Tips and Tricks of Faster LC Analysis Without Capital Investment It may be easier than you think!

Tips and Tricks of Faster LC Analysis Without Capital Investment It may be easier than you think! Agilent Technologies, Inc. Fall 28 Page 1

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 HB5 * Work Schedule (* HB5 Home By 5, Ray Lombardi, Agilent Technologies, Inc. 27) At What Cost and Difficulty? That Depends on What You Want to Accomplish! Page 2

How Fast Do You Want To Go? Your Answer Will Detere the Cost of Increased Speed 2 to 3 X increase in Speed Easy 5-1 X Increase May Require Instrument Tweak 1 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 3

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 4

HETP (cm/plate) Trick: Use Smaller Particles For More Efficient Separations Across a Wide Flow Rate Range.3.25.2.15.1 Small Particle Columns including Monolith ZORBAX 5.μm ZORBAX 3.5μm Vendor A 2.5μm Vendor B 2.μm ZORBAX 1.8μm Monolith Dimensions: 4.6 x 5/3/2mm Eluent: 85:15 ACN:Water Flow Rates:.5 5. ml/ Temp: 2 C Sample: 1.μL Octanophenone in Eluent.5...5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. Volumetric Flow Rate (ml/) Page 5

Page 6 ISOCRATIC ELUTION

Tip: Short Column/Smaller Particle Maintains Rs Trick: Tailor Column Dimensions and Particle Size to Analysis Needs Column Length (mm) Resolving Power N(5 µm) Resolving Power N(3.5 µm) Resolving Power N(1.8 µm) Typical Pressure Bar (1.8 µm) Analysis Time* 15 1 12,5 8,5 21, 14, 32,5 24, >4 >4 Analysis Time -33% 75 5 6 4,2 1,5 7, 17, 12, 32 21 Peak Volume -5% -67% 3 15 N.A. N.A. 4,2 2,1 6,5 2,5 126 55 Solvent Usage -8% -9% * Reduction in analysis time compared to 15 mm column pressure detered with 6:4 MeOH/water, 1ml/, 4.6mm ID Page 7

Goal: Run Current Method 2X Faster Trick: ½ Column Length and Smaller Particle = ½ Time StableBond SB-C18 4.6 x 15 mm, 5 μm 1 2 3 Mobile Phase: 45% 25 mm NaH 2 PO 4, ph 3. 55% MeOH Flow Rate: 2. ml/. Temperature: 35 C Detection: UV 254 nm Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine Analysis Time: 11.7 4 5 Rapid Resolution StableBond SB-C18 4.6 x 75 mm, 3.5 μm 1 2 Analysis Time: 5.2 3 4 5 1 2 3 4 5 6 7 8 9 1 11 12 13 14 Time () 1 2 3 4 5 6 Time () Page 8

Goal: Run Current Method 3X Faster Trick: 1/3 Column Length and Smaller Particle = 1/3 Time mau 25 2 15 1 1 2 3 4 5 Original method ZORBAX LC column Extend-C18 4.6 x 15 mm, 5 μm 3 μl inj. 6 5 2 4 6 8 1 mau 12 1 8 6 1 2 3 4 5 6 3x faster ZORBAX RRHT column Extend C18 4.6 x 5 mm, 1.8 μm 1 μl inj. 4 2 Mobile phase: (7:3) MeOH: 5 mm pyrrolidine buffer Flow = 1. ml/, Temp. : ambient -2 2 4 6 8 1 Page 9

How Long Can the Column Be with MeOH? System Pressure With MeOH/Water 4.6 x 5mm Column with 1.8um particles at 1 ml/,3 o C Pr essur e cur ve Percentage of MeOH (%) Pressure (bar) pr essur e 25 2 15 1 5 2 4 6 8 1 per cent age of MeOH 142 1 169 2 23 3 229 4 249 5 252 6 24 Use P of 4.6 x 5mm as baseline Multiply P by L col2 / L col1 If P is Lower than Max P of Instrument you can run that Length 7 216 8 184 9 145 1 98 Page 1

How Long Can the Column Be with ACN? System Pressure with ACN/Water 4.6 x 5mm Column with 1.8um particles at 1 ml/,3 o C Syst em pr essur e wi t h t he change of per cent age of ACN Percentage of ACN (%) Pressure (bar) Pr essur e 15 1 5 5 1 Per cent age of ACN 142 1 154 2 159 3 156 4 15 5 135 Use P of 4.6 x 5mm as baseline Multiply P by Lcol2 / Lcol1 If P is Lower than Max P of Instrument you can run that Length 6 12 7 14 8 88 9 7 1 59 Page 11

More Resolution in Original Method than Needed Tip: 1.8u, Very Short Column, Faster Flow 5-1X Faster Isocratic on Instrument with 4bar Pressure Limit Rs(1,2) = 4.8 1 2 3 N=21848 4.6 x 25 mm, 5 μm 29.65 4 N=2268 1 5 1 15 2 25 3 2 3 Rs(1,2) = 3.5 4.6 x 1 mm, 3.5 μm 12.71 4 N=11691. 1 5 1 15 2 25 3 2 3 N=6568 4.15 4 Rs(1,2) = 3.3 N=614 4.6 x 3 mm, 1.8 μm 1 ml/ 5 1 15 2 25 3 N=6463 2.9 N=646 Rs(1,2) = 3.1 1 2 3 4.5 1 1.5 2 2.5 4.6 x 3 mm, 1.8 μm 2 ml/ 5 1 15 2 25 3 Columns: ZORBAX SB-C18 Mobile Phase: 5% 2 mm NaH 2 PO 4, ph 2.8: 5% ACN Flow Rate: 1 ml/ Temperature: RT Detection: UV 23 nm Sample: 1. Estradiol 2. Ethynylestradiol 3. Dienestrol 4. Norethindrone Page 12

Tip: Constant Linear Velocity Maintains Efficiency Trick: Reduce Flow Rate When Reducing Column Diameter mau 175 1 Mobile Phase: 25% methanol in.4% Formic Acid 15 125 1 75 5 25 2 3 ZORBAX SB-C18, 4.6 x 25 mm, 5 μm, 1 ml/ Solvent Used: 34 ml 4 5 6 mau 175 15 125 1 75 5 1 15 2 25 3 35 ZORBAX SB-C18, 3. x 1 mm, 3.5 μm,.425 ml/ Solvent Used: 5.7 ml, decrease of 83% (decrease in analysis time of 57%) 5 25 5 1 15 2 25 3 35 Page 13

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 4.6 mm 3. mm 2.1 mm 1. mm.5 mm.3 mm.1 mm.75 mm Flow Rate 1. ml/.42 ml/.21 ml/ 47 μl/ 12 μl/ 4.2 μl/ 472 nl/ 266 nl/ Flow rate column 2 = (diameter column 2) 2 /(diameter column 1) 2 x Flow rate column 1 Maintain equivalent mobile phase linear velocity when changing column diameter. Page 14

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 3X: 15mm, 5µm (~12,N) ~ 5mm, 1.8µm (~12,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 15

Page 16 GRADIENT ELUTION

Reducing Particle Size Improves Kinetics Reduces Peak Width, Increases Height- It Does NOT Change the Gradient! mau 16 14 1.8um vs. 5um + 6% Resolution (Theory: 67%) + 16% Efficiency (Theory: 177%) 12 1 8 Pressure at start: 51bar, 5µm Rs(4,5) = 3.14 Peak Width 5σ (9) =.17 6 4 2 Pressure at start: 394bar, 1.8µm Rs(4,5) = 5.7 Peak Width 5σ (9) =.74 2 4 6 8 Chromatograms Offset for Better View Page 17

Resolution Equation for Gradient Elution Relationship of k* to Gradient Time, Flow and Column Dimensions R V N α k* 4 k* t g F S (Δ%B) V m Δ%B = S = F = t g = V m = difference between initial and final % B constant ( 4 for 1-5 Da) flow rate (ml/.) gradient time (.) column void volume (ml) Zorbax Vm= π x (Col Internal Radius) 2 x Length x.6 Page 18

Expected: Use of a Longer Gradient Time Increases Gradient Retention 1,2 Gradient Time 1. 1 2 3 Gradient Time 6. 3 4 5 6,7 4 5 6 7 Column: ZORBAX SB-C8 4.6 x 15 mm, 5 μm Gradient: 2 6% B Mobile Phase: A:H 2 O with.1%tfa, ph 2 B: Acetonitrile Flow Rate: 1. ml/ Temperature: 35 C Sample: Herbicides 8 8 5 1 15 Time () 25 5 Time () Increased gradient retention improves resolution of several peak pairs 1,2 and 4,5. Page 19

Tip: A Shorter Column (smaller Vm) Also Increases Gradient Retention and Rs Increases Gradient Retention, Increases Overall Resolution 1,2 4.6 x 15 mm, 5 μm 4.6 x 75 mm, 3.5 μm 5 3 4 5 7 2 3 4 8 Column: ZORBAX SB-C8 Gradient: 2 6% B Mobile Phase: A:H 2 O with.1%tfa, ph 2 B: Acetonitrile Flow Rate: 1. ml/ Temperature: 35 C Sample: Herbicides 6 1 6 7 8 5 1 15 Time () 5 1 Time () Page 2

Tip: Improve Resolution By Using Short Column Length ( Vm ) for BioMolecules 3SB-C8, 4.6 x 15 mm, 5µm 3SB-C8, 4.6 x 5 mm, 3.5µm 1 2 3 4 5 6 7 8 1 1 2 3 4 5 6 7 8 1 9 9 2 4 6 8 1 12 14 16 2 4 6 8 1 12 14 16 Mobile Phase: Flow Rate: Temperature: A: 95% Water : 5 % ACN,.1% TFA B: 5% Water : 95% ACN,.85% TFA Gradient: 1-6% B in 3. Sample: 1. Gly-Tyr 2. Val-Tyr-Val 3. [Gln 11 ] Amyloid-β- 6. Leu-Enk 7. Angiotensin II 8. Kinetensin 1. ml /. Protein Fragm 1-16 9. RNase Ambient 4. (TYR8) Bradykinin 1. Insulin (Eq.) 5. Met-Enk 997P1.PPT Page 21

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 189S1.PPT Page 22

Trick: Change V m and t G by Same Proportion Two Chromatograms Both Having the Same Gradient Steepness Sample: 1. Tebuthiuron 2. Prometon 3. Prometryne 4. Atrazine 5. Bentazon 6. Propazine 7. Propanil 8. Metolachlor Column: Gradient Time: StableBond SB-C8 4.6 x 15 mm, 5 μm 3. Flow Rate: 1. ml/ 4 5 8 4 5 Column: Gradient Time: 8 Rapid Resolution StableBond SB-C8 4.6 x 75 mm, 3.5 μm 15. Flow Rate: 1. ml/ Analysis Time: 24 1 2 3 6 1 2 3 6 Analysis Time: 12 7 7 5 1 15 2 25 Time () 5 1 15 Time () 1784S1.PPT Page 23

Very Fast LC on Conventional 11 HPLC G1379 Degasser G1311 Quaternary pump G1313A ALS autosampler G1316A column compartment The Instrument G1314A VWD (standard cell G1314-686, 1mm, 14uL) Acetophenone Diethyl phthalate Benzophenone Butyrophenone Valerophenone Hexanophenone Heptanophenone Octanophenone The Sample Page 24

Conventional Column - 4.6 x 15mm, 5µm, SB-C18 mau 25 2 VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-11\789SBC183.D) 3.323 5.651 8.337 Flow Rate 1. ml/ Injection Volume 15uL Temperature 3 C Wavelength 246nm Sample rate 2.5 Hz 15 6.114 Time () % Acetonitrile 1 5 1.474 5.64 6.964 2 4 6 8 1 12 14 Initial Pressure: 69 bar Final Pressure: 38 bar 9.69 1.982 1 13.5 13.6 15 5 9 9 5 5 Page 25

Tip: Shorten Column and Gradient Time by Same Factor 1/3 Column Length- 1/3 Gradient Time RRHT Column 4.6 x 5mm, 1.8µm, SB-C18 mau 14 12 1 VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-11\HDS 27-8-9 17-25-25\789SBC189.D) 1.176 2.254 2.464 3.357 Flow Rate 1. ml/ Injection Volume 5uL Temperature 3 C Wavelength 246nm Sample rate 13.74 Hz 8 6 2.4 2.811 3.871 4.343 Time () % Acetonitrile 4 2 1 2 3 4 5 6 Initial Pressure: 132 bar Final Pressure: 74 bar 3.33 4.5 4.53 5 5 9 9 5 5 Page 26

The Comparison of chromatogram with the same RRHT column in 11 and 12 SL Overlay the two Chromatograms Norm. 14 VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-11\HDS 27-8-9 17-25-25\789SBC188.D) *DAD1 A, Sig=246,4 Ref=36,1 (WORKSHOP-TAIWAN\7815SBC183.D) 1.179 2.257 3.361 12 1 2.468 8 6 2.7 2.816 3.874 4.343 4 2 1 1.5 2 2.5 3 3.5 4 4.5 5 The red one is 12, the blue one is 11. Page 27

Tip: Increase Column Flow-Reduce Gradient Time Double Flow (2mL/) ½ Gradient Time RRHT 4.6 x 5mm, 1.8µm, SB-C18 mau 14 12 1 8 VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-11\HDS 27-8-1 8-22-16\781SB.587 1.146 1.26 1.715 Flow Rate 2. ml/ Injection Volume 5uL Temperature 3 C Wavelength 246nm Sample rate 13.74 Hz 6 4 2 1.2 1.437.5 1 1.5 2 2.5 3 Initial Pressure: 266 bar Final Pressure: 146 bar 1.97 2.21 Time () 1.67 2.25 2.27 3.34 % Acetonitrile 5 9 9 5 5 Page 28

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 29

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 3

Agilent Method Translator Advanced Mode More Detaled Information, But Still Easy to Use Detailed Input Detailed Output Original Method New Method Page 31

Does it work? - Example Analysis of impurities of an active pharmaceutical ingredient by conventional HPLC (4.6mm ID x 25 mm, 5. µm): mau H3C N CH3 H 4 OH OCH 3 Main Compound 3 H 3 C N CH 3 H 2 H 3C N CH3 H OH OH OCH 3 Impurity A H3 C N CH3 H H 3C N CH 3 Br OH 1 Impurity D OCH 3 OCH 3 O CH 3 Impurity C Impurity B Bromanisole 2.5 5 7.5 1 12.5 15 17.5 2 Page 32

Converting to a 4.6 mm ID x 1 mm, 1.8µm column: Page 33

Does it work? Yes! mau 4 Conventional HPLC 3 2 1 mau 14 12 1 8 6 4 2 2.5 5 7.5 1 12.5 15 17.5 2 Resolution optimized 1.8µm -2 1 2 3 4 5 6 7 8 Added Benefit of Increased Sensitivity! Page 34

mau 35 mau 3 2 mau 4 Does it work? Conventional HPLC 25 3 15 2 15 1 2 5 1 5 1.5 1 2.51 2 51.5 3 7.5 2 4 1 2.5 5 12.5 3 6 3.5 15 7 417.5 84.52 4.6 mmid x 1 25 mm, 1.8µm 5.µm Zorbax SB C18. 5% B 2. 4.33 8. 9% B 23. 4.98 9.2 9% B 23.1 4.99 9.21 5% B 12. 3. 6.5 5% B Speed Optimized Simple Conversion Page 35

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-C18 Bidentate-C18 Double endcapping SB-C18 Diisobutyl-C18 Rx/SB-C8 Diisopropyl-C8 SB-Phenyl Diisopropyl- Phenyl SB-CN Diisopropyl-CN SB-C3 Triisopropyl SB-AQ Diisopropyl Eclipse XDB-C18 Eclipse XDB-C8 Eclipse XDB- Phenyl Eclipse XDB-CN Eclipse Plus C18 Eclipse Plus C8 Eclipse Plus- Phenyl-Hexyl Eclipse PAH Rx-C18 Dimethyloctadecylsilane Bonus-RP Embedded amide amide Diisopropyl-C14 Triple Triple endcapped Page 36

Tip: Bonded Phase Can Change Selectivity Improve Peak Shape and Shorten HPLC Assay Time Each ZORBAX phase available in matching 1.8µm, 3.5µm and 5µm choices, most in 7µm! Over 14 RRHT,1.8um 6 bar column choices available 14 Different Column Chemistries 13 bonded phases and silica for HILIC use 7 column lengths (25*, 15, 1, 75, 5, 3 and 2 mm long) * 1.8µm as custom column 3 internal diameters (4.6, 3., 2.1 mm and Prep ID) Page 37

Tip: C8 Often Yields Same Peak Order in < Time Than C18 Trick: Evaluate Different Phases with Same M.P. RRHT Eclipse Plus C18, 4.6 x 5mm, 1.8 um 1. Oxybenzone 2. Internal Std. 3. Octylmethoxycinnamate 247 bar N: 88 Pw:.14. TF: 1.2 N: 116 Pw:.47. TF: 1.6 N: 118 Pw:.93. TF: 1.2 ) 1 2 3 4 Rapid Resolution Eclipse Plus C8, 4.6 x 5mm, 3.5 um N: 5 Pw:.19. TF: 1.9 N: 66 Pw:.37. TF:.99 N: 63 Pw:.61. TF:.98 2.2. 1 2 3 4 88 bar Conditions: Mobile Phase.: Water: Acetonitrile (3:7) Flow Rate: 2. ml/. Detection: UV 23nm Temperature: 3 C Sample: Lip balm extract in ACN (melted at 1 C ACN, cooled and.45 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 38 Group/Presentation Title Agilent Restricted Month ##, 2X

Tip: Polar Phases Can Save Analysis Time Trick: Evaluate Non-Polar and Polar Phases with Same M.P. Eclipse Plus Phenyl Hexyl 2 4 6 8 1 Elution order reversal between Phenyl-Hexyl and Alkyl chains Eclipse Plus C8 2 4 6 8 1 Eclipse Plus C18 2 4 6 8 1 Mobile Phase 4 % ACN 6 % 25 mm Sodium Phosphate Buffer ph 2.4 Flow Rate: 1.5 ml/ 4.6 x 5mm UV 21 nm 2µl Elution order for Eclipse Plus Phenyl Hexyl: (1) Piroxicam, (2) Sulindac,(3) Tolmetin, (4) Naproxen, (5) Ibuprofen, (6) Diclofenac, (7) Celebrex (equal portions of approximately 1 mg/ml solutions Page 39 Group/Presentation Title Agilent Restricted Month ##, 2X

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 4

Tip: Temperature Changes Can Alter Selectivity Trick: Optimize Col Temp For Best Speed/Rs 1 2 Salicylic acid 3 4 Coelution Salicylic acid 2 C 3 C 1 2 345 Salicylic acid 4 C 1 2 345 Salicylic acid Column: RRHT SB-C18 4.6 x 5mm, 1.8um 6 C 1 2 345 Salicylic acid Increased T Decreases Run Time, Changes Selectivity and Sharpens Peaks Improving Resolution 9 C 1 2 345 Page 41

Tips on When You Should Move to the Higher Pressure and Performance 12 SL (RRLC) System Very Fast Methods with High Velocity Mobile Phase That Increase Backpressure Above 4bar Resolution of Difficult Samples Requires Long Columns With 1.8µm Particles (>4bar 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 42

Tip: 1.8µm Particle Increases Resolution (How?) Trick: Substitute 1.8µm Particles in Same Length Column mau 35 3 25 2 15 1 5 mau 25 2 15 1 5 Agilent 11 15.65 13 14 15 16 17 18 Agilent 12 RRLC 14.787 14.937 15.159 15.411 15.79 15.324 15.164 15.555 15.472 15.79 15.967 Zorbax 2.1x15mm SB C-18, 5µm Zorbax 2.1x15mm SB C-18, 1.8µm 16.746 17.136 Conditions for both experiments Pumps Solvent A: H 2 O +.1% TFA Solvent B: ACN +.1% TFA Gradient: 1% to 95% ACN in 4, hold for 1 Flow Rate:.4ml/ Autosamplers Injection volume: 3µl Thermostatted Column Comp. Temperature: 5 C Detectors DAD 2µl cell and 2Hz, 22nm, Ref: Appl. Note 5989-456 by Edgar Naegele 13 14 15 16 17 18 Page 43

mau 2.5 2 1.5 1.5 -.5-1 Norm. 2.5 2 1.5 1.5 -.5 Tip: Small Particles Improve Detection of Low Level Impurities DAD1 A, Sig=254,4 Ref =of f (51119A\SIG13.D) 5 1 15 2 DAD1 A, Sig=254,4 Ref=off (514D\LC_J2.D) -1 5 1 15 2 4.6 x 15, 5um 93 bar N = 7259 R_S = 1.15 S/N = 42 Height = 1.25 Noise = 24uAU 4.6 x 15, 3.5um 165 bar N = 14862 R_S = 1.37 S/N = 5 Height = 1.34 Noise = 2uAU Column:15x4.6 mm 5µm Pressure: 93 bar N: 8213 Height: 1.25 mau S/N: 42.3 R s = 1.15 t r = 14.9 (1 st epimer) N ptp : 2.4 1-2 mau Column:15x4.6 mm 3.5µm Pressure: 165 bar N: 14862 Height:1,34 mau S/N: 5.7 R s = 1.37 t r = 15.3 (1 st epimer) N ptp : 2 1-2 mau N 3 25 2 15 1 5 Norm. 3 2.5 2 1.5 1.5 -.5-1 N 1 d p.2.3.4.5.6 1/dp DAD1 A, Sig=254,4 Ref=off (517D\LC_X1.D) 1 4.6 x Column:15 15, 1.8um x 4.6 mm 1.8µm Pressure: 49 bar 49 bar N: 28669 N = 28669 Height:1.78 R_S S/N: = 43.6 1.8 (+57%) S/N R= s = 44 1.8 t r = 17.2 (1 st epimer) Height = 1.8 N ptp : 3 1 Noise = 3uAU -2 Page 44

Particles Reveal More Information and Improve Detection and Integration Customer Sample, Translation of Isocratic Impurity Methods, Zoom Critical Time Range (t = 7) 4 Impurities 2 Not Baseline Separated! 7 Impurities 6 Not Baseline Separated! 7 Impurities All 7 Baseline Separated! 4.6 x 15, 5μm 93 bar N = 7259 R_S = 1.15 S/N = 42 4.6 x 15, 3.5μm 165 bar N = 14862 R_S = 1.37 S/N = 5 4.6 x 15, 1.8μm 49 bar N = 28669 R_S = 1.8 (+57%) S/N = 44 Up to 6% higher resolution without loss in sensitivity Page 45

Example of What Is Possible! Shorter Column, Smaller Particle, > Flow Rate, High Temp F= 1.2ml/ T = 4 C Analysis Time = 11 Solvent Cons. = 13.2ml Easy! High Resolution: 4.6mm x 15mm 5.µm F = 4.8ml/ T = 4 C Analysis Time = 1.5 Solvent Cons. = 5.1ml F= 1.ml/ T = 4 C Analysis Time = 1.1 Solvent Cons. = 1.1ml 2 4 6 8 1 12.2.4.6.8 1 High Speed: 4.6mm x 5mm 5.µm Easy! Easy! High Speed & Resolution: 2.1mm x 5mm 1.8µm.2.4.6.8 1 F= 2.4ml/ T = 95 C Analysis Time:.4 Solvent Cons. = 1.ml PW HH = 197msec > 2x faster! Easy, But You Will Need > 4bar! Max Speed at T = 95 o C 2.1mm x 5mm 1.8um.2.4.6.8 1 Page 46

Tip: Higher Data Improves Response and Rs Trick: Optimize Data Rate for Accurate Profile PW=.3sec PW=.33sec 8Hz 8Hz versus 1Hz Data Rate Peak Width: 55% Resolution: + 9% Peak Capacity: + 12% App. Column Eff.: + 26% PW=.42sec 4Hz Data Rate 8 Hz 4 Hz Peak Width.3.329 Resolution 2.25 2.5 Peak Capacity 6 55 PW=.67sec PW=1.24sec 2Hz 1Hz 5Hz.1.2.3.4.5 2 Hz 1 Hz 5 Hz Sample: Column: Gradient:: Flow Rate:.416.666 1.236 1.71 1.17.67 45 29 16 Phenones Test Mix Zorbax SB-C18, 4.6x3, 1.8um 5-1%ACN in.3 5ml/ Page 47

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 11 modules, existing method and stationary phase Configurable delay volume in 12 SL pump Easy to also run legacy LC methods Simple LC to RRLC method translator No tedious method development No - not at all! Page 48

Stepwise Scale-up to Rapid Resolution LC From 11 to 12 RRLC in two steps - Add What You Need When You Need It! Quat Pump Actual: 11 Quat System Degasser ALS TCC VWD/MWD/DAD Step 1: 11/12 Bin SL System µ-degasser Bin Pump SL h-als SL TCC VWD/MWD/DAD + Resolution + Speed + MS-Compatibility + Solvent Saving + Compatible with conv. HPLC Step 2: 12 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 > 5 > 6 > 3 sec 5-12, 4.6 mm 5 mm.2-1ml/ 8 C 4bar > 1.5 > 2 > 1.5 sec 5-3, 2.1-4.6 mm 5-15 mm.5-5 ml/ 8 C 6 bar >.2 >.4 >.2 sec 5-6, 2.1-4.6 mm 2-15mm.5 5 ml/ 1 C 6bar Page 49

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 2X, 3X and 5X Times Savings are Easy Greater Time Savings May Require Instrument Modifications 1-2X Increase in Speed May Require Instrument Upgrade Long Columns with 1.8µm Particles May Require >4 bar Pump and Autosampler Capability Page 5