INDIAN INSTITUTE OF TECHNOLOGY ROORKEE NPTEL NPTEL ONLINE CERTIFICATION COURSE. Biomedical Nanotechnology. Lec-05 Characterisation of Nanoparticles

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1 INDIAN INSTITUTE OF TECHNOLOGY ROORKEE NPTEL NPTEL ONLINE CERTIFICATION COURSE Biomedical Nanotechnology Lec-05 Characterisation of Nanoparticles Dr. P. Gopinath Department of Biotechnology Indian Institute of technology Roorkee Hello everyone, I welcome you all to the fifth lecture of this course. So today we are going to see the characterization of nanomaterials. So there are several methods available for characterization of nanomaterials. But in this lecture, we are going to focus mainly on UV visible spectroscopy, dynamic light scattering (DLS), zeta potential, and we are also going to focus on electron microscopy like TEM, SEM, and atomic force microscopy. When you synthesize any metal nanoparticle, the first thing that you observe is the formation of color. What is the reason for formation of such beautiful colors? The reason is surface plasmon resonance. So in the previous lectures, I have already explained about surface plasmon resonance. Usually, the bulk metal is shiny due to the electron cloud. These electrons have dual nature and so if the light of matching wavelength produces the resonance, then electron cloud starts vibrating. For nanoparticles, this wavelength lies in the visible region. That is why, you are getting different kinds of colors. When the incoming radiation induces the oscillation of conduction electrons, it leads to surface Plasmon resonance. When you study these metal nanoparticles under the UV visible spectroscopy, you will get different kinds of peaks depending on the size as well as shape of the nanoparticles. There will be a red shift with an increase in the size of the particle.

2 For example, consider the gold nanoparticles. Usually they are ruby red in color. But if they are agglomerated or aggregated, it will show purple color. When it is studied under the UV visible spectroscopy, for uniformly dispersed gold nanoparticles you will get a peak at around 550 nm and for the agglomerated or aggregated particles, you will get a peak at around 650 nm. Hence, for any metal nanoparticles, the first step of characterization is the UV visible spectroscopy. For any nanoparticle, the stability and size distribution is very very important. The stability of the nanoparticle can be studied by zeta potential measurement. So what is zeta potential? The zeta potential of the particle is the overall charge that the particle acquires in a particular medium. The zeta potential balance should be ± 30 mv, which indicates good stability of the nanoparticles. The zeta potential depends on the ph and electrolyte concentration of the particular material. For measuring the zeta potential, we have to use a particular kind of cuvette and when you put this cuvette inside this zeta sizer, it will give the zeta potential of your nanoparticles. Now, let us see about dynamic light scattering. Using this same equipment we can also measure the dynamic light scattering of the particles. This is for direct determination of particle size in the solution. If you want to measure the particle size in solution, we have to use the DLS that is called the dynamic light scattering. Here, the size of the nanoparticles will include even the stabilizers bound to the molecule, which cannot be seen by the TEM. The principle is that the scattering of laser light is due to the Brownian motion of the particles, due to which there will be a temporal fluctuation in the intensity, which measures the hydrodynamic diameter of the nanoparticle. It is very much sensitive to other biological molecules and it can measure particles of size ranging from 0.6 nm to 6 micron. But the drawback of this DLS is, we cannot get any information about the particle shape. From transmission electron microscope or TEM picture, you will get the exact size of the particle but in case of DLS, that is dynamic light scattering, you will get the hydrodynamic diameter. So

3 the particle size will be slightly bigger because you are determining the particle size in the solution. So before we understand what zeta potential is, let us see some of the definitions. This is your highly negative colloid metal particle. The particle surface is in blue color and the red color is the stern layer. The stern layer is a rigid layer of ions, which is tightly bound to the particle and these ions travel with the particle. This black color minor line is also called as slipping plane or it is a plane of hydrodynamic shape. This is the boundary of the stern layer. And ions beyond this shear plane do not travel with the particle. This green color is the defused layer, also called electrical double layer. So here, the ionic concentration is not the same as in the bulk. So, there is a gradient in concentration of ions outward from the particle until it matches with the bulk. The electrical potential that exits at the slipping plane is called as Zeta potential. This zeta potential mainly depends on the stabilizing agent as well as the coating agent or capping agent. The magnitude of the zeta potential gives an indication of the potential stability of the colloidal system. Suppose if all the particles have large zeta potential that means if it is more than + 30 or if it is more than -30, then is it will repel each other. The particles having uniform charges will repel each other and there is a dispersion stability. Suppose if the particles have lower zeta potential values, that is, no force to prevent the particles from coming together. This will cause aggregation or agglomeration. That is why they made dividing lines between stable and unstable aqueous dispersion. So particles having zeta potential values between -30 and +30 mv are highly stable. This zeta potential is highly depended on the ph. So in this example you can see here if the ph is below 4 as well as ph above 7.5, then the nanoparticles are stable. Now, let us see how we can use microscopy as the means of characterization of nanoparticles. So first what is microscopy? Microscopy is any technique that produce visible images of structures or details, which are small or otherwise cannot be seen by the human eye. So why do we need microscope? Because in order to effectively study something or build something, we need the microscope so that we can understand the structure, and shape. By understanding the structure

4 and shape, we can modify them. Because the nanoparticles are smaller in size, we need a more powerful microscope to understand their structures. What are the things that you will understand by this characterization of the nanomaterials? You will get an idea about the dimension and the structure of the nanoparticles. You will also get the idea of the kind of materials that makes up the elemental proportions. We can also know about the physical and chemical properties of the nanoparticles by characterizing them. Now, let us briefly see the origin of microscopy. In the first century, Romans invented the glass and they experimented it with various shapes and converging lenses. In 1590, a Dutch eyeglass maker made a compound microscope. In the mid of 17 th century, Anton Van Leeuwenhock first observed the bacteria and protozoa under a small, simple microscope. In the late 17 th century, Robert Hooke made a lot of modifications to the microscope and created a kind of compound microscope. For the next 200 years, this optical microscope played a major role in biology and other fields. In the late 19 th century, Ernst showed that further improvements in the magnification potential of the optical microscopes was fundamentally limited by the wave length of the light. This showed that the wave length of the light is very important and a deciding factor for the magnification of optical microscopes. So, in 1931, Ernst Ruska invented the electron microscope and in 1938 using the TEM electron microscope, they reached the resolution of 10 nanometer. In the year 1940, they were able to attain a resolution of 2.4nm and in 1945, they reached 1 nm resolution. In 1981, scanning tunneling microscope was invented and in 1986, atomic force microscope was developed by the same group. Now, let us see the basic principles of SEM and TEM. SEM, that is, scanning electron microscope is similar to a scanner. It will scan the samples and it will give the image. In transmission electron microscopy or TEM, the electrons will be transmitted and we will get the image.

5 There are two modes in TEM, bright field as well as dark field. In electron microscope, the electrons pass through a very thin sample to form the image. Transmission electron microscope is a complex viewing system equipped with a set of electromagnetic lenses. These lenses are used to control the imaging electrons in order to generate an extremely fine structural details that are usually recorded on a photographic film. Here the eliminating electrons pass through the specimens and that is why the information is said to be a transmitted image. The modern TEM can achieve magnification of even one million times with the resolution of 0.1nm. For transmission electron microscope, we need a thin specimen so that the electron beam can pass through it and give information about the size and morphology of the nanoparticles. It can also give information about the crystallographic nature as well as composition details. The sample is placed on a TEM grid. A TEM grid has two sides, a shining side and a non-shining side. The sample has to be deposited on the non-shining side, which is coated with the carbon. Now, let us see the principle behind the TEM. It is similar to an optical microscope but here instead of using light, you will be using an electron beam as the light source. The condenser apertures stop the high angle electron beam which is the first step in improving the contrast. The objective aperture and the selected area aperture are optional, but they can enhance the contrast further by blocking high angle diffracted electrons. What are the advantage of this? We can study the size and morphology of even non conducting samples like polymers, ceramics and biological samples. The TEM is made up of a number of different systems. It forms one functional unit that is capable of orienting and imaging extremely thin specimens. It has an illuminating system as well as a specimen manipulating system. The illumination system consists of an electron gun and condenser lens, which gives rise to and control the amount of radiation striking the specimen. The specimen manipulation system is composed of specimen stage and specimen holders, and also other related hardware that are necessary for orienting the specimen inside or outside the microscope.

6 The TEM grid will be placed in the variable aperture holder of TEM and the O-ring will be sealed. This permits the aperture to be sealed of inside the vacuum of the microscope column. When you study this silver nanoparticle under the transmission electron microscope, we can see black color dots which are the silver nanoparticle. We have to include the scale bar and using the scale bar in image J software, we can measure the particle size. Also, we can study the diffraction pattern of the sample by switching the sample from image mood to diffraction mood. This is done by changing this strength of the intermediate lens. So, when you see the electron diffraction pattern of the sample, this can give crystallographic information about the material. It can determine whether your material is amorphous or crystalline or polycrystalline and it can measure your crystallographic information quickly as well as effectively. In case of amorphous materials, you will get this kind of diffused ring and in case of crystalline materials, you will get this kind of spotted pattern. In case of polycrystalline materials, you will get the rings with bright spots. In case of mono crystalline materials, you will get this kind of spots and when you combine this kind of things, you will get rings with the bright spot that is called as polycrystalline material. Another example of diffraction pattern you can see is amorphous carbon. Here, you will get the diffused ring pattern. For polycrystalline aluminium, you will get rings with the spot and single crystal gold you will get this kind of bright spots. TEM is heavily used in material science as well as biological science. We can do micro structure analysis and study the crystal structure. The magnification is very high in TEM, which is a good advantage. But the drawback is, we need a very thin sample. The sample should be transparent and the thickness of the sample should be in the range of 200 nm. Now, let us move on to the other microscope that is scanning electron microscope. So as I told you earlier, here instead of transmitting the electron, the samples will be scanned and the image will be produced. So, it is a microscope that produces the image by using an electron beam, which scans the surface of the specimen inside a vacuum chamber. The SEM functions like an

7 optical microscope but as I told you earlier it uses an electron beam instead of light waves. The SEM uses a series of electromagnetic coils as lens to focus and manipulate the electron beam. Here, the sample should be in a completely dehydrated form and it should be made conductive. By using SEM, we can understand the topography and morphology of the sample. We can also understand the elemental composition, the crystallography and the orientation of the grains. In SEM, the electron beam follows a vertical path through the column of microscope. It makes its way through the electromagnetic lenses, which focus and direct the beam towards the sample. Once the electrons hit the sample, back scattered electrons and secondary electrons are ejected from the sample. The detectors will collect the secondary electron or back scattered electrons and convert them into a signal that is send to a viewing screen. This will produce the image. The voltage should be between 0.1KV to 40KV. For biological samples, the recommended voltage is 10KV and for non-biological samples, we can go for even 20KV. How the SEM works? The electron gun is passing the electron beam. This hits the sample and the back scattered electrons will be amplified and it will display the image. The samples have to be prepared and dried. It should be placed on top of the SEM stub which has a carbon tape. When the electrons hit the sample, the back scattered electrons will be detected by the detector. It will scan the sample one by one and all the back scattered electrons will be detected by the detector and it will take it to the computer. Using the software, it will give the 3 dimensional image. We can compare the optical microscope verses scanning electron microscope. In the optical microscope, we will get the two dimensional image for the compound. Under a scanning electron microscope, you will get the three dimensional structure. What are the advantages of using this scanning electron microscope? You can have an improved resolution and also a good depth of field. The limitations are that it requires a vacuum. The equipment is also expensive. We can also do energy dispersive analysis. The principle is each element has unique electronic structure and this interacts uniquely with the electromagnetic radiation. This is useful for

8 chemical characterization of a substance. You will get the details about the elements present in the sample by using this EDS analysis. Next, we will move on to the scanning probe microscope. So under this scanning probe microscope, there are two types one is scanning tunneling microscope and other one is atomic force microscope. Here, we can monitor the interaction between the probe and the sample surface. So briefly on this history of STM, the scanning tunneling microscope was invented by G. Binnig. They were awarded noble prize in the year After few years, they invented the atomic force microscope. Currently, the AFM is the most common form of the scanning probe microscopy. In the scanning tunneling microscope or STM, we are going to monitor the electron tunneling current between a probe and a sample surface. So what is electron tunneling? It occurs over a short distance. The tunneling current depends on the distance between the STM probe and the sample surface. So, we are going to measure the tunneling current between the tip as well as surface of the sample. Here the basic principle is tunneling. The tunneling current flows between the tip and the sample when separated by less than 100 nm. This tunneling current gives us atomic information about the surface when the tip scans the sample. What are the challenges with the STM? It works primarily with conducting materials and there is chances of vibration interference as soon as there is contamination by dust particles and other pollutants in the air or due to the chemical reactivity of the samples. Now we will discuss about atomic force microscopy. Using atomic force microscope, we can image any kind of surfaces including polymer, ceramics, glass and biological samples. The atomic force microscope monitors the force of attraction and repulsion between the probe and the sample surface. Here, the tip is attached to a cantilever, which moves up and down in response to the forces of attraction or repulsion with the sample surface. And this movement of the cantilever is related by the laser as well as the photo detector.

9 When the tip moves up and down over the sample surface, the position of the laser beam will be changed, which will be measured using a photo detector. The image will be formed according to this measurement. We can select the tip according to the type of sample as well as its applications. There are three types of tips- a normal tip, a super tip and an ultra-lever tip. The cantilever is designed with a very low spring constant. This means it is easier to bend and it is very sensitive to even a small force. Hence, when the laser is focused to reflect on the cantilever and on those sensors, the image will be produced. Here the position of the beam in the sensor measures the deflection of the cantilever and in turn, the force between the tip and the sample. The force is measure using the Hook s law. This cantilever tip is going up and down in the sample and the laser beam is also getting deflected according to the nature of the sample. If there is no interaction between the tip and the sample, the cantilever is not deflected. In this case, based on the position of the laser beam, the image will be produced. The AFM has three different modes-contact mode, non-contact mode and tapping mode. In the contact mode, you will get high resolution image but the problem is, it will damage the sample as the cantilever tip is touching the sample. It can also measure the frictional forces. In the noncontact mode, the image is of lower resolution. But it will not cause any damage to the sample as it is not touching the sample. In the tapping mode, it will give a better resolution with a minimal damage to the sample. In the contact mode, the distance between the tip and surface is 0.5 nm and in the tapping mode the distance is approximately like 2 nm. In the non-contact mode, the tip-surface separation can even be 10 nm. So in case of contact mode, this cantilever tip is touching your samples. In case of tapping mode the repulsive force will be high and in case of non-contact mode the attractive force will be low.

10 Let us see these modes in detail. In the contact mode, the tip will be in continuous contact with your sample. This is your tip and this is your sample. It is touching your sample and it can give the details of your sample. It is mainly useful for hard samples. The imaging can be done in air and liquid and it will give a high resolution image. However, the drawback is, it can damage the sample or it can damage the cantilever tip. Next one is force spectroscopy mode. Here there will be consecutive cycles of approach and retraction of the tip. We can measure the interaction forces here. The next mode is intermittent or tapping mode. Here, the cantilever tip will be oscillating. It will be oscillating in a uniform frequency so that the tip is touching the surface gently and frequently. It is mainly useful for the biological samples. Here, we can do the imaging in the air and liquid and it will give you a good resolution. The third one is the non-contact mode. Even here, we will be having a oscillating cantilever but here the tip is not in contact with sample. This will be mainly useful for the soft samples. In noncontact mode, the imaging is done in the vacuum. Here the range of distance will be between 50A to 150A. How do we prepare the sample for the AFM? We need a completely flat and rigid surface. We can use mica which is has atomically flat surface. We can also use that SiO2 glass. The typical sample size should be 1cm 2. For liquid samples, it should be between 1 μl to 100 μl. In AFM images, the control cells have a smooth surface. The cells treated with nanoparticles have rough surface. Due to treatment of these cells with the nanoparticles, the cell membrane get damaged and the roughness of the cells got increased. We can measure the surface roughness using this atomic force microscope. By using the AFM, we can study the topographical surface and determine the roughness of the surface sample. We can also measure the thickness of the crystal growth layer. Another advantage of using AFM is, we can image non-conducting samples such as DNA and proteins.

11 For most of biological samples we will be using the AFM. Because if you are using the SEM or any other microscope, you have to do some kind of treatment. For example, you have to sputter with gold to make the biological material into a conducting material. Only then SEM can be used to image the sample. But in case of AFM, you can even use the non-conducting proteins and DNA. We can also study the dynamic behavior of living and fixed cells in case of the electron microscopes like scanning electron microscope or transmission electron microscope. However, we cannot study a living cell. We have to fix the cell and it should be in the dried form to be studied. In case of AFM, we can study the living cells also. The samples can be in liquid or gas medium. Here for most of the modes, vacuum is not required and we do not have to do any special treatment of samples. As I told you like for SEM, we have to do gold sputtering to make the sample conductive. Here also, the resolution is very high. We can reach 0.1 nm vertically and 1nm in the X and Y direction. As a summary, so in this lecture, we have learnt how to characterize the nanomaterials using UV-visible spectroscopy how. We also learnt how to study the hydrodynamic size of the nanoparticles using DLS that is dynamic light scatting and how to study the stability of the nanoparticles using zeta potential. We have also learnt how to prepare samples for electron microscope and how to study them using transmission electron microscope and scanning electron microscope. We have also learnt how to prepare samples for atomic force microscopy and what are the various modes available in atomic force microscopy. So I will end my lecture here. I thank you all for listening this lecture. I will see you in another lecture. For Further Details Contact Coordinator, Educational Technology Cell Indian Institute of Technology Roorkee Roorkee E Mail: etcell.iitrke@gmail.com, etcell@iitr.ernet.in Website:

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