HPLCzhongdeganraofengshiguangdasepugongzuozheshenweitoutengdeshiqing,womentongguoduiganraofengdelaiyuanjinxingfenlei,duiqichanshengyuanyinjinxingfenxitanjiu,yibiandajiajinxingcankaokuaisujiejuetongleiwenti。
關於其幹擾峰,我們簡單分為三類:試劑或溶劑、器材以及液相係統。下麵我們會對每一類幹擾峰來源做簡單的分析。
試劑或溶劑
HPLC中,常用的試劑主要是有機溶劑、gezhongyanleijishui。youjirongjiyibanduoweigoumaidesepuji,suoyichuxianwentidejilvjiaoxiao。danshifenzhuangchudeyoujirongji,youyuduocishiyong,beiwurandegailvjiaoda,yinciyinruganraofengdejilvjiaogao。qicishishui,shuizhongdezazhishiganraofengdezhuyaolaiyuanzhiyi,zheshidajiajiyihushidewenti。
caijibochangjiaodidejiancefangfa,jianyishiyonggaopinzhishui,zheyangkeyibimianhenduoyinweishuizhidailaidewenti。zaicishigezhongyanlei,zheyeshisepuganraofengdezhuyaolaiyuanzhiyi。duiyuwujiyanyinrudeganraofeng,zaifangfakaifachuqiyibanjiuhuigeiyanguanzhu,xunzhaojiejuefangan。fangfaquedinghou,yandepinzhongjijibieyetongshiqueding,yinciyanduifangfadeyingxiangjibenkekong。
水(shui)和(he)無(wu)機(ji)鹽(yan)引(yin)起(qi)的(de)幹(gan)擾(rao)峰(feng),比(bi)較(jiao)快(kuai)捷(jie)的(de)解(jie)決(jue)方(fang)案(an)是(shi)安(an)裝(zhuang)鬼(gui)峰(feng)補(bu)集(ji)柱(zhu),這(zhe)樣(yang)可(ke)以(yi)有(you)效(xiao)的(de)避(bi)免(mian)此(ci)類(lei)幹(gan)擾(rao)。如(ru)果(guo)使(shi)用(yong)離(li)子(zi)對(dui)試(shi)劑(ji),則(ze)需(xu)選(xuan)擇(ze)不(bu)影(ying)響(xiang)離(li)子(zi)對(dui)試(shi)劑(ji)的(de)鬼(gui)峰(feng)補(bu)集(ji)柱(zhu)。
研創鬼峰柱
器材
幹擾峰來源的第二個方麵是流動相配製及樣品前處理過程中接觸的各種器材。
首先是流動相配製過程中接觸的器材,包括燒杯、玻璃棒、pH計、濾杯、濾膜、流動相瓶等。燒杯、玻璃棒是配製流動相首先接觸的器材,因此建議配製流動相的燒杯、bolibangzhuanyong。lvbeishiyanrenyuanzhiyaoqingxiganjingyibanbuhuidailaiwuran。xuyaozhuyideshi,ruguoguolvhanyouliziduideliudongxiang,jianyizengjiaqingxicishu,bimianqingxibuganjingwuranhouxuliudongxiang。yinci,duilvbeideqingxiyinggaijianlijibendexidiguicheng,baozhengshiyanrenyuanduishiyongdeqicaiqingxidaowei。lvmoyeshiganraofengyinruyuantouzhiyi。rutu3suoshi,yijingjingguoyoujixilvmoguolvhou,zaitiduxituozhonghuiyinrujiaodaganraofeng。jianyusepujishijichangjiayijingguomo,qieduoshuyiqidouyoutuoqiji,suoyiwomenjianyiwuxuzaiduijiachun、yijingdengchunyoujixiangguolv。liudongxiangpingyinruganraofengdexianxiangzhuyaochuxianzaixiaji,zheshiyouyuxiajiwendugao,chunshuixiangrongyizishengxijun。houxudeliudongxiangpingshiyongrenyuanqingxibudaowei,suoyidaozhiyinruganraofeng。yinciliudongxiangpingdeqingxiyaojianliyangedexidiguicheng。
再次樣品前處理過程中接觸的器材。前處理過程中接觸的器材主要包括容量瓶、濾頭、注射器及進樣瓶。容量瓶的清洗同流動相瓶、過濾裝置一樣,要建立良好的清洗規程。移液器材以及濾頭、注射器,多為塑料製品,其化學惰性較玻璃製品差,因此容易引入幹擾峰。如下圖4、圖5、圖6所示案例,此方法檢測波長為210納米,溶劑為正己烷-異丙醇(90:10),溶劑分別接觸移液槍槍頭、一次性吸管、一次性注射器後,均引入不同的幹擾峰。
進樣小瓶若為一次性使用,一般不會引入幹擾峰。如果進樣瓶反複清洗,多次使用,則應建立清洗規程並對其清洗幹淨程度進行確認。
液相係統
與(yu)前(qian)兩(liang)類(lei)幹(gan)擾(rao)峰(feng)相(xiang)比(bi),液(ye)相(xiang)係(xi)統(tong)幹(gan)擾(rao)峰(feng)的(de)解(jie)決(jue)更(geng)加(jia)困(kun)難(nan)。液(ye)相(xiang)係(xi)統(tong)是(shi)一(yi)個(ge)動(dong)態(tai)過(guo)程(cheng),所(suo)以(yi)此(ci)類(lei)問(wen)題(ti)解(jie)決(jue)過(guo)程(cheng)耗(hao)費(fei)時(shi)間(jian)長(chang),而(er)且(qie)要(yao)求(qiu)實(shi)驗(yan)人(ren)員(yuan)有(you)很(hen)高(gao)的(de)色(se)譜(pu)素(su)養(yang)。我(wo)們(men)對(dui)此(ci)類(lei)問(wen)題(ti)來(lai)源(yuan)主(zhu)要(yao)歸(gui)結(jie)為(wei)三(san)類(lei):強保留物質、離子對汙染和管路滋生細菌。
首(shou)先(xian)是(shi)強(qiang)保(bao)留(liu)物(wu)質(zhi)的(de)幹(gan)擾(rao),其(qi)主(zhu)要(yao)特(te)征(zheng)是(shi)寬(kuan)峰(feng)。此(ci)類(lei)問(wen)題(ti)多(duo)數(shu)發(fa)生(sheng)在(zai)等(deng)度(du)洗(xi)脫(tuo)中(zhong),梯(ti)度(du)洗(xi)脫(tuo)主(zhu)要(yao)出(chu)現(xian)在(zai)高(gao)比(bi)例(li)有(you)機(ji)相(xiang)處(chu)。這(zhe)是(shi)由(you)於(yu)強(qiang)保(bao)留(liu)物(wu)質(zhi)在(zai)當(dang)次(ci)進(jin)樣(yang)中(zhong)未(wei)洗(xi)脫(tuo)出(chu),在(zai)後(hou)續(xu)進(jin)樣(yang)洗(xi)脫(tuo)出(chu)來(lai)所(suo)致(zhi)。
對(dui)於(yu)此(ci)類(lei)幹(gan)擾(rao)峰(feng),可(ke)通(tong)過(guo)延(yan)長(chang)采(cai)集(ji)時(shi)間(jian)或(huo)增(zeng)加(jia)洗(xi)脫(tuo)強(qiang)度(du)來(lai)消(xiao)除(chu)對(dui)後(hou)續(xu)樣(yang)品(pin)的(de)幹(gan)擾(rao)。至(zhi)於(yu)如(ru)何(he)延(yan)長(chang)采(cai)集(ji)時(shi)間(jian),大(da)家(jia)可(ke)通(tong)過(guo)下(xia)麵(mian)的(de)公(gong)式(shi)計(ji)算(suan)得(de)到(dao)。
N=5.54*(tR/Wb/2)^2(1)
由公式(1)推導可知
tR=Wb/2 *(N/5.54)^(1/2)(2)
其中,N-塔板數,在等度洗脫中N對所有峰相同;tR-保留時間;Wb/2-色譜峰半峰寬。
由公式(2)推知,tR2=tR1* W2b/2/W1b/2。積分結果顯示,圖7中,7min處的色譜峰W1b/2=0.12min,9min處的寬峰W1b/2=0.32min,由此可推知,圖7中9min處的寬峰保留時間tR2=7*0.32/0.12=18.6min。圖8為延長采集時間的圖譜,20min處即為圖7中9min處chu的de色se譜pu峰feng,與yu公gong式shi推tui導dao結jie果guo基ji本ben接jie近jin。延yan長chang采cai集ji時shi間jian後hou,後hou續xu圖tu譜pu幹gan擾rao峰feng消xiao失shi。盡jin管guan按an照zhao此ci公gong式shi計ji算suan會hui有you一yi定ding誤wu差cha,但dan這zhe足zu以yi讓rang色se譜pu工gong作zuo者zhe對dui其qi進jin行xing預yu判pan,設she置zhi合he理li的de采cai集ji時shi間jian。
係統幹擾峰第二個方麵是離子對、螯合劑這種難以洗脫的試劑對液相係統的汙染。此類汙染物多半是由於使用完畢含有離子對、螯ao合he劑ji之zhi類lei的de流liu動dong相xiang後hou,對dui係xi統tong的de衝chong洗xi不bu徹che底di所suo致zhi。此ci類lei汙wu染ran物wu主zhu要yao對dui一yi些xie采cai集ji波bo長chang較jiao低di的de方fang法fa產chan生sheng幹gan擾rao,普pu遍bian表biao現xian是shi基ji線xian噪zao音yin大da或huo者zhe幹gan擾rao峰feng,但dan隨sui著zhe方fang法fa的de運yun行xing會hui逐zhu漸jian趨qu於yu正zheng常chang。
係(xi)統(tong)幹(gan)擾(rao)峰(feng)第(di)三(san)個(ge)方(fang)麵(mian)是(shi)係(xi)統(tong)滋(zi)生(sheng)細(xi)菌(jun)。對(dui)於(yu)係(xi)統(tong)滋(zi)生(sheng)細(xi)菌(jun),多(duo)數(shu)大(da)家(jia)可(ke)能(neng)覺(jiao)得(de)不(bu)可(ke)思(si)議(yi),因(yin)為(wei)液(ye)相(xiang)係(xi)統(tong)使(shi)用(yong)完(wan)畢(bi)都(dou)會(hui)用(yong)有(you)機(ji)相(xiang)衝(chong)洗(xi)保(bao)存(cun)。目(mu)前(qian)很(hen)多(duo)方(fang)法(fa)流(liu)動(dong)相(xiang)Adoushishuixiang,suiranshuixiangmeiridouhuixinpeizhi,danruguoyoulvtoudaobilifazheyiduanguanluchangshijianzaishuixiangzhongjiuhuizishengxijun。suizheshijiandeyunxing,tiduxituozhonghuizhujiandechanshengganraofeng。cileiwentisuiranbijiaoyinbi,danbijiaorongyijiejue,zhiyaomeiriduifangzhichunshuixiangdetongdaoyongyoujixiangchongxijikebimiancileiwenti。
以(yi)上(shang)便(bian)是(shi)對(dui)幹(gan)擾(rao)峰(feng)來(lai)源(yuan)的(de)簡(jian)單(dan)分(fen)析(xi)。然(ran)而(er)對(dui)於(yu)幹(gan)擾(rao)峰(feng)的(de)產(chan)生(sheng),要(yao)具(ju)體(ti)問(wen)題(ti)具(ju)體(ti)分(fen)析(xi),因(yin)為(wei)其(qi)還(hai)與(yu)具(ju)體(ti)色(se)譜(pu)條(tiao)件(jian)有(you)關(guan)。同(tong)一(yi)種(zhong)試(shi)劑(ji),在(zai)方(fang)法(fa)A中產生幹擾,但在方法B中可能完全沒有幹擾。因為采集波長,有機相初始比例及最終比例等因素,都可能會對幹擾峰的出現產生影響。
圖片來源於網絡,侵刪
實shi際ji工gong作zuo中zhong,色se譜pu工gong作zuo者zhe遇yu到dao的de最zui大da困kun難nan是shi如ru何he識shi別bie判pan斷duan幹gan擾rao峰feng屬shu於yu哪na一yi類lei。由you於yu找zhao不bu清qing問wen題ti根gen源yuan,所suo以yi也ye就jiu難nan以yi有you針zhen對dui性xing的de解jie決jue問wen題ti。下xia麵mian為wei大da家jia簡jian單dan分fen享xiang一yi下xia解jie決jue幹gan擾rao峰feng的de小xiao經jing驗yan:
首先,判斷幹擾峰是來自係統還是供試品。此時需要運行兩次采集:隻(zhi)運(yun)行(xing)梯(ti)度(du)與(yu)正(zheng)常(chang)進(jin)樣(yang)。如(ru)果(guo)隻(zhi)運(yun)行(xing)梯(ti)度(du),未(wei)進(jin)樣(yang)品(pin),依(yi)然(ran)有(you)幹(gan)擾(rao)峰(feng),這(zhe)種(zhong)情(qing)況(kuang)下(xia)多(duo)半(ban)是(shi)係(xi)統(tong)所(suo)致(zhi),此(ci)時(shi)解(jie)決(jue)方(fang)向(xiang)應(ying)針(zhen)對(dui)液(ye)相(xiang)體(ti)係(xi),而(er)不(bu)是(shi)針(zhen)對(dui)供(gong)試(shi)品(pin)。鑒(jian)於(yu)有(you)些(xie)型(xing)號(hao)的(de)儀(yi)器(qi),如(ru)果(guo)不(bu)進(jin)樣(yang),不(bu)會(hui)執(zhi)行(xing)進(jin)樣(yang)的(de)動(dong)作(zuo)。因(yin)此(ci),對(dui)於(yu)此(ci)類(lei)儀(yi)器(qi),建(jian)議(yi)執(zhi)行(xing)正(zheng)常(chang)進(jin)樣(yang)與(yu)減(jian)少(shao)進(jin)樣(yang)量(liang)兩(liang)次(ci)采(cai)集(ji),根(gen)據(ju)幹(gan)擾(rao)峰(feng)的(de)大(da)小(xiao)來(lai)判(pan)斷(duan)是(shi)係(xi)統(tong)原(yuan)因(yin)還(hai)是(shi)供(gong)試(shi)品(pin)原(yuan)因(yin)。
qici,tiaozhayiqishiyongjilu,yushiyanrenyuanjiaoliu。yibanjianyizhakanyiqishiyongjilu,lejiejinqiyunxingnaxiefangfayijizhexiefangfadeliudongxiangzucheng。zhakanwanxiangguanjiluhou,zaigenshiyanrenyuanjiaoliu,lejieqicaozuoguocheng。biru,duiyuA通(tong)道(dao)滋(zi)生(sheng)細(xi)菌(jun)問(wen)題(ti),我(wo)們(men)可(ke)以(yi)通(tong)過(guo)儀(yi)器(qi)使(shi)用(yong)記(ji)錄(lu)發(fa)現(xian),出(chu)現(xian)幹(gan)擾(rao)峰(feng)的(de)儀(yi)器(qi)近(jin)一(yi)周(zhou)都(dou)是(shi)在(zai)運(yun)行(xing)同(tong)一(yi)個(ge)方(fang)法(fa)。實(shi)驗(yan)記(ji)錄(lu)顯(xian)示(shi),此(ci)方(fang)法(fa)的(de)流(liu)動(dong)相(xiang)為(wei)0.1%delinsuanshuirongye,yincikenengshixitongzishengxijunsuozhi。danshiyanrenyuangaozhi,meitianwanshangdouhuiyongyoujixiangchongxisepuzhu,bukenengzishengxijun。zaicixunwendezhi,shiyanrenyuanmeiwandechongxishiqiehuanchengC通道執行,而A通道卻一直在0.1%的磷酸水溶液中。因此,切換通道衝洗係統這種不規範操應盡量避免。
disan,caiqudanyinsushiyanjiejuewenti。meicizhigaibianyigeyinsu,zhubuzhaodaoganraofengdelaiyuan。cileizuofakansibijiaofeishifeili,danquerangsepugongzuozheduigeleisepuwentichanshengyuanyinyoulixingrenzhi,zengqianglesepugongzuozhejiejuewentinengli,youzhuyuhouqiwentidejiejuepaizha。
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