Dry Column Vacuum Chromatography (DCVC) Tutorial

00:31:39
https://www.youtube.com/watch?v=lBNhu4kJ4Mc

Resumen

TLDRDaniel S. Peterson, profesor asociat la Universitatea din Copenhaga, oferă o introducere în DCVC, o tehnică eficientă de cromatografie cu coloană. Acesta discută echipamentele necesare, avantajele DCVC, cum ar fi economia de solvent și eficiența sporită în comparație cu cromatografia Flash. Peterson detaliază procesul de configurare a coloanei, inclusiv ambalarea silicei și utilizarea vacumului, și subliniază importanța siguranței în laborator. Tutorialul este adresat începătorilor, dar și celor interesați de tehnici avansate, oferind resurse suplimentare pentru aprofundarea cunoștințelor despre DCVC.

Para llevar

  • 🔬 DCVC este o tehnică de cromatografie eficientă și ecologică.
  • 💨 Este o metodă bazată pe vid, mai sigură decât presiunea în laborator.
  • 📦 Coloană de silice necesară are dimensiuni specifice pentru avansare optimă.
  • 🧪 Folosirea silicei de dimensiune corectă este esențială pentru succesul separării.
  • ⚖️ DCVC permite purificarea cantităților mari de compuși, incomparabil cu DLC.
  • 🛠️ Pregătirea echipamentului este crucială pentru obținerea unor rezultate bune.
  • ⌛ DCVC este mai rapidă decât metodele tradiționale, reducând timpul necesar purificării.
  • 📖 Tutorialul oferă linkuri utile pentru învățare suplimentară.

Cronología

  • 00:00:00 - 00:05:00

    Daniel S Peterson, profesor asociat la Universitatea din Copenhaga, oferă o introducere în cromatografia în vid pe coloane uscate (DCVC). Tutorialul este bazat pe echipamente de bază, accentuând avantajele DCVC față de cromatografia flash, inclusiv economisirea de resurse, rapiditatea și scalabilitatea procesului de purificare.

  • 00:05:00 - 00:10:00

    DCVC este o tehnică de cromatografie pe coloane bazată pe silice, care folosește o tehnică de vid și este ușor de implementat cu echipamente standard. Este mai eficientă din punct de vedere al resurselor și permite purificarea unor cantități mari de compuși, în timp ce cromatografia flash este recomandată pentru cantități mici sau compuși volatili.

  • 00:10:00 - 00:15:00

    Echipamentul necesar pentru DCVC include un funel din sticlă, un adaptor pe vid, un robinet cu trei căi și sisteme de primire. Colegiile de lucru pot varia în funcție de preferințele individului, însă siguranța este prioritară, iar sticlele trebuie verificate pentru eventuale crăpături.

  • 00:15:00 - 00:20:00

    Pentru a pregăti coloana DCVC, silicea trebuie împachetată corect. Daniel demonstrează tehnica de împachetare eficientă a coloanei, subliniind importanța menținerii unui nivel de 5 cm pentru a evita problemele de separare.

  • 00:20:00 - 00:25:00

    Odată ce coloana este pregătită, Daniel explică cum să se facă asamblarea, cum să se determine siguranța echipamentului și cum să se utilizeze diverse instrumente pentru a garanta un proces de separare corect și eficient. De asemenea, sunt subliniate erorile frecvente și cum pot fi evitate.

  • 00:25:00 - 00:31:39

    În concluzie, Daniel subliniază importanța tehnicii DCVC pentru purificarea compușilor chimici, oferind linkuri și referințe utile, inclusiv recunoașterea originalității tehnicii și recomandând vizitarea blogului său pentru informații și sfaturi suplimentare.

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Mapa mental

Vídeo de preguntas y respuestas

  • Ce este DCVC?

    DCVC este o tehnică de cromatografie cu coloană bazată pe silice, care completează cromatografia cu coloană Flash.

  • Care sunt avantajele DCVC față de cromatografia Flash?

    DCVC necesită mai puțin silice și solvent, este mai rapidă și permite purificarea unor cantități mult mai mari.

  • Ce echipament este necesar pentru DCVC?

    Pentru DCVC, sunt necesare coloane de sticlă, filtre de hârtie, adaptoare de vacuum și un robinet cu trei căi.

  • Ce tip de silice ar trebui folosită pentru DCVC?

    Silica cu dimensiunea particulelor de 15-40 microni este ideală pentru obținerea unui bun separare.

  • Când ar trebui să folosesc cromatografia Flash în loc de DCVC?

    Pentru cantități foarte mici (50 mg sau mai puțin) și pentru compuși volatili, cromatografia Flash este superioară.

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Subtítulos
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Desplazamiento automático:
  • 00:00:07
    hi my name is Daniel S Peterson and I'm
  • 00:00:09
    an associate professor in Medicinal
  • 00:00:10
    Chemistry at the University of
  • 00:00:12
    Copenhagen in Denmark I'm here today to
  • 00:00:14
    give you a short introduction to dry
  • 00:00:16
    column vacuum chromatography or dcvc for
  • 00:00:20
    shorts now um this is a very basic
  • 00:00:23
    tutorial I'm only going to show you very
  • 00:00:24
    basic equipment you need and how to set
  • 00:00:27
    up a column and run it so no fancy uh um
  • 00:00:30
    experimental details today or expert
  • 00:00:33
    tips and tricks if you're interested in
  • 00:00:35
    further details at the end of this
  • 00:00:37
    introduction there will be links to a
  • 00:00:39
    useful websites and to Publications
  • 00:00:42
    where dcvc experts have lots of useful
  • 00:00:44
    information for you right so what is
  • 00:00:47
    dcvc well dcvc is basically a silica
  • 00:00:50
    based column chromatography technique
  • 00:00:52
    that complements flesh column
  • 00:00:54
    chromatography um so the big question
  • 00:00:58
    right now for you of course is why
  • 00:00:59
    should you start using dcvc instead of
  • 00:01:01
    flash column chromatography well there
  • 00:01:03
    are many advantages uh over flash column
  • 00:01:06
    chromatography when we uh think about
  • 00:01:09
    dcvc um two minor advantages but
  • 00:01:13
    interesting and positive ones is that
  • 00:01:14
    it's a vacuum-driven technique it's much
  • 00:01:16
    nicer to work with vacuum than with
  • 00:01:18
    pressure uh in in an organic synthetic
  • 00:01:21
    organic lab um secondly uh the basic
  • 00:01:25
    equipment you need for the CVC is
  • 00:01:27
    available in all well equipped Labs you
  • 00:01:28
    don't have to have the glass slow or
  • 00:01:30
    make anything uh special for you so you
  • 00:01:32
    can just get going from immediately uh
  • 00:01:34
    if you want to now for the real
  • 00:01:36
    advantages uh first off uh it's it's a
  • 00:01:39
    huge resource saver right so you need a
  • 00:01:41
    lot less silica for dcvc and much less
  • 00:01:44
    solvent that you would use for Flash
  • 00:01:45
    colum chromatography another real
  • 00:01:47
    Advantage is it's a very fast technique
  • 00:01:49
    so in a matter of hours you're
  • 00:01:50
    completely done with dcvc whereas flash
  • 00:01:53
    column chromatography can drag on for
  • 00:01:54
    hours and hours uh finally uh I guess
  • 00:01:58
    the biggest advantage of these C VC is
  • 00:02:00
    in fact that you can scale it up very
  • 00:02:02
    easily and do hundreds of grams of
  • 00:02:04
    purification so personally I've done a
  • 00:02:07
    200 g purification on several occasions
  • 00:02:10
    and this is completely Unthinkable in a
  • 00:02:12
    standard lab uh if you're using flash
  • 00:02:14
    column
  • 00:02:15
    chromatography advantages or
  • 00:02:17
    disadvantages uh of dcvc um so when
  • 00:02:21
    would I use Flash instead of dcvc uh for
  • 00:02:24
    really small scale stuff I think it's
  • 00:02:25
    Superior so if you're working with 50
  • 00:02:27
    milligrams or less I think dcvc is
  • 00:02:29
    tedious flash column chromatography much
  • 00:02:31
    faster and easier to do uh and secondly
  • 00:02:34
    a very important detail is that if you
  • 00:02:36
    have volatile compounds dcvc is not good
  • 00:02:38
    because it's a vacuum-driven technique
  • 00:02:40
    so definitely do flash for for volatile
  • 00:02:42
    compounds furthermore uh low boiling
  • 00:02:45
    solvents are problematic for dcvc it can
  • 00:02:47
    be done but it requires a bit of
  • 00:02:49
    experience so dlor methane acetone um
  • 00:02:52
    and ether for instance that are very
  • 00:02:54
    popular for Flash column chromatography
  • 00:02:56
    are a bit harder to implement for dcbc
  • 00:02:59
    so that's the basic uh pros and cons and
  • 00:03:01
    background for dcvc and now we're ready
  • 00:03:03
    to have a look at the equipment you need
  • 00:03:05
    uh to run a dcvc
  • 00:03:11
    purification so let's have a look at the
  • 00:03:13
    equipment you will need to uh to run a
  • 00:03:15
    dcvc separation so first let's have a
  • 00:03:17
    look at the columns uh the column is
  • 00:03:19
    simply a sensor glass funnel uh that
  • 00:03:22
    most Labs will have on the Shelf unlike
  • 00:03:24
    a standard c glass funnel this one has
  • 00:03:26
    to be a little bit taller so ideally you
  • 00:03:30
    will have a column that looks something
  • 00:03:31
    like this so it has to be around 7 to 10
  • 00:03:34
    cm tall uh it has to accommodate a silic
  • 00:03:36
    column that's about 5 cm salt and there
  • 00:03:38
    has to be head space for loading a
  • 00:03:40
    compound and for loading the solvent
  • 00:03:42
    while you're running the column uh these
  • 00:03:44
    can be purchased or custom made in
  • 00:03:47
    various diameters uh and so depending on
  • 00:03:51
    how much compound you're purifying of
  • 00:03:52
    course the the diameter of the column
  • 00:03:55
    has to go up the most commonly employed
  • 00:03:57
    ones are the 4 and 6 cm diameter columns
  • 00:04:00
    these are really good for most
  • 00:04:01
    applications ranging from 100 mg to I
  • 00:04:04
    would say up to 10 G uh depending on how
  • 00:04:07
    hard the separation is of course okay so
  • 00:04:10
    now we have the column in place uh when
  • 00:04:13
    we pack the column the silica has to be
  • 00:04:14
    very firm so you need to have uh some
  • 00:04:17
    good presses and spatulas to compact the
  • 00:04:19
    silica now I will shortly show you how
  • 00:04:22
    to actually pack the column but let's
  • 00:04:24
    first have a look at the equipment
  • 00:04:25
    you'll need and of course you can do
  • 00:04:26
    countless variations on this theme uh
  • 00:04:28
    what we use uh here at the University of
  • 00:04:30
    Copenhagen is these uh plastic rods
  • 00:04:33
    presses they look like this and they are
  • 00:04:36
    really really
  • 00:04:38
    practical when you're packing your
  • 00:04:40
    silica you can of course have custommade
  • 00:04:43
    fancy stuff that looks like that for
  • 00:04:46
    instance um that's up to you of course
  • 00:04:48
    uh how much you want to invest in it one
  • 00:04:51
    more thing that you require this is very
  • 00:04:52
    simple to make yourself is a spatula
  • 00:04:55
    that has a bent tip this is for
  • 00:04:56
    compacting the edges of your column and
  • 00:04:58
    these are also
  • 00:05:02
    available in all kinds of sizes of
  • 00:05:04
    course you just have to buy the right
  • 00:05:05
    size spatula this is something that our
  • 00:05:06
    Workshop does we buy the straight
  • 00:05:08
    spatulas and they bend the tip for us
  • 00:05:11
    okay so now we have the column and the
  • 00:05:13
    packing gear in place now we have to
  • 00:05:14
    look at uh the receiver to put your
  • 00:05:17
    column in after you've packed it this is
  • 00:05:19
    the most basic setup you can imagine so
  • 00:05:21
    we already have the column in place all
  • 00:05:23
    you need is a SE a separatory funnel
  • 00:05:26
    like this so just a standard SE funnel
  • 00:05:28
    nothing fancy uh um then you need a some
  • 00:05:31
    type of vacuum adapter and these are
  • 00:05:33
    normally made of glass uh here at our
  • 00:05:35
    department we use these made of Teflon
  • 00:05:37
    so it's basically a vacuum adapter with
  • 00:05:39
    a sidearm right just plunk that in there
  • 00:05:42
    next you need a three-way tap this is
  • 00:05:44
    essential because you want to be able to
  • 00:05:46
    uh release the vacuum while you're
  • 00:05:47
    running the column so you can get your
  • 00:05:48
    fractions out at atmospheric pressure uh
  • 00:05:51
    and that's simply attached
  • 00:05:55
    here like that and that's your basic
  • 00:05:58
    assembly now you you just need to pack
  • 00:06:00
    your
  • 00:06:01
    column put it on top and you're ready to
  • 00:06:04
    go that was the basic setup um there are
  • 00:06:08
    nicer ways to do this and and the
  • 00:06:10
    preferred setup that we have in our lab
  • 00:06:12
    looks like this so we have these
  • 00:06:16
    receivers made by the glass blower and
  • 00:06:19
    it's essentially a SE funnel but now uh
  • 00:06:21
    the vacuum adapter is integrated into
  • 00:06:22
    the glass wire so it's just nice and
  • 00:06:24
    convenient to work with and it has a
  • 00:06:26
    good size uh and of course we have these
  • 00:06:28
    made even bigger if we're doing very
  • 00:06:29
    large scale
  • 00:06:31
    separations uh we we prefer to not have
  • 00:06:34
    ground glass joints for these but of
  • 00:06:35
    course that's an option it's up to you
  • 00:06:37
    what you would like to work with uh but
  • 00:06:39
    we prefer this and we simply have these
  • 00:06:40
    rubber bungs we put in and like before
  • 00:06:43
    we simply put our column on top and
  • 00:06:45
    we're ready to go again essential with
  • 00:06:47
    the three-way tap otherwise you will
  • 00:06:49
    lose a lot of time because you'll have
  • 00:06:50
    to turn off the vacuum all the time so
  • 00:06:52
    always have a three-way tap on right uh
  • 00:06:55
    again you can see here I'm using the
  • 00:06:56
    plastic one obviously you can use a
  • 00:06:58
    glass three-way tap doesn't make any
  • 00:06:59
    difference um we like these because they
  • 00:07:01
    don't break when we drop them uh on the
  • 00:07:03
    floor okay let's have a look at a
  • 00:07:06
    sophisticated solution at Advance
  • 00:07:07
    solution so one of my colleagues has
  • 00:07:10
    been kind enough to borrow me his
  • 00:07:12
    personal dcvc equipment and it looks
  • 00:07:14
    like this and this is a a different way
  • 00:07:17
    of doing
  • 00:07:18
    it where he actually has the vacuum uh
  • 00:07:22
    attached to the cented funnel and he has
  • 00:07:23
    a ground glass joint and now he's
  • 00:07:25
    actually collecting fractions in Erland
  • 00:07:27
    me flasks so he has a lot of these
  • 00:07:28
    standing in the hood and he simply
  • 00:07:30
    collects a fraction and then he moves to
  • 00:07:31
    the next ear my flas collects another
  • 00:07:33
    fraction and he moves along like that so
  • 00:07:35
    that's another option there's countless
  • 00:07:36
    ways of doing this um you just have to
  • 00:07:38
    find the way you prefer but I mean if
  • 00:07:40
    you just want to give this a shut first
  • 00:07:42
    to see if you like it or not I would
  • 00:07:43
    just go for the basic setup with a SE
  • 00:07:44
    funnel and it's just as good if you
  • 00:07:47
    won't get a better separation with this
  • 00:07:48
    than you will with a SE funnel set okay
  • 00:07:52
    so what kind of vacuum will you need for
  • 00:07:55
    dcvc and what kind of Silica should you
  • 00:07:56
    use we need a soft vacuum so anything
  • 00:07:59
    between 10 and 50 Ms will do the trick
  • 00:08:02
    and this is very easy to achieve in a
  • 00:08:04
    synthetic organic lab a water aspirator
  • 00:08:06
    will do the
  • 00:08:07
    trick a diaphragm pump like this one
  • 00:08:11
    will do the trick alternatively you have
  • 00:08:13
    vacuum built into your fume Hood so
  • 00:08:15
    house vacuum which is also quite uh fine
  • 00:08:17
    for this type of U column chromatography
  • 00:08:20
    under no circumstances should you use a
  • 00:08:22
    high vacuum pump or oil pump something
  • 00:08:23
    like that because the vacuum is too
  • 00:08:25
    strong and there's a real risk of
  • 00:08:26
    implosions and it's completely
  • 00:08:28
    unnecessary it will and just solent will
  • 00:08:29
    evaporate so it's I I would not do that
  • 00:08:31
    under any
  • 00:08:32
    circumstances okay so vacuum that's
  • 00:08:34
    easily done in all
  • 00:08:38
    Labs finally the silica and this is
  • 00:08:40
    extremely important the silica particle
  • 00:08:42
    size is essential to get it right if you
  • 00:08:44
    want to get be successful with your
  • 00:08:45
    separation uh no I'm not being paid by
  • 00:08:48
    Merc to promote their products but this
  • 00:08:49
    particular product works really well uh
  • 00:08:52
    so I'm just going to show this I'm sure
  • 00:08:54
    there are many other brands of silica
  • 00:08:56
    gel that will do the same trick as long
  • 00:08:57
    as you get the particle size right uh
  • 00:08:59
    you need 15 to 14 micrometer particle
  • 00:09:02
    size to get u a good column and good
  • 00:09:05
    separation if you get finer particles um
  • 00:09:08
    your column will be eluding very slowly
  • 00:09:10
    so I wouldn't recommend it it becomes
  • 00:09:11
    too compact if you have corser particles
  • 00:09:14
    like what you would use for standard
  • 00:09:15
    flashh column chromatography it's very
  • 00:09:17
    hard to pack the column uh hard so you
  • 00:09:20
    get poor separation so very important
  • 00:09:22
    that you get the right particle size and
  • 00:09:24
    so now we've had a look at all the
  • 00:09:26
    equipment you need I think we are ready
  • 00:09:27
    to uh demon rate how to actually pack
  • 00:09:30
    the column and get started with your
  • 00:09:35
    purification okay so now we're ready to
  • 00:09:38
    pack the column and uh before we start
  • 00:09:40
    doing that we have to think about safety
  • 00:09:42
    so obviously you're going to wear a nap
  • 00:09:44
    coat plastic gloves and you'll be
  • 00:09:46
    working in a fume Hood also before you
  • 00:09:49
    start check all the glassware you'll be
  • 00:09:50
    using for cracks and so on because
  • 00:09:52
    implosion is a real danger when we were
  • 00:09:54
    working with vacuum so you want to have
  • 00:09:56
    nice clean sturdy glasswar before you
  • 00:09:58
    start this with no cracks
  • 00:10:00
    okay I'll be packing a 4 cm diameter
  • 00:10:03
    dcvc column
  • 00:10:05
    now it looks like that and so what we
  • 00:10:08
    are aiming for here is a packed column
  • 00:10:10
    that's about 5 cm tall it's not so
  • 00:10:13
    important whether it's 4 and 1/2 or 5
  • 00:10:14
    and 1/2 cm tall just the rough ballpark
  • 00:10:17
    around five if it's significantly
  • 00:10:19
    shorter than five you really lose
  • 00:10:21
    separation power and becomes more like a
  • 00:10:23
    filtration if it becomes much taller uh
  • 00:10:26
    than 5 6 cm it doesn't really help your
  • 00:10:29
    purification so you're just wasting
  • 00:10:31
    resources and time so try to get it
  • 00:10:33
    around 5
  • 00:10:34
    cm so to achieve this you have to load a
  • 00:10:37
    few cm more of loose silica so if you
  • 00:10:41
    load 7 cm of loose silica that will end
  • 00:10:44
    up being around 5 cm uh when you finish
  • 00:10:47
    packing it so
  • 00:10:49
    now just to help myself I'm going to
  • 00:10:52
    indicate on my
  • 00:10:55
    funnel where 5 cm is like that
  • 00:11:04
    and then I will require a p a flask for
  • 00:11:08
    the actual packing of my column and I
  • 00:11:10
    have a rubber bong in there so you
  • 00:11:13
    really have to uh make sure that the
  • 00:11:15
    equipment is fastly secure before you
  • 00:11:17
    start so you use a clamp of
  • 00:11:19
    course just to avoid it falling over so
  • 00:11:22
    now it's nicely positioned there with
  • 00:11:24
    the rubber
  • 00:11:25
    buk we can put our sensor funnel in like
  • 00:11:28
    that and I'm using another clamp I'm not
  • 00:11:30
    actually tightening it I'm just using it
  • 00:11:32
    to stabilize it so I don't accidentally
  • 00:11:33
    knock it over finally I need my vacuum
  • 00:11:37
    and here I have a three-way tap for my
  • 00:11:38
    vacuum and I've already actually opened
  • 00:11:40
    up for vacuum which you may be able to
  • 00:11:43
    hear or not believe
  • 00:11:47
    me it is there as you'll see in a minute
  • 00:11:50
    when we apply vacuum to the Silicon
  • 00:11:53
    right so right now there's no vacuum on
  • 00:11:54
    the system it's very important that when
  • 00:11:56
    you pack you don't apply vacuum to the
  • 00:11:58
    system okay okay so now we should add
  • 00:12:00
    the silica
  • 00:12:02
    and I've got the Merk silica here the 15
  • 00:12:05
    to 40 microm silica and we're aiming for
  • 00:12:08
    about 7 cm of blue silica I think I'll
  • 00:12:11
    just move this up so you can better see
  • 00:12:12
    what I'm
  • 00:12:13
    doing so here we
  • 00:12:20
    go and it's really important that you do
  • 00:12:22
    this in the fum with silica is actually
  • 00:12:24
    really bad for you so
  • 00:12:30
    pull down the sash while you're doing
  • 00:12:31
    this now we almost
  • 00:12:37
    there there we go that's around 7 cm a
  • 00:12:41
    bit more maybe not that
  • 00:12:43
    important uh so now we just have to tap
  • 00:12:46
    the surface of the sensor to get a nice
  • 00:12:48
    level uh surface of the silica and to
  • 00:12:49
    compact it a little bit uh and for that
  • 00:12:52
    you should just use something of plastic
  • 00:12:53
    some people like to use a rubber tube
  • 00:12:55
    I've always preferred to use a uh
  • 00:12:57
    plastic funnel it really does doesn't
  • 00:12:59
    matter what to do here just find a
  • 00:13:00
    solution something nice and soft like
  • 00:13:02
    plastic is good because it will not
  • 00:13:04
    break the glass so you can see how the
  • 00:13:06
    silica is
  • 00:13:10
    compacting when I'm doing this and I'm
  • 00:13:12
    getting a nice and level surface and
  • 00:13:14
    already now we probably down to less
  • 00:13:15
    than 6 cm of silicon okay so now I'm
  • 00:13:18
    ready to apply vacuum so here we go and
  • 00:13:22
    you can see the silica being compressed
  • 00:13:23
    even further and now I'm going to use my
  • 00:13:26
    presses so I uh I have two sizes here I
  • 00:13:29
    think I'm going to go for the for the
  • 00:13:30
    bigger size for
  • 00:13:33
    this and this is not supposed to take a
  • 00:13:35
    long time if you spend more than 5
  • 00:13:36
    minutes packing your column you're doing
  • 00:13:38
    something wrong right you should be it
  • 00:13:41
    should be a
  • 00:13:42
    fast packing it only takes a few minutes
  • 00:13:46
    to do this so first you compress the
  • 00:13:48
    column really nice and tight like
  • 00:13:52
    this and now we're ready to do the edges
  • 00:13:55
    this is very important because the edges
  • 00:13:57
    uh of the of the column is actually way
  • 00:13:59
    you get uh problems most frequently
  • 00:14:01
    because you have channels forming so
  • 00:14:03
    it's very important to pack these hard
  • 00:14:05
    and now my the little Mark I made on my
  • 00:14:07
    column is going to help me because I
  • 00:14:09
    will be able to know when I've done one
  • 00:14:10
    round of packing right as I'll be
  • 00:14:13
    turning so turning the funnel as I
  • 00:14:17
    [Music]
  • 00:14:18
    go so I'm going to twist it a bit like
  • 00:14:21
    that and keep going
  • 00:14:29
    depending
  • 00:14:32
    on how pessimistic you are you might do
  • 00:14:35
    this
  • 00:14:36
    twice personally I always just do this
  • 00:14:40
    once um the exception would be very big
  • 00:14:42
    columns because they're a bit harder to
  • 00:14:44
    pack Sly and you might repeat it a few
  • 00:14:47
    times for a very big column but we're
  • 00:14:48
    talking very big column I mean 20 cm
  • 00:14:51
    diameter that sort of
  • 00:14:54
    thing so
  • 00:14:56
    there it's packed nicely
  • 00:15:03
    and it's ready to go so now we are going
  • 00:15:06
    to put a piece of filter paper on top
  • 00:15:07
    simply to protect the surface uh when we
  • 00:15:10
    running the
  • 00:15:11
    column and of course I have the wrong
  • 00:15:13
    size filter
  • 00:15:17
    paper this is the classic problem so you
  • 00:15:19
    just have
  • 00:15:21
    to cut it
  • 00:15:23
    all of course you can buy the right
  • 00:15:27
    size there we go uh and now I have to
  • 00:15:31
    check if I pack the column properly and
  • 00:15:33
    I simply do this by pouring a apolar
  • 00:15:35
    solvent on and I I always use heane and
  • 00:15:37
    what I want to see is when I put a lot
  • 00:15:38
    of heane on I want to see the solvent
  • 00:15:40
    front descend
  • 00:15:42
    horizontally uh down the column right so
  • 00:15:44
    that's what we're looking for so I have
  • 00:15:46
    some hip Tain here
  • 00:15:59
    I'm just going to pour that on and
  • 00:16:02
    hopefully you'll see the Solen front
  • 00:16:04
    descent
  • 00:16:06
    nicely it extremely
  • 00:16:09
    rare in fact I cannot
  • 00:16:11
    remember when I lasted a column that
  • 00:16:14
    wasn't packed properly I mean it must be
  • 00:16:16
    before 2000 so with a bit of experience
  • 00:16:19
    this very you can see how the Solon
  • 00:16:20
    front is descending very nicely all now
  • 00:16:23
    we just have to suck the the column
  • 00:16:26
    dryish not super dry just until it stops
  • 00:16:28
    dripping and then we are ready to load
  • 00:16:30
    our
  • 00:16:30
    sample so while this is
  • 00:16:33
    happening um I'm going to show you how
  • 00:16:36
    to load your sample
  • 00:16:39
    now now for this purpose I would always
  • 00:16:41
    recommend uh that you use dry loading uh
  • 00:16:44
    it's foolproof it's a very efficient way
  • 00:16:46
    uh to load your compound without having
  • 00:16:47
    any problems you can do wet loading if
  • 00:16:49
    you like uh but I wouldn't recommend it
  • 00:16:51
    because there are some problems
  • 00:16:52
    associated with that so let's pretend
  • 00:16:54
    our compound is in this flask now we
  • 00:16:57
    want to dissolve it in a polar Sol
  • 00:16:59
    something that doesn't have too high
  • 00:17:01
    boiling point so that we can remove it
  • 00:17:02
    again on the root re evaporator and I'm
  • 00:17:05
    I'm going to use ethyl acetate for my
  • 00:17:06
    demo here so put a funnel
  • 00:17:09
    in put some ethal aate in there we
  • 00:17:17
    go and now my imaginary compound has
  • 00:17:20
    been uh
  • 00:17:22
    dissolved and now I will add Seine
  • 00:17:25
    because I will absorb my compound onto
  • 00:17:27
    oh sorry that was silica gel I will
  • 00:17:29
    absor my compound onto seite it's very
  • 00:17:32
    important that you use the seite and not
  • 00:17:34
    the silica
  • 00:17:36
    gel cuz the sea line doesn't really bind
  • 00:17:38
    the compound very strongly so it will
  • 00:17:41
    not compromise resolution whereas silica
  • 00:17:43
    gel of course will compromise resolution
  • 00:17:45
    uh quite dramatically because the
  • 00:17:47
    compound will be strongly associated
  • 00:17:48
    with
  • 00:17:49
    silic right you can add as much seelite
  • 00:17:52
    as you like uh as long as you can spit
  • 00:17:54
    it on top of your column it's fine
  • 00:17:56
    because the compound really has very low
  • 00:17:58
    affinity for for the sealine so you have
  • 00:18:01
    your compound dissolved with some sea
  • 00:18:03
    light in now you simply put it on the
  • 00:18:04
    rotary evaporator and concentrate it to
  • 00:18:06
    dryness remember to use a splash guard
  • 00:18:08
    because it has a tendency to bump so
  • 00:18:10
    this way you will prevent putting your
  • 00:18:12
    sea like and your compound in the entire
  • 00:18:13
    rotary evaporator so use a bump G all
  • 00:18:16
    right so um let's pretend that I've uh
  • 00:18:20
    concentrated this on the root of
  • 00:18:24
    evaporator so here I have my dry sea
  • 00:18:27
    light I taken a off the road evaporator
  • 00:18:30
    and I put it I have put it on a high
  • 00:18:32
    vacuum uh manifold house vacuum
  • 00:18:34
    something like that to remove tray
  • 00:18:35
    solvents so now it's reasonably dry and
  • 00:18:38
    it's ready to be loaded onto my silica
  • 00:18:40
    column again I will disconnect the
  • 00:18:42
    vacuum so I'll let air into the
  • 00:18:45
    system during the
  • 00:18:47
    packing now I
  • 00:18:49
    simply lo my
  • 00:18:52
    compound and of course you can go to
  • 00:18:55
    Great Lengths at trying to scrape out
  • 00:18:56
    everything and so on uh generally this
  • 00:18:59
    is not necessary because you will use
  • 00:19:01
    quite a lot of sea light to compound so
  • 00:19:04
    if there's a little bit of sea light
  • 00:19:05
    left it's it's a tiny amount of your
  • 00:19:07
    compound so it's not really that
  • 00:19:08
    important right the seite does not have
  • 00:19:11
    to be compacted as well as the silica
  • 00:19:13
    gel so um just tap up bit like
  • 00:19:18
    this you just need a nice uh firm
  • 00:19:22
    um
  • 00:19:24
    layer of sea light on top
  • 00:19:31
    so no need to exaggerate this so press
  • 00:19:36
    it a bit this
  • 00:19:38
    guy like that do the
  • 00:19:47
    edges like
  • 00:19:49
    that and now you need another piece of
  • 00:19:51
    filter paper to put on to to protect the
  • 00:19:53
    surface
  • 00:19:58
    so that the surface doesn't
  • 00:20:02
    get disrupted uh during coling so you
  • 00:20:06
    get a nice even surface and that's
  • 00:20:08
    basically the packing it's a very quick
  • 00:20:10
    you can see it doesn't take long um and
  • 00:20:13
    we're basically ready to do our column
  • 00:20:18
    out okay so now we've packed the column
  • 00:20:21
    and we are ready to actually run our DC
  • 00:20:23
    VC separation and for that purpose I'm
  • 00:20:25
    going to use my own preferred setup so
  • 00:20:27
    these customade receivers that we have
  • 00:20:29
    our glass blower made for us and I'm
  • 00:20:31
    using this small little beauty
  • 00:20:33
    here now I have to mount that on my ret
  • 00:20:37
    stand here with a clamp so I simply
  • 00:20:40
    attach it like
  • 00:20:43
    this at this stage it's quite important
  • 00:20:46
    that you adjust the height so it will
  • 00:20:48
    fit to your test
  • 00:20:49
    tube it's rather annoying if you forget
  • 00:20:52
    to do this at that's and forget to do
  • 00:20:54
    this because then you have to start
  • 00:20:55
    messing around with this when you
  • 00:20:56
    actually started running the column so
  • 00:20:58
    this is good I can receive my fractions
  • 00:21:00
    easily get it in and out
  • 00:21:02
    right I'm going to run a column using a
  • 00:21:04
    20 ml fractions so just standard test
  • 00:21:07
    tubes in a rack you can tell that I only
  • 00:21:09
    have around 33 test tubes here it's more
  • 00:21:13
    than enough a standard separation dcvc
  • 00:21:17
    you will be running around 25 fractions
  • 00:21:19
    and then you're done otherwise you've
  • 00:21:21
    done something wrong I would say right I
  • 00:21:24
    have my three-way tap here as before and
  • 00:21:26
    I attach it to my reer like
  • 00:21:29
    this I need a rubber bang again I put
  • 00:21:32
    that in the top like that and then I
  • 00:21:34
    have my column that I
  • 00:21:37
    packed put that in the
  • 00:21:40
    top and then you have to sort of adjust
  • 00:21:42
    it here to get a reasonably levels uh
  • 00:21:44
    like get
  • 00:21:45
    the the
  • 00:21:47
    column
  • 00:21:51
    reasonably well aligned so the Sol will
  • 00:21:54
    go down in a horizontal line and not at
  • 00:21:56
    an weird angle so something like that
  • 00:21:58
    and we can to apply vacuum just to
  • 00:22:00
    check how it looks and it looks good
  • 00:22:03
    yeah it looks nice like this so we're
  • 00:22:05
    basically ready to run the column now uh
  • 00:22:07
    the really great thing about the CVC the
  • 00:22:09
    reason that you get such amazing
  • 00:22:12
    separation uh compared to flash column
  • 00:22:14
    chromatography is that uh you basically
  • 00:22:16
    always do gradient illusion I mean you
  • 00:22:18
    could do isocratic illusion but it's
  • 00:22:20
    simply so easy to do gradient illusion
  • 00:22:22
    that that's what you end up doing and
  • 00:22:23
    you get excellent separation uh in fact
  • 00:22:26
    I would argue that you get better than
  • 00:22:27
    CLC separation using this
  • 00:22:31
    technique so sometimes you will have a
  • 00:22:33
    single spot on CC then you column it and
  • 00:22:34
    you discover that in fact that was two
  • 00:22:37
    things I'm going to run a column using
  • 00:22:39
    uh ethy acetate and heptane I'm going to
  • 00:22:43
    go from 0 to
  • 00:22:44
    100% ethy acetate inate and I'll be
  • 00:22:48
    doing 5%
  • 00:22:49
    increments and as I said I will be doing
  • 00:22:52
    20 M fractions so that means I will just
  • 00:22:55
    have to add 1 ml more of each acetate
  • 00:22:59
    for each fraction until I hit 100% and
  • 00:23:02
    then I'm
  • 00:23:03
    done so for it's a bit more convenient
  • 00:23:07
    to have the solvents in these small
  • 00:23:08
    beers so I'll do
  • 00:23:10
    that uh I use some measuring cylinders I
  • 00:23:12
    like these plastic ones because you tend
  • 00:23:14
    to knock them over and these don't break
  • 00:23:15
    so that's quite convenient so I will put
  • 00:23:18
    the ethal acetate
  • 00:23:22
    into this guy and then have in here now
  • 00:23:27
    interesting uh detail about dcvc is
  • 00:23:30
    that the first two to four fractions are
  • 00:23:34
    simply simply disappear right so uh they
  • 00:23:37
    are absorbed on the
  • 00:23:39
    silica the system gets saturated Vapors
  • 00:23:41
    and so on so you start off with just
  • 00:23:43
    running straight hepan through uh for a
  • 00:23:46
    while so I typically do three 100% heane
  • 00:23:50
    fractions just to get the system
  • 00:23:52
    equilibrated and ready to go vacuum is
  • 00:23:54
    already applied I'm just going to lower
  • 00:23:56
    this a bit so you can better see when I
  • 00:23:57
    add the solvent
  • 00:24:00
    so I'm just going to pour on the first
  • 00:24:02
    fraction of heane like
  • 00:24:05
    that and I'm just immediately going to
  • 00:24:07
    put the next one on because these two
  • 00:24:10
    guys are just going to get
  • 00:24:11
    absorbed I'll prepare the
  • 00:24:15
    next two fractions of HP here
  • 00:24:19
    so and when it starts coming
  • 00:24:22
    through and it you you seem like you're
  • 00:24:24
    recovering all most of your solent then
  • 00:24:26
    you're ready to start the call you can
  • 00:24:28
    see the first bit of heane is coming
  • 00:24:30
    through now it's dripping uh relatively
  • 00:24:32
    slowly already so this is about as much
  • 00:24:35
    you will get through in a reasonable
  • 00:24:36
    time span so you just stop now you let
  • 00:24:39
    air into the system vacuum is still
  • 00:24:41
    applied but not on your Reservoir and
  • 00:24:44
    you remove this tiny bit so this is
  • 00:24:46
    actually all that you get out from the
  • 00:24:48
    first two
  • 00:24:50
    fractions and now the column is pretty
  • 00:24:52
    much ready to go so let's pour on
  • 00:24:54
    another fraction and see what happens
  • 00:24:57
    never throw these fractions out you
  • 00:24:58
    never never know if something really bad
  • 00:24:59
    happened during your loading or packing
  • 00:25:02
    you might be really unlucky that your
  • 00:25:03
    compound's in here and it's actually
  • 00:25:05
    never happened to me but you never know
  • 00:25:07
    I never throw these out until I have my
  • 00:25:09
    compound and now you can tell that the
  • 00:25:11
    column is actually going very
  • 00:25:14
    nicely see the tap at the bottom here is
  • 00:25:16
    leaking a bit so you get a bit of
  • 00:25:17
    bubbling but that's no problem and you
  • 00:25:20
    shouldn't be waiting for a long time you
  • 00:25:21
    don't have to wait until it stops
  • 00:25:23
    dripping you you have to sort of move on
  • 00:25:24
    and just keep collecting fractions so
  • 00:25:26
    we're just going to stop there collect
  • 00:25:28
    the next
  • 00:25:30
    one and now I'm doing my final fraction
  • 00:25:32
    of pure
  • 00:25:33
    heane and now I will start doing
  • 00:25:37
    my ethy acetate ethy acetate
  • 00:25:40
    gradient so I'm going to do
  • 00:25:45
    um 1 ml
  • 00:25:48
    of ethy acetate to start with and 19 M
  • 00:25:53
    of heane right
  • 00:26:02
    and this is not a super exact science
  • 00:26:04
    you just have to get it about right so
  • 00:26:07
    no need to be super precise about
  • 00:26:09
    this so this first one is 19 peptine one
  • 00:26:14
    M of ethal
  • 00:26:16
    acetate and we're
  • 00:26:24
    off and the nice thing about the CVC is
  • 00:26:27
    that while the fra are going through you
  • 00:26:29
    have a bit of time to work so I always
  • 00:26:32
    make my fractions undergo so I never uh
  • 00:26:36
    oh some my solvent mixes on go I never
  • 00:26:38
    make these beforehand because you have
  • 00:26:40
    time for it and with a bit of
  • 00:26:42
    practice you will even be able to
  • 00:26:45
    squeeze in running TCS in
  • 00:26:47
    this uh during running the column so
  • 00:26:49
    basically you get everything
  • 00:26:51
    done and one go
  • 00:26:58
    so I think that's about right
  • 00:27:01
    yep so I hope you're getting sort of a
  • 00:27:03
    sense
  • 00:27:07
    of how easy this is you're not supposed
  • 00:27:10
    to sit here with a measuring cylinder
  • 00:27:13
    and measure this out super
  • 00:27:17
    accurately I like to use the measuring
  • 00:27:19
    cylinder for the eth state here because
  • 00:27:22
    it makes me keep track of how far I've
  • 00:27:24
    come in my gradient because I can simply
  • 00:27:26
    do basic math and calculate my way back
  • 00:27:29
    if I all of a sudden o sorry I have to
  • 00:27:31
    remove this one first if I'm uncertain
  • 00:27:34
    about my
  • 00:27:36
    progress right collect that
  • 00:27:40
    one let me do two more here here we
  • 00:27:43
    go now a question I often get is when is
  • 00:27:46
    my compound going to
  • 00:27:48
    elute and uh as a very rough rule of
  • 00:27:50
    thumb uh compounds a Lo in the T in the
  • 00:27:54
    in the solvent where they would have a
  • 00:27:56
    TC RF value of about 5 so let's imagine
  • 00:28:00
    that our compound that we're trying to
  • 00:28:01
    isolate here had a RF value of
  • 00:28:04
    0.5 in uh in 1 to one ethy aate heptane
  • 00:28:08
    then I would expect it to elude around
  • 00:28:11
    fraction 10 to 12 so halfway through my
  • 00:28:17
    purification right so uh now I'm
  • 00:28:20
    basically uh putting on my last fraction
  • 00:28:23
    that was 100% eate I just put on and um
  • 00:28:27
    I'm done with my uh
  • 00:28:29
    column if I had been doing a real column
  • 00:28:32
    and not just a demo like this I would
  • 00:28:33
    have been running my TLC while I was
  • 00:28:36
    going and I would already know which
  • 00:28:38
    fractions I would have to combine uh on
  • 00:28:39
    the road rap maybe I have even started
  • 00:28:42
    doing that already uh kind of depends on
  • 00:28:44
    U how good and experience you get with
  • 00:28:46
    this technique uh but I would say that
  • 00:28:49
    uh with practice and experience um
  • 00:28:53
    provided you have your compound loaded
  • 00:28:54
    on the sea to start with uh and you just
  • 00:28:58
    start with the equipment in front of you
  • 00:29:00
    it will take you 2 hours from start to
  • 00:29:02
    finish uh and I when I say finish I mean
  • 00:29:05
    your compound has been concentrated on
  • 00:29:06
    rotary evaporator so 2 hours from start
  • 00:29:09
    to finish uh when working with 4 to six
  • 00:29:11
    uh CM diameter columns so that's really
  • 00:29:13
    fast uh you can do a lot of columns in a
  • 00:29:16
    day I wouldn't recommend doing more than
  • 00:29:17
    four you start losing your mind around
  • 00:29:19
    number five um but I mean personally and
  • 00:29:22
    it was a very long day I came in very
  • 00:29:24
    early and I left extremely late uh but
  • 00:29:26
    I've done nine columns in a day
  • 00:29:28
    uh so this is I mean I cannot imagine
  • 00:29:30
    doing nine flash columns in a day that
  • 00:29:32
    would just be impossible U unless you
  • 00:29:35
    were doing them in parallel or something
  • 00:29:36
    like this so this was serial I did nine
  • 00:29:38
    columns in a
  • 00:29:39
    row right
  • 00:29:42
    so that basically
  • 00:29:46
    concludes my demonstration of uh dry
  • 00:29:49
    column vacuum
  • 00:29:51
    chromatography I hope you found it
  • 00:29:53
    helpful and uh thanks for watching
  • 00:29:59
    right so to conclude this uh
  • 00:30:01
    introduction to dcvc I have a few uh
  • 00:30:03
    practical uh links and references for
  • 00:30:05
    you and some information about the
  • 00:30:07
    history of dcvc so uh I get a lot of
  • 00:30:10
    emails about the dry column vum
  • 00:30:11
    chromatography from enthusiasts and uh
  • 00:30:14
    most people tend to ascribe the
  • 00:30:15
    technique the invention of the technique
  • 00:30:17
    to me but this is in fact not true this
  • 00:30:19
    is an old technique and we basically U
  • 00:30:22
    refined it and wrote a better protocol
  • 00:30:24
    for it uh the original publication of
  • 00:30:27
    this was by Lawrence Howard and uh the
  • 00:30:29
    reference to his original work you can
  • 00:30:31
    find in our paper on dry column vacuum
  • 00:30:33
    chromatography that we published uh back
  • 00:30:35
    in 2001 in synthesis uh below me you can
  • 00:30:38
    see uh a link to uh to this paper and
  • 00:30:42
    the reference I'll be grateful if you
  • 00:30:44
    could cite this reference if you have a
  • 00:30:45
    published work uh where you used dcvc
  • 00:30:48
    because I would like to spread the word
  • 00:30:50
    I think it's a very powerful and uh I
  • 00:30:52
    would say en environmentally very benign
  • 00:30:54
    alternative to flash column
  • 00:30:55
    chromatography so I would really hope
  • 00:30:56
    people would start using this
  • 00:30:59
    more um finally uh if you really want
  • 00:31:01
    some like real expert advice on uh on
  • 00:31:04
    dcvc and have some really technical
  • 00:31:07
    questions I would recommend that you
  • 00:31:08
    visit my blog curly Arrow you can find
  • 00:31:10
    the link below as well uh curly arrow.
  • 00:31:14
    .org um there's two blog posts on uh
  • 00:31:16
    dcvc there long ones with lots of
  • 00:31:19
    questions and comments tips and advice
  • 00:31:22
    from experts and users around the world
  • 00:31:24
    it's very useful uh and if you have your
  • 00:31:26
    own tips and tricks and ideas please
  • 00:31:28
    write them there I will respond to all
  • 00:31:30
    comments and
  • 00:31:31
    questions so All That Remains is for me
  • 00:31:34
    to thank you for watching this and good
  • 00:31:36
    luck with your cuming in the future
Etiquetas
  • DCVC
  • cromatografie
  • silice
  • echipament
  • universitate
  • Copenhaga
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