HILIC_2009 09

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Successful Polar Compound Retention: Hydrophilic Interaction (HILIC) Chromatography : 

Successful Polar Compound Retention: Hydrophilic Interaction (HILIC) Chromatography

Today’s Focus : 

Today’s Focus Defining the problem HILIC retention mechanisms and characteristics Impact on mass spectrometry sensitivity Impact on sample handling steps The promise of smaller particles

Today’s Focus : 

Today’s Focus Defining the problem What is a polar molecule? Methods of retaining polar molecules Reversed-phase retention and pore-dewetting Introduction of HILIC HILIC retention mechanisms and characteristics Impact on mass spectrometry sensitivity Impact on sample handling steps The promise of smaller particles

What is a Polar Molecule? : 

General chemistry definition: A molecule whose centers of positive and negative charges do not coincide The degree of polarity is measured by the dipole moment of the molecule What is a Polar Molecule?

Comparison of Chromatographic Methods for Retention of Polar Compounds : 

Comparison of Chromatographic Methods for Retention of Polar Compounds

Example of Polar Compounds Analysis – 멜라민 분석 - 식품공전 중 (식약청고시 제 2009-7호) : 

Example of Polar Compounds Analysis – 멜라민 분석 - 식품공전 중 (식약청고시 제 2009-7호) LC/MS/MS 분석법 정제용 카트리지 : 설폰산기(-SO3H)를 갖는 강산성 양이온교환수지 카트리지로서 Oasis MCX(150 mg 함유, 6 mL용) 또는 이와 동등한 것 칼럼 : HILIC Silica (2.1 × 150 mm, 5 μm) 또는 이와 동등한 것 이동상은 A 및 B 용액의 농도 구배조건에 따라 사용한다. 이동상 A : 0.1% 개미산․아세토니트릴(5:95, v/v) 이동상 B : 20 mM 개미산암모늄․아세토니트릴(50:50, v/v) HPLC 분석법 정제용 카트리지 : 위와 같음 칼럼 : C18 (4.6 × 250 mm, 5 μm) 또는 이와 동등한 것 이동상 : 완충용액․아세토니트릴 (85:15, v/v) 완충용액 : 무수구연산 1.92 g과 옥탄설폰산나트륨 [C8H17O3SNa] 2.16 g을 950 mL의 물에 녹이고 1 M 수산화나트륨을 사용하여 pH 3.0으로 한 후 물을 가하여 전량을 1 L로 한다.

Reversed-Phase Chromatography for Polar Compound Retention : 

Reversed-Phase Chromatography for Polar Compound Retention 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 C18 phases designed for improved peak shape for basic analytes: polar compound retention Sudden loss of retention in 100% aqueous mobile phase Poor retention of polar analytes on a high coverage, non-polar C18 stationary phase

Pore Dewetting: Relationship to Stationary Phase Design : 

1 2 3 4 5 1 2 3 4 5 Minutes 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 1 2 3,4 5 1 2,3,4 5 Atlantis® T3 4.6 x 100 mm, 3 µm Zorbax® Eclipse Plus C18 4.6 x 100 mm, 3.5 µm < 7% Thymine Dewet Thymine Dewet: ~100% Compounds: 1. Thiourea 2. 5-Fluorocytosine 3. Adenine 4. Guanosine-5’-monophosphate 5. Thymine Isocratic Conditions Mobile Phase: 10 mM NH4COOH, pH 3.0 Flow Rate: 1.2 mL/min Injection Vol: 7 µL Temperature: Ambient Detection: UV @ 254 nm Pore Dewetting: Relationship to Stationary Phase Design 100% retention loss with conventional C18 With proper design considerations, pore-dewetting can be minimized

Polar Retention:Why Does Atlantis® T3 Work? : 

Polar Retention:Why Does Atlantis® T3 Work? Dominant retention mechanism is reversed-phase (van der Waals forces – hydrophobic attraction) Retention maximized using 100% aqueous mobile phases Retention maximized by using reduced C18 coverage Secondary interactions due to residual silanols that are more accessible due to reduced C18 coverage Cation-exchange interactions Hydrogen bonding interactions

Scalability to/from UPLC® Technology: T3 Bonding & Endcapping : 

Scalability to/from UPLC® Technology: T3 Bonding & Endcapping HPLC Separation UPLC® Separation 6 5 4 3 2 7 1 Compounds: 1. Norepinephrine 2. Epinephrine 3. Dopamine 4. 3,4-Dihydroxyphenylacetic acid 5. Serotonin (5-HT) 6. 5-Hydroxy-3-indoleacetic acid 7. 4-Hydroxy-3-methoxyphenylacetic acid (HVA) Atlantis® T3 T3 bonding and endcapping present in both HPLC and UPLC Technology

Addressing Retention and Sensitivity:What if Reversed-Phase Doesn’t Work? : 

Addressing Retention and Sensitivity:What if Reversed-Phase Doesn’t Work? Reversed-phase options for retaining polar analytes Reversed-phase low coverage C18 designed for the retention of polar analytes ACQUITY UPLC® HSS T3 or Atlantis® T3 High aqueous conditions Manipulate pH to make compound neutral ACQUITY UPLC® BEH C18 or XBridgeTM C18 Low pH for acids, High pH for bases STILL have inadequate retention or insufficient MS Response??? Try HILIC…

What is HILIC? : 

What is HILIC? HILIC - Hydrophilic Interaction Chromatography Term coined in 1990 to distinguish from normal-phase* HILIC is a variation of normal-phase chromatography without the disadvantages of using solvents that are not miscible in water “Reverse reversed-phase” or “aqueous normal-phase” chromatography Stationary phase is a POLAR material Silica, hybrid, cyano, amino, diol, amide The mobile phase is highly organic (> 80% ACN) with a smaller amount of aqueous mobile phase Water (or the polar solvent(s)) is the strong, eluting solvent *Alpert, A. J. J.Chromatogr. 499 (1990) 177-196.

Benefits of HILIC: : 

Benefits of HILIC: Retention of highly polar analytes not retained by reversed-phase Less interference from matrix components Complementary selectivity to reversed-phase Polar metabolites retain more than parent compound Enhanced sensitivity in mass spectrometry High organic mobile phases (> 80% ACN) promotes enhanced ESI-MS response Direct injection of PPT supernatant without dilution Facilitates use of lower volume samples Improved sample throughput Direct injection of high organic extracts from PPT, LLE or SPE without the need for dilution or evaporation and reconstitution

Waters HILIC Column Offerings : 

Waters HILIC Column Offerings

Today’s Focus : 

Today’s Focus Defining the problem HILIC retention mechanisms and characteristics Multimodal HILIC retention mechanisms Retention comparison between hybrid and silica particles Solvent selectivity Influence of pH on retention Chemical stability Impact on mass spectrometry sensitivity Impact on sample handling steps The promise of smaller particles

HILIC Retention Mechanisms are Complex : 

HILIC Retention Mechanisms are Complex Combination of partitioning, ion-exchange and hydrogen bonding Polar analyte partitions between bulk mobile phase and partially immobilized polar layer on material surface Secondary interactions between surface silanols and/or functional groups with the charged analyte leading to ion-exchange Hydrogen bonding between positively charged analyte and negatively charged surface silanols

Retention Characteristics: HILIC and Reversed-Phase : 

Retention Characteristics: HILIC and Reversed-Phase HILIC offers dramatically more retention than reversed-phase for very polar bases. HILIC RP

Enhanced Retention Compared to Reversed-Phase Atlantis® HILIC Silica : 

Enhanced Retention Compared to Reversed-Phase Atlantis® HILIC Silica Allantoin Grumbach Reversed-Phase Retention Factor = 0 Atlantis T3 HILIC Retention Factor = 1.1 Atlantis HILIC Silica HILIC can offer retention when no retention is achieved by reversed-phase

Complementary Selectivity to Reversed-Phase XBridgeTM HILIC : 

Complementary Selectivity to Reversed-Phase XBridgeTM HILIC Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 1 2 3 3 2 1 V0 =0.26 V0 =0.25 Reversed-Phase XBridge C18 HILIC XBridge HILIC 3. Morphine 3-ß-D-Glucuronide 2. morphine 1. 6-acetyl morphine HILIC offers complementary selectivity to reversed-phase

HILIC Selectivity Comparison:BEH Amide vs. BEH HILIC : 

HILIC Selectivity Comparison:BEH Amide vs. BEH HILIC Compounds 1. Methacrylic Acid 2. Cytosine 3. Nortriptyline 4. Nicotinic Acid AU 0.00 0.20 0.40 0.60 3 1 2 4 AU 0.00 0.20 0.40 0.60 1 2 3 4 ACQUITY UPLC BEH HILIC 2.1 x 100 mm, 1.7 µm ACQUITY UPLC BEH Amide 2.1 x 100 mm, 1.7 µm Isocratic mobile phase of 90/5/5 ACN/EtOH/10 mM CH3COONH4 in H2O with 0.02% CH3COOH, flow rate 0.5 mL/min, column temp. 25 °C, 1.5 µL injection, 60 µg/mL each compound, sample diluent 75/25 ACN/MeOH, UV 210 nm New BEH Amide offers alternative selectivity for HILIC separations

Solvent Considerations:Polar Organics and Mobile Phase Additives : 

Solvent Considerations:Polar Organics and Mobile Phase Additives To increase retention of analytes, replace some of the water with another polar solvent (i.e., methanol, isopropanol) In HILIC, these polar solvents are weaker eluters than water Peak shapes and reproducibility may be compromised by completely removing the polar solvent (e.g., water) Buffers/additives Phosphate salt buffers are not recommended due to precipitation in the highly organic mobile phase (phosphoric acid is OK) Ammonium formate, pH 3*; ammonium acetate, pH 5*; 0.2% formic acid, pH 2.5*, 0.2% phosphoric acid, pH 1.8*

Elution Strength in HILIC:Solvent Selectivity : 

Elution Strength in HILIC:Solvent Selectivity Use a less polar solvent to Increase retention of polar analytes

Considerations for Retention:Mobile Phase pH : 

Considerations for Retention:Mobile Phase pH Some analytes can exhibit increased retention with increasing mobile phase pH Silica materials are subject to particle dissolution with increasing pH Un-derivatized silica materials are subject to accelerated degradation due to lack of protection Hybrid particles have substantially improved chemical resistance* Overall usable column life is improved *O.Gara, J.E., Wyndham, K.D., J. Liq. Chromatogr. Relat. Technol. 2006, 29, 1025 - 1045,

Today’s Focus : 

Today’s Focus Defining the problem HILIC retention mechanisms and characteristics Impact on mass spectrometry sensitivity Comparison of reversed-phase and HILIC Impact on sample handling steps The promise of smaller particles

Enhanced MS Sensitivity: Hybrid Phases, Reversed-Phase vs. HILIC : 

Enhanced MS Sensitivity: Hybrid Phases, Reversed-Phase vs. HILIC 1 2 1 2 More efficient desolvation of high organic HILIC mobile phase results in higher MS response compared to reversed-phase Reversed-Phase XBridge C18 100% H2O HILIC XBridge HILIC 81 ACN: 4.5 MeOH: 14.5 H2O intensity 10.5 X response 8.6 X response

Today’s Focus : 

Today’s Focus Defining the problem HILIC retention mechanisms and characteristics Impact on mass spectrometry sensitivity Impact on sample handling steps Elimination of evaporation and reconstitution steps of SPE The promise of smaller particles

Simplified Sample Preparation: Direct Injection SPE Eluent : 

Simplified Sample Preparation: Direct Injection SPE Eluent Direct injection of SPE Eluent onto XBridge HILIC Column

Today’s Focus : 

Today’s Focus Defining the problem HILIC retention mechanisms and characteristics Impact on mass spectrometry sensitivity Impact on sample handling steps The promise of smaller particles Influence of particle size on efficiency Scalability between HPLC and UPLC® Technology

Seamless Scalability to UPLC Technology:Neurotransmitters : 

Seamless Scalability to UPLC Technology:Neurotransmitters UPLC Technology enables sharper chromatographic peaks resulting in higher sensitivity compared to HPLC HPLC XBridge HILIC, 3.5 µm F = 0.5 mL/min UPLC ACQUITY UPLC BEH HILIC, 1.7 µm F = 1.0 mL/min 1.6X Higher Response Ach 1.6X Higher Response Ch 2X Faster at Scaled Flow Rate 1 2 V0 = 0.35 min

UPLC-MS Analysis of Organic Acids at High pH : 

UPLC-MS Analysis of Organic Acids at High pH Maleic acid (1 ppm) Pyruvic acid (50 ppm) Lactic acid (50 ppm) Succinic acid (50 ppm) Fumaric acid (50 ppm) 1 2 3 4 5 ACQUITY UPLC® BEH Amide HILIC Conditions, pH 9.0 SIR Mode, Electrospray -

Summary : 

Summary ACQUITY UPLC HSS T3 and Atlantis T3 Paramount retention for polar compounds Exceptional performance and versatility for polar and hydrophobic compounds ACQUITY UPLC BEH HILIC, ACQUITY UPLC BEH Amide, XBridge HILIC and Atlantis HILIC Silica Exceptional retention for very polar compounds Increased LC/MS sensitivity Improved sample throughput with direct injection of organic extracts Selectivity Choice for HILIC separations

Slide 32: 

Guide to Retaining Polar Compounds

감사합니다 : 

감사합니다

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