多肽中非芳香氨基酸检测方案(制备液相色谱)

Sample Purification Triggered with theAgilent 1260 Infinity Evaporative LightScattering Detector Splitter solutions for columns with 4.6 to 75-mm ids Technical Overview Author Abstract Graham CleaverAgilent Technologies， Inc. This Technical Overview describes a solution to monitor and trigger the collectionof preparative samples with the Agilent 1260 Infinity Evaporative Light ScatteringDetector (ELSD) using a flow splitter. The Agilent 1260 Infinity ELSD is thedetector of choice to monitor low UV-absorbing compounds or compounds withno UV-chromophore. Furthermore， this detection method is able to detect allcompounds less volatile than the mobile phase， suitable for the detection of smallmolecule mixtures. A drug-like sample mixture has been purified on an Agilent 1260 InfinityPreparative-scale Purification System by triggering the fraction collection with theAgilent 1260 Infinity ELSD.99 % of purity and 91 % recovery were achieved. Agilent Technologies Introduction For complex mixtures， natural products，small molecules， or low-UV-wavelengthabsorbing compounds， using an ELSDis a perfect solution to achieve reliablesample purification. In addition， thisdetection method allows adjustmentsof the different detector parameterssuch as light intensity， nebulizertemperature， gas flow， and temperature.This flexibility enhances the detectorcapabilities for preparative-specificapplications.For example， in preparativeliquid chromatography， the sensitivityof the detection method is an importantcriterium. By reducing or increasing thelight intensity of the ELSD， the sensitivityof the detector can be adjusted to theconcentration used for the samplepurification. In peptide synthesis， the remainingnonaromatic amino acids cannot bemonitored by a conventional UV-detector，thereby requiring an ELSD or a MSD.The Agilent 1260 Infinity ELSD providesthe simplest way to monitor or triggerthe collection of the target compoundwithout risk of collecting impurities with the collected target peptide fractions .Another example is DMSO peak removalby increasing the gas flow or temperatureto monitor the compounds underneath ofthe DMSO signal. This Technical Overview describes aneasy way to split the preparative flowafter UV-detection to the ELSD andfraction collector for fraction collectiontriggering of the sample mixture. Collecting the sample mixture canbe done by ELSD or UV peak-basedcollection methods (Figure 1). The split is achieved by flow restrictionsgenerated by system backpressure afterthe column. Thus， for different flow rateranges and systems， different splitter kitsare required. Table 1 gives the differentsplitter configurations depending onpurification scales and systems. . Figure 1. Schematic view of the splitter configurations on the Agilent 1260 Infinity Preparative-scalePurification System， the Agilent 218 Purification System， and Agilent SD-1 Purification System. Table 1. Splitter and capillaries kits available for the different flow rate ranges of Agilent Purification Sytems Flow range extensions made possible by exchangeable pump heads ColumnAgilent ZORBAX SB-C18， Prep HTCartridge 21.2× 100 mm， 5 pm(870100-902) with end fittings(820400-901) Splitter Depending on the operating flow rate，the splitter kit part numbers are given inTable 1. Software Agilent OpenLAB CDS ChemStationEdition for LC and LC/MS Systems，Rev. C.01.05 [36] Gradient 1 minute isocratic hold at 2% B， from 2% B to 98 % B in 6 minutes and Methyl 4-hydroxy-benzoat 2.5 mg/mL 2 minutes flush time at 98 % B Flow rate 25 mL/min Agilent 1260 Infinity Multiple Wavelength Detector (G1365D) Monitored wavelength Signal A) 254 nm， bandwidth of 4 nm， Acetyl-protected sucrose 4.9 mg/mL in DMSO Reference 360 nm， bandwidth 100 nm All solvents used were LC grade， not degassed. Fresh ultrapure water was obtained from a Milli-0Integral system equipped with a 0.22-pm membrane point-of-use cartridge (Millipak) Signal B) 240 nm， bandwidth of 40 nm， no reference Agilent 1260 Infinity ELSD (G4260B) Evaporator temperature 30°C Nebulizer temperature 35C Gas flow rate 1.85 SLM Output data rate 80 Hz Smoothing 30 PMT Gain 1 Results and Discussion In this Technical Overview， we purifieda drug-like sample mixture containing atarget compound without chromophore. To prove the reliable performance ofthe splitter， the standard deviation ofthe retention time and peak area werecalculated. The Agilent 1260 InfinityPreparative-scale Purification System，combined with the Agilent 1260Infinity ELSD， gave highly reproduciblepurification results， with an averageRSD of 0.02 % for the retention times，and 2.74 % for the average peak areaprecision (Figure 2). The 21.2-mm id column was loadedwith a higher volume to determine thetypical recovery and purity of the systemcombined with the ELSD. The collection was triggered by a Booleanor a combination of the UV and ELSDsignal，each peak detector then triggeredthe collection on slope and thresholdmode. Figure 3 shows that 3.4 mg of the targetnon-UV chromophore compound waspurified，achieving a recovery of 91 %， anda purity of 99 %. Conclusion This Technical Overview shows howa simple splitter solution was used toenhance the detection method for thepurification of small molecules or non-UVchromphores with the combination of theAgilent 1260 Infinity Preparatives-scalePurification System with the Agilent 1260Infinity ELSD. The splitter kits were high reproducibleconcerning the purification results.Collection of the target compound wastriggered by the ELSD， leading to arecovery of 91 %， and a purity of 99 %. ELSD signal， Average peak %RSD UIB signal (mV) retention time (min) retention time% RSD area Figure 3. UIB/ELSD and UV signals 3D-overlays. Collection was triggered on both peak detectors.The highlighted green peak represents the fraction collected on ELSD peak-based collection， and thenon-highlighted peaks represent the UV peak-based collection. The green and red tick marks representthe beginnings and ends of collection. References 1. B. Schuhn"PerformanceCharacteristics of the Agilent 1290Infinity Evaporative Light ScatteringDetector" Agilent Technical Overview，publication number 5991-2097EN(2013). 2. B. Schuhn "Optimizing the Performanceof the Agilent 1290 Infinity EvaporativeLight Scattering Detector" AgilentTechnical Overview， publication number5991-2176EN (2013). www.agilent.com/chem This information is subject to change without notice. C Agilent Technologies， Inc.， 2011-2015Published in the USA， August 1， 2015 Abstract This Technical Overview describes a solution to monitor and trigger the collection of preparative samples with the Agilent 1260 Infinity Evaporative Light Scattering Detector (ELSD) using a flow splitter. The Agilent 1260 Infinity ELSD is the detector of choice to monitor low UV-absorbing compounds or compounds with no UV-chromophore. Furthermore, this detection method is able to detect all compounds less volatile than the mobile phase, suitable for the detection of small molecule mixtures. A drug-like sample mixture has been purified on an Agilent 1260 Infinity Preparative-scale Purification System by triggering the fraction collection with the Agilent 1260 Infinity ELSD. 99 % of purity and 91 % recovery were achieved.Introduction For complex mixtures, natural products, small molecules, or low-UV-wavelength absorbing compounds, using an ELSD is a perfect solution to achieve reliable sample purification. In addition, this detection method allows adjustments of the different detector parameters such as light intensity, nebulizer temperature, gas flow, and temperature. This flexibility enhances the detector capabilities for preparative-specific applications. For example, in preparative liquid chromatography, the sensitivity of the detection method is an important criterium. By reducing or increasing the light intensity of the ELSD, the sensitivity of the detector can be adjusted to the concentration used for the sample purification. In peptide synthesis, the remaining nonaromatic amino acids cannot be monitored by a conventional UV-detector, thereby requiring an ELSD or a MSD. The Agilent 1260 Infinity ELSD provides the simplest way to monitor or trigger the collection of the target compound without risk of collecting impurities with the collected target peptide fractions1 . Another example is DMSO peak removal by increasing the gas flow or temperature to monitor the compounds underneath of the DMSO signal. This Technical Overview describes an easy way to split the preparative flow after UV-detection to the ELSD and fraction collector for fraction collection triggering of the sample mixture. Collecting the sample mixture can be done by ELSD or UV peak-based collection methods (Figure 1). The split is achieved by flow restrictions generated by system backpressure after the column. Thus, for different flow rate ranges and systems, different splitter kits are required. Table 1 gives the different splitter configurations depending on purification scales and systems.Conclusion This Technical Overview shows how a simple splitter solution was used to enhance the detection method for the purification of small molecules or non-UV chromphores with the combination of the Agilent 1260 Infinity Preparatives-scale Purification System with the Agilent 1260 Infinity ELSD. The splitter kits were high reproducible concerning the purification results. Collection of the target compound was triggered by the ELSD, leading to a recovery of 91 %, and a purity of 99 %.