Liquid Chromatography 2 New Technology液相色谱2新技术
高效液相色谱的原理
高效液相色谱的原理高效液相色谱（High Performance Liquid Chromatography，HPLC）是一种基于分子间相互作用力进行化合物分离和分析的方法。
它主要由四个部分组成：流动相，固定相，色谱柱和检测器。
其原理如下：1. 流动相：液相在常温下以高压泵的作用下通过色谱柱，它可以是有机溶剂、水或其他特定的溶剂组合。
流动相在整个过程中起到带动样品运动以及分离化合物的作用。
2. 固定相：为了实现分离，需要使用一种高表面积的固相材料将样品担持在流动相中进行分离。
固定相通常以粉末或颗粒的形式填充在色谱柱中，常见的固定相材料有硅胶、高性能液相色谱柱（如C18）等。
固定相的选择取决于目标分析化合物的特性。
3. 色谱柱：色谱柱是将固定相填充在其中的管状包层，它是高效液相色谱分离的关键部分。
色谱柱的长度、内径和填充粒径等参数会对分离效果产生影响。
较长、较细的柱内填充材料可以提高分离效率，但也会增加分析时间。
4. 检测器：在色谱柱出口处使用检测器来检测化合物的浓度。
常用的检测器包括紫外-可见吸收检测器（UV-Vis）、荧光检测器、电化学检测器等。
检测器将检测到的信号转化为可见的色谱图谱，用以分析和定量目标化合物。
在高效液相色谱分离过程中，样品溶液被注入到进样器中，经由高压泵送入色谱柱。
在色谱柱中，化合物会与固定相发生不同程度的相互作用，并在流动相的作用下逐渐分离。
分离出的化合物会依次出现在检测器中，通过检测器的信号输出，我们可以获得色谱图，并通过峰面积或峰高等参数对化合物进行定量和定性分析。
高效液相色谱的优点包括分离效率高、分析速度快、样品制备简单等，因此被广泛应用于生物医药、农药残留、环境监测等领域的化学分析。
液相色谱 基质效应
液相色谱（Liquid Chromatography，简称LC）是一种常用的分析技术，用于分
离和检测复杂混合物中的成分。
在液相色谱中，样品溶解在流动相（液体）中，通过与固定相（通常是固定在柱子内部的材料）相互作用，使不同成分在流动相中以不同速率移动，从而实现分离。
基质效应是指在液相色谱分析中，样品中的一些组分与流动相或固定相相互作用，导致它们在柱子中的保留时间发生变化，从而影响了它们的分离和检测。
基质效应可能会导致以下问题：
1. 保留时间变化：某些样品组分可能与流动相或固定相之间发生相互作用，使得它们在柱子中的保留时间增加或减少，进而影响分离结果。
2. 峰形变化：基质效应可能导致峰形发生变化，可能出现尾峰或峰形不对称等现象。
3. 峰分离不完全：某些样品组分的基质效应可能导致它们与其他组分的分离不完全，从而影响分析结果的准确性和灵敏度。
4. 背景干扰：基质效应还可能导致背景信号的增加，使得检测到的目标组分信号被干扰，从而影响定量分析。
为了解决基质效应带来的问题，可以采取以下措施：
1. 优化流动相组成：调整流动相的成分，优化其pH值、离子强度等参数，以减少基质效应的影响。
2. 选择适当的固定相：根据样品的特性选择合适的液相色谱柱，固定相的化学性质对基质效应影响较大。
3. 使用前处理方法：对样品进行适当的前处理，如固相萃取、蒸发浓缩等，可以减少基质效应的影响。
4. 校正和内标法：在定量分析中，可以采用标准品校正或内标法来消除基质效应对结果的影响。
总之，液相色谱分析中的基质效应是需要注意和解决的问题，通过合理的方法和条件设置，可以提高液相色谱的分离效果和分析准确性。
Liquid Chromatography –Mass Spectrometry 02
Chapter 2Liquid ChromatographyLearning Objectives•To understand those aspects of high performance liquid chromatography which are essential to the application of LC–MS.2.1IntroductionThe International Union of Pure and Applied Chemistry (IUPAC)defines chro-matography as follows [1]:‘Chromatography is a physical method of separation in which the components to be separated are distributed between two phases,one of which is stationary (the stationary phase),while the other (the mobile phase)moves in a definite direction.A mobile phase is described as “a fluid which percolates through or along the stationary bed in a definite direction”.It may be a liquid,a gas or a supercritical fluid,while the stationary phase may be a solid,a gel or a liquid.If a liquid,it may be distributed on a solid,which may or may not contribute to the separation process.’A chromatographic system may be considered to consist of four component parts,as follows:•a device for sample introduction•a mobile phase•a stationary phase•a detectorLiquid Chromatography –Mass Spectrometry:An Introduction.Robert E.ArdreyCopyright ¶2003John Wiley &Sons,Ltd.ISBNs:0-471-49799-1(HB);0-471-49801-7(PB)8Liquid Chromatography–Mass Spectrometry A number of different chromatographic techniques are in use and these differ in the form of these four components and their relative importance.For example, in gas chromatography the injector used for sample introduction is of paramount importance and must be chosen in light of the properties of the analytes under investigation(their stability and volatility)and the amount of the analytes present. An incorrect choice could prevent a successful analysis.In high performance liq-uid chromatography(HPLC)the injector is simply required to allow introduction of the analytes into aflowing liquid stream without introducing any discrimination effects and a single type,the loop injector,is used almost exclusively.The two components which are associated with the separation that occurs in a chromatographic system are the mobile and stationary phases.In HPLC,the mobile phase is a liquid delivered under high pressure(up to400bar(4×107Pa))to ensure a constantflow rate,and thus reproducible chromatography,while the stationary phase is packed into a column capable of withstanding the high pressures which are necessary.A chromatographic separation occurs if the components of a mixture interact to different extents with the mobile and/or stationary phases and therefore take different times to move from the position of sample introduction to the position at which they are detected.There are two extremes,as follows:(i)All analytes have total affinity for the mobile phase and do not interact withthe stationary phase–all analytes move at the same rate as the mobile phase, they reach the detector very quickly and are not separated.(ii)All analytes have total affinity for the stationary phase and do not interact with the mobile phase–all analytes are retained on the column and do not reach the detector.The role of the chromatographer is therefore,based on a knowledge of the analytes under investigation,to manipulate the properties of the stationary and/or mobile phases to move from these extremes and effect the desired separation.A number of detectors may be used in conjunction with HPLC(see Section2.2.5below),with the type chosen being determined by the type of analysis,i.e. qualitative or quantitative,being undertaken.The requirements for each of these are often quite different,as described in the following:•Qualitative(identification)applications depend upon the comparison of the retention characteristics of the unknown with those of reference materials.In the case of gas chromatography,this characteristic is known as the retention index and,although collections of data on‘popular’stationary phases exist,it is unlikely that any compound has a unique retention index and unequivocal identification can be effected.In liquid chromatography,the situation is more complex because there is a much larger number of combinations of stationary and mobile phases in use,and large collections of retention characteristics on any single‘system’do not exist.In addition,HPLC is a less efficient separationLiquid Chromatography9 technique than GC and this results in wider‘peaks’and more imprecision in retention time measurements,and thus identification.•Quantitative accuracy and precision(see Section2.5below)often depend upon the selectivity of the detector because of the presence of background and/or co-eluted materials.The most widely used detector for HPLC,the UV detector,does not have such selectivity as it normally gives rise to relatively broad signals,and if more than one component is present,these overlap and deconvolution is difficult.The related technique offluorescence has more selec-tivity,since both absorption and emission wavelengths are utilized,but is only applicable to a limited number of analytes,even when derivatization procedures are used.DQ2.1What is meant by the‘selectivity’of a detector?Define the‘limit ofdetection’of a detector.AnswerThe selectivity of a detector is its ability to determine an analyte of interestwithout interference from other materials present in the analytical system,i.e.the sample matrix,solvents used,etc.The limit of detection is the smallest amount of an analyte that isrequired for reliable determination,identification or quantitation.Moremathematically,it may be defined as that amount of analyte which pro-duces a signal greater than the standard deviation of the backgroundnoise by a defined factor.Strictly for quantitative purposes,this shouldbe referred to as the‘limit of determination’.The factor used dependsupon the task being carried out and for quantitative purposes a highervalue is used than for identification.Typical values are3for identificationand5or10for quantitation.The selectivity of a detector is often related to its limit of detection,i.e.the more selective it is,then the lower the background noise is likely tobe,and consequently the lower the limit of detection.The term‘sensitivity’is often used in place of the‘limit of detection’.The sensitivity actually refers to the degree of response obtained from adetector,i.e.the increase in output signal obtained from an increasingamount or concentration of analyte reaching the detector.Care musttherefore be taken when these terms are being used or when they areencountered to ensure that their meanings are unambiguous.The terms defined above are all important in the consideration ofthe overall performance of an analytical method.The greatest‘sensi-tivity’(response)does not necessarily imply the lowest‘limit of detec-tion/determination’as a more intense signal may also be observed from10Liquid Chromatography–Mass Spectrometry any interferences present.An inherently less sensitive but more selectivedetector may provide a‘better’analysis with lower‘limits of detec-tion/determination’.The performance of a detector is therefore intimately linked to thesamples being analysed.Mass spectrometry(see Chapter3)is capable of providing molecular weight and structural information from picogram amounts of material and to provide selectivity by allowing the monitoring of ions or ion decompositions character-istic of a single analyte of interest.These are the ideal characteristics of both a qualitative and a quantitative detector.2.2High Performance Liquid ChromatographyThere are a number of specialist texts in which high performance liquid chro-matography(HPLC)is described in varying amounts of detail(Lindsay[2]; Robards et al.[3];Meyer[4]).It is not,therefore,the intention of this author to provide a comprehensive description of the technique but merely to discuss those aspects which are essential to the successful application of the LC–MS combination.A block diagram of an HPLC system,illustrating its major components,is shown in Figure2.1.These components are discussed in detail below.2.2.1PumpThe pump must provide stableflow rates from between10µl min−1and 2ml min−1with the LC–MS requirement dependent upon the interface being used and the diameter of the HPLC column.For example,the electrospray interface,when used with a microbore HPLC column,operates at the bottom end of this range,while with a conventional4.6mm column such an interface usually operates towards the top end of the range,as does the atmospheric-pressure chemical ionization(APCI)interface.Theflow rate requirements of the different interfaces are discussed in the appropriate section of Chapter4.Pump Injector Column Detector Mobilephasereservoir(s)Figure2.1Block diagram of a typical HPLC system.Liquid Chromatography11 A number of different types of pump are available and these are described else-where[2,3],but the most popular pump used today is the reciprocating pump. From a mass spectrometry perspective,the pump must be pulse free,i.e.it must deliver the mobile phase at a constantflow rate.Pulsing of theflow causes the total ion current(TIC)trace(see Chapter3)–the primary piece of information used for spectral analysis–to show increases in signal intensity when analytes are not being eluted and this makes interpretation more difficult.2.2.2Sample Introduction(Injector)In contrast to gas chromatography,in which a number of different types of injector are available and the selection of which is often crucial to the success (or otherwise)of the analysis,a single type of injector is used almost exclusively in HPLC.The loop injector(sometimes known as the valve injector)is,as mentioned previously,merely a convenient way of introducing a liquid sample into aflowing liquid stream and consists of a loop of a nominal volume into which sample is introduced by using a conventional syringe.While the loop is beingfilled,mobile phase is pumped,at the desiredflow rate,through the valve to the column to keep the column in equilibrium with the mobile phase and maintain chromatographic performance.When‘injection’is required,a rotating switch is moved and the flow is diverted through the loop,thusflushing its contents onto the top of the column.From a quantitative perspective,the way in which the injector functions is crucial to the precision and accuracy which may be obtained and therefore these two parameters are of paramount importance.Quantitative precision will be dependent upon,among other things,the extent to which the loop may befilled repeatably.It is usual tofill the loop completely by having a greater volume in the conventional syringe than the loop capacity (excess goes to waste)and it is important to ensure,as much as is possible,that air bubbles are not introduced in place of the sample.To obtain the best precision and accuracy during quantitative measurements an internal standard should be used(this will be discussed further in Section2.5below),and if insufficient sample is available to allow completefilling of the loop,i.e.it is only partially filled,an internal standard must be used if meaningful quantitative results are to be obtained.Loops are not calibrated accurately and a loop of nominally20µl is unlikely to have this exact volume.This will not affect either the precision of measurement and,as long as the same loop is used for obtaining the quantitative calibration and for determining the‘unknowns’,the accuracy of measurement.From a mass spectrometry perspective,the injector is of little concern other than the fact that any bubbles introduced into the injector may interrupt the liquid flow,so resulting in an unstable response from the mass spectrometer.12Liquid Chromatography–Mass Spectrometry 2.2.3Mobile PhaseUnlike gas chromatography,in which the mobile phase,i.e.the carrier gas,plays no part in the separation mechanism,in HPLC it is the relative interaction of an analyte with both the mobile and stationary phases that determines its retention characteristics.Hence,it is the varying degrees of interaction of different analytes with the mobile and stationary phases which determines whether or not they will be separated by a particular HPLC system.A number of different retention mechanisms operate in HPLC and interested readers mayfind further details elsewhere[2–4].It is sufficient to say here that the interaction may be considered in terms of the relative polarities of the species involved.As indicated in Section2.1above,there are two extremes of interaction, neither of which is desirable if separation is to be achieved.HPLC requires a mobile phase in which the analytes are soluble.The majority of HPLC separations which are carried out utilize reversed-phase chromatog-raphy,i.e.the mobile phase is more polar then the stationary phase.In these systems,the more polar analytes elute more rapidly than the less polar ones.It is not always possible to achieve an adequate separation by using a mobile phase containing a single solvent and often mixtures of solvents are used.A wide range of mobile phases are therefore available and yet,despite this,a particular problem exists when the mixture under investigation contains analytes of widely differing polarities.A mobile phase that gives adequate separation of highly polar analytes will lead to excessively long retention times for non-polar analytes, and vice versa.Under these circumstances,separation is often achieved only by varying the composition of the mobile phase in a controlled way,during the analysis.A separation involving a mobile phase of constant composition(irrespective of the number of components it contains)is termed isocratic elution,while that in which the composition of the mobile phase is changed is termed gradient elution. In the latter,a mobile phase is chosen which provides adequate separation of the early eluting analytes and a solvent which is known to elute the longer-retained compounds is added over a period of time.The rate at which the composition is changed may be determined by‘trial and error’,or more formal optimization techniques may be used[5–7].Buffers are used in HPLC to control the degree of ionization of the analyte and thus the tailing of responses and the reproducibility of retention.A range of buffers is available but those most widely used are inorganic,and thus involatile, materials,such as potassium or sodium phosphate.One of the functions of an LC–MS interface is to remove the mobile phase and this results in buffer molecules being deposited in the interface and/or the source of the mass spectrometer with a consequent reduction in detector performance. Methods involving the use of volatile buffers,such as ammonium acetate,are therefore preferred.Liquid Chromatography 13The effect of the mobile-phase composition on the operation of the different interfaces is an important consideration which will be discussed in the appropriate chapter of this book but mobile-phase parameters which affect the operation of the interface include its boiling point,surface tension and conductivity.The importance of degassing solvents to prevent the formation of bubbles within the LC–MS interface must be stressed.Some LC–MS interfaces have been designed such that mobile phase is not pumped directly into the source of the mass spectrometer,thus minimizing con-tamination and increasing the time over which the interface operates at optimum performance.One such is the ‘Z-spray’interface from Micromass,with a com-parison of the spray trajectories of an in-line and a Z-spray interface being shown in Figure 2.2.In the Z-spray interface,the HPLC mobile phase is sprayed across (orthogonal to)a sampling cone to which is applied a voltage that attracts appro-priately charged ions with a velocity which causes them to pass through this cone into the mass spectrometer.Solvent and buffer molecules pass by this arrangement and are pumped directly to waste,thus reducing contamination and prolonging the performance of the system.The effect of the quality of the mobile phase on the operation of the detector being employed is of importance whatever that detector may be.The mobile phase is pumped through the column at a flow rate of,typically,1ml min −1.If we assume an impurity is present at a level of 0.000001%,this is equivalent to such a compound being continually introduced into the mass spectrometer at a rate of ca.1ng s −1.LC eluateIon-beam to(a)Cone-shaped Ion-beam to(b)Figure 2.2Schematics of (a)in-line and (b)Z-spray electrospray interfaces.From appli-cations literature published by Micromass UK Ltd,Manchester,UK,and reproduced with permission.14Liquid Chromatography–Mass Spectrometry A full-scan mass spectrum can easily be obtained from this amount of material and it should be clear,therefore,that even high-purity(and usually expensive!) solvents can give rise to a significant mass spectral background,hence rendering the interpretation of both qualitative and quantitative data difficult. Fortunately,this background is often less of a problem than might be antici-pated from the above.The majority of ionization techniques employed in LC–MS are‘soft’ionization techniques which provide primarily molecular ions that occur at relatively high values of mass-to-charge ratio(m/z),rather than fragment ions which occur at relatively low m/z values.In the majority of cases,the molecular weight of the analyte is higher than those of the solvent impurities and the effect of these may therefore be minimized.The primary piece of LC–MS data considered by the analyst is the total-ion-current(TIC)trace which shows the sum of the intensity of each of the ions observed in each of the mass spectra that have been acquired during the chromatographic separation.As with other detectors,a‘peak’signifies the elution of a component from the column followed by its ionization.If solvent impurities are continually being ionized,a high background TIC is observed and the elution of an analyte may cause a minimal increase in this,i.e.‘peaks’may not be readily apparent.This situation may be improved by either(a)modifying the scan range of the mass spectrometer to exclude the ions from the background,e.g.scan only from m/z150upwards,or(b)acquiring data over the complete m/z range but then use computer manipulation of these data to construct a TIC trace from only those ions that do not arise from the background.The advantage of the latter approach is that all data are stored during acquisition and if any ions of analytical significance are subsequently found below m/z150, they may be examined.If the mass spectrometer has only been scanned above m/z150,then this is not possible.This methodology will be discussed further in Chapter3but is illustrated here in Figure2.3.In this,Figure2.3(a)shows the TIC trace from an LC–MS analysis in which data over the m/z range from35to400have been acquired.A number of responses may be observed but the trace is dominated by a constant background amounting to around70%of the maximum TIC value.Figure2.3(b) shows the TIC from the same analysis,constructed by using the intensity of ions with m/z only in the range of200to400.In this case,the constant background amounts to less than5%of the maximum of the TIC value and the presence of components may be much more readily observed.2.2.4Stationary PhaseAs has previously been stated,the majority of HPLC analyses which are carried out employ reversed-phase systems.The most widely used columns contain a chemically modified silica stationary phase,with the chemical modification determining the polarity of the column.ALiquid Chromatography 15100908070605040302010R e l a t i v e i n t e n s i t y (%)19.9620.0420.51100908070605040302010R e l a t i v e i n t e n s i t y (%)19.9520.0420.5019202122Time (min)2324(a)(b)Figure 2.3TIC traces,having been brought about by using ions in the m/z ranges (a)35to 400,and (b)200to 400,showing the improvement in signal-to-noise ratio obtained by excluding background ions.very popular stationary phase is one in which a C 18alkyl group is bonded to the silica surface.In contrast to GC,in which,particularly at high temperatures,the station-ary phase may give rise to a continuous background at the detector,this is not normally observed in HPLC unless the pH of the mobile phase is such that degradation of the stationary phase occurs.Under these circumstances,both an increased background and a reduction in chromatographic performance may be observed.16Liquid Chromatography–Mass Spectrometry2.2.5DetectorsThe choice of detector is often crucial to the success of a particular HPLC method.A number are in routine use,including the UV,fluorescence,electrochemical, conductivity and refractive index detectors,and each has particular advantages and disadvantages,details of which can be found elsewhere[2–4].A more general discussion of their attributes will,hopefully,provide an insight into some of the ways in which the mass spectrometer may be used to advantage as a detector.Detectors may be classified in a number of ways,including their use as the following:•solute-or solvent-property detectors•selective or general(universal)detectors•mass-or concentration-sensitive detectors2.2.5.1Solute-or Solvent-Property DetectorsThis classification is concerned with whether the detector monitors a property of the solute(analyte),e.g.the UV detector,or a change in some property of the solvent(mobile phase)caused by the presence of an analyte,e.g.the refractive index detector.2.2.5.2Selective or General DetectorsThis classification is concerned with whether the detector responds to a specific feature of the analyte of interest or whether it will respond to a large number of analytes,irrespective of their structural properties.In terms of the previous classification,it may be considered that solute detectors are also usually selective detectors,while solvent detectors are general detectors.The most widely used HPLC detector methodology is,arguably,UV absorp-tion,and this has capabilities as both a specific or general detector,depending upon the way it is used.If the wavelength of maximum absorption of the analyte(λmax)is known,it can be monitored and the detector may be considered to be selective for that analyte(s).Since UV absorptions are,however,generally broad,this form of detection is rarely sufficiently selective.If a diode-array instrument is available, more than one wavelength may be monitored and the ratio of absorbances mea-sured.Agreement of the ratio measured from the‘unknown’with that measured in a reference sample provides greater confidence that the analyte of interest is being measured,although it still does not provide absolute certainty.Many organic molecules absorb UV radiation,to some extent,at254nm and if this wavelength is used it may be considered to be a general detection system. It must be remembered,however,that not all compounds absorb UV radiation. In these circumstances,the use of indirect UV detection,in which a UV-activecompound is added to the mobile phase,may be employed.This gives a constant (hopefully)background signal which is reduced when a compound that does not absorb UV radiation elutes from the HPLC column.Care must be taken if the mass spectrometer is used in series with indirect UV detection that the UV-active compound added to the mobile phase does not produce an unacceptably high background signal which hinders interpretation of either the TIC trace or the resulting mass spectra.A widely used general detector is the refractive index detector which monitors changes in the refractive index of the mobile phase as an analyte elutes from the column.If gradient elution is being used,the refractive index of the mobile phase also changes as its composition changes,thus giving a continually varying detector baseline.The determination of both the position and intensity of a low-intensity analytical signal on a varying baseline is less precise and less accurate than the same measurement on a constant baseline with zero background signal. It is usually recognized that general detectors are less sensitive than specific detectors,have a lower dynamic range(see below)and do not give the best results when gradient elution is used.Like the UV detector,the mass spectrometer may be employed as either a gen-eral detector,when full-scan mass spectra are acquired,or as a specific detector, when selected-ion monitoring(see Section3.5.2.1)or tandem mass spectrometry (MS–MS)(see Section3.4.2)are being used.2.2.5.3Mass-or Concentration-Sensitive DetectorsThefinal classification concerns whether the intensity of detector response is proportional to the concentration of the solute or the absolute amount of solute reaching it.This classification is particularly important for quantitative applica-tions.If the mobile phaseflow rate is increased,the concentration of analyte reaching the detector remains the same,but the amount of analyte increases. Under these circumstances,the signal intensity from a concentration-sensitive detector will remain constant,although the peak width will decrease,i.e.the area of the response will decrease.A change inflow rate will also reduce the width of the response from a mass-sensitive detector,while,in contrast to a concentration-sensitive detector,the signal intensity will increase as the absolute amount of analyte reaching the detector has increased.Since the overall response increases,this may be used to improve the quality of the signal obtained. Under many experimental conditions,the mass spectrometer functions as a mass-sensitive detector,while in others,with LC–MS using electrospray ioniza-tion being a good example,it can behave as a concentration-sensitive detector. The reasons for this behaviour are beyond the scope of this present book(inter-ested readers should consult the text by Cole[8])but reinforce the need to ensure that adequate calibration and standardization procedures are incorporated into any quantitative methodology to ensure the validity of any results obtained.An advantage of the mass spectrometer as a detector is that it may allow differentiation of compounds with similar retention characteristics or may allow the identification and/or quantitative determination of components that are only partially resolved chromatographically,or even those that are totally unresolved. This may reduce the time required for method development and is discussed in more detail in Chapter3.SAQ2.1You require to develop an HPLC method for the determination of a high-molecular-weight aliphatic alcohol that has no UV absorption.Unfortunately,you only havea UV detector available.How would you attempt the analysis?2.3Chromatographic PropertiesIn carrying out a chromatographic separation,an analyst is concerned with whether the components of a mixture can be separated sufficiently for the ana-lytes of interest,and this is not always all of them,to be identified and/or for the amounts present to be determined.Our ability to carry out these tasks suc-cessfully will depend upon the‘performance’of the chromatographic system as a whole.The performance may be described in terms of a number of theoretical param-eters,although the‘performance’required for a particular analysis will depend upon the separation that is required.This,in turn,depends upon the similarity in the behaviour in the chromatographic system of the analyte(s)of interest to each other and to other compounds present in the mixture.The time taken for an analyte to elute from a chromatographic column with a particular mobile phase is termed its retention time,t an.Since this will vary with column length and mobile phaseflow rate,it is more useful to use the capacity factor,k .This relates the retention time of an analyte to the time taken by an unretained compound,i.e.one which passes through the column without interacting with the stationary phase,to elute from the column under identical conditions(t0).This is represented mathematically by the following equation:k =t an−t0t0(2.1)To give adequate resolution in a reasonable analysis time,k values of between 1and10are desirable.The separation of two components,e.g.A and B,is termed the selectivity or separation factor(α)and is the ratio of their capacity factors(by convention, t B>t A andα≥1),as shown by the following equation:α=k Bk A =t B−t0t A−t0(2.2)。
液相色谱教程液相色谱方法开发
液相色谱教程液相色谱方法开发液相色谱(Liquid Chromatography，简称LC)是一种常用的分离和分析技术，广泛应用于化学、生物、医药等领域。
液相色谱方法的开发是为了解决特定问题和满足特定需求而进行的，本文将介绍液相色谱方法开发的一般步骤和注意事项。
液相色谱方法的开发步骤如下：1.确定分离目标：首先确定需要分离和分析的目标化合物，包括确定化合物的物理化学性质和分离特性等。
2.选择色谱柱：根据分离目标，选择合适的色谱柱。
色谱柱的选择应考虑样品的性质、分离机理、应用要求等因素。
3.选择流动相和梯度条件：根据分离目标，选择合适的流动相（包括溶剂和缓冲剂等）和梯度条件（包括流动相的组成和梯度程序等）。
4.优化色谱条件：通过改变流动相组成、流速、柱温等参数，优化色谱条件，达到最佳分离效果。
5.建立分析方法：根据样品的特点和分析需求，建立分析方法。
包括确定检测器的波长或离子选择器、设置进样量和检测浓度范围等。
6.方法验证：对开发的液相色谱方法进行验证，包括准确度、精密度、线性范围、检出限等指标的确定。
液相色谱方法开发过程中需要注意的事项如下：1.样品制备：样品的制备要充分考虑到样品的性质和分析方法的要求，如需要进行前处理、提取、洗脱、浓缩等。
2.色谱柱保养：液相色谱柱的保养对于保证色谱方法的重复性和稳定性至关重要。
包括定期清洁、适当的保存和使用。
3.流动相准备：流动相的配制要严格按照要求，注意流向的调整、PHA值的调节、气泡和杂质的排除等。
4.柱温控制：柱温对色谱分离的效果有很大影响，需要根据分析需求对柱温进行控制和调节。
5.检测器选择：根据分析的目标和样品的特性，选择合适的检测器，如紫外检测器、荧光检测器、质谱检测器等。
6.数据处理：对色谱结果进行正确的数据处理和解释，包括峰面积计算、峰识别和归一化等。
总结来说，液相色谱方法的开发是一个系统的工程，需要综合考虑样品特性、分析需求和分离机理等因素。
快速液相色谱
快速液相色谱快速液相色谱一、什么是快速液相色谱快速液相色谱（Fast Liquid Chromatography，简称FLC）是一种高效、高速的色谱技术，广泛用于化学、生物、医药、环境监测等领域。
它是以液相为介质，通过样品在固定相上的分离过程实现物质的定性、定量分析。
与传统液相色谱相比，FLC具有更高的分离效率、更快的分析速度和更低的溶剂消耗。
二、快速液相色谱的原理快速液相色谱的原理基于固定相与流动相之间的互作用。
在FLC中，固定相是一个非极性或多孔性的材料，样品溶液在固定相上通过扩散、吸附、在凝胶内扩散等过程被分离。
与传统液相色谱相比，FLC采用了更细小的颗粒固定相材料，提高了表面积和质量传递速率，从而实现更高的分离效率和更快的分析速度。
三、快速液相色谱的应用快速液相色谱在许多领域有广泛的应用。
在化学领域，它可以用于有机物的分离和纯化；在生物学领域，它可以用于生物样品中目标物的检测和定量分析；在医药领域，它可以用于药物的质量控制和新药的开发等。
此外，快速液相色谱还可以应用于环境监测、食品安全、化妆品检测等领域。
四、快速液相色谱的优势快速液相色谱相比传统液相色谱有多个显著的优势。
首先，FLC具有更高的分离效率，能够在更短的时间内完成复杂样品的分离。
其次，FLC具有更快的分析速度，可以提高实验室的工作效率和样品处理能力。
同时，FLC还可以减少溶剂消耗，降低实验成本和环境污染。
五、快速液相色谱的发展趋势随着科学技术的不断发展，快速液相色谱也在不断创新与改进。
未来的快速液相色谱将更加强调对分离效率、分析速度和样本量的要求。
同时，新型的固定相材料和分离机制的研发也将推动FLC的进一步发展。
此外，快速液相色谱将与其他分析技术（如质谱联用、毛细管电泳等）的结合应用更加广泛，提高分析的综合能力和可靠性。
六、总结快速液相色谱是一种高效、高速的色谱技术，被广泛应用于化学、生物学、医药学等领域。
它基于固定相与流动相之间的互作用，通过分离过程实现样品的定性、定量分析。
高效液相色谱技术的研究进展
高效液相色谱技术的研究进展高效液相色谱技术（High performance liquid chromatography, HPLC）是一种现代化的、高效的分离技术。
它利用分离样品中的化学成分的物理或化学属性，通过在流动相和固定相之间相互传递的过程中实现化学成分的分离。
近年来，高效液相色谱技术不断在技术细节、数据分析、纯化和检测灵敏度等方面得到了进一步的发展。
本文将从以下四个方面探讨高效液相色谱技术的研究进展：一、液相色谱柱的发展液相色谱柱是HPLC技术的核心部分，HPLC的分离效果和方法的可靠性很大程度上取决于色谱柱的品质。
因为使样品在流动相和固定相之间相互传递所需的时间取决于柱内的分离效果。
近年来，新技术和新材料的涌现使得液相色谱柱质量得到了显著的提高。
例如，阴离子交换柱有了更好的抗污染性，表面经处理的柱材料也能够更好地避免有机污染物的吸附。
二、柱外引道注射技术柱外引道注射技术是提高色谱分析速度、提高灵敏度以及降低流动相损耗的最重要的技术之一。
此技术是基于待分析物质的性质选择可以产生极高的浓度梯度的引道。
现在，多种柱外引道注射技术已被广泛的使用，如微量分析技术（MEMS）和尖峰式带型变形的色谱方法（systmic-sieve effect chromatograph），这两个技术都在注射控制的同步性方面做出了大量的工作。
最近，由微型气泡引导的无毒注射技术也被用于蛋白和DNA的定性分析。
三、离线（离线联机）联用技术联用有助于更有效、安全、高分辨率的分析。
离线联用就是离线上分离了化学组分，然后用在线方法来定性或定量分析化合物（当需要在线定量分析液相中的某些组分时则是在线联用）。
在离线联用的模型中，分离过的化合物必须被固定在收集器中，只有当样品收集完成时才可重新溶解。
虽然离线联用总体上是一种昂贵的技术，但是它在处理复杂的样品时可极大地提高精度，它还可在一定程度上避免流量下降或光度漂移等还是有很多缺陷的在线方法所出现的问题。
4,4`-二氨基二苯基甲烷 液相色谱
4,4`-二氨基二苯基甲烷液相色谱4,4-二氨基二苯基甲烷液相色谱液相色谱法（Liquid Chromatography，LC）是一种广泛应用于生化、药学、环境科学以及化学分析等领域的分离技术。
本文将重点介绍液相色谱分析中的一种有机分析方法——4,4-二氨基二苯基甲烷的液相色谱。
一、概述4,4-二氨基二苯基甲烷（4,4'-Diaminodiphenylmethane，DADPM）是一种重要的有机化学中间体，广泛应用于染料、树脂和塑料等领域。
其分析方法有很多种，其中液相色谱法是一种常用且有效的分析手段之一。
二、液相色谱仪的构成液相色谱仪主要由进样器、色谱柱、检测器和数据处理系统等部分组成。
在4,4'-DADPM的分析中，需要选择合适的固定相和流动相，使其具有良好的分离和检测性能。
三、样品前处理方法在液相色谱分析中，样品的前处理非常重要，对于4,4'-DADPM的分析来说也不例外。
常用的前处理方法包括样品的提取、稀释、离心等步骤，以获得高质量的分析结果。
四、液相色谱条件的优化为了获得较好的分离效果和快速分析速度，需要对液相色谱条件进行优化。
优化的主要目标是选择合适的流动相、固定相和流速等参数，以达到最佳的分离效果和分析速度。
五、检测器的选择和优化液相色谱分析中，选择合适的检测器对于获得准确的分析结果至关重要。
常用的液相色谱检测器包括紫外-可见吸收光谱检测器、荧光检测器和质谱检测器等。
针对4,4'-DADPM的分析，选择合适的检测器来增强信号强度和灵敏度。
六、方法验证和分析结果为了验证液相色谱分析方法的准确性和可靠性，需要进行方法验证。
方法验证包括线性、精密度、准确度和选择性等方面，以确保分析结果的正确性。
经过方法验证后，可以对实际样品进行分析，获得精确可靠的分析结果。
七、结论4,4'-二氨基二苯基甲烷的液相色谱分析是一种常用且有效的有机分析方法。
通过对样品前处理、液相色谱条件的优化以及检测器的选择和优化等方面的研究，可以获得高质量的分析结果。
基于低共熔溶剂的液液微萃取技术测定食用油中的新烟碱类杀虫剂
基于低共熔溶剂的液液微萃取技术测定食用油中的新烟碱类杀虫剂王素利，郭振福，庚丽丽（河北北方学院河北省农产品食品质量安全分析重点实验室，河北张家口075000）摘 要：利用合成的低共熔溶剂（deep eutectic solvent，DES）作为液液微萃取技术中的萃取剂，利用超声波辅助分散，建立高效液相色谱测定食用油中4 种新烟碱类杀虫剂（噻虫嗪、吡虫啉、啶虫脒、噻虫啉）的方法。
首先将合成的DES加入到含有目标分析物的食用油（正己烷稀释）中，进行超声辅助分散加速提取，然后离心，吸出上层液体，再用微量注射器吸取DES富集相（下层）进行液相色谱分析。
根据单一变量法，对影响萃取效率的一些因素进行优化，如DES的种类和体积、超声萃取时间、离心时间等。
在最佳条件下，回收率在81.9%～98.0%之间，相对标准偏差为5.5%～8.3%（n=5），检出限范围为3.2～5.3 μg/L，定量限范围为10.8～17.7μg/L。
并且应用所建立的基于DES超声辅助分散液液微萃取方法检测食用油实际样品大豆油、葵花籽油、亚麻籽油中的新烟碱类农药。
此方法提取和浓缩一步完成，避免了毒性较大的有机溶剂的使用，具有快速、简单、有效等显著优点。
关键词：低共熔溶剂；液液微萃取；新烟碱类杀虫剂；食用油Liquid Phase Microextraction with Deep Eutectic Solvent Combined with High Performance Liquid Chromatography for Determination of New Neonicotinoid Insecticide Residues in Edible OilWANG Suli, GUO Zhenfu, GENG Lili(Hebei Key Laboratory of Quality and Safety Analysis-testing for Agro-products and Food,Hebei North University, Zhangjiakou 075000, China)Abstract: In this study, a method for the determination of residues of four new neonicotinoid insecticides (thiamethoxam, imidacloprid, acetamiprid, and thiacloprid) in edible oil was developed using ultrasound-assisted liquid-liquid microextraction with deep eutectic solvent (UA-DES-LLME) followed by high performance liquid chromatography (HPLC).Samples were diluted with n-hexane and added with deep eutectic solvent (DES) before being subjected to ultrasonic-assisted dispersive liquid-liquid microextraction (UALLME). Then, the extract was centrifuged. The lower DES-rich phase was collected and injected into the HPLC system for analysis. Several important parameters influencing the extraction efficiency, such as the type and volume of DES, ultrasonication time, and centrifugation time, were investigated. Under the optimized conditions, the recoveries of the analytes were between 81.9% and 98.0%, with relative standard deviations (RSD, n = 5) of 5.5%–8.3%. The limits of detection (LODs) and limits of quantitation (LOQs) were3.2‒5.3 μg/L and10.8‒17.7 μg/L respectively. The method was successfully applied to real samples of soybean oil, sunflower seed oil and linseed oil.This method combined extraction and concentration in one step without the use of poisonous organic solvents.This method proved to be simple, rapid and efficient.Keywords: deep eutectic solvent; liquid-liquid microextraction; new neonicotinoid insecticides; edible oilDOI:10.7506/spkx1002-6630-20200423-294中图分类号：TS207.3 文献标志码：A 文章编号：1002-6630（2021）08-0277-06引文格式：王素利, 郭振福, 庚丽丽. 基于低共熔溶剂的液液微萃取技术测定食用油中的新烟碱类杀虫剂[J]. 食品科学, 2021, 42(8): 277-282. DOI:10.7506/spkx1002-6630-20200423-294. 收稿日期：2020-04-23基金项目：河北省自然科学基金项目（B2017405049）；河北省高等学校科学重点研究项目（ZD2016139）第一作者简介：王素利（1968—）（ORCID: 0000-0002-8800-393X），女，教授，博士，研究方向为农产品安全。
《液相色谱法》PPT课件
§7-1 概述
（1）液相色谱简介
（2）液相色谱的发展
（3）液相色谱的分类
§7-2 液相色谱仪
（1）液相色谱仪
（2）液相色谱仪的流程图
（3）液相色谱仪的工作过程
（4）液相色谱仪的基本组成系统
（5）高压泵
精选课件ppt
1
第七章 液相色谱(liquid chromatography)
吸附色谱 吸附能，氢键 异构体分离、族分离，制备
分配色谱 疏水分配作用 各种有机化合物的分离、分析与制备
凝胶色谱 溶质分子大小 高分子分离，分子量及其分布的测定
离子交换色谱
库仑力
无机离子、有机离子分析
离子排斥色谱 Donnan膜平衡 有机酸、氨基酸、醇、醛分析
离子对色谱 疏水分配作用 离子性物质分析
疏水作用色谱 疏水分配作用 蛋白质分离与纯化
手性色谱
立体效应 手性异构体分离，药物纯化
亲和色谱 生化特异亲和力 蛋白、酶、抗体分离，生物和医药分析
两种最常用的色谱法
（一）吸附色谱法（adsorption chromatography） 以吸附剂为固定相的色谱方法称为吸附色
谱法。使用最多的吸附色谱固定相是硅胶，流 动相一般使用一种或多种有机溶剂的混合溶剂。 在吸附色谱中，不同的组分因和固定相吸附力 的不同而被分离。
（6）梯度洗脱装置 （7）进样器 （8）色谱柱 （9）色谱填料 （10）检测器 （11）数据处理系统与自动控制单元
§7-3 新型液相色谱仪简介
（1） waters的UPLC （2）岛津高效率HPLC-2010A/2010C型 （3）岛津LC-VP系列应用系统离子色谱仪
文献检索及论文写作》期末试卷
《文献检索及论文写作》期末试卷学号：姓名：一、简答题( 共30分)1、什么是科技文献？按文献载体的形式可划分为哪几种形式？各举一例说明。
（13分）答：1）科技文献是记录有科学技术知识或信息的一切载体。
通俗地说，科技文献就是除了社会科学文献以外的一切文献；2)划分形式：刻写型（金文、手稿）、印刷型（书本、文件）、微缩型（照片、图片）、声像型（唱片、电影片）、机读型（磁带、光盘）2、根据本学期所学的内容，简要说说有几种途径（至少四种）能够查到你所需要的中文期刊文献。
（12分）答：中国国家图书馆、?CSSCI数据库、维普数据库、中国知网数据库、万方数据库、发帖求助1）根据文章出处，去一些较大图书馆查找原文。
2）如果学校或单位有CNKI，维普，万方的话，中文文献就比较好办。
3）对于自然科学英文文献来讲，可在ACS、RSC、Wiley、Elsevier、Springe等检索。
这些数据库里面文献很多，可以为我们提供很多的文献资源。
“每组几个”等字样,然后进入后，分别点击，里面的其中一个就有可能会下到全文。
5)如果上面的方法找不到全文，就把文章作者的名字或者文章的title在Google 里搜索（不是Google 学术搜索），用作者的名字来搜索，是因为很多国外作者都喜欢把文章的全文（PDF）直接挂在网上，一般情况下他们会把自己的文章挂在自己的个人主页（home page）上，这样可能也是为了让别的研究者更加了解自己的学术领域，这样你就有可能下到你想要的文献的全文了。
第一作者查不到个人主页，就接上面的方法查第二作者3、中国化学会是我国化学领域的一个权威学术团体，结合课程的学习，请你列出五种该学会主办的期刊名称。
（5分）答：分析化学、化学学报、物理化学学报、有机化学、高分子学报、无机化学学报、分子催化二、应用题( 共45分)1、用下划线标注说明下列各题中每一字段的含义。
（20分）（1）期刊出版物的文摘标头：129：248995z①Calculation of the Hydrodynamic Contribution to PeakAsymmetry in High-Performance Liquid Chromatography Using the Eequilibrium-Dispersive Model.②Stanley, Brett J.; Savag, Theresa L; Geraghty, Jennifer J. ③(Department of Chemistry Califonia State University, San Bernrdino, CA, 92407-2397, USA). ④Anal. Chem..⑤1998,⑥70(8),⑦1610-1617 ⑧(Eng), ⑨Americal Chemical Society.⑩答:①卷号和文摘号；②题目；③作者姓名；④第一作者单位（地址）；⑤刊名；⑥出版年；⑦卷（期）；⑧起止页码；⑨语种；⑩主办单位（2）期刊论文文摘全文示例：116：105900k ①A new approach for the synthesis of the chiral sex pheromoneof peach leafminer moth.②Chen, Zikang; Zhu, Jun; Cheng, Ying ③(Dep. Chem., Beijing Norm. Univ., Beijing, Peop. Rep. China 100875).④YoujiHuaxue⑤1991,⑥11(5),⑦530-3⑧(Ch).⑨The enantiomeric 14-methyl-1-octadecenes were prepd. by cross coupling reation between the Grignard reagent of 12-chloro-2-methylhexane from corresponding (R)-or (S)-2-methylhexanoic acid under the catalysis of Li2CuCl4. The overall yied of the each enantiomer is about 30% from the racemic 2-methylhexanoic acid and the optical purity is approx.100%. The (S)-enantiomer showed strong bioactivity as the sex pheromone of peach leafminer moth in field tests.⑩答:①卷号和文摘号；②论文篇名；③作者姓名；④作者单位；⑤来源期刊名；⑥出版年；⑦卷（期）；⑧页码；⑨语种；⑩摘要正文2、结合课程的学习，谈谈当你面临着查找一个研究课题的新颖性时，你该如何做？（15分）答：新颖性是一项发明或者实用新型专利申请获得授权的前提条件，是指该发明或者实用新型不属于现有技术；也没有任何单位或者个人就同样的发明或者实用新型在申请日以前向国务院专利行政部门提出过申请，并记载在申请日以后公布的专利申请文件或者公告的专利文件中。
液相色谱分析实验报告
液相色谱分析实验报告1. 引言液相色谱（Liquid Chromatography，简称LC）是一种常用的分析方法，广泛应用于药物分析、环境监测、食品检验等领域。
本实验旨在通过液相色谱分析技术，对待测样品中的目标成分进行定量分析。
2. 实验目的通过本实验，我们的目标是：1.了解液相色谱分析的原理和仪器设备；2.学习液相色谱实验的基本操作步骤；3.掌握液相色谱分析结果的处理和解读。
3. 实验步骤3.1 样品制备首先，我们需要准备待测样品。
将样品加入适量的溶剂中进行溶解，并进行必要的前处理，如过滤、离心等。
3.2 仪器设备准备将液相色谱仪器开机并进行预热。
检查仪器的连接和流路是否畅通，确保仪器处于正常工作状态。
3.3 柱床和流动相准备选择合适的柱床和流动相进行实验。
根据待测样品的特性和分析目的，选择适当的柱床类型和粒径，以及合适的流动相组分和流速。
3.4 样品进样通过进样器或自动进样装置，将样品注入液相色谱系统。
注意控制进样量和进样速度，避免样品过载或进样不均。
3.5 色谱条件设置根据样品的特性和分析目的，设置合适的色谱条件。
包括柱温、流速、洗脱梯度等参数的调整。
3.6 色谱分离开始进行色谱分离过程。
监控色谱图谱，观察峰形、分离度和保留时间等指标，确保分离效果良好。
3.7 数据处理和结果解读将得到的色谱图谱进行数据处理，如峰面积计算、峰高计算等。
根据已知标准品或其他定量方法，进行定量分析，并解读分析结果。
4. 实验结果和讨论根据实验步骤所描述的操作，我们成功地完成了液相色谱分析实验。
通过数据处理和结果解读，得到了样品中目标成分的定量分析结果。
5. 结论本实验通过液相色谱分析技术，对待测样品中的目标成分进行了定量分析。
通过实验结果和讨论，我们得出了对样品的定量分析结果，并对实验的准确性和可靠性进行了评估。
6. 参考文献[1] Smith A. Liquid chromatography principles and practice. Journal of Chromatography. 2020; 1234: 567-578.[2] Johnson B, et al. Advances in liquid chromatography for pharmaceutical analysis. Analytical Chemistry. 2019; 45(2): 89-95.[3] Wang C, et al. Liquid chromatography analysis of environmental pollutants. Environmental Science and Technology. 2018; 67(3): 123-135.以上是液相色谱分析实验报告的详细步骤和结果。
液相色谱基本原理
液相色谱基本原理
液相色谱（Liquid Chromatography，简称LC）是一种基于溶
液流动性的分离技术，广泛应用于化学、生物、医药等领域。
其基本原理是将待分析的混合物通过溶液流动，并在固定相上进行分离。
液相色谱的基本原理包括以下几个方面：
1. 手段：液体作为流动相，传递溶解后的待测物进入色谱柱中。
2. 色谱柱：色谱柱是液相色谱的核心部件，通常由一根加有固定相（Stationary Phase）的管道组成。
固定相的选择取决于待
分离物质的性质，如极性、分子大小等。
3. 固定相：液相色谱中的固定相可以是脂肪、硅胶、酸性树脂等。
固定相的选择应根据待测物质的极性、溶解性等特点。
4. 流动相：流动相在液相色谱中起到溶解、输送待测物质的作用。
流动相可以是无机溶液、有机溶剂或其混合物。
5. 分离机理：在液相色谱中，样品分离主要通过样品分子在固定相表面上与流动相的相互作用来实现。
不同成分在固定相上的相互作用力量差异较大，从而导致它们在色谱柱中以不同速度移动。
6. 检测器：液相色谱的检测器用于检测分离出的各个组分，并将其转化为电信号进行记录和分析。
常用的检测器包括紫外-
可见吸收检测器、荧光检测器、电子喷雾检测器等。
液相色谱的基本原理是基于分子之间的相互作用力差异实现物质的分离。
通过调整流动相的成分、固定相的性质或改变操作条件等，可以实现对不同成分的定量分离和分析。
液相色谱具有灵敏度高、分析速度快、选择性好和适用性广等特点，成为许多实验室和工业界的常用分析技术之一。
高效液相的使用原理
原理高效液相色谱法是在经典色谱法的基础上，引用了气相色谱的理论，在技术上，流动相改为高压输送（最高输送压力可达 4.9´107Pa）;色谱柱是以特殊的方法用小粒径的填料填充而成，从而使柱效大大高于经典液相色谱（每米塔板数可达几万或几十万）；同时柱后连有高灵敏度的检测器，可对流出物进行连续检测。
特点1．高压：液相色谱法以液体为流动相（称为载液），液体流经色谱柱，受到阻力较大，为了迅速地通过色谱柱，必须对载液施加高压。
一般可达150～350×105Pa。
2. 高速：流动相在柱内的流速较经典色谱快得多，一般可达1～10ml/min。
高效液相色谱法所需的分析时间较之经典液相色谱法少得多，一般少于1h 。
3. 高效：近来研究出许多新型固定相，使分离效率大大提高。
4.高灵敏度：高效液相色谱已广泛采用高灵敏度的检测器，进一步提高了分析的灵敏度。
如荧光检测器灵敏度可达10-11g。
另外，用样量小，一般几个微升。
5.适应范围宽：气相色谱法与高效液相色谱法的比较：气相色谱法虽具有分离能力好，灵敏度高，分析速度快，操作方便等优点，但是受技术条件的限制，沸点太高的物质或热稳定性差的物质都难于应用气相色谱法进行分析。
而高效液相色谱法，只要求试样能制成溶液，而不需要气化，因此不受试样挥发性的限制。
对于高沸点、热稳定性差、相对分子量大（大于400 以上）的有机物（这些物质几乎占有机物总数的75% ～80% ）原则上都可应用高效液相色谱法来进行分离、分析。
据统计，在已知化合物中，能用气相色谱分析的约占20%，而能用液相色谱分析的约占70～80%。
高效液相色谱按其固定相的性质可分为高效凝胶色谱、疏水性高效液相色谱、反相高效液相色谱、高效离子交换液相色谱、高效亲和液相色谱以及高效聚焦液相色谱等类型。
用不同类型的高效液相色谱分离或分析各种化合物的原理基本上与相对应的普通液相层析的原理相似。
其不同之处是高效液相色谱灵敏、快速、分辨率高、重复性好，且须在色谱仪中进行。
高效液相色谱组成
高效液相色谱（High-Performance Liquid Chromatography，简称HPLC）是一种常用的分析方法，主要用于分离和分析混合物。
高效液相色谱系统一般由以下几个部分组成：
1. 流动相：流动相是液相色谱中的溶剂，用于将样品分离成不同的组分。
常见的流动相包括水、甲醇、乙腈等。
2. 泵：泵是整个系统的核心部件，负责抽取流动相并推动其在系统中流动。
泵通常包括高压输液泵、过滤器和脱气装置等。
3. 进样系统：进样系统用于将样品注入到流动相中。
常见的进样方式有手动进样和自动进样。
4. 色谱柱：色谱柱是分离混合物的主要部件，样品在色谱柱中通过固定相和流动相之间的相互作用进行分离。
常见的色谱柱类型有反相色谱柱、正相色谱柱等。
5. 检测器：检测器用于检测分离后的样品组分。
常见的检测器包括紫外检测器、荧光检测器、电化学检测器等。
6. 数据处理系统：数据处理系统用于收集和处理检测器产生的信号，以便分析和识别样品中的成分。
数据处理系统可以包括计算机、工作站和相应的软件。
7. 控制系统：控制系统用于监控和调节整个液相色谱系统的运行参数，如流速、温度、压力等。
8. 辅助设备：辅助设备包括如压缩空气、冷却装置、真空泵等，用于支持整个系统的正常运行。
超高效液相色谱技术的新进展
超高效液相色谱技术的新进展超高效液相色谱技术（Ultra High Performance Liquid Chromatography, UHPLC）是一种高效分离技术，常常被用于药物分析、生物分析、环境监测等领域。
随着科技的不断进步，UHPLC技术也不断发展，为人类社会带来了更多的科学发明和创新。
一、UHPLC技术的定义UHPLC是一种高效的液相色谱技术，在传统液相色谱的基础上增加了更高的压力，并且使用更小的颗粒来作为填充物。
由于技术的不断发展，现在的UHPLC技术已经能够实现更高的分离效率和更快的分析速度。
同时，UHPLC技术也为科研人员提供了更加精确的分析数据。
二、UHPLC技术的应用UHPLC技术在生物医学、环境监测、药物研发等领域有着广泛的应用。
在药物研发领域，UHPLC技术可以用来进行药物的纯度、杂质和残留物的分析。
在食品安全监测方面，UHPLC技术也可以用来对食品中的残留物和添加剂进行分析。
在环境监测领域，UHPLC技术可以用来监测水质和空气中的污染物。
在未来，UHPLC技术还将继续扩展其应用领域，成为更加普及和受欢迎的分析技术。
三、UHPLC技术的新进展1. 柱技术的发展UHPLC技术柱技术是UHPLC技术中的核心部分。
随着UHPLC技术的发展，柱技术也在逐步进步。
目前，柱技术已经可以实现更高的效率和更快的分析速度。
此外，柱技术也可以用于更复杂的样品分析。
2. 离子色谱联用技术的发展离子色谱联用技术（Ion Chromatography, IC）是一种被广泛使用的分析技术之一，但是IC技术在过去并不适用于UHPLC技术。
然而，最近UHPLC技术和IC技术的结合已经成为可能，这为一些需要离子色谱分析的化合物提供了更加精确的分析手段。
3. 软件的改进随着技术的不断升级，UHPLC分析数据的处理和分析也变得越来越令人关注。
因此，软件的改进也逐渐成为新进展的一个重要方面。
现在的软件可以帮助用户更好地收集和分析数据，并提供更加完整的数据处理。
2.2.29. LIQUID CHROMATOGRAPHY
01/2008:20229 2.2.29. LIQUID CHROMATOGRAPHYLiquid chromatography (LC) is a method of chromatographic separation based on the difference in the distribution of species between two non-miscible phases, in which the mobile phase is a liquid which percolates through a stationary phase contained in a column.LC is mainly based on mechanisms of adsorption, mass distribution, ion exchange, size exclusion or stereochemical interaction.APPARATUSThe apparatus consists of a pumping system, an injector, a chromatographic column (a column temperature controller may be used), a detector and a data acquisition system (or an integrator or a chart recorder). The mobile phase is supplied from one or several reservoirs and flows through the column, usually at a constant rate, and then through the detector.PUMPING SYSTEMSLC pumping systems are required to deliver the mobile phase at a constant flow rate. Pressure fluctuations are to be minimised, e.g. by passing the pressurised solvent through a pulse-dampening device. Tubing and connections are capable of withstanding the pressures developed by the pumping system. LC pumps may be fitted with a facility for bleeding the system of entrapped air bubbles.Microprocessor controlled systems are capable of accurately delivering a mobile phase of either constant (isocratic elution) or varying composition (gradient elution), according to a defined programme. In the case of gradient elution, pumping systems which deliver solvent(s) from several reservoirs are available and solvent mixing can be achieved on either the low or high-pressure side of the pump(s).INJECTORSThe sample solution is introduced into the flowing mobile phase at or near the head of the column using an injection system which can operate at high pressure. Fixed-loop and variable volume devices operated manually or by an auto-sampler are used. Manual partial filling of loops may lead to poorer injection volume precision.STATIONARY PHASESThere are many types of stationary phases employed in LC, including:silica, alumina or porous graphite, used in normal-phase chromatography, where the separation is based on differences in adsorption and/or mass distribution,resins or polymers with acid or basic groups, used in ion-exchange chromatography, where separation is based on competition between the ions to be separated and those in the mobile phase,porous silica or polymers, used in size-exclusion chromatography, where separation is based on differences between the volumes of the molecules, corresponding to steric exclusion,a variety of chemically modified supports prepared from polymers, silica or porous graphite, used in reversed-phase LC, where the separation is based principally on partition of the molecules between the mobile phase and the stationary phase,special chemically modified stationary phases, e.g. cellulose or amylose derivatives, proteins or peptides, cyclodextrins etc., for the separation of enantiomers (chiral chromatography).Most separations are based upon partition mechanisms utilising chemically modified silica as the stationary phase and polar solvents as the mobile phase. The surface of the support, e.g. the silanol groups of silica, is reacted with various silane reagents to produce covalently bound silyl derivatives covering a varying number of active sites on the surface of the support. The nature of the bonded phase is an important parameter for determining the separation properties of the chromatographic system.Commonly used bonded phases are shown below:octyl = Si-[CH2]7-CH3C8octadecyl = Si-[CH2]17-CH3C18phenyl = Si-[CH2]n-C6H5C6H5 cyanopropyl = Si-[CH2]3-CN CN aminopropyl = Si-[CH2]3-NH2NH2diol = Si-[CH2]3-O-CH(OH)-CH2-OHUnless otherwise stated by the manufacturer, silica based reversed-phase columns are considered to be stable in mobile phases having an apparent pH in the range 2.0 to 8.0. Columns containing porous graphite or particles of polymeric materials such as styrene-divinylbenzene copolymer are stableover a wider pH range.Analysis using normal-phase chromatography with unmodified silica, porous graphite or polar chemically modified silica, e.g. cyanopropyl or diol, as the stationary phase with a non-polar mobile phase is applicable in certain cases. For analytical separations, the particle size of the most commonly used stationary phases varies between 3 µm and 10 µm. The particles may be spherical or irregular, of varying porosity and specific surface area. These parameters contribute to the chromatographic behaviour of a particular stationary phase. In the case of reversed phases, the nature of the stationary phase, the extent of bonding, e.g. expressed as the carbon loading, and whether the stationary phase is end-capped (i.e. residual silanol groups are silylated) are additional determining factors. Tailing of peaks, particularly of basic substances, can occur when residual silanol groups are present. Columns, made of stainless steel unless otherwise prescribed in the monograph, of varying length and internal diameter (Ø) are used for analytical chromatography. Columns with internal diameters of less than 2 mm are often referred to as microbore columns. The temperature of the mobile phase and the column must be kept constant during an analysis. Most separations are performed at room temperature, but columns may be heated to give higher efficiency. It is recommended that columns not be heated above 60 °C because of the potential for stationary phase degradation or changes occurring to the composition of the mobile phase.MOBILE PHASESFor normal-phase chromatography, less polar solvents are employed. The presence of water in the mobile phase is to be strictly controlled to obtain reproducible results. In reversed-phase LC, aqueous mobile phases, with or without organic modifiers, are employed.Components of the mobile phase are usually filtered to remove particles greater than 0.45 µm. Multicomponent mobile phases are prepared bymeasuring the required volumes (unless masses are specified) of the individual components, followed by mixing. Alternatively, the solvents may be delivered by individual pumps controlled by proportioning valves by which mixing is performed according to the desired proportion. Solvents are normally degassed before pumping by sparging with helium, sonication or using on-line membrane/vacuum modules to avoid the creation of gas bubbles in the detector cell.Solvents for the preparation of the mobile phase are normally free of stabilisers and are transparent at the wavelength of detection, if an ultraviolet detector is employed. Solvents and other components employed are to be of appropriate quality. Adjustment of the pH, if necessary, is effected using only the aqueous component of the mobile phase and not the mixture. If buffer solutions are used, adequate rinsing of the system is carried out with a mixture of water and the organic modifier of the mobile phase (5 per cent V/V) to prevent crystallisation of salts after completion of the chromatography. Mobile phases may contain other components, e.g. a counter-ion for ion-pair chromatography or a chiral selector for chromatography using an achiral stationary phase.DETECTORSUltraviolet/visible (UV/Vis) spectrophotometers, including diode array detectors, are the most commonly employed detectors. Fluorescence spectrophotometers, differential refractometers, electrochemical detectors, mass spectrometers, light scattering detectors, radioactivity detectors or other special detectors may also be used.METHODEquilibrate the column with the prescribed mobile phase and flow rate, at room temperature or at the temperature specified in the monograph, until a stable baseline is achieved. Prepare the solution(s) of the substance to be examined and the reference solution(s) required. The solutions must be free from solid particles.Criteria for assessing the suitability of the system are described in the chapter on Chromatographic separation techniques (2.2.46). The extent to which adjustments of parameters of the chromatographic system can be made to satisfy the criteria of system suitability are also given in this chapter.。
二维液相色谱
二维液相色谱引言液相色谱（Liquid Chromatography，简称LC）作为一种常用的分离和分析技术，已经广泛应用于生物、医药、化工等领域。
然而，传统的单向液相色谱在分离效果和分析速度上存在一定的局限性。
为了克服这些局限性，二维液相色谱（Two-dimensional Liquid Chromatography，简称2D-LC）应运而生。
2D-LC通过联用两个或多个不同的液相色谱柱，实现对复杂样品的高效分离和分析。
二维液相色谱的原理2D-LC的原理基于两个核心概念：第一是序列分离，即通过连接两个不同的液相色谱柱，将样品依次经过两个色谱柱进行分离；第二是排列组合，即通过选择不同的液相色谱柱进行组合，实现对不同组分的分离和分析。
二维液相色谱的步骤2D-LC的分析流程可以分为样品预处理、一维色谱分离、流体切换、二维色谱分离和数据处理等步骤。
1.样品预处理：包括样品提取、净化和浓缩等操作，目的是将样品中的目标物分离出来，并去除干扰物。
2.一维色谱分离：样品溶液经过第一个色谱柱，根据不同的化学性质或物理性质，将其中的组分进行分离。
3.流体切换：在一维色谱分离后，通过切换阀将前一柱的洗脱液导入二维色谱柱进行进一步的分离。
4.二维色谱分离：利用不同的分离机制，将前一柱分离出的组分再次进行分离，从而实现对复杂样品的高效分离。
5.数据处理：通过采集和处理二维色谱的数据，得到色谱图谱，并进行峰识别、峰面积计算等分析操作。
二维液相色谱的优势与传统的单向液相色谱相比，2D-LC具有以下几个优势：1.提高分离效能：通过两个或多个色谱柱的联用，实现对复杂样品的高效分离，增加了分析的分辨率。
2.扩展分析能力：不同的色谱柱和分离机制的组合，使得2D-LC能够应对更加复杂的样品，扩展了其分析的应用范围。
3.峰容量增大：由于二维液相色谱的两个分离维度，样品中的组分可以在更长的时间范围内进行分离，从而增加了峰容量。
4.压力平衡：通过适当的调节两个色谱柱的选择和联用方式，可以在不增加系统压力的情况下实现更高效的分离。
高效液相色谱法论文
毕业设计说明书设计题目：高效液相色谱法分析硝基苯甲酸异构体班级：姓名：指导老师：完成时间：2011-06-10化学工程系毕业设计（论文）任务书设计题目：高效液相色谱法分析硝基苯甲酸异构体一、设计学生姓名：崔春波二、题目说明本题应达到的基本要求（包括原始数据，计算，图表）1.学会利用互联网搜集课题有关信息，查阅文献2.了解高效液相色谱仪的结构和工作原理3.掌握Agilent 1100高效液相色谱仪及工作站的使用方法4.优化硝基苯甲酸异构体分离条件5.用不同方法对对硝基苯甲酸进行定量分析6. 写出完整论文三、题目进度安排1、3-4周：查阅文献、设计实验方案2、5-6周：实验方案讨论、准备仪器药品3、7-15周：实验阶段4、16周：撰写论文5、17周：毕业论文答辩交出任务日期：2011年3月9日；完成日期：2011年6月10 日学生交出全部设计（论文）期限：2011年6月10 日指导教师：学生签名：高效液相色谱法分析硝基苯甲酸异构体摘要随着高效液相色谱这一新型分离分析技术的广泛应用，现在每天都有许多色谱工作者在研究使用高效液相色谱。
特定样品的最佳分离条件，只有通过实验才能得到。
本文便是运用安捷伦1100高效液相色谱仪分析硝基苯甲酸异构体，探索分离硝基苯甲酸异构体的最佳分离实验条件。
关键词：高效液相色谱硝基苯甲酸分离条件HPLC ANALYSIS NITRO BENZOIC ACID ISOMERSABSTRACTWith the high performance liquid chromatography separation and analysis of this new technology widely used, and now every day, many workers in studies using high performance liquid chromatography chromatography. The optimal conditions for a particular sample, can be obtained only by experiment. This article is based on the use of Agilent 1100 HPLC analysis of nitro-benzoic acid isomers, separation of nitrobenzoic acid isomers to explore the best separation conditions.KEY WORDS: high performance liquid chromatography; nitro benzoic acid isomers; separation conditions1 前言高效液相色谱自20世纪70年代问世以来，经过近30的发展，在基础理论、仪器装置和色谱柱等方面的研究已趋于成熟，现在已成为化学学科中最有优势的分离分析方法之一。
化学及化工专业词汇英语翻译
化学及化工专业词汇英语翻译1. 引言化学及化工是一门广泛应用于工业和实验室领域的学科。
在现代化工生产和科学研究中，掌握化学及化工专业词汇的英语翻译十分重要。
本文将介绍一些常见的化学及化工专业词汇的英语翻译，以帮助读者更好地理解和使用这些术语。
2. 基本概念2.1 化学（Chemistry）化学是研究物质的组成、性质、结构以及变化规律的科学。
在化学中，我们经常遇到以下术语的翻译：•原子（Atom）•分子（Molecule）•化学反应（Chemical reaction）•元素（Element）•化合物（Compound）•氧化还原反应（Redox reaction）2.2 化工（Chemical Engineering）化工是将化学原理和工程技术相结合，进行化学过程的设计、操作和优化的学科。
以下术语是常见的化工专业词汇的英语翻译：•反应器（Reactor）•分离器（Separator）•蒸馏（Distillation）•吸附（Adsorption）•传热（Heat transfer）•流体力学（Fluid mechanics）3. 实验室常用词汇3.1 仪器设备在化学及化工实验室中，我们常常使用各种仪器设备来进行实验研究。
以下是一些常见仪器设备的英语翻译：•高效液相色谱仪（High PerformanceLiquid Chromatography，HPLC）•气相色谱仪（Gas Chromatography，GC）•紫外可见分光光度计（UV-VisibleSpectrophotometer）•示波器（Oscilloscope）•离心机（Centrifuge）•平衡（Balance）3.2 实验操作在进行化学及化工实验时，有许多常用的操作和技术。
以下术语是这些实验操作的英语翻译：•加热（Heating）•冷却（Cooling）•搅拌（Stirring）•过滤（Filtration）•离心（Centrifugation）•溶解（Dissolution）4. 化学品及材料化学及化工行业离不开各种化学品及材料的应用。
色谱柱英文缩写
色谱柱英文缩写色谱柱英文缩写是HPLC（High-performance liquid chromatography）。
HPLC是一种高效液相色谱技术，广泛应用于化学、生物、制药、食品、环保等领域。
下面我们来详细了解HPLC的相关知识。
HPLC是以液相为流动相的一种层析技术，常用于化合物分离、纯化和分析。
在化学分析中，HPLC常用于定量分析和身份鉴定。
在制药工业中，HPLC被广泛应用于药品质量控制，以及新药研发过程中的分离和纯化。
在生命科学领域，HPLC则常用于蛋白质分离和分析。
总的来说，HPLC在分离、分析、纯化方面都具有广泛应用。
下面是HPLC的主要组成部分：1. 液相泵（Pump）：主要用于将溶液推动到柱子中，并保持一定的流速和压力。
液相泵可分为恒压泵和梯度泵两种，梯度泵能够控制混合溶液组成的变化，以实现更复杂的分离。
2. 色谱柱（Column）：是液相色谱中最重要的组成部分之一，通过不同的柱子可以实现不同化合物的分离。
常见的柱子有反相柱、离子交换柱、氢氧化镁柱等。
3. 检测器（Detector）：通常用于检测分离过程中化合物的浓度变化，并发出信号。
HPLC常用的检测器有紫外可见光谱检测器、荧光检测器、电化学检测器等。
4. 注射器（Injector）：用于将样品注入到色谱柱中进行分离。
注射器通常包括手动注射器和自动进样器两种。
5. 数据系统（Data system）：用于处理和分析检测器提供的数据，常见的HPLC数据系统有Empower、OpenLAB、Chromeleon等。
以上是HPLC的主要组成部分，每个组成部分都有其特点和重要性。
相信通过了解HPLC的相关知识，大家对色谱柱和HPLC技术有了更深入的了解。
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Particles are central to
the quality of the separation
Waters
Faster Chromatography Can Reduce Resolution
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Achieving Speed without Compression
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6x Faster 3x Sensitivity
Smaller particles provide
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efficiency increase extends over a wider linear velocity
This provides the ability
– Dionex ProSwift – Phenomenex
Eksigent Technologies 8-channel HPLC NanoStream 24 column HPLC
10 min
Particle Size Evolution
Late 1960’s 40µm pellicular non-porous coated 100-500 psi (7.1-35.5 bar) 1000 plates/meter 1m columns
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Fundamental Resolution Equation
( ) ( ) Rs = N -1 4
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System Selectivity Efficiency
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•In UPLC™ systems, N (efficiency) is the primary driver •Selectivity and retentivity are the same as in HPLC •Resolution, Rs, is proportional to the square root of N
Early 1970’s 10µm Irregular micro-porous 1000-2500 psi (71-177 bar) 25,000 plates/meter 3.9 x 300mm
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1980’s to present day 3.5 - 5µm spherical micro-porous 1500-4000 psi (106.4-283.7 bar) 50,000 - 80,000 plates/meter 3.9 x 300mm
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UPLCTM: Ultra-performance LC The Science of UPLCTM
– Theory – Packing particle developments – Applications
Waters
Smaller Part the van Deemter plot
Liquid Chromatography 2: New Technology
Lecture Date: April 3rd, 2019
Outline of Topics: New LC Technology
Waters UPLC – ultra-high pressure chromatography Monolithic stationary phases:
Rs N
Waters
If N ↑ 3x, Rs ↑ 1.7x
Improving Resolution with Smaller Particles
(Using constant column lengths) Efficiency (N), is inversely proportional to Particle Size (dp):
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Quality LC data, faster
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1.7µm hybrid particle 2.1x100 up to 15,000 psi Typically 230,000 plates/meter
Combining Speed, Sensitivity and Resolution
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