Optimize The Agilent 1260 Infi Nity Analytical SFC Solution .

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Optimize the Agilent 1260 InfinityAnalytical SFC Solution with theAgilent 1290 Infinity ELSDTechnical OverviewAuthorAbstractEdgar NaegeleThis Technical Overview gives a detailed description of an optimized configurationAgilent Technologies, Inc.of an instrument for supercritical fluid chromatography (SFC) when used with anWaldbronn, Germanyevaporative light scattering detector (ELSD). The influence of preheating the SFCeffluent on the signal quality of the ELSD is shown. This was done by means of aheat exchanger in an additional thermostatted column compartment. The effectof additional make-up flow prior to the ELSD is also discussed, supported bystatistical data.

IntroductionExperimentalModern supercritical fluidchromatography (SFC) instrumentsoffer huge performance advantagescompared to classical HPLC instruments.Compared to HPLC mobile phases, SFC’smobile CO2 phase has lower viscosity,increased diffusion, and better masstransfer capabilities. This enables higherspeed separation at lower backpressureby means of columns with small innerdiameter and small particle size packingmaterial. Both methods, SFC and HPLC,are comparable in terms of sensitivityand stability, but provide orthogonalselectivity for the separation. This makesSFC a valuable complementary techniqueto classical HPLC, as well as modernUHPLC.InstrumentsThe application range of SFC can bewidened by coupling with other detectors,for example, evaporative light scattering(ELSD). However, effects such asexpansion cooling from decompressedCO2, the need for splitting, and a make-upflow to equal flow rates make it lessstraightforward.This Technical Overview describes aconfiguration to connect an ELSD toan SFC instrument. The effect of CO2expansion cooling and preheating of thecolumn effluent on the peak performanceare shown and discussed. Theintroduction of a make-up flow is includedin the instrument configuration.Agilent 1260 Infinity Analytical SFCSolution (G4309A) with: Agilent 1260 Infinity SFC ControlModule Agilent 1260 Infinity SFC BinaryPump Agilent 1260 Infinity HighPerformance Degasser Agilent 1260 Infinity SFC StandardAutosampler Agilent 1260 Infinity ThermostattedColumn Compartment (TCC) introduced into the flow path. This splitteris connected to the second one by a short0.12-mm id capillary (both splitters couldalso be used independently). Here, theflow is split into the part going to theELSD, and the other part going to thebackpressure regulator (BPR) of the SFCmodule. The connection from the secondsplitter to the heat exchanger in thesecond TCC for preheating of the effluentstream is made by a newly developed50-µm id stainless steel capillary, 1 min length. The connection from the heatexchanger in the TCC to the ELSD ismade by another 0.12-mm id capillary. Thesplit ratio depends on the backpressuregenerated by this restriction capillary andthe pressure set by the BPR.ColumnAgilent ZORBAX Rx-SIL,4.6 150 mm, 5 µm (p/n 883975-901)Agilent 1260 Infinity DAD with highpressure SFC flow cellSoftware Agilent 1260 Infinity IsocraticPump (G1310B)Agilent OpenLAB CDS ChemStationEdition for LC & LC/MS Systems,Rev. C.01.05 Agilent 1290 Infinity ThermostattedColumn Compartment (TCC)Standards Agilent 1290 Infinity ELSD(G4261B)Instrument setupThe recommended configuration ofthe Agilent 1260 Infinity AnalyticalSFC Solution with the Agilent 1290Infinity ELSD is shown in Figure 1. Theexit capillary of the DAD flow cell isdirectly connected to a splitter assembly(p/n G4309-68715), which contains twocombined splitters (and an additionalcheck valve to prevent backflush of CO2into the make-up pump and a solventfilter). At the first splitter, the make-upflow coming in from an isocratic pump is2A solution of the following compoundswas used: 1) caffeine, 2) theophylline,3) cortisone, 4) prednisone,5) hydrocortisone, 6) prednisolone,7) sulfamerazine, 8) sulfaquinoxaline(stock solution at 1 mg/mL each inmethanol). This solution was dilutedwith methanol to a final concentration of10 µg/mL.ChemicalsMethanol was purchased from J. T. Baker,Germany. Chemicals were purchasedfrom Sigma Aldrich, Corp., Germany.Fresh ultrapure water was obtained froma Milli-Q Integral system equipped withLC-Pak Polisher and a 0.22-μm membranepoint-of-use cartridge (Millipak).

BPRAgilent ZORBAX Rx-SIL columnUV detectorAgilent 1260 InfinityIsocratic pumpPreheatingWasteSplitter assemblyFigure 1. Configuration of the Agilent 1260 Infinity Analytical SFC Solution with the Agilent 1290 Infinity ELSD. TheDAD flow cell is directly connected to the splitter assembly containing two splitters, a check valve, and a solvent filter(BPR backpressure regulator, splitter assembly p/n G4309-68715). Capillary from splitter assembly to preheating TCC:0.05 1,000 mm.SFC conditionsAgilent 1260 Infinity Analytical SFC Solution conditionsSolvent ACO2Modifier BMethanolSFC flow3 mL/minGradient5 % B – 0 minutes, 25 % B – 10 minutesStop time10 minutesPost time2 minutesBackpressure regulator (BPR)temperature60 CBPR pressure290 barColumn temperature40 CInjection volume5 µL, 3 loop over fillNeedle wash in vial with methanolMake-up flow0.2 mL/min, methanolDAD254 nm/band width 4 nm; ref. 360 nm/bandwidth 100 nmdata rate: 20 HzAgilent 1290 Infinity ELSD conditionsEvaporator temperature40 CNebulizer temperature55 CGas flow rate1.15 SLMData rate10 HzSmoothing2sPMT gain53

Results and DiscussionThe SFC flow was split behind the DADflow cell and before the BPR to theELSD. The BPR was adjusted to producea higher backpressure in the system todirect a large part of the flow through thesplitter towards the ELSD for improveddetection. In the initial experiment, thetransfer capillary was connected directlyto the ELSD and allowed decompressiondirectly at the nebulizer in the ELSD. Thisis also the point where decompressioncooling occurs. This effect is known tocompromise ELSD signal intensity. Underthe given conditions, the chromatographyshowed clear separation of the eightcompounds in the test mixture, wherethe first two compounds were separatedearly in the run with a valley betweenthese peaks and the other compoundswere baseline separated. The separationwas maintained from DAD detectionduring splitting and transfer through the50-µm id 1 m capillary to the ELSD(Figure 2). To avoid the negative effect ofexpansion cooling, a heat exchanger inanother TCC was connected to the 50-µmid capillary for preheating the SFC effluentbefore entering the ELSD.DAD, 254 233.987 4.327505.4035.6425.8420To demonstrate the effect, heatexchangers with different internalvolumes were kept at regulated ambienttemperature (25 C) and connected tothe 50-µm id capillary. Comparison ofthe signals immediately showed anincrease in signal height and signal areawhen the SFC effluent was preheatedbefore entering the ELSD (Figure 3).There was also a slight increase in peakwidth, but due to the much higher peakintensity and peaks area, they were inan acceptable range. Because there wasnot much difference in peak width for thedifferent heat exchanger volumes (6, 3,and 1.8 µL), the 3-µL heat exchanger wasused for further experiments.min8ELSD0123456789 minFigure 2. Separation of an eight-compound mix with SFC and DAD/ELSD detection. The splittingconnection to the ELSD was located in the flow path behind the DAD and before BPR by means of aT-piece and a 50 µm 1 m capillary.mV45040022.911No heat exchanger, direct connection of SFC to ELSDConnection of SFC to ELSD via:Heat exchanger with 6 µLHeat exchanger with 3 µLHeat exchanger with 1.8 µL12.804350300250Peak ID1. Caffeine2. Theophylline3. Cortisone4. Prednisone5. Hydrocortisone6. Prednisolone7. Sulfamerazine8. 85.6535.76650033.544.555.56minFigure 3. Effect of the introduction of a heat exchanger in the SFC effluent before entering the ESLD.Peak broadening and peak height effects are shown for different heat exchanger volumes at regulatedambient temperature of 25 C.4

Compound 3Compound 4300.00Compound 5Area250.00Compound 6200.00Compound 7150.00Compound 8100.0050.000.00DirectconnectionTCC 30 CArea RSDTCC 40 CConditionsTCC 60 CArea RSD versus heating16.00Compound 314.00Compound 412.00Compound 510.00Compound 68.00Compound 76.00Compound 30 CTCC 40 CConditionsTCC 60 CFigure 4. Peak area and peak area RSD % versus preheating temperature of split SFC effluent. Themaximum peak areas were obtained at a preheating temperature of 30 C with an RSD of approximately12 %.Peak height versus heatingPeak heightThe examination of the dependence ofpeak height on preheating temperatureshowed a similar result (Figure 5). Fromthe direct connection of SFC to ELSDwithout preheating, to preheating with aheat exchanger temperature of 30 C, thesignal height showed a clear increase.Further heating to 40 and 60 C decreasedthe signal again. The signal height RSDvalues were approximately 10 % for thedirect connection and 10 to 12 % for thepreheated examples at 30 C.Area versus heating350.0080.00Compound 370.00Compound 460.00Compound 550.00Compound 640.00Compound 730.00Compound 820.0010.000.00DirectconnectionTCC 30 CTCC 40 CConditionsTCC 60 CPeak height RSD versus heatingPeak height RSDThe effect of preheating the SFC effluenton the signal performance at the ELSDwas examined in more detail. The signalsof six compounds were compared at 30,40, and 60 C to a direct connection ofSFC to ELSD without preheating for signalarea, signal height, and signal-to-noise(S/N) ratio. The experiments wererepeated 12 times for a proper statisticalevaluation. The dependence of the peakarea showed a clear increase for allcompounds for preheating of the SFCeffluent to 30 C before entering theELSD. Further increase in preheating to40 and 60 C decreased the signal areaagain (Figure 4). The statistical evaluationshowed that relative standard deviations(RSD) were 8 to 10 % for the directlyconnected ELSD and approximately 12 %for preheating at 30 C, with a tendencyto decrease again at higher preheatingtemperatures.14.00Compound 312.00Compound 4Compound 510.00Compound 68.00Compound 76.00Compound 30 CTCC 40 CConditionsTCC 60 CFigure 5. Peak height and peak height RSD % versus preheating temperature of split SFC effluent. Themaximum peak heights were obtained at a preheating temperature of 30 C with an RSD of approximately10 to 12 %.5

The S/N ratios were at approximately thesame level for direct connection of SFCto ELSD without preheating comparedto preheating at 30 C (Figure 6). TheS/N ratio decreased with preheatingtemperature at 40 and 60 C.Compound 3Compound 450.00Compound 5Compound 6S/N40.00Compound 7Compound 830.0020.0010.000.00DirectconnectionTCC 30 CTCC 40 CConditionsTCC 60 CFigure 6. S/N ratio versus preheating temperature of split SFC effluent. The S/N ratios were at the samelevel for direct connection and a preheating temperature of 30 C.AreaArea versus make-up flow350.00Compound 3300.00Compound 4250.00Compound 5Compound 6200.00Compound 7150.00Compound 8100.0050.000.00No make-up0.2 mL/minConditions0.4 mL/minArea RSD versus make-up flowArea RSDStarting from the optimized preheatingtemperature at 30 C, the influenceof an additional make-up flow and itsdependence from the flow rate wasexamined. The peak areas decreasedduring the addition of a make-up flowdue to the dilution, but the area RSDachieved a minimum at approximately 5 %for a make-up flow rate of 0.2 mL/min(Figure 7).60.0018.00Compound 316.00Compound 414.00Compound 512.00Compound 610.00Compound 78.00Compound make-up0.2 mL/minConditions0.4 mL/minFigure 7. Peak area and peak area RSD % versus make-up flow rate with split SFC effluent at 30 C. Thepeak areas decreased during the addition of a make-up flow, but the area RSD achieved a minimum atapproximately 5 % for a make-up flow rate of 0.2 mL/min.6

Peak height80.00Compound 370.00Compound 460.00Compound 550.00Compound 640.00Compound 730.00Compound 820.0010.000.00No make-up0.2 mL/minConditions0.4 mL/minPeak height RSD versus make-up flowPeak height RSDThe S/N ratios remained almost constantduring the addition of a make-up flowof 0.2 mL/min and started to decreasefor higher make-up flow rates (Figure 9).When considering the retained S/N ata preheating temperature of 30 C, theconnection from the SFC to the ELSDwas optimum at 30 C for preheating with0.2 mL/min make-up flow.Peak height versus make-up flow18.00Compound 316.00Compound 414.00Compound 512.00Compound 610.00Compound 78.00Compound make-up0.2 mL/minConditions0.4 mL/minFigure 8. Peak height and peak height RSD % versus make-up flow rate with slit SFC effluent at 30 C.The peak heights decreased during the addition of a make-up flow but the peak height RSD achieved aminimum at about 4 to 6 % for a make-up flow rate of 0.2 mL/min.Peak height versus make-up flow60.00Compound 3Compound 450.00Compound 5Compound 640.00Compound 7S/NThe peak heights showed the samebehavior. They decreased during theaddition of a make-up flow, but the peakheight RSD achieved a minimum atapproximately 4 to 6 % for a make-up flowrate of 0.2 mL/min (Figure 8).Compound 830.0020.0010.000.00No make-up0.2 mL/minConditions0.4 mL/minFigure 9. S/N ratio versus make-up flow rate with split SFC effluent at 30 C. The S/N ratios remainedalmost constant during the addition of a make-up flow of 0.2 mL/min and started to decrease for highermake-up flow rates.7

ConclusionsThis Technical Overview describes theconnection of the Agilent 1260 InfinityAnalytical SFC Solution to an ELSDdetector by a specially designed splitter.The effects of preheating the SFC effluentafter the split identified an optimumpreheating temperature to get the bestperformance in peak height, peak area,S/N ratio, and their relative standarddeviations. Additionally, the introductionof a make-up flow at the optimizedtemperature and the dependence of theparameters was examined. Finally, anoptimized preheating temperature andmake-up flow rate was derived from thedata.The combination of SFC and ELSD isrecommended, with additional modulesand a splitting assembly to obtainoptimum performance. To obtain theoptimized performance, a second TCCfor preheating the SFC effluent beforeentering the ELSD is required. Anadditional pump for introduction of amake-up flow is highly recommended.The preheating temperature, as well asthe make-up flow rate, must be optimizedfor each analytical SFC separation.www.agilent.com/chemThis information is subject to change without notice. Agilent Technologies, Inc., 2014Published in the USA, September 1, 20145991-5144EN

Autosampler Agilent 1260 Infi nity Thermostatted Column Compartment (TCC) Agilent 1260 Infi nity DAD with high . Confi guration of the Agilent 1260 Infi nity Analytical SFC Solution with the Agilent 1290 Infi nity ELSD. The DAD fl ow cell is directly connected to the splitter

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