Evaluation of linear and step gradient performance and retention time precision of the Agilent 1200 Series Rapid Resolution LC

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Evaluation of linear and step gradient perfor-mance and retention time precision of theAgilent 1200 Series Rapid Resolution LC system

Introduction

A main performance criterion ofany LC system is precision ofretention times, which is influ-enced primarily by the pumpingdevice. It is important that theselected flow is delivered precise-ly and that for gradient operationthe mobile phases are mixed reliably over the complete gradient range. In this TechnicalNote the precision of retentiontimes for gradient and isocratic

applications is evaluated. Further,based on tracer experiments, it is demonstrated that linear and stepwise gradients are deliveredwith excellent accuracy and precision. The experiments alsoshow that the variability of thedelay volume meets the demandsof applications using narrow-borecolumns as well as standard-borecolumns.

Technical Note

min3 4 5

suited for applications where low-est noise and highest resolutionfor optimum quantification isneeded. For applications where alow delay volume is needed thedamper and the mixer can beswitched out of the flow path,resulting in a delay volume of 120 µL. This configuration is bestsuited for application wherespeed and lowest peak dispersionare needed. To enable thesechanges the pressure transduceris separated from the damper.Both pump heads have an addi-tional damping coil each of 500 µLvolume and an additional outletvalve. This does not add delayvolume during gradient analysisbecause mixing occurs later inthe flow path. The pump can beused up to pressures of 600 bars,providing for the use of sub-2-micron particle columns up to150-mm length. In the standarddelay volume configuration themixer and damper are in the flow

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path. To change to the low delayvolume configuration, the flexible0.17-mm id capillary between themixer and the upper position ofthe pressure sensor is disconnect-ed. Then, the capillary originallyattached to the mixer is connectedto the pressure sensor. Thedamper and mixer are now not inthe flow path. Degassing is recom-mended for all application rangesto ensure optimum performance.The Agilent 1200 Series microdegasser SL has 4 channels and aninternal volume of 1 mL. Eachchannel degasses the solventscontinuously. The following sec-tions discuss the performance ofboth pump configurations; withand without mixer and damper inthe flow path. The configurationwith mixer and damper in the flowpath delivers optimum resolutionand lowest pump ripple and isbest suited for the analysis ofcomplex samples where highest

Experimental

EquipmentAn Agilent 1200 Series Rapid Res-olution LC system was used withthe following modules:• Agilent 1200 Series binary pump

SL with vacuum degasser forapplications using 1.8-µm parti-cle columns up to 150-mmlength and with internal diame-ters from 2.1 to 4.6 mm

• Agilent 1200 Series high-perfor-mance autosampler SL for high-est area precision

• Agilent 1200 Series thermostat-ted column compartment SLwith wide temperature rangefrom 10 degrees below ambientup to 100 °C

• Agilent 1200 Series diode-arraydetector SL for 80-Hz operation,including new data protectiontool

• ZORBAX SB C-18 columns withdifferent internal diameters andlengths, packed with 1.8-µm particles

Pump designThe Agilent 1200 Series binarypump SL is designed to meet thedemands of high throughput andhighest performance for optimumresolution and lowest pump ripplefor a wide range of applicationson narrow or standard-borecolumns. Figure 1 shows aschematic of the 1200 Series binarypump SL. The delay volume canbe configured according to theapplication needs. The standardconfiguration is best suited forhighest performance in terms ofnoise and resolution. In this stan-dard configuration the mixer anddamper are in the flow path andthe delay volume is between 600and 800 µL, depending on thepressure. This configuration is

Figure 1Standard and low delay volume configurations of the Agilent 1200 Series binary pump SL.

resolution and lowest systemnoise is needed. The configurationwithout damper and mixer in theflow path is best suited for LC/MSor LC/UV using 2.1-mm ID columns.

Retention time precision of Agilent1200 Rapid Resolution LC system forgradient LC analysisRetention time precision was evaluated for both pump configu-rations under gradient and isocrat-ic conditions using standard boreand narrow bore columns. In thefirst example the standard delayconfiguration was tested using astandard bore column.A “phenonemix“ was analyzed using gradientconditions from 35 to 95 %. A 100 x 4.6 mm column and a flowrate of 1.5 mL/min were selected.These conditions are close to con-ditions used in conventional LCanalysis. Figure 2 shows 10 runsoverlaid, demonstrating the reten-tion time precision obtained forthis application. The run time was6 min. The evaluation of the reten-tion time precision was found tobe less than 0.07 % RSD.

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min1 2 3 4 5

Overlay of 10 runsInjection volume: 50 µLInjected amount: 80 ngRSD RT <0.07 %

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DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\55075100000013.D) DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\55075100000014.D) DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\55075100000015.D) DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\55075100000016.D) DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\55075100000017.D) DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\55075100000018.D) DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\55075100000019.D) DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\55075100000020.D) DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\55075100000021.D) DAD1 A, Sig=245,10 Ref=360,80 (E:\CHEM32\1\DATA\INJECTIONVOL2\55075100000022.D)

Chromatographic conditions:Test sample: 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

Column: 100 x 4.6 mm ZORBAX SB C-18, 1.8 µm for 600 bar operationPump: Solvent: A = Water, B = ACN

Gradient: 35 to 95 %B in 5 min, hold at 95 %B for 1 minStop Time: 6 min Post Time: 2 minFlow Rate: 1.5 mL/min

Autosampler: Injection volume: 50 µL Wash 10 s for exterior of needle

Thermostatted column compartment:Temperature: 50 °C

Detector: 13 µL cell20-Hz data acquisitionPeak width = <0.01 min Slit: 8 nmSignal: 245/10 nm, ref 360/80 nm

Figure 2Overlay of 10 chromatograms.

Retention time precision of Agilent1200 Rapid Resolution LC systemusing the low-delay volume configu-rationIn the previous section the reten-tion time precision of the stan-dard delay configuration was eval-uated. In this section the retentiontime precision of the low volumedelay configuration will be evalu-ated. The analyses were run athigh and low flow rates as well asat high and low temperatures. Forall low flow rate applications therelative standard deviation for theretention times was typically lessthan 0.07 %. For high flow rateand ultra-fast applications the relative standard deviation for theretention times was typically lessthan 0.14 %, see figure 3 and alsoliterature reference 1.

Limit QA/QC = 0.500

0.0000.0500.1000.1500.2000.2500.3000.3500.400

Flow=0.35 mL/min, T=40 °CFlow=0.35 mL/min, T=80 °CFlow=1.20 mL/min, T=40 °CFlow=2.00 mL/min, T=80 °C

0.450 RT Precision (n=10)

Acetanilide

Acetophenone

Propiophenone

Butyrophenone

Benzophenone

Valerophenone

Hexanophenone

Heptanophenone

Octanophenone

Average RSD

%

RSD

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7 %

RSD

, SD

=0.0

0107

min

0.07

0 %

RSD

, SD

=0.0

0084

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8 %

RSD

, SD

=0.0

0048

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0.13

4 %

RSD

, SD

=0.0

0031

min

Figure 3Statistical 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 1200Series Rapid Resolution LC system.

Low flow High temp.F=0.35 mL/min T=80 °C

High temp.T=80 °C

Low flow Low temp.F=0.35 mL/min T=40 °C

High flow Low temp.F=1.20 mL/min T=40 °C

High flowF=2.00 mL/min

0.5 1 1.5 2 2.5 3.0 min

mAU

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100150200250300350400

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mAU

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100150200250300

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mAU

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100150200250300350

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100150200250300350400

Figure 4Overlaid chromatograms of the repeated analysis of a 9-compound mixture under various conditions.

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Chromatographic conditions:Solvent: A = Water, B = ACN Temp.: 40 °C, 80 °CFlow: 0.35 mL/min, 1.20 mL/min, 2.0mL/minGradient: 0.00 min 35 %B

2.60 min 95%B3.20 min 95%B3.21 min 35%BTime 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 minPost time: 2.00 minWavelength: 245 nm (8), Ref. 450 nm (100)Slit: 8 nmPeak width: > 0.0025 min (0.05 s response

time), 80 HzSpectra: All, 190-500 nm,

bandwidth = 1 nmInjection vol.: 1.0 µLInjector: Overlapped injection, Automatic

delay volume reduction, Sample flush out factor = 10Needle wash = 5 s

Sample: “Phenone mix” (0.1 µg/µL each,0.2 µg/µL of Butyrophenone)

Retention time precision of Agilent1200 Rapid Resolution LC system forisocratic LC analysisIn this example isocratic condi-tions were selected, see condi-tions in figure 5. A narrow borecolumn was chosen and the flowrate was set to 0.5 mL/min. In fig-ure 5 an overlay of 10 runs isshown for the analysis of test mix-ture. Typically the relative stan-dard deviation for this type ofapplication is less than 0.07 % forretention times.

Delay volume, system ripple, precision and accuracy of gradientsTo evaluate pump performancetracer experiments are a frequent-ly used tool to evaluate the systemripple at different gradient mix-tures. The delay volume and theaccuracy and the precision of gra-dients are also evaluated usingstep gradients and linear gradi-ents. Figure 6 shows a step gradi-ent from 0 to 100 % in 10 % stepsusing the Agilent 1200 SeriesRapid Resolution LC system con-figured for standard delay volume.

min2.75 2.8 2.85 2.9 2.95 3

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Overlay of 10 runsInjection volume: 3 µLRSD for RT < 0.07 %

Figure 5Precision of retention times on 2.1-mm column, isocratic elution, overlay of 10 runs, RSD of RTless than 0.07 %.

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Delay volume with ALS in the flow path = 860 µL

210 Bar backpressureRipple on step 10: = 0.010 % Ripple on step 30: = 0.007 % Ripple on step 50: = 0.006 %Ripple on step 80: = 0.010 %

Overlay of theoretical and real step gradient from 0 to 100 % in 10 % steps

Figure 6Accuracy, delay volume and ripple using the standard delay configuration of the Agilent 1200Series Rapid Resolution LC system at 210 bar backpressure, 0 to 100 %B gradient in 10 % steps.

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 µm

Mobile phase: Water/acetonitrile = 30/70Injection vol.: 3 µLFlow rate: 0.5 mL/min

Chromatographic conditions:Column: Restriction capillariesMobile phases: A=water, B=water/0.5 % acetoneFlow rate: 1 mL/minStep gradients: 0 %B to 10 %B in 1 % steps, each step held for 5 min 0 %B to 100 %B in 10 %steps,

each step held for 5 minLinear gradient: 100 % to 0 % in 5 minTemperature: 36 °CDetection: Signal 265/20 nm, reference 360/100 nm

Slit 4 nmPeak width 0.01 min13 µL cell

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With the autosampler in the flowpath the delay volume is about 860 µL. The system ripple is lessthan or equal to 0.01 % related tothe 100 % step. The overlay withtheoretical step gradient showsexcellent agreement with the realstep gradient with regard to thestep heights. Figure 7 shows anoverlay of 3 step gradients usingthe standard delay volume config-uration of the Agilent 1200 SeriesRapid Resolution LC system. Thesteps are 1 % from 0 to 10 %.The precision for the 3-step gradi-ent runs is excellent and the sys-tem ripple for the 1 % steps is lessthan or equal to 0.011 % related tothe 100 % step. Figure 8 shows anoverlay of 3 linear gradients usingthe low delay volume configura-tion of the Agilent 1200 SeriesRapid Resolution LC system. Thelinear gradient was run from 100 %to 0 % tracer. The overlay showsthat precision for a linear gradientis also excellent for the low delayvolume configuration. The delayvolume in this configuration isabout 420 µL with autosampler inthe flow path. If the autosampleris taken out of the flow path thedelay volume is about 120 µL. Thesystem ripple with the autosam-pler in the flow path is typicallyless than 0.05 % related to the 100 % step.

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20Overlay of 3 runs Step gradient from 1 to 10% in 1% steps

210 Bar BackpressureRipple on step 1: = 0.011 % Ripple on step 3: = 0.009 % Ripple on step 5: = 0.011 %Ripple on step 6: = 0.010 %Ripple on step 9: = 0.010 %

Delay volume with ALS in the flow path = 945 µl

Figure 7Precision 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.

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Figure 8Precision using the low delay volume configuration of the Agilent 1200 Series Rapid ResolutionLC system at 200 bar backpressure, linear gradient from 100 to 0 %, 3 overlays.

References

1. “Performance characteristics ofthe Agilent 1200 Series LC sys-tem”, Agilent Application Note,

Publication Number 5989-

4489EN, 2006.

Conclusion

The data presented in this noteshow that the precision of reten-tion time of the Agilent 1200Series binary pump SL pump isexcellent for a wide range of LCapplications using either narrow-bore or standard-bore columns.Typically a precision for retentiontimes of less than 0.07 % relativestandard deviation can be expect-ed for isocratic and gradient appli-cations using the standard delayvolume configuration of thepump. For the low delay volumeconfiguration the retention timeprecision is typically less than0.07 % relative standard deviationfor more conventional flow ratesand is typically less than 0.14 %for ultra-fast applications with runtimes less than 1 min.

Accuracy and precision of linearand step gradients are excellent.Ripple for the standard delay vol-ume configuration is typically lessthan or equal to 0.01 %. The lowdelay volume configuration has asystem ripple typically less than0.05 %. The delay volume variabili-ty supports the needs for lowdelay volume applications as wellas the demands for delay volumeneeded for more conventional val-idated methods. The standarddelay volume configuration withthe autosampler in the flow pathhas a delay volume of 860 µL at200 bar backpressure. The lowdelay volume configuration has adelay volume of 120 µL when theautosampler is switched out ofthe flow path.

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Copyright © 2006 Agilent TechnologiesAll Rights Reserved. Reproduction, adaptationor translation without prior written permissionis prohibited except as allowed under thecopyright laws.

Published April 1, 2006Publication Number 5989-5031EN

www.agilent.com/chem/1200rrht

Angelika Gratzfeld-Huesgen and

Michael Frank are Application

Chemists at Agilent Technologies,

Waldbronn, Germany.