials Used for Preparative HPLC
Purification of Peptide Derivatives
With increasing demands for pharmaceutical peptides, selection and evaluation of packing materials for HPLC, especially silica- based reverse phase, are of great interest for the pharmaceutical industries. Focusing on preparative separation of peptides and their derivatives, several model peptides have been designed. These model peptides were used for separation-tests to compare the resolution of packing materials.  The results were used as a feed-back to find optimal derivatization procedures for the silica surface. In addition to efficient removal of by-products from the target peptide, recovery of desired products is also important for productivity. A GLP-1 analog, taspoglutide, consisting of 30 amino acid residues containing non-natural AAs, was chosen as a model peptide for the recovery test and for comparison of commercially available column packing materials. By-product containing peptides are often deletion peptides, which are found for sequences containing sterically hindered amino acids, cis- trans isomers of Pro-, succinimidyl Asp-, Met(O)-residues or degradation compounds caused by cleavage of Trp residues. Model peptides containing these by-products have been synthesized using conventional Fmoc-SPPS with HBTU and HOBt on an automated peptide synthesizer with low-cost Wang-resin in DMF. Cleaved peptides were characterized with a high resolution reverse phase HPLC-column, HiPep-Cadenza (3 micron ODS, 3 id. X 150 mm), using on-line ion-trap MS to identify the desired peptide and by-products. Several test columns (2 id. X 250 mm) were prepared and, for comparison, commercial ODS was also used. Based on the above tests a novel reverse phase silica for preparative separation has been developed and designated Daisogel®SP (octadesyl silica, 10 micro meter particles, 100 and 120 angstrom porosity). The recovery was calculated by the yield after re-chromatography. Both materials gave >65% recovery and the 100 angstrom material was slightly better than the 120 angstrom product for the model peptide. Yield from crude The homogeneity of the resulting purified Packing material (10 micron) Recovery peptide peptides was confirmed by LCMS using the above HiPep-Cadenza to give >95% purity Daisogel®SP-100-ODS-P (100A) 14.3% 68.2% (calculated from peak area). These materials Daisogel®SP-120-ODS-RPS (120A) 11.8% 66.4% were packed in columns (5 id. X 500 mm) and several peptides were purified on a gram Commercially available 100A-C18 11.1% 32.5% scale with flow rates of 35-50 mL/min. from a leading company ★ Presented at the 31st European Peptide Symposium in 2010 (reprint of proceedings is available upon request) HiPep Laboratories  URL：www.hipep.jp   info@hipep.jp Tel：075-813-2101  Fax：075-801-0280 from abroad voice +81-75-813-2101, FAX +81-75-801-0280
Efficient Solid-Phase Synthesis and Purification of β-Amyloid Peptides by Improved Protocols with a Novel HPLC Column Miyazato, N., Hirata, A., Kawakami, H., Kawahira, N., Ohyama, T., and Nokihara, K. (*Correspondence:info@hipep.jp) HiPep Laboratories, Kyoto and Okinawa, Japan (http://www.hipep.jp/) Synthesis protocols 3   HPLC-Profiles of crude Aβ(1-43) (mwt : calcd. 4615.1) Synthesizer : PSSM-8 (Automated assemble without monitoring) Desired peptide was monitored by LC-MS : TentaGel® (Fmoc-AA TGS PHB ), Strategy  : PyBOP / HOBt / DIEA Resin Reaction Solv. Super mix  (Ref : Int.J.Peptide Res.Therapeutics (2007) 13, 377-386. 2% PIP, 2% DBU in DMF, Resin : ca. 60 mg (15 μmol) Deprotection THE RE : β-Amyloid(1-43) Sample (mwt : caled. 4615.1, Found : [M+2H] 2307.1 respectively ) Gradient : A/B= 80/20-50/50 in 30 min Eluent : A = 0.1 % TFA, B = 0.1 % TFA- 90 % ACN Column : HiPep-Intrada WP-RPم3.0 × 150 mm ( P/N : HIWR03015 ) Flow Rate : 0.3 mL / min This column is commercially available info@hipep.jp 6 (Not efficiently characterized by the conventional ODS-column) 8 Separation of conformational isomers by HiPep Columns: Synthetic human Prion Protein (90-142) HiPep-Cadenza (ODS) Column ($3.0 × 150 mm) HiPep-Intrada  (φ3.0 × 150 mm) F   Gradient : A/B=20-50% in 30 min itな12. 19. 6 ion o Eluent : A=0.1% TFA,  B=0.1% TFA-60% ACN, Column Temp : 60 ℃,  Flow Rate : 0.5 mL / min Separation of conformational isomers the same primary structure, the same MS data, different CD spectra. (published elsewhere) HiPep-Cadenza (φ3.0 × 150 mm)
Eluent: A=NH e OH (pH 9.0), B = 90 % ACN Polymer Column (φ4.6 × 300 mm)
Eluent: A=NH,OH (pH $.0), B = 91 90 % ACN Gradient : A/B=30/10-30/70 in 30 min Gradient : A/B=50/10-80/10 in 30 min Conditions +10 mM sodium phosphate (pH 7.0) +0.1 mM peptide concentration •Room Temperature •Predicted based on Reed et al. (Anal.Biochem.,1997)

9
Conclusion
1.The synthesis and purification of Aβ peptides could be easily performed without expensive reagent, such as pseudoproline or HATU etc. The reaction solvent system plays an important role in assembling of peptides having difficult sequences. 2.HiPep-Intrada is packed by a wide pore reversed-phase silica. (3 μm for analysis and 5 μm for preparative separation) 3.Aβ(1-43) was difficult to characterize by the conventional  ODS columns, although recently developed HiPep-Intrada gave improved elution profiles. Thus Aβ(1-43) could be efficiently purified by a Prep-HiPep-Intrada (20×250 mm), which was characterized by MALDI-TOF-MS. 4.The secondary structure of the present peptides were characterized by CD spectra. Gradest : A/B=10-40% in 30 min HPLC-profiles of synthetic Aβ related peptides by the novel HiPep-Intrada The secondary structure of Aβ peptides by CD B=0.1% TFA-90% ACN Eluent : A=0.1% TFA Column :  φ 3.0 × 150 mm
Flow Rate: 0.5 mL / min A : Aβ(1-43),  B : Aβ(1-42), A ~ C  characterization → difficult by conven- tional ODS columns < 60°C > 5. The present method was applied for synthesis of prion related peptides. info@hipep.jp HiPep Laboratories  URL：www.hipep.jp Tel：075-813-2101  Fax：075-801-0280 from abroad voice +81-75-813-2101,