Preparative HPLC

1、Preparative HPLC (prepHPLC) has emerged as a mainstay of natural products isolation and purificationThe various modes available to date, eg , normal phase, reversedphase, size exclusion, and ion exchange,can be used to purify most classes of natural products This chapter presents an overview of the

2、differentmodes along with a practical guide as to how to purify a natural product using the most robust and widelyused of the modes, namely reversedphase prepHPLC Instrumentation setup and detection methods,sample preparation, method development, and sample work up？are also discussed半制备高效液相色谱仪（制备型HP

3、LC）已成为天然产物分离纯化的主流，现今可用的形式有很多。例如，正相，反相，排阻和离子交换法，可用于提纯大多数类的天然产物。本章内容有，各种色谱的概览和用于纯化大多数天然产物最有效的方法，即反相半制备HPL。本文也讨论了仪器设置和检测方法，方法建立,样品活化等问题。The term “preparative” in the context of separation or isolation of compounds from a complex matrix generally refers to a “largescale” process 从复杂物质中分离提纯化合物的方面来理解，术语“制

4、备型”,通常指的是“大规模”的过程。Thus, preparative high performance/pressure liquid chromatography (prepHPLC) usually implies large columns, large sample loading, and high flow rates in an HPLC system with the aim to purifying or isolating compounds in large quantities 因此，半制备高性能/高压液相色谱（制备型HPLC）通常意味着在HPLC系统中用大柱子，大样

5、本装载，和高流速，来纯化或分离的化合物。The use of prepHPLC has become a mainstay in the isolation of most classes of natural products over the last couple of decades 在过去几十年，利用半制备型高效液相色谱法已成为分离大多数类天然产物主要方法。The relative cost of prepHPLC systems has fallen due to increased competition, with the arrival of numerous column

6、and equipment manufacturers In addition, the constant innovation and new applications within the area of HPLC has meant that systems which were “state of the art” some 10 years ago are now within the reach of most research groups随着越来越多的柱的生产和设备制造商的出现，竞争的加剧，半制备HPLC系统的相对成本下降，。此外，在HPLC的区域内，不断的创新和应用使大多数课

7、题组都用上了大约十年前“最先进的”的色谱柱。Prep HPLC is a robust, versatile, and usually rapid technique by which compounds can be purified from complex mixtures 半制备型HPLC是一个强大的，多功能的，快速技术，此法可以从从复杂混合物纯化得到化合物。The main difference between prep HPLC and other “lower pressure” column chromatographic system is the consistency a

8、nd size of the particles in the stationary phase (see Chapter 7) 制备性HPLC与“低压”柱色谱系统之间的主要区别是一致性和固定相的颗粒大小（见第7章）。Particle size distribution is critical when trying to separate a mixture of two compounds as the smaller the particle size, the better the separating power (or resolution) between the two com

9、pounds 分离混合物中的两种化合物时，粒径越小，分离效果（或分辨率）越好。The “average” particle size of prep HPLC stationary phases is typically between 3 and 10 m, substantially smaller than other stationary phases 半制备型HPLC固定相的“平均”粒度通常在3和10米，比其它的柱固定相小得多。The particles are synthesized to be spherical and the size distribution to be n

10、arrow, which allows the stationary phase to be tightly packed in a highly uniform and reproducible manner 颗粒被制造成球形并且大小分布要窄，使得固定相被压紧时高度均匀而且可以重现。In addition, this minimizes the occurrence of voids or channels which would disrupt the mobile phase traveling uniformly through the stationary phase and lea

11、d to inefficient separation此外，这可以减少空隙或通道，空隙或通道会使流动相通过固定相不均匀并导致低效分离。 The small particle size results in having to use high pressures (up to 34,000 psi) to push the mobile phase through the system 由于颗粒尺寸变小，就必须加高压(高达 to 34,000 磅/平方英寸)才能推动流动相在柱中移动。However, the high surface area available for the solutes

12、 to interact with the stationary phase results in a chromatography with high powers of resolution that are necessary for purifying complex natural products mixtures 但是，高表面积可供洗脱剂与固定相充分反应，得到高分辨率。这对纯化复杂天然产物的混物来说是很重要的。Crude natural product extracts and mixtures may sometimes consist of hundreds of compo

13、unds, and the isolation of particular components presents their own unique problems生药提取物和混合物有时包含数百种化合物，特殊的成分分离有不同的难题。 Invariably a fast and efficient technique is required to purify the compounds of interest 用一种快速和有效的技术来纯化目标化合物一直刚需。However, the end requirement of the pure compounds is what drives th

14、e size and scale of the isolation technique used 然而，归根结底，分离出纯度好的化合物还是需要考虑分离用的材料的大小和多少。If microgram quantities of compound are needed for initial bioassay screening, then purification can sometimes be performed using analytical scale HPLC systems where the column internal diameter (id) is usually aro

15、und 46 mm 如果最初的生物鉴定需要筛选微克数量化合物时，有时用分析型HPLC系统中纯化的柱内直径（id）的46毫米。Greater quantities are usually needed for structure elucidation purposes (milligram quantities needed for NMR or X ray crystallography) or in vivo bioassays involving animals or humans (gram quantities), and a laboratory scale prep HPLC s

16、ystem is required to isolate the required amount 如果需要用作结构解析（用于核磁共振或X-射线晶体学毫克量）或者在涉及更大量的动物或人（克数量）体内生物检测时，那么就需要实验室规模的半制备型HPLC系统。Column internal diameters usually range from 10 to 100 mm 柱的内径范围通常从10至100毫米。If gram quantities are called for then typically pilot plant scale prepHPLC systems are needed (co

17、lumn id 100 mm) which has their own unique issues, though the theory behind the isolation process is essentially the same 如果分析量为克数，通常需要用中试规模prepHPLC系统（色谱柱内径 100毫米）虽然分离过程的理论是基本相同的，但其有独有的特征。The materials and methods used for isolating natural products by prep HPLC also depend upon the type of compound

18、 that is encountered in the extract, which in turn is dependent upon the extraction procedure 半制备型HPLC法分离的天然产物使用的材料和方法用于也取决于提取物中化合物的类型，反过来，提取出来的化合物是什么类型也取决于分离的过程A polar extract of a plant carried out using aqueous ethanol (EtOH) will differ substantially in compounds encountered than if the same pla

19、nt was extracted with n hexane 使用含水乙醇（EtOH）提取的植物化合物的极性与用正己烷萃取提取的到的化合物的极性不相同。Th erefore, polarity of the compound mixture is one of the major deciding factors as to which prep HPLC method is to be applied因此，该化合物的混合物的极性是决定用哪种半制备型HPLC方法是主要因素之一。This chapter concentrates on the practical aspects of perfo

20、rming a lab scale prep HPLC separation to purify natural products 本章集中进行实验室规模的制备HPLC分离纯化天然产物的实际问题。It covers the various modes of prep HPLC and selecting the right mode to achieve separation 它涵盖半制备型HPLC的各种类型，选择合适的模式，以实现分离。Instrumentation set up and detection methods, sample preparation, method develo

21、pment, and sample work up？ are also outlined 也提到了仪器设置和检测方法，样品制备，方法开发和上样。Discussion of chromatographic theory is kept to a minimum, and further information can be found in the excellent reviews listed under the Suggested Readings section at the end of this chapter。本章并没有过多讨论色谱理论，进一步的信息可以在本章结尾下推荐读物部分找到

22、。Prep-HPLC purification of natural products typically requires one of the following four chromatographic modes: normal phase, reversed-phase, gel permeation chromatography (GPC), and ion exchange chromatography The modes are determined by the stationary phase and of the preparative column being used

23、, and the solvents used for elution Deciding which mode to use depends on the compatibility of the extract or mixture with the different column modes Table 1 illustrates the different stationary phases available, and the separation modes they utilize The brand of stationary phase also plays a signif

24、icant role in the purification process Not all C 18silica gels, for example, are the same and a separation achieved using a Waters brand column may look completely different from a Merck brand This column selectivity, therefore, has to be taken into account when considering a separation strategy ( 1

25、 ) Normal phase chromatography uses a polar stationary phase (usually silica) and less polar (nonaqueous) eluting solvents Compounds are separated by adsorption onto the surface of the polar stationary phase as they are eluting down the column and the affinity they have to the eluting nonpolar solve

26、nt In general, the more polar the compound, the more likely it is to be adsorbed onto the stationary phase, and less polar compounds will be eluted first from the column Increasing the polarity of the eluting solvent reduces elution time Normal phase prep-HPLC is best suited to the separation and is

27、olation of lipophilic compounds, long-chain alkane derivatives or where the mixture of interest is sparingly soluble in aqueous conditions It is often successful in separating geometric and positional isomers though not quite successful in separating compounds differing only by alkyl groups In most

28、cases, normal phase prep-HPLC has been superseded by reversedphase prep-HPLC The eluants used in normal phase prepHPLC are usually mixtures of aliphatic hydrocarbons ( n -hexane and n -heptane), halogenated hydrocarbons (chloroform and dichloromethane), more polar oxygenated hydrocarbons (diethyl et

29、her, ethyl acetate, and acetone), or hydroxylated solvents such as isopropanol and methanol (see Note 1) Care must be taken to control the aqueous content of the solvents as water deactivates silica causing a breakdown in the separation This problem is seen particularly when using the hydroxylated s

30、olvents and they should be avoided or another separation mode used to maintain the robustness of the separation system In addition, the toxic and flammable nature of the solvents must be taken into account and the prep-HPLC system should be positioned in a fume cupboard and efforts made to make sure

31、 the system is “earthed” sufficiently to prevent the possibility of a spark being created by static electricity causing an explosion As the name indicates, this technique is the reverse on normal phase prep-HPLC whereby the stationary phase is more nonpolar than the eluting solvent Examples of rever

32、sed stationary phases are seen in Table 1 (including a non-silica-based reverse-phase prep-HPLC sorbent) Silica-based reversed-phase sorbents are also called “bonded-phase” materials whereby the silica particles are derivatized with alkylsilyl reagents The degree of silanization (or carbon loading)

33、can result in columns from different manufacturers having substantially differing chromatographic characteristics and in some cases different columns may be used for separating different mixtures ( 1 ) The cost of columns can make it prohibitive to have more than one or two different brands of prep-

34、HPLC column Therefore, a compromise may have to be struck between price and optimal separation The eluant used in reversed-phase prep-HPLC usually comprises a mixture of water and miscible organic solvents, usually acetonitrile (ACN), methanol (MeOH), or tetrahydrofuran (THF) In addition, buffers, a

35、cids, or bases may be added to suppress compound ionization or to control the degree of ionization of free unreacted silanol groups to reduce peak tailing and improve chromatography The issues of free silanol groups has been addressed in numerous other ways to improve chromatography, such as the use

36、 of inert non-silica supports so removing the silanol issue or using end-capping in an attempt to mop up the free silanol groups Each of these innovations adds to the cost of the column and in some ways may not be necessary for the particular compounds being examined Reversed-phase prep-HPLC lends i

37、tself well to the purification of most classes of natural product ( 2 ) Because of this, it is usually the first technique used when analyzing and attempting to purify compounds from a complex mixture especially when the identity of the compounds of interest is unknown GPC (also called size exclusio

38、n chromatography) is predominantly used for fractionating and purifying proteins and oligosaccharides but has been used in some cases for separating lower molecular weight molecules (see Chapter 7) The stationary phase is typically made of rigid spherical particles of macroporous polystyrene/divinyl

39、benzene copolymers The stationary phase is inherently hydrophobic (similar to reverse-phase packing materials) and is essentially chemically and physically inert The pore size in the particles is strictly controlled Compounds are separated by their ability to enter the pores; the smaller molecules a

40、re “trapped” temporarily in the pores, while larger molecules are not held up and pass through the column relatively unhindered The extent of retardation of the molecules is a function of their molecular size, and as such this type of chromatography has found a use in purifying biomolecules While na

41、tural product mixtures invariably contain many compounds of similar molecular weights, GPC has become a useful adjunct to the other modes of prep-HPLC separation of natural products where some prior knowledge of the molecular weight of the various components may be known Ion exchange chromatography

42、(see Chapter 8) uses an anionic or cationic stationary phase for the separation of acids and amines Compounds with a net charge bind reversibly to the ionisable groups on the stationary phase and are eluted through displacement of a stronger ionized species in the eluent The support in the stationar

43、y phase may be of a silica or styryl-divinylbenzene origin Again, the use of ion exchange columns assumes that there is some prior knowledge of the chemical content of the sample mixture and as such is not used as a first-line separation method Solvents used in prep-HPLC (Table 2 ) typically have to

44、 be a high purity to maintain the integrity of the system and sample, b compatible with the detector and not interfere with the observation of your target compounds, ie, “transparent,” c compatible with the sample (solubility and nonreactive), d low viscosity to keep system back pressure low, and e

45、reasonably priced (a typical prep-HPLC run may use a liter or more of solvent each time) Furthermore, the solvents need to be “degassed” to remove dissolved oxygen, which comes out of solution to form microscopic bubbles under the high pressures seen in the system These bubbles interfere with the de

46、tector causing sharp spikes to be seen There are numerous ways to degas solvents including applying a vacuum to the solvent or placing the container of solvent into an ultrasonic bath before use Most prep-HPLCs, however, come fitted with inline degassers and helium “sparge” systems that purge the so

47、lvents with helium gas, initially and periodically, during the use of the instrument and maintain the solvents in a degassed state As the ionic state of the compounds and the stationary phases are critical for producing efficient and reproducible chromatography, the pH of the eluting solvents must b

48、e controlled Unionized form of compounds is more likely to interact with the stationary phase in reversed-phase prep-HPLC Buffers have been used extensively in reversed-phase prep-HPLC to do this, and a number of buffers are listed in Table 3 Care must be taken with the use of buffers to ensure that

49、 they do not precipitate out in the presence of organic solvents and that the salts are removed from the final purified product (see Note 2) In addition, one must be careful not to use excessive amount of base as most silica-based columns are unable to operate at pHs greater than 8 The use of buffer

50、s can be bypassed to some extent by the use of straight acids or bases This is particularly useful when the compounds are unknown and the use of a small amount of acid or base during the method development phase can help greatly in achieving good chromatography Ion suppression of carboxylic acids in

51、 samples can be brought about by the addition of either mineral or organic acids to the mobile phase (Table 4 ) Peak tailing, caused by free silanol groups, can lead to poor chromatography and may be overcome to some extent by adding triethylamine (00501% v/v) to the mobile phase Care should be take

52、n to ensure the acid and/or base is removed quickly after purification takes place to avoid compound breakdown or unwanted reactions taking place Prep-HPLC systems are made up of a number of components as shown in Fig 1 It should be noted that “method development” is usually carried out on analytica

53、l HPLC systems, which require much less solvent and sample Once a suitable solvent system and method has been established, this is then scaled-up to the prep-HPLC system The level of sophistication depends on the age and the cost of the system, but they are made up of a number of essentials as follo

54、ws System controller : It is a computer (PC) that which controls the whole prep-HPLC set up and operations, eg , pump(s), flow rate, solvent composition in binary, ternary and quaternary systems in both isocratic and gradient modes, fraction collections, detection parameters, and data presentation P

55、umps : They are designed to pump solvent at high pressure with minimal pulsing Flow rates may vary from 5 to 100 mL/min dependant on the size of the pump heads and the system Degaser : This component removes any air bubbles from the mobile phase through ultra sonication When the flow rate is 10 mL/m

56、in, instead of an online degaser, a suitable pursing system may be used Autosampler : It comprises injection loop, syringe, and a sample carousel The sample is dissolved and added to sample vials, and injected as a solution in (or as close to) the mobile phase starting conditions Loop capacity tends

57、 to range from 1 to 30 mL in semi-prep and prep-HPLC systems The sample should be dissolved in the smallest volume of solvent possible and therefore the loop should be changed to match this volume Injecting a 1 mL sample into a 20 mL loop will lead to the sample “diluting” in the loop and cause band

58、 broadening and poor peak shape and chromatography Guard column : While for analytical HPLC, a guard column is often used with the main column to protect the column from particulate matter that may be in the sample, in most of the larger prep-HPLC columns, a guard column may not be used because of excessive back pressure The use of a guard may be negated by pref