色譜基礎(chǔ)知識(shí)及原理

1、會(huì)計(jì)學(xué)1色譜基礎(chǔ)知識(shí)及原理色譜基礎(chǔ)知識(shí)及原理分離是一個(gè)物理的過(guò)程流動(dòng)相（流動(dòng)相（Mobile Phase）固定相（固定相（Stationary Phase）樣品（溶解于流動(dòng)相中的溶質(zhì)）樣品（溶解于流動(dòng)相中的溶質(zhì)）色 譜液 相 色 譜柱 色 譜紙 色 譜薄 層 色 譜高高 效效 液液 相相 色色 譜譜H H P P L L C C氣 相 色 譜 色譜泵自動(dòng)進(jìn)樣器 檢測(cè)器廢液數(shù)據(jù)處理系統(tǒng)色譜圖色譜圖即色譜柱流出物通過(guò)檢測(cè)器時(shí)所產(chǎn)生的響應(yīng)信號(hào)對(duì)時(shí)間的曲線圖，其縱坐標(biāo)為信號(hào)強(qiáng)度，橫坐標(biāo)為保留時(shí)間。 色譜峰色譜峰保留時(shí)間（分）保留時(shí)間（分）基線基線峰高峰高峰寬峰寬響應(yīng)值響應(yīng)值長(zhǎng)度內(nèi)徑，進(jìn)樣量初始初始現(xiàn)在

2、現(xiàn)在初始LDLDLDLoadLoad22)()()(內(nèi)徑流速初始現(xiàn)在初始DDDFF22)()()(問(wèn)題：什么樣的分離結(jié)果是好的？分離度？分離度的公式：)(211212WWVVRR：分離程度的量度411 NkkR若若 N 增加增加 2倍倍 或或 3倍倍 , R會(huì)如何變化會(huì)如何變化?1141RN 1600 N.5516 NWV16N22100N2516NWV16N22PW = .5PW = 2理論塔板數(shù)計(jì)算公式：Ws 方法Wtan16切線法Wh5.54半峰高W3s9 3sW4s16 4sW5s25 5s21WVNs2000400060008000050100150200250300理論塔板數(shù)理論塔

3、板數(shù)峰寬（峰寬（ L）k 2，Vo 1000 L流速（流速（cm/min） 塔板數(shù)塔板數(shù) 103 123456789101112131415051015變化結(jié)果塔板數(shù) % 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 會(huì)如何變化？會(huì)如何變化？ VVVV k k 01021V2V 或 3.5 - 1.5 - 2 2V 9 .5 - 1.5 - 5 3V

4、V1 V2V3 V0時(shí)間 0.5 1 2 5120102 kkttttRR1R62:38 / 42:58 / 72:28 / 2322OHCNCHOHMeOHOHTHF若若 = 1, 2, 10 or 20, R會(huì)如何變化會(huì)如何變化? 1N141R0011VVV k1.5.5 - 1 k 1V 9.5.5 - 5k 3V V0V1 V2V3時(shí)間 0.5 1 2 5 k 值值 k 項(xiàng)項(xiàng) k 對(duì)對(duì)分離度影響分離度影響 0 0 0 1 1/2 .50 2 2/3 .67 3 3/4 .75 10 10/11 .91 20 20/21 .95 與 R 的關(guān)系60/4050/5040/60譜圖CHOOH

5、OCH3COONaCHOCOOHOCH31234Packing: 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. Pyrilami

6、ne 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溶劑

7、 自動(dòng)進(jìn)樣器 HPLC色譜柱廢液死體積/譜帶展寬體積(無(wú)色譜柱時(shí))滯后(系統(tǒng))體積滯后(系統(tǒng))體積溶劑輸送系統(tǒng)(泵)檢測(cè)器進(jìn)樣器色譜柱色譜柱梯度混合器溶劑輸送系統(tǒng)(泵)高壓梯度注意注意 滯后體積包括進(jìn)樣器、阻尼滯后體積包括進(jìn)樣器、阻尼 器、混合器及其管路器、混合器及其管路比例閥檢測(cè)器進(jìn)樣器ABCD色譜柱色譜柱溶劑輸送系統(tǒng)(泵)低壓梯度 阻尼器 混合器梯度百分比保留時(shí)間梯度曲線好的梯度滯后曲線保留時(shí)間梯度百分比不好的梯度滯后曲線固定滯后體積，改善了梯度性能普通的低壓梯度系統(tǒng)P680A LPG without pulse damperBack-pressure:60 bar, 85 bar and

8、 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固定波長(zhǎng)固定波長(zhǎng)可變波長(zhǎng)可變波長(zhǎng)光電二極管矩陣光電二極管矩陣6:1NSAdapted fro

9、m: Snyder, L.R.; Kirkland, J.J.; Glajch, J.L; Practical HPLC Method Development 2nd Ed., Wiley-Interscience, New York, 1997, p. 2通用檢測(cè)器通用檢測(cè)器 RI ELSDMS靈敏度mgngpg線性范圍104否103流速敏感是否是溫度敏感是否否破壞性否是是選擇性檢測(cè)器選擇性檢測(cè)器 ABSFLEC Cond MS靈敏度ngpgfgpgpg線性范圍105103106105103流速敏感否否是是是溫度敏感否否是是是破壞性否否是否是Separation of Lactose-Red

10、uce 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靈敏度缺乏靈敏度 和其他檢測(cè)器相比，和其他檢測(cè)器相比，RI檢測(cè)器一般靈敏度較低檢測(cè)器一般靈敏度較低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.

11、5 8.0 8.5 9.0 9.5AU0.000.020.040.060.04Refractive Index S/N 125:1流動(dòng)相：MeOH/H2O 60:40流速：1.0 mL/min色譜柱：C18色譜柱溫：30 C檢測(cè)溫度：35 C樣品： Paraben Mixture流動(dòng)相：A= CH3CN/H2O 75:25B= CH3CN流速： 1.4 mL/min色譜柱：NH2色譜柱溫：30 C檢測(cè)溫度：35 CMV-2600-2400-2200-2000-1800-1600-1400-1200-1000-800-600-400-2000200400Minutes02

12、46810 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.Thereisvirtua

13、llynoadsorptionabove230nmandbelow230nmpicksupsolventinterference.ImconsideringusingRIdetectionandafterwardsubtractingablankrun.IsthereanyhopeofthisworkingorwouldIbewastingmytime?http:/ 甲醇 0.25mL / minute檢測(cè)器溫度 35C；沒(méi)有使用柱溫箱Temp.(Deg C)18.519.019.520.0MV-1.000.001.002.00Minutes51015202530354045505560657

14、075室溫變化RI 基線AU0.000.050.100.15Minutes0.501.001.502.002.503.003.504.004.505.005.506.00Parabens at 254 nm實(shí)際濃度濃度理想10吸光度吸光度210UV Spectrum of Eluents with 0.1% TFA Against HPLC Grade H2O BlankAU0.0000.400.500.600.700.800.901.0001.401.50nm200.00210.00220.00230.00240.00250.00260.00270

15、.00280.00290.00H20 with 0.1% TFA50% ACN with 0.1% TFA色譜條件色譜條件色譜柱：C18, 5 m, 3.9x150mm at 35 C.流動(dòng)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.樣品： 水（10 L inj. vol.）檢測(cè)： 214 nm 圖1；好的梯度系統(tǒng) 圖2；一般的二元梯度系統(tǒng) 圖3；一般的四元梯度系統(tǒng)五個(gè)小肽的5ng進(jìn)樣，好的色譜系統(tǒng)非常容易鑒別出所有峰。在不好的色譜系統(tǒng)中，第一個(gè)峰則消失在基

16、線噪音中。1.2241.1831.183Caffeine 271 nm基態(tài)激發(fā)態(tài)S1S0激發(fā)（1）振動(dòng)能（2）發(fā)射（3）氨基甲酸酯類殺蟲(chóng)劑OOOOOOCH3黃曲霉毒素多環(huán)芳烴（PAH）OHCH3CH3CH3CH3CH3維生素OOONHCH3CH3CH3mV-0.100.0000.400.50Minutes5.006.007.008.009.0010.0011.0012.0013.006.8949.639OONHCH3CH3OOCH3NH2MinutesColumn - PAH Column 27 CEluent A: WaterEluent B: Acetonitrile

17、Gradient: 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充分脫氣未充分脫氣1231. Benzo(b)flouranthene - 400 ppb2. Benzo(k)fluoranthene - 200 ppb3. Benzo(a)pyrene - 200 ppbEmission Exci

18、tation 103 M 10 5 M在正己烷中From: Turro, N.J., Modern Molecular PhotochemistryMenlo Park CA, Benjamin/Cummings Publishing, 1978三維水平的吸光度檢測(cè)器三維水平的吸光度檢測(cè)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.

19、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 損失分辨率，UV最大波長(zhǎng)移動(dòng) 1.0nm3.0nm190nm5.0nmAU0.0000.0010.002nm250.00300.00266.2AU0.0000.0010.0020.003nm250.00300.00271.5312.11.2 nm14.4 nmm/z+/-質(zhì)譜（Mass Spectrometry）是一種按照分子量及其電荷分離物質(zhì)的技術(shù)，經(jīng)

20、過(guò)多年的不斷開(kāi)發(fā)及改進(jìn)，已經(jīng)使質(zhì)譜成為一種多功能、高靈敏度，并被愈來(lái)愈廣泛使用的分析技術(shù)。液相色譜與質(zhì)譜的連用。除去HPLC的溶劑；產(chǎn)生離子；分離離子；檢測(cè)離子；計(jì)算離子強(qiáng)度；處理數(shù)據(jù)離子化前的準(zhǔn)備：離子化前的準(zhǔn)備：1.霧化（Nebulization）2.蒸發(fā)溶劑或解吸附（Evaporation/ or Desorption）3.大氣壓下或減壓下（Atmosphere or Pressure reduction）4.離子化（Ionization Ion Source）LCMSCo(l)C+/-(l)C+/-(g)Co(g)EvaporationDesorptionLC/MS Interface

21、200 300 400 500 600 700 800m/z529527233489543APCI(-): 40V100200300400500m/z5797147219278515530EIOHCH3CH3CH3CH3CH3CH3OO(CH2)17CH310 410 210 110 310 5LCGC PolarityMolecular WeightVolatilityLCGCElectrospray (ESI)APCIEIGCFlow rate, mL/min00.51.01.52.050100Relative intensityAPCIPneumatically assisted ESI

22、Pure ESI流速對(duì)API源的影響Intensity1x1062x1063x1064x1065x1066x1067x1068x106Minutes2.003.004.005.006.007.008.009.0010.0011.00采集類型：采集類型： 單離子模式（SIR） 掃描模式（Scan） 總離子流（TIC或BPC）ESI +ve 180-400 m/z TICESI -ve 180-400 m/z TIC極性切換：極性切換： 正離子 負(fù)離子n離子排序方式n四極桿（Quadrupole）n離子阱（Ion Trap）n飛行時(shí)間（Time of Flight）n磁質(zhì)譜（Sector Mass

23、 Analyzers）n富里葉變換（Fourier Transform）n檢測(cè)方式n電子倍增n光電倍增n02004006008001000120014001600180002004006008001000Molecular WeightFrequency of Occurrance標(biāo)稱分子量在250的化合物的數(shù)量超過(guò)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電化學(xué)檢測(cè)器Electrochemi