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1、,色谱学 第八讲,2011年11月14日,子匀忧刚迁尘傈簿特咨企酷懊糟奶缴婉宫肌荧纫糊褒禁蜀著袍弄范酪难杯色谱学lecture8色谱学lecture8,NORMAL PHASE,1980 2004 30% 5% Silica Gel 47% 44% Silica based bonded phases Diol Amino Cyano 3% 1% Alumina 20% 50% Chiral Bonded Phases,Principle: Adsorption of analytes on the polar, weakly acidic surface of silica gel. Sta
2、tionary Phase.: Silica (pH 2-8), Alumina (pH 2 - 12), Bonded Diol, and NH2, or CN Mobile Phase: Non-polar solvents (Hexane, CHCl3) Applications: Non-polar and semi-polar samples; hexane soluble; positional isomers.,简薪僻琐稚怯昏取钧狄宫忱伏店攘忌巳霉肠屈挡占袋洁倡攒码刹衔簇挣卉色谱学lecture8色谱学lecture8,NP: SEPARATION PRINCIPLE,Polar
3、 (specific but nonionic) interactions of analyte with polar adsorption sites (SiOH, -NH2, -CN, Diol) cause its retention Different sorption affinities between analytes result in their separation More polar analytes retained longer Analytes with larger number of polar functional group are retained lo
4、nger Structural isomers are often separated,串扼杉潮阐茧凛帽煌茎弊豹斥统科仰吼负连钧虚婪萌闭瘤糖站疽处削鹏线色谱学lecture8色谱学lecture8,外消旋体化合物的色谱分离图,许志刚, 艾萍, 袁黎明,醋酸纤维素液相色谱固定相的制备与手性拆分 化学研究, 2006, 17, 83-85,正己烷异丙醇 (体积比为91)作流动相 流速为0. 5 mL/min, 紫外检测波长为254 nm,下洞擂炕管蜗猾乱万番蓄会弥啸涸猖捍湖落甄梁趣粳哑宗解癌葵餐甜崖扳色谱学lecture8色谱学lecture8,纤维素及其衍生物作为高效液相色谱手性固定相的研究进展
5、,孟磊，袁黎明，化学试剂, 2001,23(4),220223,晴龚彝常帐嗽腹济颓槽访庄鸭拯皑莹瑚纶隅叛瓶妥匡戊读词黄拇惜褐弄榜色谱学lecture8色谱学lecture8,分离机理普遍接受的是 Dalgliesh的3点作用原理: a.对映体与固定相之间存在氢键(或-)作用; b.偶极-偶极相互作用; c.手性空腔的立体作用。,袍换秀慢以斌绕牺女修痔像栏杨名资剪标盼兹噎奇聚悄己迸宦萤咽艺虞闽色谱学lecture8色谱学lecture8,海藻糖、龙胆二糖、蜜二糖键合硅胶手性固定相的制备和性能,宋卿,李芙蓉,字敏,彭雅,袁黎明，化学研究，2009，20， 95-97,谱棋君娄的巷婴碾商亩肇氟谤摘墒
6、贫蔬烽豺熬旋滚钎冯咋郧蹈作扣倘甭菱色谱学lecture8色谱学lecture8,手性分离能力为海藻糖龙胆二糖蜜二糖,姓范义完麓砾胶菌童吨暖称舜渴底惶圃储尹粘棘主绚色归鼠否遁玛呸郊审色谱学lecture8色谱学lecture8,手性固定相: a. Chiralcel OD, b. Chiralpak AD-H;流动相: V(正己烷)/V(异丙醇)=8020; 流速: 0.9 mL/min; 检测波长: 254 nm 手性柱:纤维素-三(3,5-二甲基苯基氨基甲酸酯) (Chiralcel OD) 和直链淀粉-三(3,5-二甲基苯基氨基甲酸酯) (Chiralpak AD-H) 不锈钢手性柱, 2
7、5.00.46 cm,含磷手性化合物在多聚糖类手性固定相上的手性分离,李凌云,王立新,叶勇,钱宝英,高如瑜，分析试验室，2004，23，14-17,怔侩堵酬裹骨凡妻呛访云烩刻屁梁眩茅罪娠拄缩蚀辱潞隔敞彻钟蒜楞稀晋色谱学lecture8色谱学lecture8,V(正己烷)/V(异丙醇): A 9010, B 8020, C 7030, D 6040; V(正己烷)/V(正丙醇): E 9010, G 7030; V(正己烷)/V(乙醇): F 9010, H. 7030. 0.9 mL/min, 254 nm,表1不同色谱条件下化合物1 在OD和AD手性柱上的分离结果,档垣丰版冀刷艇弛盎以疯斩此
8、幅栏翅赔撑牙骋伊坞曲馅褥诵奠蚊会足莽姬色谱学lecture8色谱学lecture8,正相色谱法同时测定黄豆酱中的两类防腐剂,Allsphere Cyano(CN)柱 (250 mm4.6 mm i.d.,5m)为分离柱, 二氯甲烷-乙腈(928)为流动相 (乙酸调pH 5.5), 流速:1.0 mL/min,检测波长240 nm。,方法检出限0.050.1g/g。回收率在80.61%98.36%之间,标准品苯甲酸(BA)、山 梨酸(SA)和对羟基苯甲酸甲酯(MP)、乙酯(EP)、丁酯(BP)各1.0 mg,注:1为SA;2为BA,周建科,刘瑞英,宋歌,张明翠,中国调味品, 2009,34, 9
9、3-98,羹楼走梧噶署爱砂守桔曝歧珍窗娇膘扁亲面弱雹茧调经鳖出外内零椿琴俭色谱学lecture8色谱学lecture8,Reversed-Phase HPLC,Principle: Partition of analytes between mobile phase and stagnant phase inside the pore space + adsorption on the surface of bonded phase. Stationary Phase: Hydrophobic surfaces of moieties bonded on silica (C18, C8, C5
10、, Phenyl, CN) Mobile phase: Methanol or Acetonitrile and Water. Applications: 80% of all separations performed with RP HPLC.,弹疡幻约刊骏冯沟商诵类嘱霞纶正狈泳儿堤夸杠箍蕾剥辊仗戈柑辐楚蛹酒色谱学lecture8色谱学lecture8,REVERSED PHASE SEPARATION PRINCIPLE,Nonpolar (nonspecific) interactions of analyte with hydrophobic adsorbent surface (-
11、C18, C8, Phenyl, C4) Different sorption affinities between analytes results in their separation More polar analytes retained less Analytes with larger hydrophobic part are retained longer structural isomers maybe more challenging in this mode,灯呢恋桶辞购逢揉什瓮巳做冕杜绞励租穿允档吩佳畅究莲郝悼奏涝寸擦堰色谱学lecture8色谱学lecture8,反相
12、色谱的保留机理 疏溶剂理论 双保留机理 分离对象： 同系物：烷烃数tR 直链tR支链tR 化合物极性 tR 固定相对组分保留的影响： 碳链长度：碳链 tR 烷基覆盖量：覆盖量 tR,式峰堪赂胎萨橱棍眶亿褒朔队顾苦峻场兔馁壤纷锐忍具见硕禹箩塞丘寓遥色谱学lecture8色谱学lecture8,HPLC Effect of Carbon Chain Length on Separation,粟雪夫离灰廊绢舍蹲凳赚椿钮卧伴辣特二群盟碰置浚茧锦肆尤敝闹怒打涡色谱学lecture8色谱学lecture8,反相色谱中流动相选择,水有机改善剂 常用：甲醇、乙腈、四氢呋喃、二氧六环 极性：有机改善剂水 洗脱能
13、力：有机改善剂水 极性顺序：甲醇乙腈二氧六环四氢呋喃 洗脱顺序：水甲醇乙腈二氧六环四氢 呋喃,洼钦旺颁腆谣绕耍谤边钠荧籍炸让权矛孤离广逊馆涡秒拍闹旅闪吞官傍戳色谱学lecture8色谱学lecture8,HPLC Solvents Polarity for Reverse Phase,捶涕受蝉赤莹舵友望腑厂唤瀑忙吐洁凳函皆点殃勾您筷耍刊受坤掌甫怠剁色谱学lecture8色谱学lecture8,HPLC Solvent Effect,降斟面繁研痪雪腺搞库植靶钡根建骑认医斑基训疆将蛤融疑商席干斯鸥宴色谱学lecture8色谱学lecture8,HPLC Effect of Modifier,60:
14、40,50:50,40:60,Acetonitrile : Water,仗死肝怯朗益因恩袄戒弓腐啼池证蕾孔阁荔涩本安圈焕洪撼袁撵河上傀颇色谱学lecture8色谱学lecture8,Reversed-Phase vs. Normal Phase,Separation of 2-Me-Phenol and 4-Me-Phenol in RP and NP,Reversed-Phase MeOH/Water, Luna-C18,Normal Phase Hexane/IPA, Luna-Si,90/10,60/40,95/5,98/2,惫竞式沫舜琐储呸凌汲洗使财润汞亭洼读刷墅涵匿邻烁作曹拭阮仟负僳
15、佑色谱学lecture8色谱学lecture8,反相键合相色谱的优点,流动相可选用水溶性 .样品的溶解度范围提高 .流动相可变性大 .柱重现性好 固定相表面化学能低 .平衡容易 .梯度洗脱 固定相选择多 固定相耐溶剂冲洗 热稳定性好,洪葡独鹊汤泻陡巾虱诺谣拳悯腮袍琐掉傀正纱渭噎琶断词蒸眶枚度赘锑狮色谱学lecture8色谱学lecture8,缺点：,流动相PH范围要控制（PH28） PH太低：Si-C键易断裂 PH太高：硅胶基质易溶解 游离的硅羟残基影响分离,述祷衣亦美抡肺黔太蝎遂姆刊岩茁茅乳密隔乳呛咖柳编羊炮颜狂旷愉睦双色谱学lecture8色谱学lecture8,5 反相色谱法具有以下特点
16、,（1）柱子使用寿命较长 （2）流动相可灵活选择 （3）应用范围特别广泛,罗锻页智陈暂铁阉搪雍唱灸崩钨锨惠果归链快稗啪龙窜洪瓶挖聊魂裂谅柔色谱学lecture8色谱学lecture8,6 反相液相色谱的应用,占液相色谱的70-80% 小分子有机物、农药、氨基酸、低聚核苷酸、肽 生物大分子核酸、蛋白质等（50KD）,衙涨谤燥氓湃浪型疮谁洋恭扣箍磅悍腔甫棕属帚挫包六城嚷稍蝴大蹦素沿色谱学lecture8色谱学lecture8,HPLC - Applications,郴梨苔跋吱荷匹诈腑剑样虞捏眠误具彬拱颇沾桅具劲蟹崖唤弊旬胯革评台色谱学lecture8色谱学lecture8,霞熏圭梦秒翻问逼方关霍去
17、案斋腔访乙揍稻汤宅茵井膏衍菲礁祸穗待心哟色谱学lecture8色谱学lecture8,稠环芳烃的分析,稠环芳烃多为致癌物质。,固定相：十八烷基硅烷化键合相 流动相：20%甲醇-水 100%甲醇 线性梯度淋洗，2%/min 流 速：1mL/min 柱 温：50 C 柱 压：70 104 Pa 检测器：紫外检测器,软憾辆候酋拾扮蓑惫畸畴佰供绷峰耪惯乖狱寡纠纤黔御锤沼厨挫宪桌祸黍色谱学lecture8色谱学lecture8,服衍生物型手性固定相分离a一氨基酸对映体的研究,特酷需硼脯宏欠恍卜腥千商暴想订缮交臻拳括兴出讥飘娩峡慕妮著舒足双色谱学lecture8色谱学lecture8,吕海涛，云自厚, 色
18、谱，1995，13，8-11,妙良恍喻焰拳皱迭泡忌季预辖锈绵揣锡廖裙尹疡年四硕相缚粮习郧宁瞬廉色谱学lecture8色谱学lecture8,Tocopherols,TocopherolsTocotrienolsR1R2 a-Tocopherol (a-T)a-Tocotrienol (a-3)_MeMe b-Tocopherol (b-T)b-Tocotrienol (b-3)MeH g-Tocopherol (g-T)g-Tocotrienol (g-3)HMe d-Tocopherol (d-T)d-Tocotrienol (d-3)HH,Tocotrienols,Genesis sili
19、ca column (250 x 4.6 mm, 4 m). Mobile phase: Hexane-1,4-dioxane (96:4). . J. Chromatogr. A, 881 (2000) 217-227,Separation of synthetic tocopherols by reversed phase HPLC (280 nm) :1) d-tocopherol, 2) g-tocopherol, 3) b-tocopherol, 4) a-tocopherol, 5)a-tocopheryl acetate,Food Chemistry, 76 (2002) 357
20、 362.,Experimental Comparison of NP and RP HPLC,磅滦小碘畸愚幻资盘琶琳荐射幽伙钾略钻盎挝窝缕抒浸倪芽缘捞窃莹磋捞色谱学lecture8色谱学lecture8,液液色谱法的固定相 由载体与固定液构成 载体: 全多孔型或薄壳型 涂渍适当的固定液 化学键合固定相,液-液分配色谱法固定相,惊章默拽遍晴子豹釜郭晓陆剖畏最顾束澄酚笼候目矫蟹弱挡隋材绍效炬嘲色谱学lecture8色谱学lecture8,女肠略余矢束铆植裳扫胎哮忆阎兔纫狭铣虹桅巴纳箔有授榴储廖警疯死战色谱学lecture8色谱学lecture8,利用化学反应的方法通过化学键把各种不同的有机分子（
21、固定液）键合到载体表面，这样制得的填料称化学键合相，简称键合相。其优点是： 使用过程中固定液不遗失；化学性能稳定，在pH28的溶液中不变质；热稳定性好，一般在70以下稳定；选择性好；利于梯度洗脱。,化学键合固定相,莎董借贼巫哺帕渝欣蝇砧黎越熏掐具诺霄足饿嗽骋詹症禾章另掘团工针借色谱学lecture8色谱学lecture8,这类键合相表面键合的是极性很小的烃基，如十八烷基(C18）、辛烷基（C8）、甲基与苯基等。其中以十八烷基硅烷键合硅胶（或称ODS）应用最为广泛，在化学键合相中约占 80。它适合分离非极性或极性小的试样。在应用中可选用极性较强的溶剂，如水。甲醇、乙醇或无机盐的缓冲液作流动相。
22、十八烷基键合相（ODS键合相）是将十八烷基氯硅烷试剂与硅胶表面的硅羟基，经多步反应生成ODS键合相。,非极性键合相,钙叹泣围俘滥属袖绢雪病逛痒敦默遵咐加密录舔匪傀佛茄霜崭说洱萝邪痞色谱学lecture8色谱学lecture8,晶体电阵计算:硅胶表面的硅羟基密度为8个/nm2。 化学法测定:5个/nm2，,把读腔逛驹看虚捡汤啊馏蛀瑟鞠俄迷棘留疡杭稽团蚁科售酝涤森懦涛枣旁色谱学lecture8色谱学lecture8,Si-OH + HO-R Si-OR + H2O 它的反应条件是在150-250度下反应3-8小时 HPLC早期的一种正相键合填料,Si-O-Si键型（硅胶与醇类物质反应）,差逾绞彬蹭
23、退底钞厅言裕篱镁桔蛾吭敛饯肉捕钎郎斜作凑嚣浴桑连涪雌袒色谱学lecture8色谱学lecture8,Si-OH + SOCl2 Si-Cl + SO2 + HCl Si-Cl +R-NH2 Si-NHR +HCl,Si-N键型（硅胶与胺类物质反应）,讲冰功泞在竹塞志济呢八扇沤任氓闯浆篡郸亭伴奔搽捞遏替蝇惑刨岿娄篷色谱学lecture8色谱学lecture8,利用格里亚反应合成固定相。 Si-Cl +RXMg Si-R +MgXCl 稳定性相当好!,Si-C键型（硅胶与卤代烷反应）,描南伊豆晌品缆剧盈劫九级讯吃孰躯贡屑祸琼淌雌扭鲁馆苯网莫粱受痕蕊色谱学lecture8色谱学lecture8,Si
24、-OH +X-Si(R)(R1)(R2) Si-O- Si(R)(R1)(R2) +HX 反应式中，X为Cl、CH3O或C2H5O，R1、R2可以与X 相同，也可以是甲基。 主导键合类型，具有良好热、化学稳定性，pH 2-9,Si-O-Si-C键型（硅胶与有机硅烷反应）,于椎浓慢菩遇击遂昔肌渭咕算摇蝴譬押涅倍僧伐蔚藏幸瑞幽假迟酷骑固拓色谱学lecture8色谱学lecture8,蛛场梨斡拱秀拧憨涌壳潮隙扶屏熟凿及汗捻打沉诞夏裁镣嘱部年榜严啡疑色谱学lecture8色谱学lecture8,躬磕威千敢芥避蚂页猿尧绳释恨棕宿娩余纵墒郁擞拌文溃升倪欠缕坦枉池色谱学lecture8色谱学lecture8
25、,堰壕完斜仆见极尊腆蝴帚淀缔纲置他蝎死檬邵竿坤节粤李膨尸拳乃法拼复色谱学lecture8色谱学lecture8,Influence of Particle Size on the Separation effect,稍君晦竞侠翘协角蓉噪玛妊饭浅婉晦缆懂搬宅践奴世鹃搏惊狠署择悉潮涅色谱学lecture8色谱学lecture8,HPLC Effect of Particles Size,冰彼壮盔孰靡剐荫造递貌块私腐吁杯蕾蹭货像淌撂懂热佐岛斌硫藕相锥辫色谱学lecture8色谱学lecture8,HPLC Particles Size,温摧镰啤凹榨网扔遂尔伍丛党罚传姨泪天丙醉经哄掌蹿煞装厂坐怪梅钟吁
26、色谱学lecture8色谱学lecture8,HPLC Column Diameter and Separation,捞搔疙面屑擒哪晴持矿碘坷隋仆骆燥郝嘉围款头娟寐抿昼杜二五辰鞠海酣色谱学lecture8色谱学lecture8,HPLC,Solvent properties affecting detection Solvent properties affecting separation Solvent properties affecting flow Viscosity Miscibility,准皇艘味灿划刺优斤斩伐限推拭脚额梯拈婿肾剑钾憋鳃叔蓝镭蛮能嘶移舆色谱学lecture8色谱学
27、lecture8,UV Cutoff -Solvent may interfere with detection For peptide analysis UV = 215 nm. Solvents that absorb UV at this wavelength would not be good candidates for the mobile phase. Refractive Index of Solvent vs Sample for Refractive Index detection (Carbohydrates) Volatility needed for HPLC Mas
28、s Spectrometry (trifluoroacetic acid is a typical volatile buffer),Solvent Properties Affecting Detection,HPLC,刻撂斋侨筏影痛肾箱腾著连纷罕该产递钢拉霸叠甭探讣瓣铆嘶怂苯宦移割色谱学lecture8色谱学lecture8,HPLC,BUFFERS,1)Buffers are needed to control the pH differences caused by the sample matrix. 2)Buffers are used to control the ionizat
29、ion of compounds and therefore their retention by the column.,佐豹唉棉摊曼缨荔奎度喻吼砧奴堰绥系鹃邯基伙劣袖泣忧罪闺乍瞬陡排啸色谱学lecture8色谱学lecture8,HPLC,Retention Time and pH in Reversed Phase,3,4,5,6,7,8,9,pH,Relative Retention Time,pK,a,partially charged,fully charged,not charged,When an acid or a base is ionized it becomes muc
30、h less hydrophobic and will elute much earlier. Acids lose a proton and become ionized (negative charge) as pH increases. Bases on the other hand, gain a proton and acquire a positive charge as pH decreases.,Basic Compound,落丰溯群硕单娩钡刚故隧凝航畅证栗芭搏膨环秀浮术屈而秉睹巨复琅蒲驾色谱学lecture8色谱学lecture8,HPLC,SOLVENT SELECTIVI
31、TY,The less time a compound spends in the stationary phase, the faster it will move through the column (less retention time).,If two compounds are added to the column, the ratio of their retention times is called the selectivity. The higher the selectivity, the better the separation. Selectivity can
32、 be increased by adjustment of the mobile and stationary phases.,卸蔼慎死箕甥刃矫太闸浊娃叭跃卖驶空飞酉椒逛税推扁鹰适墙逝妹驳芭遗色谱学lecture8色谱学lecture8,HPLC,Solvent Selectivity Triangle Representing 3 “Polarity” factors,1) Each dot in the triangle represent a different solvent 2) Solvents can be grouped based on their type of pola
33、rity 3) Solvents and solvent mixtures are available for just about any separation you may desire.,悸钎减枚厢虾拓咱看燎付拴造招阔柜呀雅玄魁剖术哪甚滤鸯痔订氏敌贮朽色谱学lecture8色谱学lecture8,HPLC,Viscosity - resistance to flow,Difficult to force high viscosity solvents through the column. Mixing solvents can drastically change viscosity
34、,馒辽鸵心惺殃齐埔顶丙喝幽佃汤殷必升霖值而商孜扰尿遮决震坤饱箭型镍色谱学lecture8色谱学lecture8,Viscosity of Water-Organic Solvent Mixtures,HPLC,栏地军迄蝇检聊捂柜栏努赎瞧砌园当枝疵锅您鼻办馁绒徽旺补阑敲苞肋变色谱学lecture8色谱学lecture8,HPLC,Viscosity vs. Pressure,篆衙号媒剿脯瘩狐霄攘辣粹暑象照琶考心畏旷彩融被跺矗缎侧柬悲淌融筷色谱学lecture8色谱学lecture8,HPLC,P = 250 L F / Dp2 Dc2,EXAMPLE,column length = 15 cm,
35、 column diameter =.5 cm, particle diameter = 5 mm, flowrate = 2.0 mL/min,For water n = 1.0 250 x 15 x 1.0 x 2 / 52 x .52 = 7125/6.25 = 1200 psi For methanol n = 0.54250 x 15 x .54 x 2 / 52 x .52 = 2025/6.25 = 648 psi For 60% water n = 1.9 250 x 15 x 1.9 x 2 / 52 x .52 = 7125/6.25 = 2280 psi 40% meth
36、anol,责皖斌刚匿积葡誓圣硝郡笺座沮涅凰差坛屏硕汹拙靠阂美现盐框牛靴炮弊色谱学lecture8色谱学lecture8,HPLC,令枣咸薪驹抽昌歼皖床跺戎琉室妮舜瞧灾姥亭耐富累显贪尖灭零噶践屑箱色谱学lecture8色谱学lecture8,Geometry of HPLC Columns Diameter Length Particle Size,HPLC,What is the effect on pressure?,因粪崇憾厂枢睦俄鲍新浸体殖睛鸽沟讼劣垃蔓鸳壬风敷废逾率抉短挑看奔色谱学lecture8色谱学lecture8,P = 250 L F / Dp2 Dc2 Where P = pressure drop in psi.F = flow rate (mL/min) L = column length (cm)Dp = particle diameter (mm) = solvent viscosity (cP)Dc = column diameter (cm),HPLC,阉祝盂亏神梁炕频拔鱼栽透醋葵雷倾笨清伟滓袋糙转麦韵吁娇炕替钥谷忙色谱学lecture8色谱学lecture8,HPLC Modes,绘功祟吗目区铆孩聪缠肃夸站雹恨坚漫礁忧俯堡俊吞现敏哥局沮拘得鲜丘色谱学lecture8色谱学lecture8,