MULTIPARAMETRIC CHARACTERIZATION OF AMINO ACIDS- AND
PEPTIDE-SILICA STATIONARY PHASES – A COLUMN SELECTION
FOR SEPARATION TARGETS
Magdalena Skoczylas, Szymon Bocian, Bogusław Buszewski
Department of Environmetal Chemistry and Bioanalytics,
Faculty of Chemistry, Nicolaus Copernicus University, Gagarina Street 7,
87-100 Torun, Poland, E-mail: magdalena_skoczylas@hotmail.com
In terms of the separation techniques that are widely used in many fields of science, the choice of stationary phase which are suitable for our separation targets represents an imperative objective. Thus, the characterization of surface properties that result from a specificity of chemically bonded ligands and their impact on the overall chromatographic behavior is essential.
The immobilization of suitable amino acids sequences on the silica surface allows obtaining stationary phases with different hydrophobicity and polarity. The appropriate selection of amino acids and peptide sequences allows the preparation of stationary phases useful in desired chromatographic systems. In order to prove this assumption and facilitate the column selection for the potential application, the description of the structure-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 stationary phases with chemically bonded amino acids and peptides. These materials were characterized in terms of the selectivity for hydrophilic and hydrophobic compounds. The applied column characterization methodology allowed the classification of the tested stationary phases according to their chromatographic properties. Based on this specification, amino acids and peptide-silica stationary phases were successfully applied in analysis of biologically significant compounds.
Chromatographic experiments were performed on the Shimadzu Prominence* and Shimadzu UHPLC Nexera** 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 were performed with LabSolutions software for HPLC. The methodology was based on the investigation of differences in selectivities of the tested materials for certain pairs of compounds, which provide specific interaction modes. Working solutions comprised selected pairs of compounds as well as toluene (HILIC) and thiourea (RP HPLC) as a void volume markers (Table 1).
INTRODUCTION
EXPERIMENTAL
Fig. 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 properties
of tested compounds
STRUCTURES
Acknowledgments
This study was supported by grant from Ministry of Science and Higher Education Iuventus Plus IP2014 003673.
RP HPLC
CONCLUSIONS
0,0 0,5 1,0 1,5 2,0 2,5 3,0 CX k (U) AX (Tb/Tp) (2dG/3dG) (V/A) (OH) (CH2) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 CX AX k (U) (Tb/Tp) (2dG/3dG) (V/A) (OH) (CH2) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 AX k (U) CX (Tb/Tp) (2dG/3dG) (V/A) (OH) (CH2) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 CX k (U) AX (Tb/Tp) (2dG/3dG) (V/A) (OH) (CH2) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 CX AX k (U) (Tb/Tp) (2dG/3dG) (V/A) (OH) (CH2) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 CX AX k (U) (Tb/Tp) (2dG/3dG) (V/A) (OH) (CH2) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 CX k (U) AX (Tb/Tp) (2dG/3dG) (V/A) (OH) (CH2) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 CX k (U) AX (Tb/Tp) (2dG/3dG) (V/A) (OH) (CH2) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 CX AX k (U) (Tb/Tp) (2dG/3dG) (V/A) (OH) (CH2) 0 1 2 3 4 5 k (PB) (B/P)2.6 (B/P)7.6 (T/O) (C/P) (PB/BB) --0 1 2 3 4 5 k (PB) (B/P)2.6 (B/P)7.6 (T/O) (C/P) (PB/BB) --0,0 0,5 1,0 1,5 k (PB) (B/P)2.6 (B/P)7.6 (T/O) (C/P) (PB/BB) --0,0 0,5 1,0 1,5 k (PB) (B/P)2.6 (B/P)7.6 (T/O) (C/P) (PB/BB) ---1 0 1 2 3 4 5 k (PB) (B/P)2.6 (B/P)7.6 (T/O) (C/P) (PB/BB)--HILIC
amino-(Gly)3 amino-(Gly)1 amino-(Asp)1
amino-(Ala)2 amino-(Phe)1 amino-(Phe)2
amino-(Leu)1 amino-(Leu)2 amino-(Leu)3
amino-(Phe)1
amino-(Phe)2
amino-(Leu)1
amino-(Leu)2 amino-(Leu)3
Fig. 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 14 leu3 leu2 leu1 phe2 phe1 asp1 gly3
Retention time (min)
ala2 gly1
Fig. 4. Chromatograms for investigations of methylene group
selectivity, α(CH2). Solutes: toluene, 5-methyluridine, uridine.
0 2 4 6 8 10 12 leu3 leu2 leu1 phe2 phe1 asp1 gly3 gly1 ala2
Retention 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 16 leu3 leu2 leu1 phe2 phe1 asp1 gly3 gly1 ala2
Retention 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 22 leu3 leu2 leu1 phe2 phe1 ala2 asp1 gly3 gly1
Retention 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 20 leu3 leu2 leu1 phe2 phe1 ala2 asp1 gly3 gly1
Retention 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.
Hydrophilic
interactions Hydrophobic
interactions Conigurationalisomerism
Regioisomerism Anion-exchange interactions Cation-exchange interactions 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 0 2000 4000 6000 8000 10000 12000 14000 Ab so rb an ce (µ AU )
Retention time (min)
1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 -10000 -5000 0 5000 10000 15000 20000 25000 Ab so rb a n ce (µ AU )
Retention time (min) 1 2
3 4
5
Fig. 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).
HILIC
RP HPLC
Fig. 10. amino-(Ala)2SP, 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 20 0 2500 5000 7500 10000 12500 15000 In te nsi ty [µ V]
Retention time [min]
D -x ylos e D-f ru cto se D-g luco se D -glu cos amine m alto se lact ose
APPLICATIONS
Carbohydrates 0 2 4 6 8 10 12 14 16 18 20 500 1000 1500 In te n si ty (µ V)Retention time (min) Trp Met Pro Gly Arg Lys Amino acids Mixed-mode behaviour
In 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 hydrophilic retention 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 polarity of 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 subtly distinguished 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 of carboxyl 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 with bonded 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 single amino 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 these research, 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 for column selection depending on HILIC or RP HPLC separation targets.
NH N H2 O NH N H O O NH O N H2 NH N H2 O NH N H NH2 O O NH N H NH N H2 O O O N H OOC O H3N NH N H NH2 O O C H3 CH3 N H NH2 O N H NH N H2 O O -+ Mix
no. Tested solutes Sign
Molecular stucture pKa LogD LogP * Variable Mobile phase composition HILIC mode 1 Uridine – 5-methyluridine α (CH2) 12.6 12.0 -1.58 -1.02 Hydrophobic interactions ACN/20 mM NH4Ac pH = 4.7 (90/10v/v) 2 Uridine – 2`-deoxyuridine α (OH) 12.6 13.9 -1.58 -1.26 Hydrophilic interactions 3 Vidarabine – adenosine α (V/A) 13.9 13.9 -1.02 -1.03 Configuratio-nal isomers selectivity 4 2`-deoxyguanosine – 3`-deoxyguanosine α (2dG/3d G) 13.5 13.5 -1.14 -1.14 Regioisomers selectivity 5 Sodium p-toluenesulfonate – uracil α (AX) -12.8 13.8 0.88 -1.08 Anion exchange selectivity 6 N,N,N-trimethylphenylammo nium chloride - uracil
α (CX) -13.8 -2.31 -1.08 Cation exchange selectivity 7 Theobromine – theophylline α (Tb/Tp) 10 8.6 -1.06 -2.51 Acidic-basic nature of stationary phase RP mode 1 Pentyl- and butylbenzene α (PB/BB) -5.0 4.4 Hydrophobi-city ACN/H2O (30/70v/v) 2 Trifenylen – o-terfenyl α (T/O) -4.9 5.5 Steric selectivity 3 Caffeine – phenol α (C/P) 10.4 9.99 -0.1 1.46 Hydrogen bonding capacity MeOH/H2O (30/70v/v) 4 Benzylamine – phenol α (B/P)7.6 9.34 9.99 1.1 1.46 Ion-exchange capacity at pH > 7 MeOH/20 mM KH2PO4 pH = 7.6 5 α (B/P)2.7 Ion-exchange capacity at pH < 3 MeOH/20 mM KH2PO4 pH = 2.7 0 2 4 6 8 10 12 14 16 18 leu3 leu2 leu1 phe2 phe1 ala2 asp1 gly3 gly1
