Sophisticated peak-based fraction collection – working with up and ...

1. Fraction triggering decisionsUp and down slopeWhat is up and down slope?The slope is the first derivative ofthe signal as shown in figure 1.The slope value equals zero at thebaseline, rises to a maximumvalue at the first inflexion point,becomes zero at the peak apex,falls to a negative maximum at thesecond inflexion point andbecomes zero again at the baselineafter the peak.Tip:Peak start and stop can only betriggered using up and downslope between the baseline andthe inflexion points because atthe inflexion points the slopereaches its positive or negativemaximum. Also, always keep inmind that the slope maximum ofthe peaks strongly depends on thepeak intensities.Fraction triggering decisionwhen using up and down slopeFigure 2 shows how the up anddown slope set-up values are usedto decide whether a peak start orstop is triggered. To trigger a peakstart the slope simply has to riseabove the value entered for upslope. As soon as a peak start isfound the system expects to find apeak stop. To find a peak stop theslope must first fall below the negativevalue of the down slopeentered and as soon as it risesagain above the negative value thepeak stop is triggered. While fractiontriggering on slope-only ispossible it can also be combinedwith the threshold using the WorkingMode Threshold/Slope in theAgilent ChemStation. Figure 3shows a chromatogram with thestart and stop tick marks for thecollected fractions. The start of afraction is triggered when the setup values for threshold and upslope are exceeded (tick mark 1).A peak stop is triggered when thesignal falls either below the0Up slopeFigure 1The slope is the first derivative of a peak0Up slopeStartFigure 2Using up and down slope values for fraction triggering2σ4σthreshold or meets the downslope criteria. At tick mark 2 thefraction was stopped since thedown slope criteria was metbecause the slope equals zero atthe local minimum. The thresholdwas still exceeded at this point.Down slopeInflexionpointStop1 st derivativeChromatogramDown slope(negative value!)3

terminated. Since the fraction triggeringparameters are activatedagain immediately after the maximumpeak duration was exceededthis feature should not be usedwhen triggering on threshold only.If the end of the fraction couldnot be found when collecting onthreshold only indicates, that thesignal did not fall back below thespecified threshold. If the maximumpeak duration terminatesthe fraction but the signal is stillabove the threshold means, thatthe collection of the next fractionstarts immediately. To avoid this itis recommended to set MaximumPeak Duration to a relatively highvalue for fraction collection onthreshold only.Tip:When Threshold only was selectedas Working Mode for fraction collectionthe Maximum Peak Durationshould be set to a high value,for example the run time.Fraction preview toolThe fraction preview tool (figure 5)can be used to determine the optimalfraction collection parametersfor repetitive purification runsand to simulate fraction collectionusing these parameters. Afterloading a signal the fraction collectionparameters threshold, upand down slope, upper thresholdand maximum peak duration canbe applied to the signal and theresulting fraction collection is displayed.With the buttons on theright hand side of the screen, severalcursors for up slope, downslope, upper threshold and threshold(from top to bottom) can beactivated and moved through theFigure 5Fraction preview toolchromatogram (black line at 1.20min). When moving the cursorthrough the chromatogram thevalue of the selected parameter isalso displayed at the bottom of thewindow. When the cursoris set to a specific point in thechromatogram a left-click with themouse copies the value at thispoint into the parameter sectionof the peak detector. While thefraction preview tool can be usedto optimize fraction collectionparameter settings for repetitivepurification runs it can also beused to get familiar with thefraction collection parametersthreshold, up and down slopeand upper threshold.2. ApplicationsSeparation of steep and shallowpeaksBy using up and down slope it ispossible to separate steep peaksfrom shallow peaks. Figure 6shows that it is easy to separatesteeper peaks from more shallowpeaks. To to do this the up anddown slope values have to beknown before starting fractioncollection. This is only possible ifa pilot run is available. In manypurification applications, especiallyin the drug discovery phase, thisis not the case. Due to the largenumber of samples a genericmethod has to be set up. Optimum5

peak tail. This means that withthe same values for up and downslope it can happen that a startcriteria for a peak was foundbut no stop criterion (figure 8a).Using a lower value for downslope than for up slope led to aproper collection of the twopeaks at about 6.5 and 8 minutes(figure 8b).Tip:The down slope value should beset to a lower value then the upslope value. Most peaks are tailingand with identical up anddown slope values a peak stopmay not be found.a) Absorbanceb)[mAU]Stop/startbecause wellwas full2000150010005000No stopfoundStopfound5.5 6.5 7.5 8.5 9.5Time [min]Absorbance[mAU]150010005000Stopfound5.5 6.5 7.5 8.5 9.5Time [min]Separation of non-baselineseparated peaksIf two peaks are not baseline-separatedthere are a few strategiesto collect the two compounds asseparate fractions, usually withgood recovery and purity 4 . One ispeak-based fraction collectionusing up and down slope. Sincethe slope in the minimumbetween the peaks equals zero thepeaks can be split into two fractionseven if the signal does notfall below the threshold value byusing a minimum down slopevalue of 1 mAU/s or even lower(figure 9). This means the peakstop criterion is found almost atthe valley point and the start ofthe next fraction is found immediatelyafterwards. However, ittakes some time for the fractioncollection needle to move to thenext position, which leads to thegap between the stop and starttick marks in figure 9. As a resultfraction one contains someamount of the second compoundwhile in the second fraction someFigure 8a) Up and down slope: 100 mAU/s, b) Up slope: 100 mAU/s, down slope 1 mAU/sAbsorbance[mAU]100080060040020003 3.5 4 4.5 5 5.5 6 6.5Figure 9Up and down slope: 1 mAU/s,Time [min]7

ConclusionIn this Application Note weexplained what up and downslope is and how it is used totrigger fractions in peak-basedfraction collection. We alsoshowed the advantages of beingable to specify two parameters, upand down slope, rather than only asingle slope parameter. For manyapplications peak-based fractioncollection based on threshold onlyis sufficient, however, we alsoshowed some application examplesthat also require up and downslope. Examples are separation ofsteep and shallow peaks, separationof compounds showing nonbaselineseparated peaks andpurification of compounds fromchromatograms with drifting baseline.We also explained how thetriggering of unwanted fractionsfor a peak in the detector saturationcan be avoided using theupper threshold parameter. Tosimulate a fraction collection runor to get familiar with parametersettings for threshold, up anddown slope, upper threshold andthe maximum peak duration thefraction preview tool wasdescribed. For those and all otherapplications requiring additionalpeak detection criteria up anddown slope offers the possibilityof sophisticated peak-based fractioncollection with the Agilent1100 Series purificationsystem.10

References1.“New dimensions for HPLC applications”,Agilent TechnologiesBrochure, publication number5988-6707EN, 2002.2.“Fluorescence-based isolation offormononetin from red cloverextract with the Agilent 1100Series purification system”,Agilent Technologies ApplicationNote, publication number5988-5749EN, 2002.3.“Peak-based fraction collectionusing an evaporative light scatteringdetector with the Agilent 1100Series purification system”, AgilentTechnologies ApplicationNote, publication number5988-5816EN, 2002.4.“Strategies for purification of compoundsfrom non-baseline separatedpeaks”, Agilent TechnologiesApplication Note, publicationnumber 5988-7460EN,2002.11

Udo Huber is ApplicationChemist at Agilent Technologies,Waldbronn, Germany.www.agilent.com/chem/purification© 2004 - 2010 Agilent TechnologiesPublished June 15, 2010Publication Number 5989-0511EN