2 Who Are Malvern Instruments? 马尔文仪器是一家英国公司，专注于设计和制造精确的测量仪器，应用于粒子尺寸及其分布粒子的电荷分子量粒子形态分散体系的流体力学性质在具体谈论仪器前，我想先简单介绍一下马尔文公司Malvern Instrument is a company oriented in UK, whcih offers innovative solutions in material charachterization.We focus on …..History of Malvern???
4 Zetasizer Nano系列: 粒子尺寸 Zeta电位 分子量 胶体颗粒，乳液，高分子溶液…… 高灵敏度 高浓度 维护简单 高灵敏度，准确率高分辨率分子量蛋白质和高分子Zetasizer粒径范围 (nm)Zeta 电位测量的尺寸范围 (nm)分子量范围 (Da)0.6 to 6000-1000 to 2 x 1075 to 10,0001 to 300010000 to 2 x 107我们可以由仪器上的标签来分辨Nano系列中不同的仪器配置。Nano-s仅有动态和静态光散射装置。散射光的测量角度为173 度。测量范围为，对粒径范围。。。对分子量。。。-z中仅配置了zeta电位测量装置。Nano zs中结合了动静态光散射装置。Nano s90中，仅配置了动静态光散射装置，散射光的测量角度为90度
7 Zetasizer Nano是如何测试粒子的粒径的? 动态光散射Dynamic Light Scattering (DLS)，也称光子相关光谱Photon Correlation Spectroscopy (PCS) ，准弹性光散射quasi-elastic scattering，测量光强的波动随时间的变化粒子的布朗运动Brownian motion导致光强的波动光子相关器correlator将光强的波动转化为相关方程相关方程检测光强波动的的速度，从而我们得到粒子的扩散速度信息和粒子的粒径d(h)从相关方程我们还可以得到尺寸的分布信息DLS also named as pcs, quasi-elastic light scattering is to measure the fluctuation of the light scattering caused by the Brownian motion of the particles.From DLS we get the dynamic properties of the solute particles known as Brownian motion.The dynamic information of the particles is derived from an autocorrelation of the intensity trace recorded during the experiment.
12 相关方程表示随时间变化的相关性质 Time g2 1 = 0 Time 1 Time g2 = 1 Time Time 1 2 g2 IntensityTimeg21 = 0Time1Timeg2Intensity = 1TimeTime12g2Intensity在起始时刻，光强乘以其自身，相关性能最好，定义为一随着时间变化，光强的相关性越来越差，最后在无限长时刻，相关性为0 = 2TimeTimeIntensityTime123g2 =
13 相关方程（曲线） 初始斜率依赖于粒子大小 衰减的过程与粒子尺寸的分布相关 基线是否归零告诉我们是否有灰尘的存在 截距 This slide shows a schematic of a correlogram and illustrates the type of information that can be gained.Firstly, the time when the correlation starts to significantly decay indicates the mean size of the sample.Secondly, the gradient of the decay indicates the polydispersity of the sample. The steeper the gradient more monodisperse the sample is. Conversely, the more extended the decay becomes, the greater the sample polydispersity.Thirdly, the baseline of the correlation function gives information about the presence of large particles and or aggregates in the sample.The extrapolation of the data to zero time results in an intercept on the Y axis. This is the signal to noise ratio of the measurement. In the example shown in this slide, the intercept value is approximately zero point seven six. That is, the signal to noise ratio is zero point seven six for this measurement. As discussed earlier, a perfect signal would give an intercept value of 1. This is not possible during a real measurement as there is inherently a certain amount of noise present which reduces the intercept value obtained. The various reasons for the reduction in the intercept will discussed in more detail in future modules.基线是否归零告诉我们是否有灰尘的存在
14 光强波动，相关函数和粒径分布 Small Particles Large Particles Apply Algorithm CorrelateApplyAlgorithmTime (s)Intensity (kcps)SmallParticlesCorrelateApplyAlgorithmTime (s)Intensity (kcps)LargeParticles
15 STOKES-EINSTEIN EQUATION D 为扩散系数d(h) 为流体力学直径kB 为波尔兹曼常数T 为绝对温度h 为粘度这里我们通过。。。。。方程将粒子的运动速度和尺寸联系起来2
30 Zetasizer Nano 软件中的尺寸分布分析 由DLS而得的基本的尺寸分布，是一个根据光强的贡献率，并使用 (NNLS) 分析方法得到的分布尺寸分布被表示为一个散射光相对光强对于对应的粒子尺寸的曲线默认地，在尺寸分布中最多可以出现70个等级The primary size distribution obtained from a dynamic light scattering measurement is the intensity-weighted distribution obtained from the chosen non-negative least squares analysis.This size distribution is displayed as a plot of the relative intensity of light scattered by particles (on the Y axis) versus various size classes (on the X axis).By default, there are 70 size classes which are logarithmically spaced.
31 Zetasizer Nano 软件中的尺寸分布分析 Here is an example of an intensity size distribution obtained from a dynamic light scattering measurement.The table shows the 70 size classes which are logarithmically spaced between a lower limit of zero point four nanometres and an upper limit of 10 microns on the X axis. The Y axis consists of the relative percentage of light scattered by particles in each of the size classes.The plot at the top right of the slide is the graphical representation of this data as a histogram. By default, the distribution is displayed as a frequency curve in the Nano software. An example is shown in the bottom right of the slide.There are various size distributions available in the Zetasizer Nano software and we will now look at each one in more detail.
32 光强粒度分布 体积粒度分布 数量粒度分布 从动态光散射得到最初的结果 结果基于粒子的散射光强度 对于大的粒子和灰尘十分敏感 分析样品的特性仅仅需要媒体的粘度和折光指数体积粒度分布使用光强分布数据应用Mie theory演算而来等同于质量粒度分布换算过程需要粒子的光学性质粒子的折光指数粒子对光的吸收率The number size distribution is also derived from the intensity size distribution using Mie theory and the optical properties of the particles are also required to make this transformation.数量粒度分布使用光强分布数据应用Mie theory演算而来换算过程需要粒子的光学性质粒子的折光指数粒子对光的吸收率
33 动态光散射 DLS 的粒子尺度分布 光强分布，体积分布和数量分布之间的相互转换基于以下前提: 所有的粒子都是球型的所有的粒子都是均匀的，且密度相同光学性质已知（折光指数，吸收率）动态光散射 DLS 技术往往高估分布峰的宽度，这个影响可以从体积分布和数量分布的相互转换过程中体现体积和数量分布中，峰的平均值和分布宽度只能用来估计成分的相对量。While the transformation of the measured intensity distribution to volume or number seems straightforward, DLS users are strongly cautioned to be careful not to over analyse the results.Transformation from intensity to volume or number makes the following assumptions: (pause) all particles are spherical (pause) all particles have an homogenous and equivalent density (pause) the optical properties are known – that is the particle refractive index and absorbance and (pause) there is no error in the intensity distribution.The DLS technique tends to overestimate the width of the peaks in the distribution and this effect is magnified in the transformations to volume and number.The volume and number size distributions should only be used for estimating the relative amounts of material in separate peaks as the means and particularly the widths are less reliable. This will be illustrated with an example later in this module.
34 动态光散射 DLS 的粒子尺度分布 如果光强分布是一个相对平滑的峰，那么光强分布和转化得到的体积分布以及数量分布将会比较相似 如果光强分布中有一条非常明显的尾巴，或是多于一个分布峰，那么转化得到的体积和数量分布将会非常不同，并且会更真实地展现尾巴和其它峰总的来说 d(intensity) > d(volume) > d(number)Let us go on to discuss the relationship between the intensity, volume and number size distributions.If the intensity distribution is a fairly smooth peak, conversion into volume will give a very similar distribution.If the intensity plot shows a substantial tail, or more than one peak, then the volume distribution will be quite different and give a more realistic view of the importance of the tail or second peak.In general the intensity distribution will always be larger than the volume distribution which, in turn, will be larger than the number distribution.
36 光强，体积和数量分布: 例子 60nm 和 220nm 聚苯乙烯乳液标样1:1 体积混合 z-均直径 = 168nm PDI = 0.215 Peak 1Peak 2Mean (nm)%Intensity23186.365.813.7Volume23250.361.849.7Number1842.658.297.4To illustrate the relationship between intensity, volume and number size distributions, here is an example of a mixture of different sized polystyrene latex standards. These latex standards were mixed in equal volumes. This record is contained in the example results file which is provided with the Zetasizer Nano software. The z-average diameter obtained was one hundred and sixty eight nanometres with a polydispersity index value of zero point two one five.If we first look at the intensity size distribution, we find that the main peak has a mean diameter of two hundred and thirty one nanometres. This peak constitutes around 86 percent of the distribution. This is because the larger particles in the sample are scattering more light compared to the smaller populationWhen the distribution is viewed in volume, the percentage of each peaks is approximately 50%, which corresponds with the ratio at which the 2 latex standards were mixed at.When the result is viewed in number, the vast majority of the distribution is contained in the smaller sized peak. So if this sample was viewed under an electron microscope, the smaller particles would be dominant. That is, on a number basis, the sample appears to consist mainly of the smaller population of particles. The intensity distribution however, detects the presence of larger particles which may be missed by a number based technique.
37 体积和数量分布: 建议 建议在报告个分布峰所对应的尺寸时，使用光强分布曲线的结果 在报告各个峰的相对数量时，使用体积或者是数量分布 Peak As we have discussed in a previous slide, DLS tends to overestimate the width of the peaks in the distribution and this will become even more significant upon transformation into volume or number. In addition, we have seen that a small amount of large particles present in the sample will dominate the intensity size distribution obtained from a DLS measurement. Therefore, it is recommended to use the intensity size distribution for reporting the size of each mode in the distribution but to the use volume or number data for reporting the relative amounts of each particle family in the sample.This recommendation can be highlighted with the example shown here. The intensity and volume particle size distributions shown in this slide were derived from a 2 to 1 volume mixture of 60 and 220nm polystyrene latex standards. The intensity particle size distribution shows a bimodal with peak means of 59 and 220 nanometres respectively. The relative percentages of each mode based upon the intensity data are 21 and 79 percent respectively with the larger peak being due to the larger sized population. However, conversion to volume gives relative percentages of 67 and 33 percent respectively. Therefore, the recommended way of reporting this result is to use the peak mean diameters from the intensity size distribution together with the relative percentage values obtained from the volume distribution.PeakDI (nm)% Int% Wt159216722207933
46 International Standard ISO 13321 (1996) 样品要求样品应该较好的分散在液体媒体中理想条件下，分散剂应具备以下条件:透明和溶质粒子有不同的折光指数应和溶质粒子相匹配 (也就是：不会导致溶胀, 解析或者缔合掌握准确的折光指数和粘度，误差小于0.5%干净且可以被过滤International Standard ISO (1996)Let’s begin by discussing the sample requirements.The sample should consist of well-dispersed particles in a liquid medium.Ideally, the dispersant should meet the following requirements:It should be transparent.It should have a different refractive index from the particles.It should be compatible with the particles (i.e. not cause swelling, dissolution or aggregation).Its refractive index and viscosity should be known with an accuracy better than 0.5%.It should be clean and filterable.These recommendations are made in the International Standard on dynamic light scattering I S O one three three two one.
47 动态光散射对粒子尺寸的下限 依赖于： 粒子相对于溶剂产生的剩余光散射强度 仪器敏感度 折光指数样品浓度仪器敏感度激光强度和波长检测器敏感度仪器的光学构造We now discuss what the lower and upper size limits of dynamic light scattering are.We start with the lower size limit of the technique. It is dependent upon the amount of excess scattered light the particles generate compared to the suspending medium and on the sensitivity of the instrument.The excess scattering generated will depend on the relative refractive index and sample concentration. The relative refractive index is the particle refractive index compared to the dispersant refractive index.The sensitivity of the instrument will be governed by the laser power and wavelength, the sensitivity of the detector and optical configuration of the system. A backscatter instrument such as the Nano S will be able to measure smaller particles more easily than a conventional 90 degree instrument such as the Nano S90.The lower size limit is typically 2nm
48 动态光散射对粒子尺寸的上限 DLS 测量粒子无规则的热运动/ 布朗运动 （Brownian motion ） 若粒子不进行无规则运动，则仪器无法应用粒子尺寸的上限定义于沉淀行为的开始因此上限取决于样品 – 应考虑粒子和分散剂的密度使用更高粘度的分散剂去阻止或者降低粒子的沉淀速度没有任何优势，因为布朗运动的速度将会被等同的降低We move on to discuss what the upper size limit of the technique is.Dynamic light scattering measures the random motion of particles undergoing Brownian motion and the technique will not be applicable when the particle motion is not random.Therefore, the upper size limit is normally defined by the onset of particle sedimentation and is sample dependent. Both the particle and dispersant density have to be considered. For example, a uranium colloid with a density approximately equal to 19 may have an upper size limit of only 50nm, whereas for a liposome, whose density is virtually the same as water, this upper limit may be up to 5 microns.There is no advantage in suspending a material in a more viscous medium to try to reduce or prevent sedimentation. The benefit of doing this is offset by the fact that Brownian motion is reduced by exactly the same amount.
49 样品浓度总结 从动态光散射得到的样品尺寸应该不依赖于浓度 (ISO 13321) 每种样品都有其理想的测试浓度范围 如果浓度太低，可能散射光强不足以进行试验这种状况不太可能出现在Nano S/Nano ZS系列中，除非在一些极端条件下如果样品浓度太高，实验结果可能会依赖于浓度为了得到正确的尺寸信息，可能会需要在不同的浓度下检测样品尺寸We now move on to discuss the lower and upper concentration limits of dynamic light scattering.The result obtained from a DLS measurement should be independent of sample concentration – this is highlighted in I S O one three three two one.Each type of sample material has its own ideal range of concentration where measurements should be made.If the concentration is too low, there may not be enough light scattered to make a measurement.This is unlikely in the Nano S/Nano ZS except in extreme circumstances.If the concentration is too high, the result may not be independent of sample concentration.During method development, determining the correct sample concentration may involve several size measurements at different concentrations.
50 样品浓度下限 依赖于： 粒子相对于溶剂产生的剩余光散射强度 仪器敏感度 折光指数样品浓度仪器敏感度激光强度和波长检测器敏感度仪器的光学构造Let us begin by looking at the lower concentration limit of dynamic light scattering. The factors influencing the lower concentration limit of the technique are the same as for the lower size limit. They are the amount of excess scattered light the particles generate compared to the suspending medium and the sensitivity of the instrument.A backscatter instrument such as the Nano S will be able to measure lower sample concentrations than a conventional 90 degree instrument such as the Nano S90 due to the increased sensitivity associated with backscatter detection.The lower size limit is typically 2nm
51 样品浓度上限 对于高浓度样品，由动态光散射测得的表观尺寸可能会受到不同因素的影响 多重光散射 – 检测到的散射光经过多个粒子散射 扩散受限 – 其他粒子的存在使得自由扩散受到限制聚集效应 – 依赖于浓度的聚集效应应电力作用 – 带电粒子的双电层相互重叠，因而粒子间有不可忽视的相互作用。这种相互作用将影响平移扩散At high sample concentrations, the apparent size reported from a dynamic light scattering measurement may be influenced by various factors.Firstly, there could be multiple scattering effects. This is where light scattered from diffusing particles is re-scattered by other particles.Secondly, there may be restricted diffusion. This is where the presence of other particles blocks or hinders free particle diffusion.Thirdly, aggregation effects might be present where there is a concentration dependent aggregation of primary particles.Finally, there may be electrostatic interactions occurring. This is where overlapping electric fields lead to soft particle interactions that can influence the translational diffusion.
52 样品浓度上限 表观 z-Average 直径 (nm) 样品浓度 低 高 可测量浓度 多次光散射 Nano S 可测量浓度 When multiple scattering is insignificant the size will be independent of concentration.The plot shown in this slide illustrates the effect of sample concentration on the mean diameter obtained for both a 90 degree and backscatter instrument.In both instruments, the size obtained is independent of concentration when the sample concentration is low. However, as the concentration of the sample is increased, the presence of multiple scattering will begin to influence the size obtained. In a backscatter instrument such as the Nano S, the concentration over which the sample can be measured correctly is greatly extended compared to the 90 degree system due to the reduction of multiple scattering effects.Instruments which have backscatter detection extend the concentration over which samples can be measured before seeing the effect of multiple scattering.Nano S90低高样品浓度
53 二氧化硅浆料的粒径以及分散系数随浓度的变化 样品浓度上限二氧化硅浆料的粒径以及分散系数随浓度的变化When multiple scattering is insignificant the size will be independent of concentration.The plot shown in this slide illustrates the effect of sample concentration on the mean diameter obtained for both a 90 degree and backscatter instrument.In both instruments, the size obtained is independent of concentration when the sample concentration is low. However, as the concentration of the sample is increased, the presence of multiple scattering will begin to influence the size obtained. In a backscatter instrument such as the Nano S, the concentration over which the sample can be measured correctly is greatly extended compared to the 90 degree system due to the reduction of multiple scattering effects.Instruments which have backscatter detection extend the concentration over which samples can be measured before seeing the effect of multiple scattering.
54 推荐样品浓度 粒子尺寸 最小推荐浓度 最大推荐浓度 Nano S90/ZS90 Nano S/ZS < 10nm 5mg/ml Only limited by the sample material interaction (gelation, aggregation)10nm to 100nm1mg/ml0.1mg/ml0.1% w/v5% w/v (assuming a density of 1gcm-3)100nm to 1μm0.01mg/ml0.01% w/v1% w/v (assuming a density of 1gcm-3)> 1μmAn important factor in determining the maximum and minimum concentrations the sample can be measured at is the size of the particles.This table is an approximate guide for obtaining results which are independent of concentration for samples with a density near to 1 gramme per cubic centimetre.If such concentrations cannot be selected easily, it is recommended that various concentrations of the sample should be measured in order to determine if concentration dependent effects such as particle-particle interactions or multiple scattering, are present.
56 注入溶液 只用干净的样品池！ 缓慢注入溶液以避免气泡 如果使用注射管滤膜过滤样品，请放弃开始的几滴溶液以避免在滤膜下面的灰尘进入样品池 使用滴液管，同时倾斜样品池如果使用注射管滤膜过滤样品，请放弃开始的几滴溶液以避免在滤膜下面的灰尘进入样品池用盖子将样品池封住Now we discuss the filling of a sizing cell. When filling the cell, there are several actions to consider. It is recommended that only clean cells should be used and all size cells should be rinsed or cleaned with filtered dispersant.The cell should be filled slowly to avoid air bubbles. Using a pipette and tilting the cell at an angle will aid with this.If using syringe filters for the dispersant, discard the first few drops in case of any residual dust particles in the filter that may contaminate the dispersant.A minimum sample volume must be provided in the cell for successful measurements to be made. This minimum volume depends upon the actual cell type and it is easier to ensure a certain depth of the sample in the cell. This minimum is 10 millimetres from the bottom of the cell. The measurement is made 8 millimetres from the bottom of the cuvette.It is recommended that the maximum level should be no more than 15 millimetres from the bottom of the cell. This is to minimise thermal gradients within the sample which will reduce the accuracy of the temperature control.The level of the sample in the cuvette can be checked against the figure on the inside of cell area lid.Finally, when the cell has been filled with the sample, it is advisable to cap the cell to avoid dust contaminating the sample.
57 将样品池放入仪器The size cell is inserted into the instrument by firstly pushing the button on the front to open the cell area lid.Secondly, the cell is pushed into the cell holder until it stops. The orientation of the cell in the instrument is very important as some cells have opaque surfaces as well as polished optical surfaces. A polished optical surface must be facing the front of the instrument. The polystyrene disposable cells have a small triangle at the top to indicate the side that should face the front of the instrument.If a flow cell is used, insert the sample tubes into the threaded inserts and screw into the top of the flow cell and then push both tubes down into the pinch valve on the side of the cell area.Thirdly, place the thermal cap over the cell. Do not fit it if you are using the flow cell.Fourthly, close the cell area lid.It is worth noting here that some cells, particularly the glass and quartz ones, can feel quite tight when being inserted into the cell area. It is important that the cell is inserted all of the way into the cell holder. To ensure that this is the case, press firmly down on the top of the cell.
58 样品制备: 稀释 如果样品浓度很高，则需要将溶液稀释 稀释样品时须注意保证保持样品原来的性质，如吸附在粒子表面的物质和原溶液之间的化学/物理平衡稀释溶液应和原来的样品溶液保持相同的性质如果样品很多，稀释液可以由过滤或者离心原来的样品溶液除去溶质而得到如果样品很少，稀释液应尽量按原溶液性质制备We now move on to discuss some important points in the preparation of samples for successful dynamic light scattering measurements.If the sample is too concentrated, it will need to be diluted.Dilution needs to be carefully performed to ensure that the equilibrium of any absorbed species between the particle surface and bulk solution is preserved.The diluent should be the same as the continuous phase of the original sample.The diluent could be obtained by filtering or centrifuging the original sample and obtaining a clear supernatant suitable for dilution.If this is not possible, the continuous phase should be made up to be as close as possible to that of the sample.
59 样品制备: 过滤 灰尘是光散射实验最主要的问题之一，灰尘的存在可能导致测试失败 为了避免灰尘的影响，样品溶液在测试之前应该被适当的过滤 商业化的注射管过滤膜网眼的尺寸通常从 1μm 到 20nmDust is one of the major problems in DLS measurements and may bias the results obtained.To avoid any possible dust contamination during dilution, the medium should be ideally filtered.Commercial syringe filters are available for use with pore sizes ranging from 1 micron down to 20 nanometres.
60 校准和检查 动态光散射Dynamic light scattering是一种绝对测试，因此不需要校准 然而光学仪器如光路，有时会因环境（如温度，外力）改变而改变，应当定时检查检查的频率以用户的使用方式和需求而定检查可由检测标样（聚苯乙烯乳液）来完成马尔文公司推荐Duke Scientific Corporation (www.dukesci.com) 的聚苯乙烯乳液为标准样品标准起源于 NIST – the National Institute of Standards and Technology (www.nist.gov)Read slide
90 静电力排斥（ELECTROSTATIC） 维持分散体系的稳定性粒子稳定的存在于溶液中主要基于以下两种机制:静电力排斥（ELECTROSTATIC）Easy to measure the controlling parameter (zeta potential)ReversibleMay only require change in pH or ion concentration（位阻效应）STERICSimple, but limited optionsIrreversibleAn extra component
93 什么是Zeta电位? Zeta电位同时依赖于粒子表面和分散剂的化学性质 对于静电力稳定的分散体系，通常是Zeta电位越高，体系越稳定 体系稳定与否通常以Zeta电位是否大于 30mV为标准Zeta电位是粒子间静电力相互作用的标尺，可以被用来预测分散体系的稳定性以及存储时间The one idea to remember about zeta potential, is that it is not the same parameter as surface charge or surface potential. This difference may not be very important for simple systems such as a dispersed mineral oxide in demineralised water, but as soon as there are other components in the dispersion that interact with the surface.These components can change the magnitude of the zeta potential or even reverse the sign of the charge.The zeta potential is the value of the potential at a distance from the surface known as the slipping plane.This is the distance within which the ions move as part of the particle, and from the charge point of view defines the particle.Particles interact according to the magnitude of the zeta potential not their surface change.
107 M3 测试技术 高频电场转换1000Hz能够准确地测量zeta电位的平均值，但是分辨率较低 -50mV-110低频电场的转换可以给出更好的分辨率但是受到电渗的影响This is the measurement from fast filed reversalBy combining the fast mode and slow mode measurement, we get better resolution and also the speed of the electroosmotic flow通过M3测试技术，结合高频和低频电场的转换，我们既得到准确地平均zeta电位，又得到了较高的分辨率