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