HPLC Separation and Identification of Parabens Homologs

Transcription

1 HPLC Separaton and Identcaton o Paraben Hoolog Experent Objectve By experent, learn the bac prncple o chroatographc analy and the bac operaton o the ntruent, and undertand the proce o data analy. Learn about chroatographc quanttatve ethod and copare the advantage and dadvantage o derent ethod. HPLC Introducton Hgh peed: HPLC ue hgh-preure nuon equpent, the low rate greatly ncreaed, and the analy peed extreely at, only a ew nute generally; whle the clacal ethod depend on gravty eedng, t take everal hour to coplete an analy. Ecent: The ller partcle are very ne and regular, the coatng on the tatonary phae unor, and the a traner retance all, o the colun ecency very hgh. It can coplete the eparaton o hundred o ateral wthn nute. Hgh entvty: The detector extreely entve: UV-10-9 g, luorecence detector g. Coparon HPLC wth GC Object and cope o analy: GC analy lted to gae and low-bolng table copound, whch account or only 20% o the total organc atter; HPLC can analyze hgh-bolng, hgh-olecular-weght table or untable copound that account or 80% o the total organc atter. The choce o oble phae: GC ue lted optonal knd o "nert" gae a the oble phae, carry only, all eect on coponent. HPLC ung the oble phae a xture o lqud or varou lqud, there are any knd o optonal oble phae. In addton to t role a a carrer, t can alo nteract wth the coponent and copete wth the eect o tatonary phae, that, the contrbuton o oble phae uch ore or eparaton, and the eparaton can be controlled and proved by the olvent.

2 Operatng teperature: GC requre hgh teperature; HPLC uually perored at roo teperature. pplcaton eld Due to t hgh entvty, quanttatve accuracy, and utable or analy o nonvolatle and therally untable coponent, HPLC eparaton analy extreely veratle n ndutral and centc reearch, epecally n bology and edcne. For exaple, analy o ano acd, proten, nuclec acd, hydrocarbon, carbohydrate, drug, polyaccharde, polyer, petcde, ub-antbotc, choleterol, and etal organc are otly done by HPLC. HPLC tructure and prncple Fg.1 HPLC Structure HPLC cont o Pup yte, njecton yte, eparaton yte, detector and data proceng yte. Hgh-preure nuon yte 1) Moble phae bottle: 1-2L gla bottle wth olvent lter (N alloy), t pore are approxately 2 μ, preventng partculate ro pupng. 2) Degaer: ultraonc degang or vacuu heatng degang. The olvent pae through the degang ebrane n the degaer, and the relatvely all olecular weght ga reoved ro the olvent through the ebrane.

3 3) Hgh-preure pup: The requreent or the nuon pup: good ealng, table low wthout nlux, wde adjutable range, and corroon retance. Injecton yte Manual njector or auto-apler Fg.2 Injecton prncple

4 Colun eparaton yte Fg.2 Separaton prncple Detector Fg.3 UVD

5 Separaton prncple Chroatographc proce: In yte wth two cble phae, the oble phae and the tatonary phae, the thrd coponent (e the olute or adorbate) contnuouly alternant dtrbuton between the two phae. Due to the derence n the property o the oble phae, the tatonary phae, and the olute xture, the coponent how derent chroatographc behavor durng the chroatographc proce, o that the coponent are eparated ro each other. Reoluton The reoluton o a eluton a quanttatve eaure o how well two eluton peak can be derentated n a chroatographc eparaton. It dened a the derence n retenton te between the two peak, dvded by the cobned wdth o the eluton peak. Correcton actor The quanttatve analy o chroatography baed on that the aount o coponent proportonal to the peak area: = ' ' peak area abolute weght correcton actor

6 Weght correcton actor: = ', = ', Quanttatve ethod External tandard ethod Calbraton curve can be etablhed accordng to ollowng equaton: c c % % = c % c % = ppled to regular analy dvantage: eay operaton and calculaton Shortage: the accuracy o reult depend on the repeatablty o njecton and tablty o operatng condton. Quanttatve njecton requred or th ethod.

7 Internal tandard ethod dd certan aount o pure ubtance nto the aple a nternal tandard. = 1 C % = 100 = 100 =, 100 pplcaton range: only deterne everal coponent o the aple, and not all the peak o coponent can be lowed dvantage: le nluence o operatng condton, uch precer, and le retrcton than noralzaton ethod, t or trace analy Shortage: unt or rapd analy. Syrnge The needle o the GC yrnge harp and the needle o the LC yrnge lat. The two cannot exchange to ue. Experent Experental condton 1)Inject 10μL x tandard oluton (ethyl paraben, propyl paraben, and butyl paraben) n derent rato oble phae o ethanol:water = 90:10, 80:20, and 70:30 to tet, record retenton te and the reoluton between the varou

8 coponent, wth the reoluton greater than 1.5 and the hortet retenton te a the prncple o electng the bet oble phae atchng rato. Note: Each te you change the oble phae, you need to wat about 5 nute to balance the chroatographc yte (both gnal and preure lne are balanced). 2) Inject 10 μl x tandard oluton (ethyl paraben, propyl paraben, and butyl paraben) at derent oble phae low rate o 0.8, 1.0, and 1.5 L/n and optal oble phae rato njecton analy, the ae prncple elected the bet oble phae low rate. Qualtatve analy Under the optal experental condton, nject 10 μl each o ethyl paraben, propyl paraben, and butyl paraben and the retenton te o each peak recorded. Copared wth the reult obtaned n tep 2). Quanttatve analy Calbraton curve Under the optal experental condton, 2, 5, 8, 10, 15, and 20 μl o the xed tandard aple wa njected and analyzed, and the tandard curve etablhed by njecton volue and peak area. It requred that the correlaton coecent o th curve greater than I t not ated, retetng can be perored on experental pont that devate gncantly. Unknown aple analy Inject 10 μl unknown aple to tet, quanttatve analy by calbraton curve. ter analy, wah the HPLC yte wth pure ethanol about 10n, then hut o worktaton and power wtch. Queton Baed on the experental data, dcu the derence n analy ethod between ga chroatography and lqud chroatography. Explore the applcaton value o HPLC analy. Whether the UV detector utable or detectng all organc copound? Why?