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Chromatography for Visual Learners

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  1. at its core chromatography is a technique used to separate out the components of a mixture this isn't always as easy as it seems if we have a
  2. beaker containing a solution with an unknown number of unknown compounds dissolved into it how can we go about identifying what's in there the most
  3. basic form of chromatography is paper chromatography you've probably done this experiment yourself at school at some point let's have a quick reminder of how
  4. it works because the principles involved here are exactly the same for more sophisticated types of chromatography with paper chromatography we take a
  5. piece of chromatography paper and we draw a pencil line across the bottom to act as our start line on that line we place dots of the inks we're analyzing
  6. we then suspend the paper in a solvent making sure that the top of the solvent is below our inks over time the solvent soaks up the paper and we can see the
  7. different dyes in the inks separate out now why does this happen you probably originally learned that this was related solubility of the different dyes in the
  8. solvent the more soluble the dye the further it moves with the solvent this is true but we can give this a more General explanation that will hold true
  9. for all other types of chromatography let's imagine we have two immiscible liquids an organic layer and an aqueous layer immiscible means they don't mix
  10. together like oil and water if we mix in a solute of some kind the solute will always have a greater affinity for one solvent over the other depending on the
  11. intermolecular forces it can form with the solvent molecules this means it will dissolve more in one of the liquids compared to the other
  12. this separation will also happen if our two phases are a liquid and an adsorbent solid or a liquid and a gas or an absorbent solid and a gas the solute
  13. will bind to one more strongly than the other no matter what the two phases are the solute is partitioned between the two phases it will establish a dynamic
  14. equilibrium between the two phases whereby the rate at which it moves from one phase into the other will be equal to the rate at which it moves back just
  15. like any equilibrium we can establish an equilibrium constant which we call the partition coefficients coming back to our paper chromatography there are two
  16. phases that the dye molecules can interact with here the paper and the solvent the paper is still so we call it the stationary phase and the solvent is
  17. moving so we call it the mobile phase any given molecule in these mixtures will interact with both the stationary phase and the mobile phase however
  18. they'll each have different affinities for each phase days and will be partitioned between them accordingly if a given molecule binds more strongly to
  19. the stationary phase than it does to the mobile phase it won't move as far if another molecule binds more strongly to the mobile phase than it does to the
  20. stationary phase it will move further so relative Affinity to the stationary phase and to the mobile phase is what separates or partitions the components
  21. in the mixture once they're separated we can measure the retention Factor RF of each component this is given by distance moved by the spot divided by distance
  22. moved by the solvent the RF values allow us to compare chromatograms that might have been left for different amounts of time even if the spots had a chance to
  23. move further the RF value should remain constant we can also compare RF values to those of known compounds to help identify the components in our mixture
  24. now a more sophisticated form of paper chromatography is thin layer chromatography or TLC C this follows the exact same principles as before except
  25. that the stationary phase is a thin layer of alumina or silica which has been coated onto a plate of glass metal or plastic
  26. the alumina or silica thin film is polar with oh groups at the surface so it can form hydrogen bonds and dipole-dipole interactions with the molecules passing
  27. over it this means it will bind more strongly to polar molecules than it will to non-polar molecules apart from that TLC works in the same way as paper
  28. chromatography the mixtures are dotted onto the pencil line the plate is suspended in a solvent and over time the spots move up the film just like with
  29. paper chromatography we can measure RF values however another useful feature of TLC is that the stationary phase can often be made to fluoresce under UV
  30. lights this is particularly useful if the spots from our mixture can't be seen under visible light we can shine UV light on the plate and watch it
  31. fluoresce the regions which are obscured by the spots from our mixture will appear darker than the rest of the plate alternatively We can spray the plate
  32. with a separate die that can make our spots easier to see and another useful feature of TLC is that unlike chromatography paper TLC plates can be
  33. washed and reused however what if we wanted to properly separate a mixture so that we ended up with separate Solutions of each component maybe we want to
  34. quantify the amount of each component as well it would be very difficult to do all this with TLC since we use such small quantities and since there's no
  35. good way to accurately transfer the spots to a solution for quantitative chromatography of larger quantities we would use column chromatography with
  36. column chromatography we'd use a glass column which has a tap at the bottom very similar to a buret first we place a bit of cotton wool at the bottom with a
  37. thin layer of sand on top to prevent the stationary phase from getting into the tap we then pack the column with the same material that's used as the
  38. stationary phase for TLC alumina or more commonly silica to do this we'd create a slurry of silica with a solvent and pour it into the column the silica will
  39. settle at the bottom how do we know what solvent to use for column chromatography for that we can quickly run a few tests using TLC to see which solvents best
  40. separate the components of our mixture it's important to ensure that the silica is kept wet at all times so the solvent level will need to be continually topped
  41. up if the solvent drops below the top of the silica the silica can dry out leading to cracking and poor separation to protect the silica we place another
  42. layer of sand on top now we'll be able to top up the solvent without worrying about messing up our horizontal levels if the layers become uneven the
  43. components won't separate out evenly underneath the column we'll place a test tube or a flask depending on how big the column is we'll need to have several
  44. empty test tubes nearby so that we can swap them out once the current test tube gets filled up before we load our mixture we'll need to let some solvent
  45. out so that the level is around the top of the sand layer now we'll gently drop a solution of our mixture on top using a pipette pouring it all around the edge
  46. of the column to create an even layer on top of the sand layer we can also wash around the inside of the column using a little bit more solvent to make sure
  47. none of the mixture is left on the glass now we'll gently open the tap to allow the mixture to settle at the top of the silica layer once this is all settled at
  48. the top of the silica we can close the tap and gently top up the solvent layer so that there's plenty to work with and we're ready to start separating with the
  49. tap open running into the test tube below the solvent will start to pour out causing our mixture to move down as well as one test tube fills up we'll close
  50. the tap and switch it out for an empty test tube then open the tap again it's important to remember to keep topping up the solvent so that the silica doesn't
  51. dry out the different components in the mixture will have different affinities for the stationary silica phase and the mobile solvent phase more polar
  52. molecules in the mixture will be more strongly attracted to the polar silica and hence they won't move as fast more non-polar molecules in the mixture will
  53. be less attracted to the polar silica and will move faster over time the different components will separate out these different components will end up
  54. in different tubes TLC can be used to check that each test tube only contains one component we can also get more sophisticated types of column
  55. chromatography which have a detector fitted to the outlet this can produce a chromatogram which shows Peaks when each component passes through the detector
  56. the time taken for a component to pass all the way through the column is called the retention time comparing the retention time of an unknown component
  57. with that of a known compound which has been run under the same conditions can help to identify it a more advanced form of column chromatography is high
  58. performance liquid chromatography or hplc rather than relying on gravity this involves pumping a solvent mobile phase through a thin column that has been
  59. packed with a silica stationary phase using a polar stationary phase like this is called normal phase hplc and we typically use a non-polar solvent such
  60. as hexane as the mobile phase the silica can also be coated with non-polar hydrocarbons in in this case we typically use a more polar solvent such
  61. as methanol as the mobile phase this is called reverse phase hplc either way we inject a solution of our mixture into the high pressure solvent stream and the
  62. components of the mixture are separated by their relative affinities to the stationary phase and to the mobile phase since high pressures are required to
  63. force the solvent through the column hplc is sometimes called high pressure liquid chromatography the high pressures make hplc a lot faster than column
  64. chromatography each component is detected as it makes its way out of the column the most common type of detector used with hplc is a UV absorbance
  65. detector this involves passing a beam of UV light through the solvent as it exits the column A detector opposite measures the intensity of UV light that makes it
  66. through the solvents as a compound in the mixture flows in front of the UV detector it will absorb some of the UV radiation so the detector measures a
  67. decrease in UV and intensity giving us a peak just like with column chromatography we can end up with a chromatogram which shows the retention
  68. time of the different components and we can compare our retention times to those of known compounds that have been run under the same conditions to identify
  69. the components in our sample column chromatography and hplc are both great techniques but what if we're more interested in a gas phase mixture for
  70. that we'd use gas chromatography gas chromatography uses a long thin column which is packed with a solid such as silica which can act as the stationary
  71. phase alternatively the solid can act as a support for a non-volatile unreactive liquid cyloxane polymer which can act as the stationary phase instead the column
  72. can be up to 100 meters long and is coiled inside a computer-controlled oven an inert carrier gas such as argon helium or nitrogen continuously flows
  73. through the column and acts as the mobile phase at the end there will be a detector of some kind the there are two common types of detector used for gas
  74. chromatography the first is a flame ionization detector or FID which contains a small flame that combusts each component that passes through it
  75. this forms ions which are attracted to electrodes resulting in a small current which is detected to give a peak the second type of detector is a mass
  76. spectrometer which I'll go into more in another video there are many ways to prepare a sample with gas chromatography our mixture can be a gas a volatile
  77. solution or even an extract of the headspace above a volatile solution no matter what our sample is we first inject a small amount of it into the
  78. injector oven this is a smaller oven located before the main column oven the injector oven is set to a high temperature usually between 200 to 300
  79. degrees C this ensures our sample vaporizes so that the carrier gas can transport it into the column the column oven usually starts off at much lower
  80. temperature inches so much of our sample will often condense back into a liquid as soon as it enters the column now the computer-controlled column oven will
  81. gradually increase its temperature over a set amount of time let's say it increases from 30 degrees C to 200 degrees C over the course of 30 minutes
  82. one by one as each component in the mixture reaches its boiling points they will evaporate allowing the carrier gas to push them through the column this is
  83. similar to separation via fractional distillation however an advantage with gas chromatography is that if two components have similar boiling points
  84. gas chromatography can further separate them based on their Affinity to the mobile and stationary phases again just like with column chromatography and hplc
  85. with gas chromatography we can end up with a chromatogram which shows the retention time of the different components which we can compare to those
  86. of known compounds that have been run under the same conditions to identify the components in our sample an important thing to note with any type of
  87. chromatography that involves a detector is that the detector will often respond differently to different compounds so how can we quantify the amount of a
  88. given component in our mixture for that we'll need to use a calibration curve let's say we've identified that one of our components is butyl propanoate based
  89. on its retention time but we want to figure out the concentration of butyl propanoate in our original sample we'd need to prepare several different
  90. solutions of butyl propanolate each with a different concentration we then perform chromatography on each solution we should see that each solution
  91. produces a Peak at the same retention time but with higher concentrations the detector response will increase the type of response depends on the type of
  92. detector but let's just call it Peak area for now by plotting concentration against Peak area we should get a straight line this is our calibration
  93. curve Now by finding the area of the butyl propanoate peak in our original sample we can simply go cross to our line and then down to find the
  94. concentration of butyl propanoate so as we've seen chromatography is a very powerful and very versatile separation technique with a wide range of
  95. applications if you found this video helpful please consider subscribing to support the channel and let me know in the comments if you have any questions

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