Using Hydrophilic Interaction Chromatography (HILIC) for the Retention of Highly Polar Analytes Eric S. Grumbach,, Diane M. Diehl and Jeffrey R. Mazzeo 2003 Waters Corp.
Outline Introduction HILIC Definition and Benefits Important considerations for HILIC Mobile phase considerations Solvent selectivity Column performance Column reproducibility Complementary selectivity to reversed-phase Enhanced ionization in mass spectrometry Low limits of detection Direct injection of SPE eluent Summary
Introduction Traditional options for retaining polar analytes Ion pairing or ion exchange Only works if analytes ionize Often not compatible with mass spectrometry (MS) Manipulate ph to make compound neutral Reversed-phase columns designed for the retention of polar analytes (such as the Atlantis dc 18 ) Used under highly aqueous conditions, often resulting in poor MS sensitivity What happens when you ve tried all of these options and you STILL have inadequate retention??? Let s take a look at 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, cyano, amino, diol The mobile phase is highly organic (> 80%) 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.
HILIC Retention on Silica Polar analyte partitions into and out of adsorbed water layer Charged polar analyte can undergo cation exchange with charged silanol groups Combination of these mechanisms results in enhanced polar retention
HILIC vs. Reversed-Phase Retention Characteristics H N O N Retention NH 3 + Cytosine 0 10 20 30 40 50 60 70 80 90 % MeCN HILIC offers dramatically more retention than reversed-phase for very polar bases. HILIC RP Alden, Iraneta
Benefits of HILIC Retention of highly polar analytes not retained by reversed-phase Complementary selectivity to reversed-phase Enhanced sensitivity in mass spectrometry High organic mobile phases (> 80%) promotes enhanced ESI-MS response Shortens sample preparation procedure Elimination of evaporation/reconstitution step by directly injecting the organic phase
Outline Introduction HILIC Definition and Benefits Important considerations for HILIC Mobile phase considerations Solvent selectivity Column performance Column reproducibility Complementary selectivity to reversed-phase Enhanced ionization in mass spectrometry Low limits of detection Direct injection of SPE eluent Summary
Wash solvents Mobile Phase Considerations: Before You Start Seal wash: 10% acetonitrile in water Needle wash: 1:1 acetonitrile:water Purge Solvent: initial mobile phase conditions (without salt, additive or buffer) Initial conditions: 95% to 70% acetonitrile Remember, acetonitrile is the weaker solvent At least 5% should be a polar solvent (i.e., water or methanol) Where do I start? Initial scouting gradient from 95 to 50% acetonitrile If analytes are not retained, try 95% acetonitrile isocratic
Mobile Phase Considerations: Before You Start 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* For optimum performance and reproducibility it is recommended that a concentration of 10 mm buffer or 0.2% of an additive be introduced ON COLUMN 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) Let s take a closer look *The actual ph of the mobile phase may be 1 ph unit higher due to the highly organic mobile phase Canals, I.; Oumada, F. Z.; Roses, M.; Bosch, E. J. Chromatogr. A. 911 (2001) 191-202.
Solvent Strength: HILIC Solvent Water Methanol Ethanol Isopropanol Acetonitrile Acetone Tetrahydrofuran Strongest Weakest In HILIC, utilizing a less polar solvent can increase the retention of polar analytes.
Compounds 1. 5-Fluorouracil 2. Uracil 3. 5-Fluorocytosine 4. Cytosine Increased Retention with a Less Polar Solvent Initial 90 ACN: 5 H 2 O: 5 buffer Final 50 ACN: 45 H 2 O: 5 buffer 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 1 1 Initial 90 ACN: 5 MeOH: 5 buffer Final 50 ACN: 45 MeOH: 5 buffer 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 1 2 2 2 3 3 4 Minutes Minutes 4 3 4 Initial 90 ACN: 5 IPA: 5 buffer Final 50 ACN: 45 IPA: 5 buffer Replace H 2 O with MeOH Replace MeOH with IPA 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes Retention increases by using a less polar solvent. Grumbach
Outline Introduction HILIC Definition and Benefits Important considerations for HILIC Mobile phase considerations Solvent selectivity Column performance Column reproducibility Complementary selectivity to reversed-phase Enhanced ionization in mass spectrometry Low limits of detection Direct injection of SPE eluent Summary
S 1 2 3 4 Reproducibility of Atlantis TM HILIC Silica Injection 1 Injection 100 Injection 200 Injection 300 Analytes: S. Solvent Peak 1. 5-Fluorouracil 2. Uracil 3. 5-Fluorocytosine 4. Cytosine Injection 400 Injection 500 Injection 600 Injection 700 Injection 800 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes Continuous injections of polar bases under gradient conditions yield excellent reproducibility. Grumbach
Retention Benefits of HILIC AU 0.10 0.05 0.00 0.15 0.10 AU 0.05 0.00 k = 0 V o = 0.65 min 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 V o = 1.15 min Minutes k = 1.09 Atlantis TM dc 18 4.6 x 50 mm, 3 µm 100% Formate Buffer, ph 3 1.0 ml/min H 2 N Atlantis TM HILIC Silica 4.6 x 50 mm, 3 µm 95:5 ACN:Formate Buffer, ph 3 1.0 ml/min O NH O H N N H Allantoin O 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes HILIC offers retention when there is no retention by reversed-phase. Grumbach
Complementary Selectivity to Reversed-Phase HO 1 2 V o = 0.5 min Atlantis TM HILIC Silica 4.6 x 50 mm, 3 µm 90% to 50% ACN HO O H N 1. Morphine CH 3 1.00 2.00 3.00 4.00 5.00 2 Minutes V o = 0.65 min 1 Atlantis TM dc 18 4.6 x 50 mm, 3 µm 2% ACN 2. Morphine 3-ß-D-Glucuronide 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes Grumbach
Reversed-Phase Conditions: Separation of Cytochrome C Tryptic Digest 100 % 204 (T2) 261 (T6,11) 361 (T18) 434 (T21) 678 (T14) 779 (T15) Peak Annotation M/Z (Fragment ID) Atlantis TM dc 18 4.6 x 50 mm, 3 µm 634 (T4) 729 (T10) 964 (T19) 1005 (T12) 585 (T8) 0 Extremely polar peptides are not retained under reversed-phase conditions even with the aid of Atlantis dc 18, a column specifically designed for the retention of polar analytes Atlantis HILIC Silica can aid in the separation and identification of these very polar peptides Rainville
Complementary Selectivity to Reversed-Phase: Separation of Cytochrome C Tryptic Digest 100 % 204 (T2) 261 (T6,11) 361 (T18) 434 (T21) 678 (T14) 779 (T15) Peak Annotation M/Z (Fragment ID) Atlantis TM dc 18 4.6 x 50 mm, 3 µm 634 (T4) 729 (T10) 964 (T19) 1005 (T12) 585 (T8) 100 0 634 (T4) Atlantis TM HILIC Silica 4.6 x 50 mm, 3 µm % 779 (T15) 678 (T14) 434 (T21) 585 (T8) 261 (T6,11) 204 (T2) 964 (T19) 729 (T10) 1005 (T12) 361 (T18) 00 45 Polar peptides not retained by reversed-phase can be retained by HILIC. Rainville
Enhanced Sensitivity in Mass Spectrometry LC/MS conditions were optimized individually for both HILIC and RP. 100 % 0 Reversed Phase 100 % 0 1 2 1.84 2.18 1.84 1 2 2.18 1.75 e3 1.00 2.00 3.00 4.00 5.00 Time ES+ 239.9 209.9 2.8e5 0-50% ACN Atlantis TM dc 18 Peak Area 2.1 x 50 mm, 3 µm 10.0 µl injection volume 1. Albuterol 100 pg/µl 21 (ND) 2. Bamethan 50 pg/µl 14 (ND) 100 % 0 HILIC 2 1.53 1 1.92 ES+ 239.9 209.9 2.8e5 95-50% ACN 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Time Atlantis TM HILIC Silica Peak Area 2.1 x 50 mm, 3 µm 10.0 µl injection volume 2. Bamethan 50 pg/µl 110085 1. Albuterol 100 pg/µl 19567 HILIC requires high volatility solvents which increase sensitivity compared to high-aqueous mobile phases used in reversed-phase.* *Naidong, W.; Shou, W.; Chen, Y-L.; Jiang, X. J. Chromatogr. B. 754 (2001) 387-399. Grumbach
Mass Spectrometer Detection Limits Approximate Limit of Detection Single Quadrupole MS (SIR) mode ES+ Triple Quadrupole MS (MRM) mode ES+ (Analyte Dependant) Reversed-phase 10 ng/ml 1 ng/ml HILIC 1 ng/ml 0.1 ng/ml [100 fg/µl] HILIC-ESI-MS will achieve at least 10x higher sensitivity with high organic mobile phases than typical RP-ESI-MS with high aqueous mobile phases.
Extremely Low Detection Limits on a Single Quadrupole Mass Spectrometer 100 % ACh H 3 C H 3 C N + CH 3 O CH 3 O ES+ 146.4 3.19e5 Atlantis TM HILIC Silica 2.1 x 50 mm, 3 µm 20.0 µl injection volume Waters ZQ single quadrupole MS SIR m/z: 104.2, 118.3, 146.4 100 % Ch H C 3 N + H C 3 CH 3 OH ES+ 104.2 1.78e5 Analyte Concentration 1. Acetylcholine (Ach) 100 fg/µl 2. Choline (Ch) 100 fg/µl 3. Betaine (I.S.) 5000 fg/µl 0 100 % CH 3 H C 3 H 3 C O N + O - Bet (I.S.) ES+ 118.3 1.32e6 86% ACN Detect 100 fg/µl on a single quadrupole MS! 0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Time
Traditional SPE Methods for Reversed-Phase Chromatography Traditional SPE methods often contain an elution step that consists of high organic content To make this extracted sample compatible with your mobile phase, you must first evaporate the high organic eluent then reconstitute in some portion of aqueous Evaporation and reconstitution are often the most lengthy steps in an SPE procedure* In HILIC, the high organic eluent can be directly injected on the column, thus eliminating the need for evaporation and increasing your throughput *Jemal, M., Teitz, D., Ouyang, Z., J.Chromatogr. B, 732 (1999) 501.
*Oasis HLB µelution Plate Generic Oasis HLB SPE Procedure for Polar Bases Using HILIC Condition/Equilibrate* 200 µl methanol/200 µl water Load 75 µl spiked plasma sample 75 µl internal standard with 2% ammonium hydroxide Wash 200 µl 5% methanol in water Elute 75 µl 40% acetonitrile/60% isopropanol with 2% formic acid Inject eluent directly onto column Eliminate Evaporation and Reconstitution Step
Generic Oasis HLB SPE: Direct Injection onto HILIC Column HO HO OH Albuterol H 3 C CH 3 NH CH 3 H 3 C NH Bamethan OH OH Atlantis TM HILIC Silica 2.1 x 50 mm, 3 µm 1. Bamethan 10 pg/µl 2. Albuterol 50 pg/µl 100 1.49 1.84 1 2 SIR of 2 Channels ES+ 239.8 209.9 8.97e4 % Polar species from matrix elute after analytes (normally seen in solvent front using RP) 0 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Time SPE eluent injected directly onto Atlantis TM HILIC Silica column Grumbach
Summary: When to Use HILIC Atlantis TM dc18 or XTerra Yes 2 Can it be retained by RP? Yes 1 Is the polar compound a base? Start Here 3 Sufficient Mass Spec sensitivity? No Yes No Atlantis TM HILIC Silica No 2 Can it be retained by RP? Yes Atlantis TM dc 18 Note: Atlantis TM dc 18 = High aqueous, Low ph XTerra = High aqueous, High ph Atlantis TM HILIC Silica = Low aqueous, Low ph No Alternate separation technique such as ion exchange, ion-pair, etc.
Summary Atlantis TM HILIC Silica columns offer: Retention of highly polar basic analytes Complementary selectivity to reversed-phase Excellent reproducibility Enhanced sensitivity in ESI-MS Shorter sample preparation procedures Elimination of the evaporation and reconstitution steps by directly injecting the final organic phase of the sample prep procedure
References 1. Alpert, A. J. J. Chromatogr. 499 (1990) 177-196. 2. Canals, I.; Oumada, F. Z.; Roses, M.; Bosch, E. J. Chromatogr. A. 911 (2001) 191-202. 3. Naidong, W.; Shou, W.; Chen, Y-L.; Jiang, X. J. Chromatogr. B. 754 (2001) 387-399. 4. Naidong, W.; Rapid Commun. Mass. Spectrom. 16 (2002), 1613-1621.
Acknowledgements Bonnie Alden Pamela Iraneta Paul Rainville Uwe Neue Doug McCabe Tom Walter Michael Savaria Susan Karn