quatrnary pump agilent

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Scope of low and high pressure mixing Comparing the Agilent 1260 Infinity Quaternary and Binary Pumps Abstract The Agilent 1260 Infinity LC family offers two analytical scale LC gradient pump types: a quaternary low pressure mixing pump and a binary high pressure mixing pump. Both pumps show excellent performance within their design range. The optimum gradient range for the quaternary pump is between 5% to 95% organic. The binary pump shows optimum performance from 0% to 100% organic, which is advantageous for compounds eluting at very low or at very high organic percentage. In the range of 5% to 95% organic both pumps show comparable performance. Another advantage for the binary pump is the high mixing efficiency, which is necessary if TFA is used as a modifier. At high flow rates and fast gradients, both pumps show excellent retention time precision. The quaternary pump has the advantage that ternary and quaternary gradients can be applied. Author Angelika Gratzfeld-Huesgen Agilent Technologies Waldbronn, Germany Technical Overview Agilent 1260 Infinity Quaternary Pump min 0 2 4 6 8 10 mAU 0 10 20 30 40 50 60 Agilent 1260 Infinity Binary Pump min 0 2 4 6 8 10 mAU 0 10 20 30 40 50 60

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Page 1: Quatrnary Pump Agilent

Scope of low and high pressure mixingComparing the Agilent 1260 Infinity Quaternary andBinary Pumps

Abstract

The Agilent 1260 Infinity LC family offers two analytical scale LC gradient pump types:

a quaternary low pressure mixing pump and a binary high pressure mixing pump. Both

pumps show excellent performance within their design range. The optimum gradient

range for the quaternary pump is between 5% to 95% organic. The binary pump

shows optimum performance from 0% to 100% organic, which is advantageous for

compounds eluting at very low or at very high organic percentage. In the range of 5%

to 95% organic both pumps show comparable performance. Another advantage for

the binary pump is the high mixing efficiency, which is necessary if TFA is used as a

modifier. At high flow rates and fast gradients, both pumps show excellent retention

time precision. The quaternary pump has the advantage that ternary and quaternary

gradients can be applied.

Author

Angelika Gratzfeld-Huesgen

Agilent Technologies

Waldbronn, Germany

Technical Overview

Agilent 1260 Infinity Quaternary Pump

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IntroductionOne of the most important performancecriteria for a pump is precise retentiontimes. The precision of retention timefor gradient runs and isocratic runswhere the solvent compositions isformed out of two solvent channelsdepends mainly on:

• Flow precision

• Mixing performance

• Compressibility settings

• Stroke volumes

• Primary channel selection (Agilent1260 Infinity Quaternary Pump only)

• Composition accuracy and precision

Two main pump types are typically usedin analytical LC: low pressure gradientpumps and high pressure gradientspumps.

In this Application Note, we will dis-cuss the application range of the twopump types comparing the Agilent 1260Infinity Binary Pump to the Agilent 1260Infinity Quaternary Pump.

ExperimentalAn Agilent 1260 Infinity LC system wasused including:

• Agilent 1260 Infinity Quaternary Pump(G1311B)

• Agilent 1260 Infinity Binary Pump(G1312B) and Degasser (G1379B)

• Agilent 1260 Infinity Autosampler(G1367E)

• Agilent 1260 Infinity ThermostattedColumn Compartments (G1316C)

• Agilent 1260 Infinity Diode ArrayDetector (G4212B) with 10 mm flowcell

• Several Agilent ZORBAX columns

• Agilent ChemStation B.04.02

the quaternary pump only one valuecan be entered for all channels, whichcan cause lower retention time preci-sion.

• The stroke volume (see Appendix)setting influences the mixing perfor-mance. As the flow rate decreases,the stroke volume should decrease aswell. Typically the AUTO setting isappropriate for both pumps.

The following experiments verified theperformance and demonstrated the dif-ferences between the two pumps.Retention time precision was measuredfor the following conditions:

• approximately 50% acetonitrile/50% aqueous

• slow generic gradient usingMeOH/water

• slow generic gradient usingACN/water and TFA as modifier

• low organic percentage

• high organic percentage

• 3 mL/min flow rate and gradienttime of 1 min

Results and discussionThe Agilent 1260 Infinity Binary andQuaternary Pumps are designed forspecific application ranges. The Agilent1260 Infinity Quaternary Pump is the“work horse” for applications using iso-cratic and conventional gradient condi-tions, and a practical flow rate rangefrom 0.3 up to 10 mL/min. The Agilent1260 Infinity Binary Pump is typicallyused for more demanding applications,especially for gradients from 0% to10%, 90% to 100% and a practical flowrate range from 0.05 to 5 mL/min. Thispump is the best choice for TFA as amodifier in the mobile phase because ofits excellent mixing performance.

Table 1 presents an overview of theimportant parameters and specifica-tions for the two pump types.

Some specific pump parameters mustbe set by the user and the settings havean effect on pump performance:

• The compressibility (see Appendix),which influences the flow accuracyover the complete pressure range, canbe set to its optimum for each solventchannel using the binary pump. For

Agilent 1260 Infinty Quaternary PumpPrimary channel = A (default)

Agilent 1260 Infinity Binary Pump

Low pressure gradient pump High pressure gradient pump with standard and low delay volume configuration

One compressibility setting for all used mobile phases

Compressibility setting individually related to theselected mobile phase

Maximum flow rate is 10 mL/min Up to 600 bar for 5 mL/min

Maximum flow rate is 5 mL/min up to 600 bar

Ternary and quaternary gradients Binary gradient

Flow precision: ≤ 0.07% RSD* Flow precision: ≤ 0.07% RSD*

Composition precision: < 0.2% RSD* Composition precision: < 0.15% RSD*

Optimum gradient range: 5% to 95% Optimum gradient range: 0% to 100%

Table 1Design and specifications*measured for retention times under conditions described in the manuals

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Retention time precision at approximately 50% acetoni-trile/50% aqueousFor most pumps the mixing perfor-mance at approximately 50% organicand 50% aqueous is a critical perfor-mance point. Experimental conditionswere selected to optimize performanceof both types and the analgesicsantipyrine, phenobarbital, andphenacetin were analyzed (Figure1).The mobile phases were mixed out oftwo channels. The different designs ofhigh and low pressure mixing pumpsproduce different mixing compositions,which result in different retention timesfor both pumps. The quaternary pumpadds slightly more organic phase to thecomposition mixture resulting in shorterretention times.

The results are presented in Figure 2.The binary pump shows slightly betterresults for the first two peaks, but over-all the results are comparable.

Agilent 1260 Infinity Binary Pump

Agilent 1260 Infinity Quaternary Pump

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Chromatographic conditions

Compounds: Antipyrine, phenobarbital, phenacetin Concentration: 0.3 ng/µL eachColumn: Agilent ZORBAX SB C8 RRHT, 100 mm × 4.6 mm, 1.8 µmMobile phases: Water/acetonitrile = 55/45, isocratic from two channels Flow rate: 1.2 mL/minStop time: 5 minColumn temperature: 40 °CInjection volume: 5 µLDAD: 220/10 nm Ref. 360/100, 20 Hz

Figure 1Analysis of analgesics using isocratic conditions and 55/45 water/ACN mixture.

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Precision of retention times for isocratic run at 50/50%

Agilent 1260 Infinity Quaternary Pump

Agilent 1260 Infinity Binary Pump

Specification Agilent 1260 Infinity Quaternary Pump

Specification Agilent 1260 Infinity Binary Pump

Figure 2Comparable precision of retention times using isocratic conditions.

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Retention time precision for aconventional generic gradientusing MeOH/water as eluentsBoth pumps typically show comparableresults for conventional gradients fromabout 5% to 95% in 10 min. Mixing per-formance and pump ripple do not differsignificantly. The experiments weredone using a paraben standard (Figure 3).

The delay volume of an LC systemdetermines how fast a gradient changecan reach the head of the column ingradient applications. Typically highpressure mixing pumps have smallerdelay volumes than low pressure mix-ing systems. This is also true for thedelay volumes of the Agilent 1260Infinity Quaternary and Binary Pumps.A peak analyzed with the Agilent 1260Infinity Binary Pump elutes earlier thanon the Agilent 1260 Infinity QuaternaryPump.

The results for both pumps are shownin Figure 4. As expected, both pumpsshowed excellent and comparableresults with acetonitrile as mobilephase (data not shown).

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Agilent 1260 InfinityBinary Pump

Agilent 1260 Infinity Quaternary Pump

Chromatographic conditions

Compounds: Uracil, phenol, methyl-, ethyl-, propyl-, butyl- and heptylparaben Column: Agilent ZORBAX Eclipse C18 RRHT, 100 mm × 4.6 mm, 1.8 µmMobile phases: Water/methanolGradient: 10% to 90% in 10 minFlow rate: 1.2 mL/minStop time: 18 minColumn temperature: 40 °CInjection volume: 3 µLDAD: 254/10 nm Ref. 360/100 nm, 20 Hz

Figure 3Analysis of paraben sample using a conventional gradient from 10% to 90% organic (methanol) in 10 min.

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Precision of retention times for gradient from 10% to 90% in 10 min

Agilent 1260 Infinity Quaternary Pump

Agilent 1260 Infinity Binary Pump

Specification Agilent 1260 Infinity Quaternary Pump

Specification Agilent 1260 Infinity Binary Pump

Figure 4Comparable precision of retention times for conventional gradients using methanol as organic phase.

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Retention time precision forconventional generic gradientusing ACN/water and TFA asthe modifier In the following experiment, TFA wasadded to both mobile phases. TFA isknown to cause problems for the preci-sion of retention times if mixing of theaqueous and organic phases during agradient run is not sufficient.

For the binary pump, mixing of themobile phases takes place at the highpressure side using a 420-µL mixer,where turbulences occur due to thepacking material. This provides veryeffective mixing and reduces the TFAproblems significantly. For the quater-nary pump, mixing occurs in pumpheads and capillary connections.Typically mixing is less effective and theprecision of retention times might beworse.

In Figure 5 the chromatograms obtainedusing the binary and quaternary pumpare overlaid. TFA was added at 0.05% tothe aqueous and the organic (acetoni-trile) phase.

In Figure 6, the results show that thebinary pump produces slightly betterretention time precision due to thehigher mixing performance.

Agilent 1260 InfintiyBinary Pump

Agilent 1260 InfinityQuaternary Pump

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Compounds: Uracil, phenol, methyl-, ethyl-, propyl-, butyl- and heptylparaben Column: Agilent ZORBAX Eclipse Plus C18 RRHT, 100 mm × 4.6 mm, 1.8 µmMobile phases: Water/acetonitrile + 0.05% TFA eachGradient: 10% to 90% in 10 minFlow rate: 1.2 mL/minStop time: 11 minPost time: 4 minColumn temperature: 40 °CInjection volume: 3 µLDAD: 254/10 nm Ref. 360/100 nm, 20 Hz

Figure 5Analysis of the paraben sample using TFA as modifier in both mobile phases.

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Precision of retention times using TFA as modifier

Agilent 1260 Infinity Quaternary Pump

Agilent 1260 Infinity Binary Pump

Specification Agilent 1260 Infinity Quaternary Pump

Specification Agilent 1260 Infinity Binary Pump

Figure 6Better precision of retention times for the binary pump using TFA as modifier.

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Retention time precision at loworganic percentage In this experiment, the precision ofretention time was tested running agradient from 2% to 12% organic(Figure 7). During the isocratic run, thequaternary pump adds slightly moreorganic phase to the mixture resultingin earlier elution.

In this low organic percentage rangethe use of the binary pump is advanta-geous (Figure 8). The difference in pre-cision is significant for the early elutingpeak (theophylline), which elutesbetween 4% and 5% organic. The caf-feine, which elutes at higher organicpercentage, shows similar precision onboth pumps.

Although the range of 0% to 5% organ-ic phase is not specified for the quater-nary pump, the RSD for retention timesis still below specification.

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Agilent 1260 Infinity Binary Pump

Agilent 1260 Infinity Quaternary Pump

Chromatographic conditions

Compounds: Theophylline and caffeine Column: Agilent Poroshell 120 EC 18, 50 mm × 4.6 mm, 2.7 µmMobile phases: Water/acetonitrileGradient: 2% to 12% ACN in 5 minFlow rate: 2 mL/minStop time: 5 minPost time: 4 minColumn temperature: 40 °CInjection volume: 5 µLDAD: 254/10 nm Ref. 360/100 nm, 20 Hz

Figure 7Analysis of theophylline and caffeine at low organic percentage.

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Agilent 1260 Infinity Quaternary Pump

Agilent 1260 Infinity Binary Pump

Specification Agilent 1260 Infinity Quaternary Pump

Specification Agilent 1260 Infinity Binary Pump

Figure 8Better results for the binary pump at low organic percentage.

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Retention time precision at highorganic percentage In this experiment Sudan Red 1 andSudan Red 3 were analyzed using agradient of 90% to 100% organic(Figure 9). The results show that at thehighest organic percentage the binarypump provides three to four times bet-ter precision (Figure 10).

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Agilent 1260 Infinity Binary Pump

Agilent 1260 Infinity Quaternary Pump

Chromatographic conditions

Compounds: Sudan Red 1(50 ng/µL) and 3 (300 ng/µL) Column: Agilent ZORBAX Eclipse Plus C18 RRHT,

100 mm × 4.6 mm, 1.8 µmMobile phases: Water/acetonitrileGradient: 90% to 100% in 5 minFlow rate: 1.2 mL/minStop time: 6 minPost time: 4 minColumn temperature: 40 °CInjection volume: 3 µLDAD: 490/20 nm Ref. off, 20 HzPrimary channel for quanternary pump: A

Figure 9Analysis of Sudan Red 1 and 3 at high organic percentage.

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Precision of retention times at high organic percentage

Agilent 1260 Infinity Quaternary PumpAgilent 1260 Infinity Binary Pump

Specification Agilent 1260 Infinity Quaternary Pump

Specification Agilent 1260 Infinity Binary Pump

Figure 10Better results for the binary pump at high organic percentage.

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Retention time precision at 3 mL/min flow rate and a gradient time of 1 minSufficient resolution in the shortestpossible run time is important in chro-matography. This means that shortcolumns, high flow rates, and short gra-dient times are applied. Chromato-graphic conditions were selected todemonstrate that the Agilent 1260Infinity Quaternary and Binary Pumpscan provide optimum performance. APoroshell 120 50 mm × 4.6 mm columnwas selected and operated at a flowrate of 3 mL/min. Gradient time wasset to 1 min. Using these conditions thefirst peaks elute at approximately 0.5 min and the last one at approxi-mately 1.5 min (Figure 11).

The results are presented in Figure 12and show that both pumps performwell. The precision for retention timesis < 0.05% RSD overall.

Agilent 1260 InfinityBinary Pump

Agilent 1260 InfinityQuaternary Pump

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Compounds: RRLC checkout sample (p/n 5188-6529)Column: Agilent Poroshell 120 EC C18, 50 mm × 4.6 mm, 2.7 µmMobile phases: Water/acetonitrileGradient: 20% to 90% in 1 minFlow rate: 3 mL/minStop time: 2 minPost time: 1 minColumn temperature: 40 °CInjection volume: 3 µLDAD: 245/10 nm Ref. 360/100 nm, 20 Hz

Figure11Analysis of phenone sample using high flow rates.

Agilent 1260 Infinity Quaternary PumpAgilent 1260 Infinity Binary Pump

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Specification Agilent 1260 Infinity Quaternary Pump

Specification Agilent 1260 Infinity Binary Pump

Figure 12Comparable precision of retention times applying fast chromatographic conditions.

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ConclusionThe performances of the Agilent 1260Infinity Binary and Quaternary Pumpswere tested and compared using sever-al different chromatographic conditions.These conditions include isocratic runsand conventional gradients with andwithout TFA as modifier; low and highorganic percentage; and fast chromato-graphic conditions. The results showthat the binary pump has advantages ifthe mixing performance is demanding,such as when TFA is used as modifier.The retention time precision was com-pared in the ranges of 0% to 10%organic and 90% to 100% organic andshowed that the performance of thebinary pump is better. For conventionalgradients between 5% and 95% organicand isocratic runs, the performance ofboth pumps is comparable. Results arealso comparable for high flow rates andshort gradient times in the range of10% to 90% organic.

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CompressibilityWhen a solvent is metered at ambientpressure and compressed to a higherpressure, the volume decreases. This isdue to an effect known as solvent com-pressibility. Solvent compressibility is anonlinear function of pressure and tem-perature. It is unique to every solvent.In order to deliver the desired flowaccurately at all pressures, Agilentpumps use compressibility compensa-tion. Usually, an average compressibilityvalue for the solvent is used across thewhole pressure range of the pump,such as that applied for the quaternarypump. The Agilent 1260 Infinity BinaryPump has a different compressibilitycompensation concept. The compress-ibility of a solvent is determined at dif-ferent pressures between 0 to 600 bar.This pump uses the obtained nonlinearfunction to select the correct compress-ibility value for the actual pump pres-sure. Compressibility data for the mostcommon solvents are readily availablein the pump firmware.

Stroke Volume The smaller the solvent volume in thepump chamber, the faster it can berecompressed to operating pressure.The Agilent 1260 Infinity pumps allowmanual or automatic adjustment of the

pump stroke volume of the first pistonin the range of 20 – 100 µL. Due to thecompression of the solvent volume inthe first pump chamber, each pistonstroke of the pump will generate asmall pressure pulsation, influencingthe flow ripple of the pump. The ampli-tude of the pressure pulsation is mainlydependent on the stroke volume andthe compressibility compensation forthe solvent in use. Small stroke vol-umes generate less pressure pulsationthan larger stroke volumes at the sameflow rate. In addition, the frequency ofthe pressure pulsation is higher. Thisdecreases the influence of flow pulsa-tions on quantitative results. In gradientmode, smaller stroke volume results inless flow ripple and improves the com-position ripple. The Agilent pumps usea processor-controlled ball screw sys-tem to drive its pistons. The normalstroke volume is optimized for theselected flow rate. Small flow rates usea small stroke volume while higher flowrates use a higher stroke volume. Thestroke volume for the pump is bydefault set to AUTO mode. This meansthat the stroke is optimized for the flowrate in use. A change to larger strokevolumes is possible but not recommended.

Appendix

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www.agilent.com/chem/lc

© Agilent Technologies, Inc., 2011Published January 1, 2011Publication Number 5990-7143EN