Bocian, Szymon

Buszewski, Bogusław

Skoczylas, Magdalena

English

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MULTIPARAMETRIC CHARACTERIZATION OF AMINO ACIDS- AND PEPTIDE-SILICA STATIONARY PHASES – A COLUMN SELECTION FOR SEPARATION TARGETSMagdalena Skoczylas, Szymon Bocian, Bogusław BuszewskiDepartment of Environmetal Chemistry and Bioanalytics,Faculty of Chemistry, Nicolaus Copernicus University, Gagarina Street 7, 87-100 Torun, Poland, E-mail: magdalena_skoczylas@hotmail.comIn terms of the separation techniques that are widely used in many fields of science, the choice of stationaryphase which are suitable for our separation targets represents an imperative objective. Thus, thecharacterization of surface properties that result from a specificity of chemically bonded ligands and theirimpact on the overall chromatographic behavior is essential.The immobilization of suitable amino acids sequences on the silica surface allows obtaining stationaryphases with different hydrophobicity and polarity. The appropriate selection of amino acids and peptidesequences allows the preparation of stationary phases useful in desired chromatographic systems. In order toprove this assumption and facilitate the column selection for the potential application, the description of thestructure-selectivity relationships for newly synthesized stationary phases must be performed.The aim of the research was to carry out a multiparametric characterization of nine home-made stationaryphases with chemically bonded amino acids and peptides. These materials were characterized in terms of theselectivity for hydrophilic and hydrophobic compounds. The applied column characterization methodologyallowed the classification of the tested stationary phases according to their chromatographic properties. Basedon this specification, amino acids and peptide-silica stationary phases were successfully applied in analysis ofbiologically significant compounds.Chromatographic experiments were performed on the Shimadzu Prominence* and Shimadzu UHPLCNexera** LC systems (Kyoto, Japan) equipped with ternary* and binary** gradient pump, an autosampler,a diode array detector, and column thermostat. Instrument control, data acquisition, and processing wereperformed with LabSolutions software for HPLC. The methodology was based on the investigation ofdifferences in selectivities of the tested materials for certain pairs of compounds, which provide specificinteraction modes. Working solutions comprised selected pairs of compounds as well as toluene (HILIC) andthiourea (RP HPLC) as a void volume markers (Table 1).INTRODUCTION EXPERIMENTALFig. 1. Structures of chemically bonded stationary phases:A – amino-(Leu)1, B - amino-(Leu)2, C -amino-(Leu)3,D - amino-(Gly)1, E - amino-(Gly)3, F - amino-(Phe)1,G - amino-(Phe)2, H - amino-(Asp)1, I – amino-(Ala)2.RESULTS* Value presented in this column correspond to logD of particular solutes in the case of HILIC test,while in RP conditions logP values were provided.Table 1 Operation parameters of chromatographic tests and propertiesof tested compoundsSTRUCTURESAcknowledgmentsThis study was supported by grant from Ministry of Science and Higher Education Iuventus Plus IP2014 003673.RP HPLCCONCLUSIONS0,00,51,01,52,02,53,0CXk (U)AX(Tb/Tp)(2dG/3dG)(V/A)(OH)(CH2)0,00,51,01,52,02,53,0CXAXk (U)(Tb/Tp)(2dG/3dG)(V/A)(OH)(CH2)0,00,51,01,52,02,53,0AXk (U)CX(Tb/Tp)(2dG/3dG)(V/A)(OH)(CH2)0,00,51,01,52,02,53,0CXk (U)AX(Tb/Tp)(2dG/3dG)(V/A)(OH)(CH2)0,00,51,01,52,02,53,0CXAXk (U)(Tb/Tp)(2dG/3dG)(V/A)(OH)(CH2)0,00,51,01,52,02,53,0CXAXk (U)(Tb/Tp)(2dG/3dG)(V/A)(OH)(CH2)0,00,51,01,52,02,53,0CXk (U)AX(Tb/Tp)(2dG/3dG)(V/A)(OH)(CH2)0,00,51,01,52,02,53,0CXk (U)AX(Tb/Tp)(2dG/3dG)(V/A)(OH)(CH2)0,00,51,01,52,02,53,0CXAXk (U)(Tb/Tp)(2dG/3dG)(V/A)(OH)(CH2)012345k (PB)(B/P)2.6(B/P)7.6 (T/O)(C/P)(PB/BB)----------012345k (PB)(B/P)2.6(B/P)7.6(T/O)(C/P)(PB/BB)----------0,00,51,01,5k (PB)(B/P)2.6(B/P)7.6(T/O)(C/P)(PB/BB)----------0,00,51,01,5k (PB)(B/P)2.6(B/P)7.6(T/O)(C/P)(PB/BB)-----------1012345k (PB)(B/P)2.6(B/P)7.6 (T/O)(C/P)(PB/BB)----------HILICamino-(Gly)3    amino-(Gly)1           amino-(Asp)1amino-(Ala)2 amino-(Phe)1           amino-(Phe)2amino-(Leu)1    amino-(Leu)2        amino-(Leu)3amino-(Phe)1amino-(Phe)2amino-(Leu)1amino-(Leu)2 amino-(Leu)3Fig. 2. Radar plots of characterization data – the comparison of investigated stationary phases.Fig. 3. Radar plots of hydrophobic stationary phases – the comparison of RP-characteristic properties.0 2 4 6 8 10 12 14leu3leu2leu1phe2phe1asp1gly3Retention time (min)ala2gly1Fig. 4. Chromatograms for investigations of methylene group selectivity, α(CH2). Solutes: toluene, 5-methyluridine, uridine.0 2 4 6 8 10 12leu3leu2leu1phe2phe1asp1gly3gly1ala2Retention time (min)Fig. 5. Chromatograms for test of hydroxy group selectivity, α(OH). Solutes: toluene, 2`-deoxyuridine, uridine. 0 2 4 6 8 10 12 14 16leu3leu2leu1phe2phe1asp1gly3gly1ala2Retention time (min)Fig. 6. Chromatograms for test of configurational isomers selectivity, α(V/A). Solutes: toluene, adenosine, vidarabine. 0 2 4 6 8 10 12 14 16 18 20 22leu3leu2leu1phe2phe1ala2asp1gly3gly1Retention time (min)Fig. 7. Chromatograms for test of regioisomers selectivity, α(2dG/3dG). Solutes: toluene, 3`-deoxyguanosine, 2`-deoxyguanosine. 0 2 4 6 8 10 12 14 16 18 20leu3leu2leu1phe2phe1ala2asp1gly3gly1Retention time (min)Fig. 8. Chromatograms for test of anion exchange interactions, α(AX). Solutes: toluene, uracil, sodium p-toluenesulfonate (SPTS). Fig. 9. Chromatograms for test of cation exchange interactions, α(CX). Solutes: toluene, uracil, N,N,N-trimethylphenylammonium chloride.  HydrophilicinteractionsHydrophobicinteractionsConigurationalisomerismRegioisomerismAnion-exchange interactions Cation-exchange interactions0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1802000400060008000100001200014000Absorbance (µAU)Retention time (min)12345678910110 1 2 3 4 5 6 7-10000-50000500010000150002000025000Absorbance (µAU)Retention time (min)12 3 45Fig. 11. Stationary phase: amino-(Leu)2, flow rate 1.0 mL min-1, 30ºC,λ=254 nm; HILIC: ACN/H20 (95/5) 2-deoxythymidine (1), uridine (2), 1-methyladenosine (3), 1-methylinosine (4), adenosine (5), pseudouridine(6), 1-methylguanosine (7), 8-bromoguanosine (8), cytidine (9), N2-methylguanosine (10), guanosine (11), RP: MeOH/H20 (50/50),benzene (1), toluen (2), ethylbenzene (3), propylbenzene (4),butylbenzene (5).HILICRP HPLCFig. 10. amino-(Ala)2 SP, carbohydrates: mobile phase: ACN/H2O 0-5 min 90% ACN, 5-10 min 85%ACN, flow rate: 1mL min-1, Detector ELSD: 40ºC, 320 kPa, Gain 1; amino acids: mobile phase:ACN/H2O (80/20, v/v), flow rate: 1mL min-1, Detector ELSD: 40ºC, 320 kPa, Gain 1.0 2 4 6 8 10 12 14 16 18 200250050007500100001250015000Intensity [µV]Retention time [min]D-xyloseD-fructoseD-glucoseD-glucosaminemaltoselactoseAPPLICATIONS Carbohydrates0 2 4 6 8 10 12 14 16 18 2050010001500Intensity (µV)Retention time (min)TrpMetProGly ArgLys Amino acids Mixed-mode behaviourIn terms of the absolute hydrophilicity, tested materials may be divided into two groups. Stationary phases containing glycine, alanine, and aspartic acid in amino acids sequence demonstrated higher hydrophilicretention than modifications with leucine and phenylalanine. These correlations were in compliance with the hydrophilic/hydrophobic nature of bonded amino acids. It should emphasized that despite of the lower polarityof the second group of materials, they are compatible for HILIC applications. The discrimination of configurational isomers was comparable for all the investigated stationary phases, while the regioisomers was subtlydistinguished by materials with immobilized hydrophilic amino acids and peptides. The anion-exchange capability was observed for all the tested columns except the stationary phase with aspartic acid. The presence ofcarboxyl group in the side chain of such amino acid plays as cation-exchange functionality, simultaneously causes the electrostatic repulsion with anionic compound.Stationary phases with chemically bonded hydrophobic amino acids and peptides (leucine and phenylalanine) demonstrated also the RP-compatible character. Among the stationary phases investigated, material withbonded dipeptide of phenylalanine exhibits the greatest hydrophobicity. Moreover, the retention of hydrophobic solutes increased with the elongation of peptide chain. The steric selectivity was slightly higher for singleamino acid modification compared to peptide ligands. In addition, the ion-exchange capacity (caused by residual silanols) was reduced, whereas the peptide chain of particular amino acid was elongated. Judging from theseresearch, stationary phases with immobilized hydrophobic sequence of amino acids could be applied both in RP and HILIC systems.As a result of the research, it was evident to realize how the sequence of amino acids - their type and length influences on the overall chromatographic properties. This format may comprise convenient approach forcolumn selection depending on HILIC or RP HPLC separation targets.NHNH2ONHNHOONHONH2NHNH2ONHNHNH2OONHNHNHNH2OOONHOOCOH3NNHNHNH2OOCH3CH3NHNH2ONHNHNH2OO -+Mix no.Tested solutes SignMolecularstucturepKaLogDLogP *VariableMobile phasecompositionHILIC mode1Uridine –5-methyluridineα (CH2)12.612.0-1.58-1.02Hydrophobic interactionsACN/20 mM NH4Ac pH = 4.7 (90/10v/v)2Uridine –2`-deoxyuridineα (OH)12.613.9-1.58-1.26Hydrophilic interactions3Vidarabine –adenosineα (V/A)13.913.9-1.02-1.03Configuratio-nal isomers selectivity42`-deoxyguanosine –3`-deoxyguanosineα(2dG/3dG)13.513.5-1.14-1.14Regioisomers selectivity5Sodium p-toluenesulfonate –uracilα (AX)-12.813.80.88-1.08Anion exchange selectivity6N,N,N-trimethylphenylammonium chloride - uracilα (CX)-13.8-2.31-1.08Cation exchange selectivity7Theobromine –theophyllineα(Tb/Tp)108.6-1.06-2.51Acidic-basic nature of stationary phaseRP mode1Pentyl- andbutylbenzeneα(PB/BB)-5.04.4Hydrophobi-cityACN/H2O (30/70v/v)2Trifenylen –o-terfenylα (T/O) -4.95.5Steric selectivity3Caffeine –phenolα (C/P)10.49.99-0.11.46Hydrogen bonding capacityMeOH/H2O (30/70v/v)4Benzylamine –phenolα (B/P)7.69.349.991.11.46Ion-exchange capacity at pH > 7MeOH/20 mM KH2PO4pH = 7.65α(B/P)2.7Ion-exchange capacity at pH < 3MeOH/20 mM KH2PO4pH = 2.70 2 4 6 8 10 12 14 16 18leu3leu2leu1phe2phe1ala2asp1gly3gly1Retention time (min)

MULTIPARAMETRIC CHARACTERIZATION OF AMINO ACIDS- AND PEPTIDESILICA STATIONARY PHASES – A COLUMN SELECTION FOR SEPARATION TARGETS

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MULTIPARAMETRIC CHARACTERIZATION OF AMINO ACIDS- AND PEPTIDESILICA STATIONARY PHASES – A COLUMN SELECTION FOR SEPARATION TARGETS

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