Agilent Evaluation of linear step gradient performance retention time precision of the Agilent 1200 Series Rapid Resolution LC system Manual
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1. Water B ACN Gradient 35 to 95 B in 5 min hold at 95 B for 1 min Stop Time 6min Post Time 2 min Flow Rate 1 5 mL min Injection volume 50 pL Wash 10 s for exterior of needle Thermostatted column compartment Detector Temperature 50 C 13 pL cell 20 Hz data acquisition Peak width lt 0 01 min Slit 8 nm Signal 245 10 nm ref 360 80 nm ee Retention time precision of Agilent 1200 Rapid Resolution LC system using the low delay volume configu ration In the previous section the reten tion time precision of the stan dard delay configuration was eval uated In this section the retention time precision of the low volume delay configuration will be evalu ated The analyses were run at high and low flow rates as well as at high and low temperatures For all low flow rate applications the relative standard deviation for the retention times was typically less than 0 07 For high flow rate and ultra fast applications the relative standard deviation for the retention times was typically less than 0 14 see figure 3 and also literature reference 1 Chromatographic conditions Solvent A Water B ACN Temp 40 C 80 C Flow 0 35 mL min 1 20 mL min 2 0 mL min Gradient 0 00 min 35 B 2 60 min 95 B 3 20 min 95 B 3 21 min 35 B Time values for F 0 35 mL min For all other flow rates times are scaled so that time x flow 0 90 mL Stop time 3 20 min Post time 2 00 min W2. e Agilent 1200 Series diode array detector SL for 80 Hz operation including new data protection tool e ZORBAX SB C 18 columns with different internal diameters and lengths packed with 1 8 7m particles Pump design The Agilent 1200 Series binary pump SL is designed to meet the demands of high throughput and highest performance for optimum resolution and lowest pump ripple for a wide range of applications on narrow or standard bore columns Figure 1 shows a schematic of the 1200 Series binary pump SL The delay volume can be configured according to the application needs The standard configuration is best suited for highest performance in terms of noise and resolution In this stan dard configuration the mixer and damper are in the flow path and the delay volume is between 600 and 800 pL depending on the pressure This configuration is 2 Flow Path D Std delay volume 600 800 uL delay Low delay volume 120 uL delay S J Figure 1 Standard and low delay volume configurations of the Agilent 1200 Series binary pump SL suited for applications where low est noise and highest resolution for optimum quantification is needed For applications where a low delay volume is needed the damper and the mixer can be switched out of the flow path resulting in a delay volume of 120 pL This configuration is best suited for application where speed and lowest peak dispersion are needed To enable these chan
3. Evaluation of linear and step gradient perfor mance and retention time precision of the Agilent 1200 Series Rapid Resolution LC system Technical Note Introduction A main performance criterion of any LC system is precision of retention times which is influ enced primarily by the pumping device It is important that the selected flow is delivered precise ly and that for gradient operation the mobile phases are mixed reliably over the complete gradient range In this Technical Note the precision of retention times for gradient and isocratic applications is evaluated Further based on tracer experiments it is demonstrated that linear and stepwise gradients are delivered with excellent accuracy and precision The experiments also show that the variability of the delay volume meets the demands of applications using narrow bore columns as well as standard bore columns EE Agilent Technologies Experimental Equipment An Agilent 1200 Series Rapid Res olution LC system was used with the following modules e Agilent 1200 Series binary pump SL with vacuum degasser for applications using 1 8 ym parti cle columns up to 150 mm length and with internal diame ters from 2 1 to 4 6 mm e Agilent 1200 Series high perfor mance autosampler SL for high est area precision e Agilent 1200 Series thermostat ted column compartment SL with wide temperature range from 10 degrees below ambient up to 100 C
4. UV using 2 1 mm ID columns Retention time precision of Agilent 1200 Rapid Resolution LC system for gradient LC analysis Retention time precision was evaluated for both pump configu rations under gradient and isocrat ic conditions using standard bore and narrow bore columns In the first example the standard delay configuration was tested using a standard bore column A phenone mix was analyzed using gradient conditions from 35 to 95 A 100 x 4 6 mm column and a flow rate of 1 5 mL min were selected These conditions are close to con ditions used in conventional LC analysis Figure 2 shows 10 runs overlaid demonstrating the reten tion time precision obtained for this application The run time was 6 min The evaluation of the reten tion time precision was found to be less than 0 07 RSD mA 25 THT TL OMMI AAAA OUA AAMA TAAL AIII AAA oe eee PERPRREEEE u i i r u Overlay of 10 runs Injection volume 50 uL Injected amount 80 ng RSD RT lt 0 07 Figure 2 Overlay of 10 chromatograms Chromatographic conditions Test sample Column Pump Autosampler Set of 9 compounds 100 ng uL each dissolved in water ACN 65 35 1 Acetanilide 2 Acetophenone 3 Propiophenone 4 Butyrophenone 200 ng mL 5 Benzophenone 6 Valerophenone 7 Hexanophenone 8 Heptanophenone 9 Octanophenone 100 x 4 6 mm ZORBAX SB C 18 1 8 um for 600 bar operation Solvent A
5. avelength 245 nm 8 Ref 450 nm 100 Slit 8 nm Peak width gt 0 0025 min 0 05 s response time 80 Hz Spectra All 190 500 nm bandwidth 1 nm Injection vol 1 0 pL Injector Overlapped injection Automatic delay volume reduction Sample flush out factor 10 Needle wash 5 s Sample Phenone mix 0 1 pg L each 0 2 ug uL of Butyrophenone g N Limit QA QC 0 500 0 45 n E ES RT Precision n 10 EES 0 350 Flow 0 35 mL min T 40 C 5 S ea 0900 Flow 0 35 mL min T 80 C SG a A 0 250 Flow 1 20 mL min T 40 C 2262 S Flow 2 00 mL min T 80 C aags a 0 200 5 2233 0 150 Seat 0 100 BEs oo a it HN lt wt a ii 0 000 il til all nll ali amp O amp Sv s Ss w Se S lt SS rs amp oF SF oe O R Q SX SR S amp s amp We S os SF aw om et Ca Average RSD J Figure 3 Statistical evaluation of the retention time precision for the repeated analysis of a nine com pound mixture under various conditions using the low delay configuration of the Agilent 1200 Series Rapid Resolution LC system Low flow Low temp mAU F 0 35 mL min T 40 C Low flow High temp mAU F 0 35 mL min T 80 C 05 1 15 2 25 High flow High temp F 2 00 mL min T 80 C High flow Low temp F 1 20 mL min T 40 C 350 01 02 03 O4 05 min Figure 4 Overlaid chromatograms of the repeated analysis of a 9 compound mixture under various conditio
6. ges the pressure transducer is separated from the damper Both pump heads have an addi tional damping coil each of 500 pL volume and an additional outlet valve This does not add delay volume during gradient analysis because mixing occurs later in the flow path The pump can be used up to pressures of 600 bars providing for the use of sub 2 micron particle columns up to 150 mm length In the standard delay volume configuration the mixer and damper are in the flow path To change to the low delay volume configuration the flexible 0 17 mm id capillary between the mixer and the upper position of the pressure sensor is disconnect ed Then the capillary originally attached to the mixer is connected to the pressure sensor The damper and mixer are now not in the flow path Degassing is recom mended for all application ranges to ensure optimum performance The Agilent 1200 Series micro degasser SL has 4 channels and an internal volume of 1 mL Each channel degasses the solvents continuously The following sec tions discuss the performance of both pump configurations with and without mixer and damper in the flow path The configuration with mixer and damper in the flow path delivers optimum resolution and lowest pump ripple and is best suited for the analysis of complex samples where highest resolution and lowest system noise is needed The configuration without damper and mixer in the flow path is best suited for LC MS or LC
7. itched out of the flow path References 1 Performance characteristics of the Agilent 1200 Series LC sys tem Agilent Application Note Publication Number 5989 4489EN 2006 Angelika Gratzfeld Huesgen and Michael Frank are Application Chemists at Agilent Technologies Waldbronn Germany www agilent com chem 1200rrht Copyright 2006 Agilent Technologies All Rights Reserved Reproduction adaptation or translation without prior written permission is prohibited except as allowed under the copyright laws Published April 1 2006 Publication Number 5989 5031EN os i a Agilent Technologies
8. ns Chromatographic conditions Sample Isocratic standard sample 1 dimethylphthalate 0 15 wt 2 diethylphthalate 0 15 wt 3 biphenyl 0 01 wt 4 o terphenyl 0 03 wt Column 2 1 x 100 mm ZORBAX SB C 18 1 8 pm Mobile phase Water acetonitrile 30 70 Injection vol 3 pL Flow rate 0 5 mL min Retention time precision of Agilent 1200 Rapid Resolution LC system for isocratic LC analysis In this example isocratic condi tions were selected see condi tions in figure 5 A narrow bore column was chosen and the flow rate was set to 0 5 mL min In fig ure 5 an overlay of 10 runs is shown for the analysis of test mix ture Typically the relative stan dard deviation for this type of application is less than 0 07 for retention times Delay volume system ripple precision and accuracy of gradients To evaluate pump performance tracer experiments are a frequent ly used tool to evaluate the system ripple at different gradient mix tures The delay volume and the accuracy and the precision of gra dients are also evaluated using step gradients and linear gradi ents Figure 6 shows a step gradi ent from 0 to 100 in 10 steps using the Agilent 1200 Series Rapid Resolution LC system con figured for standard delay volume Overlay of 10 runs Injection volume 3 uL RSD for RT lt 0 07 0 L 05 1 15 2 25 3 35 min Figure 5 Precision of retention times on 2 1 mm column isocratic elu
9. nt 1200 Series Rapid Resolution LC system at 200 bar backpressure linear gradient from 100 to 0 3 overlays Conclusion The data presented in this note show that the precision of reten tion time of the Agilent 1200 Series binary pump SL pump is excellent for a wide range of LC applications using either narrow bore or standard bore columns Typically a precision for retention times of less than 0 07 relative standard deviation can be expect ed for isocratic and gradient appli cations using the standard delay volume configuration of the pump For the low delay volume configuration the retention time precision is typically less than 0 07 relative standard deviation for more conventional flow rates and is typically less than 0 14 for ultra fast applications with run times less than 1 min Accuracy and precision of linear and step gradients are excellent Ripple for the standard delay vol ume configuration is typically less than or equal to 0 01 The low delay volume configuration has a system ripple typically less than 0 05 The delay volume variabili ty supports the needs for low delay volume applications as well as the demands for delay volume needed for more conventional val idated methods The standard delay volume configuration with the autosampler in the flow path has a delay volume of 860 pL at 200 bar backpressure The low delay volume configuration has a delay volume of 120 pL when the autosampler is sw
10. rom 0 to 10 The precision for the 3 step gradi ent runs is excellent and the sys tem ripple for the 1 steps is less than or equal to 0 011 related to the 100 step Figure 8 shows an overlay of 3 linear gradients using the low delay volume configura tion of the Agilent 1200 Series Rapid Resolution LC system The linear gradient was run from 100 to 0 tracer The overlay shows that precision for a linear gradient is also excellent for the low delay volume configuration The delay volume in this configuration is about 420 pL with autosampler in the flow path If the autosampler is taken out of the flow path the delay volume is about 120 pL The system ripple with the autosam pler in the flow path is typically less than 0 05 related to the 100 step fia mAU 204 Overlay of 3 runs Step gradient from 1 to 10 in 1 steps 17 54 154 12 54 J 104 i Delay volume with ALS in the flow path 945 ul 7 54 210 Bar Backpressure Ripple on step 1 0 011 Ripple on step 3 0 009 Ripple on step 5 0 011 254 Ripple on step 6 0 010 Ripple on step 9 0 010 r r T r T Xe 0 10 20 30 40 50 min Figure 7 Precision and ripple using the standard delay configuration of the Agilent 1200 Series Rapid Resolution LC system at 210 bar backpressure 0 to 10 in 1 steps 74 min 62 64 66 68 70 72 Figure 8 Precision using the low delay volume configuration of the Agile
11. tion overlay of 10 runs RSD of RT less than 0 07 Norm 2004 1504 100 Figure 6 Accuracy delay volume and ripple using the standard delay configuration of the Agilent 1200 Series Rapid Resolution LC system at 210 bar backpressure 0 to 100 B gradient in 10 steps Overlay of theoretical and real step gradient from 0 to 100 in 10 steps 210 Bar backpressure Ripple on step 10 0 010 Ripple on step 30 0 007 Ripple on step 50 0 006 Ripple on step 80 0 010 Delay volume with ALS in the flow path 860 uL Chromatographic conditions Column Mobile phases Flow rate Step gradients steps Linear gradient Temperature Detection Restriction capillaries A water B water 0 5 acetone 1 mL min 0 B to 10 B in 1 steps each step held for 5 min 0 B to 100 B in 10 each step held for 5 min 100 to 0 in 5 min 36 C Signal 265 20 nm reference 360 100 nm Slit 4 nm Peak width 0 01 min 13 uL cell With the autosampler in the flow path the delay volume is about 860 pL The system ripple is less than or equal to 0 01 related to the 100 step The overlay with theoretical step gradient shows excellent agreement with the real step gradient with regard to the step heights Figure 7 shows an overlay of 3 step gradients using the standard delay volume config uration of the Agilent 1200 Series Rapid Resolution LC system The steps are 1 f
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Agilent Evaluation of linear step gradient performance retention time precision of the Agilent 1200