Practical Applications of Method Translation Using the Agilent Method Translation Tool Rita Steed Inside Application Engineer Agilent Technologies

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1 Practical Applications of Method Translation Using the Agilent Method Translation Tool Rita Steed Inside Application Engineer Agilent Technologies Title

2 Objectives Demonstrate Agilent Method Translation tool For fast, easy, and successful method transfer to smaller volume columns Review important method variables Isocratic Gradient Review method transfer examples using the Agilent Method Translation Tool

3 Successful Method Translation Separation Goals and Method Performance Criteria Isocratic separations Instrumentation Which instrument and method parameters afford optimal results Considerations for successful implementation Agilent Method Translator for isocratic separations Gradient separations Gradient retention parameters Instrument considerations Agilent Method Translator for gradient separations

4 Separation Goals and Method Performance Criteria Separation Goals and System Suitability Resolution: 2 Peak shape: USP T f close to 1 (<2) Injection Repeatability: areas, T f, etc. (RSD %) Absolute retention: 1< k<10 Relative Retention: α or k 2 /k 1 Signal-to-Noise Ratio: >10 AVOID THESE for System Suit. Criteria Column efficiency (theoretical plates) Absolute retention Method Performance Criteria Accuracy Precision Ruggedness Repeatability Intermediate precision Reproducibility Robustness Selectivity/Specificity Linearity Range Quantitation Limit (LOQ, 10x S/N) Detection Limit (LOD, 3x S/N)

5 An Approach for Isocratic Method Translation Assess current method performance and parameters Set performance goals for method to be translated Determine column geometry for necessary efficiency Instrument needs vs. method performance goals Depend on requirements for column size and particle size to get desired R s Instrument ECV, detector data rate System pressure limitations Adjust injection volume for smaller column volume Assess injection repeatability and sample solvent composition robustness Adjust flow rate vs. system max. pressure relative to method performance goal for analysis time

6 Isocratic Method: Document current method performance, parameters, and instrument configuration Current Method Performance Limiting resolution for critical pair(s) Peak shape(s) (USP T f ) Injection repeatability Signal-to-Noise ratio Instrument Configuration Extra Column Volume Tubing ID and length Flow cell volume Detector data rate Flow cell pathlength System maximum pressure Method Parameters Column length, id and particle size Flow rate Mobile phase composition (viscosity) Column temperature Injection volume Sample concentration and sample solvent composition Nominal backpressure

7 Isocratic Method Example Situation: You have isocratic method for tocopherols developed for 4.6 mm i.d. columns in 150 mm length. Run time is ~14 min. Pump: Autosampler: TCC: Detector: Flow Cell: Flow Rate: Column temp. 23ºC Goals: Agilent 1100 quaternary system Standard autosampler 1100 standard 1100 DAD, max. data rate 20 Hz Typical setting, PW = 0.05 min. 13 µl, 10 mm path length 1.0 ml/min. Decrease run time and improve throughput (5X, if possible) Save solvent usage and waste (implies smaller column id or shorter run at higher flow rate) Can anything be done to speed up these methods with existing equipment? What modifications can be made and which are most important?

8 Isocratic method on Conventional Column Tocopherols mau RRHT 4.6 x 50 mm 1.8 µm Flow Rate: 3 ml/min Pressure = 229 bar Column: ZORBAX Eclipse XDB-C18 Mobile Phase: 95% ACN: 5% Water Temp: 23ºC Injection volume: 1 ul Conventional 4.6 x 150 mm 5 µm Flow Rate: 1 ml/min P = 37 bar R s ~ 5.2 R s ~ min 13.5 min Sample: Vitamin E α, β, γ-tocopherols in gel cap Eclipse XDB-C18 is a good first choice for many methods. min

9 Assess Your Current Method Assess your current method 4.6 x 150 mm, 5 µm column 1.0 ml/min RT last = 14 minutes Questions to ask? What is the mobile phase composition? What is the current backpressure? Injection volume? Data rate/peak width? What is your limiting resolution with current method? What size column can deliver the resolution you need? Can your current instrument be used with a shorter column with smaller particle size? Which changes in method parameters are necessary and can you get the same or similar performance and results?

10 Efficiency Ranking of Various Column Geometries and Typical Backpressures This RRHT column Replaces These Longer Columns 50 mm, 1.8 µm 150 mm, 5 µm, 100 mm, 3.5 µm 100 mm, 1.8 µm 250 mm, 5 µm

11 Tocopherol method translation Current Method 4.6 x 150 mm, 5 µm XDB-C18 Viscosity of 95:5 ACN/water at 23ºC is ~0.43 cp Flow Rate is 1 ml/min Backpressure is 37 bar Standard flow cell (13 µl) Standard 0.17 mm tubing throughout Limiting Resolution ~4.4 Peak Width required 0.1 min Response Time = 2 sec or Data Rate = 2.5 Hz is adequate

12 Isocratic Method: Translation Tool 13 ul flow cell and 0.17 mm tubing 21.6 ul tubing vol + 13 ul flow cell Effective N hurt by EC vol. For isocratic runs the 2 nd row must be set to same %B as row 1

13 Use 5 ul flow cell and 0.12 mm id tubing Improvement in N effective

14 Speed Optimized at 3 ml/min Adjust % max. pressure until desired flow rate Adjust to 3 ml/min

15 Tocopherol Method Translation Translated Method 4.6 x 50 mm, 1.8 um RRHT XDB-C18 Column length in shorter dimensions with 1.8 µm particles is 4.6 x 50 mm RRHT At 1 ml/min expected backpressure is 79 bar + ~10 bar (a/s and flow cell) or ~90 bar Expected run time will be ~1/3 of 14 minutes or 4.67 minutes Try 3 ml/min for run time of 1/9 of 14 min. or 1.55 min. Predicted pressure is 238 bar Limiting resolution will be approximately the same (4.4) or 4.4 x SQRT(13043/12077) = 4.2, If no band broadening due to extra column volume or data rate. Standard DAD or MWD at fastest setting (20 Hz) with 0.17 mm id tubing adequate but not optimum Choose 0.12 mm i.d. tubing and 5 µl flow cell for better results

16 Comparison of Conventional Isocratic Method vs. Translated Method at 3 ml/min mau RRHT 4.6 x 50 mm 1.8 µm Flow Rate: 3 ml/min Pressure = 229 bar Column: ZORBAX Eclipse XDB-C18 Mobile Phase: 95% ACN: 5% Water Temp: 23ºC Injection volume: 1 ul Conventional 4.6 x 150 mm 5 µm Flow Rate: 1 ml/min P = 37 bar Solvent used 15 ml Solvent used 5.1 ml R s ~ 5.2 R s ~ min 13.5 min Sample: Vitamin E α, β, γ-tocopherols in gel cap Eclipse XDB-C18 is a good first choice for many methods. min

17 Flow Cells for RRLC 13 µl Standard Flow Cell: For highest sensitivity High-demanding quantitative work, e.g. analytical method development, QA/QC 2 µl Micro Flow Cell: For highest resolution Ultra-fast semi-quantitative work, e.g. Screening Experiments, HT LC/MS/UV Dimension Sensitivity* Resolution* 13 µl / 10 mm µl / 6 mm µl / 3 mm µl Semi-micro Flow Cell: Best compromise of sensitivity and resolution For good quantitative and qualitative results, e.g. Screening, HT LC/MS/UV, Early Formulation Studies * Depends on analytical conditions and column dimension

18 Effect of Detector Response Time on Fast Gradient Analyses Response Time 0.1 sec 1 st peak = 1.2 sec At 20 pts/sec = 24 pts/sec 1 st peak = 1.2 sec At 5 pts/sec = 6 pts/sec 0.2 sec 0.5 sec 1.0 sec 2.0 sec Time (min) 1.0 Agilent 1100 DAD Agilent 1100 WPS with ADVR Column: Poroshell 300SB-C x 75 mm, 5 mm Mobile Phase: A: 95% H 2 O, 5% ACN with 0.1% TFA B: 5% H 2 O, 5% ACN with 0.1% TFA Flow Rate: 2 ml/min Temperature:70 C Detector: Piston stroke: 20 UV 215 nm Sample: 1. Neurotensin3. Lysozyme 2. RNaseA 4. Myoglobin You may have to adjust the response rate of your detector for rapid peak detection. Page 18

19 High Speed LC with RRLC and RRHT Columns Maintaining Resolution at High Analysis Speed PW=0.30sec PW=0.33sec 80Hz 80Hz versus 10Hz (20Hz) Data Rate Peak Width: 55% ( 30%) Resolution: + 90% (+ 30%) Peak Capacity: + 120% (+ 40%) App. Column Eff.: + 260% (+ 70%) PW=0.42sec 40Hz Data Peak Resolution Peak Rate Width Capacity 80 Hz Hz PW=0.67sec 20H z 10Hz PW=1.24se 5Hz c min 20 Hz Hz Hz Sample: Column: Gradient:: Flow Rate: Phenones Test Mix Zorbax SB-C18, 4.6x30, 1.8um %ACN in 0.3min 5ml/min Page 19

20 Translating Gradient Methods

21 Advantages of Gradient Elution Complex samples are analyzed in a single HPLC run Analysis time can be reduced Peaks elute with the same bandwidth More peaks can be baseline resolved per unit time higher peak capacity than isocratic method Signal-to-Noise ratios and LOD/LOQ are relatively the same during a gradient run (barring ghost peaks, anomalies, etc.!) peaks don t broaden with increasing retention time as they do in an isocratic separation)

22 Resolution Relationship for Gradient Elution R V N 4 α k* k* - represents the fact that k changes constantly during a gradient k* = t g F S ( %B) V m %B = S = F = t g = V m = difference between initial and final % B values constant ( 4 for Da) flow rate (ml/min.) gradient time (min.) column void volume (ml) Title of Presentation Date Agilent Restricted

23 This Relationship Says that to Keep Relative Peak Position in the Chromatogram Unchanged Any Decrease in Can be Offset by a Proportional Column length Decrease in t G or F Increase in Φ Column volume (i.d.) Φ (same column) Decrease in t G or F Increase in Φ Decrease in t G or F t G F k* = S Φ Vm

24 100% B t g = 5 Gradient Steepness Affects Retention (k*) and Resolution 0% B 0% B 0% B 100% B t g = % B t g = % B k* = t g F Φ V m S 1/k* gradient steepness = b Φ =change in volume percent of B solvent (%) S = property of sample compound F =flow rate (ml/min.) t g = gradient time (min.) V m =column void volume (ml) 0% B t g = 40 S 4 5 for small molecules 10 < S < 1000 for peptides and proteins Time (min) P1.PPT

25 Transferring a Gradient Method to a Small(er) Column Examine the current method Column length and i.d., particle size, N Injection volume Injection precision Gradient program Initial Hold Time Linear gradient segments Isocratic holds during gradient Delay Volume Resolution of critical pair(s) Backpressure It s much easier to transfer a linear gradient than one with multiple segments and hold times Can you trade excess resolution for time or can you get the same efficiency (N) with a shorter column? Calculate critical pair resolution on shorter column(s) with smaller particle size(s) Calculate expected pressure at one or more flow rates on shorter column

26 Gradient Separations: Considerations When Translating Existing Gradient methods Isocratic Separations Sample load; V inj, analyte Sample solvent strength Extra column volume Flow cell volume Injection volume Tubing volume Injector precision Can vary with V inj Data Rate Too fast, too much noise Too slow, loss of N Gradient Separations Same as Isocratic Separations plus Delay Volume Same instrument (different pressures) Different instrument (for example, Capillary 1200 vs. Binary 1200) Gradient Time Adjust relative to equation for gradient retention Keep k* constant Gradient Delay Time Gradient delay time must be same as for larger column separation Ratio of gradient volume/column volume must be same as for larger column Column Equilibration Time (Post Time)

27 Gradient Separations What is Delay Volume? Also known as Dwell Volume Delay Volume Delay Volume = volume from formation of gradient to the column Behaves as isocratic hold at the beginning of gradient.

28 Seamless Method Transfer What if critical specifications change...? * Optimized for mm ID ** Optimized for 2.1mm ID 1260 Infinity 1100/ Series 1260 Infinity 1290 Infinity Quaternary Series HPLC RRLC Std. Binary LC Binary LC LC Max Flow Rate 5 ml/min 5 ml/min 5mL/min 5 ml/min 5 ml/min Delay Volume µl µl µl* µl* µl* Capillary ID 0.17mm 0.17mm 0.17mm 0.17mm 0.12mm* Disp. Vol. w/o cell 15µl 15µl 15µl 15µl 7.5µL* Injection Principle Variable Loop Variable Loop Variable Loop Variable Loop Variable Loop Inj.Vol. Std/Ext. 100 / 1500 µl 100 / 1500 µl 100 / 1500 µl 100 / 1500 µl 100 µl Area RSD <0.25 % <0.25 % <0.25 % <0.25 % <0.25 % Oven Design A A A A A Column Length 300 mm 300 mm 300 mm 300 mm 300 mm * Smaller delay volumes possible

29 Delay volume, cont d Configuration Delay Volume (µl)** 1290 Pump Pump + Fixed Loop * (for MS) Pump + Jet Weaver + Fixed Loop* Pump + Autosampler (for MS) Pump + Jet Weaver * + Autosampler RRLC (low delay volume) RRLC (standard delay volume) * 5 µl loop ** 10 % step gradient definition Binary Pump 29

30 Agilent Method Translator

31 Agilent Method Translator

32 Features of the Agilent Method Translator Basic mode with certain pre-set parameters: Enter the parameters of your existing method and the parameters of the desired column you would like to convert to.

33 Features of the Agilent Method Translator Advanced mode all calculation parameters in your hands: More to enter but much more information returned 3

34 Does it work? - Example Analysis of impurities of an active pharmaceutical ingredient by conventional HPLC (4.6mmID x 250mm, 5.0µm): mau H3C N CH3 H 40 OH OCH 3 Main Compound 30 H3C N CH3 H 20 H3C N CH3 H OH OH OCH 3 Impurity A H C CH 3 N 3 H H3C N CH3 Br OH 10 Impurity D OCH 3 OCH 3 O CH 3 Impurity C Impurity B Bromanisole min

35 Does it work? Converting to a 4.6 x 100 mm, RRHT column:

36 Does it work? YES mau mau 35 mau Conventional HPLC min min 4.6 mm ID x mm, 1.8µm 5.0µm Zorbax SB C min 5% B min 90% B min 90% B min 5% B min 5% B Speed Optimized Simple Conversion

37 Advanced Mode: Select worst case viscosity for ACN/water at 40ºC 0.75 cp

38 Advanced mode, Simple Conversion

39 Advanced mode, Resolution Optimized

40 Using RRHT and other Low Volume HPLC Columns Effectively on Agilent 1200 Infinity Series and 1100 Use data acquisition rate of 0.1 sec Use DAD SL for 80 Hz data acquisition Short lengths of 0.12 mm i.d. tubing or smaller (watch pressure) Thermostated column compartment plumbed through 3 µl side For 2.1 mm id columns at elevated temps, use low vol. heat exchangers For gradients - 80 µl (p/n ) or no mixer and injector bypass (not relevant for quaternary systems) Recommend micro and well plate autosamplers (ADVR on ) Otherwise, use injector program to reduce delay volume

41 1100 System Configuration for Ultra-fast LC Recommendations for System Setup and Connecting Capillaries 1100 Binary Pump (G1312A) 1100 WPS (G1367A) 3 µl heat exchanger 4.6mm ID, 1.8um 1100 DAD SL (G1315C) Waste 1100 TCC (G1316A) RRHT Column Replace standard mixer of Binary Pump with 80 µl filter (p/n ) to reduce delay volumne Use low volume, 3ul heat exchanger of TCC G1316A to thermostate eluent For 4.6 and 3mm columns use shortest possible 0.17mm ID connecting capillaries Note: In ultra-fast applications the typical flow rate range using 4.6 and 3mm ID columns is 1-5 ml/min. At such higher flow rates the larger delay volume of 0.17mm ID capillaries doesn t have a measurable negative impact on chromatographic performance. For 2.1 and 1mm columns use shortest possible 0.12 or 0.1mm ID capillaries Note: In ultra-fast application the typical flow rate range using 2.1 and 1 mm ID columns is between ml/min. At these lower flow rates smaller ID connecting capillaries should be used to minimize system delay volume and extra column peak dispersion/band broadening. Inlet tubing of the flow cell should be directly connected to the column. Note: If this is not possible an appropriate low-volume connection should be used (capillary of small ID, i.e mm or 0.17mm and ZDV-union).

42 Optimizing Gradient Separations With 1.8 um RRHT Columns: 10 X Faster Analysis Conditions: Column: SB-C18, Dimensions listed below, Gradient: 10 90% ACN/25mM H 3 PO 4, Gradient time: t G, as noted CPAH s = Chlorphenoxyacid herbicides environmental sample C. RRHT SB-C x 50mm, 1.8um Temp: 50 C Flow: 1 ml/min Gradient (t G ): 2.4 min Rapid Resolution SB-C x 150mm, 3.5um Temp: 25 C Flow: 1.0 ml/min Gradient (t G ) : 18 min B. Key Parameters Particle size Flow Rate Gradient Time Column Length Column ID Temperature A. SB-C x 250mm, 5um Temp: 25 C Flow: 1mL/min Gradient (t G ): 30 min Sample: CPAH= Chlorophenoxy herbicides : Picloram, Chloramben, Dicamba, Bentazon, 2,4-D, Dichlorprop, 2,4,5-TP, Acifluorfen. min min R s optimized

43 Translation to 3.0 x 150 mm, 3.5 um 18 min gradient

44 3.0 x 150 mm, 3.5 um, Resolution Optimized

45 mau Scaling Gradients from 4.6 mm I.D. Columns to Solvent Saver Plus Column-Organic Acids mau x 250 mm SB-C18, 5-um x 150 mm SB-C18, 3.5-um mau x 100 mm SB-C18, 3.5-um 7 57 ml solvent used 25 ul std injection 1.5-mL/min; t g = 38 min 8 min 33 ml solvent used 15 ul std injection 1.0-mL/min; t g = 33 min min 10.5 ml solvent used 6 ul injection with INJ Program 0.5-mL/min; t g = 21min min Analytes 1) gallic acid 3) protocatechuic acid 2) hydrocaffeic acid 4) gentisic acid 5) syringic acid 6) sinapinic acid 7) salicylic acid 8) caffeic acid

46 1200 Infinity Calculator

47 Key Equations Flow Conversion Pressure estimate Injection volume conversion Column efficiency Gradients

48 How conversion works for flow Flow modification, for columns of different diameters 2 Diam. Flow column2 = col. 1 Diam. column1 Flow = Flow col. 2 i.e mm 1.0ml/min = 0. 21ml/min 4.6mm Page 48 of 26 RRLC How-to Guide Agilent Restricted June 11, 2008

49 Reference for Common Column Diameters Maintain Equivalent Linear Velocity for Different Column IDs Column Type Column ID Flow Rate Analytical 4.6 mm 1.0 ml/min Solvent Saver 3.0 mm 0.42 ml/min NarrowBore 2.1 mm 0.21 ml/min MicroBore 1.0 mm 47 µl/min Capillary 0.5 mm 12 µl/min Capillary 0.3 mm 4.2 µl/min Nano 0.1 mm 472 nl/min Nano mm 266 nl/min Flow rate column 2 = (diameter column 2) 2 /(diameter column 1) 2 x Flow rate colum Maintain equivalent mobile phase linear velocity when changing column diamete Page 49

50 Conversion for injection volume Keep Injection volume proportional to column volume Volumecolumn2 Inj.Vol. col. 1 = Volume column1 Inj.Vol. col. 2 Zorbax column volume = 3.14 x r 2 x L x 0.6 (r and L in cm) 0.4mlcolumn2 i.e. 20µl col. 1 = 4µl 2.0ml column1 col. 2 Page 50 of 26 RRLC How-to Guide Agilent Restricted June 11, 2008

51 How conversion works for time Run Time or Gradient Segment Time Adjustment Length Time column2 col. 1 = Time Length column1 col. 2 i.e. 150mm 25min. = 15min. 250mm *assumes flow is proportional for columns 1 and 2 Page 51 of 26 RRLC How-to Guide Agilent Restricted June 11, 2008

52 Gradient Separations Any Decrease in Can be Offset by a Proportional Column length Column volume (i.d.) Φ (same column) Decrease in t G or F Increase in Φ Decrease in t G or F Increase in Φ Decrease in t G or F t G F k* = S Φ Vm Separation Fundamentals Agilent Restricted December 11, 2007

53 Efficiency N - Number of theoretical plates This is one case where more is better! Plates is a term inherited from distillation theory. For LC, it is a measure of the relative peak broadening for an analyte during a separation N = 16 t 2 R w or N = L H Column length HETP A Number of Theoretical Plates Separation Fundamentals Agilent Restricted December 11, 2007

54 What About Pressure? Pressure Increases Exponentially with Decreasing Particle Size P η L v d θ Equation For Pressure Drop Across an HPLC Column p P = η L v θ d p 2 Many parameters influence column pressure = Pressure Drop = Fluid Viscosity = Column Length = Flow Velocity = Particle Diameter = Dimensionless Structural Constant of Order 600 For Packed Beds in LC Particle size and column length are most critical Long length and smaller particle size mean more resolution and pressure Separation Fundamentals Agilent Restricted December 11, 2007

55 Summary Method conversions are an opportunity to increase lab productivity significantly. The Agilent Method Translator is easy to use and can make your method translations to smaller columns much quicker and successful. Maintain resolution and avoid any change of selectivity Proper choice of column size and efficiency, Careful selection of method parameters. System optimization may be required to use smaller columns and/or smaller particle sizes (tubing, flow cell, delay volume, data rate) Increased operating pressure may result ensure that system has adequate capacity for standard and increased pressure operation across the flow range of routine and optimized methods

56 Appendix Method translator link Step-by-step Upgrade of 1100 to 1200 RRLC Pt. 1, 2.1mm ID columns, Pub. No EN Pt. 2, 4.6mm ID columns, Pub. No EN Optimize Data Sampling Rate, Pub. No EN Agilent 1200 Series RRLC and RRLC/MS Optimization Guide, Pub. No. G Plug & Play Fast & Ultra-fast Separations Using 3.5um RR and 1.8um RRHT Columns, Pub. No EN

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