制备超临界流体色谱的新注射方法

我们开发了一种用于制备超临界流体色谱的新型注射方法（Prep SFC），当将大体积样品装载到柱上以获得更高的产率时，该方法非常有效地提高了分离效率。我们将这种方法命名为双线注射（DLI）方法，该方法的单元包括样品注射器中的旁通线。该方法对于具有较短保留时间的峰特别有效。对反式氧化二苯乙烯受体进行手性分离，以评价DLI方法。计算了两种对映体的纯度，并与传统的直接注射法进行了比较。峰引导得到改善，第二级分的纯度从87±1%提高到98±1%。本应用中结论：一种新的注射方法，双线注射（DLI），已成功地开发成为一种强大的方法，可提供更高的生产率和纯度。DLI方法改进了相邻峰的峰的形状和分离度。它对保留时间较短的峰值尤其有效。我们将该方法应用于反式氧化二苯乙烯外消旋体的手性分离和分馏。第二组分的纯度从87 ± 1%提高到98 ± 1%。Packed-Column SFC 2008， Switzerland JASCO global homepage： http：//www.j ascoinc.com Summary W e hav e d e vel op ed a novel i nj ec tio n me thod f o r p r e p a r a tive supe r e r i t i cal f l u i d c h r om ato g r ap hy (Pre p -S FC )， whic h i s v e ry iv e t o im p r ove s epa r at i on e f ficienc y w he n a l arg e v o l ume sa m ple i s l oad e d onto a c o lu mn f or h igher prod ucti v i t y. We e n n i m thi s me t hod Dual L in e I njec t ion (DL I) m e t hod*， o f which uni t i nclude s a bypas s l i n e in t he s amp le in j ec t or . The n es p ec i ally ef f ec t ive for the peaks hav i n g s horter retention t ime s .C h iral se par ati on o f t r a ns -st i lb ene o x i d e rec emat e was pe r f o rm ed t o evaluat e t he DL I m e th o d . e p I S we r e calcula t ed and compa r ed with t h ose by t h e conventiona l d i rect inj ec t i on m et hod . P ea k l eadin g w a s im prov ed a nd t he p urity of t t l he sec o nd frac t ion was i ncreased f rom 87±1%to98±1%. *Patent p ending Introduction Re cently ， f ract i onation a n d p urification by Prep-SFC has been widely u sed a s i t of f er s l ow r unning cost. h igh-t hroughput a nd Cen v ironment a lly fr iendly gre e n c hemical p r ocess . E s pe c i a lly ， i t i s e ffec t i ve for t he se p aration an d f ract i onati o n o f r ac e c hir a l com p oun ds ， whi c h a r e i m p o r t a nt i ngre d i en t s f or me dic i n es . I n 1995， J A SC O ’s g ro u p s ucc e ssf u l l y demo n str at ed p re p a rat i ve c h iral s e p ar a ti o n b y p r e p -SFC with stack cocti o ns，go showed t he method was very effic i ent a nd suit a b l e for f r a c ti onation o l oorted P T CP p-SFC w ith c i rc u l a r dichr o is m (CD) detection. They s howed that g -f a ctor， which is a si gnal of CD s ig nal div i d ed by U V V s ign al ， t r i g ge r ed f r ac t i o n ati o n m et h o d w as e x c el l e nt to a cc ur ate l y con t r o l t h e f racti on a t i on ti ming ， off ering coll ec t io n o f e na nt i omer i c p urit y 2， no l st ilbe n e o x i d e Experimental Mal 250s. A pr e p -S IRALPAK I A (20 mm Carbon d i oxide (99.99%) w a s s up p lied by Ni p po n Ta n sa n ， Co.， L t d.， T okyo， J ap an. H P LC -grade methanol used as a modiher Ind u A Volu m Fig ur e 1. Pr ep arat ive SFC Sy st e r r F ig ur e 2. L a rge V olu me Inj ector (LV I ) c o， Cyl in der Modi f ie r So l v e ntPU -2088.C 02 C O. D e livery Pame 单 PU -2088 Modi f ier De li v e r y P um p Pr c heue r 6. AS-2059SF S F C Auto S ampleror L a r ge V o l um e I n ject o r (L.V I ) 1. D ual L i n e I n je e ti o n (DL I)C o lum n 10 CO-2060 Colum a O vem U V-2075 U V Detecto r 11 FC -2088-08 F ra ction C o l ke l or 2 BP -2080 Back P ressure R eg al i t o 0E Selector a nd Frac t ion va l v e Gas-liquid S e para ter 15. C o llect ion B o ttk e s 1E3 Supercr i tic a l F l uid Extrac t i o n /C h romatog h y Sy s t em Sy ste m C o nt r o l Monitor P rogr a m SF -NAV IN is m e ra e d with the sol vent o f t h e bypas s l ine. The b ypas l i ne a lso p r e ven ts t he p re ssure s ur ge when t he i nje ct or valv e is t ur m ed . The S F -NAV so ftw are ma na ge d t he t ot a l syst e m co n tr o l . Results and Discussion Determination of di l ution r a tio i n the DL I system It is i m portant to determine t he dilution r atio when the injected sa m ple is me r ged with t h e m ob i l e pha se fl ui d (a m ix tu r e o f C O， a nd m o d if i er s o lve nt ) i n t he b ypass l i ne .Because the DL I sy stem ta k es a pa rt in c a rr ying a s am ple in to t he ma i n l ine o f t he f l u id hav i ng weaker e l ut ti i or p ow er b y d iluti ng t he bypass li ne fl ui d and climinating the pe a k slipping in a col umn b y t he sa mple sol ve nt .We measured t he d ilut i on ratios with di f ferent le ngths o f bypas s line t ube s by flow injec t ion method. F i g ure 4shows t he f low i njec ti on tr aces when tube s wit h different lengt hs we r e used. A pe a k width b ecomes larg er a s th e l e ngt h o f byp ass li ne t u b e be com es shor t e r . Thi s shows that t he dilution r a tio changed according to the f l ow r esistance o f bypass line tube . Pe a k width s (p eak s tart -e nd ti me ) of the t rac e s s hown in F i gur e 4 wer e c al c ulated a nd t he di l ut i on ra t i o s wer e c al c ula ted us i n g Eq uat i on 1.Table I s how s t he p e a k wid t hs a nd t he calcul a t e d dilut i on ratios with v a r i ous le ngt hs of t he tube. We s elec t ed t he t u b e l e n g t h t ha t gives t he d i l ut i on r a ti o o f 1.2 t o 2.0 t hat g a v e t he goo d r es ults . The l e n gth sho uld b e c h a n g ed cord i ng t o t he solubi l it y o f t he s am ple i n the solvent t o obt a in t he be s t resu lt S s . Fi gure 4. Change in pe a k wi d t h when the l e ngth of b yp as s l ine t ube is ch a nged i te of mL/mi n ； pressu r e =20 MPa； wavelength @ 254 nm； samp l e， 0.015 m g’mL E t hy 14.h y d r oxybenzoa t e； i njec ti on vol u me = 2mL ； b ypas s l i n e - 1.59 mm OD x0.8mm ID ie an d Ta b l e 1. Pe a k w i dth a nd D i l ution R atio when t he l ength o f by p as s l ine t ubing wa s c h anged De一 Pw i ((Equation 1) Pwo D i l u tio n R atio ，P e ak w idth w it h ou t a bypas s l i ne (min) Pea k width with a bypas s l ine (m i n) Bypa s s line l eng th (m)6 4 2 1 0.15 0Peak width 0.1750.1920.255 0.298 0.3640.154(mi n )Dilut i on Rati 1o 0 1.14 1.25 1.66 1.94 2.36 Comparison of peak widths W e c o mpa re d t he pe a k wi d th s wi th a nd wit ho ut the DL I met h o d . Firs t ， we e va luat e d t he p en k wi d th a nd a symmetry facto r by usi n g a s ilica column and e thy l 4-hydroxybenzoate as a test sample. T ota i l peak s are l isted in Table 2.A s show n in F ig u r e 5， p e ak l ead in g was d r a stica l l y r ed uc ed and ev e n exh i bit e d a l i tt e t a iling . Therefore， t h e p e ak widt h was e duce d f ro m 2.0 t o 1.5 an d t he a s y mme t r y f a c t o r wa s i mpr oved f r o m 0.85 to 1.09 as sh o w n i n T able 2. Next， we ev a l uated the degre e of s e paration by t h e chiral s ep a ration o f tr a n s-s t i l bene oxide usin g a prep arativ e c h iral colu m n. F ig i 6 shows t he obtained c h rom a tograms with and without the DL I . To express t he d eg re e o f se par a ti o n ， t he reso e re s olut i on could n ot b e appl i ed b ec a u se i t is ex p ressed by (the d ifference i n re t ention t i me ) /(ave r age hs S)).As show n i n F i gu r e 6， t he d if f er e nc e in r ete nti o n t i me a nd t he p e a k wi d ths ar e s im il a r in bot h c hromat o gram s a n d R s ofp uld not d i f ferent i ate t he de g r ee of s eparation. Therefor e ， we i n t rod u ced a f orm u la that ex pres ses the degr ee o f sep arati on in su c a se . W e defined a fa c tor of degre e o f separat io n D， as de s cribed i n the follow i ng par a g raph. Table 3 s hows t h e p e ak s tart -end ti me of two a dj a cent p e a k s and t h e f a c t or of degre e of s epa r a tion D A s shown in F ig v er be ca u s e l e ad in g f o r eac h e nan t i ome r p e a k was d ra st i c a ll y r educed . Theref or e， t he p eak s tar t-e nd t ime wa s r ed u ced f r om 2.5 t o 1.7 a nd t h e f act o r D. was i ncrea s ed fr om 30.1 t o 88.1 a s shown i n Table 3. (1) Single pe a k wi t h o u t D L I w i t h D L I H O -< 8-0cH C H Peak width (start-end time)(min) Asymmetry factor without DLI 2.0 0.85 with DLI 1.5 1.09 Peak start-end time of two peak (min) A fasctor D， of valley depth/ peak height (%) without DLI 2.5 30.1 with DLI 1.7 88.1 F i g u re 9. C h romatcog r a m o f p r e p anatve s ereral e c o o n u n hn tts g reum forthe byna ss l ine tubing (L =1m， D ，=1.94a n d (B ) a r Conclusion 1A move infection method. Dunl L ine Iniec ti on (DL I )， h a s s u cessf u lly been d e ve lo p ed a s a po w e rf tul m e t h od t hit o f f ers hig her produ c t i v it y a nd p ur it y i n Pre p -S FC . 2 The Du I nechod inaroyes neak sthares of adiac en t p eaks and t he d egrec o f s eparation. I t i s especially c ffective f or t he peaks h av i n g s horter r et e nt ion t imes. 3.We annlied the method to the chiral separation a nd f inacti on at i on of t r a ns st i bene o xid e r ac emat e . T he p ur ity of t he s eco nd f ruc t i on w a s increased from 87±1% to 98±1%. References 1) Saito e t . al . “Prepa r at ive superc r i ti c a l f lui d c h romatog r aphy a nd i t s a p plicati o H a t a no and H a nai Eds .， lnt. S ym p. C hr omat o gr . 35th A nni v. Res . G ro up L i y. Ch ro man t ogr . 1995.p p 863-86566 2) T . K an o mata e u a l ."Ad va snt a ge s o fCir e u l ar i c h are i n u u EPnu P a t eqt i opnh .ye r hpi s rc y f o rc h