Building a Nanodrop Style UV/Vis Spectrometer

I think it's safe to say we're all pretty familiar with rainbows you've either seen them after a good rain while you're washing your car or maybe just through some glass when the Sun comes through at just the right angle visible light is part of the electromagnetic spectrum which means it's made of photons whose wavelengths range from about 400 nanometers to about 700 nanometers when you shine a white light through a prism different wavelengths of light react differently some being redirected more than others as such the white light spreads out into all of its component colors at a rainbow forms but what makes a light white well for humans so long as it's got a bit of blue green and red it doesn't really matter what else is in there to us it looks white so long as you get that balance right take the Sun for example light from the Sun is a pretty good mixture of all the various colors but a large amount of it is concentrated around the greenish wavelengths this is why humans have more green color receptors we're set up to capture the maximum amount of usable light from the Sun but we don't see it as greenish we see it as white so how do we know that there's more green well we use prisms or other methods of splitting the normally mixed white light into all the various colors and then measure the intensities of each this is called spectrometry however when we do this we suddenly see all sorts of other details if we look closely for the solar spectrum there's these thin lines of color missing these correspond to specific elements in the Sun that absorbed those specific wavelengths we figured out which is which by taking individual elements putting them in a special tube and then putting enough electricity through them that they start to emit light we then measure that light with a spectrometer and see what lines show up a classic example is hydrogen pure hydrogen releases light at a few different specific wavelengths which is known as its emission spectrum these lines correspond to the way that the electrons orbiting the hydrogen atoms can move around electrons are confined to specific areas called orbitals we used to think that they literally orbited at different distances from the nucleus but now we know it's a bit more complicated than that for the sake of this discussion just think of them as discrete energy levels that are the only allowed places for electrons to exist when we put electricity through the hydrogen gas were essentially bombarding it with high-energy electrons which can pass off some of their energy to the electrons orbiting the hydrogen atoms this energy makes them jump to a higher orbital but that high-energy orbital is unstable and the electron wants to get rid of its new energy and return to what's called the gran State but because of conservation of energy they can't just do that so it packages and releases the extra energy as a photon and jumps down to the lower orbital it came from the energy of the released photon and subsequently its color is directly proportional to the difference in energy between the high and low energy state if we give the electrons even more energy they may jump two or three orbitals at once and when they decay they'll release higher and higher energy photons which are bluer and bluer depending on how far they jumped for hydrogen there are five of these potential jumping points that release photons we can see and they show up as these five bands in its spectra it's also important to note that electrons don't need to jump straight down to the ground state they can take smaller jumps on the way down which makes photons in the infrared range or larger jumps that produce UV light so there are way more than just five spectral lines this process also works in Reverse if a photon of the right wavelength hits a hydrogen atom it can absorb it and its electron will get bumped up to the corresponding high energy State remember those missing lines in the solar spectrum that's why they're there most of the photons emitted by the Sun or due to blackbody radiation because it's extremely hot which is a continuous spectrum think of it like an old incandescent light bulb the hot filament produces a smooth spectrum of light that looks white when that spectrum interacts with the ionized elements in the Sun any spots that correspond to elemental spectral lines get absorbed as a quick aside we can actually see elements in space this way if you put special filters on your telescope you can block out all the light except that thin band from one of the elements this red line on the hydrogen spectrum for example is called the hydrogen alpha line and this is what it looks like if you look at the Sun at only that wavelength this is literally the distribution of hydrogen in the Sun this also works for oxygen calcium and most other elements though it can be finicky if the lines overlap between elements which is possible and there's some other physical phenomena that can you shift these lines but we'll talk about that at the end of the video okay that's enough theory for now let's actually build a spectrometer so we can see all the different ways we can use it as simply looking at glowing things to see spectral lines are hardly all they're good for in the lab we use spectrometers all the time and all good labs should have at least a basic one I got the design for this build from hacked area and they have a PNG file of all the various parts you'll need I had these cut out on a laser cutter but having now built this it's safe to say you could have made the my hand with a little extra patience the design is far from perfect and the stock design was built around looking pretty rather than actual function but I think it's a good starting point as such I made several changes to the stock design that really improved the results the other parts you'll need are a good quality webcam that you can take apart some pieces of mirror here I'm using high quality first surface mirror some microscope slides and coverslips an audio fiber optic splitter a white LED a 405 nanometers v milliwatts diffraction grating and some black paint you also need some small nuts and bolts for holding the parts together and some hot glue or other adhesive after a quick test fit to see how everything goes together I gave all the wooden pieces a quick paint job I'm using black 2.0 the world's darkest commercially available matte black paint but regular black acrylic should work just fine we're just trying to cut down on reflections inside the device so the rainbow we make is as clear as possible off-camera I had already taken my webcam apart but I'll link to the one I used in the description moving on to the fiber optic splitter we'll be using this so that we can pipe two different light sources through the spectrometer but this isn't in the original design if you don't care about UV functionality you can just mount the white LED to the top part of the arm but I need both so I glued a white LED into one hole and later the UV laser in the other I modified the laser first so that I could power it with my bench power supply the design has a spot to mount the webcam but the author used a different model of webcam so I had to modify this piece to fit mine by first drilling a hole in the part of the mounting plate that is marked to be removed and then gluing it back into the main plate I ended up using a file to make this a tight fit and had to temporarily remove the camera lens to get it to fit nicely keep in mind that when you do this do not get anything inside the camera housing dust can ruin the sensor and/or the final image for the mirror I have a big pain of first surface mirror which means the mirrored surface is the one you can touch there's no glass between it this helps keep the light path clear and minimize losses I scored the mirror and tried to cut a nice piece but the glass itself was really thick so I had to go with artistically smashing it to get a workable piece it still cracked mostly along the score line just not as nicely as I wanted two more scores and cuts and I had a reasonably rectangular piece to work with be careful not to scratch the mirror when you do this which I realize is ironic considering I just smashed mine but I used an undamaged first surface mirror is fairly delicate because it doesn't have that protective glass mount the piece of mirror onto the mirror mounting plate with some hot glue I made sure not to glue the back but rather hold the mirror flat onto the wood and then tack on glue on the sides I didn't want it aimed slightly to the left or right so this gave a nice flat mounting that's still strong one last piece we need to make before assembling everything is a small acrylic cylinder to mount on the top arm I just cut a small square in the bandsaw and used a filed around it this is then painted black around the edges but leave the faces untouched finally it's assembly time thanks to the prep and laser-cut pieces this is actually really quick five of the sides can all be put together and held in place by bolts which leave the main chamber open so that we can install the rest of the hardware though it makes sure to feed the camera wire through the spot on the back first the camera mount is then just press fit into place and the mirror mount is held in with a bolt a few more details and this will be done before we get to the grading let's focus on the upper part of the assembly to make sure the fiber optic splitter fit and modified the mounting plate on the top arm so that it fit the square end of the splitter the plate was then attached to the arms of the fiber optic shines down through the hole to the underside of the arm I added both a coverslip to keep the fiber optic and protected and then added the acrylic nub on top which will guide the light to the sample and also squish the sample flat so it can be measured evenly to the lower part of the assembly I added a piece of microscope slide to act as a waterproof cover to put samples on before I glue it down permanently I used some aluminium tape on the underside to only leave a sliver for the light to shine through don't glue it down till the very end though so you can adjust its position this will make the rainbow quality way nicer by making the light into a thin beam but no matter what the stock hole in the design is way too big and must be reduced I may switch the tape for an adjustable set of razor blades to make the beam even cleaner later and finally with most of the light path done we can complete this by adding the grating at the beginning of the video I mostly talked about prisms as we're used to the idea of a prism splitting light well you could use a prism here diffraction gratings are convenient because they do the same job but are a flat sheet on a microscopic scale a diffraction grating is essentially a series of tiny prisms denoted by how many lines per millimeter that grading has think of the number of lines for a millimeter is sort of the focal distance of the grating the lower the number the closer the focal distance for this we want a 1000 line per millimeter grading eye right others but had the best results with the thousand line version if you can't find a diffraction grading you could use an old piece of CD but honestly getting diffraction gratings online is so easy I really wouldn't advise that when you go to mount the grading which is just done with a drop of hot glue make sure it's facing the right direction the lines on the grading go in a particular direction and the rainbow comes out at an angle relative to that direction so it's best to have the camera plugged in and look at what it sees as you're mounting it turn on the white LED on top make sure the light is shining down through the hole and start adjusting things adjust the mirror until it's shining the light through the grading and adjust the grading until it makes a good rainbow in the camera once that's done focus the camera so the rainbow is sharp and carefully close everything up and add all the remaining bolts and the spectrometer is basically done you can actually use the power supply for the camera to also power the LEDs and laser with the appropriate switch but for now I'll just be using my bench power supply it's obviously more bulky this way but I can fine-tune the amount of light carefully to make sure the sensor isn't being overwhelmed eventually I will get around to giving the LED and laser their own power supply and potentiometers so I don't need the bench of supply I also mounted the fiber optic splitter to the bar at the back here with a small hinge so it isn't flopping around a final touch is to go through and plug any remaining holes to reduce the amount of stray light that gets into the box - as close to zero as possible okay with the speck built let's talk more about what it can do and also why I chose this variety and not the kind you may be more used to seeing if you've worked in a lab this speck is what I would call nanodrop style and is modeled after thermo Fisher's nanodrop spectrometers those specs are great because they're designed to test very tiny volumes of liquid so in a bio lab where every micro liter of liquid counts being able to only test a small portion without wasting is super important the smallest sample I've tested with this device so far was about three microliters which seemed to be more than enough to actually use this we're going to need some software here I'm using a program called thermi know which is pretty basic but it works fairly well there are other pieces of software like spectrograph but I couldn't get that one working so I stuck with her me know for now to calibrate the software I use three different lasers of known wavelength and adjusted everything until they lined up with the correct parts of the spectrum a fun note is the green laser you can see the expected spike at 532 nanometers but look at the far end in the univers of the spectrum you can see a distinct spike this is the pump infrared beam that the laser uses to produce the green light which we've talked about in a previous video that's also why these cheap green lasers are so dangerous you can see that there's just as much infrared light is green light which can really damage your eyes if you get hit with a reflection so what else can we do with this now that it's calibrated well let's look at a few different common uses of specs if we keep the light source constant rather than looking at different light sources we can see how the light is affected when we put things in the light path and from that we can gain lots of useful information one of the most common examples is determining the concentration of a solution first we make a series of solutions of the thing we want to test that have known concentrations we place each in the path of the light and measure how much a particular wavelength is affected this is known as the absorbance value the more stuff in solution and the more light will be absorbed when we graph this to form what's called a calibration curve we should end up with a nice straight line we're increasing solute concentration directly increases absorbance then if we measure an unknown concentration of the same reagent we can work backwards take its absorbance put it on the graph and see how much stuff is in solution this basic concept is built upon for doing things like an Eliza assay where we can use a mixture of antibodies one of which is labeled with a colorful molecule to determine how much of a particular protein or other target is in a solution will look more analyzed assays in a future video another similar test is the look at optical density when we grow bacteria in a liquid medium as more of them grow the solution gets more and more cloudy if we measure the absorbance of the solution at about 600 nanometers we can estimate how many bacteria are there by how much light is absorbed this is how we know if our cultures are at the optimum cell count for doing various experiments more simply we can just look at the whole spectrum and see how the substance affects it here I've got some different lighting gels which are just colorful pieces of plastic when I put each in the light path you can see that different parts of the spectrum are absorbed more or less heavily based on which colour sheet I used and finally let's switch from the white LED to the UV laser now we're not looking at the light being absorbed instead we're looking at how the test material responds to the UV light and emits new lower energy photons a simple example is this piece of green fluorescent plastic here's the spectrum of just the UV light and here's what I had the plastic you can see a sharp peak from the dyeing the plastic emitting photons again if we do some calibration we could measure how much fluorescent stuff is in the light path one of the main reasons I built the speck this way is for this functionality rather than random green plastic we want to quantify DNA if we stay in some DNA with the same fluorescent dye that we use to make and run gels when the two combine they'll start to fluoresce under UV light by measuring how much light we see we can quantify how much DNA is present assuming we make a calibration curve like before I actually tried this but there's a bit of a problem the amount of laser light required to produce a glow makes an absolute mess on the camera image from stray scatter and overwhelming the sensor but the glow is very distinct and with a bit of Photoshop adjustment to remove the purple you can see a stripe of fluorescence clearly but since thermi no can't do this for us we can't currently quantify DNA properly so to help solve this I've ordered a special glass filter which will remove all of this purple light in a future video once it arrives I'll be making another mod to the speck so that the filter can move in and out of the light path as needed when we want to measure DNA once that's done assuming it works the speck will actually be pretty great ask any biologist and they'll tell you that aspect that can measure DNA normally cost upwards of $10,000 but this whole build including that new filter cost less than a hundred but other than that that's basically all there is to spectrometers I'm so excited to start putting this news back to lots of good use and we'll keep you posted on how well it functions even if it isn't good enough for DNA quantification yet it's still good enough for lots of other applications and I look forward to increasingly accurate quantification of data as I said earlier links to everything will be in the description if you'd like to make one of your own before I end this episode it's once again time to talk about nerd thunder which is a chance to share some other awesome YouTube channels today that channel is applied science first if you haven't already go subscribe because Ben's channel is amazing but more specifically he's done a bunch of videos about different optical phenomena one of which is how you can shift spectral lines using powerful magnets he did a really great visual demo of this and I've put some links to his videos in the description and that's where I'll end this video if you enjoyed then be sure to subscribe and ring that bell to see when I post new videos also be sure to head over to my other social media pages especially Instagram to see these projects long before they end up in videos and as always a massive thank you to my amazing patrons and channel members who make these videos possible that's all for now and I'll see you next week

Channel: The Thought Emporium

Views: 222,037

Keywords: spectrometer, spectrum, emission, absorption, od600, wavelength, uv vis, uv, visible, LED, nanodrop, qubit, spectrophotometer, calbration curve, diy, tutorial, build, concentration, black 2.0, diffraction grating, solar, fraunhofer lines, theremino, spectragryph

Id: pIk8I10ZmYY

Length: 15min 41sec (941 seconds)

Published: Fri Jan 18 2019

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[u/Roofofcar]

Thought Emporium is a fantastic channel with a wide range of videos on a ton of topics. I’ve been subbed for years, and I recommend it to anyone interested in chemistry, genetics, electronics, or space.

Great stuff.