 In current module, we will discuss about separation technique, a brief introduction. Separation technique, which we were discussing in the previous slide on sample preparation technique. As a part of sample preparation, we have to separate our analyte in first phase. Our metrics may be gas, solid, semi-solid, or liquid, and our analyte may be soluble in the metrics and may be as a suspended solid or a coagulate. Separation techniques may be the most basic technique called filtration. If you want to separate solid from liquid, it is called filtration. Filtration is done through simple filter paper, and if the vacuum is applied for it, then we use bookner funnel etc. In this way, the filter paper is folded and used as a one-quarter filter paper. If it is folded further, then we prepare a fluted filter paper through which we can increase the surface area so that our separation becomes better. If your sample or your analyte is insoluble, then we can separate it through filtration. But if it is a suspended solid and has very thin particles, then it will need to be coagulated first, and then we will perform an analysis. Second technique called sublimation. If you want to separate solid from a solid material, it is called sublimation. If our analyte may be sublime, i.e. on a particular temperature, a direct gaseous form can come from a solid material, then we use sublimation. This small apparatus that we are seeing, in which we place our sample in the battery dish, and after heating it, the ammonium chloride vaporizes and sticks with this funnel. If we want to separate the ammonium chloride from the sodium chloride, then it is a very easy method called sublimation. The third technique is called solvent extraction. If you want to separate our liquid sample or solid sample in between two immiscible liquids, it is called solvent extraction. For solvent extraction, then there will be need of immiscible liquids. For example, one liquid is usually water, while the other liquid is used as organic liquid. If organic liquid is denser than water, then its layer will be placed on the bottom side of the separation funnel. If it is lighter, then the layer of water will come down and the layer of our organic solvent will be placed on top. Our matrix or our analyte can also be soluble in water and our analyte can also be soluble in organic solvent. On the basis of its solubility, we choose a particular water or organic layer after which it is used for analysis. Solid phase extraction is another technique through which we enrich the minute and light concentration. For example, if we want to do an analysis of pesticide residues and water is our matrix, then we use this technique to separate the minute quantity of analyte in microgram from 100 of litters. There are four steps through which we separate it. The first step is conditioning. In the second step, we load our sample, i.e. litters of water. Our particular analyte and other than that, some constituents stay on this matrix or column. Through washing, we keep the particular analyte present there, In addition, we separate the rest of the different ions out of it. And in the final step, we get an illusion through which we get pure analyte. To know the concentration of analyte, it is necessary to separate it from the rest of the matrix. That is why the process of this concentration is called solid phase extraction. Chromatographic techniques are also used to separate. There are different types of chromatographic techniques which we will discuss in the next slides or in the next module in detail. For example, paper chromatography, thin layer chromatography, column chromatography. In different types of chromatography, the common thing is our mobile phase or our stationary phase. Mobile phase and stationary phase, sometimes we use liquid, sometimes we use gaseous form. Similarly, stationary phase, sometimes liquid and sometimes solid stationary phase are also used. On this basis, we give different names to our techniques. If liquid, mobile phase and liquid stationary phase are used, then we call it partitioning. If solid stationary phase is used, then it is called adsorption. Paper chromatography is a very basic technique through which we separate and concentrate different analytes. And then we know their quantity. Paper chromatography is also in ascending order in which liquid is in the pool and flows upwards. In this way, two-dimensional chromatography is also done. Flat or horizontal paper chromatography is also performed. And these different techniques we will use to separate. Column chromatography is another type in which our silica gel, which is a stationary phase, is used to separate. Column chromatography is another type in which our silica gel, which is a stationary phase, is packed in a glass column and we pass our analytes or our matrix through this column which is distributed in different parts. As you can see in the color band in this picture, in the glass column too, this band will be separated at their own time and we will perform the work to quantify them. Instrumental techniques that we use are HPLC or gas chromatography. HPLC is based on the same chromatographic technique. That is, if our liquid-mobile phase and liquid-heat stationary phase are there, then HPLC is called high-pressure or high-performance liquid chromatography. And if we are using a gaseous mobile phase and using liquid or solid surface as a stationary phase, then it is called gas chromatography. Apart from these two basic techniques, there are many other spectrophotometric techniques that we use for analysis. In the next module, we will discuss them in detail.