T009E

Handbook of HiPep-Columns for Proteins & Peptides
Catalog and Technical Notes

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T009E

HiPep Columns: Peptide focused high resolution Separation
HiPep-Cadenzaz, a C18 reverse phase packing
BACKGROUND The founder of HiPep Laboratories, Dr. Nokihaara has continued to research peptides and their derivatives since1973. In the meantime, he anticipated HPLC which was introduced in bio-research field, and invested a lot of efforts topurification 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 ofpacking materials with different derivatization and optimizedby using specially designed synthetic peptides.
A column sized 20 id X 150 mm can separate around 10 mgof 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
P/N

PurposeColumn performances
for peptides had been
compared with different
derivatization for inhouse useSize（mm）

Flow rate

HC18-375

High-throughput analysis

3.0 id X 75

0.3 mL/min

HC18-4675

High-speed analysis

4.6 id X 75

0.5 mL/min

HC18-2150

LC-MS for peptide-mapping

2.0 id X 150

0.15～0.2 mL/min

HC18-3150

Solvent-saving analyses

3.0 id X 150

0.5 mL/min

HC18-46150

Regular analysis

4.6 id X 150

1.0 mL/min

HC18-20150

Semi-preparative purification

20 id X 150

10～20 mL/min

HC18-10250

Tissue extraction, prep.purification

10 id X 250

2～3 mL/min

HC18-20250

Prep. purification

20 id X 250

10～20 mL/min

HC18-28250

Prep. purification

28 id X 250

20～40 mL/min

All columns above have greater performances than the others in theoretical plate number, resolution, residual silanol, acidresistance and lower nonspecific adsorption. For designing, we gave priority to highly-efficient separation for peptide andrelated 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 ofeluent 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 filteredwith membrane-filters of 0.45 μm or 0.2 μm. Floating materials or precipitates may cause pressure increase trouble byclogged columns.
Normal operation pressure: approximately 20 MPa (maximum). In consideration of solvent's viscosity, please set flowvolume 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 andstructure 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 fromnatural resources: resolution of peaks patteren
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2T009E
検出器 A (210nm)
TO6016-586H-TFA221002
40224AC05150125200Standard Column

150

100

150

7550mABS

Identicalpatterns

75mABSmABS

100

100

50

50

0050

25

25

000

2

468

1012141618202224262830

024681012min

14161820222426

2830min

HiPep-Cadenza-C18, 4.6 id X 150 mmFlow 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)

検出器 A (210nm)

TO6016-586H-TFA221002
C18
column of the other40223AC03
manufactures’ products(Identical size, identicalconditions)

120

100

80

120

100

Broad andseparation
is difficult

4.6 id X 150 mm
Flow rate: 1.0 mL/min(1% = 1 min)

60

40

60

40

20

200002468101214

16182022242628

Comparison of Efficiency in Separation/Resolution: Analyte as an example: Solidphase syntheses peptides before purification

80mABSmABS

200

Solvent-saving type

125100mABS

検出器 A (210nm)
TO6016-586HTFA221002 030304C
40308AC02150mol. weight: approximately 3200 (LinearPeptide 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.

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 separation by HiPep-Intrada 20 id x 250 mm(60℃): sharper peakPreparative purification with C18 column(20 id x 150 mm)

Calcd. : 3118.87★

15Found : 1560.4 (M+2H)
Desired

３０４５

Calcd. : 3118.87★

１０Found : 1560.4 (M+2H)
Desired

２０３０４０★ 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.

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3T009E
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. Thesephenomena 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 thesample 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.
P/N

PurposeSize (mm)Flow rate

HiWR-2150

LC-MS for peptide-mapping

2.0 id X 150

0.15～0.2 mL/min

HiWR-3150

Solvent-saving

3.0 id X 150

0.5～0.75 mL/min

HiWR-46150

Regular analysis

4.6 id X 150

0.75～1.25 mL/min

HiWR-10150

Semi-preparative purification

10 id X 150

2～3 mL/min

HiWR-10250

Tissue extraction, prep.purification

10 id X 250

2～3 mL/min

HiWR-20250

Prep. purification

20 id X 250

10～20 mL/min

HiWR-28250

Prep. purification

28 id X 250

20～40 mL/min

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 theresearch 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 Aβ(1-40), Aβ(1-42), Aβ(1-43), hence sample preparations is important.
Hirata, A., Miyajima, M., and Nokihara, K. Separation of peptides having structures and derivatives of mimosine, a nonproteinogenic amino acid, by a novel reverse-phase HPLC column packed with wide-pore silica, J. Liquid Chromatography &Related Technologies, 36, 2960-2967, 2013.

HiPep-Cadenza C18 vs HiPep-Intrada for Aβ peptides

Structured peptides by theconventional C18 columgives broad peak anddifficult to separate,
although HiPep-Intrada
Give a sharp peak andeasyer to characterize(a) HiPep-Cadenza C18(b) HiPep-Intrada(c) HiPep-Intrada for
mixture of Aβ(d) Enlarged (c) at 60℃

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4Sample Preparation in Purification for Poorly-soluble Peptides
Example 1. Peptide A
Peptides which havestructures, cause
aggregation are/orpoor solubility show
often broad peak onHPLC elution profiles.
In these cases
purification is difficultand after purification
not only purity but
also recovery
generally low due tobroad peaks. To
improve thesepurification, 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 ºC)

Example 2. Peptide B

Example 3. Peptide C (peak was not detected in DMSO preparation)

Analysis of Aβ(1-43) by HiPep-Intrada 3.0 φ x 150 mmmAU
210nm,4nm (1.00)
200

at 60℃

Voyager Spec #1[BP = 4616.1, 344]
4616.1269

100

4617.0778

904618.0602

80

150% Intensity

50344.3

4615.0981

70

100

MALDI-TOF
MS Range:
4611-4625

60
50

4619.0729

4614.0431

4030
200

100.0

0
4611.0

5.0

10.0

15.0

20.0

25.0

min

4620.0979
4612.1498

4613.84621.8344
4616.64619.44622.204625.0

Mass (m/z)

Efficient separation for structured peptides, peptides having aggregation tendency using HiPep-Intrada. HiPep-Intradadiscriminate structure of peptides and proteins.
Reference: Peptide Science 2007: S. Aimoto and S. Ono (Eds) The Japanese Peptide Society (2008) page 143-146.

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5T009E

Prion protein related fragment peptides for research(a), (b); PrP(106-126), (c), (d); PrP[(169-176)+180-195)]
Columns (a), (c); HiPep-Cadenza C18（RT）; (b), (d); HiPep-Intrada （60℃）(a)(b)(c)(d)

HiPep-Cadenza C18 gave single component, although HiPep-Intrada gave multiple components, which was been identifile byLC-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.

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6T009E

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 andderivatization 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 silicabased reverse phase, are of great interest for the pharmaceutical industries. Focusing on preparative separation of peptides andtheir 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 forproductivity. A GLP-1 analog, taspoglutide, consisting of 30 amino acid residues containing non-natural AAs, was chosen as amodel peptide for the recovery test and for comparison of commercially available column packing materials. By-productcontaining peptides are often deletion peptides, which are found for sequences containing sterically hindered amino acids, cistrans isomers of Pro-, succinimidyl Asp-, Met(O)-residues or degradation compounds caused by cleavage of Trp residues. Modelpeptides containing these by-products have been synthesized using conventional Fmoc-SPPS with HBTU and HOBt on anautomated 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, 10micro meter particles, 100 and 120 angstrom porosity). The recovery was calculated by the yield after re-chromatography. Bothmaterials 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)
Recoverypeptidepeptides was confirmed by LCMS using theabove 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 gramscale with flow rates of 35-50 mL/min.

Commercially available 100A-C18
from a leading company

11.1%

32.5%

★ Presented at the 31st European Peptide Symposium in 2010 (reprint of proceedings is available upon request)

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7Efficient Solid-Phase Synthesis and Purification of -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/)

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