This work was supported by UWS and ThermoFisher Scientific. One of the authors (DK) acknowledges the receipt of an Australian Postgraduate Award.

<ol>
	<li>Srijaranai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+1-(2-pyridylazo)-2-naphthol+as+the+post+column+reagent+for+ion+exchange+chromatography+of+heavy+metals+in+environmental+samples.">Use of 1-(2-pyridylazo)-2-naphthol as the post column reagent for ion exchange chromatography of heavy metals in environmental samples.</a> Microchem. J. 99, 152-158 (2011).</li><li>Kubickova, A., Kubicek, V., Coufal, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV-VIS+detection+of+amino+acids+in+liquid+chromatography:+online+post-column+solid-state+derivatization+with+Cu(II)+ions.">UV-VIS detection of amino acids in liquid chromatography: online post-column solid-state derivatization with Cu(II) ions.</a> J Sep Sci. 34, 3131-3135 (2011).</li><li>Quinto, M., Spadaccino, G., Palermo, C., Centonze, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+aflatoxins+in+cereal+flours+by+solid-phase+microextraction+coupled+with+liquid+chromatography+and+post-column+photochemical+derivatization-fluorescence+detection.">Determination of aflatoxins in cereal flours by solid-phase microextraction coupled with liquid chromatography and post-column photochemical derivatization-fluorescence detection.</a> J. Chromatogr. A. 1216, 8636-8641 (2009).</li><li>Lee, M., Lee, Y., Soltermann, F., von Gunten, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+N-nitrosamines+and+other+nitro(so)+compounds+in+water+by+high-performance+liquid+chromatography+with+post-column+UV+photolysis/Griess+reaction.">Analysis of N-nitrosamines and other nitro(so) compounds in water by high-performance liquid chromatography with post-column UV photolysis/Griess reaction.</a> Water Res. 47, 4893-4903 (2013).</li><li>Niu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+isoflavonoids+in+Radix+Puerariae+for+quality+control+using+on-line+high+performance+liquid+chromatography-diode+array+detector-electrospray+ionization-mass+spectrometry+coupled+with+post-column+derivatization.">Identification of isoflavonoids in Radix Puerariae for quality control using on-line high performance liquid chromatography-diode array detector-electrospray ionization-mass spectrometry coupled with post-column derivatization.</a> Food Res Int. 48, 528-537 (2012).</li><li>Zacharis, C. K., Tzanavaras, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+chromatography+coupled+to+on-line+post+column+derivatization+for+the+determination+of+organic+compounds:+a+review+on+instrumentation+and+chemistries.">Liquid chromatography coupled to on-line post column derivatization for the determination of organic compounds: a review on instrumentation and chemistries.</a> Anal. Chim. Acta. 798, 1-24 (2013).</li><li>Dousa, M., Brichac, J., Gibala, P., Lehnert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+hydrophilic+interaction+chromatography+determination+of+lysine+in+pharmaceutical+preparations+with+fluorescence+detection+after+postcolumn+derivatization+with+o-phtaldialdehyde.">Rapid hydrophilic interaction chromatography determination of lysine in pharmaceutical preparations with fluorescence detection after postcolumn derivatization with o-phtaldialdehyde.</a> J Pharm Biomed Anal. 54, 972-978 (2011).</li><li>Iijima, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+an+Online+Post-Column+Derivatization+System+for+Ultra+High-Performance+Liquid+Chromatography+(UHPLC)+and+Its+Applications+to+Analysis+of+Biogenic+Amines.">Optimization of an Online Post-Column Derivatization System for Ultra High-Performance Liquid Chromatography (UHPLC) and Its Applications to Analysis of Biogenic Amines.</a> Anal Sci. 29, 539-545 (2013).</li><li>Cunico, R. L., Schlabach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Ninhydrin+and+o-Phthalaldehyde+Postcolumn+Detection+Techniques+for+High+Performance+Liquid+Chromatography+of+Free+Amino.">Comparison of Ninhydrin and o-Phthalaldehyde Postcolumn Detection Techniques for High Performance Liquid Chromatography of Free Amino.</a> J. Chromatogr. A. 1983, 461-470 (1983).</li><li>Donahue, E. P., Brown, L. L., Flakoll, P. J., Abumrad, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Measurement+of+Leucine-specific+Activity+in+Biological+Fluids+by+Ion-exchange+Chromatography+and+Post-column+Ninhydrin+Detection.">Rapid Measurement of Leucine-specific Activity in Biological Fluids by Ion-exchange Chromatography and Post-column Ninhydrin Detection.</a> J. Chromatogr. A. 571, 29-36 (1998).</li><li>Udenfriend, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescamine:+A+Reagent+for+Assay+of+Amino+Acids,+Peptides,+Proteins+and+Primary+Amines+in+the+Picomole+Range.">Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins and Primary Amines in the Picomole Range.</a> Science. 1972, 871-872 (1972).</li><li>Samejima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+Fluorescamine+Derivitices+of+Aliphatic+Diamines+and+Polyamines+by+High+Speed+Liquid+Chromatography.">Separation of Fluorescamine Derivitices of Aliphatic Diamines and Polyamines by High Speed Liquid Chromatography.</a> J. Chromatogr. A. 96, 250-254 (1974).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+antioxidant+activity+of+ten+compounds+in+different+tea+samples+by+means+of+an+on-line+HPLC-DPPH+assay.">Evaluation of antioxidant activity of ten compounds in different tea samples by means of an on-line HPLC-DPPH assay.</a> Food Res Int. 53, 847-856 (2013).</li><li>Niu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+anti-oxidants+in+Flos+Chrysanthemi+by+HPLC-DAD-ESI/MS(n)+and+HPLC+coupled+with+a+post-column+derivatisation+system.">Identification of the anti-oxidants in Flos Chrysanthemi by HPLC-DAD-ESI/MS(n) and HPLC coupled with a post-column derivatisation system.</a> Phytochem Anal. 24, 59-68 (2013).</li><li>Raudonis, R., Bumblauskiene, L., Jakstas, V., Pukalskas, A., Janulis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+validation+of+post-column+assay+for+screening+of+radical+scavengers+in+herbal+raw+materials+and+herbal+preparations.">Optimization and validation of post-column assay for screening of radical scavengers in herbal raw materials and herbal preparations.</a> J. Chromatogr. A. 1217, 7690-7698 (2010).</li><li>Raudonis, R., Raudone, L., Jakstas, V., Janulis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+post-column+free+radical+scavenging+and+ferric+reducing+antioxidant+power+assays+for+screening+of+antioxidants+in+strawberries.">Comparative evaluation of post-column free radical scavenging and ferric reducing antioxidant power assays for screening of antioxidants in strawberries.</a> J. Chromatogr. A. 1233, 8-15 (2012).</li><li>Zakrzewski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Methimazole+in+Pharmaceutical+Preparations+using+an+HPLC+Method+Coupled+with+an+Iodine-Azide+Post-Column+Reaction.">Determination of Methimazole in Pharmaceutical Preparations using an HPLC Method Coupled with an Iodine-Azide Post-Column Reaction.</a> J. Liq. Chrom. Rel. Technol. 32, 383-398 (2008).</li><li>Zakrzewski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+reversed-phase+HPLC+method+with+post-column+iodine-azide+reaction+for+the+determination+of+thioguanine.">Development and validation of a reversed-phase HPLC method with post-column iodine-azide reaction for the determination of thioguanine.</a> J. Anal. Chem. 64, 1235-1241 (2009).</li><li>Gritti, F., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+measurements+of+the+true+column+efficiency+and+of+the+instrument+band+broadening+contributions+in+the+presence+of+a+chromatographic+column.">Accurate measurements of the true column efficiency and of the instrument band broadening contributions in the presence of a chromatographic column.</a> J. Chromatogr. A. 1327, 49-56 (2014).</li><li>Stewart, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post+cotumn+derivatization+methodology+in+high+performance+liquid+chromatography+(HPLC).">Post cotumn derivatization methodology in high performance liquid chromatography (HPLC).</a> Trends Anal. Chem. 1, 170-174 (1982).</li><li>Rigas, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-column+labeling+techniques+in+amino+acid+analysis+by+liquid+chromatography.">Post-column labeling techniques in amino acid analysis by liquid chromatography.</a> Anal. Bioanal. Chem. 405, 7957-7992 (2013).</li><li>Frei, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+Detectors+in+Modern+Liquid+Chromatography.">Reaction Detectors in Modern Liquid Chromatography.</a> Chromatographia. 15, 161-166 (1982).</li><li>Pravadil-Cekic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Reaction+Flow+Chromatography+for+the+Analysis+of+Amino+Acid:+Derivatisation+With+Fluorescamine+Reagent.">Using Reaction Flow Chromatography for the Analysis of Amino Acid: Derivatisation With Fluorescamine Reagent.</a> Microchem. J. , (2015).</li><li>Camenzuli, M., Ritchie, H. J., Dennis, G. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+segmented+flow+chromatography+columns+with+multiplexed+detection:+An+illustration+using+antioxidant+screening+of+natural+products.">Parallel segmented flow chromatography columns with multiplexed detection: An illustration using antioxidant screening of natural products.</a> Microchem. J. 110, 726-730 (2013).</li><li>Koleva, I. I., Niederlander, H. A. G., van Beek, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+On-Line+HPLC+Method+for+Detection+of+Radical+Scavenging+Compounds+in+Complex+Mixtures.">An On-Line HPLC Method for Detection of Radical Scavenging Compounds in Complex Mixtures.</a> Anal Chem. 72, 2323-2328 (2000).</li><li>Selim, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Two-component+Post-column+Derivatisation+Method+Utilsing+Reaction+Flow+Chromatography.">A Two-component Post-column Derivatisation Method Utilsing Reaction Flow Chromatography.</a> Microchem. J. 116, 87-91 (2014).</li><li>Bigley, F. P., Grob, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Phenols+in+Water+and+Wastewater+by+Post-column+Reaction+Detection+High-performance+Liquid+Chromatography.">Determination of Phenols in Water and Wastewater by Post-column Reaction Detection High-performance Liquid Chromatography.</a> J. Chromatogr. A. 350, 407-416 (1985).</li><li>Camenzuli, M., Ritchie, H. J., Dennis, G. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+flow+chromatography+for+rapid+post+column+derivatisations:+The+analysis+of+antioxidants+in+natural+products.">Reaction flow chromatography for rapid post column derivatisations: The analysis of antioxidants in natural products.</a> J. Chromatogr. A. 1303, 62-65 (2013).</li></ol>

The authors have nothing to disclose.

RF-PCD מאפשר ערבוב יעיל של מגיב derivatization עם הפוסט-טור בשפכים HPLC ללא שימוש סלילי התגובה, למזער את ההשפעות של הרחבת הלהקה ושיפור ביצועים ההפרדה. שיטות RF-PCD גם הראו שיפורים בתגובה האות ביחס שיטת הזיהוי. Camenzuli et al. 28 היה הראשון לדווח על השימוש בעמודי זרימת תגובה עם DPPH...

כרומטוגרפיה נוזלית ביצועים גבוהים (HPLC) בשילוב עם פוסט טור derivatization (PCD) היא כלי רב עצמה כי הוא שימושי בפתרון מספר בעיות במעבדה האנליטית. זה יכול לשמש כדי לזהות תרכובות שאינן אחר לגילוי עם החבילה של גלאי זמין 1,2, להגדיל את האות של אנליטי היעד, המאפשר גבולות נמוכים של גילוי quantitation 3-5 או סלקטיבי derivatize אנליטי יעד על מנת למנוע תופעות מטריקס 6. בשימוש נפוץ התגובות PCD כוללים התגובה של אמינים, כגון חומצות אמינו, עם-phthaladehyde אורתו 7-9, ninhydrin 9,10 או fluorescamine 11,12, את derivatization של מינים חמצן תגובתי (ROS) עם 2,2-diphenyl- 1-picrylhydrazil הרדיקלי (DPPH •) 13,14 או 2,2&#39;-azino-bis (3-ethylbenzothiazoline-6-sulphonic חומצה (ABTS) 15,16, ושימוש מגיב-אזיד יודיד כדי derivatize ג גופריתתרכובות ontaining 17,18. יש, עם זאת, חסרונות רבים לשימוש תגובות PCD עם מערכות HPLC 6. בעיקר בקרב אלה הוא השימוש סליל תגובה בין נקודת תוספת של מגיב derivatization (ים) ו הגלאי, המאפשר זמן לערבוב והתגובה להתרחש 8. יש תגובה אלה לולאות לעתים קרובות כרכים של 500 μl או יותר, אשר גבוה משמעותית לעומת היקף שאר המערכת HPLC 19. שימוש תגובה בנפח גבוה אלה לולאות תוצאות רחבות שיא גדל לעומת מה יהיה ציין ללא הנוכחות של לולאת התגובה. התוצאה היא קצרות, פסגות רחבות יותר כי יש גבולות גבוהים של quantitation וזיהוי ואפקטים שלילי רזולוצית chromatographic. איורי 1 ו -2 להדגיש את ההידרדרות של צורת שיא הנגרמות כתוצאה מהתוספת של כרכי לולאת תגובה שונים פוסט עמודה. ניתוח זהבוצע באמצעות רכב שלב נייד של מתנול 94% ו -6% מי מילי- Q. קצב הזרימה של השלב הנייד היה 1 מ&quot;ל / דקה, את עוצמת הזריקה היה 20 μl ואת אורך הגל הניתוח היה 265 ננומטר. סלילי של כמויות משתנות מת מ -20 μl 1,000 μl הוכנסו בין הטור לבין הגלאי כדי לדמות את ההשפעה של נפח מת לולאת תגובה בשיטות PCD. לולאות אלה הוכנו מ צינורות נירוסטה של ​​קוטר פנימי 0.5 מ&quot;מ. הניסוי בוצע על מערכת HPLC המורכבת של בקר (SCL-10AVP), בלחץ נמוך Gradient Valve (FCL-10ALVP), משאבה (LC-20AD), מזרק (SIL-10ADVP), וגלאי PDA ( SPD-M10ADVP). השלב הנייד נשאב באמצעות מסלק גזים לפני המבוא למערכת HPLC. ההפרדה בוצעה באמצעות טור 250 מ&quot;מ x 4.6 מ&quot;מ id 5 מיקרומטר. תנאי הניסוי נבחרו להיות אופייני של תגובות PCD אשר פורסמו לאחרונה בספרות. הפשוט, התקנת כור טור פוסט הנפוץ ביותר נקראת כור צינורי הלא מפולח אשר הוא למעשה צינור ארוך, דק שדרכו הנוזל יכול לזרום ואת התגובה יכולה להתרחש. בשנת שיא במערכת זו רחבה תלויה לא רק את הנפח מת להוסיף למערכת, אלא גם את הקוטר הפנימי של הצינור כפי מודגש על ידי Iijima et al. 8. יתר על כן, גיאומטרית סליל ממלא תפקיד הרחבת המותג ציינה. סטיוארט 20 נאמר כי התפתלות של הכור משנה את פרופילי זרימה משני, וכתוצאה מכך ערבוב טוב יותר, כלומר הנפח המת יכול להיות ממוזער. זה כבר נאמר כי הרחבת השיא הוא לא משמעותי בעת שימוש צינורי פתוח סרוגים סליל 21. כאשר הרחבת השיא היא גדולה מדי, סוגים אחרים של כורים יכולים גם להיחשב 20,22. אלה עשויים לכלול כורי מיטה או כורי תזרים מפולחים. כורים אלה הם שימושיים במיוחד עבור תגובות איטיות שאחרת requirלולאות התגובה הגדולה הדואר. כמו כורי צינורי הלא מפולחים הם הסוגים הנפוצים ביותר של כורים המשמשים ליישומי PCD, שאר מאמר זה עוסק בסוג זה של התקנת כור. העיצוב של זרימת תגובה (RF) טור משלבת הולמת סוף מרובה יציאות מאפשרת בשלב נייד כדי לצאת (או להיכנס) בעמודה או דרך יציאה אחת הממוקמת באזור המרכז הרדיאלי של העמודה או שלוש יציאות הממוקמים בחלק החיצוני קיר באזור של הטור (ראה איור 3). זרמים אלה שני מופרדים באמצעות הולם סוף המכיל frit נקבובי מרכזי מוקפת טבעת חדירה כי הוא בתורו המוקף frit הנקבובי חיצוני המשתרע החוצה אל קיר העמודה. בשל זרימת צלב טבעת המרכזית החדירה אינו אפשרי בין שני האזורים הנקבוביים. במהלך כרומטוגרפיה זרימת תגובה, מגיב derivatization (ים) נשאבים נגד כיוון זרימה בשלב נייד לאחת או TWo הנמלים החיצוניים של טור זרימת תגובה. Eluent הטור הוא מעורבב עם מגיב derivatization (הים) frit החיצוני והעביר אל הגלאי דרך יציאה חיצונית בחינם. זרימת תגובה יכולה לשמש אחת מעמד derivatization מגיב יחיד (1 נמלית מגיב derivatization, 1 יציאה להעביר את eluent העמודה אל הגלאי ו -1 היציאה החסומה) או מערכת מגיב כפולה (2 יציאות עבור ריאגנטים derivatization ו -1 יציאה להעביר את eluent עמודה אל הגלאי). תזרים מן הזרם המרכזי או שניתן להשתמש בהם כדי לזהות את eluent טור underivatized, ריבוב ביעילות זיהוי 23, או עבר לבזבז. טכניקת כוונון אחד עיקרית זמינה בעת הפעלת כרומטוגרפיה RF-PCD הוא היחס של התזרים המרכזי ואת הפריפריה. היחס האופטימלי עבור כל derivatization תלוי במספר גורמים כגון האם הזרם המרכזי יזוהו או עבר לבזבז. לכן פעם היחס האופטימלי נקבע, יש להבטיח את זה כי יחס התזרים הנכון מושגת לפני כל סיבוב מתבצע. זה כבר נמצא כי השימוש של frit לערבב את זרם eluent עמודה מגיב derivatization בתוצאות RF-PCD ב ערבוב יעיל יותר לעומת טכניקות ערבוב המסורתי שבדרך כלל להעסיק נפח מת אפס מת נפח T-piece או נמוך פ- חתיכה לערבב שני הזרמים. זו אפשרה את השימוש של לולאות תגובה קטנות יחסית, או אפילו החיסול של לולאת התגובה כלילה. צמצום תוצאות גודל לולאת תגובת פסגות חדות בהשוואה לשיטות derivatization טור פוסט מסורתיות. משמעות דבר היא כי, על אף העובדה כי לא כל eluent העמודה derivatized, אות גדולה יחס רעש הם נצפו ולכן גבולות נמוכים של גילוי quantitation יכולים להיות מושגת. כרומטוגרפיה זרימת תגובה פותחה כדי להתגבר על קשיים עם התאמת תגובת PCD<html> ים לעמודות ומערכות HPLC מודרניים, במיוחד מההפסד היעיל הנגרם על ידי להקת הרחבה בשל כרכים מתו טור פוסט גדולים הנובעים מהצורך להעסיק תגובת נפח גדולה לולאות. תהליכי הערבוב היעילים יותר ב RF-PCD לעומת PCD הקונבנציונלי אומרים כרכי לולאת תגובה קטנה יכולים להיות מועסקים מובילים לעלייה יעילה הפרדה נצפית. יתר על כן כרומטוגרפיה RF-PCD מראה הן גדלו אות וירידת רעש לעומת טכניקות PCD קונבנציונליות וכתוצאה מכך גבולות נמוכים של גילוי quantitation בהשוואה לשיטות PCD קונבנציונליים. יתרון נוסף של RF-PCD בהשוואה לשיטות PCD קונבנציונאלי היא היכולת לנטר את זרם underivatized כי elutes מהנמל המרכזי של הטור RF כמו גם את זרם derivatized כי elutes מאזור הפריפריה של הטור. RF-PCD היא טכניקה חדשה יחסית אך מבטיח המציג יתרונות רבים על פני שיטות PCD המסורתית. &gt; חיבור של טור RF מושג כמעט באותו אופן כמו טור HPLC קונבנציונלי עם ההבדל העיקרי להיות מספר אבזרי קצה על עמודת RF. אבזרים המשמשים לחיבור טור HPLC תקן למערכת HPLC מסוגלים לשמש לחיבור טור RF למערכת HPLC.

<table><tbody><tr><td>HPLC instrument</td><td>Agilent</td><td>1290 Series HPLC</td><td></td></tr><tr><td>Additional Pump(s) for derivatization system</td><td>Shimadzu</td><td>LC-20A</td><td></td></tr><tr><td>RF colum</td><td></td><td></td><td>Non-commercial</td></tr><tr><td>PEEK tubing</td><td>Sigma Aldrich</td><td>Z227307</td><td></td></tr><tr><td>Column stoppers</td><td></td><td></td><td>Provided with column</td></tr><tr><td>PEEK tube cutter</td><td>Sigma Aldrich</td><td>Z290882</td><td></td></tr><tr><td>Analytical Scale Balance</td><td></td><td></td><td>4-point analytical balance</td></tr><tr><td>Stop watch</td><td></td><td></td><td>Non-Scientific equiptment</td></tr><tr><td>Eluent collection vials</td><td></td><td></td><td>Any Small vial with a flat bottom will do, &lt;em&gt;e.g.&lt;/em&gt;, HPLC vials</td></tr><tr><td>HPLC Vials</td><td></td><td></td><td>Will depend on instrument used</td></tr><tr><td>Vessels for mobile phase and derivatization solution(s)</td><td>Sigma Aldrich</td><td>Z232211</td><td></td></tr><tr><td>General Laboratory glassware</td><td></td><td></td><td>Volumetric Flasks, pippettes, &lt;em&gt;etc.&lt;/em&gt; Quantity and volumes will depend on sample preparation method.</td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>34860</td><td></td></tr><tr><td>DPPH</td><td>Sigma Aldrich</td><td>D9132</td><td></td></tr><tr><td>Ammonium Acetate</td><td>Sigma Aldrich</td><td>17836</td><td></td></tr><tr><td>Ammonia</td><td>Sigma Aldrich</td><td>320145</td><td>Corrosive</td></tr><tr><td>Acetonitrile</td><td>Sigma Aldrich</td><td>34998</td><td></td></tr><tr><td>Fluorescamine</td><td>Sigma Aldrich</td><td>F9015</td><td></td></tr><tr><td>4-aminoantipyrene&amp;nbsp;</td><td>Acros Organics BVBA</td><td>AC103151000</td><td></td></tr><tr><td>Potassium ferricyanide&amp;nbsp;</td><td>AnalaR</td><td>B10204-30</td><td></td></tr></tbody></table>

post column derivatization using reaction flow high performance liquid chromatography columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

שיטת PCD הראשונה הותאמה לשימוש על ידי RF-PCD הייתה derivatization של נוגדי חמצון באמצעות 2,2-diphenyl-1-picrylhydrazil הרדיקלי (DPPH •) 24. תגובה זו הונהגה על ידי ואח Koleva. 25 כבר בשימוש נרחב מאז. הגילוי מסתמך על הַדהָיָה של DPPH • הרדיקלי בנוכחות של מיני חמצן תגובתי, ומכא...

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Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

הודעת הטור derivatization שימוש ביצועים גבוהים תזרים תגובת עמודות נוזלת כרומטוגרפיה

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

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15.0K Views.  University of Western Sydney. A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

Video: Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms of efficiency and sensitivity and to enable multiplexed detection. This form of AFT uses a parallel segmented flow (PSF) column. A PSF column outlet end-fitting consists of 2 or 4 ports, which can be multiplexed to connect up to 4 detectors. The PSF column not only allows a platform for multiplexed detection but also the combination of both destructive and non-destructive detectors, without additional dead volume tubing, simultaneously. The amount of flow through each port can also be adjusted through pressure management to suit the requirements of a specific detector(s). To achieve multiplexed detection using a PSF column there are a number of parameters which can be controlled to ensure optimal separation performance and quality of results; that is tube dimensions for each port, choice of port for each type of detector and flow adjustment. This protocol is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

Here, we present a protocol for the operation and tuning of parallel segmented flow chromatography columns to enable multiplexed detection.

כוונון טור מקוטע זרימה מקבילה והפעלת איתור Multiplexed

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms ...

Curtain Flow Column: Optimization of Efficiency and Sensitivity

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially purpose built multiport end-fitting(s). Curtain Flow (CF) columns belong to the AFT suite of columns, specifically the CF column is designed so that the sample is injected into the radial central region of the bed and a curtain flow of mobile phase surrounding the injection of solute prevents the radial dispersion of the sample to the wall. The column functions as an 'infinite diameter' column. The purpose of the design is to overcome the radial heterogeneity of the column bed, and at the same time maximize the sample load into the radial central region of the column bed, which serves to increase detection sensitivity. The protocol described herein outlines the system and CF column set up and the tuning process for an optimized infinite diameter 'virtual' column.

Here, we present a protocol for the operation and optimization of Active Flow Technology (AFT) column in Curtain flow (CF) mode for enhanced separation performance.

הרמת מסך טור זרימה: אופטימיזציה של יעילות ורגישות

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially ...

Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach

Capillary ultra-high performance liquid chromatography (UHPLC) is currently a method of choice for the sample separation step in LC-MS-based proteomics. However, capillary columns are much less robust in comparison to their higher flow countertypes. Because of easy contamination and blocking, they often need replacement. That makes them a markedly expensive part of the total LC-MS analysis cost. In-house packing of UHPLC capillary columns saves a lot of money and allows customization. However, the standard packing procedure in the 100-bar pressure bomb works well only for HPLC columns but is too slow for UHPLC sorbents. Here we provide a description of an optimized FlashPack protocol applied to the same 100-bar pressure bomb setup. The method is based on packing from ultra-high sorbent concentration slurry and is developed for in-house manufacturing of UHPLC capillary columns of unlimited length in reasonable time.

Here we present a protocol for the optimized FlashPack capillary column packing procedure. Application of an optimized protocol to a common 100-bar pressure bomb setup allows 10-times faster packing and manufacturing of long ultra-high performance capillary columns.

ייצור עמודות נימי פשוטות עם ביצועים גבוהים במיוחד עם גישת FlashPack

Capillary ultra-high performance liquid chromatography (UHPLC) is currently a method of choice for the sample separation step in LC-MS-based proteomics. ...

Biochemistry

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

כרומטוגרפיה נוזלית בשילוב לאינדקס שבירה או זיהוי ספקטרומטרי מסה ליצירת פרופיל מטבוליט במערכות ללא תאים מבוססות ליסאט

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...

Bioengineering

High-Performance Liquid Chromatography (HPLC)

Source: Dr. Paul Bower - Purdue University
High-performance liquid chromatography (HPLC) is an important analytical method commonly used to separate and quantify components of liquid samples. In this technique, a solution (first phase) is pumped through a column that contains a packing of small porous particles with a second phase bound to the surface. The different solubilities of the sample components in the two phases cause the components to move through the column with different average velocities, thus creating a separation of these components. The pumped solution is called the mobile phase, while the phase in the column is called the stationary phase.
There are several modes of liquid chromatography, depending upon the type of stationary and/or mobile phase employed. This experiment uses reversed-phase chromatography, where the stationary phase is non-polar, and the mobile phase is polar. The stationary phase to be employed is C18 hydrocarbon groups bonded to 3-&#181;m silica particles, while the mobile phase is an aqueous buffer with a polar organic modifier (acetonitrile) added to vary its eluting strength. In this form, the silica can be used for samples that are water-soluble, providing a broad range of applications. In this experiment, the mixtures of three components frequently found in diet soft drinks (namely caffeine, benzoate, and aspartame) are separated. Seven prepared solutions containing known amounts of the three species are used, and their chromatograms are then recorded.

Operation of High-Performance Liquid Chromatography HPLC

כרומטוגרפיה נוזלית בעלת ביצועים גבוהים (HPLC)

Source: Dr. Paul Bower - Purdue University
High-performance liquid chromatography (HPLC) is an important analytical method commonly used to separate and ...

JoVE Science Education

Purification of a Total Lipid Extract with Column Chromatography

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The product of an organic solvent extraction, a total lipid extract (TLE), is often a complex mixture of hundreds, if not thousands, of different compounds. The researcher is often only interested in a handful of compounds. The compounds of interest may belong to one of several classes of compounds, such as alkanes, ketones, alcohols, or acids (Figure 1), and it may be useful to remove the compound classes to which it does not belong in order to get a clearer view of the compounds you are interested in. For example, a TLE may contain 1,000 compounds, but the Uk&#39;37 sea surface temperature proxy is based on only two compounds (alkenones) and the TEX86 sea surface temperature proxy is based on only four (glycerol dialkyl glycerol tetraethers). It would behoove the researcher to remove as many of the compounds they are not interested in. This makes the instrumental analysis of the compounds of interest (alkenones or GDGTs) less likely to be complicated by other extraneous compounds.
In other cases, an upstream purification technique may have produced compounds you wish to now remove from the sample, such as the production of carboxylic acids during saponification in our previous video. In both of the above cases the purification technique called column chromatography is very useful.
<img alt="Figure 1" src="/files/ftp_upload/10159/10159fig1.jpg" /> 
Figure 1. Geochemically important functional groups. From Killops and Killops1.

טיהור תמצית שומנים כוללת עם כרומטוגרפיה של עמוד

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The product of an organic solvent extraction, a total lipid extract (TLE), ...

Environmental Sciences

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Column Chromatography

Source: Laboratory of Dr. Jimmy Franco - Merrimack College
Column chromatography is one of the most useful techniques for purifying compounds. This technique utilizes a stationary phase, which is packed in a column, and a mobile phase that passes through the column. This technique exploits the differences in polarity between compounds, allowing the molecules to be facilely separated.1 The two most common stationary phases for column chromatography are silica gel (SiO2) and alumina (Al2O3), with the most commonly used mobile phases being organic solvents.2 The solvent(s) chosen for the mobile phase are dependent on the polarity of the molecules being purified. Typically more polar compounds require more polar solvents in order to facilitate the passage of the molecules through the stationary phase. Once the purification process has been completed&#160;the solvent can be removed from the collected fractions using a rotary evaporator to yield the isolated material.

Column Chromatography: Principle, Separation of Compounds from a Mixture

כרומטוגרפיה של עמודה

Source: Laboratory of Dr. Jimmy Franco - Merrimack College
Column chromatography is one of the most useful techniques for purifying compounds. This ...

Organic Chemistry

Principles Of Column Chromatography

The chromatography technique was first invented in 1901 by Michael S. Tswett, a Russian botanist, to separate plant pigments using organic solvents. Further, in 1941, Archer John Porter Martin and R. L. M. Synge modified the technique by packing silica gel into a column. A mixture of amino acids was then separated on the packed column using chloroform and water mixture as the mobile phase. This was the first report on column chromatography. At present, column chromatography is a widely used technique for separating various types of compounds from a sample mixture.
Factors Influencing Efficient Separation of Proteins 
Various parameters like column material, packing, and operational conditions such as flow rates and temperature determine the efficiency of separation by column chromatography.
The choice of column material or matrix determines the extent of interaction with the sample. The matrix material must be tightly and uniformly packed in the column. Air bubbles, debris, large particles, and precipitates interfere with the uniform flow of the solvent through the column, affecting its separation efficiency. The column should also be free from particulate matter.
The sample injected into the column should be clear and free from aggregates that might clog the column, hindering solvent flow. The flow rate of solvent also affects the separation. Very high or very low flow rates of solvents result in inefficient separation of compounds and impure preparations. Very high rates may also disturb the column packing, affecting process efficiency. In addition, the composition of elution buffer is also an important factor. It should be non-corrosive and compatible with the sample and also the column material to prevent in situ precipitation or dissolution.
Another operational parameter, temperature, also plays an important role in the process. It decides the stability of the sample, column material, and solvent buffer. Also, a constant temperature throughout the column efficiently resolves compounds. After completing the separation process, the columns must be washed thoroughly by repeatedly passing a suitable solvent to avoid sample contamination in further runs. Occasionally, the solvent is passed in a column in the reverse direction to remove any clogged material.
Limitations
Though a very widely used technique, the method still has some limitations. It is a very time-consuming method as the flow rates need to be slower for better resolution of the compounds. Also, large quantities of highly pure solvents required in the mobile phase make the process expensive. This also raises the scaling-up cost when higher yields of pure compounds are needed.

עקרונות כרומטוגרפיית טורים

The chromatography technique was first invented in 1901 by Michael S. Tswett, a Russian botanist, to separate plant pigments using organic solvents. ...

Cell Biology

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KEY TERMS AND CONCEPTS

High-Performance Liquid Chromatography: Introduction

High-performance liquid chromatography(HPLC), formerly referred to as High-pressure liquid chromatography, is a powerful technique used to separate, identify, and quantify components in complex mixtures. The term &#34;high pressure&#34; refers to using high pressure to push the liquid mobile phase through the tightly packed columns.
In HPLC, two phases play a critical role in the separation process:
<ol>
	<li>Mobile Phase: This is a liquid solvent that flows through the system, carrying the sample with it. The solvent is chosen based on its polarity to optimize the interaction with the analytes and the stationary phase. The &#34;polarity index&#34; serves as a guide to selecting the suitable solvent for separation.</li>
	<li>Stationary Phase: The stationary phase is a solid material, often silica-based, packed within the column. In reverse-phase HPLC, the stationary phase is typically derivatized silica, such as octadecyl (C18) silica. The small particle size of the packed material creates a high surface area, enhancing interactions with the analytes. To reduce undesirable interactions, such as those with unreacted silanol groups, the stationary phase is often end-capped with reagents like trimethylchlorosilane</li>
</ol>
Separation occurs as the analytes interact differently with both the mobile and stationary phases, depending on their chemical properties, which leads to differential migration through the column.
Sometimes, solute molecules bind irreversibly to the stationary phase, clogging the analytical column and decreasing its performance. To prevent this, a guard column is placed before the analytical column. A precolumn called a scavenger column may also be placed between the mobile phase reservoir and the injector. This ensures that the solvent enters the analytical column, which is already saturated with silica from the scavenger column, preventing unwanted reactions and extending the lifetime of the analytical column.
After the separation, analytes are detected by various detectors, depending on the characteristics of the compounds. The results are visualized as chromatograms, with peaks corresponding to the different components of the mixture. This provides both qualitative and quantitative data about the sample.

כרומטוגרפיה נוזלית בעלת ביצועים גבוהים: מבוא

High-performance liquid chromatography(HPLC), formerly referred to as High-pressure liquid chromatography, is a powerful technique used to separate, ...

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כרומטוגרפיה נוזלית בעלת ביצועים גבוהים (HPLC)

טיהור תמצית שומנים כוללת עם כרומטוגרפיה של עמוד

כרומטוגרפיה של עמודה

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כרומטוגרפיה נוזלית בעלת ביצועים גבוהים: מבוא

כרומטוגרפיה נוזלית בעלת ביצועים גבוהים: מבוא