title: Capillary liquid chromatography fraction collection and postcolumn reaction using segmented flow microfluidics.
abstract: A challenge for capillary LC (cLC) is fraction collection and the manipulation of fractions from microscale columns. An emerging approach is the use of segmented flow or droplet technology to perform such tasks. In this work, a fraction collection and postcolumn reaction system based on segmented flow was developed for the gradient cLC of proteins. In the system, column effluent and immiscible oil are pumped into separate arms of a tee resulting in regular fractions of effluent segmented by oil. Fractions were generated at 1 Hz corresponding to 5 nL volumes. The fraction collection rate was high enough to generate over 30 fractions per peak and preserve chromatographic resolution achieved for a five-protein test mixture. The resulting fractions could be stored and subsequently derivatized for fluorescence detection by pumping them into a second tee where naphthalene dicarboxyaldehyde, a fluorogenic reagent, was pumped into a second arm and added to each fraction. Proteins were derivatized within the droplets enabling postcolumn fluorescence detection of the proteins. The experiments demonstrate that fraction collection from cLC by segmented flow can be extended to proteins. Further, they illustrate a potential workflow for protein analysis based on postcolumn derivatization for fluorescence detection.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Nie
authors.forename: Robert T
authors.initials: RT
authors.lastname: Kennedy
mesh_terms: D000327:Adsorption; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D010316:Particle Size; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013499:Surface Properties
chemical_list: D011506:Proteins
keywords: Capillary liquid chromatography; Postcolumn derivatization; Protein separation; Segmented flow microfluidics
references.citation: Anal Chem. 2010 May 1;82(9):3449-53
references.pmid: 20373759
references.citation: Anal Chem. 2005 Feb 1;77(3):785-96
references.pmid: 15679345
references.citation: Lab Chip. 2012 Aug 7;12(15):2625-9
references.pmid: 22695710
references.citation: Anal Chem. 2006 Jul 15;78(14):4839-49
references.pmid: 16841902
references.citation: Langmuir. 2009 Feb 17;25(4):2153-8
references.pmid: 19152256
references.citation: Anal Chem. 2012 Jul 3;84(13):5794-800
references.pmid: 22656268
references.citation: Anal Chem. 1989 Mar 1;61(5):436-41
references.pmid: 2719258
references.citation: Chem Commun (Camb). 2009 Nov 7;(41):6159-61
references.pmid: 19826654
references.citation: Electrophoresis. 1997 Nov;18(12-13):2214-27
references.pmid: 9456036
references.citation: J Chromatogr. 1992 Mar 20;595(1-2):319-25
references.pmid: 1577911
references.citation: Anal Chem. 1997 Nov 15;69(22):4560-5
references.pmid: 9375517
references.citation: J Neurosci Methods. 2010 Jun 30;190(1):39-48
references.pmid: 20447417
references.citation: Angew Chem Int Ed Engl. 2009;48(15):2719-22
references.pmid: 19142923
references.citation: J Chromatogr A. 1999 Sep 24;856(1-2):117-43
references.pmid: 10526786
references.citation: J Chromatogr B Biomed Sci Appl. 1997 Jul 18;695(1):27-38
references.pmid: 9271126
references.citation: J Chromatogr A. 2004 Jun 25;1040(2):273-82
references.pmid: 15230534
references.citation: J Chromatogr A. 2004 Jan 2;1022(1-2):201-6
references.pmid: 14753787
references.citation: J Chromatogr. 1989 Oct 20;480:157-68
references.pmid: 2592478
references.citation: Annu Rev Biomed Eng. 2001;3:335-73
references.pmid: 11447067
references.citation: J Chromatogr A. 2005 Mar 4;1067(1-2):73-83
references.pmid: 15844511
references.citation: Anal Chem. 2010 Jun 15;82(12):5260-7
references.pmid: 20491430
references.citation: Lab Chip. 2004 Aug;4(4):257-64
references.pmid: 15269790
references.citation: Anal Chem. 1998 Jul 1;70(13):2493-4
references.pmid: 21644770
references.citation: Anal Chem. 2007 Apr 1;79(7):2756-61
references.pmid: 17338503
references.citation: Anal Chem. 2011 Jul 1;83(13):5207-13
references.pmid: 21604670
references.citation: J Sep Sci. 2007 Jul;30(11):1589-610
references.pmid: 17623443