Part 2. Overview of HPLC Media

Transcription

1 Part 1. General Chromatographic Theory Part 2. Overview of HPLC Media Part 3. The Role of the Mobile Phase in Selectivity Part 4. Column Care and Use 1

2 HPLC Particle Technology Core-Shell Particle Fully porous Particle 2

3 Fully Porous Silica Polymerization Alkaline Tetraethoxysilane Silica Sol-Gel 99.9% of reactive surface area is internal 3

4 There is a high amount of variability in the final physicochemical qualities of silica made by different manufacturers. These differences in the base silica itself help account for the wide variance in the behavior of different brands of HPLC columns. The principle variables are: Particle Shape (Irregular versus spherical) Purity (metal content) Surface area Pore size Carbon load Fully Porous Silica 4

5 Fully Porous Silica Advantages: Ability to derivatize with numerous bonded phases High mechanical strength Excellent efficiency Highly amenable to modulation of material characteristics (pore size, surface area, etc.) Disadvantages: Dissolution of silica at ph > ~7.5 (may extend with bonded phase) Hydrolysis of bonded phase at ph <1.5 5

6 Organosilica Hybrid Particle Conventional Silica Particle Organosilica Hybrid Particle Siloxane Bridge Ethane linkage Dissolution at ph > 7.5 Stable to ph ~12 6

7 Organosilica Hybrid Particle Advantages: Extended ph range from 1-12 Performance and strength of conventional silica particle Unique selectivity Disadvantages: Fewer stationary phases available compared to conventional silica (e.g. cyano, amino) 7

8 Organosilica Hybrid Particle LC/MS Analysis of Nicotine & Metabolites Gemini-NX 3 µm C x 2.0mm MP: A = 10mM NH 4 HCO 3 B = Acetonitrile Gradient: %B in 3 min Flow rate: 500 µl/min 1. Nornicotine 2. 3-OH-Cotinine 3. Anabasine 4. Cotinine 5. Nicotine 8

9 Monolithic Silica Rod Advantages: Extremely low pressure Macroporous structure allows direct injection of dirty samples No bed shifting/instability of packed columns Disadvantages: Fewer stationary phases Efficiency less than conventional particles (< 3 m performance) 9

10 Monolithic Silica Rod Onyx C x 4.6mm 15 µl injections of human plasma; filtered; no PPT 1. H = Hypoxanthine 2. U = Uric acid 3. X = Xanthine 4. A = Adenosine 5. I = Inosine 10 Journal of Chromatography B, Volume 854, Issues 1-2, 1 July 2007, Pages

11 Core Shell Particle 0.35 µm Porous Shell 2.6 µm Core-Shell Particle 1.9 µm Solid Core 11

12 Core Shell Particle Conventional Core-Shell 2.6 m: 2.6 m total particle diameter Pressure ~50% less than sub-2 m fully-porous Efficiency = ,000 P/m UPLC efficiency using conventional HPLC systems* UHPLC Core-Shell 1.7 m: 1.7 m total particle diameter Efficiency = ,000+ P/m Highest efficiency media currently available 12

13 Core Shell Particle Traditional 3 µm C x 4.6 mm N = 166,502 p/m Core-Shell 2.6 µm C x 4.6 mm N = 295,343 p/m Increased peak height Agilent 1100 Agilent Agilent is a registered trademark of Agilent Technologies, Inc. Phenomenex is in no way affiliated with Agilent Technologies, Inc..

14 Core Shell Particle Fully porous 1.7 µm C18 50 x 2.1 mm 0.6 ml/min Efficiency = 272,080 p/m Core-Shell 1.7 µm C18 50 x 2.1 mm 0.6mL/min Efficiency = 318,680 p/m Core-Shell 2.6 µm C18 50 x 2.1 mm 0.6mL/min Efficiency = 267,720 p/m 14

15 Core Shell Particle Advantages: 3x the efficiency of 5 m fullyporous media & 2x the efficiency of 3 m media Pressures compatible with conventional HPLC systems* Disadvantages: Pressure is still higher than 3 m media More sensitive to system extracolumn volumes More sensitive to overload in some cases 15

16 Core Shell Particle Drug Impurity Profiling: MP: A = 40mM KH 2 PO 4 ph 7.2, B = Acetonitrile Gradient: 5-35%B over 30min Flow rate: 0.2 ml/min Fully porous 1.7 µm C18 150x2.1mm 634 Bar AU Minutes Core-Shell 2.6 µm C18 150x2.1mm Bar AU Minutes

17 Review Fully-porous Silica Particle: General workhorse Many particle sizes and stationary phases Low sensitivity to system dead volume Core-Shell Particle: UPLC performance using conventional HPLC systems Should be the new go-to choice for method development in place of fully-porous media Organosilica Hybrid Particle: Expanded ph stability range Same performance characteristics as fully-porous Optimal for high ph applications 17 Monolithic Rod: Low back-pressure; reduced susceptibility to physical clogging Ideal for analysis of dirty samples (e.g. plasma) Efficiency ~3um fully-porous spherical silica

18 Reversed Phase Bonded Phases 18 Granick Research Group University of Illinois at Urbana-Champaign

19 Bonded Phases Once you have decided upon the optimal HPLC particle, the next decision will be to choose an appropriate stationary phase. This choice will greatly influence the final selectivity of your separation. Choice of bonded phase: Alkyl-bonded phases Phenyl phases Polar-embedded phases Choice of particle technology: Fully-porous Core-shell Monolithic rod UPLC 19

20 The Bonding Reaction HCl 1. Bonding main stationary phase ligand 2. Endcapping residual silanols 20

21 RP Stationary Phase Classes Alkyl bonded phases (C18, C8, C4): Polar-embedded phases: Fusion Phenyl phases (Phenyl, PFP): Polar-endcapped phases: F F F Hydro F F 21

22 Alkyl Bonded Phases Primary Mode of Interaction 22

23 Hydrophobic Interactions The primary mechanism of retention in reversed-phase chromatography is based upon hydrophobic interactions between the analytes and the bonded phase. Therefore, bonded phases that exhibit strongly hydrophobic nature tend to perform well. CH CH 3 CH3 H C 3 3 OH CH 3 CH 3 H3C N CH 3 O C H 3 Si H Si 3 C H O CH 3 CH 3 C O 3 O - OH OH O Si H 3 C Si CH 3 CHO 3 O CH 3 Si CH 3 CH 3 C H 3 Si Si Si Si Si Si Si O O O O O O H O OH H O OH OH OH H O O Si Si Si O O OH OH OH OH 23

24 Methylene Selectivity We use the methylene selectivity test to determine the ability of stationary phase to separate molecules based upon differences in their hydrophobic character. In general, very hydrophobic bonded phases (e.g. C18) will display higher levels of methylene selectivity than less hydrophobic phases. mau 250 C m A U min Phenyl m in 24

25 Methylene Selectivity C18 > C8 > C5 Phenyl > CN > Amino Slope of log k vs. # -CH2- units Luna C18(2) Synergi Hydro-RP Jupiter C18 Synergi Max-RP Luna C8(2) Luna C5 Luna Phe-Hex Jupiter C4 Synergi Polar-RP Prodigy Phenyl Luna Cyano Luna Amino 25

26 Methylene Selectivity Columns: Mobile phase: Flow rate: Components: 5 m C18 150x4.6mm 5 m C8 150x4.6mm 5 m Phenyl 150x4.6mm 65:35 Acetonitrile:Water 1 ml/min Two steroids: 1. Testosterone 2. Methyltestosterone CH 3 OH CH 3 OH CH 3 CH 3 H CH 3 H O H H O H H 26 Testosterone Methyltestosterone

27 Methylene Selectivity Testosterone Met-Testosterone C18 R s = 3.39 High Selectivity C8 R s = 1.78 Medium Selectivity Phenyl R s = 1.06 Low Selectivity 27

28 Phenyl Phases Phenyl phases are significantly less hydrophobic than C18 phases, but offer a potential selectivity that can be quite distinct due to the capacity of the phase to engage in pi-pi interactions with analyte molecules. It should be considered a complementary selectivity to conventional alkyl-bonded (e.g. C18) phases. Modes of Interaction F F F F F 28

29 Phenyl Selectivity While C18 phases excel at separating molecules that differ primarily in the hydrophobic character, phenyl phase often display an enhanced ability to separate molecules that differ in polar functional groups. This enhanced polar selectivity is probably attributable to interactions with the pi electron cloud of the phenyl ring. 29

30 Phenyl Selectivity Columns: C18 Phenyl Dimensions: Mobile phase: Flow rate: 150 x 4.6 mm 75:25 Methanol:water 1 ml/min OH CH 3 O CH 3 Components: 1. Estrone 2. Estradiol HO H H H HO H H H Estradiol Estrone 30

31 Phenyl Selectivity OH CH 3 O CH 3 H H C18 mau C18 HO H Estradiol H HO H H Estrone min Phenyl mau 100 Phenyl min 31

32 Polar Embedded Phases The residual silanol groups of any silica-based RP sorbent give some degree of hydrophilic interaction. The presence of additional polar-functional groups (endcapping or embedded within the bonded phase) further increases interactions with polar compounds. These phases are typified by: a. Polar selectivity for some polar analytes b. Stability in 100% aqueous mobile phases c. Improved peak shape for basic drugs (minimal silanols) 32

33 Nucleic Acid Bases: Aqueous Stability of Embedded Phases Luna C18(2) Day 1 Polar-Endcapped C18 Day Day Day 6 Phase Collapse! min

34 Aqueous Stability of Embedded Phases LC/MS/MS Analysis of ETG & ETS in Urine: Polar-Endcapped 2.5 µm C18 100x3.0mm XIC of -MRM (6 pairs): / Da ID: ETG-1 from Sample 6 (P-2_Hyro-RP_100x4.6_4u_FR e5 9.50e4 9.00e4 8.50e4 Max cps. 10mM Ammonium formate 8.00e4 7.50e4 7.00e4 6.50e4 ETG ETS 6.00e4 1. Ethyl glucuronide 2. Ethyl sulfate Intensity, cps 5.50e4 5.00e4 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 2.00e4 1.50e4 1.00e Time, min XIC of -MRM (6 pairs): / Da ID: ETG-1 from Sample 6 (P-2_Hyro-RP_100x4.6_4u_FR Max cps. 1.10e4 ETG 1.00e ETG ETS Intensity, cps ETS Time, min 34

35 Enhanced Retention of Polar Analytes mau 175 DAD1 A, Sig=210,4 Ref=360,100 (DJ031111\CATECHOL D) C18 150x4.6mm min mau 140 DAD1 A, Sig=210,4 Ref=360,100 (DJ031111\CATECHOL D) Polar-Embedded Phenyl 150x4.6mm Epinephrine Norepinephrine Dopamine min 35

36 Enhanced Polar Selectivity Oxymetazoline and oxidation product: Oxymetazoline mau 4 3 C mau 4 3 Phenyl mau 6 5 Polar-Embedded Phenyl R s R s R s min min min Improved R s with Phenyl Optimal R s with polarembedded and endcapped Phenyl Phase 36

37 Stationary Phases Summary 1. Choice of Particle Technology will have a dramatic effect on of critical components. Particle selection will usually be either fully-porous silica or core-shell media Monolithic rod for extremely dirty samples Organosilica hybrid for high ph Differences in pore size and surface area may give columns from different manufacturers very different performance characteristics 2. The most common stationary phase for RP methods is C18, and it is logical to begin screening with a C18 phase Phenyl phases offer an alternative selectivity, especially for polar aromatics Polar-endcapped or polar-embedded phases offer another selectivity option, and stability in 100% aqueous mobile phases 37

38 Column Selection Model Does sample require 97+% aqueous mobile phase? No Does sample require basic ph mobile phase? No Is your matrix extremely dirty? No Yes Yes Yes Polar-embedded or Polar-endcapped phase Organosilica hybrid particle Monolithic HPLC Column Particle size & Length Bonded phase screening Conventional or Core-Shell HPLC Column Insufficient Resolution? Alternate SP 38

39 39 Method Development Exercise 2: Media Selection and Phase Screening

40 Testosterone Epimer Analysis Epimers = stereoismers that differ in configuration of one stereogenic center (Carbon 17) Identical mass, so much be separated by HPLC for accurate quantitation using MS CH 3 OH CH 3 H H O Testosterone, C 19 H 28 O 2 Monoisotopic mass: Da Epitestosterone, C 19 H 28 O 2 Monoisotopic mass: Da 40

41 Particle Selection Does sample require 97+% aqueous mobile phase? No Does sample require basic ph mobile phase? No Is your matrix extremely dirty? No Particle size & Length Bonded phase screening Conventional or Core-Shell HPLC Column 100x2.1mm; C18, XB-C18, C8 Core-shell Particle 41

42 Alkyl Phase Screening Core-Shell C18 Testo Epi- Core-Shell XB-C18 Core-Shell C8 42

43 Alternative Column Screening Does sample require 97+% aqueous mobile phase? No Does sample require basic ph mobile phase? No Is your matrix extremely dirty? No Polar-embedded, or polar-endcapped phase Organosilica hybrid particle Particle size & Length Flow rate Bonded phase screening Mobile phase optimization Conventional or Core-Shell HPLC Column Insufficient Resolution? Alternate SP 43

44 Evaluation of Phenyl Phase Core-Shell XB-C18 Core-Shell PFP 44

45 Evaluation of Embedded and Hybrid Core-Shell XB-C18 Polar-endcapped C18 Organosilica Hybrid C18 45

46 Final Method Core-Shell XB-C18 (Primary) Organosilica Hybrid C18 (Back-up) Columns: Core-Shell 2.6 µm XB-C18 100x2.1mm Organosilica Hybrid 3 µm C18 100x2.1mm 46 MP A: 0.1%FA, 1 mm Ammonium Formate in Water MP B: 0.1% FA, 1 mm Ammonium Formate in ACN Flow rate 0.4 ml/min Step Time, min %B

47 47 End of Part II