RPLC/HILIC/API-MS: polarity extended analysis for organic molecules in water bodies Sylvia Grosse and Thomas Letzel Analytical Research Group Ixia, Rhodes, 1st September, 2015
content polarity extension RPLC with HILIC HILIC retention mechanisms: adsorption distribution (water layer) electrostatic interaction stationary phases and mobile phases in HILIC serial HILIC-RP-MS coupling application conclusion 2
Why polarity extension? Polar and nonpolar molecules Partition coefficient (P): Distribution coefficient (D): (For charged molecules) log P = log ([solute] oct /[solute] wat ) log D = log ([solute] oct /([solute] wat ionized + [solute] wat neutral ) Log P < 0 or Log D < 0 Log P > 0 or Log D > 0 Hydrophilic compounds Hydrophobic compounds 0-2 -1 1 2 Log P HILIC RPLC 3
HILIC Separation of polar and nonpolar compounds Hydrophilic analytes Hydrophobic analytes RPLC hydrophylic interaction liquid chromatography NP stationary phases RP eluents ACN/H 2 Reversed phase liquid chromatography Typical mobile phase H 2 /ACN RPLC vs. HILIC: rthogonal Use of the same solvents MS compatible 4
Retention mechanisms ADSRPTIN Analyte - Stationary Phase Hydrogen bonding Dipole - Dipole Si Si Si Si 5
Retention mechanisms DISTRIBUTIN increasing H 2 -content at least 2-3% water are essential in the mobile phase! Greco et al., J. Chrom. Sci, 2013 6
Retention mechanisms ELECTRSTATIC INTERACTIN Electrostatic attraction Electrostatic repulsion 7
Stationary Phases Silica Diol, Amide, Cross-linked diol Aminopropyl Greco und Letzel, www.sepscience.com HILIC Solutions #3 uillarme, HILIC: a critical evaluation, 2014 Sulfobetaine 8
Stationary Phase: neutral Distribution Electrostatic interaction Adsorption R R NH 2 Amide n m H H Diol H H Cross-linked Diol H Greco et al., J. Chrom. Sci, 2013 9
Stationary Phase: ionic Aminopropyl Distribution Electrostatic interaction Adsorption + NH 3 Anion exchange Si Si - H Silica ph < 4/5 ph > 4/5 Cation exchange Greco et al., J. Chrom. Sci, 2013 10
Stationary Phase: zwitterionic Sulfobetaine (ZIC-HILIC) Distribution Electrostatic interaction Adsorption + N S - P N + - Phosphorylcholine (ZIC-cHILIC) weak Greco et al., J. Chrom. Sci, 2013 11
rthogonality RPLC and HILIC 12
RPLC-HILIC coupling 13
No HILIC Polarity Extension RPLC HILIC RPLC 14
HILIC-RPLC application I: Amino acids 1234 5 6 7 89 10 1 Phe; 2 Leu; 3 Trp; 4 Ile; 5 Val; 6 Tyr; 7 Pro; 8 Thr; 9 Ala; 10 Asn 15
HILIC-RPLC application II: pharmaceuticals and neurotransmitter Gabapentin Betaine Vigabatrin 16
HILIC-RPLC application II: pharmaceuticals and neurotransmitter gamma aminobutyric acid? Acetylcholine? 17
HILIC-RPLC application II: pharmaceuticals and neurotransmitter smrm gamma-aminobutyric acid Standard EPI Fragment Spectra ratio Quan/Qual: 3.6 gamma-aminobutyric acid real sample ratio Quan/Qual: 4.1 18
HILIC-RPLC application II: pharmaceuticals and neurotransmitter smrm Acetylcholine Standard EPI Fragment Spectra ratio Quan/Qual: 8.9 Acetylcholine real sample ratio Quan/Qual: 4.2 r r 19
HILIC-RPLC application III: Sweetener and industrial chemicals, herbicide Cyanuric acid Acesulfam Isopentylamine Glyphosate 20
HILIC-RPLC application IV: An xidation Scenario with Diclofenac BDD electrode Diclofenac (DCF) MilliQ water Synthetic hard water Real wastewater effluent Sample collection: 0-60 min Rajab, Greco, et al., J. Sep. Sci. 2013. 21
HILIC-RPLC application IV: Diclofenac oxidation Literature proposed transformation products Rajab, Greco, et al., J. Sep. Sci. 2013. 22
HILIC-RPLC application IV: Diclofenac oxidation Rajab, Greco, et al., J. Sep. Sci. 2013. 23
HILIC-RPLC application IV: Diclofenac oxidation TP_34 TP_27 rganic transformation products TP_41 TP_26_A TP_46 TP_4 TP_8 TP_6 DCF Cl 4 - Inorganic compounds Cl 3-5 10 15 20 25 30 35 Counts vs. Acquisition Time (min) 24
conclusion Understanding of HILIC mechanisms HILIC valid with logd value < 0 extended polarity with serial RPLC-HILIC coupling Combination of different chromatographic techniques in just one technique (RP, GC, IC) A new field of molecules is taped and will give a lot more interesting results 25
Dr. Giorgia Greco Dr. Mohamad Rajab Sofia Veloutsou Prof. Dr. Drewes and the whole team Thanks... Funding: This work was partially financed by the German Federal Ministry of Education and Research within the RiSKWa program, funding code 02WRS1354A. Funded by: 26
Thanks... And for your attention... Any questions??? 27