色谱基础知识及原理

分离是一个物理的过程流动相（流动相（Mobile Phase）固定相（固定相（Stationary Phase）样品（溶解于流动相中的溶质）样品（溶解于流动相中的溶质）色谱液相色谱柱色谱纸色谱薄层色谱高高效效液液相相色色谱谱H HP PL LC C气相色谱 色谱泵自动进样器 检测器废液数据处理系统色谱图色谱图即色谱柱流出物通过检测器时所产生的响应信号对时间的曲线图，其纵坐标为信号强度，横坐标为保留时间。色谱峰色谱峰保留时间（分）保留时间（分）基线基线峰高峰高峰宽峰宽响应值响应值长度内径，进样量初始初始现在现在初始LDLDLDLoadLoad22)()()(内径流速初始现在初始DDDFF22)()()(问题：什么样的分离结果是好的？分离度？分离度的公式：)(211212WWVVRR：分离程度的量度411 NkkR若若 N 增加增加 2倍倍 或或 3倍倍,R会如何变化会如何变化?1141RN 1600 N.5516 NWV16N22100N2516NWV16N22PW=.5PW=2理论塔板数计算公式：Ws 方法Wtan16切线法Wh5.54半峰高W3s9 3sW4s16 4sW5s25 5s21WVNs2000400060008000050100150200250300理论塔板数理论塔板数峰宽（峰宽（L）k 2，Vo 1000 L流速（流速（cm/min）塔板数塔板数 103 123456789101112131415051015变化结果塔板数%N N 73%54.73000 3000N 41%44.72000 2000N 31.61000 1000N 73%122.415,000 15,000N 41%10010,000 10,000N 70.7 5,000 5,000N 1N41R1 若若 =1.1 or 1.4，R 会如何变化？会如何变化？VVVV k k 01021V2V 或 3.5-1.5-2 2V 9 .5-1.5-5 3V V1 V2V3 V0时间 0.5 1 2 5120102 kkttttRR1R62:38 /42:58 /72:28 /2322OHCNCHOHMeOHOHTHF若若 =1,2,10 or 20,R会如何变化会如何变化?1N141R0011VVV k1.5.5-1 k 1V 9.5.5-5k 3V V0V1 V2V3时间 0.5 1 2 5 k 值值 k 项项 k 对对分离度影响分离度影响 0 0 0 1 1/2 .50 2 2/3 .67 3 3/4 .75 10 10/11 .91 20 20/21 .95 与 R 的关系60/4050/5040/60谱图CHOOHOCH3COONaCHOCOOHOCH31234烷基长度不同，形成对离子的能力不同通常有：B5，B6，B7，B8 后面的数字是碳链的长度Packing:Bondapak C18Column:4 mm ID x 30cmSolvent:Methanol/H2O with 0.005M PENTANE Sulfonic Acid&1%HOAc(50/50)Flow Rate:2.0 ml/minDetector:UV,254 nm,0.1 AUFS1.Maleic Acid2.Phenylephrine-HCl 3.Phenylpropanolamine-HCl 4.Naphazoline-HCl 5.Phenacetin6.Pyrilamine MaleatePacking:Bondapak C18Column:4 mm ID x 30cmSolvent:Methanol/Water with 0.005M HEXANE Sulfonic Acid&1%HOAc(50/50)Flow Rate:2.0 ml/minDetector:UV,254 nm,0.1 AUFS1.Maleic Acid2.Phenylephrine-HCl 3.Phenylpropanolamine-HCl 4.Naphazoline-HCl 5.Phenacetin6.Pyrilamine Maleate溶剂 自动进样器 HPLC色谱柱废液死体积/谱带展宽体积(无色谱柱时)滞后(系统)体积滞后(系统)体积溶剂输送系统(泵)检测器进样器色谱柱色谱柱梯度混合器溶剂输送系统(泵)高压梯度注意注意 滞后体积包括进样器、阻尼滞后体积包括进样器、阻尼 器、混合器及其管路器、混合器及其管路比例阀检测器进样器ABCD色谱柱色谱柱溶剂输送系统(泵)低压梯度 阻尼器 混合器实际梯度会比设置值晚 2 分钟响应，没有问题实际梯度会比设置值晚20 分钟响应，不能接受梯度百分比保留时间梯度曲线好的梯度滞后曲线保留时间梯度百分比不好的梯度滞后曲线固定滞后体积，改善了梯度性能普通的低压梯度系统P680A LPG without pulse damperBack-pressure:60 bar,85 bar and 105 barVariation:2%Acclaim 120,C18,5 m,4.6 x 100 mm;0.5 mL/min;25C;5 L injection volume;UV detection at 256 nm;Eluent(A)Water and(B)Acetonitril;Gradient:30%b to 100%B in 10 min;Methyl-,Ethyl-,Propyl-and Butylparabene,10 mg/L each固定波长固定波长可变波长可变波长光电二极管矩阵光电二极管矩阵6:1NSAdapted from:Snyder,L.R.;Kirkland,J.J.;Glajch,J.L;Practical HPLC Method Development 2nd Ed.,Wiley-Interscience,New York,1997,p.2通用检测器通用检测器 RI ELSDMS灵敏度mgngpg线性范围104否103流速敏感是否是温度敏感是否否破坏性否是是选择性检测器选择性检测器 ABSFLEC Cond MS灵敏度ngpgfgpgpg线性范围105103106105103流速敏感否否是是是温度敏感否否是是是破坏性否否是否是碳水化合物（Carbohydrates）、聚合物（Polymers）脂肪酸（Fatty Acids）及油脂类（Lipids）Separation of Lactose-Reduce MilkMV-20-100102030405060708090100110Minutes4.04.55.05.56.06.57.07.58.08.59.09.510.0 10.5 11.0 11.5 12.0 12.5葡萄糖乳糖半乳糖缺乏灵敏度缺乏灵敏度 和其他检测器相比，和其他检测器相比，RI检测器一般灵敏度较低检测器一般灵敏度较低UV254 nm S/N 15000:1MV-30.00-25.00-20.00-15.00-10.00-5.000.005.0010.0015.00Minutes2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5AU0.000.020.040.060.080.100.120.14Refractive Index S/N 125:1流动相：MeOH/H2O 60:40流速：1.0 mL/min色谱柱：C18色谱柱温：30 C检测温度：35 C样品：Paraben Mixture流动相：A=CH3CN/H2O 75:25B=CH3CN流速：1.4 mL/min色谱柱：NH2色谱柱温：30 C检测温度：35 CMV-2600-2400-2200-2000-1800-1600-1400-1200-1000-800-600-400-2000200400Minutes0246810 12 14 16 18 20 22 24 26 28 300%B25%BChromatography Forum:Is RI with a Reverse Phase gradient possible?ByXXXXXXXXX:Ihaveagoodseparationofamulti-substituted,cyclohexanethatfullyresolves12differentoligomers.IcandetectthecomponentsbyMS,butIwouldliketouseastandardLCdetectorinstead.Thereisvirtuallynoadsorptionabove230nmandbelow230nmpicksupsolventinterference.ImconsideringusingRIdetectionandafterwardsubtractingablankrun.IsthereanyhopeofthisworkingorwouldIbewastingmytime?http:/ 甲醇 0.25mL/minute检测器温度 35C；没有使用柱温箱Temp.(Deg C)18.519.019.520.0MV-1.000.001.002.00Minutes51015202530354045505560657075室温变化RI 基线AU0.000.050.100.15Minutes0.501.001.502.002.503.003.504.004.505.005.506.00Parabens at 254 nm实际浓度浓度理想10吸光度吸光度210UV Spectrum of Eluents with 0.1%TFA Against HPLC Grade H2O BlankAU0.000.100.200.300.400.500.600.700.800.901.001.101.201.301.401.50nm200.00210.00220.00230.00240.00250.00260.00270.00280.00290.00H20 with 0.1%TFA50%ACN with 0.1%TFA色谱条件色谱条件色谱柱：C18,5 m,3.9x150mm at 35 C.流动相：A:0.1%TFA in Water.B:0.1%TFA in Acetonitrile.梯度：5-40%B in 35 min.流速：1.0 mL/min.样品：水（10 L inj.vol.）检测：214 nm 图1；好的梯度系统 图2；一般的二元梯度系统 图3；一般的四元梯度系统五个小肽的5ng进样，好的色谱系统非常容易鉴别出所有峰。在不好的色谱系统中，第一个峰则消失在基线噪音中。1.2241.1831.183Caffeine 271 nm基态激发态S1S0激发（1）振动能（2）发射（3）氨基甲酸酯类杀虫剂OOOOOOCH3黄曲霉毒素多环芳烃（PAH）OHCH3CH3CH3CH3CH3维生素OOONHCH3CH3CH3mV-0.100.000.100.200.300.400.50Minutes5.006.007.008.009.0010.0011.0012.0013.006.8949.639OONHCH3CH3OOCH3NH2MinutesColumn-PAH Column 27 CEluent A:WaterEluent B:AcetonitrileGradient:60%B to 100%B using curve 9 in 12 minutesHold 11 minutesBack to initial conditionsFlow Rate 1.2 ml/minInjection:20ulTime programmed wavelength changes20.521.022.01000 MV充分脱气未充分脱气1231.Benzo(b)flouranthene -400 ppb2.Benzo(k)fluoranthene -200 ppb3.Benzo(a)pyrene -200 ppbEmission Excitation 103 M 10 5 M在正己烷中From:Turro,N.J.,Modern Molecular PhotochemistryMenlo Park CA,Benjamin/Cummings Publishing,1978三维水平的吸光度检测器三维水平的吸光度检测器AU0.0000.0020.0040.0060.0080.0100.0120.0140.0160.0180.0200.0220.0240.0260.0280.0300.032nm200.00220.00240.00260.00280.00300.00320.00283.5240.9245.6AU0.0000.0050.0100.015Minutes2.002.503.003.504.004.505.005.506.006.507.002.1934.3106.448230.00250.00270.00nmBenzene 3.0nm vs 1.0nm 损失分辨率，UV最大波长移动 1.0nm3.0nm190nm5.0nmAU0.0000.0010.002nm250.00300.00266.2AU0.0000.0010.0020.003nm250.00300.00271.5312.11.2 nm14.4 nmm/z+/-质谱（Mass Spectrometry）是一种按照分子量及其电荷分离物质的技术，经过多年的不断开发及改进，已经使质谱成为一种多功能、高灵敏度，并被愈来愈广泛使用的分析技术。液相色谱与质谱的连用。除去HPLC的溶剂；产生离子；分离离子；检测离子；计算离子强度；处理数据离子化前的准备：离子化前的准备：1.雾化（Nebulization）2.蒸发溶剂或解吸附（Evaporation/or Desorption）3.大气压下或减压下（Atmosphere or Pressure reduction）4.离子化（Ionization Ion Source）LCMSCo(l)C+/-(l)C+/-(g)Co(g)EvaporationDesorptionLC/MS Interface200300400500600700800m/z529527233489543APCI(-):40V100200300400500m/z5797147219278515530EIOHCH3CH3CH3CH3CH3CH3OO(CH2)17CH310 410 210 110 310 5LCGC PolarityMolecular WeightVolatilityLCGCElectrospray(ESI)APCIEIGCFlow rate,mL/min00.51.01.52.050100Relative intensityAPCIPneumatically assisted ESIPure ESI流速对API源的影响Intensity1x1062x1063x1064x1065x1066x1067x1068x106Minutes2.003.004.005.006.007.008.009.0010.0011.00采集类型：采集类型：单离子模式（SIR）扫描模式（Scan）总离子流（TIC或BPC）ESI+ve 180-400 m/z TICESI-ve 180-400 m/z TIC极性切换：极性切换：正离子 负离子离子排序方式四极杆（Quadrupole）离子阱（Ion Trap）飞行时间（Time of Flight）磁质谱（Sector Mass Analyzers）富里叶变换（Fourier Transform）检测方式电子倍增光电倍增02004006008001000120014001600180002004006008001000Molecular WeightFrequency of Occurrance标称分子量在250的化合物的数量超过了1500！Adapted from:R.Willoughby et al A Global View of LC/MS,1998Intensity0.05.0 x1051.0 x106294.20Intensity0500000294.20Intensity01x1062x106m/z240.00250.00260.00270.00280.00290.00300.00310.00320.00278.22Intensity3x1064x1065x106Minutes0.001.002.003.004.005.006.007.008.009.0010.002.4592.8039.5722.60 min2.80 min9.60 mintrans-10-hydroxyamitriptyline(EHAT)cis-10-hydroxyamitriptyline(ZHAT)amitriptyline电化学检测器Electrochemical电导检测器ConductivityWRC有机及无机离子儿茶酚胺 Catecholamines亚硝胺 Nitrosamines有机酸 Organic acids碳水化合物 Carbohydrates氨基酸 Amino AcidsN2/S2RadiochemicalNMR