WEBVTT 1 00:00:00.032 --> 00:00:05.520 2 00:00:05.520 --> 00:00:09.266 Hello everyone, welcome to Swayam, MOOCs course, 3 00:00:09.266 --> 00:00:11.901 this course is on Analytical Techniques. 4 00:00:11.901 --> 00:00:16.306 I am Rajeshwari Morganti, professor at the Department of biochemistry, 5 00:00:16.306 --> 00:00:19.790 All India Institute of Medical Sciences New Delhi. 6 00:00:19.790 --> 00:00:23.837 , this course, analytical techniques starts with the week one 7 00:00:23.837 --> 00:00:27.734 and this topic is the week one, tutorial 1. 8 00:00:27.734 --> 00:00:29.893 which is on aqueous solutions, 9 00:00:29.893 --> 00:00:37.020 acids ,bases,pH and buffers as well as about pH meter. 10 00:00:37.020 --> 00:00:40.710 Before we go into the objective of this tutorial, 11 00:00:40.710 --> 00:00:43.314 I would like to make you appreciate 12 00:00:43.314 --> 00:00:46.028 that this is the most fundamental topic 13 00:00:46.028 --> 00:00:50.028 because any experiment you do your solution has to be 14 00:00:50.028 --> 00:00:54.385 or the buffer has to be correct and the concentration has to be correct. 15 00:00:54.385 --> 00:00:59.290 in this what I'm going to talk about is aqueous solutions. 16 00:00:59.290 --> 00:01:03.671 And what is Aqueous solution? Aqueous solution is typically given 17 00:01:03.671 --> 00:01:07.179 a term for water, where the water is a solvent. 18 00:01:07.179 --> 00:01:11.782 ,what is a solvent ? what is a solution and what is a solute? 19 00:01:11.782 --> 00:01:14.139 These are the terms you will be introduced. 20 00:01:14.139 --> 00:01:18.147 And also the concentration. concentration can be expressed 21 00:01:18.147 --> 00:01:21.004 in n number of ways depending on the application, 22 00:01:21.004 --> 00:01:25.552 sometimes you must have heard of ppm parts per million, parts per billion 23 00:01:25.552 --> 00:01:27.718 or you can even use it in the concentration 24 00:01:27.718 --> 00:01:30.980 in molar concentrations or normality and so on. 25 00:01:30.980 --> 00:01:34.806 ,theseterms will all be introduced to you, 26 00:01:34.806 --> 00:01:38.060 so, that you will recollect and refresh your.. 27 00:01:38.084 --> 00:01:41.403 what you have learned at the UG level? 28 00:01:41.403 --> 00:01:45.332 And also to remind you that the preparing 29 00:01:45.332 --> 00:01:49.990 a buffer is the most important one and aqueous solutions are important. 30 00:01:49.990 --> 00:01:53.967 , coming to the first thing that what is a 31 00:01:53.967 --> 00:01:59.459 solution. a solution can have any number of more than two 32 00:01:59.459 --> 00:02:03.949 components and component is the mixture of two components. 33 00:02:03.949 --> 00:02:06.020 It could be homogeneous mixture 34 00:02:06.020 --> 00:02:08.353 or it could be heterogeneous mixture. 35 00:02:08.353 --> 00:02:10.417 ,the homogeneous mixture can be 36 00:02:10.417 --> 00:02:13.393 for example, liquids to alcohol, and water 37 00:02:13.393 --> 00:02:15.957 or it could be any the heterogeneous mixture, 38 00:02:15.957 --> 00:02:20.457 it can be a solid and a solute and a solvent. 39 00:02:20.457 --> 00:02:22.648 ,these are the terms we will be discussing. 40 00:02:22.648 --> 00:02:26.893 And based on this kind of small brief introduction. 41 00:02:26.893 --> 00:02:29.790 The objectives that are laid out are 42 00:02:29.790 --> 00:02:33.631 expresse the concentration of a solution in different units. 43 00:02:33.631 --> 00:02:36.131 Describe the properties of solutions. 44 00:02:36.131 --> 00:02:39.360 Distinguish between acidic and basic solutions. 45 00:02:39.360 --> 00:02:42.336 Application of Henderson–Hasselbalch equation 46 00:02:42.336 --> 00:02:46.670 to determine the pHand explain the functions of buffers. 47 00:02:46.670 --> 00:02:50.336 Explain the titration curves and also the PK values 48 00:02:50.336 --> 00:02:54.090 with the examples of amino acids etc. 49 00:02:54.090 --> 00:02:57.892 Solution is in chemistry , not a problem and solution 50 00:02:57.892 --> 00:03:01.161 in general english terminology. What When a solution in 51 00:03:01.161 --> 00:03:07.146 came in terms of chemistry, it is a homogeneous mixture of two or it could be more than two 52 00:03:07.146 --> 00:03:10.415 chemically non-interacting substances 53 00:03:10.415 --> 00:03:13.915 whose composition can be vary , within certain limits. 54 00:03:13.915 --> 00:03:17.090 , both the two components took should not chemically 55 00:03:17.090 --> 00:03:20.042 react otherwise there will be chemical reaction. 56 00:03:20.042 --> 00:03:23.296 ,in a solution the two components are non interacting, 57 00:03:23.296 --> 00:03:26.177 but the concentrations of each one can be different. 58 00:03:26.177 --> 00:03:28.320 ,it's a homogeneous mixture 59 00:03:28.320 --> 00:03:32.844 of all the particles are of molecular size that is up to 10 to the power 60 00:03:32.844 --> 00:03:36.503 minus nine meters or a nanometer the size, 61 00:03:36.503 --> 00:03:38.598 because otherwise what will happen they can lead to 62 00:03:38.598 --> 00:03:41.098 coagulation and so on precipitation. 63 00:03:41.098 --> 00:03:45.392 ,in a normal solution, the particle size is less than 64 00:03:45.392 --> 00:03:48.574 or about one nanometer 65 00:03:48.574 --> 00:03:53.606 and also every solution is made up of a solvent and a solute. 66 00:03:53.606 --> 00:03:55.669 , when a Solve, what is a solvent? 67 00:03:55.669 --> 00:03:59.312 Solvent is mainly the larger component in a solution, 68 00:03:59.312 --> 00:04:03.439 and which is present in large amount by weight 69 00:04:03.439 --> 00:04:07.027 compared to the other component which we call it as a solute. 70 00:04:07.027 --> 00:04:09.987 ,you have a solute and solvent and then what is a solute? 71 00:04:09.987 --> 00:04:15.209 It is obviously in a solution lesser amount or the minimum concentration. 72 00:04:15.209 --> 00:04:18.241 ,if we define it you can call it as 73 00:04:18.241 --> 00:04:20.159 it is a component of this solution. 74 00:04:20.160 --> 00:04:23.335 Which is present in smaller amounts by weight 75 00:04:23.335 --> 00:04:27.890 as compared to that of the other weight that is solvent. 76 00:04:27.890 --> 00:04:30.470 , after giving these two terms 77 00:04:30.470 --> 00:04:32.668 ,when we talk about an aqueous solution, 78 00:04:32.668 --> 00:04:37.843 the term aqueous solution is only used for where the solvent is water, 79 00:04:37.843 --> 00:04:41.358 ,solution can be any alcohol in a normal term, 80 00:04:41.358 --> 00:04:45.184 the solvent can be alcohol or any other medium. 81 00:04:45.184 --> 00:04:47.604 which can not necessarily be water, 82 00:04:47.604 --> 00:04:50.081 but the term aqueous solution is particularly given 83 00:04:50.081 --> 00:04:54.358 to only when there is a medium or the solvent is water. 84 00:04:54.358 --> 00:04:56.890 And of course, it needs a different kind of 85 00:04:56.890 --> 00:04:59.168 understanding and properties. 86 00:04:59.168 --> 00:05:04.493 Therefore, all the other substances other than the water, we call it a solute. 87 00:05:04.493 --> 00:05:09.874 Since water isionic, rather it's a polar solvent, 88 00:05:09.874 --> 00:05:12.438 these solutes are normally ionic, 89 00:05:12.438 --> 00:05:16.787 it could be strong n or a weak n components, 90 00:05:16.787 --> 00:05:20.620 and these aqueous acids ,bases, 91 00:05:20.620 --> 00:05:23.977 molecular compounds, all these compounds can be called as solutes 92 00:05:23.977 --> 00:05:26.128 in a water in the aqueous solution. 93 00:05:26.128 --> 00:05:28.954 ,the aqueous solution can be very dilute or 94 00:05:28.954 --> 00:05:33.946 it could be medium, medium dilute or it could be a strong, 95 00:05:33.946 --> 00:05:36.446 like I have shown you in the picture that 96 00:05:36.446 --> 00:05:39.938 the extreme this thing is dilute, the central one is 97 00:05:39.938 --> 00:05:43.382 not so dilute, but then again you have a concentrated solution, 98 00:05:43.382 --> 00:05:45.160 or a strong solution. 99 00:05:45.160 --> 00:05:47.263 ,the water structure is given therefore 100 00:05:47.263 --> 00:05:50.120 any water because water in aqueous solutions, 101 00:05:50.120 --> 00:05:53.549 the water is a solvent, any solute will interact by forming here 102 00:05:53.549 --> 00:05:56.041 polar water molecule interactions 103 00:05:56.041 --> 00:05:58.470 that could be charge-charge interactions or 104 00:05:58.470 --> 00:06:02.343 it could be hydrophilic interactions hydrogen bonding and so on and so forth. 105 00:06:02.343 --> 00:06:05.216 For example, you have a sodium chloride salt, it will, 106 00:06:05.216 --> 00:06:08.993 sodium will interact and as well as an Na plus as well as 107 00:06:08.993 --> 00:06:11.263 CL minus interact Na solution. 108 00:06:11.263 --> 00:06:13.581 How to express the concentration of a solution 109 00:06:13.581 --> 00:06:15.835 and a large number of students usually 110 00:06:15.835 --> 00:06:19.136 may commit a lot of mistakes and understanding the molarity 111 00:06:19.136 --> 00:06:24.266 and molality, normality, although that supposed to be very commonly used terms. 112 00:06:24.266 --> 00:06:28.610 When it comes to a kind of for describing them or understanding them. 113 00:06:28.610 --> 00:06:32.357 It's not very clear therefore, introduce them here all the terms. 114 00:06:32.357 --> 00:06:35.079 ,the some of the important terms is that 115 00:06:35.079 --> 00:06:37.801 how to express? you can express it in a number of ways, 116 00:06:37.801 --> 00:06:42.690 but a number of ways that can be used in different units also. 117 00:06:42.690 --> 00:06:45.690 ,the some of the important terms is a molecular weight 118 00:06:45.690 --> 00:06:48.206 that is awhenever you have a concentration 119 00:06:48.206 --> 00:06:50.682 that means obviously how much amount is there, 120 00:06:50.682 --> 00:06:53.730 ,it can be concentration presented in grams, 121 00:06:53.730 --> 00:06:58.603 but in true terms, it has to be expressed in daltons not grams, 122 00:06:58.603 --> 00:07:02.047 and also mole that is amount of the substance 123 00:07:02.047 --> 00:07:04.523 that has a mass in grams, 124 00:07:04.523 --> 00:07:07.134 numerically equivalent to that of a molecular weight. 125 00:07:07.134 --> 00:07:10.825 Then also, of course, Avogadro's number which was introduced to you earlier, 126 00:07:10.825 --> 00:07:13.079 that one mole of the substance has the 127 00:07:13.079 --> 00:07:15.714 number of molecules of as a mole of 128 00:07:15.714 --> 00:07:19.154 any other substance, that is 6 into 10 to the power 23, 129 00:07:19.154 --> 00:07:21.281 that is Avogadro's number. 130 00:07:21.281 --> 00:07:25.115 ,let's come to different units that we express . 131 00:07:25.115 --> 00:07:28.361 One of the terms which is not so common in 132 00:07:28.361 --> 00:07:30.758 terms of the biological systems, 133 00:07:30.758 --> 00:07:34.567 but nevertheless, a one unit that is commonly known is the 134 00:07:34.567 --> 00:07:38.940 ppm, or ppb that is a parts per million, parts per billion, 135 00:07:38.940 --> 00:07:42.837 the concentration of the solution is amount of the solute dissolved 136 00:07:42.837 --> 00:07:46.488 in a known amount of the solvent or a solution ppm 137 00:07:46.488 --> 00:07:48.916 is defined as a mass of the solute present 138 00:07:48.916 --> 00:07:53.400 in 1 million that is 10 to the power six parts of the solution. 139 00:07:53.400 --> 00:07:57.599 Therefore, if your solute is 'A ' is PPM A, 140 00:07:57.599 --> 00:08:01.797 that means it's mass is present in ten to the power six 141 00:08:01.797 --> 00:08:05.504 mass of the solution and not solvent 142 00:08:05.504 --> 00:08:08.123 when we talk about the total one is a mass of the solution. 143 00:08:08.123 --> 00:08:12.075 Similarly, you can express it by weight also. 144 00:08:12.075 --> 00:08:16.210 ,some of the biologists like to express in percentage weight, 145 00:08:16.210 --> 00:08:20.678 which is not very nice, but in some cases it is 146 00:08:20.678 --> 00:08:24.353 routinely used by for example, microbiology and so on. 147 00:08:24.353 --> 00:08:26.662 ,the weight of this resolute present in 148 00:08:26.662 --> 00:08:30.004 hundred grams of the solution again it is a solution not the solvent. 149 00:08:30.004 --> 00:08:33.297 percentage by volume also can be expressed 150 00:08:33.297 --> 00:08:35.766 that is percentage by volume means 151 00:08:35.766 --> 00:08:37.980 it means the weight of the solute in grams 152 00:08:37.980 --> 00:08:41.051 dissolved in hundred cc of the solution. 153 00:08:41.051 --> 00:08:45.289 , this is the volume and that is a by the weight. 154 00:08:45.289 --> 00:08:48.583 Because when you have to determine the concentrations 155 00:08:48.583 --> 00:08:51.266 or make solutions for example,h2so4, 156 00:08:51.266 --> 00:08:55.416 you have to take it from their density you have to determined from their 157 00:08:55.416 --> 00:08:59.329 , concentrated solution may be present in 158 00:08:59.329 --> 00:09:02.289 grams per percentage wise. 159 00:09:02.289 --> 00:09:06.639 Then you have to use that to convert it into your own requirement. 160 00:09:06.639 --> 00:09:11.242 And in case of the solid dissolved in a liquid is usually taken and 161 00:09:11.242 --> 00:09:15.210 but if you take it in case of a liquid to liquid 162 00:09:15.210 --> 00:09:19.369 that means concentration of the one liquid which is present in another 163 00:09:19.369 --> 00:09:21.821 then it is called the percentage by volume 164 00:09:21.821 --> 00:09:26.280 that means both are present in volume earlier it is solute in liquid. 165 00:09:26.280 --> 00:09:30.129 , it is grams by solution and here it is actually 166 00:09:30.154 --> 00:09:34.558 both will be in volume by volume UX. 167 00:09:34.558 --> 00:09:36.569 , we come to the molarity 168 00:09:36.570 --> 00:09:38.332 which is one of the most common one. 169 00:09:38.332 --> 00:09:39.856 , you should be able to do this 170 00:09:39.856 --> 00:09:44.149 in simple terms simple, the 171 00:09:44.149 --> 00:09:46.443 solutes like sodium chloride these molecular weights 172 00:09:46.443 --> 00:09:49.181 also should be in at the tip of your tongue. 173 00:09:49.181 --> 00:09:53.094 ,what is molarity is defined as the number of moles 174 00:09:53.094 --> 00:09:56.951 of the solute dissolved per liter of the solution. 175 00:09:56.951 --> 00:09:59.641 Always it is solution unless it is specified 176 00:09:59.641 --> 00:10:02.887 most of the time it is a total volume of the solution. 177 00:10:02.887 --> 00:10:05.824 , it is represented by capital M molar 178 00:10:05.824 --> 00:10:08.776 and is given by the number of moles of solute 179 00:10:08.776 --> 00:10:11.951 per volume of the solution present in liters. 180 00:10:11.951 --> 00:10:15.475 Example,I have given sodium chloride you can calculate yourself. 181 00:10:15.475 --> 00:10:18.142 The next one is the molality. 182 00:10:18.142 --> 00:10:22.285 , a lot of you can get confused with these two terms, 183 00:10:22.285 --> 00:10:25.856 this molality is defined as a number of moles of solute 184 00:10:25.856 --> 00:10:28.515 present per kilogram of solvent 185 00:10:28.515 --> 00:10:31.546 solvent not solution., please remember this is the 186 00:10:31.546 --> 00:10:36.777 only difference. here to express the total volume total weight of the solvent. 187 00:10:36.777 --> 00:10:40.324 , the molality can say as moles of solute 188 00:10:40.324 --> 00:10:42.538 per kilogram of the solvent. 189 00:10:42.538 --> 00:10:45.301 ,this is a different that molarity is a measurement 190 00:10:45.301 --> 00:10:47.864 of the moles in total volume of the solution. 191 00:10:47.864 --> 00:10:50.991 Whereas, the molality is measurement of the moles of the 192 00:10:50.991 --> 00:10:53.936 relation to the mass of the solvent. 193 00:10:53.936 --> 00:10:56.396 examples also I have given here 194 00:10:56.396 --> 00:10:59.785 that you can calculate of the same 195 00:10:59.785 --> 00:11:03.864 that is CCL4 you can calculate both in molar and molal. 196 00:11:03.864 --> 00:11:07.920 Normality has may not be so common, 197 00:11:07.920 --> 00:11:10.824 but has its own advantages in expressing some of the 198 00:11:10.824 --> 00:11:13.888 acids where you have the equivalent weight is taken. 199 00:11:13.888 --> 00:11:17.634 ,normality is defined as a number of gram euivalence 200 00:11:17.634 --> 00:11:20.348 remember it is a gram equivalence of the solute. 201 00:11:20.348 --> 00:11:24.674 ,therefore, you can denote it by capital N 202 00:11:24.674 --> 00:11:27.380 and the number of gram equivalence per 203 00:11:27.380 --> 00:11:31.197 of the solute per volume of the solution in one liter. 204 00:11:31.197 --> 00:11:34.142 ,this is given by N molarity into 205 00:11:34.142 --> 00:11:38.639 if you want to calculate normality, molarity into equivalent will be N 206 00:11:38.639 --> 00:11:40.909 that is the relation 207 00:11:40.909 --> 00:11:45.329 and for example,if you take h2so4 because two moles of 208 00:11:45.368 --> 00:11:49.298 hydrogen ions are released by h2so4 it will become N equal to 209 00:11:49.298 --> 00:11:53.203 it will become the gram equivalent will be two. 210 00:11:53.203 --> 00:11:57.369 , the mole fraction one it's important when you are doing in a 211 00:11:57.369 --> 00:12:00.695 kind of mole fraction. It has its own advantage, 212 00:12:00.695 --> 00:12:03.393 especially when you're doing in mixtures 213 00:12:03.393 --> 00:12:05.671 and you want to express it in 214 00:12:05.671 --> 00:12:08.941 terms of the fraction but in moles, 215 00:12:08.941 --> 00:12:13.354 how they are related to each other and you will be using this in DNA experiments 216 00:12:13.354 --> 00:12:15.985 and also in absorption experiments a lot 217 00:12:15.985 --> 00:12:18.596 that mole fraction has its own advantages. 218 00:12:18.596 --> 00:12:21.382 >Now<>,what is as the name suggests that it is a 219 00:12:21.382 --> 00:12:25.469 constituent of the fraction obtained by dividing the number of the 220 00:12:25.469 --> 00:12:29.398 constituentswhether it is say for example, N 221 00:12:29.398 --> 00:12:31.485 by the total number of the equivalent 222 00:12:31.485 --> 00:12:34.112 constituent that is N1 one plus n two plus n three 223 00:12:34.112 --> 00:12:37.374 mole fraction need not be a mixture of two components, 224 00:12:37.374 --> 00:12:39.779 but it can have a number of components 225 00:12:39.779 --> 00:12:43.778 x y z and so on that are x a, x b and so on. 226 00:12:43.778 --> 00:12:46.437 , if you have two components simple 227 00:12:46.437 --> 00:12:51.175 homoge.. binary mixture, a plus b then you have Xa equal to 228 00:12:51.175 --> 00:12:54.294 'a' by moles of 'a' by total that is 'a' plus 'b' 229 00:12:54.294 --> 00:12:58.873 or B imputing Xb will be b moles of b by the total. 230 00:12:58.873 --> 00:13:02.286 ,when you actually combine mole fraction Xa plus Xb, 231 00:13:02.286 --> 00:13:06.524 it has to be one., this is useful when you're talking about a 232 00:13:06.524 --> 00:13:09.635 experiments like DNA drug interaction, 233 00:13:09.635 --> 00:13:11.929 when where you want to keep both moles 234 00:13:11.929 --> 00:13:15.333 one is to one, then you have to have equal fraction. 235 00:13:15.333 --> 00:13:19.746 ,there you use or if you want one drug molecule is binding to 236 00:13:19.746 --> 00:13:22.913 six molecules are binding to one molecule of DNA, 237 00:13:22.913 --> 00:13:25.476 then you need to maintain the constant other ratio of 238 00:13:25.476 --> 00:13:30.071 mole fraction six to one., there you use this more frequently. 239 00:13:30.071 --> 00:13:32.611 ,depending on the need and necessity, 240 00:13:32.611 --> 00:13:35.865 the unit of concentrations differ. 241 00:13:35.865 --> 00:13:39.143 ,this is actually giving a table where you, 242 00:13:39.143 --> 00:13:42.643 I have given all the units and definition summarizing 243 00:13:42.643 --> 00:13:47.373 both molarity ,ppm, ppb, normality, mole fraction. 244 00:13:47.373 --> 00:13:50.563 ,the term acids and bases come in picture. 245 00:13:50.563 --> 00:13:54.152 Acid is of is can be easily 246 00:13:54.152 --> 00:13:58.183 termed as which use hydrogen, and hydrogen ions 247 00:13:58.183 --> 00:14:01.104 when dissolved in water 248 00:14:01.104 --> 00:14:04.913 and the substance that reduces the hydrogen ion concentration 249 00:14:04.913 --> 00:14:07.683 in a solution we call it as a base because 250 00:14:07.683 --> 00:14:12.810 hydrogen and OH , you can even say which gives out the hydroxyl ions. 251 00:14:12.810 --> 00:14:17.627 A strong acid when you talk about it could be weak acid or A strong acid. 252 00:14:17.627 --> 00:14:22.802 A strong acid is a thing that actually completely dissociates in water 253 00:14:22.802 --> 00:14:25.260 whenever like hydrochloride when you HCL 254 00:14:25.260 --> 00:14:28.117 when you put it in water, when you add in water, 255 00:14:28.117 --> 00:14:30.704 it actually completely dissociates into hydrogen 256 00:14:30.704 --> 00:14:34.308 and chloride but whereas a weak acid actually 257 00:14:34.308 --> 00:14:37.577 partially dissociate, there is an equilibrium generated 258 00:14:37.577 --> 00:14:41.133 between the dissociated and undissociated form. 259 00:14:41.133 --> 00:14:46.395 ,the acid can be weak or strong similarly base can be strong or weak. 260 00:14:46.395 --> 00:14:51.836 Though therefore, need to introduce the term when we have 261 00:14:51.836 --> 00:14:55.701 how much it is dissociation then how do you express the 262 00:14:55.701 --> 00:15:01.582 strength of any acid than it comes the term synonymous is the pH scale. 263 00:15:01.582 --> 00:15:04.788 The pH is generally defined as a 264 00:15:04.788 --> 00:15:07.257 negative logarithm of hydrogen and concentration. 265 00:15:07.257 --> 00:15:12.915 , when you take water actually it dissociates into H+ OH- 266 00:15:12.915 --> 00:15:17.090 ,the pH can be minus logarithm of H+ 267 00:15:17.090 --> 00:15:23.018 to the base 10. And if you want to add if you have to add 2.303 into as, 268 00:15:23.018 --> 00:15:28.612 base of the if you converting it from the logarithmic scale of the exponential. 269 00:15:28.612 --> 00:15:31.406 ,the most of the biological fluids 270 00:15:31.406 --> 00:15:35.787 actually are in the pH range of six to eight,not very far. 271 00:15:35.787 --> 00:15:38.716 Of course, in a stomach, you have an acidic and then 272 00:15:38.741 --> 00:15:41.509 in a saliva you have a basic condition. 273 00:15:41.509 --> 00:15:45.180 ,the scale actually tells you about the 274 00:15:45.180 --> 00:15:47.561 different regions of the pH's, 275 00:15:47.561 --> 00:15:50.767 which are associated with the biological fluids. 276 00:15:50.767 --> 00:15:54.513 ,a small change in pH actually 277 00:15:54.513 --> 00:15:58.013 indicates a substantial change in hydrogen and concentration 278 00:15:58.013 --> 00:16:02.280 and hydroxyl concentration., the pH is an indicator because it's 279 00:16:02.280 --> 00:16:06.248 a logarithmic scale it tells you how much change in the concentration. 280 00:16:06.248 --> 00:16:08.580 You should be able to know that if a 281 00:16:08.580 --> 00:16:11.334 pH is given you should be able to know how much is the 282 00:16:11.334 --> 00:16:13.890 hydrogen and concentration and ,the reverse 283 00:16:13.890 --> 00:16:17.763 hydrogen concentration is known, we should be able to know the pH. 284 00:16:17.763 --> 00:16:21.255 , the pH and buffers. 285 00:16:21.255 --> 00:16:24.707 ,what is the buffer.A buffer is that, 286 00:16:24.707 --> 00:16:30.294 which actually eliminates a large, sudden changes in pH, 287 00:16:30.294 --> 00:16:34.358 or which is resistant to a change in pH. 288 00:16:34.358 --> 00:16:38.612 ,when you want to do any experiment under a constant pH, 289 00:16:38.612 --> 00:16:41.763 or a certain a hydrongen concentration, 290 00:16:41.763 --> 00:16:45.150 there may be reaction going on 291 00:16:45.150 --> 00:16:47.325 with various interacting molecules, 292 00:16:47.325 --> 00:16:50.809 then you do not want the buffer to change the pH to change of 293 00:16:50.809 --> 00:16:55.539 the medium you're working on. experiment, you're doing an enzyme kinetics. 294 00:16:55.539 --> 00:16:57.930 You don't want the buffer because the enzyme function will 295 00:16:57.930 --> 00:17:01.327 change as the pH changes .you want a condition, 296 00:17:01.327 --> 00:17:04.232 where the pH is more or less maintained. 297 00:17:04.232 --> 00:17:06.724 ,that is what the buffer does. 298 00:17:06.749 --> 00:17:10.517 A buffer helps the you know, the organism also 299 00:17:10.517 --> 00:17:15.406 maintains the pH of the body fluids within a narrow certain range. 300 00:17:15.406 --> 00:17:17.970 It doesn't mean that it takes care of a largely, 301 00:17:17.970 --> 00:17:22.009 a certain to some extent well, within that narrow range. 302 00:17:22.009 --> 00:17:25.882 A buffer actually maintains the how does it maintain it actually, 303 00:17:25.882 --> 00:17:30.240 a resist because it has its own it can sort of act 304 00:17:30.240 --> 00:17:35.017 as a sinkto either two hydrogen ions on hydroxyl ions 305 00:17:35.017 --> 00:17:40.287 and therefore, it maintains a pH at a given adjusted pH whatever it was. 306 00:17:40.287 --> 00:17:42.819 ,each buffer has its own actually 307 00:17:42.819 --> 00:17:48.240 a range of buffer pH where it can maintain can be used. 308 00:17:48.240 --> 00:17:51.120 a combination of hydrogen ions 309 00:17:51.120 --> 00:17:54.716 acceptors and donors actually forms 310 00:17:54.716 --> 00:17:57.390 a solution of weak acid or a weak base. by work accepting the hydrogen ion 311 00:18:00.000 --> 00:18:05.250 from the solution when they are in excess and diluting the 312 00:18:05.250 --> 00:18:09.240 hydrogen ion ,when they have to be displaced. , this is what 313 00:18:09.240 --> 00:18:13.050 actually buffer does , the term itself explain so, well, 314 00:18:13.410 --> 00:18:18.450 that it takes care of the ,it acts actually sink or it will give it 315 00:18:18.510 --> 00:18:22.890 if it is required in a small range that has to be remembered. 316 00:18:23.940 --> 00:18:28.470 , then comes the how do you say which is a good 317 00:18:28.470 --> 00:18:32.760 buffer or a bad buffer or it can be that comes the term called 318 00:18:32.760 --> 00:18:36.870 buffer capacity. , the capacity means, it tells that 319 00:18:36.930 --> 00:18:40.920 how efficient it is to maintain you want to maintain it seven 320 00:18:40.920 --> 00:18:44.550 pH, how efficiently this buffer is working, which buffer you 321 00:18:44.550 --> 00:18:48.030 have to choose under what conditions, whether it needs it 322 00:18:48.030 --> 00:18:51.990 serves your needs or not. , the buffer capacity is actually 323 00:18:52.020 --> 00:18:57.180 a measure of the efficiency of a buffer in resisting the changes 324 00:18:57.180 --> 00:19:02.610 in the pH because sometimes the reaction you're doing, there may 325 00:19:02.610 --> 00:19:05.190 be release of hydrogen ions or there may be release of hydroxyl ions 326 00:19:05.190 --> 00:19:08.520 , which actually can change the pH. , the 327 00:19:08.520 --> 00:19:11.520 buffer should be able take care of these conditions. 328 00:19:11.610 --> 00:19:15.630 , conventionally what is happening is a buffer capacity 329 00:19:15.630 --> 00:19:19.860 which is expressed in bita is as expressed as amount of the 330 00:19:19.860 --> 00:19:25.380 strong acid or base in gram eqivalence that must be added to 331 00:19:25.380 --> 00:19:30.540 one liter of the solution, so as to change the pH to its by one 332 00:19:30.540 --> 00:19:34.410 unit. , that is what is the definition by buffer capacity. 333 00:19:34.620 --> 00:19:37.620 Then you have a number of buffers, which are actually 334 00:19:37.620 --> 00:19:41.070 normally used in biological systems, i'm only talking about 335 00:19:41.070 --> 00:19:45.930 the biological systems. Suppose you need to use say for example, 336 00:19:45.930 --> 00:19:49.500 acidic condition one to two, then you choose HCL and 337 00:19:49.500 --> 00:19:53.580 potassium chloride as a buffer or you want to use two to three 338 00:19:53.580 --> 00:19:57.570 or two to four then the glycine is a best buffer. Glycine -HCL 339 00:19:57.570 --> 00:20:02.010 buffer because glycine has as a pH a pk of about seven and 340 00:20:02.010 --> 00:20:06.510 therefore, it takes care of your pH range of two to four. you 341 00:20:06.510 --> 00:20:09.870 want to change it to a higher one, slightly acidic, but you 342 00:20:09.870 --> 00:20:13.350 need to change from three to six, then you use, you better 343 00:20:13.350 --> 00:20:17.040 use a Citrate- buffer or citrate phosphate buffer if you really 344 00:20:17.040 --> 00:20:20.940 want to use from a large range of two to seven, or a three to 345 00:20:20.940 --> 00:20:25.680 seven. when it comes to the slightly basic side, phosphate 346 00:20:25.680 --> 00:20:29.490 buffer is the ideal one where you can take, it can work very 347 00:20:29.490 --> 00:20:34.140 well from six to eight. I mean, it's not exactly six to seven, 348 00:20:34.140 --> 00:20:37.740 but it can go up to eight also. phosphate buffer also has 349 00:20:37.740 --> 00:20:41.070 some advantages because it doesn't interact with so many 350 00:20:41.070 --> 00:20:45.060 other in biological situations it is better to use 351 00:20:45.060 --> 00:20:49.200 phosphate buffer. Again Tris-HCL buffer is also good, but tris 352 00:20:49.200 --> 00:20:52.950 gives sometimes problems if it can interfere with some of the 353 00:20:52.950 --> 00:20:57.450 spectroscopic methods experiments. But Tris-HCL 354 00:20:57.450 --> 00:21:00.690 can be used if you are not using any by spectroscopy.The 355 00:21:00.690 --> 00:21:04.560 range again can be phosphate and Tris, seven to nine, seven to 356 00:21:04.560 --> 00:21:08.520 10. They're very good then glycine sodium hydroxide buffer. 357 00:21:08.730 --> 00:21:12.000 That is again in the basic one or you can use a carbonium 358 00:21:12.000 --> 00:21:16.380 bicarbonate buffer 9 to 10 if you're in the higher side of the 359 00:21:16.380 --> 00:21:20.460 basic range., these are the commonly used buffer and the 360 00:21:20.460 --> 00:21:25.680 reason they choose is because of their buffer capacity and the 361 00:21:25.680 --> 00:21:30.870 range the buffer they work on. , then after knowing the pH, 362 00:21:31.290 --> 00:21:35.970 there is the terms we need to know about the PK and so on. PK 363 00:21:35.970 --> 00:21:37.650 is ionization constant when 364 00:21:37.650 --> 00:21:40.140 you come across of the amino acid 365 00:21:40.710 --> 00:21:46.350 or any other as I just mentioned about glycine. Henderson 366 00:21:46.350 --> 00:21:50.820 hasselbalch equation is the >You , the the most 367 00:21:50.820 --> 00:21:54.270 fundamental equation which you use, rather you don't even 368 00:21:54.270 --> 00:21:57.210 realize that you are using that equation, but you still 369 00:21:57.240 --> 00:22:02.850 determine the PKs of the acids bases,amino acids routinely. 370 00:22:02.850 --> 00:22:07.830 what does it describe, what is Henderson Hasselbalch equation,. this describes the 371 00:22:07.860 --> 00:22:12.060 derivation of this pH as a measure of the acidity in 372 00:22:12.060 --> 00:22:16.230 biological systems. , the equation actually tells you a.. 373 00:22:16.260 --> 00:22:21.450 the useful for estimating the pH of a buffer and it is widely 374 00:22:21.450 --> 00:22:27.030 used to calculate isoelectric point that is 'Pi'. What is a 'Pi'? 375 00:22:27.030 --> 00:22:32.490 'Pi' is an isoelectric point at which the charge on the this 376 00:22:32.490 --> 00:22:37.350 thing is zero whether it can have a charge, it can have both 377 00:22:37.350 --> 00:22:41.670 charges positive and negative at the same time it can have a net 378 00:22:41.670 --> 00:22:44.550 charge will be zero that is the , wonder thing that there is no 379 00:22:44.550 --> 00:22:49.590 charge at 'Pi' the molecule will have a charge but the net charge 380 00:22:49.590 --> 00:22:52.380 is zero it will have some positive negative but the net 381 00:22:52.380 --> 00:22:56.400 charge is zero that has to be remembered very well. there 382 00:22:56.400 --> 00:23:00.570 comes the Henderson Hasselbalch equation. this Henderson Hasselbalch 383 00:23:00.570 --> 00:23:03.630 equation is very important, particularly when you're 384 00:23:03.630 --> 00:23:07.260 studying in the amino acids in the context of biological 385 00:23:07.260 --> 00:23:11.220 molecules. And in this what you are going to do is this 386 00:23:11.400 --> 00:23:17.310 determination of the pH or the PK and also the Pi of PK of a 387 00:23:17.310 --> 00:23:21.600 acid or a PK of the base .Though the Henderson Hasselbalch equation I 388 00:23:21.600 --> 00:23:25.800 have shown for example, you can do for acidic or basic. For 389 00:23:25.800 --> 00:23:31.710 example, HA any acid in water it gives a , hydroxonium ion and A minus. And 390 00:23:31.710 --> 00:23:36.060 therefore the pH can be calculated as PK A, that is the 391 00:23:36.060 --> 00:23:42.030 acid ionization, PK is ionization, K is ionization constant and PK 392 00:23:42.060 --> 00:23:47.340 plus log A minus by HA. this is the Henderson equation. 393 00:23:47.490 --> 00:23:51.930 Similarly you can write for a base also B plus H2O gives BH 394 00:23:51.930 --> 00:23:56.580 plus and OH- minus.Therefore POH that is the hydroxyl ion 395 00:23:56.700 --> 00:24:03.600 concentration, pK plus log BH Minus plus by H. , the base of 396 00:24:03.600 --> 00:24:08.670 BH plus by B that is the base unionized base. so ionized by 397 00:24:08.670 --> 00:24:13.770 the unionized form. this is the way you use the Henderson 398 00:24:13.860 --> 00:24:18.810 Hasselbalch equation for determining the 'Pi'. For example, I've shown 399 00:24:18.810 --> 00:24:23.220 you here how to determine the buffer of a solution made from 400 00:24:23.220 --> 00:24:29.460 from point two molar acetic acid, and point five molar . it has to 401 00:24:29.580 --> 00:24:33.090 dissociation constants. Therefore, you can determine the 402 00:24:33.120 --> 00:24:37.710 total PK pH of this mixture of these acids. This you can do 403 00:24:37.710 --> 00:24:42.060 yourself . comming to the amino acids, these are zwitterions 404 00:24:42.090 --> 00:24:46.680 or the dipolar ions, for example, glycine .you have an amino group 405 00:24:46.680 --> 00:24:50.880 and as well as a carboxyl group and therefore both of them have 406 00:24:50.880 --> 00:24:56.160 a different PKs. the amino acid will have a Pi based on the 407 00:24:56.190 --> 00:25:01.560 strength of these two groups. However, in some amino acids you also 408 00:25:01.560 --> 00:25:06.210 have a ionic group in the side chain for example glutamic acid 409 00:25:06.450 --> 00:25:10.590 and aspartic acid or a lysine or arginine, where the side group 410 00:25:10.620 --> 00:25:13.890 also contributes to the charge. Therefore, in the 411 00:25:13.920 --> 00:25:17.790 amino acid you will have like glutamic acid, where you have 412 00:25:17.790 --> 00:25:22.560 three chargeable groups and therefore, the PK or the Pi of 413 00:25:22.560 --> 00:25:27.270 the amino acid will depend on the PK of three groups, which is a 414 00:25:27.900 --> 00:25:30.900 contribution from three different groups. , I would 415 00:25:30.900 --> 00:25:34.650 like to summarize the, what you have learned in this class 416 00:25:34.650 --> 00:25:38.250 is, what are aqueous solutions, what is it, how to 417 00:25:38.250 --> 00:25:41.730 determine the concentration, how to express their concentration 418 00:25:41.910 --> 00:25:45.870 and also how to determine the pH and by using the 419 00:25:45.870 --> 00:25:49.920 Hasselbalch equation ,how to determine the Pk of amino acid as well as 420 00:25:49.920 --> 00:25:53.520 the Pi of the amino acids. 421 00:25:54.240 --> 00:25:58.350 , the summary comes in front of you, that is solutions are 422 00:25:58.350 --> 00:26:03.090 defined as homogeneous mixture of two or more pure substances 423 00:26:03.330 --> 00:26:06.120 and the solvent is that component of the solution which 424 00:26:06.120 --> 00:26:09.990 is present in large amount by weight then the other component 425 00:26:10.020 --> 00:26:13.950 that is termed as solvent. , the concentration and pH are two 426 00:26:13.950 --> 00:26:17.760 important quantitative properties of aqueous solutions. 427 00:26:18.060 --> 00:26:21.270 The concentration of a solution is the amount of solute 428 00:26:21.480 --> 00:26:25.230 dissolved in a known amount of the solvent or solution. 429 00:26:26.430 --> 00:26:30.180 Therefore, the concentration is expressed in terms of a molarity 430 00:26:30.390 --> 00:26:35.400 it could be molality or normality or a mole fraction and 431 00:26:35.400 --> 00:26:39.600 parts ratio, percentage weight etc. This depends on the need 432 00:26:39.660 --> 00:26:44.970 and the application where we require. We have also discussed 433 00:26:44.970 --> 00:26:49.230 in this what are buffers, what is the buffer capacity and what are 434 00:26:49.260 --> 00:26:51.570 zwitterion ions in terms of amino acids? 435 00:26:51.960 --> 00:26:54.840 And what are their PKs and how do they influence the 436 00:26:54.840 --> 00:26:58.950 Pi' of different amino acids. The most important equation the 437 00:26:58.950 --> 00:27:03.180 Henderson Hasselbalch equation is most frequently used equation. 438 00:27:03.420 --> 00:27:04.680 And I have also explained 439 00:27:04.680 --> 00:27:08.640 how to use this in biological systems. The Henderson-Hasselbalch 440 00:27:08.640 --> 00:27:12.930 equation is also useful in estimating the pH or a 441 00:27:12.960 --> 00:27:17.040 buffer solution. And buffers are also discussed in a number of 442 00:27:17.040 --> 00:27:19.380 different buffers, where you can use what 443 00:27:19.380 --> 00:27:21.240 are the ranges and things like that. 444 00:27:21.660 --> 00:27:24.990 And it is also used to calculate the isoelectric point of 445 00:27:24.990 --> 00:27:29.220 proteins. it's examples, I've shown you for the proteins 446 00:27:29.280 --> 00:27:33.120 amino acids, but you can also determine the Pi of a protein 447 00:27:33.510 --> 00:27:35.490 where and also 448 00:27:35.520 --> 00:27:37.470 it using the titration curves. 449 00:27:37.710 --> 00:27:44.760 with this, this topic, I have explained the the fundamentals 450 00:27:44.760 --> 00:27:49.140 of aqueous solutions and so on. And thank you, we'll see 451 00:27:49.140 --> 00:27:49.680 next time.