Agilent Video Notes for Poroshell Method Transfer Video Manual
Contents
1. You ll see the injector needle move away Gently pry up the injector seat with a small screwdriver Do not pry the ring at the top of the injector seat Change capillary by pulling it through and threading the new needle seat in Properly tighten the capillary fitting with a small wrench Close the door and select end in ChemStation window The needle will reposition Capillary kits are available packaged with an assortment of usefully sized For RRHT optimization look for these kits For the 1100 use capillary kit 5065 9947 For the 1200 use capillary kit G1316 68716 To view kits go to www agilent com chem capillarykits Ensure Proper Connections Poorly installed fittings can lead to poor chromatographic results due to ECVs and unswept dead volumes as this example shows ilent Technologies Inc 2010 Page 5 of 7 LC troubleshooting poor peak shape improperly swaged tubing DADI A SF 54A RED 100 MERCURY 20 DATA MERCURY 2010 ECUPSE PLUS 3 4100 1 BUSUYBOM6 00007 0 J j Before troubleshooting Problem All peaks tail a Py Says k TN oc Somes 1 J DADIA a a een mn AND PRESS LOAD ECUPSE PLUS C38 a SUT ED 440 D i After troubleshootin a Solution g Replace tubing Cause Capillary tubing connecting ALS and column was swaged improperly on the ALS end ferrule was flush w2. 2 y Agilent Technologies Video Notes Transferring a Method from a Conventional 3 5 pm or 5 pm to an Agilent Poroshell 120 Column to Improve Analytical Speed and Resolution Ron Majors is Senior Scientist for Agilent s Columns and Supplies Division John Henderson is an Agilent Applications Scientist Instrument pressure limits are getting higher to take advantage of trends to new smaller particles and all the speed and resolution advantages they provide New instrumentation can provide significant new features and sensitivity to improve analyses Find out more about the latest advances at Agilent at www agilent com chem infinity However if you cannot upgrade your instrument in the nearterm transferring your methods from conventional 3 5 or 5 um columns to new Agilent Poroshell 120 columns can save your lab time and money today What is Poroshell 120 Poroshell 120 particles are manufactured by Agilent using a proprietary process that creates a superficially porous shell around a solid core These are different than sub 2 um particles re 0 5um shell EUER Poroshell 120 particles are 2 7 um in total size with a 1 7 2 7 um total particle size presi um solid core and a 0 5 um porous outer shell So you get analytical performance of a sub 2 um particle but at the pressure of a 2 7 um particle usually less than 400 bar eaee oa oP aats ea see 0 5 um shell eS Poroshell 120 columns will work on virtuall
3. ant to pay attention to the instrument configuration and method parameters to ensure you get the best results The key instrument parameters to address for optimized performance with Poroshell 120 are 1 Detector response time Data Collection Rate 2 Flowcell volume 3 Sample flow path tubing volume 4 Flow path fitting 5 Flow rate Set the Proper Data Collection Rate Set the data collection rate to the fastest setting such that signal to noise S N is not affected This may not be the fastest setting on the detector but should be close For best results when using small columns collect data at 40 Hz or more Newer Agilent detectors have data rates up to 80 Hz or 160 Hz and are great for Poroshell 120 columns too Slower data collection rate is practical when analytes retain longer e g k gt 4 Page 2 of 7 Comparison of Peak Efficiency on Poroshell 120 EC C18 4 6 x 100 mm with Different Data Collection Rates 0 es that illustrates how the data collection N 25 705 rate affects results If the data collection rate is too slow there Is a loss of N79 amin Nets E N 25 706 i J SDa N Here is a comparison of chromatograms efficiency and differences in peak shape aaa 24 2 may result an 60 40 MeCN Water 80 Hz 2 ul flow Cell 1200 SL Looking at it another way the table below illustrates the same point Response Hz Uracil Phenol 4 Chloro Naph
4. bing Once you have optimized LC flow with the lower volume tubing you don t need to change it back for higher volume columns See the Agilent Essential Chromatography and Supplies Catalog for tables that indicate which tubing is appropriate for the LC connections Agilent Technologies Inc 2010 Page 4 of 7 1 2 3 4 o 6 1 8 Ag Extra Column Effects on 2 1 x 100 mm Poroshell 120 Columns in See here on this example using a 2 1 x 100 mm Poroshell 120 column early eluters k lt 3 are affected more severely by extra z S f o wi z oS column broadening Tubing Volume ul 1 ul QC Mix Uracil Phenol k 0_5 4 Chloronitrobenzene k 2 Napthalene k 3 8 55 MeCN 45 Water 0 55 ml min micro flow cell column part number 695775 902 The autosampler needle assembly is an important part of the flow path that can be reduced in volume It s easy to do this yourself If you are using Lab Advisor select your instrument from the opening menu and select tools Select the autosampler module and a window will open that includes the needle seat option Skip to Step 5 For older instruments using ChemStation go to the Method amp Run Control view then to the Diagnosis view use top tabs Under the Maintenance menu scroll down to the Wellplate Sampler WPS then over to WPS Maintenance Positions Select start under Change needle seat
5. ical support lc column Support agilent com for assistance You can request a catalog at www agilent com chem getguides Flow Cell Choices evaluated with a 2 1 x 100 mm Poroshell 120 EC C18 Column 25000 20000 Efficiency N Column part number k 695775 902 1 ul QC Mix Uracil Phenol k 0 4 4 Chloronitrobenzene k 1 6 Napthalene k 3 55 MeCN 45 Water 0 55 ml min micro flow cell Flow Cell Choices evaluated with a 3 0 x 100 mm Poroshell 120 EC C18 Column 25000 20000 15000 10000 Efficiency N 5000 0 80 Hz 40Hz 20Hz 10Hz S5Hz Data Collection Rate Hz 2 ul QC Mix Uracil Phenol k 0_5 4 Chloronitrobenzene k 2 Napthalene k 3 8 60 MeCN 40 Water 0 8 ml min micro flow cell Column part number 695975 302 Minimize Extra Tubing Volume Larger flow cells used at fast data Baai collection speeds can be used with large eee columns as shown with this 3 x 100 mm i Poroshell 120 EC C18 column 15000 There is a 30 loss of efficiency with a 10 mm 13 pl 10 mm 13 uL standard flow cell With 10000 m 6 mm Sul o 3mm 2 ul 2 1mm ID columns it is best to use a 3 5000 d mm 2 uL micro flow cell 0 kK 0 4 k 1 6 k 3 Another ECV that can reduce column efficiency are the connecting capillary tubes Use the shortest length of tubing possible for all connections Use Red 0 12 mm id tubing instead of Green 0 17 mm id as it is only half the volume of the larger internal diameter ID green tu
6. ith end of tubing causing a void Be sure the tubing Is not too long or too short into the ferrule Wrong too long Wrong too short Ferrule cannot seat properly Mixing Chamber lf Dimension X is too short a dead volume or x lf Dimension X is too long leaks will occur re mixing chamber will occur Alternately finger tight fittings are more convenient allowing for adjustment of the end fitting properly without wrenches Use polyketone fittings on column connections with Poroshell 120 PN 5042 gM 8957 Polyketone fittings and Poroshell 120 columns can withstand 600 bar pressures Note that PEEK fittings can only withstand pressures up to 400 bar Optimize the Flow Rate Optimize the flow rate to achieve the desired efficiency N or peak capacity Pc Suggested starting flow rates for Poroshell 120 2 1 mm ID start with 0 42 mL min 3 0 mm ID start with 0 85 mL min 4 6 mm ID start with 2 mL min Agilent Technologies Inc 2010 Page 6 of 7 You ll want to use a higher flow rate with Poroshell 120 Increase flow rate F 10 and compare efficiency N to the starting flow rate If efficiency has increased continue to increase F and compare efficiency N to determine the best flow rate Van Deemters 60 40 CH CN H O with RRLC measuring heptanophenone 0 015 0 005 Other chromatographic considerations Agilent Poroshell 120 EC C18 4 6 mmx 50 mm 2 7 um CFU 1161 Agilen
7. t ZORBAX Eclipse Plus C18 4 6 mm x 50 mm 1 8 um USUXG04047 Agilent JORBAX Eclipse Plus C18 4 6 mm x 50 mm 3 5 um USBH006534 ote Agilent ZORBAX Eclipse Plus C18 4 6 mm x 50 mm 5 0 um USFAD07925 As this van Deemter plot illustrates the optimal flow rate for Poroshell 120 is faster than for 5 um or 3 5 um columns that they replace Properly scale the gradient conditions and the injection volume to the new smaller column volume to quickly transfer the method and avoid overloading Be sure to minimize sample dispersion in the column To do this a Use an injection solvent that is weaker than the mobile phase especially when using an isocratic method b Gradients can minimize dispersion but be aware of possible effects Poroshell 120 columns can achieve similar efficiencies as sub 2 um columns with substantially less pressure Excellent results can be achieved on Agilent 1200 RRLC or 1290 Infinity Summary In order to achieve best performance 1 Optimize data collection rate 40 Hz detector with fast response time 2 Use the smallest flow cell available 3mm micro flow cell works best when using 2 1 I D columns 3 Minimize extra column volume flow cell and connecting tubing are the biggest contributors but needle seats should be replaced with smaller volume seats Red tubing has half the volume of green tubing 4 Take care to make proper tubing flowpath connections 5 Optimize flow rates for bes
8. t performance Start with flow rate 2 ml min on a 4 6 mm column 0 85 ml min on a 3 0 or 0 42 ml min on 2 1 mm column If you need more help contact Agilent technical support Ilc column support agilent com for assistance Agilent Technologies Inc 2010 Page 7 of 7
9. thalene Time Nitrobenzene Peak Width 0 008 Peak Width 0 011 Peak Width 0 021 Peak Width 0 031 0 02 80 16 240 21 641 25 706 25 061 40 15 414 22 028 25 705 25 080 12 755 18 647 24 699 24 506 6 965 12 658 21 712 23 872 Column Poroshell 120 EC C18 4 6 x 100 mm Instrument 1200 SL 2um flow cell Flow Rate 2 00 mL min Sample 2 uL injection Mobile Phase 60 40 MeCN Water For narrower 2 1 mm ID columns the same principles apply Again you ll see that you should collect data at at least 40 Hz Slower data collection is practical when analytes retain longer k gt 4 Flow Cell Selection Extra column volumes ECVs in your sample flow path can reduce the efficiency observed for Poroshell 120 columns The flow cell volume is one of these ECVs Smaller volume flow cells such as the semi micro 6 mm 5 ul or micro 3 mm 2 pl are recommended for best performance With 2 1 mm columns it is best to use the 3 mm micro flow cell Standard flow cell with RFID tag 10 mm p n G1315 60022 Semi micro flow cell with RFID tag 6 mm p n G1315 60025 Micro flow call with RFID tag 3 mm p n G1315 60024 There are a number of different flow cells available and your needs may vary depending on whether you are using an Agilent 1100 or 1200 Consult the Essential Chromatography and Supplies Catalog under Agilent Technologies Inc 2010 Page 3 of 7 LC and LC MS Detectors for more information Contact Agilent techn
10. y any HPLC and enable you to scale methods from conventional 3 or 5 um columns with a few minor modifications to the instrument Today you can purchase Poroshell 120 columns in an endcapped EC C18 phase and a non endcapped StableBond SB C18 phase The selectivity will be the same or very similar to your current C18 column More phases are coming soon Realizing Time Savings with Poroshell 120 See the following comparison examples of an analysis of Naproxen using a conventional 5 um column then two different Poroshell 120 columns Ultimately the time savings was 450 for this analysis Additional savings include a 77 reduction in solvent usage Agilent Technologies Inc 2010 Page 1 of Agilent Technologies Inc 2010 USP Method for Naproxen Tablets 4 5X Faster Analysis on Poroshell 120 Method Requirement N gt 4000 Rs better than 11 5 Common Conditions 4 6 x 150 mm L1 Eclipse Plus oe e 5um Mobile Phase 50 49 1 MeCN H20 Acetic Acid i PN 959993 902 i Flow Rate 1 2 mL min 20 ul injection Peak 1 Naproxen 2 Butyrophenone Rs 14 9 N 14 885 6 7 8 9 4 6 x 100 mm L1 Poroshell 120 EC C18 2 7 um N 21 046 PN 695975 902 _N 20 676 13 67 ul injection t 2X Faster P 238 bar 6 7 4 6 x 50 mm L1 N 11 281 Poroshell 120 EC C18 2 7 um 5 7 PN 699975 902 4 5X Faster N 12 051 6 7 ul injection Rs 12 6 P 133 bar Transferring Your Method to Poroshell 120 It s import
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Agilent Video Notes for Poroshell Method Transfer Video Manual