# Zetasizer Nano 系列: 培训课程

## Presentation on theme: "Zetasizer Nano 系列: 培训课程"— Presentation transcript:

Zetasizer Nano 系列: 培训课程

Who Are Malvern Instruments?

Zetasizer Nano 能够测量什么参数？

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

Contents 动态光散射（第一天） 静态光散射及分子量的测定（第二天） Zeta 电位 测量原理（第二天） 测量原理
Nano系列的优化测量位置 由相关曲线得到粒径信息 – 预算法则 样品要求 样品制备 数据解释 静态光散射及分子量的测定（第二天） Zeta 电位 测量原理（第二天）

Zetasizer Nano是如何测试粒子的粒径的?

Nano 的光学构造

Intensity Time g2 1  = 0 Time 1 Time g2 Intensity  = 1 Time Time 1 2 g2 Intensity 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  = 2 Time Time Intensity Time 1 2 3 g2  = 

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

Correlate Apply Algorithm Time (s) Intensity (kcps) Small Particles Correlate Apply Algorithm Time (s) Intensity (kcps) Large Particles

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

STOKES-EINSTEIN EQUATION

2c/b2 为分布系数

z-均 直径 z-均直径(ZD)的定义: 累积距法得到的粒子平均尺寸对应于不同尺寸粒子散射光强的贡献 这里“平均”的概念特指用于光散射试验中

General Purpose Multiple Narrow Modes 这两种算法的差异在于所得到的分布曲线的平滑程度 general purpose 算法适用于大部分分布状况未知的样品 multiple narrow mode 算法适用于分布状况不连续的样品

Zetasizer Nano 软件中的尺寸分布分析

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.

Peak 1 Peak 2 Mean (nm) % Intensity 231 86.3 65.8 13.7 Volume 232 50.3 61.8 49.7 Number 184 2.6 58.2 97.4 To 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 population When 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.

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. Peak DI (nm) % Int % Wt 1 59 21 67 2 220 79 33

Nano 系列中的优化测量位置

Nano 的光路系统

NIBS: 可调整检测位置 小粒子/ 稀溶液 高浓度溶液 减小散射光体积 较大的散射提体积 降低多次散射影响 检测器 激光 凸透镜 样品池

Zetasizer Nano自动水衰减器有11个光学衰减镜片涵盖100% 到 % 的透射率 透射率指到达样品的激光的强度占光源激光强度的百分比 在测量粒子大小的过程中，自动衰减器会自动调节透射光的强度，直到检测器检测到的光强小于 500kcps

0.01 4 99.97 0.03 5 99.9 0.1 6 99.7 0.3 7 99 8 97 9 90 10 70 30 11 100

International Standard ISO 13321 (1996)

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 样品浓度

Only limited by the sample material interaction (gelation, aggregation) 10nm to 100nm 1mg/ml 0.1mg/ml 0.1% w/v 5% w/v (assuming a density of 1gcm-3) 100nm to 1μm 0.01mg/ml 0.01% w/v 1% w/v (assuming a density of 1gcm-3) > 1μm An 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.

z-均直径重复性 多次z-均直径的测试结果误差应在1%-2%之内 z-均直径增长意味着: z-均直径下降意味着: 粒子聚集

Contents 动态光散射 静态光散射及分子量的测定 测量原理 应用实例 Zeta 电位

Mixtures NO

dn/dc = 折光指数对浓度的增量 Rg = 均方旋转半径  = 检测角度 IA = 绝对光强 (I样品 – I溶剂) no = 溶剂 IT = 标准物光强 (toluene) nT = 标准物 (toluene) 折光指数 RT = 标准物瑞利比 (toluene) 者是静态光散射检测方程。 对于小粒子，同场小于激光波长的1/10 p(o)约等于一，也就是说散射光强不再有角度依赖性

1/截距 = 14.6KDa 斜率 = x 10-4

Contents 动态光散射 静态光散射及分子量的测定 Zeta 电位 测量原理 样品制备 样品测试 测试中的选择 数据解释

Zeta电位 Zeta Potential 电泳光散射 Electrophoretic Light Scattering (ELS) 激光多普勒电泳 Laser Doppler Electrophoresis (LDE) 理论概述

Zeta电位(Zeta Potential)
Slipping plane 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 Particle with negative surface charge 影响Zeta电位的因素 Stern layer 对于一个带点粒子，通常对电位由三个定义。由于粒子带点，在粒子的表面的电势称为表面zeta电势。在粒子表面有一层抗衡离子紧密地和表面结合在一起，这个边缘的电势称为stern电势，在外层还有一些离子和带电粒子松散的结合在一起，但是这层离子区别于溶剂中自由运动的离子,这一层电势称作zeta电势 Diffuse layer 影响Zeta电位的因素有： pH变化, 电导率 (浓度，盐的类型) 组成成分浓度的变化 (如高分子，表面活性剂) -100 - { Surface potential Stern potential mV Zeta potential Distance from particle surface

（ELECTROPHORETIC MOBILITY）

Henrys 方程 F(ka) 非极性溶剂 极性溶剂 Huckel 近似 F(ka) = 1.0 Smoluchowski 近似

Nano的光学构造 参考光不通过样品池 衰减镜片调整入射光的光强 这样仪器可以检测很宽浓度范围内的样品

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
-50 mV -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 reversal By 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

FFR（高频电场转换） SFR（低频电场转换） 通过相分析和复利叶变换得到电位的分布 通过相分析得到平均电位 (Ep)

PALS only to obtain mean (Ep) – no distribution or width

Zeta电位（Zeta Potential）

zeta电位试验中的样品测试 Zeta电位测试中对样品的要求不像尺寸测试对样品的要求那样严格，样品可以看上去不很透明

zeta电位试验中的样品测试（SiO2 浆料）

Zeta电位和pH 自动滴定确定等电点 Zeta Potential (mV) pH 等电点 （IEP） 9 2 3 4 5 6 7 8 1
10 11 12 13 14 pH Zeta Potential (mV) +30 -30 等电点 （IEP） 自动滴定确定等电点

TiO2 的pH滴定 zeta size Aggregation at Iso-electric point leads to a size increase.

zeta电位试验中的样品测试 在稀释溶液的过程中应该保持粒子表面的性质 你有稀释溶液吗？ 过滤或者离心一些原溶液，取上清夜作为稀释溶液

Zeta电位的测试中，激光必须穿过样品，因为散射光在向前的角度被检测到 如果样品的浓度太高，被检测到的粒子散射的激光强度会有很大的衰减 为了抵消这些影响，仪器中衰减镜片的位置将被调整到一个比较高的指数，也就是比较高的透射率

Zeta电位（Zeta Potential）

Phase Plot Report Voltage/Current Report Frequency Report 参数-PARAMETERS Conductivity Attenuator

Zeta质量报告 Zeta质量报告对任何一个实验结果进行六项测试

Zeta 电位分布范围限制 默认的Zeta 电位分布范围限制在 +150 到 –150mV

Zeta 电位分布范围限制 有几种原因可能导致这个问题的出现 样品的zeta电位分布很宽 较高的导电率导致施加的电压自动降低

Zeta 电位分布范围限制 Mean zeta = +63mV Wall zeta = +61mV Voltage = 150V

Zeta 电位分布范围限制 Voltage = 100V

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