Protein & peptide Shodex TM columns

Transcription

1 HPLC Columns Protein & peptide Shodex TM columns Technical notebook No. 7

2 Contents. Introduction. Separation of Proteins and Peptides by HPLC. Separation modes -. Size exclusion chromatography --. Separation mechanism --. Introduction of Shodex columns PROTEIN KW-800 series KW00 series -. Ion exchange chromatography --. Separation mechanism --. Introduction of Shodex columns ---. Anion exchange Strong anion exchange Weak anion exchange ---. Cation exchange Strong cation exchange Weak anion exchange IEC QA-8 IEC DEAE-8, Asahipak ES-0N 7C IEC SP Non-porous gel (Fast analysis) Anion exchange DEAEN-T Cation exchange SP-0N (Appendix) Selection of a suitable buffer -. Reversed phase chromatography --. Separation mechanism --. Introduction of Shodex columns IEC CM-8, Asahipak ES-0C 7C Asahipak ODP, C8P, CP series ODP HP series RSpak RP8, DS, DE series -. Hydrophobic interaction chromatography --. Separation mechanism --. Introduction of Shodex columns HIC PH-8 -. Affinity chromatography --. Separation mechanism --. Introduction of Shodex columns AFpak series -. Multi mode --. Separation mechanism --. Introduction of Shodex columns Asahipak GS series

3 . Introduction The analysis of proteins and peptides is essential to food science, biomolecular, and pharmaceutical studies. One of the most useful tools for this purpose is HPLC, which relies on wide variety of separation techniques. The main separation modes used for the analysis of proteins by HPLC are: Size exclusion chromatography, Reversed phase chromatography, Ion exchange chromatography, Hydrophobic interaction chromatography and Affinity chromatography. In addition, Multi mode chromatography, which combines Size exclusion and Reversed phase chromatography (or Ion exchange chromatograpgy) can also be used. In this technical notebook, we will first review each separation mechanism, and then introduce the corresponding Shodex columns along with relevant application data.. Separation of Protein and Peptide by HPLC In order to plan the separation and purification of proteins or peptides, it is necessary to study the character of the target substances and select a suitable separation mode. Accordingly to choose a suitable separation mode, one must first consider which of the following case best describes the analytes: () The case where there is large difference of molecular weight between the target substance and the matrix Size exclusion chromatography () The case where the difference in isoelectric points between the target substance and the matrix is greater than Ion exchange chromatography () The case where there is the difference in hydrophobicity between the target substance and the matrix Reversed phase chromatography, Hydrophobic interaction chromatography () The case where a resin with affinity to the target substance is available Affinity chromatography () The ph, salt concentration, and thermostability of the target substance When analysis is the main purpose, studying points () () is enough for the selection of a suitable separation mode. If purification is the main purpose, studying aspect () is also necessary in order to prevent protein denaturation. Usually, a combination of several separation modes is used for separation and purification. A model purification shceme is shown in Figure. For an unknown sample, Size exclusion chromatography is usually suitable because all substances are eluted within a limited time in order of molecular size. Fig. Model separation & purification scheme Salting out Desalting ( Gel filtration or Ultrafiltration ) Hydrophobic interaction chromatography Size exclusion chromatography Ion exchange chromatography Affinity chromatography Organic solvent fractionation Ion exchange chromatography Size exclusion chromatography ( Removing organic solvent ) Affinity chromatography Hydrophobic interaction chromatography Isoelectric precipitation Ion exchange chromatography Hydrophobic interaction chromatography Size exclusion chromatography Affinity chromatography - -

4 Shodex provides columns corresponding to each of the above mentioned-separation modes for the analysis of proteins and peptides. Figures - & - can be used for the selection of a suitable column, and each separation mode is explained in chapter. Fig. - Shodex column selection guide (Size exclusion, Reversed phase) Separation mode Suitable Shodex columns Protein, Peptide GFC MW 0 KW-80., KW0. 0 KW-80, KW0 0 KW-80, KW0 0 KW0 0 7 Reversed phase MW Low RP8-, RP8- DE-, DE- ODP-0, ODP-0 C8P-0 CP-0 High RP8- Fig. - Shodex column selection guide (Ion exchange, Hydrophobic interaction, Multi mode, Affinity) Separation mode Suitable Shodex columns Protein Peptide Ion exchange PI QA-8 DEAE-8 DEAEN-T ES-0N 7 9 SP-8 SP-0N CM-8 ES-0C Hydrophobic interaction PH-8 Multi mode MW 0 0 GS-0 HQ 0 GS-0 HQ 0 GS-0 HQ 0 GS-0 HQ 0 7 Affinity AFpak series - -

5 . Separation modes -. Size exclusion chromatography --. Separation mechanism Size exclusion chromatography (SEC) relies on the separation of molecules based on their size whereby larger molecules, which have a larger -dimensional volume in the eluent, elute faster. The principle of size exclusion chromatography is shown in Figure. Fig. The principle of SEC SEC : Size Exclusion Chromatography The largest substance Packing material The smallest substance Exclusion limit Elution volume Substances are eluted in decreasing order of molecular size. There is no interaction with the packing material. The packing material has a network of small wedged holes called pores. When proteins are introduced into a column, they diffuse into pores by capillary action. Proteins which are larger than the pore size cannot penetrate into the pore and move forward with eluent, therefore eluting from the column earlier. On the other hand, proteins smaller than the pore size can get into the pore and elute from the column later. With SEC, proteins are eluted from the largest one on. SEC has a disadvantage, which is a low loading capacity and the impossibility to increase the capacity factor (k ) above.0. SEC is therefore not suitable for the separation of complex samples. In spite of that, SEC is frequently used as the first screening method for the separation of unknown proteins, because of the following three reasons: () The eluent can be easily selected. () It is possible to separate by molecular size. () The purification ratio is effective. --. Introduction of Shodex columns Two types of base materials can be packed into a column, i.e. silica-based, and polymer-basedmaterials. Generally speaking for proteins and peptides analysis, silica-based stationary phases show the best separation performance because the base material has a smaller pore size distribution. On the other hand, polymer-based stationary phases are at an advantage in alkaline eluents of ph higher than 7., because silica gel dissolves in alkaline eluents. Figure shows the comparison chromatograms of a Polyethylene glycol (PEG) mixture between the polymer-based column OHpak SB-80 HQ and the silica-based PROTEIN KW-80 & KW-80.. OHpak SB-80 HQ is suitable for the analysis of synthetic polymers with a wider molecular range thanks to its wider pore size distribution. On the other hand, the PROTEIN KW-80 & KW-80. columns are suitable for the analysis of proteins thanks to their narrower pore size distribution. - -

6 Fig. Comparison between silica-based columns KW-80 & KW-80. and polymer-based column SB-80 HQ Fig. - Comparison of chromatograms Sample :. Poly(ethylene glycol) (MW: 0,000). Poly(ethylene glycol) (MW:,000). Poly(ethylene glycol) (MW:,000). Poly(ethylene glycol) (MW: 00). EG, Ethylene glycol (MW: ) SB-80 HQ KW-80 KW min Columns : Shodex OHpak SB-80 HQ or PROTEIN KW-80. or KW-80 (8.0mmID x 00mm each) Eluent : H O Flow rate : 0.mL/min Detector : Shodex RI Column temp. : 0 C Fig. - Comparison of calibration curves 0000 KW-80. KW MW SB-80 HQ Elution time (min) - -

7 PROTEIN KW-800 series (Silica-based) The packing material of the Shodex PROTEIN KW-800 series is based on silica particles whose surface is coated with a hydrophilic polymer. This column is for aqueous SEC, and indexed in the Shodex catalogue under Gel filtration chromatography (GFC). It enjoys a positive reputation thanks to its high recovery ratio of proteins and peptide. The PROTEIN KW-800 series comprises three kinds of columns, differing in pore and particle size. Figure shows the analysis of a mixture of 0 standard proteins with the PROTEIN KW-800 series. Fig. Calibration curve of the PROTEIN KW-800 series Molecular Weight (Protein) KW-80. KW Elution volume (ml) KW-80 Sample. Thyroglobulin (Mw: 9,000). Aldolase (Mw: 8,000). BSA (Mw: 7,000). Ovalbumin (Mw:,000). Peroxidase (Mw: 0,00). Adenylate kinase (Mw:,000) 7. Myoglobin (Mw: 7,000) 8. Ribonuclease A (Mw:,700) 9. Aprotinin (Mw:,00) 0. Vitamin B (Mw:,0) Column : Shodex PROTEIN KW-800 series (8.0mmID x 00mm) Eluent : 0mM Sodium phosphate buffer (ph7.0) + 0.M NaCl Flow rate :.0mL/min Detector : UV (0nm) PROTEIN KW-80. PROTEIN KW-80 PROTEIN KW min min min The PROTEIN KW-800 series line-up PROTEIN KW-800 series Size (mm) I.D. x L Plate Number (TP/column) Exclusion Limit Particle Size Pore Size (Å) Pullulan Protein (µm) Max. Avg. KW x 00 >,000 0,000 0, KW x 00 >,000 70, ,000, KW x 00 >,000 00,000,000,000 7,00 00 KW-G.0 x 0 (Guard column)

8 KW00 series (Silica-based) The Shodex KW00-F series is the high-performance semi-micro column version of the KW-800 series. The theoretical plate number and the sensitivity are improved by reducing the diameter of the packed particle to micrometers. KW0-F, the newest addition to the KW00 line-up, has a larger pore size than any columns in the existing KW-800 series, and can analyze proteins whose molecular weight is larger than,000,000. It enables the scientist to carry out the analysis of protein complexes. Figure shows the calibration curves of the KW00 series with proteins. Fig. Calibration curves of the KW00-F series Fig. 7 Comparison between KW0.-F and KW-80. columns Molecular Weight (Protein) KW0-F KW0-F KW0.-F KW0-F Sample : 0µL. Blue dextran mg/mL. γ-globulin 0.8mg/mL. Ovalbumin 0.8mg/mL. Myoglobin 0.mg/mL. Uridine 0.0mg/mL (A) KW0.-F Elution volume (ml) (B) KW min Figure 7 shows the comparison between the highperformance semi-micro column KW0.-F and the original PROTEIN KW-80. column for protein analysis. The KW00-F series exhibits approximately a. times higher theoretical plate number and times better sensitivity compared to the KW-800 series. Columns : (A) Shodex KW0.-F (.mmid x 00mm) (B) Shodex PROTEIN KW-80. (8.0mmID x00mm) Eluent : 0mM Sodium phosphate buffer (ph7.0) + 0.M NaCl Flow rate : (A) 0.mL/min, (B).0mL/min Detector : UV (80nm) ( C) The KW00 series line-up KW00 series Size (mm) I.D. x L Plate Number (TP/column) Exclusion Limit Particle Size Pore Size (Å) Pullulan Protein (µm) Max. Avg. KW0.-F. x 00 >,000 0,000 0, KW0-F. x 00 >,000 0,000 00, KW0-F. x 00 >,000 00,000,000,000,00 00 KW0-F. x 00 >,000,00,000 0,000,000,000,000 KW00G-A. x 0 (Guard column) - The following semi-micro and micro columns can be prepared. Inner diameter :.0mm,.0mm Length : 0mm, 0mm, 0mm - -

9 -. Ion exchange chromatography --. Separation mechanism Ion exchange chromatography is an analytical method relying on the interaction of ion-charges between the surface of proteins and the surface of the packing material. Packing materials with a positive surface charge are called anion exchangers and those with a negative surface charge are called cation exchangers. Obtaining some information about the isoelectric point (pi) of proteins is useful for selecting suitable analytical conditions. Proteins consist of amino acids, and are amphoteric compounds, so there is ph at which the total sum of electrical charges becomes zero in the solution. That ph is called isoelectric point (pi). Around the pi, the electrical charge on the surface of proteins is neutralized and such proteins cannot easily adsorb on the surface of the packing material. In some cases, proteins precipitate around the pi, a phenomenon known as isoelectric precipitation. In order to adsorb the target protein, the ph of the eluent must be adjusted at more than.0 ph unit from pi. When the ph is more acidic than the pi, a cation exchanger should be used, and when the ph is more alkaline than the pi, an anion exchanger should be selected. Figure 8 shows the mechanism of an anion exchanger. Fig. 8 The mechanism of anion exchange separations [Anion exchange] Anions (-) in the eluent are bound to the anion exchanger groups (+)on the packing material. Anion in eluent Anionic substances in sample Anions (-) in the eluent compete with the anionic substances (-) in the sample. Anionic substances which interact more strongly with the anion exchange group are better retained. Hereinafter, we describe the mechanism of an ion exchange separation through the example of a Diethylaminoethyl (DEAE)- based anion exchange resin. In the case of the separation of a protein whose pi is 7, the ph of the eluent should be adjusted to 8, to make the protein anionic. The DEAE ion exchanger should be equilibrated with a buffer solution with chloride (Cl - ) as the counter ion. When the protein, which has negative charge as an anion, is introduced into the column, it binds to the DEAE ion exchanger while removing Cl -. If a protein with a pi of 9 is mixed in the sample, that protein exists as cation and goes through the DEAE ion exchanger without adsorption. In order to elute the adsorbed proteins, there are two methods. One elution method is the gradual concentration increase of salts such as sodium chloride (NaCl) in the eluent, and another method is the gradual change of ph in the eluent. The first method is used more prevalently. Hereinafter we will discuss the salt concentration gradient method. When the concentration of NaCl increases in the eluent, the concentration of Cl - increases and competes for adsorption against previously adsorbed negatively charged proteins,thus removing proteins from the anion exchanger. The situation of the separation also depends on the type of ion exchanger. An Ion exchanger is classified not only by anion or cation exchanger type, but also by the strength of the exchanger resulting from differences in functional groups. The difference between a strong and a weak exchanger is not a difference in the binding strength for proteins but the difference in the effect of ph change in the eluent, i.e. the difference in buffering capacity. Strong ion exchangers can keep the same electrical charge and adsorb proteins in a wider ph range, while week ion exchangers are affected by ph changes in the eluent, readily changing the ion exchange capacity. In the next chapter, we show how to optimize analytical conditions using Shodex ion exchange chromatography columns

10 --. Introduction of Shodex columns ---. Anion exchange Strong anion exchange IEC QA-8 Weak anion exchange IEC DEAE-8, Asahipak ES-0N 7C Fig. 9 IEC QA-8 (Quaternary ammonium) ph Amount of HCI added (meq/g) A strong anion exchange resin with a quaternary ammonium functional group is packed into the IEC QA-8 column. This column can be used at a wider ph range because the pka of the ion exchange base is.7 (Figure 9). Figure 0 shows the separation of five kinds of proteins with IEC QA-8. Conalbumin is eluted earlier at a ph higher than.0 because the pi of Conalbumin is between.0 and.8. On the other hand, the isoelectric points of other proteins, namely Transferin, Ovalbumin and Trypsin inhibitor are lower than.0 so these elute consecutively throughout the gradual the salt concentration increase. Fig. 0 QA-8 Sample :. Conalbumin. Transferrin. Ovalbumin. Trypsin inhibitor 0 0 min Column : Shodex IEC QA-8 (8.0mmID x 7mm) Eluent : (A) 0mM Piperazine-HCl buffer (ph.0) (B)= (A) + 0.M NaCl Linear gradient : 0min to 0min, 00% (A) to 0% (B) Flow rate :.0mL/min Detector : UV (80nm) Fig. IEC DEAE-8/Asahipak ES-0N 7C (DEAE base) ph 0 8 A weak anion exchange resin with a diethylaminoethyl (DEAE) functional group is packed into IEC DEAE-8 and Asahipak ES-0N columns. These columns are suitable for the separation of acidic proteins because the pka of the ion exchanger is about 7.8 (Figure ) Amount of HCI added (meq/g) - 8 -

11 Figure shows the effect of ph on the separation of proteins on weak anion exchange chromatography column IEC DEAE-8. Under acidic eluents, the four kinds of protein can be separated well but under alkaline eluents, the separation worsens with increasing ph. It is due to the ion exchanger not being properly ionized at alkali ph. Weak anion exchangers (DEAE) are preferably used for protein analysis, even though the ph range for protein retention is narrower than the range for a strong anion exchanger, because milder analytical conditions can be used. Fig. ph.0 ph7.0 ph8.0 ph9.0 ph0.0, 0 0 min 0 0 min 0 0 min 0 0 min 0 0 min Column : Shodex IEC DEAE-8 (8.0mmID x 7mm) Eluent : (A) 0mM Piperazine-HCl buffer (ph.0) 0mM Tris-Trispropanol-HCl buffer (ph7.0) 0mM Tris-HCl buffer (ph8.0) 0mM Ethanolamine-HCl buffer (ph9.0) 0mM,-Diaminopropane-HCl buffer (ph0.0) (B)=(A) + 0.M NaCl Linear gradient : 0min to 0min, (A) to (B) Flow rate :.0mL/min Sample:. Conalbumin. Transferrin. Ovalbumin. Trypsin inhibitor Figure shows the analysis of Lipoxidase (reagent grade) with the IEC QA-8 (strong anion exchange resin) and IEC DEAE-8 (week anion exchange resin). Enzymatic activity was detected in the range delimited by double arrows. Fig. Comparison between QA-8 and DEAE-8 Sample : Lipoxidase 0.% 0µL QA-8 DEAE-8 Lipoxidase.0% 00µL Columns Eluent Linear gradient Flow rate Detector Column temp. : Shodex IEC QA-8 (8.0mmID x 7mm) : (A); 0mM Ethanolamine-HCl buffer (ph9.0) (B); (A) + 0.M NaCl : 0min to 0min, (A) to (B) :.0mL/min : UV(80nm) : Room temp min min Sample : Lipoxidase from soybean Columns Eluent Linear gradient Flow rate Detector Column temp. : Shodex IEC DEAE-8 (8.0mmID x 7mm) : (A); 0mM Tris-HCl buffer (ph8.0) (B); (A) + 0.M NaCl : 0min to 0min, (A) to (B) :.0mL/min : UV(80nm) : Room temp. Anion exchange Product Type Size (mm) ID x L Base material Ion Exchange Capacity (meq/g) Pore size (Å) Particle size (µm) IEC QA-8 Strong 8.0 x 7 Polyhydroxymethacrylate 0.,000 IEC DEAE-8 Weak 8.0 x 7 Polyhydroxymethacrylate 0.,000 8 Asahipak ES-0N 7C Weak 7. x 00 0.,000 9 The following semi-micro and micro columns can be prepared. Inner diameter :.0mm,.0mm, 0.8mm, 0.mm, 0.mm Length : 0mm, 0mm, mm - 9 -

12 ---. Cation exchanger Strong cation exchange IEC SP-8 Weak cation exchange IEC CM-8, Asahipak ES-0C 7C Fig. IEC SP-8 (Sulfopropyl base) ph Amount of NaOH added (meq/g) A strong cation exchange resin with a sulfopropyl functional group is packed into the IEC SP-8 column. This column can be used at a wider ph range because the pka of the ion exchange base is. (Figure ). Figure shows the effect of ph on the separation of proteins with IEC SP-8. The proteins can be retained at a wide ph range, and the five kinds of proteins were separated particularly well at ph 7.0 and ph 8.0. The retention time of Ribonuclease A increases at lower ph and it is eluted after α Chymotrypsinogen A under ph.0. At ph.0 the separation between Ribonuclease A and α Chymotrypsinogen A is insufficient, while at ph.0 and ph.0, the peak shape of Myoglobin is not sharp. Fig. Effect of ph (SP-8) ph.0 ph.0 ph.0 ph7.0 ph8.0 Sample :. Myoglobin. Ribonuclease A. α Chymotrypsinogen A. Cytochrome c. Lysozyme min 0 min 0 min 0 min 0 min Column : Shodex IEC SP-8 (8.0mmID x 7mm) Eluent : (A) 0mM Sodium formate buffer (ph.0) 0mM Sodium malonate buffer (ph.0 and.0) 0mM Sodium phosphate buffer (ph7.0) 0mM HEPES (ph8.0) (B)= (A) + 0.M NaCl Linear gradient : 0min to 0min, (A) to (B) Flow rate :.0mL/min Fig. IEC CM-8/Asahipak ES-0C 7C (Carboxymethyl base) ph 0 8 IEC CM-8 and Asahipak ES-0C are packed with a weak cation exchange resin with Carboxymethyl as the functional group. These columns are suitable for the separation of basic proteins because the pka of the ion exchange base is.7 (Figure ) Amount of NaOH added (meq/g) - 0 -

13 Figure 7 shows the separation of six kinds of proteins including Trypsinogen with the IEC CM-8 (week cation exchange resin) and IEC SP-8 (strong cation exchange) columns. The elution patterns are similar but the elution is faster with SP-8 than with CM-8. Fig. 7 Comparison between CM-8 and SP-8 CM-8 SP-8 Sample :. Myoglobin. Trypsinogen. Ribonuclease A. α Chymotrypsinogen A. Cytochrome c. Lysozyme 0 0 min 0 0 min Columns : (Left) Shodex IEC CM-8 (8.0mmID x 7mm) (Right) Shodex IEC SP-8 (8.0mmID x 7mm) Eluent : (A) 0mM Sodium phosphate buffer (ph7.0) (B)= (A) + 0.M NaCl Linear gradient : 0min to 0min, (A) to (B) Flow rate :.0mL/min Detector : UV (80nm) Cation exchange Product Type Size (mm) ID x L Base material Ion Exchange Capacity (meq/g) Pore size (Å) Particle size (µm) IEC SP-8 Strong 8.0 x 7 Polyhydroxymethacrylate 0.,000 8 IEC CM-8 Weak 8.0 x 7 Polyhydroxymethacrylate 0.,000 8 Asahipak ES-0C 7C Weak 7. x 00 0.,000 9 The following semi-micro and micro columns can be prepared. Inner diameter :.0mm,.0mm, 0.8mm, 0.mm, 0.mm Length : 0mm, 0mm, mm - -

14 ---. Non-porous gel (Fast analysis) Anion exchange DEAEN-T Cation exchange SP-0N For fast analysis, columns packed with a non-porous gel are effective. A non-porous gel is made of packing material that does not have pores. The particle diameter of a non-poous gel can be shortened and the compressive strength of the gel is high, so it can be used for fast analysis with increased eluent flow rates. <Maximum flow rate> DEAE-8, SP-8 (porous resin) :.ml/min DEAEN-T, SP-0N (non-porous resin) :.0ml/mim [Packing material schematic] A disadvantage though, is that the maximum protein loading volume of non-porous gel is reduced. <Maximum protein loading volume> DEAE-8, SP-8 (porous resin) :,000µg/column DEAEN-T, SP-0N (non-porous resin) : 0µg/column Porous gel Non porous gel Figure 8 shows the separation of proteins with IEC DEAEN-T (non-porous weak anion exchange resin). The analytical time with DEAEN-T is shorter than the time with IEC DEAE-8, which is packed with a porous weak anion exchange resin. Fig. 8 Comparison between DEAE-8 and DEAEN-T (Non-porous) DEAE-8 00µL inj. DEAE-N-T 0µL inj. Sample :. Conalbumin (0.mg/mL). Ovalbumin (.mg/ml). Trypsin inhibitor (.0mg/mL) min 0 min Columns : Shodex IEC DEAE-8 (8.0mmID x 7mm) Eluent : (A) 0mM Piperazine-HCl buffer (ph.0) (B)= (A) + 0.M NaCl Linear gradient : 0min to 0min, (A) to (B) Flow rate :.0mL/min Detector : UV (80nm) Columns : Shodex IEC DEAEN-T (.mmid x mm) Eluent : (A) mm Piperazine-HCl buffer (ph.0) (B) (B) = (A) + 0.M NaCl Linear gradient : 0min to 0min, (A) to (B) Flow rate :.ml/min Detector : UV (80nm) - -

15 Non-porous Product Type Size (mm) ID x L Base material Ion Exchange Capacity (meq/g) Pore size (Å) Particle size (µm) IEC DEAEN-T Strong anion. x Polyhydroxymethacrylate 0.. IEC SP-0N Strong cation. x Polyhydroxymethacrylate 0.. The following semi-micro and micro columns can be prepared. Inner diameter :.0mm,.0mm, 0.8mm, 0.mm, 0.mm Length : 0mm, mm (Appendix) Selection of a suitable buffer for the eluent A buffer with a sufficient buffering power at the ph of the eluent and with an ion of identical electrical charge to that of the ion exchanger should be selected for the eluent. The table below shows some examples. We recommend the use of a salt solution of NaCl, KCl, NaSO or KSO, possibly in combination with a buffer solution. The total salt concentration should remain within the 0mM to 00mM range. PH Cation Exchange Resin PH Anion Exchange Resin.8 ~. Sodium formate.8 ~.0 N-Methylpiperazine HCl..8 ~. Sodium succinate Sodium acetate.0 ~.0.8 ~.. ~ 7. Piperazine HCl Bis Tris HCl Bis Tris Propane HCl.0 ~.0 Sodium malonate 7. ~ 7.7 Triethanolamine HCl. ~.7 MES 7. ~ 8.0 Tris HCl.7 ~ ~ 8. Sodium phosphate HEPES 8.0 ~ ~ ~ 9.0 N-Methyldiethanolamine HCl Diethanolamine HCl,-Diaminopropane HCl 8. ~ 8.7 BICINE 9.0 ~ 9. Ethanolamine HCl MES HEPES BICINE Tris Bis Tris : -(N-morpholino)ethanesulfonic acid : -(-hydroxymethyl)--piperazine ethanesulfonic acid : N,N-bis(-hydroxyethyl)glycine : tris(hydroxymethyl)aminomethane : Bis(-hydroxyethyl)iminotris(hydroxymethyl)methane Bis Tris propane :,-Bis[tris(hydroxymethyl)methylamino]propane - -

16 -. Reversed phase chromatography --. Separation mechanism The separation mode of reversed phase chromatography is based on the hydrophobic interactions governing the distribution/adsorption equilibrium between nonpolar amino acids in proteins and the surface of the packing material (See Figure 9). The surface of the packing material s polymer- or silica-based gel is bound with a hydrophobic functional group such as octadecyl (C8). Some types of polymer gels can be used for reversed phase chromatography without the need for a functional group because the base material in itself is hydrophobic. Proteins with a higher hydrophobicity are retained more strongly, so proteins are eluted from the most hydrophilic one on. Fig. 9 Separation mechanism of reversed phase chromatography Low polarity area Packing material : Low polarity (ex.c 8 ) High polarity area Eluent : Highly polar solvent (ex. CH CN/H O) Packing material polarity Eluent polarity Retention strength : Highly polar substance Low polarity substance Elution order : From the most polar substance on --. Introduction of Shodex columns Asahipak ODP, C8P, CP series ODP HP series RSpak RP8, DS, DE series In order to separate the target substance well, several kinds of columns are available with varying polarity and gel pore sizes (Figure 0). A smaller pore size is suitable for peptides, and larger pore size is suitable for proteins. The RSpak RP8- column has the largest pore size (about 0 Å) in the Shodex lineup and is suitable for protein analysis. The pore size of Asahipak ODP, C8P and CP columns is about 0 Å and these can be used for analyzing both proteins and peptides. The RSpak DE column has a smaller pore size (about Å) and is suitable for analyzing smaller molecules like oligopeptides. The above-mentioned columns are polymer-based but can be used with similar analytical condition as silica-based ODS columns. In case the adsorption of the target substance is not sufficient with DE-, DE- or ODS columns, RSpak RP8- and RP8- are recommended. Fig. 0 Characteristics and applications of packing materials for normal & reversed phase columns Large Pore size of packing materials Small Silicapak SIL Saccharides Sugar alcohols Vitamins Polycyclic aromatic compounds Peptides NH CN TMS C C8 ODS NHP-0 DC- DM- CP-0 C8P-0 ODP HP Proteins ODP-0, 0 DE Low molecular weight substances, medicines, organic acids, food additives, ets. RP8- DS-, RP8-, High Polarity of packing materials Low - -

17 The chromatogram and recovery rate of proteins with the polymer-based reversed phase chromatography column RSpak RP8- are shown in Figure. The figure shows that the proteins and peptides are analyzed in minutes with a high recovery rate. The maximum durable pressure is MPa, so the flow rate can be increased for faster analysis. Fig. 7 Recovery of proteins (%). Ribonuclease A 9. Insulin 98. Cytochrome c 00. Lysozyme 00. BSA 98. Myoglobin Ovalbumin min Column : Shodex RSpak RP8- (.mmid x 0mm) Eluent : (A) 0.% TFA aq./ch CN=99/ (B) 0.% TFA aq./ch CN=/9 Linear gradient : 0 to min, 0% (B) to 0% (B) Flow rate :.0mL/min Detector : UV (0nm) Figure shows the chromatogram of a mixed sample including proteins and peptides with gradient elution. The polymer-based Asahipak ODP-0 D column is used for its excellent elution volume reproducibility for proteins and peptides. In addition, the results show a high recovery rate. Fig. 0%CH CN %CH CN Proteins Peptides. Lys-Bradykinin. Bradykinin. Met-Enkephalin. Neurotensin. Leu-Enkephalin. Substance P 7. Bacitracin 8. Insulin 9. Insulin B chain 0. Lysozyme. Mastoparan. Myoglobin Mw VR(mL) First Analysis VR(mL) Second Analysis Recovery (%) min Column : Shodex Asahipak ODP-0 D (.0mm x 0mm) Eluent : 0.0% TFA aq./ch CN Linear gradient : 80/0 to 0/0, 0min Flow rate :.0mL/min Detector : UV (0nm) Column temp. : 0 C - -

18 Figure shows the separation of dipeptides with the polymer-based RSpak DE-. Fig. Sample : Dipeptides. Gly-val. Phe-Gly. Ala-Gly. Val-Phe. Tyr-Leu min Column : Shodex RSpak DE- (.0mmID x 0mm) Eluent : (A) 0.0% TFA aq./ch CN=9/ (B) 0.0% TFA aq./ch CN=7/ Linear gradient : 0 to 0min, (A) to (B) Flow rate :.0mL/min Detector : UV (0nm) Figure shows the comparison of the retention of kinds proteins with the polymer-based reversed phase chromatography columns Asahipak ODP-0, C8P-0 and CP-0. The Octadecyl functional group (C8) is bonded onto the base material of ODP-0, the Octyl group (C8) is on C8P-0, and the Butyl group (C) is on CP-0. The shorter alkyl chain has the weakest hydrophobic interaction. If the retention of the ODP-0 column is too strong, it is recommended to use C8P-0 or CP-0 because the ratio of organic solvent in the eluent can be reduced and the analytical conditions for proteins and peptides can be milder. Fig. Sample :. β-endorphin. Big gastrin. Insulin CP C8P ODP Columns : Shodex Asahipak ODP-0, C8P-0, CP-0 (.mmid x 0mm each) Eluent : 0.0% TFA aq./ch CN=7/8 Flow rate : 0.mL/min Detector : UV (0 nm) Column temp. : 0 C min - -

19 The Shodex Asahipak series line-up Product Size (mm) ID x L Functional group Base material Pore size (Å) Plate number (TPN/column) Particle size (µm) ODP-0 D. x 0 Octadecyl 0 >,000 ODP-0 E. x 0 Octadecyl 0 > 7,000 ODP-0 D.0 x 0 Octadecyl 0 > 9,000 ODP-0 E.0 x 0 Octadecyl 0 >,000 ODP-0G A.0 x 0 Octadecyl (Guard column) ODP-0 B. x 0 Octadecyl 0 >,00 ODP-0 D. x 0 Octadecyl 0 > 9,000 ODP-0 E. x 0 Octadecyl 0 >,000 ODP-0G A. x 0 Octadecyl (Guard column) ODP-0 D.0 x 0 Octadecyl 0 >,000 ODP-0G A.0 x 0 Octadecyl (Guard column) C8P-0 D. x 0 Octyl 0 > 7,000 C8P-0 E. x 0 Octyl 0 >,000 C8P-0G A. x 0 Octyl (Guard column) CP-0 D. x 0 Butyl 0 >,000 CP-0 E. x 0 Butyl 0 > 9,000 CP-0G A. x 0 Butyl (Guard column) The following semi-micro and micro columns can be prepared for ODP-0. Inner diameter :.0mm,.0mm, 0.8mm, 0.mm, 0.mm Length : 0mm, 0mm, 0mm The Shodex RSpak series line-up Product Size (mm) ID x L Functional group Base material Pore size (Å) Plate number (TPN/column) Particle size (µm) RP8-. x 0 Styrene divinylbenzene copolymer 0 >,000 RP8-.0 x 0 Styrene divinylbenzene copolymer 00 >,000. RP8-. x 0 Styrene divinylbenzene copolymer 00 >,000. RP8-G. x 0 Styrene divinylbenzene copolymer (Guard column) DS-.0 x 0 Styrene divinylbenzene copolymer 00 >,00 DS-. x 0 Styrene divinylbenzene copolymer 00 >,000 DS-G. x 0 Styrene divinylbenzene copolymer (Guard column) 0 DE-.0 x 0 Polymethacrylate > 7,000 DE-. x 0 Polymethacrylate >,000 DE-L. x 0 Polymethacrylate > 7,000 DE-S. x 0 Polymethacrylate >,000 DE-G. x 0 Polymethacrylate (Guard column) 0 DE-.0 x 0 Polymethacrylate >,000 DE-SG.0 x 0 Polymethacrylate (Guard column) The following semi-micro and micro columns can be prepared for DE-. Inner diameter :.0mm,.0mm, 0.8mm, 0.mm, 0.mm Length : 0mm, 0mm, 0mm - 7 -

20 -. Hydrophobic interaction chromatography --. Separation mechanism The mechanism of hydrophobic interaction chromatography is based on the hydrophobic interaction between proteins and the surface of the packing material. It is like reversed phase chromatography, however eluent conditions differ. As the salt concentration increases, the hydrophobicity of the protein surface also increases and the protein adsorbs onto the packed material. This effect is similar to the salting out of proteins, a procedure usually carried out with ammonium sulfate. After adsorption, the adsorbed protein is eluted by decreasing the salt concentration thus weakening the hydrophobic interaction with the packing material. The protein is adsorbed under high salt concentration, so this separation mode is usable after ammonium sulfate precipitation or ion exchange chromatography. Moreover a less hydrophobic base like phenyl is used as the functional group, hence the analytical conditions for proteins are milder than the conditions of reversed phase chromatography, preserving the bio-activity of protein. --. Introduction of Shodex columns HIC PH-8 Shodex HIC PH-8 is a column for hydrophobic interaction chromatography, with a phenyl group bound to the packing material as a hydrophobic ligand. Figure shows the separation of kinds of proteins with HIC PH-8. Fig. Sample :. Cytochrome c. Myoglobin. Ribonuclease A. Ovalbumin. Lysozyme. α Chymotrypsinogen min Column : Shodex HIC PH-8 (8.0mmID x 7mm) Eluent : (A)=(B) +.8M Ammonium sulfate (B) 0.M Phosphate buffer (ph7.0) Linear gradient : 0min to 0min, (A) to (B) Flow rate :.0mL/min Detector : UV (80nm) - 8 -

21 The effect of the ammonium sulfate concentration in the eluent on protein separation with HIC PH-8 is illustrated in Figure. The retention of proteins is controlled not only by the salt concentration but also by ph and temperature. The retention is stronger at higher ph or temperatures and weaker under lower ph and temperatures. Fig. Retention time (min) Sample :. α Chymotrypsinogen A. α Chymotrypsin. Lysozyme. Ovalbumin. Ribonuclease A. Myoglobin 7. Cytochrome c Ammonium Sulphate Concentration (M) Column Eluent Linear gradient : (A) to (B) Flow rate :.0mL/min : Shodex HIC PH-8 (8.0mmID x 7mm) : (A)=(B) + Ammonium sulfate (B) 0.M Phosphate buffer (ph7.0) The Shodex HIC PH-8 column Product Size (mm) ID x L Functional group Base material Pore size (Å) Particle size (µm) HIC PH x 7 Phenyl Polyhydroxymethacrylate,

22 -. Affinity chromatography --. Separation mechanism Affinity chromatography is a highly selective separation method relying on biochemical interaction. The mechanism is explained in Figure 7. Fig. 7 Adsorption of Washing out target substance of impurities injection of mixture Elution of the target substance Adsorption Washing Elution Regeneration Matrix (Shodex GEL) spacer ligand Target substance ) Inject sample into an initially equilibrated affinity chromatography column (AFpak). ) Only the substances with affinity for the ligand are retained in the column. ) Substances with no affinity for the ligand are eluted from the column without interacting with the stationary phase. ) The substances retained in the column can be eluted by changing the ph or salt or organic solvent concentrations of the eluent. --. Introduction of Shodex columns AFpak series The rigid polymer-based packing material used with the various ligands for Shodex AFpak columns, has the following advantages. ) High-speed and high-pressure (~0 MPa) analysis ) Minimizes the detachment of ligands, ensuring highly reproducible analyses Application data with each AFpak column is shown below

23 Fig. 8 Ovalbumin Fig. 9 α Antitrypsin Ligand : Concanavain A Sample : Ovalbumin Ligand : Dextran sulfate Sample : Antitrypsin (Human plasma SIGMA) 0% 0% min min Column : Shodex AFpak ACA-89 (8.0mmID x 0mm) Eluent : (A) 0.0M Tris-HCl buffer (ph7.) + 0.M NaCl (B)= (A) + 0.0M α-methyl-d(+)-glucoside Linear gradient : (A) to (B) for 0min Flow rate : 0.mL/min Detector : UV (80nm) Column : Shodex AFpak ADS-89 (8.0mmID x 0mm) Eluent : (A) 0.0M Tris-HCl (ph7.) (B) = (A) +.0M NaCl Linear gradient : (A) to (B) for 0min Flow rate :.0mL/min Detector : UV ( 80nm) Fig. 0 Bovine serum albumin Fig. Ovalbumin / Conalbumin / Lysozyme Ligand : Cibacron blue Ligand : Hepanin Sample: Bovine serum albumin Sample :. Ovalbumin. Conalbumin. Lysozymea Eluent A Eluent B min min Column : Shodex AFpak ACB-89 (8.0mmID x 0mm) Eluent : (A) 0.M Potassium phosphate buffer (ph.0) (B) 0.M Potassium phosphate buffer (ph7.) +.M KCl Step gradient : (A) to (B) Flow rate :.0mL/min Detector : UV (80nm) Column : Shodex AFpak AHR-89 (8.0mmID x 0mm) Eluent : (A) 0.0M Tris-HCl (ph7.) + 0.0M NaCl (B) = (A) + 0. M NaCl Linear gradient : (A) to (B) Flow rate : 0.mL/min Detector : UV ( 80nm) Column temp. : C - -

24 Fig. Glutathione S-transferase Fig. L-lactate dehydrogenasepsin Ligand : Glutathione Sample : Glutathione S-transferase Eluent A Eluent B Eluent A Eluent B min min Column : Shodex AFpak AGT-89 (8.0mm ID x 0mm) Eluent : (A) mm Phosphate buffer (ph7.0) (B) 0mM Tris buffer (ph9.) + mm Glutathione Step gradient : (A) to (B) Flow rate :.0mL/min Detector : UV ( 80nm) Column : Shodex AFpak ANA-89 (8.0mmID x 0mm) Eluent : (A) 0.0M Potassium phosphate buffer (ph7.0) (B) = (A) + 0.M KCl Step gradient : (A) to (B) Flow rate :.0mL/min Detector : UV (80nm) Fig. Super oxide dismutase Fig. Fibrinogen (Sigma) Eluent A Eluent B Eluent A Eluent B min min Column : Shodex AFpak AIA-89 (8.0mmID x 0mm) Eluent : (A) 0.M Sodium acetate buffer (ph7.7) + 0.M NaCl (B) 0.0M Tris-HCl (ph8.0) + 0.M NH Cl Step gradient : (A) to (B) Flow rate : 0.mL/min Detector : UV (80nm) Column : Shodex AFpak ALC-89 (8.0mmID x 0mm) Eluent : (A) 0.0M Tris-HCl (ph7.) + 0.M NaCl + 0.mM CaCl + 0.mM MnCl (B) = (A) + M0. M Methyl mannoside Step gradient : (A) to (B) Flow rate : 0.mL/min Detector : UV (80nm) - -

25 Fig. Plasminogen (from bovine plasma, Sigma) Fig. 7 Human serum Eluent A Eluent B Eluent A Eluent B min 0 8 min Column : Shodex AFpak ALS-89 (8.0mmID x 0mm) Eluent : (A) 0.M Sodium acetate (ph7.) (B) 0.M Sodium acetate + 0.M -aminohexanoic acid (ph.0) Step gradient : (A) to (B) Flow rate :.0mL/min Detector : UV (80nm) Column : Shodex AFpak APG-89 (8.0mmID x 0mm) Eluent : (A) 0.M Sodium phosphate buffer (ph.0) (B) 0.M Sodium citrate buffer (ph.) Step gradient : (A) to (B) Flow rate :.0mL/min Detector : UV (80nm) Fig. 8 Globulin Bovine γ-globulin (Sigma) Fig. 9 Bovine γ-globulin Globulin Sample : Bovine γ-globulin Globulin Eluent B 00% 0 0 Eluent B Eluent A Eluent B 0 0 min min min Column : Shodex AFpak APR-89 (8.0mmID x 0mm) Eluent : (A) 0.0M Sodium phosphate buffer (ph.) (B) = (A) +.0M NaCl Flow rate :.0 ml/min Detector : UV (80nm) Column : Shodex AFpak APA-89 (8.0mmID x 0mm) Eluent : (A) 0.M Sodium phosphate buffer (ph.) (B) 0.M Sodium phosphate buffer (ph.0) Flow rate :.0mL/min Detector : UV (80nm) - -

26 Fig. 0 Globulin Human γ-globulin (Sigma) Fig. Human IgM (Sigma) Eluent 00% Eluent 00% min min Column : Shodex AFpak APR-89 (8.0mmID x 0mm) Eluent : (A) 0.0M Sodium phosphate buffer (ph.) (B) = (A) +.0M NaCl Linear gradient : (A) to (B) Flow rate :.0mL/min Detector : UV (80nm) Column : Shodex AFpak APR-89 (8.0mmID x 0mm) Eluent : (A) 0.0M Sodium phosphate buffer (ph.) (B) (B) = (A) +.0M NaCl Linear gradient : (A) to (B) Flow rate :.0mL/min Detector : UV (80nm) Fig. Human serum albumin (Sigma) Fig. C-Reactive protein (Sigma) Eluent A Eluent B Eluent A Eluent B 0 8 min min Column : Shodex AFpak APD-89 (8.0mmID x 0mm) Eluent : (A) 0.0M Tris-HCl buffer (ph.0) (B) = (A) +.0M KCl Step gradient : (A) to (B) Flow rate :.0mL/min Detector : UV (80nm) Column : Shodex AFpak APE-89 (8.0mmID x 0mm) Eluent : (A) Tris-HCl (ph7.0) + 0.M NaCl + mm CaCl (B) = (A) + 0.M Phosphorylcholine Step gradient : (A) to (B) Flow rate :.0mL/min Detector : UV (80nm) - -

27 Fig. Trypsin Fig. Adenosine phosphate Sample :. Adenosine-'-monophosphate, AMP. Adenosine-'-diphosphate, ADP. Adenosine-'-triphosphate, ATP Eluent A Eluent B min Column : Shodex AFpak AAB-89 (8.0mmID x 0mm) Eluent : (A) 0. M Sodium acetate buffer (ph7.0) + 0.M NaCl (B) 0. M Sodium acetate buffer (ph.8) + 0.M NaCl Step gradient : (A) to (B) Flow rate :.0 ml/min Detector : UV (80 nm) min Column : Shodex AFpak AAF-89 (8.0mmID x 0mm) Eluent : 0.M Ethylmorpholine-acetate buffer (ph7.0) Flow rate :.0mL/min Detector : UV (0nm) Fig. 0.% Avidin, 0µL Fig. 7 Chymotrypsin Sample : 0µL Avidin 0.% Sample : Chymotrypsin (Sigma) B=00% Eluent A 00% Eluent B 00% B=0% min Column : Shodex AFpak ABT-89 (8.0mmID x 0mm) Eluent : (A) 0.0M Sodium phosphate buffer (ph7.0) (B) = (A) +.0M NaCl Linear gradient : 0min to 0min, (A), 0min to 0min, (A) to (B) Flow rate : 0.mL/min Detector : UV (0nm) min Column : Shodex AFpak AST-89 (8.0mmID x 0mm) Eluent : (A) 0.M Sodium acetate buffer (ph7.7) + 0.M NaCl (B) 0.M Sodium acetate buffer (ph.8) + 0.M NaCl Linear gradient : 0min to 0min, (A), 0min to 0min, (A) to (B), after 0min, (B) Flow rate :.0 ml/min Detector : UV (80 nm) - -

28 Fig. 8 Lysozyme Fig. 9 Lysozyme Sample : Lysozyme Sample : Lysozyme Eluent A Eluent A Eluent B Eluent B min min Column : Shodex AFpak AGA-89 (8.0mmID x 0mm) Eluent : (A): 0.0M Tris-HCl buffer (ph7.) + 0.0M NaCl (B) 0.0M Tris-HCl buffer (ph7.) + 0.M NaCl Step gradient : (A) to (B) Flow rate : 0.mL/min Detector : UV (80nm) Column : Shodex AFpak AHR-89 (8.0mmID x 0mm) Eluent : (A) 0.0M Tris-HCl buffer (ph7.) + 0.0M NaCl (B) 0.0M Tris-HCl buffer (ph7.) + 0.M NaCl Step gradient : (A) to (B) Flow rate :.0 ml/min Detector : UV (80nm) Fig. 0 Fibronectin Fig. Trypsin (type I) Sample : Fibronectin (Human plasma, Green Cross) Sample : Trypsin type I Sigma Eluent A Eluent B min Eluent A Eluent B min Column : Shodex AFpak AGE-89 (8.0mmID x0mm) Eluent : (A) 0mM Tris-HCl buffer (ph7.) + 0.0M NaCl (B) = (A) + M Urea Step gradient : (A) to (B) Flow rate : 0.mL/min Detector : UV (80nm) Column : Shodex AFpak AOV-89 (8.0mmID x 0mm) Eluent : (A) 0.M Sodium acetate buffer (ph7.7) + 0.M NaCl (B) 0.M Sodium acetate buffer (ph.8) + 0.M NaCl Step gradient : (A) to (B) Flow rate :.0mL/min Detector : UV (80nm) - -

29 The Shodex AFpak (Column size : I.D. 8.0mm x L 0mm) Product AFpak Ligand Application Ligand load /gel(g) Capacity Particle size (µm) AAB-89 Aminobenzamidine Serine protease 00µmol 8 AAM-89 'AMP NAD, ATP dependent enzymes 0µmol Lactic dehydronase.mg/g 8 AAP-89 Aprotinin Serine proteases mg Trypsin mg/g 8 ABT-89 Biotin Avidin Avidin 9mg/g 8 ACA-89 Concanavalin A (ConA) Glycoprotein, polysaccharides mg 8 ACB-89 Cibacron Blue Albumin, NDA dependent enzymes 0µmol BSA 0mg/column 8 ADS-89 Dextran sulfate Lipoproteins, blood coagulation factors 0mg LDL mg/g 8 AED-89 Ethylenediamine diacetic acid Nucleic acids, serum proteins 0µmol 8 AGA-89 N-acetyl-glucosamine Lectins, carbohydrate metabolizing enzymes Lysozyme 0.mg 8 AGE-89 Gelatin Fibronectin 0mg Fibronectin 0mg/g 8 AGT-89 Glutathione Enzymes related to glutathione 0µmol 8 AHR-89 Heparin Lipoproteins, blood coagulation factors mg Lysozyme mg/column 8 AIA-89 Iminodiacetic acid Interferon, serum proteins 70µmol BSA 70mg/column 8 ALC-89 Lentil lectin Glycoproteins, polysaccharides -7mg 8 ALS-89 Lysine (LCA) Plasminogen, plasminogen activator, RNA, DNA Plasminogen µg/g 8 ANA-89 NAD NAD-Dependent dehydrogenase 0µmol Lactic dehydronase.mg/g 8 AOV-89 Ovomucoid Trypsin-like protease 0mg Trypin 00mg/g 8 APA-89 Protein A Human IgG, immune complexes mg IgG Human 0mg/g 8 APB-89 Aminophenyl boronic acid Nucleic acids and cathecholamines 800µmol Sorbitol 0.mg/column 0 APD-89 Procion red NAD, NADP, interferon enzymes 0-0µmol BSA 7mg/g 8 APE-89 Phosphorylethanolamine C-reactive protein, enzymes C-reactive protein 0.9mg/g 8 APG-89 Protein G IgG immune complex -mg IgG Humnan 0mg/g 8 APH-89 Phenyl alanine Subtilisin Carlsberg 0µmol Subtilisin Carlsberg 9mg/g 8 APR-89 Proteamine IgM -mg IgM Human.9mg/column 8 ARC-89 RCA-I Glycoproteins, polysaccharides 0mg 8 AST-89 Soybean trypsin inhibitor Trypsin-like proteases 0mg Trypsin 00mg/g 8 AWG-89 Wheat germ agglutinin (WGA) Glycoproteins, polysaccharides mg 8 ACH-9 Choline oxydase, acetylcholine esterase Choline, acetylcholine 8 The following semi-micro and micro columns can be prepared. Inner diameter :.0mm,.0mm, 0.8mm, 0.mm, 0.mm Length : 0mm, mm - 7 -

30 -. Multi mode chromatography --. Separation mechanism Multi mode chromatography is an analytical method combining several separation modes. When a single separation mode is not enough for separating the target substance, an improvement can be expected by using multi mode chromatography. The balance between the separation modes involved can be controlled by changing the eluent composition. --. Introduction of Shodex columns Asahipak GS series The Shodex Asahipak GS-HQ series mainly relies on size exclusion chromatography but also works in multi mode with the combination of reversed phase and ion exchange modes One can therefore expect different separation patterns from those obtained by standard size exclusion chromatography. The GS-HQ series is particularly suited for the separation of hydrophilic peptides, especially acidic peptides and basic peptides (See Figure ). The base material is a polymer, resulting in the following advantages; ) usable at a wide ph range, ) washable with alkaline solutions, ) column durability. Fig. Sample : No. MW Σf pl GFC Val-Glu-Glu-Ala-Glu Glu-Ala-Glu Val-Glu-Ser-Glu Arg-Asp Gly-His-Lys Arg-Pro-Lys-Pro Σf : Hydrophobic parameter pl : Isoelectric point GFC Molecular weight 0 RPC IEX 0 ODS column Ion exchange column min min 0 0 min Column : Shodex Asahipak GS-0 HQ (7.mmID x 00mM) Eluent : 0mM Ammonium acetate buffer (ph.7) Flow rate : 0.mL/min Detector : UV (0nm) Column temp. : 0 C - 8 -

31 Metal-bonded amino acids can be analyzed by multi mode chromatography. Figure shows the investigation of metal-bonded amino acids which is important for the study of how metals are metabolized in the human body. Selenized amino acids are analyzed using the micro column GS0A-MD (0. mm ID x 0 mm) and ICP/MS. The sensitivity of the analysis using this micro column is higher than with a conventional type column. Fig Sample : 00 nl. γ-glutamylmethylselenocysteine, γglumesecys. Methylselenocysteine, MeSeCys Intensity (counts) HN COOH O H N COOH SeCH y-glutamylmethylselenocysteine (γglumesecys) 0 COOH Retention time (min) HN SeCH methylselenocysteine (MeSeCys) Column : Shodex GS0A-MD (0.mmID x 0mm) Eluent : 0mM CH COONH buffer (ph.) Flow rate :.0µL/min Detector : ICP-MS (Se at m/z=8) Courtesy of Dr. Yasumitsu Ogra, Graduate School of Pharmaceutical Sciences, Chiba University The Shodex Asahipak GS series Product Size (mm) ID x L Plate Number (TP/column) Exclusion Limit (Pullulan) Particle size (µm) Pore size (Å) Max. Avg Usable ph range GS-0 HQ 7. x 00 > 9,000,000 0 to 9 GS-0 HQ 7. x 00 > 9,000 0, to GS-0 HQ 7. x 00 > 8,000 00,000 7,000 to GS-0 HQ 7. x 00 >8,000,000, ,000 to GS-G 7B 7. x 0 (Guard column) 9 to Max. usable organic solvent concentrations: GS-0 HQ, GS-0 HQ, GS-0 HQ : 00% Methanol, 0% Acetonitrile GS-0 HQ : 0% Methanol, 0% Acetonitrile The following semi-micro and micro columns can be prepared. Inner diameter :.mm,.0mm,.0mm, 0.8mm, 0.mm, 0.mm Length : 0mm, 0mm, 0mm - 9 -

32 HPLC Columns To discover other Shodex technical notebooks and much more visit our website: For any questions, requests or technical support you can contact us at: +9(0) Be up to date with Shodex events and news in our social media Manufactured by SHOWA DENKO K.K. Shodex offices (HEADQUARTERS) Showa Denko K.K Shodex (Separation & HPLC) Group (NORTH & LATIN AMERICA) Showa Denko America, Inc. (EUROPE, MIDDLE EAST & AFRICA) Showa Denko Europe GmbH (CHINA) Shodex China Co., Ltd. (JAPAN) Shoko Co., Ltd. (KOREA) Shoko Korea Co., Ltd F Muza Kawasaki Central Tower, 0, Omiya-cho, Saiwai-ku, Kawasaki, Kanagawa -00 JAPAN Tel: +8(0)080 Fax: +8(0)08 sdk_shodex@sdk.co.jp Web: 0 Lexington Avenue, Suite 80, New York, NY 070 USA Tel: Fax: support@shodex.net Web: Konrad-Zuse-Platz, 889 Munich, GERMANY Tel: +9(0)89999 Fax: +9(0) info@shodex.de Web: 8F, Wang Wang Building, No. Shi Men Yi Road, Jing An, Shanghai, 000, CHINA Tel: +8(0)7 Fax: +8(0) support@shodexchina.com Web: -, Shibakohen -chome, Minato-ku, Tokyo, 0-8, JAPAN Tel: +8(0)90 Fax: +8(0)908 shodex.tokyo@shoko.co.jp Web: Chungjeong Rizion,, Chungjeongno -ga, Seodaemun-gu, Seoul 0-0, KOREA Tel: +8(0)78 Fax: +8(0)78 shoko.korea@shokokorea.com Web: