Understanding (and Creating) Polar Retention Using ...

1 2003 Waters CorporationUnderstanding (and creating ) Polar Retention Using Reversed-Phase hplc and hydrophilic Interaction ChromatographyPittcon March 2003 2003 Waters CorporationIntroduction Introduction The Problem Background hydrophilic Interaction chromatography Reversed-Phase hplc for Polar Molecules Applications Summary 2003 Waters CorporationIntroduction - The Problem Conventional hplc columns often do not provide adequate Retention and separation of highly Polar compounds Drug Discovery Gradient - High-throughput screening (HTS) does not work for Polar compounds Elute un-retained in the void volume Co-elute at the beginning of the run if retained at all Method Development Isocratic- Many RP columns cannot be run under the 100% aqueous conditions necessary for Polar compound Retention columns dewet (also termed -hydrophobic collapse) 2003 Waters CorporationBackground Introduction Background Polar molecules Chromatographic methods for retaining Polar compounds hydrophilic Interaction chromatography Reversed-Phase hplc for Polar Molecules Applications Summary 2003 Waters CorporationWhat is a Polar Molecule?

2 General chemistry definition: A molecule whose centers of positive and negative charges do notcoincide The degree of polarity is measured by the dipole momentof the molecule Dipole moment is the product of the charge at either end of the dipole times the distance between the chargesHOH +2 - +Dipole moment = DThis is a Polar Molecule!Electro-static attraction opposite charge locations on different molecules attract 2003 Waters CorporationExamples of Polar MoleculesHNNHOOU racil (N)HNNHOOT hymine (N)OAcetone (N)NNHNH2 OCytosine (B)HNHNFOOOHO5-Fluoroorotic acid (A)OHOHNHOHE pinephrine (B)Ascorbic acid (A)OOHOHOOHOH 2003 Waters CorporationComparison of Chromatographic Methods for Retention of Polar Compounds Dewetting under aqueous conditions Poor Retention of Polar compounds with non- Polar stationary phases Familiar technique High efficiency Rapid equilibration Wide selection of columnsReversed-Phase Long equilibration times Difficult to run gradients Mostion pairing agents not compatible with MS Difficult method development Can retain any ionizable compound Can perform ion pairing chromatography and reversed-phase chromatography on same columnIon Pairing Sample and mobile phase solubility problems Long re-equilibration times High percentage organic mobile phases give higher sensitivity in ESI-MSHILIC Does not work well if the compound does not ionize High salt buffers or pH gradients for elution not compatible with MS Can retain any ionizable compoundIon ExchangeDisadvantagesAdvantages 2003 Waters CorporationReversed-Phase chromatography for Polar Compound Retention

3 Reversed-Phase chromatography Non- Polar stationary phase with >80% aqueous mobile phases Strengths Familiar, well understood technique Many stationary phase choices Good reproducibility, stable equilibration High efficiency Weaknesses of modern C18phases designed for improved peak shape for basic analytes: Polar compound Retention Sudden loss of Retention in 100% aqueous mobile phase Poor Retention of polaranalytes on a high coverage, non-polarC18stationary phase 2003 Waters CorporationHydrophilic Interaction chromatography for Polar Compound Retention HILIC hydrophilic Interaction chromatography Polar stationary phase with >80% organic mobile phases Strengths Good Retention of verypolar molecules Volatile mobile phases Increased ESI-MS sensitivity Lower backpressure Complementary selectivity vs reversed-phase Weaknesses Not many stationary phases available Poor Retention of hydrophobic compounds 2003 Waters CorporationHydrophilic Interaction chromatography Introduction Background hydrophilic Interaction chromatography (HILIC) What is it?

4 Why would someone use it? AtlantisTMHILIC Silica Reversed-Phase hplc for Polar Molecules Applications Summary 2003 Waters CorporationHILIC: What is it? HILIC hydrophilic Interaction chromatography Reverse reversed-phase (water is the eluting solvent) Verypolar analytes are retained and separated on a Polar , hydrophilic stationary phase Analytes elute in order of increasing hydrophilicity High organic/low aqueous conditions Mobile phases used >70% ACN (with water/buffer) Very volatile (very ESI-MS-friendly ) Stationary phases SiO2, diol, amino, amide, cyano, ion-exchange, SEC, Zwitterion, etc. 2003 Waters CorporationHILIC: What is it? Polar PhaseHILICR etention Polar analyte partitions into and out of adsorbed water layer The larger the water layer (greater aqueous concentration), the faster the compound(s) will elute Analytes elute in order of increasing hydrophilicity High Acetonitrile (>70%) with aqueous Polar Phases (Bonded and Unbonded)HNNHOOHNNHOOOH80% acetonitrile20% aqueous,( ,pH )uracil 2003 Waters CorporationHILIC: What is it?

5 Polar PhaseWeak Cation ExchangeRetention Charged Polar analyte can undergo cation exchange with charged silanol groups High Acetonitrile (>70%) with aqueousO-80% acetonitrile20% aqueous, ( ,pH )HILIC is defined as the high organic conditionswhere Polar compounds are retained longer(reversereversed-phase)NNH3+NHONNH 3+NHOcytosine 2003 Waters CorporationHILIC Retention CharacteristicsCytosineHILIC:Increased Retention occurs when Using greater than 70% organic for Polar 102030405060708090% MeCNLN (Tr min/cm)pH 5pH 4pH 3 AqueousRetention 2003 Waters 102030405060708090% MeCNLN (Tr min/cm)pH 5pH 4pH 3 Reversed-Phase Retention CharacteristicsCytosineReversed-phase:In creased Retention occurs when Using less than 20% organic for Polar basesNNHONH2 AqueousRetention 2003 Waters CorporationHILIC:Why would someone use it? Why would someone use HILIC? Retains verypolar compounds not retainable by RP Volatile mobile phase Increased ESI-MS sensitivity (lower LODs) Increased flow rates Increased sample throughput Lower backpressures Facilitates sample prep (SPE, liquid/liquid, protein precipitation) as the final elution/extraction solvent does not have to be evaporated/reconstituted 2003 Waters CorporationVo= min x 50 mm, 3 m95:5 ACN:Formate Buffer, pH mL/minAtlantisTMHILIC Silica: Retains very Polar compounds not retainable by x 50 mm, 3 m100% Formate Buffer, pH mL/minVo= min NHNHNHOOONH2 AllantoinHILIC offers Retention when there is no Retention by reversed-phase k = = 2003 Waters CorporationAtlantisTMHILIC Silica : Enhanced ESI-MS SensitivityAtlantisTMdC18 Peak x 50 mm, 3 m1.

6 Albuterol 100 pg/ L782. Bamethan 20 pg/ L2 AtlantisTMHILIC Silica Peak x 50 mm, 3 m2. Bamethan 20 pg/ L93871. Albuterol 100 pg/ L13131 HILIC requires high volatility solvents which increase ESI-MS sensitivity vs. high-aqueous mobile phases used in RP + + 2003 Waters CorporationAtlantisTMHILIC Silica :Simplify Sample PreparationCondition/Equilibrate*200 L methanol/200 L waterLoad150 L spiked plasma sample150 L internal standardwith 2% ammonium hydroxideWash200 L 5% methanol in waterElute300 L 40% acetonitrile/60% isopropanolwith 2% formic acid*Oasis HLB Elution PlateInject eluent directly onto columnNoEvaporation and Reconstitution Step 2003 Waters CorporationAtlantisTMHILIC Silica :Simplify Sample of 2 Channels ES+ x 50 mm, 3 m1. Bamethan 10 pg/ L 2. Albuterol 50 pg/ L12 SPE eluent injected directlyonto AtlantisTMHILIC Silica column 2003 Waters CorporationAtlantis HILIC SIlica:Summary AtlantisTMHILIC Silica Provides Retention of some Polar analytes not retained under RP conditions Enhanced sensitivity in ESI-MS is enjoyed due to the highly volatile mobile phases (> 80% organic) used Sample preparation procedures are shortened by eliminating the evaporation and reconstitution steps and directly injecting the eluent AtlantisTMHILIC Silica columns are an alternative to RP chromatography for the Retention of very Polar basic analytesNewat Pittcon 2003 2003 Waters CorporationReversed-Phase hplc for Polar Molecules Introduction Background hydrophilic Interaction chromatography Reversed-Phase hplc for Polar Molecules The objective and challenge Dewetting (nothydrophobic collapse) What doesn t work (and why)

7 AtlantisTMdC18- an intelligent solution Applications Summary 2003 Waters CorporationThe Challenge for Stationary Phase Manufacturers The Objective Create a stationary phase that retains all analytes, gives good peak shape for bases, and is compatible with all LC detectors The Challenge To retain a hydrophilic ( Polar ) analyte on a hydrophobic (non- Polar ) stationary phaseWhy is this so difficult? 2003 Waters CorporationReversed-Phase ChromatographyO-SiO-SiOHO-SiO-SiOHO-SiOH O-SiO-SiO-SiOHO-SiO-SiNon-polarstationar yphasePolar mobilephaseAnalyte XPolarAnalyte YNon-PolarChromatographically how does oneattempt to forcepolarAnalyte X to retain onthe non-polarstationary phase?FlowPoorlyRetained (like attracts like)Well retained(like attracts like) 2003 Waters CorporationChallenge for Reversed-Phase chromatography of Polar Compounds To retain Polar compounds on this non- Polar surface we reduce the amount of organic in the mobile phase( ,make mobile phase weaker, ,100% aqueous) PROBLEM: Risk of dewetting(hydrophobic collapse) the particle surface the chromatographic pores dry-out (non- Polar pore surface expels the pure aqueous, Polar mobile phase)What is dewetting and why does it happen?

8 2003 Waters CorporationMinutes0246810 Initial(Column was wetted first with organic)After Flow Stoppage (Pores dewet 100%)Mobile phase: Acetic AcidAmoxicillinVo: Noretention of analyte1,500psiNote: Column is not broken --It just stopped workingWhy?1,500psiConventional C18 Column Dewetting 2003 Waters CorporationPore Dewetting MechanismFlow stoppage relieves the pressure that was forcing the aqueousmobile phase into the pores. When pressure is reduced ( ,pump stopped), the hydrophobic pore surfacecan expel the Polar mobile phase and dewet the flow, with pressure on the mobile phase. Stopped flow with no pressureon the mobile de-wet restart flow pores still dewettedand analytes never enter pores resulting in no Retention . AnalytesAnalytes properly retainedRemember: Most of the surface area (> 95%) is insidethe silica pores! 2003 Waters CorporationWateron C18:d = 100 = dynes/cm = **B. Janczuk, T. Bialopiotrowicz and W. Wojcik, Colloids and Surfaces36(1989) 391-403 Methanolon C18:d = 100 = 22 dynes/cm = *Pc< 0 psiPc= 1,500 psi Note: Self-wetting No pressure required Equation of Young and Laplace =4 Pc=4 d cosPressure on C18pore requiredto force water back into the poreWhere.

9 Pc= Capillary pressure = Surface tensiond= Capillary diameter = Contact angleStationary Phase WettingSolvent dependentH2 OMeOH Contact Angle 2003 Waters CorporationRe-wetting a Stationary Phase Use a mobile phase containing > 40% methanol or other Polar organic solvent (other organic solvents may vary in % required for wetting) This works by reducing the contact angle ( ) The use of pressure alonecannot force aqueous mobile phase back into silica pores Not practical because column outlet is at atmospheric pressure not all pores see required pressure (1,500 psi)1,500 psi1 atm = psiPressure gradient inside column1,500 psiAtmospheric pressure 2003 Waters CorporationDewetting Summary Dewetting Waters research suggests that dewetting - not hydrophobic collapse is the reason for sudden loss of Retention under highly aqueous conditions The observed reduction in V0and sudden loss in Retention after a pressure drop indicate that the pores expel the aqueous solvent (vs. ligand chains folding or matting as described by hydrophobic collapse) Rewetting with an organic solvent can rewet the pores by reducing the contact angle and surface tension Ideally, it is best to avoid dewetting have chromatographers attempted to do this?

10 2003 Waters CorporationEmbedded Polar Groups What Doesn t Work Embedded Polar groups were designed to: Improve peak shapes for basic compounds Provide complementary selectivity as compared to straight-chain alkyl stationary phases Examples of embedded Polar groups include carbamate, ether, amide, urea, etc. Another feature of embedded Polar groups is 100% aqueous mobile phase compatibilityThis feature is confused with enhanced Polar compound retentionWhy? 2003 Waters CorporationEmbedded Polar Groups What Doesn t Work Embedded polargroups resist pore dewetting by increasing the water layer at the surface of the poresEmbedded Polar group behaveslike a Polar Hook holding onto Retention requires these very weak polarmobile ,highly aqueous mobile phases with little-or-no organic modifierThis aqueous compatibility is confused with Polar compound is false! 2003 Waters CorporationCarignan, IranetaEmbedded Polar Groups What Doesn t Thiourea2. 5-Fluorocytosine3. Adenine4.