Innovative fraction collection with the Agilent 1100 Series purification platform Application Note
Contents
1. a second detector the so called delay sensor is integrated into the frac tion collector Whenever a delay calibrant is injected into the flow path both detectors record a signal The time delay between the two signals minus the migration time between diverter valve and fraction delay sensor that is calculated inter nally is the delay time Depending on the flow rate used for the cali bration procedure the exact delay volume is automatically calculated by the system and stored in the frac tion collector memory The precise delay time can now be calculated by the system for every flow rate Re calibration is not necessary Delay calibrant 23 4 5 6 Time min Figure 1 UV detector Fraction collector Diverter valve Fraction delay sensor Principle of Agilent s unique delay volume calibration Dispersion An often overlooked phenomenon that impacts the compound distri bution during migration from the detector to the fraction collector is dispersion Dispersion equates with peak broadening and there fore impairs chromatographic resolution tremendously According to the Aris Taylor equation band broadening is directly proportion al to flow rate and tubing length but proportional to the fourth power of the tubing inner diameter This effect is impressively shown in figure 2 Consequently connecting the detector with the fraction collector by tubing with an in appropriate i d will lead to bad purifi2. cation results such as a poor recovery or even a remixing of compounds Therefore a non specific fraction collector designed for a broad flow range might give acceptable results at high flow rates column i d larger than 25 mm but will sacrifice progressively recovery and purity when approaching low flow rates column with i d smaller than 9 mm Agilent therefore offers fraction collectors especially tuned for various purification scales Each fraction collector type has been manufactured to provide optimal performance at dedicated flow rate ranges and column i d for highest compound purity and recovery Additionally it should be considered that each flow disturbance contributes to additional dispersion and there fore deteriorates chromatographic resolution Sources for distur bances are columns fittings flow cells valves etc Their impact on purification results is in particular crucial at low flow rates In order to keep the disturbance of the analyte composition on its way from the detector cell to the fraction collector needle tip as unchanged as possible distur bance sources within the flow path should be avoided A key part of every fraction collector and disturbance source is the diverter valve The task of the diverter valve is to switch the flow from the column either to the waste or to the fraction collector needle tip The impact on dispersion of such a valve type solenoid valve membrane type conv
3. e detector to the fraction collector that may tremendously impact the composi tions of the collected fractions In this Application Note we described some of the innovative features of the Agilent 1100 Series purification system that ensure a nearly unperturbed analyte trans port to the fraction device Agi lents patented delay volume cali bration guarantees a convenient and precise determination of the delay volume between detector and fraction collector which is of significant importance for high purities and recoveries Further more we explain the sophisticat ed flow path design that keeps dis persion low and therefore con tributes to high chromatographic resolution without remixing of previously separated peaks Final ly Agilent s system integrated intelligence principle provides real time data processing for fast reliable and precise fraction col lection Altogether Agilent pro vides high performance purifica tion systems for a broad applica tion range and proven recoveries of nearly 100 References I Optimizing Fraction Collection with LC MSD Systems Using an Active Splitter and Delay Sensor Agilent Application Note publication number 5988 7610EN 2002 2 Patent US6106710 Al Ralf Moritz is Application Chemist at Agilent Technologies GmbH Waldbronn Germany www agilent com chem purification Copyright 2003 Agilent Technologies All Rights Reserved Reproduction adaptat
4. entionally built into many commercially available fraction collectors is displayed in figure 3 The consider able gain in peak broadening is clearly visible Depending on flow rate and analyte concentration this effect tremendously impairs purification performance In contrast the Agilent diverter valve practically has no influence on the peak shape due to its innovative design figure 4 Additionally the Agilent diverter valve distinguishes from other commercially available valves by its robustness Since it can be used at pressures up to 6 bar it guarantees leak free and reliable operation under almost all conditions 0 25 mm wo sr ee ee a a ae ee a ae aE a a a a aA a 29 4 4 5 5 55 Time min Figure 2 Impact of tubing i d on chromatographic resolution Sample volume tubing length and flow rate remained constant during the experiments SS SS SS 3 4 3 6 3 8 4 4 2 4 4 4 6 4 8 5 Time min Figure 3 Impact of conventional diverter valve solenoid valve membrane type on dispersion flow rate 1 mL min gm ee ree ieee Fee ere emer ener 4 3 4 3 6 3 8 4 4 2 44 46 48 Time min Figure 4 Impact of Agilent diverter valve patented design on dispersion same conditions as in figure 3 System integrated intelligence System integrated intelligence not only allows users a tailored modu lar system set up from the wide choice of Agilent 1100 Series mod ules but also accomplishes real time data pr
5. eo o c ee a B e e r e Abstract Due to its high separation efficiency preparative HPLC is usually employed for compound purifica tion in the pharmaceutical industry In such purification processes it is often overlooked that not only the development of an appropriate method accounts for a successful purification but also the choice of right instrumentation Only a perfectly designed instrument can ensure that the composition of a collected fraction is really in accordance with the corresponding section indicated in the chromato gram As a consequence when purifying compounds with such a system re analyses would become Innovative fraction collection with the Agilent 1100 Series purification platform Application dispensable in most instances In this Application Note we pro vide some technical insights into the innovative design of the Agilent 1100 Series purification platform Fully automated delay volume cali bration low dispersion due to a low delay volume and real time data processing contribute to a reli able system for high performance purification tasks Introduction Ideally the analyte composition that elutes as a fraction from the fraction collector needle tip corre sponds in its composition exactly to the detector signal that is if an Ralf Moritz additional chromatogram would be recorded at the fraction collec tor needle tip it would be identical to the ch
6. ion or translation without prior written permission is prohibited except as allowed under the copyright laws Printed April 1 2003 Publication Number 5988 9250EN Agilent Technologies
7. ocessing Real time data processing is of particular importance for instantaneous frac tion collection and safe and reli able system operation even in the case of PC power or network breakdown In order to maintain such a flexible and safe system operation all Agilent 1100 Series modules communicate via a Con troller Area Network CAN The outcome of this is fully PC inde pendent system operation Frac tion triggering still proceeds in real time even if the communica tion between system and PC is disturbed by heavy network traffic a busy CPU or or gets totally lost Because of this fractions are col lected precisely as indicated in the corresponding chromatogram Besides CAN connection each Agi lent 1100 Series module bears its own intelligence that starts becoming active as soon as the system receives a task from the PC The PC just represents the interface between user and instru ment Its functionality is reduced to monitoring and evaluating experimental results Conclusion Chromatographic purification sys tems have a crucial impact on purity and recovery of the target compounds Generally re analyses of the purified samples are per formed in order to confirm purity of these compounds Users expe riences have shown that the com position of the purified samples is usually different from what the corresponding chromatograms suggest The eluent flow is suscep tible to distrubances during its migration from th
8. romatogram measured by a detector that is located after the column However in order to match this requirement the follow ing effects that impact compound recovery and collection reliability have to be considered delay volume dispersion and system response This note outlines how the innovative Agilent 1100 Series purification platform design takes into account the influence of these effects on the chromatographic results to provide reliable com pound purification with high recoveries j Agilent Technologies Delay volume calibration The delay time is referred to as the time it takes for an analyte mole cule to migrate from the detector cell to the fraction collector In order to trigger start and stop of fraction collection precisely the delay time has to be determined Later on this delay time can easily be converted to the flow rate inde pendent delay volume Conven tionally the delay time is determined by injecting a dye and stopping the time until the dye appears at the fraction collector needle tip Measuring the delay time in such a manner is not only laborious but also imprecise Therefore Agilent 1100 Series purification systems comprise an innovative delay volume calibration functionality This patented feature performs the measurement of the delay volume fully automatically and precisely The principle of the delay volume calibration is illustrated in figure 1 In addition to the UV detector
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Innovative fraction collection with the Agilent 1100 Series purification platform Application Note