Tips and Tricks of Faster LC Analysis Without Capital Investment It may be easier than you think! Agilent Technologies, Inc. Fall 8 Page There Are Many Reasons to Reduce Analysis Time Run More Samples/Day Reduce Mobile Phase Use Reduce Mobile Phase Waste Disposal Better Utilize Existing Resources in Instruments and Personnel Implement HB * Work Schedule (* HB Home By, Ray Lombardy, Agilent Technologies, Inc. 7) At What Cost and Difficulty? That Depends on What You Want to Accomplish! Page
How Fast Do You Want To Go? Your Answer Will Detere the Cost of Increased Speed to X increase in Speed Easy - X Increase May Require Instrument Tweak X + Increase Will Require Instrument Upgrade and Tweak What Are the Paths to LC Method Speed Gains? Most Speed From Column and Particle Size Optimization Instrument Optimization Will Enable or Boost Those Gains The Following Discussion is Designed to Simplify the Process - Often at No Cost! Page How Do Small Particles Help Speed Up Methods? Increase Kinetics of the separation Increase Efficiency (N) in Same Length Column Allow Shorter Column to Match Efficiency of Longer Column Provide Expanded Flow Rate Range Without Loss of Efficiency Page
HETP (cm/plate) Trick: Use Smaller Particles For More Efficient Separations Across a Wide Flow Rate Range..... Small Particle Columns including Monolith ZORBAX.μm ZORBAX.μm Vendor A.μm Vendor B.μm ZORBAX.8μm Monolith Dimensions:.6 x //mm Eluent: 8: ACN:Water Flow Rates:.. ml/ Temp: C Sample:.μL Octanophenone in Eluent............. Volumetric Flow Rate (ml/) Page ISOCRATIC ELUTION Page 6
Tip: Short Column/Smaller Particle Maintains Rs Trick: Tailor Column Dimensions and Particle Size to Analysis Needs Column Length (mm) 7 Resolving Power N( µm), 8, 6, N.A. N.A. Resolving Power N(. µm),,, 7,,, Resolving Power N(.8 µm),, 7,, 6,, Typical Pressure Bar (.8 µm) > > 6 Analysis Time Peak Volume Solvent Usage Analysis Time* -% -% -67% -8% -9% * Reduction in analysis time compared to mm column pressure detered with 6: MeOH/water, ml/,.6mm ID Page 7 Goal: Run Current Method X Faster Trick: ½ Column Length and Smaller Particle = ½ Time StableBond SB-C8.6 x mm, μm Mobile Phase: % mm NaH PO, ph. % MeOH Flow Rate:. ml/. Temperature: C Detection: UV nm Sample: Cardiac Drugs. Diltiazem. Dipyridamole Analysis Time:. Nifedipine.7. Lidoflazine. Flunarizine Rapid Resolution StableBond SB-C8.6 x 7 mm,. μm Analysis Time:. 6 7 8 9 Time () 6 Time () Page 8
Goal: Run Current Method X Faster Trick: / Column Length and Smaller Particle = / Time Original method ZORBAX LC column Extend-C8.6 x mm, μm μl inj. 6 6 8 8 6 6 x faster ZORBAX RRHT column Extend C8.6 x mm,.8 μm μl inj. Mobile phase: (7:) MeOH: mm pyrrolidine buffer Flow =. ml/, Temp. : ambient - 6 8 Page 9 How Long Can the Column Be with MeOH? System Pressure With MeOH/Water.6 x mm Column with.8um particles at ml/, o C pressure Pressure curve 6 8 percentage of MeOH Use P of.6 x mm as baseline Multiply P by L col / L col If P is Lower than Max P of Instrument you can run that Length Percentage of Pressure (bar) MeOH (%) 69 9 9 6 7 6 8 8 9 98 Page
. How Long Can the Column Be with ACN? System Pressure with ACN/Water.6 x mm Column with.8um particles at ml/, o C Pressure System pressure with the change of percentage of ACN Percentage of ACN Use P of.6 x mm as baseline Multiply P by Lcol / Lcol If P is Lower than Max P of Instrument you can run that Length Percentage of Pressure (bar) ACN (%) 9 6 6 7 8 88 9 7 9 Page More Resolution in Original Method than Needed Tip:.8u, Very Short Column, Faster Flow -X Faster Isocratic on Instrument with bar Pressure Limit Rs(,) =.8 N=88.6 x mm, μm 9.6 N=68 Rs(,) =..7.6 x mm,. μm N=69 N=668. Rs(,) =. N=6.6 x mm,.8 μm ml/ N=66.9 N=66 Rs(,) =.....6 x mm,.8 μm ml/ Columns: ZORBAX SB-C8 Mobile Phase: % mm NaH PO, ph.8: % ACN Flow Rate: ml/ Temperature: RT Detection: UV nm Sample:. Estradiol. Ethynylestradiol. Dienestrol. Norethindrone Page 6
Tip: Constant Linear Velocity Maintains Efficiency Trick: Reduce Flow Rate When Reducing Column Diameter 7 Mobile Phase: % methanol in.% Formic Acid 7 ZORBAX SB-C8,.6 x mm, μm, ml/ Solvent Used: ml 6 7 ZORBAX SB-C8,. x mm,. μm,. ml/ Solvent Used:.7 ml, decrease of 8% (decrease in analysis time of 7%) 7 Page Quick Reference for Changing to Common Column Diameters Maintains Equivalent Linear Velocity for Different Column ID s: Column Type Analytical Solvent Saver NarrowBore MicroBore Capillary Capillary Nano Nano Column ID.6 mm. mm. mm. mm. mm. mm. mm.7 mm Flow Rate. ml/. ml/. ml/ 7 μl/ μl/. μl/ 7 nl/ 66 nl/ Flow rate column = (diameter column ) /(diameter column ) x Flow rate column Maintain equivalent mobile phase linear velocity when changing column diameter. Page 7
Isocratic Tips and Tricks Summary Increased Isocratic Speed Does Not Necessarily Require Sub-Two Micron Particles Particle Diameter and Column Length are Major Factors in Resolving Power Chose New, Shorter Column to Approximate Original Column Efficiency - Chose Length Based on Time Savings Goal Example X: mm, µm (~,N) ~ mm,.8µm (~,N) Usually Only Need to Reduce Col. Length and Particle Size Not Necessary to Change Flow, Injection Vol. or Organic Content of M.P. Altered Column Diameter Requires Change in Flow and Injection Volume Page GRADIENT ELUTION Page 6 8
Reducing Particle Size Improves Kinetics Reduces Peak Width, Increases Height- It Does NOT Change the Gradient! 6.8um vs. um + 6% Resolution (Theory: 67%) + 6% Efficiency (Theory: 77%) 8 6 Pressure at start: bar, µm Rs(,) =. Peak Width σ (9) =.7 Pressure at start: 9bar,.8µm Rs(,) =.7 Peak Width σ (9) =.7 6 8 Chromatograms Offset for Better View Page 7 Resolution Equation for Gradient Elution Relationship of k* to Gradient Time, Flow and Column Dimensions R V N α k* k* t g F S (Δ%B) V m Δ%B = difference between initial and final % B S = constant ( for - Da) F = flow rate (ml/.) t g = gradient time (.) V m = column void volume (ml) Zorbax Vm= π x (Col Internal Radius) x Length x.6 Page 8 9
Expected: Use of a Longer Gradient Time Increases Gradient Retention, Gradient Time. Gradient Time 6. 6,7 6 7 Column: ZORBAX SB-C8.6 x mm, μm Gradient: 6% B Mobile Phase: A:HO with.%tfa, ph B: Acetonitrile Flow Rate:. ml/ Temperature: C Sample: Herbicides 8 8 Time () Time () Increased gradient retention improves resolution of several peak pairs, and,. Page 9 Tip: A Shorter Column (smaller Vm) Also Increases Gradient Retention and Rs Increases Gradient Retention, Increases Overall Resolution,.6 x mm, μm.6 x 7 mm,. μm 7 8 Column: ZORBAX SB-C8 Gradient: 6% B Mobile Phase: A:HO with.%tfa, ph B: Acetonitrile Flow Rate:. ml/ Temperature: C Sample: Herbicides 6 6 7 8 Time () Time () Page
Tip: Improve Resolution By Using Short Column Length ( Vm ) for BioMolecules SB-C8,.6 x mm, µm SB-C8,.6 x mm,.µm 6 7 8 6 7 8 9 9 6 8 6 6 8 6 Mobile Phase: Flow Rate: Temperature: A: 9% Water : % ACN,.% TFA Sample:. Gly-Tyr 6. Leu-Enk B: % Water : 9% ACN,.8% TFA. Val-Tyr-Val 7. Angiotensin II Gradient: -6% B in.. [Gln ] Amyloid-β- 8. Kinetensin. ml /. Protein Fragm -6 9. RNase Ambient. (TYR8) Bradykinin. Insulin (Eq.). Met-Enk 997P.PPT Page Tip: Maintain k* To Keep Relative Peak Position in a Chromatogram Unchanged While Reducing Time Any Decrease in Vm-Column length Can be Offset by a Proportional Decrease in t G or F Vm-Column (i.d.) Decrease in t G or F k* t G F S Δ%B Vm 89S.PPT Page
Trick: Change V m and t G by Same Proportion Two Chromatograms Both Having the Same Gradient Steepness Sample:. Tebuthiuron. Prometon. Prometryne. Atrazine. Bentazon 6. Propazine 7. Propanil 8. Metolachlor Column: StableBond SB-C8.6 x mm, μm Gradient Time:. Flow Rate:. ml/ 8 Column: Rapid Resolution StableBond SB-C8.6 x 7 mm,. μm Gradient Time:. Flow Rate:. ml/ 8 Analysis Time: 6 6 Analysis Time: 7 7 Time () Time () 78S.PPT Page Very Fast LC on Conventional HPLC G79 Degasser G Quaternary pump GA ALS autosampler G6A column compartment The Instrument GA VWD (standard cell G-686, mm, ul) Acetophenone Diethyl phthalate Benzophenone Butyrophenone Valerophenone Hexanophenone Heptanophenone Octanophenone The Sample Page
Conventional Column -.6 x mm, µm, SB-C8 VWD A, Wavelength=6 nm (D:\SAMPLE TEST\RRHT-\789SBC8.D)..6 8.7 Flow Rate. ml/ Injection Volume ul Temperature C Wavelength 6nm Sample rate. Hz 6. Time () % Acetonitrile.7.6 6.96 6 8 Initial Pressure: 69 bar Final Pressure: 8 bar 9.69.98..6 9 9 Page Tip: Shorten Column and Gradient Time by Same Factor / Column Length- / Gradient Time RRHT Column.6 x mm,.8µm, SB-C8 VWD A, Wavelength=6 nm (D:\SAMPLE TEST\RRHT-\HDS 7-8-9 7--\789SBC89.D).76..6.7 Flow Rate. ml/ Injection Volume ul Temperature C Wavelength 6nm Sample rate.7 Hz 8 6..8.87. Time () % Acetonitrile 6 Initial Pressure: bar Final Pressure: 7 bar... 9 9 Page 6
The Comparison of chromatogram with the same RRHT column in and SL Overlay the two Chromatograms Norm. VWD A, Wavelength=6 nm (D:\SAMPLE TEST\RRHT-\HDS 7-8-9 7--\789SBC88.D) *DAD A, Sig=6, Ref=6, (WORKSHOP-TAIWAN\78SBC8.D).79.7.6.68 8 6.7.86.87..... The red one is, the blue one is. Page 7 Tip: Increase Column Flow-Reduce Gradient Time Double Flow (ml/) ½ Gradient Time RRHT.6 x mm,.8µm, SB-C8 8 VWD A, Wavelength=6 nm (D:\SAMPLE TEST\RRHT-\HDS 7-8- 8--6\78SB.87.6.6.7 Flow Rate. ml/ Injection Volume ul Temperature C Wavelength 6nm Sample rate.7 Hz 6..7... Initial Pressure: 66 bar Final Pressure: 6 bar.97. Time ().67..7. % Acetonitrile 9 9 Page 8
Gradient Tips and Tricks Summary k* t g F S (Δ%B) V m Gradient Retention Relationship Formula Is the Key to - Improving Resolution Longer Gradient Time (t g ) Smaller Column Volume (Vm) Length or Inner Diameter Keeping All other Parameters Constant Increasing Speed Without Losing Resolution Shorter Column with Proportionally Shorter Gradient Time Shorter Gradient Time with Proportionally Faster Flow Rate Page 9 Tip: Method Translator Makes Changes Easy Trick: Let Agilent Method Translator Do the Math Basic Mode for Easy Transfer of Conventional Method to RRLC Injection Volume Conversion Detector Settings recommendation Gradient and Isocratic Method Conversion (autodetected) Page
Agilent Method Translator Advanced Mode More Detaled Information, But Still Easy to Use Detailed Input Detailed Output Original Method New Method Page Does it work? - Example Analysis of impurities of an active pharmaceutical ingredient by conventional HPLC (.6mm ID x mm,. µm): HC N CH H OH OCH Main Compound HC N CH H HC N CH H OH OH OCH Impurity A H C CH N H HC N CH Br OH Impurity D OCH OCH O CH Impurity C Impurity B Bromanisole. 7.. 7. Page 6
Converting to a.6 mm ID x mm,.8µm column: Page Does it work? Yes! Conventional HPLC 8 6. 7.. 7. Resolution optimized.8µm - 6 7 8 Added Benefit of Increased Sensitivity! Page 7
Does it work? Conventional HPLC... 7... 6. 7 7. 8..6 mmid x mm,.8µm.µm Zorbax SB C8. % B.. 8. 9% B..98 9. 9% B..99 9. % B.. 6. % B Speed Optimized Simple Conversion Page ZORBAX Selectivity Choices Options for Improved Speed and Resolution Reversed-Phase Chromatography Specialty products StableBond Diisopropyl Diisobutyl bonding Eclipse XDB Dimethyl bonding Double endcapping Eclipse Plus Dimethyl bonding Double endcapping Extend-C8 Bidentate-C8 Double endcapping SB-C8 Diisobutyl-C8 Rx/SB-C8 Diisopropyl-C8 SB-Phenyl Diisopropyl- Phenyl SB-CN Diisopropyl-CN SB-C Triisopropyl SB-AQ Diisopropyl Eclipse XDB-C8 Eclipse XDB-C8 Eclipse XDB- Phenyl Eclipse XDB-CN Eclipse Plus C8 Eclipse Plus C8 Eclipse Plus- Phenyl-Hexyl Eclipse PAH Rx-C8 Dimethyloctadecylsilane Bonus-RP Embedded amide amide Diisopropyl-C Triple Triple endcapped Page 6 8
Tip: Bonded Phase Can Change Selectivity Improve Peak Shape and Shorten HPLC Assay Time Each ZORBAX phase available in matching.8µm,.µm and µm choices, most in 7µm! Over RRHT,.8um 6 bar column choices available Different Column Chemistries bonded phases and silica for HILIC use 7 column lengths (*,,, 7,, and mm long) *.8µm as custom column internal diameters (.6,.,. mm and Prep ID) Page 7 Tip: C8 Often Yields Same Peak Order in < Time Than C8 Trick: Evaluate Different Phases with Same M.P. RRHT Eclipse Plus C8,.6 x mm,.8 um. Oxybenzone. Internal Std.. Octylmethoxycinnamate 7 bar N: 88 Pw:.. TF:. N: 6 Pw:.7. TF:.6 N: 8 Pw:.9. TF:. ) Rapid Resolution Eclipse Plus C8,.6 x mm,. um N: N: 66 Pw:.9. Pw:.7. TF:.9 TF:.99 N: 6 Pw:.6. TF:.98.. 88 bar Conditions: Mobile Phase.: Water: Acetonitrile (:7) Flow Rate:. ml/. Detection: UV nm Temperature: C Sample: Lip balm extract in ACN (melted at C ACN, cooled and. um filtered) Less retention can save significant time the C8 is a good choice here. The RRHT column is delivering the efficiency and resolution expected, but the C8 bonded phase may be the best choice. Page 8 Group/Presentation Title Agilent Restricted Month ##, X 9
Tip: Polar Phases Can Save Analysis Time Trick: Evaluate Non-Polar and Polar Phases with Same M.P. Eclipse Plus Phenyl Hexyl 6 8 Elution order reversal between Phenyl-Hexyl and Alkyl chains Eclipse Plus C8 6 8 Eclipse Plus C8 6 8 Mobile Phase % ACN 6 % mm Sodium Phosphate Buffer ph. Flow Rate:. ml/.6 x mm UV nm µl Elution order for Eclipse Plus Phenyl Hexyl: () Piroxicam, () Sulindac,() Tolmetin, () Naproxen, () Ibuprofen, (6) Diclofenac, (7) Celebrex (equal portions of approximately mg/ml solutions Page 9 Group/Presentation Title Agilent Restricted Month ##, X Tip: Higher Temperature Can Improve Speed and Resolution Higher Temperature should always be considered as a parameter during speed optimization Provides more rapid mass transfer: Improves Efficiency enhances resolution Decreases analysis time faster separations with no loss in resolution Decreases Mobile Phase Viscosity Lowers backpressure allows for higher flow rates, faster separations, greater efficiency Can change selectivity optimize resolution Not Necessary to Go to the Limit-Midrange Offers Benefits Page
Tip: Temperature Changes Can Alter Selectivity Trick: Optimize Col Temp For Best Speed/Rs Salicylic acid Coelution Salicylic acid C C Salicylic acid C Salicylic acid Column: RRHT SB-C8.6 x mm,.8um 6 C Salicylic acid Increased T Decreases Run Time, Changes Selectivity and Sharpens Peaks Improving Resolution 9 C Page Tips on When You Should Move to the Higher Pressure and Performance SL (RRLC) System Very Fast Methods with High Velocity Mobile Phase That Increase Backpressure Above bar Resolution of Difficult Samples Requires Long Columns With.8µm Particles (>bar Backpressure) Labs Required to Run Both Traditional and New, Higher Performance LC Methods Need Faster Data Acquisition for Narrow, High Efficiency Peaks Generated in Higher Speed Methods Page
Tip:.8µm Particle Increases Resolution (How?) Trick: Substitute.8µm Particles in Same Length Column Agilent.6 6 7 8 Agilent RRLC.787.97.9..79.7.79..6..967 Zorbax.xmm SB C-8, µm Zorbax.xmm SB C-8,.8µm 6.76 7.6 Conditions for both experiments Pumps Solvent A: H O +.% TFA Solvent B: ACN +.% TFA Gradient: % to 9% ACN in, hold for Flow Rate:.ml/ Autosamplers Injection volume: µl Thermostatted Column Comp. Temperature: C Detectors DAD µl cell and Hz, nm, Ref: Appl. Note 989-6 by Edgar Naegele 6 7 8 Page... -. - Norm.... -. Tip: Small Particles Improve Detection of Low Level Impurities DAD A, Sig=, Ref =of f (9A\SIG.D) DAD A, Sig=, Ref=off (D\LC_J.D) -.6 x, um 9 bar N = 79 R_S =. S/N = Height =. Noise = uau.6 x,.um 6 bar N = 86 R_S =.7 S/N = Height =. Noise = uau Column:x.6 mm µm Pressure: 9 bar N: 8 Height:. S/N:. R s =. t r =.9 ( st epimer) N ptp :. - Column:x.6 mm.µm Pressure: 6 bar N: 86 Height:, S/N:.7 R s =.7 t r =. ( st epimer) N ptp : - N Norm.... -. - N d p.....6 /dp DAD A, Sig=, Ref=off (7D\LC_X.D).6 x Column:,.8um x.6 mm.8µm Pressure: 9 bar 9 bar N: 8669 N = 8669 Height:.78 R_S S/N: =.6.8 (+7%) S/N R= s =.8 t r = 7. ( st epimer) Height =.8 N ptp : Noise = uau - Page
Particles Reveal More Information and Improve Detection and Integration Customer Sample, Translation of Isocratic Impurity Methods, Zoom Critical Time Range (t = 7) Impurities Not Baseline Separated! 7 Impurities 6 Not Baseline Separated! 7 Impurities All 7 Baseline Separated!.6 x, μm 9 bar N = 79 R_S =. S/N =.6 x,.μm 6 bar N = 86 R_S =.7 S/N =.6 x,.8μm 9 bar N = 8669 R_S =.8 (+7%) S/N = Up to 6% higher resolution without loss in sensitivity Page Example of What Is Possible! Shorter Column, Smaller Particle, > Flow Rate, High Temp F=.ml/ T = C Analysis Time = Solvent Cons. =.ml F =.8ml/ T = C Analysis Time =. Solvent Cons. =.ml F=.ml/ T = C Analysis Time =. Solvent Cons. =.ml F=.ml/ T = 9 C Analysis Time:. Solvent Cons. =.ml 6 8...6.8...6.8 High Resolution:.6mm x mm.µm High Speed:.6mm x mm.µm Easy! Easy! Easy! High Speed & Resolution:.mm x mm.8µm Easy, But You Will Need > bar! PW HH = 97msec Max Speed at T = 9 o C > x faster!.mm x mm.8um...6.8 Page 6
Tip: Higher Data Improves Response and Rs Trick: Optimize Data Rate for Accurate Profile PW=.sec PW=.sec PW=.sec 8Hz Hz 8Hz versus Hz Data Rate Peak Width: % Resolution: + 9% Peak Capacity: + % App. Column Eff.: + 6% Data Rate 8 Hz Hz Peak Width..9 Resolution.. Peak Capacity 6 Hz PW=.67sec Hz PW=.sec Hz..... Hz Hz Hz Sample: Column: Gradient:: Flow Rate:.6.666.6.7.7.67 9 6 Phenones Test Mix Zorbax SB-C8,.6x,.8um -%ACN in. ml/ Page 7 Moving From Conventional LC to RRLC: Moving to RRLC is simple and easy! Doesn t this cost a fortune? Same software (e.g. ChemStation, EZChrom) No additional training effort Limit capital investment by modular step-wise upgrade Leverage existing modules, existing method and stationary phase Configurable delay volume in SL pump Easy to also run legacy LC methods Simple LC to RRLC method translator No tedious method development No - not at all! Page 8
Stepwise Scale-up to Rapid Resolution LC From to RRLC in two steps - Add What You Need When You Need It! ALS TCC VWD/MWD/DAD Actual: Quat System Degasser Quat Pump h-als SL TCC VWD/MWD/DAD Step : / Bin SL System µ-degasser Bin Pump SL + Resolution + Speed + MS-Compatibility + Solvent Saving + Compatible with conv. HPLC Step : Rapid Resolution System u-degasser Bin Pump SL h-als SL TCC SL DAD SL + Speed + Resolution + Sensitivity + MS-Robustness + Data Security & Traceability + Qualification (Degasser, ACE) + Compatible with conv. HPLC Analysis Time Cycle times Peak Width N Column ID Column Length Flow rates Temperature Pressure > > 6 > sec -,.6 mm mm. - ml/ 8 C bar >. > >. sec -,. -.6 mm - mm. - ml/ 8 C 6 bar >. >. >. sec - 6,. -.6 mm - mm. ml/ C 6bar Page 9 SUMMARY Increasing Speed of HPLC Separations Need Not be Difficult or Costly Changes in Columns and Equipment Depend on Your Goals Majority of Time Saved Will Be Based on Column and Particle Size Isocratic and Gradient X, X and X Times Savings are Easy Greater Time Savings May Require Instrument Modifications -X Increase in Speed May Require Instrument Upgrade Long Columns with.8µm Particles May Require > bar Pump and Autosampler Capability Page