High Performance Liquid Chromatography Chapter 3 High Performance Liquid Chromatography (HPLC)

Outline of chapter 3 Requirements: 要求掌握HPLC法的基本原理 Characteristics of HPLC(特点) Structure of high performance liquid chromatographer (仪器构造） Types and separation principles of HPLC methods(方法的类型及其分离原理） Factors influencing broadening of chromatographic peaks and separation(影响色谱峰扩展及色谱分离的因素) Stationary phase of HPLC(液相色谱固定相) Mobile phase of HPLC(液相色谱流动相) Selection of chromatographic methods(色谱方法的选择) Requirements: 要求掌握HPLC法的基本原理 Emphases: 重点学会色谱分析方法的选择

§3-1 Characteristics of HPLC ●High pressure Because carrier liquid moving through the column endures high resistance, high pressure must be provided Usually The pressures for supplying carrier liquid(供液压力）and for injection sample(进样压力）reach 150 ~ 350×105 Pa ●High velocity Flow rate of carrier liquid: 1 ~ 10mL.min-1, much faster than classical liquid chromatography. Analytical velocity is much faster than classical LC. It usually takes less than 1 hour

●Wide application fields ●High efficient Due to use of new-type of stationary phases, column efficiency of HPLC improved further (number of theoretical plates per meter is more than 3×104/m ●High sensitivity Highly sensitive detectors’ application improves the analytical sensitivity. Minimum detectable quantity UV-detector: down to 10-9g Fluorescence detector: 10-11g Sample amount needed: Very little, micro-liter of test sample is enough for full analysis ●Wide application fields Available for organic compounds with high boiling point, poor thermal stability and high molecular weight(>400) and some of inorganic compounds

● 由于柱内色谱峰扩展所引起的塔板高度的变化为： §3-2 Factors influencing broadening of chromatographic peaks and separation Diffusion coefficient Dm/cm2.s-1: 10-1(gas)/10-5(liquid) Density /g.cm-3: 10-3(gas)/1(liquid) Viscosity /gcm-1.s-1: 10-4(gas)/10-2(liquid) = 2l dp + Cd Dm u + Cm dP2 CSmdP2 Cs df2 Dm Dm Ds H = A + + Cu B ● 由于柱内色谱峰扩展所引起的塔板高度的变化为：

He = 2ldP Dm Hd ∝ u Cd Dm Hd = 1.涡流扩散项He 2.纵向扩散项Hd 上式的含义及对峰扩展的影响同气相色谱 Due to very little diffusion coefficient of the molecules in liquid phase, when linear velocity is higher than 0.5cm.s-1， contribution of longitudinal diffusion term to peak broadening is neglectable.

3. 传质阻力项 ●Stationary phase mass transfer resistance term 固定相传质过程：试样分子从流动相进入固定液进行质量交换 Hs = Cs df2 Ds u Cs is a coefficient depending on k How to eliminate or reduce peak broadening induced by mass transfer of stationary phase ?

▲Use the stationary liquid with a big diffusion coefficient ▲ Improve mass transfer to quicken desorption of solute molecules on stationary phase ▲ Use thin layer of stationary phase for liquid-liquid partition chromatography ▲Use small grains with a little radii (小颗粒）as filling materials(填料）for adsorption, size-exclusion and ion-exchange chromatography ▲Use the stationary liquid with a big diffusion coefficient ▲Reduce rate of mobile phase However, too low mobile phase rate will deduce enhancement of molecular diffusion term and prolonging(延长）of analytical time.

●Mass transfer resistance term of mobile phase Mass transfer in moving mobile phase Mass transfer in retardation(滞留） mobile phase Mass transfer resistance term Hm Flow rate of mobile phase is not identical on whole column, slower near packing particles Hm ∝ dp2 Dm u Cm = constant depending on k , diameter and shape of applied column and structure of filling materials packed. Hm = Cm dp2 Dm u

Mass transfer resistance term in retardation mobile phase Due to porosity of stationary phase particles, some mobile phase stays in the aperture(孔），and these stationary phase usually do not move(滞留区的流动相通常是不动的）. column Solute molecules must diffuse into these holes(滞留区） to carry out mass transfer with stationary phase (试样分子必须先扩散到滞留区才能完成质量交换） Solute molecule The smaller and deeper the holes, the lower the rate of mass transfer, the more remarkable the influence on peak broadening(微孔越深和越小，传质速率越慢，对峰扩展的影响越显著）. Mobile phase Stationary phase particle The smaller the grain of stationary phase and the bigger the aperture(孔径）， the shorter the distance of mass transfer, the faster the mass transfer rate and the higher the column efficiency(固定相粒度越小，微孔孔径越大，传质途径越小，传质速率越高，柱效越高）.

dp2 Dm u Hs m ∝ Cs m dp2 Dm u Hs m = 滞留区传质阻力项为： 对于液相色谱柱，柱效的影响因素主要为传质阻力项，分子扩散项的影响可以忽略

提高高效液相色谱分离效率的措施 提高柱内填料装填的均匀性 减小粒度 1.选择薄壳形担体 30 ~ 40mm的实心核上覆盖1 ~ 2mm厚的多孔硅胶 孔径大，孔道浅且大小均匀 2.采用微粒形填料（粒径小于10mm) 3.选用低粘度的流动相 4.适当提高柱温

其它影响色谱峰扩展的因素： 柱外扩展（超柱效应） 柱前峰展宽：由进样引起（How to eliminate?)

§3-3 Main types of HPLC methods and their separation principle Liquid-liquid partition chromatography(液-液分配色谱法) Liquid-solid adsorption chromatography(液-固吸附色谱法） Ion-exchange chromatography(离子交换色谱法） Ion-pair chromatography(离子对色谱法） Ion chromatography(离子色谱法） Steric exclusion chromatography（空间排阻色谱法）

●Liquid-liquid partition chromatography (液-液分配色谱法) and chemically bonded phase chromatography (化学键合相色谱法） Figure Schematics showing the principle of liquid-liquid partition chromatography, the solute represented by the solid circle(●) is the more strongly retained

Partition of solutes in two phases based on the difference of their relative solubility. Following formula is also true: cS cm K = = k Vm Vs =b k Differently from in GC, K is relational to properties of mobile phases in LC

In order to reduce or eliminate loss of stationary liquid, following measures are usually employed: ●Normal phase liquid-liquid chromatography: Stationary liquid is more polar than mobile phase Stationary liquid: hydrophilic(亲水的） Mobile phase: hydrophobic（疏水的） ●Reverse phase liquid-liquid chromatography: Mobile phase is more polar than stationary liquid Stationary liquid: hydrophobic(疏水的) Mobile phase: hydrophilic(亲水的） Wider applications ●Chemically bonded stationary phase: 将各种不同有机基团（group)通过化学反应键合到硅胶(silica gel)担体表面的游离羟基(dissociative hydroxyl)上，代替机械涂敷的液体固定相。 应用广泛，可用于normal-, reverse-, ion-exchange-, ion pair色谱等技术中。

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●Liquid-Solid Adsorption Chromatography Figure Schematics showing the basis of separation in adsorption chromatography. The solute represented by the solid circle(●) is the more strongly retained. Mobile Phase: Liquid Stationary phase: Sorbent (吸附剂）

▲Mechanism of liquid-solid adsorption chromatography Separation based on difference of adsorptive actions of substances Competitive adsorption of solute molecule X and solvent molecule S on active surface of sorbent (溶质分子与溶剂分子在吸附剂活性表面的竞争吸附）： Xm + nSa Xa + nSm where subscripts m and a respectively stand for mobile phase and adsorption phase When adsorption reaches a equilibrium, following formula is true: K = [ Xa][ Sm]n [Xm][Sa]n Where K denotes adsorption equilibrium coefficient or partition coefficient

▲Shortcoming: back ▲Applications: 1. Oil-soluble samples with a medium molecular weight(中等相对分 子质量） 2. Compounds linked with various functional groups and isomers(异 构体） Applicable to the compounds which can be successfully separated by thin layer chromatography ▲Shortcoming: Tailing peak appears usually due to nonlinear isothermal(等温) adsorption back

●Ion Exchange Chromatography + - Figure Schematics showing the basis of separation in ion-exchange chromatography, the solute represented by the solid circle(●) is the more strongly retained.

M+ + (Na+ -O3S-resin) (M+ -O3S-resin) + Na+ X- + (Cl- +R3N-resin) 基于离子交换树脂上可电离的离子与流动相中具有相同电荷的溶质离子进行可逆交换，根据这些离子与交换剂亲合力的不同而实现分离的色谱分析方法。 凡是在溶剂中能够电离的物质都能够用离子交换色谱法来进行分离。 M+ + (Na+ -O3S-resin) (M+ -O3S-resin) + Na+ (In Solvent) (In resin) X- + (Cl- +R3N-resin) (X- +R3N-resin) + Cl- (In Solvent) (In resin)

KX = [-NR4+X-][Cl-] [-NR4+Cl-][X-] KM = [-SO3-M+][Na+] [-SO3-Na+][M+] Equilibrium constant for ion-exchange reaction: KX = [-NR4+X-][Cl-] [-NR4+Cl-][X-] KM = [-SO3-M+][Na+] [-SO3-Na+][M+] Or Partition coefficient of anionic exchange DX DX = [-NR4+X-] [X-] = KX [-NR4+Cl-] [Cl-] Partition coefficient of cationic exchange DM DM = [-SO3-M+] [M+] = KM [-SO3-Na+] [Na+]

Used to separate various ions or dissociable(可离解的) compounds The higher the affinity of solute molecules with ion-exchange centre , the stronger the interaction of solute ions to ion-exchanger, the bigger partition coefficient and rotention value(溶质分子与离子交换中心的亲合力越高，溶质的离子与离子交换剂的相互作用越强，分配系数越大，保留值越大) ▲Applications: Used to separate various ions or dissociable(可离解的) compounds Inorganic species Organic species Biological molecules: for example, amino acids, nucleic acid, proteins ect.

back Ion-Exchange Groups Type Active group Application example pH range of operation Application example Strongly acidic cation exchanger -SO3- 1-14 Amino acids, Inorganic separations weakly acidic cation exchanger -COO- 5-14 Transition elements, organic bases Strongly basic anion exchanger For example, -N+(CH3)3 1-12 Alkaloids(生物碱）, Fatty acids For example, DEAE, -C2H4N(C2H5)2 1-9 Organic acids, Amino acids back

●Ion pair chromatography (IPC) X+ Stationary phase Mobile phase 将一种或多种与溶质分子电荷相反的离子（对离子或反离子ion-pair reagents）加入到流动相或固定相中，使其与溶质离子结合形成疏水型离子对化合物，从而控制溶质离子的保留行为。 Counter ions(对离子） For anionic separation: alkyl ammonium. Eg. tetrabutyl ammonium hydroxide, (CH3(CH2)3)4N+OH-, cetyl trimethylammonium hydroxide, (CH3(CH2)15(CH3)3N+OH-, Tetrapropylammonium hydroxide,[(CH3CH2CH2)4N+OH- , etc. For cationic separation: alkyl sulfonic acid Eg. Hexane sulfonic acid, CH3(CH2)5SO3Na

X+org + Y-aq X+Y-org KXY = [X+Y-]org [X+]aq[Y-]aq DX = [X+Y-]org ▲Separation principle X+org + Y-aq X+Y-org KXY Where X, Y denotes respectively analyte and ion-pair reagent, subscripts org and aq respectively organic phase and aqueous phase, KXY equilibrium constant of partition process KXY = [X+Y-]org [X+]aq[Y-]aq Partition coefficient of solute DX DX = [X+Y-]org [X+]aq = KXY [Y-]aq

VS VM 1 b k = DX = KXY[Y-]aq TR/TM = u / uS =1+ k = 1+ KXY[Y-]aq 1 b Capacity factor k is expressed as follows: VS VM 1 b k = DX = KXY[Y-]aq According to eq(2-11) to eq (2-16), TR/TM = u / uS =1+ k = 1+ KXY[Y-]aq 1 b TR = TM (1 + KXY[Y-]aq ) 1 b = (1 + KXY[Y-]aq ) L u

■ Reverse phase ion-pair chromatography is more common It is obvious that rotention value increases with increasing KXY and [Y-]aq, KXY depends on properties of ion-pair reagent and organic phase 改变对离子浓度是控制反相离子对色谱溶质保留值的主要措施，可在较大范围内改变分离的选择性。 ▲Applications ■Separate acids, bases, and some ionic, nonionic mixtures, especially, biochemical test samples. ■Easy to introduce some ultraviolet-adsorbed groups (chromaphore) or fluorescent groups to sample molecules to improve detection sensitivity ■ Reverse phase ion-pair chromatography is more common back

●Ion Chromatography (IC) 　　离子色谱法是以离子交换树脂为固定相，以电解质溶液为流动相，用于分离阴阳离子的一种色谱分析方法。 　　通常在来自色谱中，应用最为广泛的的检测器为电导检测器。为了消除流动相中强电解质背景离子对检测器的干扰，常设置一个抑制柱（suppressor)，称为双柱型离子色谱法或化学抑制型离子色谱法。 示意图见下页 如果选择低电导的洗脱液（流动相），则不仅洗脱效果好，而且由于背景电导低，不干扰待测离子的测定，因此，可以不安装抑制柱，称为单柱型或非抑制型离子色谱法。 离子色谱法可用于分离测定无机、有机阴阳离子，尤其对于阴离子的测定是目前最有效的方法

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●Steric Exclusion Chromatography Figure Schematics showing the basis of separation in size-exclusion chromatography. The solute represented by the solid circle(●) is the more strongly retained.

空间排阻色谱法 Size exclusion chromatography (SEC) Gel permeation chromatography (GPC) Gel filtration chromatography Exclusion chromatography 空间徘阻色谱法以凝胶作为固定相，液体作为流动相。分离机制与其它色谱法不同，它类似干分子筛的作用，但凝胶的孔 径比分子筛要大得多，一般为几百至几千埃。色谱柱内填充以凝 胶，凝胶内具有一定大小的孔穴。试样进入色谱柱后，随流动相在凝胶外部间隙，以及凝胶孔穴旁流过，体积大的分子不能渗透到凝 胶孔隙里去而受到排阻，较早地被冲洗出来。中等体积的分于产生部分渗透作用，小分子可渗透到孔穴里去，因有一个平衡过 程而较晚被冲洗出来。这样，试样组分基本上是按其分子大小受 到不同排阻而先后由柱中流出，从而实现分离的。对同系物来说， 洗脱体积是分子量的函数。洗脱次序决定于分子量大小，分子 量大的先流出色谱柱，分子量小的后流出。

A点：排斥极限，Vo 分子量更大的物质均在此出峰 Vo< Ve<Vm,分级范围，当化合物分子大小不同，且位于此分级范围内时，可以实现分离。 D点：全渗透极限,VM，分子量更小的物质，如溶剂等均在此出峰

Easy to chose stationary phase & mobile phase ●Characteristics All peaks located before solvent peak, Shorter rotention time, less peak broadening-in-column(柱内) Thinner peak Advantageous to detection Easy to chose stationary phase & mobile phase Any soluble compounds with a molecular weight ranging 102 to 8×105 Inapplicable to the separation of the molecules with close mol. wt Incapable of separating the polymers with a continuous mol. Wt distribution, but capable of determining molecular weight distribution Characteristics SEC

●Stationary and Mobile Phases of HPLC Self-study Problems What kinds of stationary phases do each HPLC method have? What characteristics do each stationary phase have? How to select mobile phase in HPLC? 详细内容

§3-6 Chromatograph 贮液器 高压泵 梯度洗脱装置 进样器 色谱柱 恒温器 检测器 记录仪

●高压泵 Provide a stable and pulseless pressure of 15 ~ 35MPa, a stable flux(流量),& a adjustable flow rate. 往复式柱塞泵(恒流泵)

优点: 缺点 不受整个色谱体系中其余部分阻力微小变化的影响,连续供给恒定体积的流动相. 易于通过调节冲程的大小或柱塞往复频率来调节流量 死体积小,溶剂更换方便,适合于梯度洗提 缺点 输出有脉冲波动,会干扰某些检测器的响应(如折光检测器),可通过使用脉冲阻尼器,或能对输出流量相互补偿的具有两个泵头的双头泵来消除脉冲干扰(见下图).不过这种现象对紫外检测器无影响.

气动放大泵(恒压泵) Air inlet Air outlet Solvent out 单向阀 Sealed underlay (密封垫) Solvent let Solvent out 单向阀 Sealed underlay (密封垫) 柱塞 气动放大泵(恒压泵)

输出流量稳定, 无脉冲 可提供大的输出流量，适用于匀浆法装填色谱柱 泵体一次吸入的液体体积取决于液缸容量 液缸体积大, 流动相更换不方便, 不便于梯度洗脱实验, 及需要频繁更换流动相的溶剂选择实验

●Gradient elution setup 梯度洗提就是载液中含有两 种(或更多)不同极性的溶剂，在分离过程中按一定的程序连续改 变载液中溶剂的配比和极性,通过载液中极性的变化来改变被分 离组分的分离因素，以提高分离效果。应用梯度洗提还可以使分离时间缩短，分辨能力增加，由于峰形的改善还可以提高最小检测量和定量分析的精度。 低压梯度系统：在常压下预先按一定 的程序将溶剂混合后再用泵输入色谱柱，也称外 梯度。 高压梯度系统：先将溶剂用高压泵增压以后输入色谱系统的梯度混合 室，加以混合后再送入色谱柱，液称内梯度系统。

●Injection sample system

Syringe: 不能承受高压 可采用停流方式进样 峰形重现性较差，保留时间难以精确确定 High pressure quantificational sampling valves 通过进样阀直接向压力系统内进样而不 必停止流动相流动。由于每次进样量都是用定量管来计算的，因此重现性较好(可达0.5％)。它可以在高压(可到200kg／cm2)下迅速送进相当大体积(＞50ml)的试样。这种进样方式适用于高压下自动进祥而试样量较大的情况。缺点是在进样中要排悼一部分试 样，另外，如要变更进祥体积时，需要更换不同的定量管。进样阀 进样对峰的展宽的影响比注射器进样要大一些。

不过，现在的进样阀可以通过更换定量管来调整进样量，也可以采用体积较大的的定量管进少量试样，而进样量则由注射器控制，只是试样不充满定量管，仅填充其中一部分体积。

●Column(色谱柱) 液相色谱法常用的色谱柱大都采用内径为I~6mm的厚壁 玻璃管或内壁抛光的不锈钢管（标准柱型内径为3.9或4.6mm)。柱子长度一般为0.5m(大多为0.15 ~ 0.3m)，柱子的形 状多采用直形往，这样装柱换柱都比较方便。螺旋形柱子柱效将 明显降低，而且很难采用干式装柱。直形的玻璃柱管和内壁抛光的不锈钢柱管具有较高的校效，易于在于式填充时保证填充均匀。 装柱方法 干法、湿法（匀浆法）

●Detectors 理想的检测器应该具有灵敏度高，重复性好，响应快，线性范围宽，适用范围广，对流量和温度的变化不敏感等特性。但目 前还没有一种很理想的液体色谱检测器。 从目前商品仪器来看， 最广泛应用的是紫外光度检测器和差示折光检测器。

■Ultraviolet photometric detector Minimum detectable concentration is down to 10-9g.ml-1 Simple apparatus setup Insensitive to changes of temperature and flow rate, and useable for gradient elution

UV detector: Photoelectric cell Optically sensitive resistance Phototube Photomultiplier tube Photodiode

§3-7液相色谱分离类型的选择 （自学，通过自学初步学会如何根据样品的性质选择合适的色谱分离方法）

§3-8 液相色谱法应用举例 §3-9 高效毛细管电泳 Self-study