A Brief Overview of HPLC & UHPLC Method Development and Optimization. Dr. Chris Message UHPLC/HPLC Product Specialist Phenomenex

Transcription

1 A Brief Overview of HPLC & UHPLC Method Development and Optimization Dr. Chris Message UHPLC/HPLC Product Specialist Phenomenex

2 Poll Question 1 What learning objectives most attracted you to this webinar? 1. Tips and tricks on how to develop NEW methods 2. Tips and tricks on how to optimize/improve EXISTING methods 3. How core-shell columns compare to fully porous columns 2 2

3 Outline I. Principles of RP Method Development II. HPLC Column Selection III. The Core-Shell Advantage IV. Optimizing Method Performance using Core-Shell Columns 3 3

4 HPLC/UHPLC Method Development 4

5 Method Development Method Development = changing stationary phase and mobile phase conditions to modulate and control these interactions and achieve a specific separation goal. Separate analyte molecules from each other Separate target molecules from matrix interferences Separation -> Accurate quantitation LC/MS -> Separate isomeric molecules and isobaric interferences; move analytes out of suppression zones 5 5

6 Resolution: The Goal of Chromatography Resolution is a measure of how well two peaks are separated from each other. It is calculated as the difference in retention time of two eluting peaks divided by the average width of the two peaks at the baseline. R (resolution) = RT B - RT A /.5 (width A + width B ) 6 6

7 Resolution: The Goal of Chromatography To understand the various factors that contribute to the ability of a method to resolve target analytes, we can use the equation below: Efficiency Capacity factor Selectivity 7 7

8 Column Efficiency (N) The amount of band (peak) broadening or dispersion that occurs in the column is measured by calculating the column efficiency (N) expressed as the number of theoretical plates in the column: tr Columns that cause a lot of peak broadening have few theoretical plates. Columns that produce very narrow peaks (little peak broadening) have a large number of theoretical plates. W 1/2 High N = Narrow Peaks = Better Rs 8 W Peak Width (W) 8

9 Capacity Factor (k ) The capacity factor of the eluting compound is its elution volume (time) relative to the elution volume (time) of an unretained compound. The k value for a given analytes will be determined by its relative affinity for the stationary phase and mobile phase. Capacity factor (k ) = t R t 0 t 0 t 0 9 9

10 Selectivity (α) Selectivity is a measure of the difference in the interactions of two compounds with the stationary phase. Selectivity is a function of both the stationary phase and the mobile phase. α = k 2 /k

11 Resolution: The Goal of Chromatography Efficiency Capacity factor Selectivity It is important to note that you (the analyst) have control over each of those factors through your choice of HPLC column and running conditions: 1. Efficiency (N) Particle size/morphology and column length 2. Selectivity (α) Stationary phase and mobile phase 3. Capacity factor (k ) Mobile phase strength (e.g. %AcCN) 11 11

12 The Effect of k, α and N on Resolution Most important determinant of resolution!! Constant increase in resolution Ineffective after k ~

13 Adjusting k, α, and N 1. Change stationary phase 2. Change mobile phase 1. Higher efficiency media (smaller or core-shell) 2. Longer columns 3. System optimization 1. Adjust mobile phase strength (%AcCN) 2. Adjust gradient rate 13 13

14 Outline I. Principles of RP Method Development II. Column Selection I. Media Supports II. Stationary Phases III. The Core-Shell Advantage 14 IV. Optimizing Method Performance using Core- Shell Columns 14

15 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 <

16 Organo-silica Particle Conventional Silica Particle Organo-silica Particle Siloxane Bridge Ethane linkage Dissolution at ph > 7.5 Stable to ph ~

17 Organo-silica 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) 17 17

18 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 silica particles ph range 18 18

19 Core-Shell Particle Advantages: Higher efficiency than fullyporous particles of same diameter Pressures compatible with conventional HPLC systems Potential for UHPLC performance on HPLC systems More resistant to UHPLC brand broadening from frictional heating Disadvantages: More sensitive to system extra-column volumes More sensitive to sample overload in some cases 19 19

20 HPLC Media Supports Fully-porous Silica Particle: General workhorse Many particle sizes and stationary phases Low sensitivity to system dead volume Core-Shell Particle: Better performance than UHPLC performance using conventional HPLC systems The new go-to choice for method development in place of fullyporous media Organo-silica Particle: Expanded ph stability range Same performance characteristics as fully-porous Optimal for high ph applications Monolithic Rod: Low back-pressure; reduced susceptibility to physical clogging Ideal for analysis of dirty samples (e.g. plasma) 20 20

21 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 21 21

22 RP Stationary Phase Classes Alkyl bonded phases (C18, C8, C4): Polar-embedded phases: Fusion Phenyl phases (Phenyl, PFP): Polar-endcapped phases: Hydro 22 22

23 Phenyl C18 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 mau min min 23 23

24 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 24 Testosterone Methyltestosterone 24

25 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 25 25

26 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 26 26

27 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 27 27

28 Poll Question 2 What factor is of utmost importance when developing or optimizing an LC method? 1. Separation / Resolution 2. Run time / Throughput 3. Cost 28 28

29 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 29 29

30 I. Principles of RP Method Development II. Column Selection III. The Core-Shell Advantage IV. Optimizing Method Performance using Core-Shell Columns 30 30

31 Kinetex Core-Shell Particle 0.35 µm Porous Shell 1.9 µm Inner Core Fully Porous Particle Kinetex 2.6 µm Core-Shell Particle 31 31

32 The Core-Shell Advantage Columns packed with core-shell particles will deliver significantly higher efficiency (N) than columns packed with fully-porous particles of the same diameter.* Fully Porous Particles w 1/2 32 Injected Sample Band * Gritti et al., Journal of Chromatography A, 1217 (2010) t R

33 The Core-Shell Advantage Columns packed with core-shell particles will deliver significantly higher efficiency (N) than columns packed with fully-porous particles of the same diameter.* Kinetex Core-Shell Particles w 1/ t R min 33 * Gritti et al., Journal of Chromatography A, 1217 (2010)

34 Efficiency( p/m) The Core-Shell Advantage Columns packed with core-shell particles will deliver significantly higher efficiency (N) than columns packed with fully-porous particles of the same diameter Fully Porous Core-Shell *Farcas et al., HPLC 2012 Conference / / Particle Diameter (µm) 34

35 The Kinetex Family Kinetex Particle 1.3 m 1.7 m 2.6 m 5 m System Compatibility UHPLC UHPLC UHPLC & HPLC HPLC & PREP LC Typical Efficiency (p/m) Fully Porous Efficiency (p/m) >400, , , ,000 N/A 280,000 ~180, ,

36 Kinetex for HPLC Methods Kinetex 5 µm A simple and effective way to improve existing 5 µm and 3 µm methods Efficiency greater or equal to a fully porous 3 µm media, but at the pressure of a 5 µm Typical Efficiency = 180,000 plates/m Typical operating pressure <250 Bar The only core-shell particle available for labscale purification 36 36

37 Kinetex 5 µm Improved resolution and sensitivity versus fully porous 5 µm: mau VWD1 A, Wavelength=295 nm (JL052412\JEFF \PAR D) VWD1 A, Wavelength=295 nm (JL052412\JEFF \PAR D) 20 Agilent ZORBAX 5 µm XDB-C x 4.6 mm 17.5 *Unoptimized HPLC (Agilent 1100) Height = 120 mau mau Rs = a b c d min VWD1 A, Wavelength=295 nm(jl052412\jeff \PAR D) VWD1 A, Wavelength=295 nm (JL052412\JEFF \PAR D) mau mau Kinetex 5 µm C x 4.6 mm Height = 165 mau Rs = Agilent and ZORBAX are registered trademarks of Agilent Technologies, Inc. Phenomenex is not affiliated with Agilent Technologies. Comparative separations may not be representative of all separations. min min

38 Kinetex 5 µm 3 µm performance at one-half the pressure: mau VWD1 A, Wavelength=210 nm (JL051612\JEFF \LCOL D) L-Column 3 µm ODS 150 x 4.6 mm Rs = 1.59 BP = 223 Bar min mau VWD1 A, Wavelength=210 nm (JL051612\JEFF \KNX D) Kinetex 5 µm C x 4.6 mm *Optimized HPLC (Agilent 1100) Rs = 1.57 BP = 120 Bar ~ 1 / 2 Pressure Agilent is a registered trademark of Agilent Technologies, Inc. Phenomenex is not affiliated with Agilent Technologies. Comparative separations may not be representative of all separations. 38 min

39 Improved performance and lifetime for LC/MS work: Heterocyclic amines from salmon by LC/MS/MS Kinetex 5 µm XIC of +MRM (13 pairs): / Da ID: Harman from Sample 19 (Sunfire 3.5um C18 50x2_amines) of DataSET1.wiff (Turbo... Max. 2.6e6 cps. XIC of +MRM (13 pairs): / Da ID: Harman from Sample 16 (Kinetex 5u C18 C18 50x2_amines) of DataSET1.wiff (Turbo... Max. 2.7e6 cps. 2.6e6 2.4e6 2.2e6 2.0e6 1.8e Waters SunFire 3.5 µm C18 50 x 2.1 mm 140 Bar 2.7e6 2.6e6 2.4e6 2.2e6 2.0e6 1.8e6 Kinetex 5 µm XB-C18 50 x 2.1 mm 72 Bar 2.98 In te n s ity, c p s 1.6e6 1.4e6 1.2e6 In te n s ity, c p s 1.6e6 1.4e6 1.2e6 1.0e6 1.0e6 8.0e5 8.0e5 6.0e5 6.0e5 4.0e5 4.0e5 2.0e5 2.0e Time, min XIC of +MRM (13 pairs): / Da ID: 7,8-/4,8-DiMeIQx from Sample 19 (Sunfire 3.5um C18 50x2_amines) of DataSET1.wiff... Max. 1.3e6 cps Time, min XIC of +MRM (13 pairs): / Da ID: 7,8-/4,8-DiMeIQx from Sample 16 (Kinetex 5u C18 C18 50x2_amines) of DataSET1.wif... Max. 1.7e6 cps. 1.3e6 1.3e6 1.2e6 1.1e6 1.0e6 9.0e e6 1.6e6 1.5e6 1.4e6 1.3e6 1.2e6 1.1e Better Resolution, 1 / 2 Pressure! In te n s ity, c p s 8.0e5 7.0e5 6.0e5 5.0e In te n s ity, c p s 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 4.0e5 3.0e5 2.0e5 4,8- & 7,8-DiMe-IQx e5 4.0e5 3.0e5 2.0e5 1.0e5 1.0e Time, min 39 SunFire is a trademark and Waters is a registered trademark of Waters Corporation. Phenomenex is not affiliated with Waters Corporation. Comparative separations may not be representative of all separations Time, min

40 Kinetex for HPLC Methods 40 40

41 Kinetex 2.6 µm UHPLC performance on a conventional LC system: mau VWD1 A, Wavelength=210 nm (JL051112\JEFF \KNX D) Kinetex 2.6 µm C x 4.6 mm *Optimized Agilent 1100 HPLC UPLC Performance on an HPLC System! min mau VWD1 A, Wavelength=210 nm (JL051112\JEFF \ZORB D) ZORBAX 1.8 µm C x 4.6 mm Agilent 1290 UHPLC Agilent and ZORBAX are registered trademarks of Agilent Technologies, Inc. Phenomenex is not affiliated with Agilent Technologies. Comparative separations may not be representative of all separations. 41 min

42 Kinetex 2.6 µm Improve productivity and resolution: DAD1 A, Sig=240,10 Ref=off (Z:\1\DATA\MT050211\DJGSK \MUPIROCIN D) mau µm Fully Porous C8 250 x 4.6 mm Rs 3 & 4 = 0.63 ~16 min min Column: Fully Porous 5 µm C8 250 x 4.6 mm Mobile phase: 70/ M Ammonium acetate ph 5.7/THF Flow rate: 1.0 ml/min 42 Comparative separations may not be representative of all separations. 42

43 Improve productivity and resolution: mau 250 VWD1 A, Wavelength=240 nm (JL050211\MUPI0006.D) Kinetex 2.6 µm C8 100 x 4.6 mm 7 5 Kinetex 2.6 µm Rs 3 & 4 = min min 43 Column: Kinetex 2.6 µm C8 100 x 4.6 mm Mobile phase: 80/ M Ammonium acetate ph 5.7:THF Flow rate: 1.5 ml/min *Optimized HPLC (Agilent 1100) Rs increased from 0.6 to 2.3 Run time decreased from 20 min to 8 min Agilent is a registered trademark of Agilent Technologies, Inc. Phenomenex is not affiliated with Agilent Technologies. Comparative separations may not be representative of all separations. 43

44 Kinetex for UHPLC Methods Kinetex 1.7 µm 44 44

45 Kinetex 1.7 µm Improve chromatography and decrease analysis time: XIC of +MRM (17 pairs): / Da ID: Lor-Gluc from Sample 13 (Sunfire 3.5u) of DataSET1.wiff (Turbo Spray) Max. 1.1e4 cps. In te n s ity, c p s 1.09e4 1.05e4 1.00e Time, min XIC of +MRM (17 pairs): / Da ID: Lor-Gluc from Sample 10 (Halo 50x2.1mm) of DataSET1.wiff (Turbo Spray) Max. 1.2e4 cps. 1.15e4 1.10e4 1.00e Fully-porous 3.5u 50x2.1mm Fused-core 2.7u 50x2.1mm In te n s ity, c p s Time, min XIC of +MRM (17 pairs): / Da ID: Lor-Gluc from Sample 9 (KNX 1.7u C18 30x2.1mm) of DataSET1.wiff (Turbo Spray) Max. 1.4e4 cps. 1.4e4 1.3e4 1.2e4 1.1e4 Kinetex 1.7u 30x2.1mm e In te n s ity, c p s Time, min Fused-Core is a registered trademark of Advanced Materials Technology, Inc. Phenomenex is not affiliated with Advanced Materials Technology. Comparative separations may not be representative of all separations.

46 Kinetex 1.7 µm Improved resolution versus Waters ACQUITY BEH 1.7 µm: Gritti and Guiochon, Chemical Engineering Science, Volume 65, Issue 23, 1 December 2010, Pages BEH Technology is a trademark and, ACQUITY and Waters are registered trademarks of Waters Corporation. Phenomenex is not affiliated with Waters Corporation. Comparative separations may not be representative of all separations. 46

47 Kinetex for UHPLC Methods 47 ACQUITY and UPLC are registered trademarks of Waters Corporation. Phenomenex is not affiliated with Waters Corporation. 47

48 Kinetex 1.3 m versus Waters ACQUITY 1.7 m BEH mau VWD1 A, Wavelength=214 nm (JL072512\JEFF \BSA D) N o 1 rm. ACQUITY 1.7 m BEH C18 Peak Capacity = 170 * Kinetex 1.3 µm (Agilent 1290) 50 * Kinetex 1.3 m BEH 1.7 m -20 mau min VWD1 A, Wavelength=214 nm (JL072512\JEFF \BSA D) Kinetex 1.3 m C18 * Peak Capacity = Agilent is a registered trademark of Agilent Technologies, Inc. BEH Technology is a trademark and, ACQUITY and Waters are registered trademarks of Waters Corporation. Phenomenex is not affiliated with Agilent Technologies or Waters Corporation. Comparative separations may not be representative of all separations. 48 min m in

49 Poll Question 3 What core-shell particle size seems the most applicable to your work and separation goals? µm µm µm 4. 5 µm 49 49

50 Kinetex Particle Selection What Type of LC System? HPLC UHPLC Kinetex 5 m Bar BP = 5 m 170,000 p/m Kinetex 2.6 m 270,000 p/m Kinetex 1.7 m >300,000 p/m Kinetex 1.3 m >400,000 p/m 50

51 Kinetex Particle Selection Kinetex 1.7 m Kinetex 5 µm Kinetex 2.6 m Kinetex 1.3 m 51 Shimadzu LC-20A Waters Alliance Agilent 1100 Agilent 1290 Waters ACQUITY UPLC All trademarks belong to their respective owners. Phenomenex is not affiliated any of these companies. 51

52 Method Development Tips for Kinetex Core-Shell Technology 52 52

53 Efficiency (Plates per meter) 1. System Optimization Hypersil GOLD 5 µm ODS 18.4% 3.9% Luna 3 µm C18(2) Kinetex 2.6 µm C % 33.8% ~50% Increase! Unoptimized Acquisition Rate Tubing Flow Cell UHPLC Agilent 1290 Agilent is a registered trademark of Agilent Technologies, Inc. Phenomenex is not affiliated with Agilent Technologies. Comparative separations may not be representative of all separations. Hypersi GOLDl is a registered trademark of Thermo Hypersil Keystone. Agilent is a registered trademark of Agilent Technologies, Inc. Phenomenex is not affiliated with Agilent Technologies or Thermo Hypersil Keystone. Comparative separations may not be representative of all separations. 53

54 Detector Acquisition Rate Increase acquisition rate to improve performance: Detector Rate = 1HZ Detector Rate = 10HZ 150 mau 70 mau 54 Comparative separations may not be representative of all separations. 54

55 2. Retention of Core-Shell A B 55 55

56 Retention of Core-Shell Isocratic 65:35 Acetonitrile:Water V W D 1 A, W avelength=254 nm (JL071112\JE FF \H YP D ) m A U 400 mau 300 VWD1 A, Wavelength=254 nm(jl071612\jeff \HYP D) tr = 6.5 min Kinetex 5 m C m in mau VWD1 A, Wavelength=254 nm (JL071112\JEFF \HYP D) Low surface area Low surface area Hypersil 5 5um ODS tr = 7.2 min tr = 10.1 min min 14 Symmetry and Waters are registered trademarks of Waters Corporation. Phenomenex is not affiliated with Waters Corporation. Comparative separations may not be representative of all separations. 56 mi

57 Retention of Core-Shell Isocratic 65:35 Acetonitrile:Water V W D 1 A, W avelength=254 nm (JL071112\JE FF \H YP D ) m A U 400 mau 300 VWD1 A, Wavelength=254 nm(jl071612\jeff \HYP D) tr = 6.5 min Kinetex 5 m C m in mau VWD1 A, Wavelength=254 nm (JL071112\JEFF \HYP D) Low surface area Low surface area Hypersil 5 5um ODS tr = 7.2 min tr = 10.1 min min 14 Symmetry and Waters are registered trademarks of Waters Corporation. Phenomenex is not affiliated with Waters Corporation. Comparative separations may not be representative of all separations. 57 mi

58 Retention of Core-Shell Gradient 10% 85% AcCN in 20min mau VWD1 A, Wavelength=254 nm (JL \JEFF \HYP D) Kinetex 5 m C tr = 17.8 min min mau VWD1 A, Wavelength=254 nm(jl071612\jeff \HYP D) Waters Symmetry 5 m C tr = 19.9 min Symmetry and Waters are registered trademarks of Waters Corporation. Phenomenex is not affiliated with Waters Corporation. Comparative separations may not be representative of all separations. 58 min

59 2. Retention of Core-Shell Gradient 10% 50% B 2.5 min; Fully porous 3 m C18 50x2.1mm Gradient 5% 10% 50% B B min; Kinetex 55u m C18 50x2.1mm Reduce starting %B to improve early-eluters 59 Comparative separations may not be representative of all separations. 59

60 3. Sample Solvent Strength Sample e4 in 50% Methanol Max. 2.4e5 cps. Sample e5 in 100% Methanol Max. 2.4e5 cps. 7.0e4 2.2e5 6.5e4 6.0e4 Hydromorphone 2.0e5 5.5e4 1.8e5 In te n s ity, c p s 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 1.6e5 Breakthrough! In te n s ity, c p s 1.4e5 1.2e5 1.0e e4 Fronting 2.0e4 1.5e4 1.0e Morphine Norhydrocodone Time, min 6.0e4 4.0e4 2.0e Time, min Strong solvents distort early-eluting peaks 60 Comparative separations may not be representative of all separations. 60

61 61 61 Agilent and ZORBAX are registered trademarks of Agilent Technologies, Inc. Phenomenex is not affiliated with Agilent Technologies. Comparative separations may not be representative of all separations. 3. Sample Solvent Strength System Suitability: Agilent ZORBAX 5 m C18 50 x 2.1 mm Kinetex 5 m C18 50 x 2.1 mm System suitability looks great!

62 3. Sample Solvent Strength Sample Injection Sample injection Is weird! Solvent = 90% Methanol Too Strong! System suitability Solvent = 50% Methanol 62 Comparative separations may not be representative of all separations. 62

63 4. Sample Loading 63 XBridge is a trademark of Waters Corporation. Phenomenex is not affiliated with Waters Corporation. Comparative separations may not be representative of all separations. 63

64 min 4. Sample Loading Reduce sample load for bases that exhibit tailing: mau DAD1 C, Sig=254,4 Ref=300,100 (DJ040611\DJGSK \GSK D) 12 g on column; USP Tailing = 1.87 pka mau DAD1 C, Sig=254,4 Ref=300,100 (DJ040611\DJGSK \GSK D) 5 g on column; USP Tailing = min mau DAD1 C, Sig=254,4 Ref=300,100 (DJ040611\DJGSK \GSK D) 2.5 g on column; USP Tailing = Comparative separations may not be representative of all separations min

65 Core-Shell Tips: 1. Optimize your system for maximum performance Detector acquisition rate System Dwell Volume Tubing, Injector Needle Seat, and Flow cell 2. Under isocratic conditions, core-shell columns will often give less retention than fully-porous columns Reduce the % organic if you need to regain lost retention In gradient mode, the lower retention of core-shell columns is less of an issue You can start your gradient at a lower % B if necessary 3. Be aware of strong solvent effects, particularly for early-eluting components Early-eluting components are more strongly affected than late-eluters More obvious on core-shell columns, which tend to have less retention than fullyporous media Sample solvent should be the same or weaker than initial mobile phase Be aware of sample overloading, particularly for basic analytes If you notice unacceptable peak tailing, reduce injection volume and re-evaluate 65

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