0:00:00.290,0:00:01.620 - [Instructor] So let's[br]say that I have a vial 0:00:01.620,0:00:03.840 of some mystery liquid right over here, 0:00:03.840,0:00:06.080 and I want to start figuring[br]out what's going on there. 0:00:06.080,0:00:08.070 And the first step is to think about, 0:00:08.070,0:00:09.730 is it just one substance 0:00:09.730,0:00:12.210 or is it a mixture of multiple substances? 0:00:12.210,0:00:15.230 And the focus of this video is a technique 0:00:15.230,0:00:17.540 to separate out the substances 0:00:17.540,0:00:19.860 to understand at least how many there are, 0:00:19.860,0:00:23.030 and this technique generally[br]is called chromatography, 0:00:23.030,0:00:25.680 but we'll focus on thin[br]layer chromatography 0:00:25.680,0:00:27.570 which is the most common[br]that you might see, 0:00:27.570,0:00:29.590 but other variations of chromatography 0:00:29.590,0:00:31.150 like paper chromatography 0:00:31.150,0:00:33.890 operate on very similar principles. 0:00:33.890,0:00:37.780 So what we're going to do is set up 0:00:37.780,0:00:40.790 on top of something like glass or plastic, 0:00:40.790,0:00:45.790 we're going to put a thin layer[br]of a solid polar substance. 0:00:46.800,0:00:48.040 Now, what you typically do 0:00:48.040,0:00:50.650 is put a thin layer of silica gel, 0:00:50.650,0:00:55.140 that's the most common solid[br]polar substance that folks use. 0:00:55.140,0:00:56.430 And it's also porous. 0:00:56.430,0:00:58.500 And the fact that it's[br]porous is really important 0:00:58.500,0:01:01.290 because we're going to want[br]liquid to have capillary action 0:01:01.290,0:01:03.480 and travel up through it. 0:01:03.480,0:01:05.600 Now, the silica gel, as I mentioned, 0:01:05.600,0:01:08.983 this thing is very polar. 0:01:10.440,0:01:11.273 Now, what we're going to do 0:01:11.273,0:01:12.970 is take some of our mystery substance, 0:01:12.970,0:01:14.900 let's say it's this color right over here, 0:01:14.900,0:01:19.900 and we're going to place a[br]dot of it on that silica gel. 0:01:19.940,0:01:21.370 You then want to take this plate 0:01:21.370,0:01:23.180 that has the silica gel on it 0:01:23.180,0:01:25.670 and that little dot of[br]our mystery substance, 0:01:25.670,0:01:30.060 and then you want to dip just[br]one end of it in a solution. 0:01:30.060,0:01:31.077 And what's really important is that 0:01:31.077,0:01:35.120 the solution is less[br]polar than the silica gel. 0:01:35.120,0:01:37.430 Less polar here. 0:01:37.430,0:01:39.620 And we'll talk a little[br]bit about what happens 0:01:39.620,0:01:41.600 depending on how polar this is. 0:01:41.600,0:01:44.810 Now, usually this is going[br]to be a very shallow amount 0:01:44.810,0:01:47.530 of this solution, which, as we'll see, 0:01:47.530,0:01:49.360 will be something of a solvent. 0:01:49.360,0:01:51.940 And you usually want to put[br]it in a closed container 0:01:51.940,0:01:52.773 like this 0:01:52.773,0:01:55.980 so that this fluid down[br]here doesn't evaporate out. 0:01:55.980,0:01:58.160 And then what do you[br]think is going to happen? 0:01:58.160,0:02:00.970 Well, as I mentioned, this[br]is a porous substance here. 0:02:00.970,0:02:03.160 And so you're going to[br]have capillary action. 0:02:03.160,0:02:05.960 This fluid at the bottom[br]is going to move upwards 0:02:05.960,0:02:07.260 through the silica gel, 0:02:07.260,0:02:09.320 through those little[br]pores in the silica gel. 0:02:09.320,0:02:11.570 This is the stationary phase.[br]Why do we call it that? 0:02:11.570,0:02:12.670 Well, 'cause it's not moving. 0:02:12.670,0:02:16.270 And you can imagine we would[br]call this less polar solvent 0:02:17.170,0:02:18.610 the mobile phase, 0:02:18.610,0:02:21.970 because that is traveling[br]through the silica gel 0:02:21.970,0:02:25.010 and it's picking up some[br]of this mystery substance 0:02:25.010,0:02:26.700 and it's transporting it. 0:02:26.700,0:02:28.280 And let's say this mystery substance 0:02:28.280,0:02:30.610 is made up of two different things. 0:02:30.610,0:02:33.200 If something is more polar, 0:02:33.200,0:02:35.210 that means it's going to be more attracted 0:02:35.210,0:02:37.600 to the stationary phase[br]which is very polar. 0:02:37.600,0:02:40.490 And so it's not going to travel that far, 0:02:40.490,0:02:42.210 while the parts of our mystery substance 0:02:42.210,0:02:43.450 that are less polar, 0:02:43.450,0:02:46.500 they're not going to be attracted[br]to the silica gel as much. 0:02:46.500,0:02:49.410 So they're going to travel[br]further with the solvent. 0:02:49.410,0:02:51.910 So maybe it might go like that. 0:02:51.910,0:02:53.530 And you would run this[br]until your mobile phase 0:02:53.530,0:02:56.940 makes a good way to the[br]top of your silica gel 0:02:56.940,0:02:58.190 right over here. 0:02:58.190,0:02:59.380 Now, just looking at this, 0:02:59.380,0:03:01.160 and the reason why it was[br]called chromatography is 0:03:01.160,0:03:02.280 when they originally did this, 0:03:02.280,0:03:04.760 they were actually separating[br]out various tissues 0:03:04.760,0:03:07.610 in vegetation that had different colors. 0:03:07.610,0:03:10.160 The chroma is referring[br]to the various colors, 0:03:10.160,0:03:11.960 but it doesn't necessarily[br]even have to refer to things 0:03:11.960,0:03:13.040 that have different colors 0:03:13.040,0:03:15.630 or sometimes you might need[br]a UV light to see them. 0:03:15.630,0:03:17.440 But when you run thin[br]layer chromatography, 0:03:17.440,0:03:20.780 you will see that your[br]original dot will have traveled 0:03:20.780,0:03:22.660 to various degrees with your solvent 0:03:22.660,0:03:24.860 and then will now be multiple dots 0:03:24.860,0:03:28.520 depending on how many things[br]were in your original mixture. 0:03:28.520,0:03:31.670 And as I just mentioned,[br]this thing right over here, 0:03:31.670,0:03:35.520 this is the less polar thing[br]is going to travel further 0:03:35.520,0:03:40.380 than the more polar thing, more[br]polar constituent substance, 0:03:40.380,0:03:42.850 because the more polar[br]thing is more attractive 0:03:42.850,0:03:45.570 to the silica gel, which is stationary, 0:03:45.570,0:03:49.440 and there is a way to quantify[br]how far these things traveled 0:03:49.440,0:03:51.660 relative to your solvent. 0:03:51.660,0:03:54.700 And that's called a retention factor. 0:03:54.700,0:03:57.853 Retention factor. 0:03:59.010,0:04:02.780 Which the shorthand is R subscript f. 0:04:02.780,0:04:07.200 And it's just defined[br]as the distance traveled 0:04:08.170,0:04:13.170 by the solute divided[br]by the distance traveled 0:04:18.690,0:04:22.080 by the solvent. 0:04:22.080,0:04:23.090 And we need to be clear. 0:04:23.090,0:04:26.000 It's not the distance traveled[br]by the solvent in total, 0:04:26.000,0:04:29.810 it's the distance traveled by[br]the solvent from this origin, 0:04:29.810,0:04:32.750 from where we applied[br]this dot right over here. 0:04:32.750,0:04:36.353 So, past the origin. 0:04:37.320,0:04:39.893 And let me label that as the origin. 0:04:40.730,0:04:42.730 So what would it be in this situation? 0:04:42.730,0:04:46.930 Well, to help us there, we[br]would have to get out a ruler. 0:04:46.930,0:04:51.930 So the retention factor for[br]substance A right over here, 0:04:52.400,0:04:54.890 so I'll put that dot there, label that A, 0:04:54.890,0:04:58.560 would be equal to the distance[br]traveled by the solute, 0:04:58.560,0:05:03.340 which we can see, it[br]traveled one centimeter, 0:05:03.340,0:05:05.040 one centimeter, 0:05:05.040,0:05:09.110 over the distance traveled by[br]the solvent past the origin. 0:05:09.110,0:05:11.810 And so that is going to be, 0:05:11.810,0:05:15.110 we see it traveled five[br]centimeters past the origin. 0:05:15.110,0:05:17.810 So one centimeter over five centimeters, 0:05:17.810,0:05:19.813 which is the same thing as 0.2. 0:05:20.700,0:05:24.630 And then the retention[br]factor for substance B 0:05:26.190,0:05:29.750 is going to be equal to,[br]how far did it travel? 0:05:29.750,0:05:31.850 Well, it traveled three centimeters 0:05:33.300,0:05:36.480 out of a total of five[br]centimeters for the solvent, 0:05:36.480,0:05:37.820 past this origin, 0:05:37.820,0:05:40.470 past where we put the[br]sample right over there. 0:05:40.470,0:05:45.410 Five centimeters, which is equal to 0.6. 0:05:45.410,0:05:47.240 So notice, in this situation, 0:05:47.240,0:05:50.660 the more polar substance[br]had a lower retention factor 0:05:50.660,0:05:54.550 than the less polar substance,[br]and that makes sense. 0:05:54.550,0:05:59.490 Because our stationary phase[br]is more polar than our solvent, 0:05:59.490,0:06:03.500 and so the things that are[br]more polar were harder to move 0:06:03.500,0:06:06.253 by the less polar solvent.