My solutions aren’t just cool, they’re SUPERCOOL! 😎 And then they’re glass!🤯 First, let me do you a a SOLID & describe what makes a solid 👇
If you you imagine a molecular “prom dance” – in a liquid the dancers are free to glide around & swap dance partners – they just can’t leave the dance hall. But in a solid the molecules can only stand in place and do that awkward sway thing with their partner(s). The difference between them is that in a solid the molecules don’t have the energy to “un-hug” their dance partners and go find someone else. What the molecules really want to do is leave the dance hall all together (escape as a gas) but that requires making their way to the exit without having new dance partners ask for a dance. So that takes a lot of energy.
The stronger the partners are hugging the more energy is needed to un-hug and water’s really huggy because the oxygen doesn’t share fairly – it hogs electrons, making it partly positive & the H’s partly negative – and opposites attract so they like to hang with oxygens from other water molecules.
Molecules would rather be on their own (where they have more entropy), but if they have to be there, and they have to get stuck in the super slow dance of solid-ness they’d rather be stuck with dance partners they like. So if they have “advance warning” that a slow song is coming they can link up to their preferred partners and doing so generates a rigid orderly arrangement of molecules we call a crystal. And that linking-upness may require pushing out the “noncool kids” (🙋♀️). So with slow freezing you end up freezing with different “cliques” in different areas of the dance hall instead of the nice evenly-distributed molecular crowd that is a homogenous solution. And if you’re dealing with water, the molecules literally get “pushed” because water expands when it freezes (even if the water’s inside pockets & channels of your protein!)
But if you can switch on the slow song without that advance warning, the molecules get stuck in place – this is the principle behind “flash freezing” And if you add cryoprotectants like glycerol that act like “dance chaperones” that break up the dance partners and make it harder for partners to find each other, you buy yourself more time
In a SOLID, molecules are “stuck in place” & only have enough energy to vibrate (as opposed to liquids where they can slide around & gases where they can go wherever they want). In some solids (like ice & diamonds) 💍, the molecules are arranged in an orderly lattice, & we call this a CRYSTAL or CRYSTALLINE SOLID
But going LIQUID ➡️ CRYSTAL (CRYSTALLIZATION) takes a lot of coordination It requires NUCLEATION. You need “trendsetting” molecules to freeze together in the “right” orientation (NUCLEATE) to form a “seed” & then others join in & those others have to be able to find that seed & get there before they run out of energy themselves
If there’s not enough time for this to happen (which can happen if you cool it really quickly &/or your solution’s really viscous (syrupy & hard to move through) the molecules will halt where they are & you get a disordered, AMORPHOUS SOLID 👉 we call this a GLASS & the process of forming it is VITRIFICATION 👍
the GLASS TRANSITION TEMPERATURE (Tg) is the range of temperatures in which a glass can form & it’s ⬇️ than the freezing temperature (when you go liquid ➡️ crystal). So in order to get to it you have to quickly skip past the freezing point to get to a SUPERCOOLED STATE (which you can do bc of the coordination required for crystallization) 👍
Our protein crystals are already in the crystalline form (thanks to a lot of hard work…) & we want them to stay this way! ⚠️ But they’re surrounded by (& full of) a LIQUID solution (which we call the mother liquor)
Today I purified a lot of protein. And I want to store it away for safe keeping in the -80°C freezer. But my protein is bathed in water and when I talk about “freezing my protein” I don’t want that water to crystallize (form ice) 😬
Why don’t we want the ice to crystallize? As we saw http://bit.ly/2U8XwRo water expands when it crystallizes, which can distort our protein 😬. And when water crystallizes, it pushes out solute, leading to uneven composition.
So how to get our protein cold enough?🤷♀️ Get the solution to form a VITREOUS GLASS 🤗 Since the molecules stay where they were as a liquid, you don’t get the expansion you get with ice👍 & the solutes can stay put too so the environment stays the same 👍
How do we prevent it from crystallizing? 🤔
🔸 add CRYOPROTECTANTS (like ethylene glycol)http://bit.ly/2U8XwRo 👉 make it harder to crystallize by
🔸🔸 ⬇️ freezing point & ⬆️ Tg (so u have less time in “danger zone” between them) – to form a crystal the water molecules must “push away” the glycerol molecules in between them and link together in their exclusive “clique.”
🔸🔸 preventing nucleation
🔸🔸 ⬆️ viscosity
🔹 freezing quickly 👉 dunk tubes of protein in liquid nitrogen to race through the freezing point & outrun ice formation
Liquid nitrogen is sometimes abbreviated LN2 , referring to the fact that the atoms of nitrogen (N) are present in pairs (diatomic). Nitrogen has a freezing point of −210 °C (−346 °F) so it can exist in a liquid form at very very cold temperatures (temperatures at which water w/a freezing point of 0°C (32 °F) would be frozen solid). So even though I’m using it as a liquid, it’s really really cold! (remember to take safety precautions – freeze your samples not your skin!)
With water, I’m worried about freezing, but with LN2, the bigger concern is *boiling*! When it comes into contact with warmer things (such as my samples), the heat from the warmer thing is transferred to the LN2. As a result, the sample is rapidly frozen, but the LN2 evaporates into N2 gas. This looks really cool but also means you’ll need more of the liquid form than you expect (our lab goes through a ton of it!). more here: http://bit.ly/30JJBni
So back to our prom analogy – we have a bunch of students (water molecules) dancing around & remove their energy (cool them) until they can only sway in place 👉 If they have “heads-up” they can all link together nicely (form a crystal) 💎
The more time they have before they run out of energy (slower the cooling rate), the more likely they are to do this & if there are already pairs or groups formed (nucleation sites), these can serve as coordinating centers
But if you have strict chaperones (cryoprotectants) that break up these pairs it’s harder 👉 & the more chaperones there are compared to # of dancers there are (⬆️ molar fraction of solute) the harder for the dancers to find each other & keep away the chaperones. It’s also harder if the room were filled w/jello instead of air (higher viscosity).
When you thaw a frozen sample, you’re adding energy that lets the molecules break free from one another & seek out new binding partners. But if you remove the energy again, they get stuck in place again (re-freeze). You can keep doing this freeze-thawing, but each time you do this you have more chances of crystals forming and damaging your sample. So you want to avoid multiple freeze-thaws.
But I have way more protein than I’d ever want to use at once – and I want to store it frozen to prevent degradation – so what to do? Make aliquots! These are like “single-portion” packages so that when I want to use some I only have to unfreeze what I want to use.
But since I usually don’t need to use a lot each time, I want to make fairly small aliquots but if I were to do this to all of my sample I’d have a lot a lot of aliquots. So instead I aliquot out 10-15 portions and then store the rest in bigger portions. If I go through the small aliquots I can dip into these “stocks” & aliquot those out.
We have a great machine shop down the hall that has a laser cutter and they made us these plastic “freezing aids” (don’t know what their technical term is) with spaced out holes to hold eppendorf tubes on ice (*on* but not *in*!) while I’m making the portions – then I pull it off the ice and stick it in the nitrogen. Let it freeze then whisk the tubes off to the -80.