Practical Applications of Method Translation Using the Agilent Method Translation Tool. eseminar and Workshop
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1 Practical Applications of Method Translation Using the Agilent Method Translation Tool eseminar and Workshop Thomas J. Waeghe, Ph.D. Inside Application Engineer Agilent Technologies Life Sciences and Chemical Analysis Title
2 Objectives for Today s e-seminar and Workshop Demonstrate the practical use of the Agilent Method Translation tool for fast, easy, and successful method transfer to smaller volume columns Review and discuss the variables that are most important for successful translation of isocratic and gradient methods Review several completed method transfer examples using the Agilent Method Translation Tool Work through several real examples submitted by customers
3 Agenda for Today Successful Method Translation Separation Goals and Method Performance Criteria Isocratic separations Which instrumentation must you have to get started Which instrument and method parameters afford optimal results Considerations for successful implementation Agilent Method Translator for isocratic separations Gradient separations Review of gradient retention parameters Instrument considerations Agilent Method Translator for gradient separations Workshop with submitted examples
4 Separation Goals and Method Performance Criteria Separation Goals and System Suitability Resolution ( 2) Method Performance Criteria Accuracy 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) Precision Repeatability Intermediate precision Reproducibility Robustness Selectivity/Specificity Linearity aka Figures of Merit AVOID THESE for System Suit. Criteria Column efficiency (theoretical plates) Absolute retention Range Quantitation Limit (LOQ, 10x S/N) Detection Limit (LOD, 3x S/N)
5 An Approach for Isocratic Method Translation Assess and document current method performance and parameters Assess current instrument configuration Set performance goals for method to be translated Determine which column geometry will provide necessary efficiency Instrument needs vs. method performance goals will depend on requirements for column size and particle size to get desired R s Instrument extracolumn volume, 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 and parameters, and instrument configuration Current Method Performance Limiting Resolution for Critical Pair(s) Peak Shape(s) (USP Tf) Injection Repeatability (pooled RSD duplicate injs) Signal-to-Noise Ratio Instrument Configuration Extracolumn 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: Agilent 1100 quaternary system Autosampler: Standard autosampler TCC: 1100 standard Detector: 1100 DAD, max. data rate 20 Hz Typical setting, PW = 0.05 min. Flow Cell: 13 μl, 10 mm path length Flow Rate: 1.0 ml/min. Column temp. 23ºC Goals: 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 to apply the 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 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 extracolumn 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
12 Agilent Method Translator
13 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
14 Use 5 ul flow cell and 0.12 mm id tubing Improvement in N effective
15 Speed Optimized at 3 ml/min Adjust % max. pressure until desired flow rate Adjust to 3 ml/min Click radio button to allow % max pressure adjustment
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 Choosing the flow cell size
19 Peak Width Setting Response Time Data Rate and Sensitivity Don t use for > 0.15 sec peak width! > 0.15 sec > 0.3 sec > 0.6 sec > 1.2 sec > 3 sec > 6 sec > 12 sec > 24 sec > 51 sec Peak Width = Peak Width at 50% Peak Height Set at fastest rate and then decrease data rate until peak width increases and S/N is optimum Recommended settings in ultra-fast LC with 50% peak width between 0.15 and 0.6 sec For 50% peak width between 0.6 and 1.2 sec Notes: Noise level changes ~ proportional to the square root of the change in data rate. For optimum selectivity and sensitivity the Peak Width should not be chosen smaller than necessary. For 50% peak width between 0.3 and 0.6 seconds Peak Width of > min is recommended, which correspondes to 40Hz data rate. Only for peaks narrower than 0.3sec at half height, Peak Width of > min (80Hz data rate) should be used. For highest sensitivity in ultra-fast LC the slit can be increased to 8 or 16nm.
20 Detector Data Acquisition Rates Effects on Peak Width, Resolution and Peak Capacity in UFLC Peak Widths / sec Peak Width [s] Peak Capacity Data Rate [Hz] Peak Capacity Data Rate Peak Width Resolution Peak Capacity 80 Hz Hz Hz Hz Hz Peak Widths / sec Peak Width [s] Resolution (4,5) Data Rate [Hz] Resolution 80Hz versus 20Hz Data Rate: 40% Peak Width => +40% Peak Capacity + 30% Resolution => + 70% Apparent Column Efficiency 80Hz versus 10Hz Data Rate: 120% Peak Width => +120% Peak Capacity + 90% Resolution => +260% Apparent Column Efficiency
21 Data Rate and Slit Width Effect on S/N Ratio (DAD and MWD, VWD data rate) S/N can be optimized with data rate Slit width can be increased to improve S/N (2 ul and 5 ul cells)
22 Break 1: Gradient Methods Next Questions?
23 Translating Gradient Methods
24 Advantages of Gradient Elution Complex samples are analyzed in a single HPLC run Analysis time is reduced All 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)
25 100% B t g = 5 Gradient Steepness Affects Retention (k*) and Resolution 0% B 0% B 0% B 100% B t g = 10 t g = 20 This equation governs gradient retention and selectivity 100% B 87t g F k* = ΔΦ V m S 1/k* gradient steepness = b ΔΦ = 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) 100% B 0% B t g = 40 S 4 5 for small molecules 10 < S < 1000 for peptides and proteins Time (min) P1.PPT
26 To Increase Gradient Resolution by Changing Gradient Retention (k*) Use: A longer gradient time A shorter column A higher flow rate A shorter organic range t G V m F %B k* = 87 t g F S (Δ%B) V m
27 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
28 Transferring Gradient Methods to Smaller Diameter Columns and to Different Instruments To Transfer Gradient Separations, Average retention factor for k* must match, and Effective delay times must match (or ratio of gradient volume/column volume must be same) Also Important for Gradient Separations Column re-equilibration time (post time) System/Dwell volume volume from point of mixing to column How to measure and account for it Correct for differences between instruments
29 Gradient Separations: Considerations When Translating Existing Gradient methods Isocratic Separations Sample load (V inj, [analyte]) Sample solvent strength Extracolumn 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 k* = 87 t g F S (D%B) V m Gradient Separations Same as Isocratic Separations plus Delay Volume Same instrument (different pressures) Different instrument (for example, Capillary 1100 vs. Binary 1100) 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)
30 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.
31 Comparison of System Delay Volumes Quat Bin. Pump w/o mixer w/ mixer n/a n/a Mixer 750 n/a n/a 420 Autosampler Standard Bypass V (loop) N/A V (inj) V (inj) V (inj) 6.2 Column compartment Standard Bypass 4.1 or ul 0 3 or or 6 0 Min Range Max Range
32 Delay Volume Comparison: 1100/1200 Series Binary Pump vs Series Binary Pump SL Binary pump SL (pressure range up to 600 bar): Standard delay volume configuration: μL (incl. damper and mixer) Low delay volume configuration: 120μL (virtual damper) Damper volume: μl Binary pump (pressure range up to 400 bar): Standard delay volume configuration: μL (incl. damper and mixer) Reduced delay volume configuration: ~200μL (damper needed) Damper volume: 180μl + 1μl per bar
33 All scaling calculations to transfer methods to RRHT, 1.8um particles are done using the Agilent Method Translator
34 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.
35 Features of the Agilent Method Translator Advanced mode all calculation parameters in your hands: [ml] [ml] More to enter but much more information returned
36 Does it work? - Example Analysis of impurities of an active pharmaceutical ingredient by conventional HPLC (4.6mmID x 250mm, 5.0µm): mau H 3C N CH 3 H 40 OH OCH 3 Main Compound 30 H3C N CH3 H 20 H3C N CH3 H OH OH OCH 3 Impurity A H3 C N CH3 H H3C N CH3 Br OH 10 Impurity D OCH 3 OCH 3 O CH 3 Impurity C Impurity B Bromanisole min
37 Does it work? Converting to a 4.6 x 100 mm, RRHT column:
38 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
39 Advanced Mode: Select worst case viscosity for ACN/water at 40ºC 0.75 cp
40 Advanced mode, Simple Conversion
41 Advanced mode, Resolution Optimized
42 How to Use Rapid Resolution HT and other Low Volume HPLC Columns Effectively on Agilent 1200 and 1100 HPLCs 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
43 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).
44 Stepwise Scale-up to Rapid Resolution LC From 1100 to 1200 RRLC in two steps Example 1 50 mm or shorter cols with 3.0 or 4.6 mm IDs 20 Hz max Quat Pump ALS (WPS?) Actual: 1100 Quat System Degasser TCC VWD/MWD/DAD 20 Hz max Step 1: 1100/1200 Bin SL System Degasser Bin Pump SL h-als SL TCC VWD/MWD/DAD + Speed + Resolution + MS-Compatibility + Solvent Saving + Compatible with conv. HPLC 80 Hz max Step 2: 1200 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 150 mm or shorter cols with 2.1, 3.0, 4.6 mm IDs Analysis Time Cycle times Peak Width N Column ID Column Length Flow rates Temperature Pressure > 5 min > 6 min > 3 sec 5-12, mm 50 mm ml/min 80 C 400bar > 1.5min > 2min > 1.5 sec 5-30, mm mm ml/min 80 C 600 bar > 0.2min > 0.4min > 0.2 sec 5-60, mm mm ml/min 100 C 600bar
45 Stepwise Scale-up to Rapid Resolution LC From 1100 to 1200 RRLC in two steps Example 2 50 mm or shorter cols with 3.0 or 4.6 mm IDs Actual: 1100 Bin System Degasser BinPump ALS ColCom VWD/MWD/DAD 80 Hz max Step 1: 1100/1200 DAD SL System Degasser binpump h-als SL ColCom DAD SL + Speed + Data Security & Traceability + Compatible with conv. HPLC 80 Hz max Step 2: 1200 Rapid Resolution System u-degasser binpumpsl h-als SL ColCom SL DAD SL + Speed + Resolution + Sensitivity + Solvent Saving + MS-Robustness + MS-Compatibility + Qualification (Degasser, ACE) + Compatible with conv. HPLC 150 mm or shorter cols with 2.1, 3.0, 4.6 mm IDs Analysis Time Cycle times Peak Width N Column ID Column Length Flow rates Temperature Pressure > 1 min > 2 min > 1.5 sec 8-12, mm 50 mm ml/min 80 C 400bar > 0.2min > 0.4min > 0.3 sec 5-22, mm mm ml/min 80 C 400 bar > 0.2min > 0.4min > 0.2 sec 5-60, mm mm ml/min 100 C 600bar
46 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 B. A Rapid Resolution SB-C x 150mm, 3.5um Temp: 25 C Flow: 1.0 ml/min Gradient (t G ) : 18 min SB-C x 250mm, 5um Temp: 25 C Flow: 1mL/min Gradient (t G ): 30 min min Sample: CPAH= Chlorophenoxy herbicides : Picloram, Chloramben, Dicamba, Bentazon, 2,4-D, Dichlorprop, 2,4,5-TP, Acifluorfen. min Key Parameters Particle size Flow Rate Gradient Time Column Length Column ID Temperature R s optimized
47 Translation to 3.0 x 150 mm, 3.5 um 18 min gradient
48 3.0 x 150 mm, 3.5 um, Resolution Optimized
49 mau Scaling Gradients from 4.6 mm I.D. Columns to Solvent Saver Plus Column-Organic Acids mau x 150 mm SB-C18, 3.5-um mau x 250 mm SB-C18, 5-um 3.0 x 100 mm SB-C18, 3.5-um 57 ml solvent used 25 ul std injection 1.5-mL/min; t g = 38 min 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
50 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
51 Workshop Examples Isocratic Gradient
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