通过自动柱前OPA衍生进行氨基酸分析 JASCO

柱前衍生化方法对多组分具有较高的选择性和灵敏度，已广泛应用于反相HPLC。许多衍生化试剂都是市售的，人们可以选择最适合应用的试剂。其中，orthophtalaldehyde（OPA）是最常用的试剂之一，因为它在室温下与氨基酸反应非常快（秒），并且可以通过荧光检测器检测衍生氨基酸，从而提高灵敏度。在该应用中，通过使用JASCO自动进样器的自动柱前衍生功能，证明了OPA柱前衍生，其再现性比手动进样好得多。2/3Application Note Analysis of Amino Acids by Automated Pre-column Derivatization with OPA Ap plication Note USGO H Analysis of Amino Acids by Automated Pre-column Derivatization with OPA Pre-column derivatization methods for amino ac i d analysis have been widely used in reversed-phase HPLC， as they offer high selectivity and sensitivity for multiple components. Many derivatizat i on reagents are commercially available and one can choose the most suit a ble reagent for the application. Among t hem， orthophtal a ldehyde (OPA) i s one of the most commonly used reagents because it reacts with amino acids very quickly (seconds) at room te m perature and derivatized amino acids can be d e tected by a fluorescence detector offering increased sensitiv i ty . In this application，the OPA pre-column der i vatization is demonstrated by using the automated pre-column derivatizatio n function of JASCO's autosampler for much better reproducibility than manua l sampling. Condi t ions Eluent Pump： PU-2080 Degasser： DG-2080-54 Gradient Unit： LG-2080-04 Column Oven： CO-2060 Autosampler： AS-2057 Detector： FP-2020 Column： CrestPak C18S (4.6mmlDx 150mmL，5pm) Eluent A： Sodium acetate buffer (Ph 6.0)/Methanol/THF(89/10/1) Eluent B： Methanol/THF(90/10) Gradient condition： (A/B)， 0min (85/15)， 7 min (80/20) 29min(48/52) 30 min (0/100) Flow rate： 1.0mL/min Column Temperature： 20°C Wavelength： Ex. 345 nm，Em.455 nm，Gain x100 Injection Volume： 10 pL Standard 18 amino acids 1 nmol/mL each in0.01N hydrochloricacid Sample： Re t en t i o n Ti m e [min ] F i gure 1. Ch r omatogram o f a sta n da r d m ixtu r e of am i n o acids . 1： Aspara t ic a c i d ， 2：G l utamicac i d ， 3： Aspa r agine， 4： Hi stidine ， 5： Ser i ne ， 6： G lu tamine， 7： A r ginine， 8： Glys i n e， 9： Thr eon in e， 10： Ta u r i ne， 11：Ala ni ne ， 12： T yr osi n e， 13：Methio n ine ， 14：Valine ， 15：P henyla l a n ine ，16： I soleu c ine， 17：L eucine， 18： Lysi n e Pr e-column D eri v atization Fu n ction o f the Autosa m per T he ope r ati n g pr in ciple o f the p re -co l um n de ri va t iza t i o n fu n c t i on of m o del AS -2057 autosam pl er is a s s h own in f i gure2.By u s i ng t h is fu n ct i on ，the pr e -colum n derivatizat i on can be pe r f o r med automat i cally . Fi gure 2. Pre -co l u mn de r i v a t iza t ion f u n ct i on o f m o de l AS -2057 A ut osample r. C ond it ions for Pre-column D eri v a t ization Volume of sample solution：50 pL Volume of reagent solution：10pL Reaction time：0.1min Reagent solution： 0.4Mborate/ 1% OPA solution/2-mercaptoethanol (1/0.5/0.01)