Column Selection Guide

Transcription

1 Column Selection Guide USP Column selection guide (Biochromatography) Comparison of separation mode ~6 Reversed-phase separation of peptides and proteins ~ Column selection guide (Low molecular weight organic compounds)----- Application (Fat-soluble vitas, Water-soluble vitas)-- Application (Water-soluble vitas, rganic acids, Ao acids)--- Reversed-phase column selection guide----

2 USP USP CLASS o. L L L7 L8 L L L L L L6 USP Description ctadecyl silane chemically bonded to porous or nonporous silica or ceramic microparticles,. to μm in diameter, or a monolithic silica rod. Porous silica particles,. to μm in diameter, or a monolithic silica rod. ctylsilane chemically bonded to totally porous or superficially porous silica particles,. to μm in diameter, or a monolithic silica rod. An essentially monomolecular layer of aopropylsilane chemically bonded to totally porous silica gel support,. to μm in diameter. itrile groups chemically bonded to porous silica particles,. to μm in diameter. Phenyl groups chemically bonded to porous silica particles,. to μm in diameter. Trimethylsilane chemically bonded to porous silica particles, to μm in diameter. Dihydroxypropane groups chemically bonded to porous silica or hybrid particles,. to μm in diameter. Polyvinylalcohol chemically bonded to porous silica particle, μm in diameter. Butyl silane chemically bonded to totally porous silica particles,. to μm in diameter. Functional group C8 Silica C8 YMC product page YMC-Triart C8 9~6 YMC-Triart C8 ExRS 6 Meteoric Core C8 Meteoric Core C8 BI 7~7 YMC-UltraT Pro C8 8 YMC-UltraT ydrosphere C8 8 YMC-Pack Pro C8 8 ydrosphere C8 8 YMC-Pack Pro C8 RS 86 YMC-Pack DS-A 87 YMC-Pack DS-AM 87 YMC-Pack DS-AQ 88 YMC-Pack DS-AL 88 J'sphere DS-8 J'sphere DS-M8 J'sphere DS-L8 YMC-Pack SIL YMC-Pack SIL-6 89 YMC-Triart C8 6 Meteoric Core C8 7~7 YMC-Pack Pro C8 96 YMC-Pack C 8 97 YMCbasic YMC-Pack 8 C YMC-Pack C 99 Phenyl YMC-Triart Phenyl 6 YMC-Pack Ph 98 C YMC-Pack TMS 98 Diol YMC-Triart Diol-ILIC 66 YMC-Pack Diol-P YMC-Pack Diol-6 YMC-Pack Diol- YMC-Pack Diol- YMC-Pack Diol-, 6 Polyvinylalcohol YMC-Pack PVA-Sil C YMC-Pack Pro C 96 YMC-Pack C 97 YMC-Pack PRTEI-RP 99 L7 Porous silica particles, to μm in diameter. Silica YMC-Pack SIL-G, L L L L L9 L6 Packing having the capacity to separate dextrans by molecular size over a range of, to, Da. It is spherical, silica-based, and processed to provide p stability. Cellulose tris-,-dimethylphenylcarbamate coated porous silica particles, to μm in diameter. Pentafluorophenyl groups chemically bonded to silica particles by a propyl spacer,. to μm in diameter. Amylose tris-,-dimethylphenylcarbamate-coated, porous, spherical, silica partilces, to μm in diameter. Packing for the size-exclusion separation of proteins (separation by molecular weight) over the range of to 7, kda. The packing is a spherical.-to -μm, silica or hybrid packing with a hydrophilic coating. C silane bonded phase on a fully porous spherical silica, to μm in diameter. Diol Cellulose tris-,- dimethylphenylcarbamate YMC-Pack Diol-6 YMC-Pack Diol- YMC-Pack Diol- YMC-Pack Diol-, 6 CIRAL ART Cellulose-C 6~9 PFP YMC-Triart PFP 6 Amylose tris-,- dimethylphenylcarbamate Diol CIRAL ART Amylose-C 6~9 YMC-Pack Diol-6 YMC-Pack Diol- YMC-Pack Diol- YMC-Pack Diol-, 6 C YMC Carotenoid

3 Column selection guide (Biochromatography) Proteins Peptides Ion exchange Molecular weight, or less YMC-BioPro YMC-Pack Diol For separation of biomolecules by the difference in surface charge For separation of biomolecules by molecular weight P.7~9 P.~7 YMC-Triart C8 Suitable as the first choice DS column P.9~6 Meteoric Core C8 Core-shell column with ultra fast analysis P.7~7 Molecular weight, or more YMC-Triart C8 Meteoric Core C8 BI For separation of biomolecules with molecular weight of up to, using high temperature Core-shell column for separation of biomolecules with molecular weight of up to, P.9~6 P.7~7 Wide-Pore Columns Column with wide pore size useful for separation of macromolecules P.7 YMC-Pack PRTEI-RP Specialized column with excellent acid resistance for separation of proteins or peptides P.99 ILIC YMC-Triart Diol-ILIC For separation of polar compounds with poor retention on reversed-phase columns P.66 ucleic acids Ion exchange ligonucleotides ucleic acids YMC-BioPro For separation of biomolecules by the difference in surface charge P.7~9 ligonucleotides ucleic acids YMC-Pack Diol For separation of biomolecules by molecular weight P.~7 ucleic acid bases ucleosides ucleotides YMC-Triart C8 ydrosphere C8 Usable with % aqueous mobile phase P.9~6 P.8, 8 ligonucleotides YMC-Triart C8 ydrosphere C8 Wide-Pore Columns Usable with % aqueous mobile phase Column with wide pore size useful for separation of macromolecules P.9~6 P.8, 8 P.7 ILIC ucleic acid bases ucleosides ucleotides YMC-Triart Diol-ILIC YMC-Pack Polyae II For separation of polar compounds P.66 P.6, 7 Sugars YMC-Pack Diol For separatiion or molecular weight deteration of sugars P.~7 YMC-Triart C8 ydrosphere C8 Usable with % aqueous mobile phase P.9~6 P.8, 8 ILIC YMC-Triart Diol-ILIC YMC-Pack Polyae II For separation of polar compounds P.66 P.6, 7

4 Comparison of separation mode Separation of proteins by different mode uman serum Ion exchange YMC-BioPro QA μm, X.6 mmi.d. IgG Albu Transferrin YMC-Pack Diol- + Diol- μm, X 8. mmi.d. X Albu Transferrin IgG 8E 866A 6 Eluent : A) mm Tris-Cl (p 8.6) B) mm Tris-Cl (p 8.6) containing. M acl -%B (- ), -%B (- ) Flow rate :. ml/ Temperature : C Detection : UV at 8 nm Injection : μl ( μl/ml) Eluent :. M K P -K P (p 7.) containing. M acl Flow rate :. ml/ Temperature : ambient ( C) 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 a ) Ion exchange YMC-BioPro QA-F μm, X.6 mmi.d. a Mouse monoclonal IgG (Anti-human IgG) P8A P8A YMC-Pack Diol- μm, X.6 mmi.d. Mouse monoclonal IgG (Anti-human IgG) a Eluent : A) mm Tris-Cl (p 8.) B) mm Tris-Cl (p 8.) containing. M acl -%B (-8 ) Flow rate :. ml/ Temperature : C Detection : UV at nm Injection : μl (. mg/ml) Eluent :. M K P -K P (p 7.) Flow rate :.7 ml/ Temperature : ambient ( C) 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) YMC-Pack Diol- μm, X 8. mmi.d.. Rabbit IgG. Mouse IgG Fc fragment Reversed-phase YMC-Pack C ( Å) μm, X.6 mmi.d. Mouse IgG Fc fragment R86D R869B Eluent :. M K P -K P (p 6.9) containing. M acl Flow rate :. ml/ Temperature : ambient (7 C) Detection : UV at nm Injection : μl (. mg/ml) Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -%B (- ) Flow rate :. ml/ Temperature : 7 C Detection : UV at nm Injection : μl (. mg/ml) chromatography (SEC) is useful for separation of substances which have distinct differences in molecular weight, like between IgG and its fragments. n 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.

5 Separation of proteins by different mode Tryptic digests of BSA Ion exchange YMC-BioPro QA μm, X.6 mmi.d. * Eluent : A) mm Tris-Cl (p 8.6) B) mm Tris-Cl (p 8.6) containing. M acl -%B (- ), -6%B (-6 ) Flow rate :. ml/ Temperature : C Detection : UV at nm Injection : μl 8B 6 6 YMC-Pack Diol- + Diol-6 μm, X 8. mmi.d. X 86L * MW 6 Calibration curve of peptides and proteins. Myoglobin (MW 7,). Insulin (Bovine) (MW,7). eurotensin (MW,67). Tetraglycine (MW 6). Glycine (MW 7) Eluent :. M K P -K P (p 7.) containing. M acl/acetonitrile (7/) Flow rate :.7 ml/ Temperature : ambient ( C) Detection : UV at nm Injection : μl Reversed-phase YMCbasic μm, X. mmi.d. 88B 6 * Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -%B (- ), -%B (- ), %B (-6 ) Flow rate :. ml/ Temperature : 7 C Detection : UV at nm Injection : μl * undigested BSA 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 by different mode Pyridylao (PA) -Sugar chains Reversed-phase YMC-Pack DS-A ( Å) μm, X.6 mmi.d..pa-sugar Chain ormal-phase YMC-Pack Polyae II μm, X.6 mmi.d..pa-sugar Chain.PA-Sugar Chain G967A G96A Eluent : methanol/ mm P (/9) Flow rate :. ml/ Temperature : 7 C Detection : FLS at Ex. nm, Em. nm Injection : μl (. pmol/ml) Sample : PA-Sugar Chain Series, manufactured by TAKARA BI IC. Eluent : methanol/ mm P (8/) Flow rate :. ml/ Temperature : 7 C Detection : FLS at Ex. nm, Em. nm Injection : μl (. pmol/ml) Sample : PA-Sugar Chain Series, manufactured by TAKARA BI IC. Pyridylao (PA) sugar chains are often analyzed for structural deteration of sugar chain in glycoproteins and glycolipids. Separations of PA sugar chains in reversed-phase (RP) mode and normal-phase (P) mode are shown. Two dimensional PLC combining two different modes, such as RP mode and P mode, is useful tool for structural deteration of sugar chain.

6 Comparison of separation mode Separation of nucleic acids by different mode DA fragments Kb DA ladder (7 -,6 bp) Ion exchange YMC-BioPro QA-F μm, X.6 mmi.d. Eluent : A) mm Tris-Cl (p 8.) containing.7 M acl B) mm Tris-Cl (p 8.) containing. M acl -%B (- ) Flow rate :. ml/ Temperature : C Detection : UV at 6 nm Injection : μl 8G DA 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 YMC-BioPro QA-F μm, X.6 mmi.d. Plasmid pbr ae III digests (8-87 bp) Plasmid pbr (,6 bp) Eluent : A) mm Tris-Cl (p 8.) B) mm Tris-Cl (p 8.) containing. M acl 7-8%B (- ), 8%B (- ) Flow rate :. ml/ Temperature : C Detection : UV at 6 nm Injection : μl 86F YMC-Pack Diol- + Diol- μm, X 8. mmi.d. X Plasmid pbr ae III digests (8-87 bp) P867A Plasmid pbr (,6 bp) Eluent :. M K P -K P (p 7.) containing. M acl Flow rate :.7 ml/ Temperature : ambient ( C) 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. ligonucleotide (mi RA) YMC-Triart C8 μm, X. mmi.d. -pugg AGU GUG ACA AUG GUG UUG- ( nt, MW 689.) -pugg AGU GUG ACA AUG GUG UUG U- ( nt, MW 796.) UV MS nt nt MIC (m/z 7., m/z 798.) nt nt Eluent : A) mm DBAA* (p 7.) B) mm DBAA* (p 7.)/acetonitrile (/) 6-7%B (- ) Flow rate :. ml/ Temperature : C Detection : UV at 6 nm and ESI-negative mode Injection : μl ( nmol/ml) Instrument : LC) Shimadzu Proence MS) Shimadzu LCMS * di-n-butylae-acetic acid This figure shows LC/MS analysis of oligonucleotides in reversed-phase mode. YMC-Triart C8 columns are useful for oligonucleotides and they can achieve excellent separation by onenucleotide difference and sufficient intensity in UV and ESI-MS Courtesy of M.Yamada, SIMADZU CRPRATI 6

7 Reversed-phase separation of peptides and proteins ow to select reversed-phase columns To separate proteins or peptides, it is important to select columns based on the molecular weight of the compounds to be separated. 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, PRTEI-RP and C type columns with different selectivity are also useful. Molecular weight of sample,,, Functional group Pore size Å Å Å C8 C8 C Separation of peptides (MW 7 -,6) Excellent peak shapes for basic peptides ydrosphere C8 ( Å) μm, X.6 mmi.d. 6 7 A8B Brand E ( Å) μm, X.6 mmi.d. (DS column for hydrophilic compounds) 6 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 (- ), %B (- ) Flow rate :. ml/ Temperature : 7 C 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 Triart and Pro series DS columns, which are processed with advanced endcapping technology, are ideal for separation of basic peptides. As shown in the above, ydrosphere C8, a Pro series column, exhibits excellent separations and superior peak shapes of basic peptides (peak and 7), in contrast to the commercial DS 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 Pore size 6 C8, Å Functional group 6 76.D, 79.D C8, Å 6 C, Å 6 8.D 7.D ptimized combination of pore size and functional group C8, Å 6 8.D. Cytochrome c (MW,). Insulin (Bovine) (MW,7). Amyloid β-protein (MW,). Lysozyme (MW,). α-lactalbu (MW,) 6. Myoglobin (MW 7,) Column : μm, X.6 mmi.d. Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) -6%B (- ) Flow rate :. ml/ Temperature : 7 C 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 above, 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. 7

8 Reversed-phase separation of peptides and proteins Separation of proteins (MW 66, - 96,) ptimization of eluent conditions (C, Å) A)water/TFA (/.) B)acetonitrile/TFA (/.).D -propanol addition A)water/TFA (/.) B)acetonitrile/-propanol/TFA (//.) 97.D. BSA (MW 66,). Conalbu (MW 77,). Lipoxidase (MW 96,) Column : YMC-Pack C ( μm, Å) X.6 mmi.d. Gradient : -7%B (- ), 7%B (- ) Flow rate :. ml/ Temperature : 7 C 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. BSA (MW 66,). Conalbu (MW 77,). Lipoxidase (MW 96,) Column : μm, X.6 mmi.d. Eluent : A) water/tfa (/.) B) acetonitrile/-propanol/tfa (//.) -7%B (- ), 7%B (- ) Flow rate :. ml/ Temperature : 7 C Detection : UV at nm 97.D 8 6 Pore size Functional group C, Å C8, Å 7.D 78.D C, Å C8, Å 7.D 8.D 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. 8

9 Effect of column temperature on separation of peptides and proteins C Condition A (MW -8,) Condition B (MW,-,7) MPa (,6-,7 psi) Peak capacity =, MPa (,7-, psi) Peak capacity = 67 7 C MPa (,9-,68 psi) Peak capacity = 79 F8B * MPa (,97-, psi) Peak capacity = 7 F6B * F8A * F6A * Analytes MW Peak width ½( ) C 7 C Condition A. xytocin,7.7.. Leu-Enkephalin 6... β-endorphin,6.6. Insulin,7.. β-lactoglobulin A 8,.. Condition B. Lysozyme,.69.. α-chymotrypsinogen, β-lactoglobulin A 8,.8.8 Column : YMC-Triart C8 (.9 μm, Å), X. mmi.d. Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.) - condition A B) acetonitrile/-propanol/tfa (//.) - condition B Gradient : -8%B (- ) - condition A -6%B (- ) - condition B Flow rate :. ml/ Detection : UV at nm Injection : μl ( μg/ml) - condition A μl ( μg/ml) - condition B System : Agilent SL PC (peak capacity) = + (gradient time/peak width*) *peak width = W.h average The effect of temperature on separation of peptides and proteins with a variety of molecular weight (MW) is estimated. The separations at C and 7 C are compared. By increasing column temperature to 7 C, selectivity change is observed, and peaks become sharper. Thus, improved resolution especially for larger molecules is obtained. Generally, larger molecules diffuse very slowly compared to small molecules. An elevated temperature can improve efficiency and peak shape by lowering mobile phase viscosity and improving mass transfer. Temperature is a simple and effective tool to increase resolution in separation of proteins and peptides. 9

10 Reversed-phase separation of peptides and proteins Improvement of resolution by increasing column temperature and coupling of.9 μm columns C.9 µm, X. mmi.d. gradient MPa (6,7-7, psi) Peak capacity = 6 P7A C Peak capacity =.9 µm, X. mmi.d. gradient MPa (,-, psi) Coupling of two columns P78A 6 8 Two coupled.9 µm, X. mmi.d. gradient MPa (8,-8,9 psi) Peak capacity = 6 9 P77A Column : YMC-Triart C8 (.9 μm, Å) Eluent : A) water/tfa (/.) B) acetonitrile/tfa (/.8) -%B (- ) for a single column -%B (- ) for two coupled columns Flow rate :. ml/ Detection : UV at nm Injection : μl for a single column μl for two coupled columns Sample : Tryptic digest of Bovine emoglobin System : Agilent 9 % more peaks can be resolved by increasing the column temperature to 7 C in the separation of tryptic digest of emoglobin. The outstanding efficiency obtained by a coupling of two mm length of Triart.9 μm columns reduces co-elution peaks and allows the precise separation in an analysis of complicated samples, such as peptide mapping.

11 Column selection guide (Low molecular weight organic compounds) Pharmaceutical products Agricultural chemicals Metabolites Food additives atural products thers ormalphase ILIC YMC-Triart C8 YMC-Triart (C8, C8 ExRS, C8, PFP, Phenyl) YMC-Pack SIL, SIL-6 YMC-Pack Diol-P YMC-Triart Diol-ILIC Suitable as the first choice column for reversed-phase separation Effective for method screening with chemistries Standard normal-phase column ormal-phase column providing separation characteristics different from bare silica gel For separation of polar compounds with poor retention on reversed-phase columns P.9~6 P.9~6 P. P. P.66 Vitas Water-soluble vitas YMC-Triart C8 Usable with % aqueous mobile phase (For separation under a buffered or ion pairing mobile phase) P.9~6 Fat-soluble vitas YMC-Triart C8 YMC-Pack DS-AL YMC Carotenoid (C) Suitable as the first choice DS column on-endcapped DS, suitable for separation of compounds with similar structure Separation behavior different from DS P.9~6 P.88 P. ILIC Water-soluble vitas YMC-Pack Polyae II, YMC-Triart Diol-ILIC For separation of water-soluble vitas such as vita C under ILIC mode For simultaneous separation of water-soluble vitas P.6~8 P.66 ormalphase Fat-soluble vitas YMC-Pack SIL, SIL-6 YMC-Pack Polyae II For separation of fat-soluble vitas such as tocopherol P. P.6, 7 rganic acids Fatty acids YMC-Triart C8 Usable with % aqueous mobile phase P.9~6 ormalphase YMC-Pack SIL, SIL-6 Standard normal-phase column P. Phospholipids YMC-Triart C8 For separation of molecular species P.9~6 ormalphase YMC-Pack SIL, SIL-6 YMC-Pack PVA-Sil YMC-Pack Diol-P For separation of phospholipid classes P., Ao acids ILIC Free ao acids YMC-Triart Diol-ILIC For simultaneous separation of ao acids under ILIC mode P.66 Free ao acids YMC-Triart C8 ydrosphere C8 Usable with % aqueous mobile phase For separation of hydrophobic ao acids P.9~6 P.8, 8 Labeled ao acids YMC-Triart C8 Suitable as the first choice DS column P.9~6 Structural isomers YMC-Triart C8 ExRS YMC Carotenoid (C) YMC-Triart C8 YMC-Triart PFP CIRAL ART igh-density bonding for excellent ability to recognize planar structure For carotenoids separation For separations of isomers or structural analogs For separations of polar compounds or isomers For separations of isomers or structural analogs P.6 P. P.6 P.6 P.6~9 ormalphase YMC-Pack SIL, SIL-6 CIRAL ART Standard normal-phase column For separations of isomers or structural analogs P. P.6~9 ptical isomers CIRAL ART YMC CIRAL EA For separation of optical isomers P.6~ ormalphase CIRAL ART YMC CIRAL EA For separation of optical isomers P.6~

12 C P Application (Fat-soluble vitas, Water-soluble vitas) Reversed-phase Vita D Separation of structurally similar compounds. C C C C C C ormal-phase Vita E (Tocopherols) Separation of tocopherol homologues. C C C C C C C C α-tocopherol C. C C C C C C β-tocopherol Ergocalciferol (Vita D). C C C C C C C γ-tocopherol. C C C. C C C C C C C δ-tocopherol C Cholecalciferol (Vita D) (P89C) (S977A) Column : YMC-Pack DS-AL ( μm, Å) X.6 mmi.d. Eluent : acetonitrile/water (9/) Flow rate :. ml/ Temperature : C Detection : UV at 6 nm Injection : μl (. mg/ml) Column : YMC-Pack SIL ( μm, Å) X.6 mm I.D. Eluent : n-hexane/-propanol/acetic acid (/6/) Flow rate :. ml/ Temperature : C Detection : FLS at Ex 98 nm, Em nm Injection : μl (~ μg/ml) Reversed-phase Water-soluble vitas Simultaneous separation of water-soluble vitas under ion pairing mobile phase 6 7. Thiae Cl (Vita B). Pyridoxine Cl (Vita B6). icotinamide. Cyanocobala (Vita B). L-Ascorbic acid -glucoside 6. L-Ascorbic acid (Vita C) 7. Erythorbic acid 8. Riboflavin (Vita B) 9. icotinic acid 8 9 (RD) ILIC Water-soluble vitas Simultaneous separation of water-soluble vitas under ILIC mode * (FA) Caffein icotinamide Pyridoxine hydrochloride Cl Riboflavin rotic acid Erythorbic acid (D-Isoascorbic acid) L-Ascorbic acid icotinic acid --α-d-glucopyranosyl -L-ascorbic acid (Ascorbic acid -glucoside) Thiae hydrochloride - + S Co + Cyanocobala Cl - Cl Column : YMC-Triart C8 ( μm, Å) X.6 mmi.d. Eluent : phosphate buffer*/acetonitrile (9/) *Dissolve. g K P in 8 ml water add 6 ml % TBA adjust p. by % P add water to make ml Flow rate :.8 ml/ Temperature : C Detection : UV at 6 nm Injection : μl ( μg/ml) Column : YMC-Triart Diol-ILIC ( μm, Å) X. mmi.d. Eluent : A) acetonitrile/ mm C-C (p.6)/water (9//) B) acetonitrile/ mm C-C (p.6)/water (//) -7%B (- ) Flow rate :. ml/ Temperature : C Detection : UV at nm Injection : μl ( μg/ml)

13 Application (Water-soluble vitas, rganic acids, Ao acids) ILIC Vita C (Ascorbic acid) Separation of ascorbic acid under ILIC mode. icotinic acid. Erythorbic acid. L-Ascorbic acid Reversed-phase rganic acids Simultaneous separation of organic acids under % aqueous mobile phase, xalic acid. Tartaric acid. Glycolic acid. Formic acid. L-Malic acid 6. Malonic acid 7. Lactic acid 8. Acetic acid 9. Maleic acid. Citric acid. Succinic acid. Fumaric acid. Acrylic acid. Propionic acid (A976A) 6 8 (UQ) Column : YMC-Pack Polyae II X.6 mmi.d. Eluent : acetonitrile/ mm P (7/) Flow rate :. ml/ Temperature : C Detection : UV at nm,.6 AUFS Injection : μl (.~. mg/ml) Column : YMC-Triart C8 ( μm, Å) X. mmi.d. Eluent : mm phosphoric acid Flow rate :. ml/ Temperature : 7 C Detection : UV at nm Injection : μl (.~. mg/ml) ILIC Ao acids Simultaneous separation of ao acids under ILIC mode Reversed-phase Ao acids Separation of hydrophobic ao acids under highly aqueous mobile phase (JP method) pa * Standard solution* (. mg/ml L-Valine,.9 mg/ml L-Isoleucine,.8 mg/ml L-Leucine) * ** Phenylalanine (Phe) Tryptophan (Trp) Leucine (Leu) Isoleucine (Ile) Methionine (Met) Tyrosine (Tyr) Valine (Val) Cysteine Cl (Cys) Proline (Pro) System suitability requirement Result Threonine (Thr) Resolution (, )..68 Alanine (Ala) γ-aobutyric acid (GABA) Serine (Ser) Glycine (Gly) Relative standard deviation of the retention time (each of the peaks).%.%.%.% Asparagine (Asn) Glutae (Gln) Citrulline (Cit) Glutamic acid (Glu) 6 istidine (is) * Aspartic acid (Asp) Arginine Cl (Arg) * Lysine (Lys) rnithine Cl (rn) *Chloride ion contained in the sample solution **Sodium ion contained in the sample solution (F68A)... C C C C C C C C L-Valine L-Isoleucine L-Leucine C (UA) Column : YMC-Triart Diol-ILIC ( μm, Å) X.6 mmi.d. Eluent : A) mm C-C (p.6) B) acetonitrile 8-8%B (- ), 8-68%B (- ) Flow rate :. ml/ Temperature : C Detection : Corona CAD (Charged Aerosol Detector) Injection : μl (. mg/ml) Corona and CAD are trademarks of Thermo Fisher Scientific. Column : YMC-Triart C8 ( μm, Å) X.6 mmi.d. Eluent : phosphate buffer (p.8)* /acetonitrile (97/) * Dissolve. g of a P in ml of water and adjust p.8 with P Flow rate :.9 ml/ (adjust the flow rate so that the retention time of L-Valine is about. ) Temperature : C Detection : UV at nm Injection : μl (The Japanese Pharmacopoeia 6th; Identification) * Standard solution was prepared from L-Valine, L-Isoleucine and L-Leucine supplied as a reagent for laboratory use.

14 Reversed-phase column selection guide st choice! Versatile column for separation of both hydrophilic and hydrophobic compounds Triart C8 Mobile phase Temperature Change Effective for separation of compounds with difficulty on other C8 column!! nly weak retention Poor peak shape p limit for mobile phase etc. Similar structures (Isomers) Improvement of resolution Improvement of retention nd choice! Triart C8 ExRS Triart C8 YMC-Pack DS-AL YMC Carotenoid ptical Isomer Separation Columns With typical structure or functional group Too much retention/resolution (Long run time) Weak retention (ighly polar compounds) Aromatic compounds (π-electron acceptor ) Conjugated compounds Basic compounds Triart Phenyl Aromatic compounds (π-electron donor) Cis-trans isomers Polar compounds alogenated compounds Triart PFP Triart C8 Triart PFP ILIC mode (Triart Diol-ILIC) Comparison of hydrophobicity and hydrogen-bonding capacity of various columns ydrogen binding capacity [α(caffeine/benzene)] Pro C ydrosphere C8 CAPCELL PAK C8 AQ Pro C8 Gei C8 Develosil C-UG Atlantis dc8 ZRBAX SB-C8 Atlantis T TSKgel DS-8Ts Develosil DS-MG Pro C8 CAPCELL PAK C8 MGIII CAPCELL PAK C8 MG Inertsil DS- Unison UK-C8 ypersil GLD CAPCELL PAK C8 MGII Gei-X C8 Luna C8() Sunfire C8 Inertsil DS- XTerra MS C8 XBridge C8 Eternity-C8 Mightysil RP-8 L-column DS Meteoric Core C8 Triart C8 Meteoric Core C8 ydrophilic DS ydrophobicity: Low ydrogen-bonding capacity: igh Triart C8 Symmetry C8 Cadenza CD-C8 TSKgel DS-S L-column DS ZRBAX Extend-C8 CAPCELL PAK C8 ACR Standard DS column ydrophobicity: Moderate ydrogen-bonding capacity: Moderate InertSustain C8 Develosil DS-SR Pro C8 RS ydrophobic DS ydrophobicity: igh ydrogen-bonding capacity: Low Triart C8 ExRS Eluent: methanol/water (8/) [Amylbenzene] methanol/water (/7) [Caffeine, Benzene] (S8A) ydrophobicity [k (Amylbenzene)]