1 00:00:00,290 --> 00:00:01,620 - [Instructor] So let's say that I have a vial 2 00:00:01,620 --> 00:00:03,840 of some mystery liquid right over here, 3 00:00:03,840 --> 00:00:06,080 and I want to start figuring out what's going on there. 4 00:00:06,080 --> 00:00:08,070 And the first step is to think about, 5 00:00:08,070 --> 00:00:09,730 is it just one substance 6 00:00:09,730 --> 00:00:12,210 or is it a mixture of multiple substances? 7 00:00:12,210 --> 00:00:15,230 And the focus of this video is a technique 8 00:00:15,230 --> 00:00:17,540 to separate out the substances 9 00:00:17,540 --> 00:00:19,860 to understand at least how many there are, 10 00:00:19,860 --> 00:00:23,030 and this technique generally is called chromatography, 11 00:00:23,030 --> 00:00:25,680 but we'll focus on thin layer chromatography 12 00:00:25,680 --> 00:00:27,570 which is the most common that you might see, 13 00:00:27,570 --> 00:00:29,590 but other variations of chromatography 14 00:00:29,590 --> 00:00:31,150 like paper chromatography 15 00:00:31,150 --> 00:00:33,890 operate on very similar principles. 16 00:00:33,890 --> 00:00:37,780 So what we're going to do is set up 17 00:00:37,780 --> 00:00:40,790 on top of something like glass or plastic, 18 00:00:40,790 --> 00:00:45,790 we're going to put a thin layer of a solid polar substance. 19 00:00:46,800 --> 00:00:48,040 Now, what you typically do 20 00:00:48,040 --> 00:00:50,650 is put a thin layer of silica gel, 21 00:00:50,650 --> 00:00:55,140 that's the most common solid polar substance that folks use. 22 00:00:55,140 --> 00:00:56,430 And it's also porous. 23 00:00:56,430 --> 00:00:58,500 And the fact that it's porous is really important 24 00:00:58,500 --> 00:01:01,290 because we're going to want liquid to have capillary action 25 00:01:01,290 --> 00:01:03,480 and travel up through it. 26 00:01:03,480 --> 00:01:05,600 Now, the silica gel, as I mentioned, 27 00:01:05,600 --> 00:01:08,983 this thing is very polar. 28 00:01:10,440 --> 00:01:11,273 Now, what we're going to do 29 00:01:11,273 --> 00:01:12,970 is take some of our mystery substance, 30 00:01:12,970 --> 00:01:14,900 let's say it's this color right over here, 31 00:01:14,900 --> 00:01:19,900 and we're going to place a dot of it on that silica gel. 32 00:01:19,940 --> 00:01:21,370 You then want to take this plate 33 00:01:21,370 --> 00:01:23,180 that has the silica gel on it 34 00:01:23,180 --> 00:01:25,670 and that little dot of our mystery substance, 35 00:01:25,670 --> 00:01:30,060 and then you want to dip just one end of it in a solution. 36 00:01:30,060 --> 00:01:31,077 And what's really important is that 37 00:01:31,077 --> 00:01:35,120 the solution is less polar than the silica gel. 38 00:01:35,120 --> 00:01:37,430 Less polar here. 39 00:01:37,430 --> 00:01:39,620 And we'll talk a little bit about what happens 40 00:01:39,620 --> 00:01:41,600 depending on how polar this is. 41 00:01:41,600 --> 00:01:44,810 Now, usually this is going to be a very shallow amount 42 00:01:44,810 --> 00:01:47,530 of this solution, which, as we'll see, 43 00:01:47,530 --> 00:01:49,360 will be something of a solvent. 44 00:01:49,360 --> 00:01:51,940 And you usually want to put it in a closed container 45 00:01:51,940 --> 00:01:52,773 like this 46 00:01:52,773 --> 00:01:55,980 so that this fluid down here doesn't evaporate out. 47 00:01:55,980 --> 00:01:58,160 And then what do you think is going to happen? 48 00:01:58,160 --> 00:02:00,970 Well, as I mentioned, this is a porous substance here. 49 00:02:00,970 --> 00:02:03,160 And so you're going to have capillary action. 50 00:02:03,160 --> 00:02:05,960 This fluid at the bottom is going to move upwards 51 00:02:05,960 --> 00:02:07,260 through the silica gel, 52 00:02:07,260 --> 00:02:09,320 through those little pores in the silica gel. 53 00:02:09,320 --> 00:02:11,570 This is the stationary phase. Why do we call it that? 54 00:02:11,570 --> 00:02:12,670 Well, 'cause it's not moving. 55 00:02:12,670 --> 00:02:16,270 And you can imagine we would call this less polar solvent 56 00:02:17,170 --> 00:02:18,610 the mobile phase, 57 00:02:18,610 --> 00:02:21,970 because that is traveling through the silica gel 58 00:02:21,970 --> 00:02:25,010 and it's picking up some of this mystery substance 59 00:02:25,010 --> 00:02:26,700 and it's transporting it. 60 00:02:26,700 --> 00:02:28,280 And let's say this mystery substance 61 00:02:28,280 --> 00:02:30,610 is made up of two different things. 62 00:02:30,610 --> 00:02:33,200 If something is more polar, 63 00:02:33,200 --> 00:02:35,210 that means it's going to be more attracted 64 00:02:35,210 --> 00:02:37,600 to the stationary phase which is very polar. 65 00:02:37,600 --> 00:02:40,490 And so it's not going to travel that far, 66 00:02:40,490 --> 00:02:42,210 while the parts of our mystery substance 67 00:02:42,210 --> 00:02:43,450 that are less polar, 68 00:02:43,450 --> 00:02:46,500 they're not going to be attracted to the silica gel as much. 69 00:02:46,500 --> 00:02:49,410 So they're going to travel further with the solvent. 70 00:02:49,410 --> 00:02:51,910 So maybe it might go like that. 71 00:02:51,910 --> 00:02:53,530 And you would run this until your mobile phase 72 00:02:53,530 --> 00:02:56,940 makes a good way to the top of your silica gel 73 00:02:56,940 --> 00:02:58,190 right over here. 74 00:02:58,190 --> 00:02:59,380 Now, just looking at this, 75 00:02:59,380 --> 00:03:01,160 and the reason why it was called chromatography is 76 00:03:01,160 --> 00:03:02,280 when they originally did this, 77 00:03:02,280 --> 00:03:04,760 they were actually separating out various tissues 78 00:03:04,760 --> 00:03:07,610 in vegetation that had different colors. 79 00:03:07,610 --> 00:03:10,160 The chroma is referring to the various colors, 80 00:03:10,160 --> 00:03:11,960 but it doesn't necessarily even have to refer to things 81 00:03:11,960 --> 00:03:13,040 that have different colors 82 00:03:13,040 --> 00:03:15,630 or sometimes you might need a UV light to see them. 83 00:03:15,630 --> 00:03:17,440 But when you run thin layer chromatography, 84 00:03:17,440 --> 00:03:20,780 you will see that your original dot will have traveled 85 00:03:20,780 --> 00:03:22,660 to various degrees with your solvent 86 00:03:22,660 --> 00:03:24,860 and then will now be multiple dots 87 00:03:24,860 --> 00:03:28,520 depending on how many things were in your original mixture. 88 00:03:28,520 --> 00:03:31,670 And as I just mentioned, this thing right over here, 89 00:03:31,670 --> 00:03:35,520 this is the less polar thing is going to travel further 90 00:03:35,520 --> 00:03:40,380 than the more polar thing, more polar constituent substance, 91 00:03:40,380 --> 00:03:42,850 because the more polar thing is more attractive 92 00:03:42,850 --> 00:03:45,570 to the silica gel, which is stationary, 93 00:03:45,570 --> 00:03:49,440 and there is a way to quantify how far these things traveled 94 00:03:49,440 --> 00:03:51,660 relative to your solvent. 95 00:03:51,660 --> 00:03:54,700 And that's called a retention factor. 96 00:03:54,700 --> 00:03:57,853 Retention factor. 97 00:03:59,010 --> 00:04:02,780 Which the shorthand is R subscript f. 98 00:04:02,780 --> 00:04:07,200 And it's just defined as the distance traveled 99 00:04:08,170 --> 00:04:13,170 by the solute divided by the distance traveled 100 00:04:18,690 --> 00:04:22,080 by the solvent. 101 00:04:22,080 --> 00:04:23,090 And we need to be clear. 102 00:04:23,090 --> 00:04:26,000 It's not the distance traveled by the solvent in total, 103 00:04:26,000 --> 00:04:29,810 it's the distance traveled by the solvent from this origin, 104 00:04:29,810 --> 00:04:32,750 from where we applied this dot right over here. 105 00:04:32,750 --> 00:04:36,353 So, past the origin. 106 00:04:37,320 --> 00:04:39,893 And let me label that as the origin. 107 00:04:40,730 --> 00:04:42,730 So what would it be in this situation? 108 00:04:42,730 --> 00:04:46,930 Well, to help us there, we would have to get out a ruler. 109 00:04:46,930 --> 00:04:51,930 So the retention factor for substance A right over here, 110 00:04:52,400 --> 00:04:54,890 so I'll put that dot there, label that A, 111 00:04:54,890 --> 00:04:58,560 would be equal to the distance traveled by the solute, 112 00:04:58,560 --> 00:05:03,340 which we can see, it traveled one centimeter, 113 00:05:03,340 --> 00:05:05,040 one centimeter, 114 00:05:05,040 --> 00:05:09,110 over the distance traveled by the solvent past the origin. 115 00:05:09,110 --> 00:05:11,810 And so that is going to be, 116 00:05:11,810 --> 00:05:15,110 we see it traveled five centimeters past the origin. 117 00:05:15,110 --> 00:05:17,810 So one centimeter over five centimeters, 118 00:05:17,810 --> 00:05:19,813 which is the same thing as 0.2. 119 00:05:20,700 --> 00:05:24,630 And then the retention factor for substance B 120 00:05:26,190 --> 00:05:29,750 is going to be equal to, how far did it travel? 121 00:05:29,750 --> 00:05:31,850 Well, it traveled three centimeters 122 00:05:33,300 --> 00:05:36,480 out of a total of five centimeters for the solvent, 123 00:05:36,480 --> 00:05:37,820 past this origin, 124 00:05:37,820 --> 00:05:40,470 past where we put the sample right over there. 125 00:05:40,470 --> 00:05:45,410 Five centimeters, which is equal to 0.6. 126 00:05:45,410 --> 00:05:47,240 So notice, in this situation, 127 00:05:47,240 --> 00:05:50,660 the more polar substance had a lower retention factor 128 00:05:50,660 --> 00:05:54,550 than the less polar substance, and that makes sense. 129 00:05:54,550 --> 00:05:59,490 Because our stationary phase is more polar than our solvent, 130 00:05:59,490 --> 00:06:03,500 and so the things that are more polar were harder to move 131 00:06:03,500 --> 00:06:06,253 by the less polar solvent.