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1 Hydrophilic Interaction Chromatography (HILIC) Bill Champion Agilent Technologies, Inc opt 3/opt3/opt 2 Page 1

2 HILIC A method of recent attention Bill Champion Agilent Technologies, Inc opt 3/opt3/opt 2 Page 2

3 HILIC but not the universal answer. Bill Champion Agilent Technologies, Inc opt 3/opt3/opt 2 Page 3

4 Objective - Outline Overview - Why HILIC is used Advantages/Disadvantages g How HILIC works Examples Method development Comments concerning use of HILIC Page 4

5 Why HILIC Is Used Retention of polar components Similar to NPLC but with aq mobile phase (General reversal of elution order from RPLC) Several different stationary phases available MS compatible May simplify sample prep Page 5

6 but there is always a price Slower equilibration than RPLC Peak distortion with mp-sample solvent mismatch (i.e., too much water in sample solvent) Poor retention of anions on silica Mechanism not well understood Page 6

7 Additional Benefits, Comments ACN has low viscosity allows high flow rates Van Deemter plot similar to RPLC Not truly orthogonal to RPLC, but dissimilar not just inverse separation complementary technique Type B Silica, high purity, less acidic less active but more reproducible Page 7

8 Type of stationary phase Parameters to Choose Organic solvent concentration ti Type of buffer Buffer (salt) concentration ph Temperature 8

9 Typical Stationary Phases - Polar Silica Amine Diol Amide Zwitterionic Page 9

10 Some Typical Polar Stationary Phases (-) Silica, Si-OH Si-O H (+) Amine, -(CH 2 ) 3 -NH 2 Diol, -(CH 2 ) 3 -O-CH 2 -(CHOH)CH 2 OH Amide, -(CH 2 ) n -(CO)NH 2 (+) (-) Zwitterionic, -(CH 2 ) n -N(Me) 2 -(CH 2 ) n -SO 3 Page 10

11 Typical Mobile Phase Water /ACN Buffer ph Page 11

12 Typical Mobile Phase Water (at least 2-3%, ~ 25%)/CAN Solvent strength: Water > MeOH > EtOH > IPA > ACN Buffer AmOAc, 5to20mM Buffer must be soluble in high CAN concentration ph 3 8 Page 12

13 Mobile Phase Need some water for hydration Water is strong solvent Increasing water, decreases retention 13

14 Buffer Controls Ionization of Silica Controls Ionization of Analyte Ammonium acetate, formate good solubility, MS friendly Phosphate - poor solubility at high % ACN 14

15 ph Controls Ionization of Silica Controls Ionization of Analyte Organic solvent affects the actual [H + ] 15

16 Temperature Higher Temperature Decreases Ret n Time Higher Temperature Increases Column Efficiency (increases N) Lower Temperature May Increase Selectivity 16

17 Separation Mechanism Page 17

18 Retention in Reversed-Phase Separations: Mechanism Similar to Liquid-Liquid Extraction In RPLC decrease retention by decreasing polarity of mobile phase Heptane Non-Polar Heptane Non-Polar H 2 0 Polar H 2 0/MeOH Less Polar Solvent Polarity: H2O > MeOH, ACN > EtOH > IPA >> Heptane Relatively non-polar analyte (e.g., toluene) favors non-polar phase. Decreasing polarity of aqueous phase increases affinity for analyte in H 2 O/MeOH phase. Page 18

19 Retention in Reversed-Phase Separations: Mechanism Similar to Liquid-Liquid Extraction In RPLC decrease retention by decreasing polarity of mobile phase Heptane Non-Polar Heptane Non-Polar H 2 0 Polar H 2 0/MeOH Less Polar Mobile Phase Strength: H2O < MeOH, ACN < EtOH < IPA << Heptane Relatively non-polar analyte (e.g., toluene) favors non-polar phase. Decreasing polarity of aqueous phase increases affinity for analyte in H 2 O/MeOH phase. Page 19

20 RPLC Comparison of HILIC and RPLC Non-polar stationary phase (e.g., C18) Polar mobile phase (i.e., H 2 0/MeOH, H 2 0/ACN, etc.) Decrease retention by decreasing polarity of mobile phase (e.g., increase ACN in mobile phase to decrease retention) HILIC Polar stationary ti phase (e.g., silica) Polar mobile phase (i.e., H 2 0/ACN) Decrease retention by increasing polarity of mobile phase (i.e., increase H 2 O in mobile phase to decrease retention) Page 20

21 Typical Elution Order for Test Compounds on HILIC Column RPLC Column Test Mixture mau chloronitrobenzene Column 4.6 x 150 mm Mobile phase 75% ACN: 25% H2O Flow Rate 0.5 ml/ml Phenol Col Temp 25 C 30 Injection Volume 5 µl Naphthalene Uracil 1. Notice Uracil normally unretained in RPLC retained ed in HILIC min 2. Notice completely different retention order than in RPLC 21

22 How Does HILIC Work on Silica Based Columns? A water layer must be adsorbed onto the stationary phase. The polar analyte partitions into and out of this adsorbed layer. A charged polar analyte can also undergo ion exchange with the charged silica molecules (i.e., cation exchange with amines) The combination of these mechanisms drives retention in HILIC. Retention/elution is from least to most polar the opposite of reversed-phase LC. HILIC offers more retention than reversed-phase for very polar bases. 22

23 How Does HILIC Work on Silica Based Columns? Potential Mechanisms H 2 O ACN ACN ACN ACN 1. ACN ACN ACN CH 2 CHCH 3 ACN NH + 3 H 2 O CH 2 CHCH 3 H 2 O H 2 O H 2 O - H 2 O H 2 O H 2 O -NH + O - O 3 2. O O - H 2 O ACN ACN 1. Partitioning in and out of adsorbed water layer 2. Ion exchange with silanols 23

24 Examples Page 24

25 Some Typical Analytes for HILIC Amino acids (when only a few are of interest) Nucleobases (purines and pyrimidines, adenine, guanine, thymine, thymine, cytosine, uracil) Nucleosides (Adenosine, cytosine etc.) Alkaloids Carbohydrates Polar compounds, small basic compounds Cytosine NH 2 CH 2 CHCH 3 N N Melamine Amphetamine NH 3 + H 2 N N NH 2 25

26 Comparison of Reversed-Phase and HILIC Structures of Drug Compounds Studied a) Paroxetine Antidepressant MW F O N O O b) Ranitidine Antiulcerative MW N O S N N NO 2 Basic portion of molecule, impacts HILIC retention 26

27 Chromatographic Conditions RPLC Instrument: Column: Mobile phase: Gradient: Column temp: 40 C Sample volume: Flow rate: Agilent Series 1100 HPLC ZORBAX Eclipse XDB-C18 (2.1 mm 150 mm, 5 μm) A: 8-mM HCO 2 NH 4 in water; B: 8-mM HCO 2 NH 4 in 95% ACN/5% water 5% B to 90% B in 10 min 5 μl 0.3 ml/min HILIC Everything same as for RPLC except for column and gradient conditions: Column: Gradient: ZORBAX Rx-SIL (2.1 mm 150 mm, 5 μm) 100% B to 50% B in 10 min 27

28 LC/MS/MS Separation of Paroxetine & Ranitidine on ZORBAX Eclipse XDB-C18 Column (RPLC Mode) RPLC separation ZORBAX Eclipse XDB-C18 (2.1mm 150mm) 100 ppb 5% B 90% B (10 min) gradient Paroxetine Abundanc ce RPLC has poor peak shape and limited sensitivity for Ranitidine! N Ranitidine O S N N m/z= F N O m/z= NO 2 O O min

29 LC/MS/MS Separation of Paroxetine and Ranitidine on ZORBAX Rx-SIL Column (HILIC Mode)- 100 ppb Level HILIC separation Zorbax RX-SIL Silica column (2.1mm 150mm) 100 ppb 100% B 50% B (10 min) gradient Paroxetine m/z= HILIC Separation has better peak shape and more sensitivity for ranitidine! F O O O N Ranitidine m/z= O N S N N NO min

30 HILIC Separations are Ideal for LC/MS Analysis of Melamine HILIC Separation Using ZORBAX Rx-Sil NH 2 Melamine 50ppt Melamine N N H 2 N N NH 2 HPLC system : Agilent 1200 RRLC Column : Agilent ZORBAX Rx-Sil, mm, 5 um Injection Volume :10μL Temp : 40 Flow rate : 0.2 ml/min Mobile phase : A - 5 mm Ammonium acetate in Water : B - 5 mm Ammonium acetate in ACN Isocratic : 95%B 30

31 @ACN 1uL Effect of Injection Solvent & Volume on Melamine uL Don t inject in strong solvent!!!! 31

32 Mobile Phase Page 32

33 Effect of Increasing % ACN on Cyanuric Acid Retention 60% Decreasing strong solvent (increasing AACN) increases retention. 70% 80% Same behavior as reversed-phase but strong and weak solvents are opposite! 90% 95% 33

34 Retention Time Changes with Changes in Mobile Phase Composition me, min ention Tim Rete Mobile phase: A= 5 mm NH 4 OAc/H20 B= 5 mm NH 4 OAc/ACN 4 % Acetonitrile 34

35 Effect of Ammonium Acetate Concentration on Cyanuric Acid Retention Buffer strength can dramatically 5 mm affect retention and resolution and should be a key parameter in method development and optimization on HILIC columns. 10 mm 20 mm 35

36 16.0 Effect of ph, Buffer on Retention on Silica 14.0 Strong Acids 12.0 Ret'n Tim me Amines Neutral 2-nsa p-xsa phenol caffeine nortrip diphhyd procain Ref: McCalley, J. Chromatogr. A 1171 (2007) mM 1.52mM 153mM.. 0.1% 4 ph 3.5 ph 3.0 ph 3.0 TFA Page 36

37 16.0 Effect of ph, Buffer on Retention on Silica procainamide Ret'n Tim me Amines benzylamine diphenhydramine nortrip diphhyd procain A, B, C - HCOONH4/85% ACN; D 95% ACN Ref: McCalley, J. Chromatogr. A 1171 (2007) 46 0 A mm B mm C - 153mM.. D - 0.1% 4 ph 3.5 ph 3.0 ph 3.0 TFA Page 37

38 Effect of ph, Buffer on Retention on Silica p-xylenesulfonic acid A, B, C - HCOONH4/85% ACN; D 95% ACN 2-naphthalenesulfonic acid Strong Acids Ret'n Tim me caffeine phenol Neutral 2-nsa p-xsa phenol caffeine 0.0 Ref: McCalley, J. Chromatogr. A 1171 (2007) 46 0 A mm B mm C mm.. D -40.1% ph 3.5 ph 3.0 ph 3.0 TFA Page 38

39 Effect of Buffer, ph Controls Ionization of Silica Controls Ionization of Analyte Organic solvent affects the actual [H + ] Acids can be repulsed (reduces ret n time) by anionic silica Buffer can mask anionic sites (reduce ret n time of amines, increase for acids) 39

40 Effect of Temperature Higher temperature improves kinetics sharper peaks, shorter ret n times Lower temperature improves selectivity Affects buffer equilibrium (effective ph) Page 40

41 Columns Page 41

42 Comparison of Silica and Amino HILIC Columns HPLC system Agilent 1200 Column 1 Agilent Zorbax-NH2, mm, 5 um Column 2 Agilent Zorbax-Rx Sil, mm, 5 um Injection Volumn 2 ul Temp 25 Flow rate 0.4 ml/min Mobile phase A - 5 mm Ammonium acetate in Water Isocratic 95%B B - 5 mm Ammonium acetate in ACN 42

43 HILIC Separation Using Zorbax NH 2 5 mm Ammonium acetate in 5% H 2 O/ACN x MRM ( > ) C_M_-mrm-reversed-NH2-11.d Melamine Cyanuric Acid Counts (%) vs. Acquisition Time (min) 43

44 HILIC Separation Using Zorbax Rx-Sil 5 mm Ammonium acetate in 5% H 2 O/ACN x MRM ( > ) C_M_Si_01.d Cyanuric Acid Melamine Counts (%) vs. Acquisition Time (min) 44

45 HILIC Separation of Sugars Using Zorbax Rx-NH 2 Comparison 70/30 and 75/25 ACN/H 2 O p 2 glucose 45

46 Method Development Page 46

47 Method Development/Optimization Systematic Approach to Method Development Stationary phase Mobile Phase Buffer, Buffer concentration ph 47

48 Method Development/Optimization Systematic Approach to Method Development Separations are too complicated to expect to find optimum conditions using Random Walk Investigate t effects of buffer, ph, buffer concentration ti Recommend use of experimental design cf: B. Dejaegher, D. Mangelings, Y. Vander Heyden, J. Sep. Sci., 31 (2008)

49 Method Development/Optimization Starting Conditions Silica Ammonium formate, 5, 10, 20 mm ph 3.0, 3.5, 4.0 (5, 6?) ACN/H 2 O 97%, 95%, 90%, 85%, 75% TFA? 49

50 Typical Stationary Phases - Polar Silica Amine Diol Amide Zwitterionic Page 50

51 Elution Order Inversely Related to RPLC, BUT Not Opposite Mechanism, Retention ti is not exactly opposite of RPLC RPLC cannot be used as predictive model Not truly orthogonal, but dissimilar separation, complementary technique Acids often poorly retained Page 51

52 Summary - Advantages of HILIC Retention of polar compounds Good peak shape for basic compounds where RPC may give tailing and/or low efficiency Higher flow rates and/or long columns can be used due to low viscosity mobile phases with high organic content; greater efficiency Enhanced detection sensitivity with MS Efficient spraying and de-solvation in electrospray MS As much as 3X sensitivity Can directly inject extracts from C18 SPE cartridges or acetonitrile precipitated plasma supernatant Dissimilar to RPLC (2D separations); elution order reversal, Complementary separations 52

53 References A.J. Alpert, J. Chromatogr. 499 (1990) 177 BA B.A. Bidlingmeyer, et. al., Anal. Chem. 54 (1982) 442 D.V. McCalley, J. Chromatogr. A 1171 (2007) 46 D.V. McCalley, J. Chromatogr. A 1217 (2010) 858 B. Dejaegher, D. Mangelings, Y. Vander Heyden, J. Sep. Sci., 31 (2008) 1438 B. A. Olsen, J. Chromatogr. A 913 (2001) 113 R.Li, Junxiong Huang, J. Chromatogr. A 1041 (2004) 163 T. Langrock, P. Czihal, R. Hoffmann, Amino Acids 30 (2006) 291 Page 53

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