The One Minute Chromatographer

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1 The One Minute Chromatographer Agilent Technologies, Inc Page 1

2 Sub 1 Minute Separations with RRHD Columns Agilent ZORBAX RRHD SB-C18, 2.1 x 100mm, 1.8 µm H2O (0.05% Formic acid) / 2-98 % Acetonitrile T = 80 C V_i = 0.5 µl WL = 210 nm DR = 80 Hz F = 2 ml/min P = 1085 bar

3 Plate Height H Smaller Particles Reduce In Column Dispersion Increased Efficiency H = A + B/u + C u Large Particle Small Particle Sum Curve: van-deemter H min Longitudinal diffusion Resistance to Mass Transfer Eddy Diffusion u opt Linear Velocity u Page 3

4 Two Advances For Better Mass Transfer SOLID CORE Sub 2um Particle Superficially Porous Particle

5 Poroshell 120 Columns for HPLC and UHPLC: Poroshell 120 columns have: 80-90% efficiency of sub 2um At ~40-50% lower pressure 2X efficiency of 3.5um (totally porous) A 2.7um particle size A 2um frit to reduce clogging A 600 bar pressure limit The particle has a solid core (1.7um) and porous outer layer with a 0.5um diffusion path 0.5um 1.7um 0.5um Confidentiality Label 5 July 17, 2013

6 Poroshell 120 Several Different Phases EC-C18 EC-C8 EC-CN Stablebond C18 Stablebond C 8 Stablebond Aq Bonus RP HILIC Phenyl Hexyl Page 6

7 HETP (cm) Van Deemter Redux van Deemter Eclipse Plus C18, 3.5 µm Eclipse Plus C18, 1.8 µm Poroshell 120 C18, 2.6um Poroshell 120 C18, 2.6 µm Poroshell 120 C18, 2.7 µm Flow rate (ml/min) 7

8 High Peak Capacity Maximizes Resolution with Complex Samples 1.8u Particles

9 High Peak Capacity Maximizes Resolution with Complex Samples Poroshell 120

10 Smaller Particles Improve Detection and Integration 4 Impurities 2 Not Baseline Separated! 7 Impurities 6 Not Baseline Separated! 7 Impurities All 7 Baseline Separated! 4.6 x 150, 5μm 93 bar 4.6 x 150, 3.5μm 165 bar Customer Sample, Impurity Method 4.6 x 150, 1.8μm 490 bar Page 10

11 What is Sensitivity? Limit of detection (LOD): The lowest concentration, or smallest mass flow, which can be distinguished from the noise by a certain predefined probability (Signal/Noise). Signal Noise Limit of detection (LOD): h Signal = 3(2) x h Noise Page 11

12 Analysis of 15 Analgesic Compounds Same Method for all Three Columns ZORBAX Eclipse Plus C18, 3 x 100 mm, 3.5 µm Ibuprofen: PW 1/2 =0.014 S/N=182 n c =43 ZORBAX RRHD Eclipse Plus C18, 3 x 100 mm, 1.8 µm Ibuprofen: PW 1/2 =0.012 S/N=353 n c =54 Poroshell 120 EC-C18, 3 x 100 mm, 2.7 µm Ibuprofen: PW 1/2 =0.012 S/N=256 n c =56 2 min June, 2011

13 Comparison of 4.6 x 250 mm 5 um to Poroshell 120 EC-C x 100 mm, 2.7um mau Time %B Column: Eclipse Plus C x 250mm, 5um Flow Rate: 1 ml/min 110 bar Mobile Phase: A: 0.1% formic acid in Water B: 0.1% formic acid in ACN Sulfadiazine, Sulfathiazole Sulfapyridine Sulfamerazine, Sulfamethazine, Sulfamethazole, Sulfamethoxypyridazine, Sulfachloropyridazine Sulfamethoxazole, Sulfadimethoxine 0 mau Time %B Column: 4.6 x 100mm Poroshell 120 EC-C18, 2.7um Flow Rate: 1 ml/min 325 bar min min

14 Expand High Speed Chromatograms for True Comparison to Slower Separation See conditions previous slide mau bar 20 mau min bar min

15 Rapid Resolution LC Ultra Fast, Resolution Maintained, Sensitivity Improved Optimized Gradient 5 Run Time 1.8 min Equilibration 1 min Time Total Analysis 2.8 min Time 480 Samples/day Column: Rapid Resolution Eclipse XDB-C8, 4.6 x 50 mm, 3.5 mm Mobile Phase: A: 55% 25 mm Na 2 HPO 4, ph 3 B: 45% MeOH Gradient: 42 90% B in 2 min Flow Rate: 3 ml/min Temperature: 35 C Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine Time (min) Page 15

16 US. Pharmacopeia 621 Column length: ± 70 % Column internal diameter: ± 25 % Column material particle size: Reduction of up to 50 %, no increase Flow rate: ± 50 % Injection volume: Changes are allowed as long as system suitability testing (SST) criteria are met. Column temperature: ± 10 % ph of mobile phase: ± 0.2 UV wavelength: no change outside manufacturer specifications Concentration of salts in buffer: ± 10 % Composition of mobile phase (adjustment of the minor component is allowed ±30 % or ± 10 % absolute whichever is smaller) Page 16

17 USP and FDA Method Adjustment Criteria Method Adjustment Criteria for Column Dimensions Parameter Maximum Specifications Comments/Examples Column Length ± 70% 250mm 75mm 150mm 50mm ±25% (FDA ORA-LAB 5.4.5*) 4.6 mm 3.0 mm (-35%) Column Internal Diameter USP ± 25% Column ID can be adjusted provided linear velocity is constant** Flow Rate ±50% Injection Volume Particle Size Reduce as much as needed must still meet detection limits and precision Reduce by up to 50% (can not be increased) 4.6 mm 2.1 mm (-54%) 3.0 mm 2.1 mm (-30%) If you change to a smaller/ shorter column make the appropriate change in injection volume You can change column length and particle size to keep Rs same 5um 3.5um (-30%) 5um 2.7um (-46%) *Changed on 2/24/09 - For the current and official copy, go to ** USP 30 Second Supplement Revisions, PF34(5), in process expected to be final Dec 2009 See Stimuli article in Pharmacopeial Forum 2009; 35(6) Page 17

18 USP Method-Naproxen Page 18

19 Naproxen 100 mm column Page 19

20 NAPROXEN 50 mm Column Page 20

21 Agilent 1200 Rapid Resolution System Speed Conventional LC UFLC RRLC F= 1.20ml/min T = 40 C Analysis Time = 11min Solvent Cons. = 13.2ml High Resolution: 4.6mm x 150mm 5.0µm min F = 4.80ml/min T = 40 C Analysis Time = 1.05min Solvent Cons. = 5.1ml High Speed: 4.6mm x 50mm 5.0µm min F= 1.00ml/min T = 40 C Analysis Time = 1.1min Solvent Cons. = 1.1ml High Speed & Resolution: 2.1mm x 50mm 1.8µm min F= 2.40ml/min T = 95 C Analysis Time: 0.4min Solvent Cons. = 1.0ml PW HH = 197msec > 20x faster! Max Speed at T = 95 o C 2.1mm x 50mm 1.8um min Page 21

22 100% B t g = 5 Changing Gradient Time to Affect Retention (k*) and Resolution 0% B 100% B k* = t g F S D%B V m t g = 10 1/k* = gradient steepness = b 0% B 100% B DF = change in volume fraction of B solvent 0% B t g = 20 S = constant F = flow rate (ml/min.) t g = gradient time (min.) V m = column void volume (ml) 100% B t g = 40 0% B Time (min) S 4 5 for small molecules 10 < S < 1000 for peptides and proteins P1.PPT Group/Presentation Title Agilent Restricted Month ##, 200X

23 Very Fast LC on Conventional 1100 HPLC G1379 Degasser G1311 Quaternary pump G1313A ALS autosampler G1316A column compartment The Instrument G1314A VWD (standard cell G , 10mm, 14uL) Acetophenone Diethyl phthalate Benzophenone Butyrophenone Valerophenone Hexanophenone Heptanophenone Octanophenone The Sample Page 23

24 Conventional Column x 150mm, 5µm, SB-C18 mau VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\070809SBC D) Flow Rate 1.0 ml/min Injection Volume 15uL Temperature 30 C Wavelength 246nm Sample rate 2.5 Hz 150 Time (min) % Acetonitrile Initial Pressure: 69 bar Final Pressure: 38 bar min Page 24

25 Shorten Column and Gradient Time by Same Factor 1/3 Column Length- 1/3 Gradient Time RRHT Column 4.6 x 50mm, 1.8µm, SB-C18 mau VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS \070809SBC D) Flow Rate 1.0 ml/min Injection Volume 5uL Temperature 30 C Wavelength 246nm Sample rate Hz Time (min) % Acetonitrile Initial Pressure: 132 bar Final Pressure: 74 bar min Page 25

26 Increase Column Flow-Reduce Gradient Time Double Flow (2mL/min) ½ Gradient Time RRHT 4.6 x 50mm, 1.8µm, SB-C18 mau VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS \070810SBC D) Flow Rate 2.0 ml/min Injection Volume 5uL Temperature 30 C Wavelength 246nm Sample rate Hz Time (min) % Acetonitrile min Initial Pressure: 266 bar Final Pressure: 146 bar Page 26

27 What Length Column Yields Highest Peak Capacity? Flow rate vs. Gradient time vs. Peak capacity For small molecules(mw < ~1000) 150mm 100mm 50mm Different Column Lengths Broken lines are isobar (800 bar)

28 Shorter Columns with Fast Gradients Yield Higher Peak Capacity Shorter Gradient (5 min) 150mm 100mm 50mm Peak Capacity: 258 for 50 mm 240 for 100 mm 221 for 150 mm

29 Longer Columns with Long Gradient Times Yield Greater Peak Capacity Long Gradient (40 min) Peak Capacity: 422 for 50 mm 150mm 100mm 50mm 510 for 100 mm 525 for 150 mm

30 Constant Particle Size, Grad Time More Resolution mau Rs: 0 RRHD SB-C x 50 mm, 1.8um Pmax=366 bar n c = mau min Rs: 1.37 RRHD SB-C x 100 mm, 1.8um Pmax=595 bar n c = mau Rs: 2.40 min RRHD SB-C x 150 mm, 1.8um Pmax=768 bar n c = min Page 30 Group/Presentation Title Agilent Restricted Month ##, 200X

31 Poroshell 120, 150mm Columns HPLC or UHPLC Conditions: Column: Poroshell 120 EC-C18, 4.6 x 150mm, 2.7um Mobile Phase: Solvent A: Water with 0.1% Formic Acid Solvent B: Acetonitrile 1200 SL controlled temperature at 25 C 2 ul flow cell mau ml/min P = 538 bar 1. Hydroquinone 2. Resourcinol 3. Catechol 4. Phenol 5. 4-Nitrophenol 6. p-cresol 7. o-cresol 8. 2-Nitrophenol 9. 3,4 di methyl phenol 10. 2,3 di methyl phenol 11. 2,5 di methyl phenol napthol min mau ml/min P = 285 bar Gradient: 1mL/min Time %B 6.0 5% 51 60% Gradient: 2mL/min Time %B 3.0 5% % min

32 UHPLC/TOF (1290/6230) Can Identify More Compounds in Less Time 224 pesticides at 50 pg each 217 ionized & detected in positive mode (97%, Find by Formula) 2.1 x 50 mm x 1.8 micron Eclipse Plus C bar 1.5 ml/min 1290 Infinity Time Composition 1.5 min % ACN % ACN TOF fast acquisition rates (20Hz) ensure maximum throughpu Page 32

33 Ultrafast LC/MS Analysis for 15 Analyte Subset RRHD Eclipse Plus C18 2.1x 50 mm, 1.8 um 750 bar 1 minute Peak Width 0.7 sec Time Composition % ACN % ACN Ultimate speed on a short column with ballistic gradient 1290 Infinity Applications

34 Method Translator Makes Changes Easy 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 34

35 Agilent Method Translator Advanced Mode More Detaled Information, But Still Easy to Use Detailed Input Detailed Output Original Method New Method Page 35

36 Page 36 Systems/Liquid-Chromatography/pages/1200infinity_cost_calculator.aspx

37 RULE OF THUMB Set of Approximations based on chromatographic behavior and mathematical relationships Will deliver nearly the desired goal Probably need to be tweaked to deliver best results

38 What Is Your Current Column Efficiency and How Fast Do You Want To Run? 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* ,500 21,000 32, ,500 14,000 24, ,500 17, ,200 7,000 12, N.A. 4,200 6, N.A. 2,100 2, Analysis Time Peak Volume Solvent Usage -33% -50% -67% -80% -90% * Reduction in analysis time compared to 150 mm column pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID Page 38

39 First, Reduce Column Length 5um 1.8um Reduce column length by factor of 3 Quite often original method will have more resolution than is actually needed and a reduction by 5 may be possible 3.5 um 1.8um Reduce column length by factor of 2 Quite often original method will have more resolution than is actually needed and a reduction by 3 is possible Page 39

40 Flow Modification - Different Diameter Columns Flow col. 1 Diam. Diam. column2 column1 2 Flow col. 2 i.e mm 1.0ml/min 0. 21ml/min 4.6mm Page 40

41 Decreased Column Volume May Require Conversion for Injection Volume Inj.Vol. col. 1 Volume Volume column2 column1 Inj.Vol. col. 2 Zorbax column volume = 3.14 x r 2 x L x 0.6 (r and L in cm) i.e. 20μl 0.4ml column2 col ml column1 μl col. 2 Page 41

42 Reduce injection volume Reduction to allow for diameter change 4.6 mm 3 mm = 0.4 x Original 4.6 mm 2.1 mm = 0.2 x Original x Reduction to allow for length change 150 mm 50 mm = 0.33 x Original 150 mm 1100 mm = 0.67 x Original 100 mm 50 mm = 0.5 x Original e.g. Original 4.6mm x 150mm transferred to 2.1mm x 100mm = 0.2 x 0.67 =0.13 x original injection volume Page 42

43 Simple Isocratic Method Transfer for Speed Step 1 Reduce Column Length and Particle Size Maintain Flow Rate Step 2 Faster Increase Flow Rate Pressure too High? Increase Temperature to Lower Pressure Increase Flow Rate Step 3 Even Faster (Need Instrument Optimization) Decrease Column Diameter (2.1mm), Reduce Flow Rate Proportionately Reduce Injection Volume Based on Ratio of Column Volumes Increase Flow Rate Page 43

44 Simplified Gradient Method Transfer Step 1 Reduce Column Length and Particle Size Adjust Gradient Time by same Factor Maintain Flow Rate Step 2 Faster Increase Flow and Reduce Gradient Time Stop When Reach Flow Limits of Instrument Step 3 Even Faster Decrease Diameter of Column (2.1mm) Match Flow to New Column Diameter Reduce Injection Volume Repeat Step 2 Until Reach % Instrument Pressure Limit Page 44

45 Let s Look at Method Development of These Compounds propranolol pindolol dipyridamole β-blocker Anti-arrhythmic Vasodilator Ca+ channel blocker disopyramide diltiazem Group/Presentation Title Agilent Restricted Month ##, 200X

46 Start at Low ph, Adjust Organic Eclipse Plus C18 Cardiac Drugs with Acetonitrile mau mau mau Column: ZORBAX RRHT Eclipse Plus C18, 4.6 x 50 mm, 1.8 mm Mobile Phase: A: 25 mm NaH 2 PO 4, ph 3.0 B: ACN Flow Rate: 2.0 ml/min Temperature: 30 C Detection: UV 240 nm Sample: Cardiac Drugs 1.Pindolol 2. Diisopyridamide 3.Propranolol 4.Dipyridamole 5. Diltiazem 40% ACN % ACN RRHT Eclipse Plus C18 allows for very fast optimization of % organic in mobile phase. 20% ACN Good resolution Fast analysis No time wasted To get this k on a 25cm column at 2 ml/min would require 1.5 hours run time!! k= 44!! min Group/Presentation Title Agilent Restricted Month ##, 200X

47 Start at Low ph on Eclipse Plus C18 Cardiac Drugs with Methanol, Changing Organic Modifier mau mau Column: ZORBAX RRHT Eclipse Plus C18, 4.6 x 50 mm, 1.8 mm Mobile Phase: A: 25 mm NaH 2 PO 4, ph 3.0 B: MeOH Flow Rate: 2.0 ml/min Temperature: 30 C Detection: UV 240 nm Sample: Cardiac Drugs 1.Pindolol 2. Diisopyridamide 3.Propranolol 4.Diltiazem 5. Dipyridamole 70% MeOH % MeOH Adjusting MeOH influences retention like adjusting ACN Solvents can be compared quickly with RRHT columns MeOH changes selectivity and gives longer analysis time for these analytes mau % MeOH k= 49!! min Group/Presentation Title Agilent Restricted Month ##, 200X

48 Viscosity When to Stop!? Viscosity When flow limit of pump is reached Consider using a smaller i.d. column When approaching about 90% pressure limit of instrument 540 bar for 1200SL pump 360 bar for 1100/1200 pump Remember to allow for increase in pressure at non-ideal mix When resolution is no longer satisfactory ACN/water 40C Methanol/water 40C %ACN %Methanol

49 Agilent 1.8u and Poroshell 120 Columns Make High Resolution, Fast LC Possible on All Instruments Reduce Analysis Time Speed Up Method Development Improves Methods on HPLC Provides Best Benefits on UHPLC Coupled with UHPLC Instruments Allow Higher Flow Rates for Higher Resolution Without Long Run Times Page 49

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