Spectroscopy allows us to spy on molecules invisible to the naked eye. Through a cuvette the spectrophotometer allows us to peer and measure concentrations with the help of Beer! It lets you measure concentrations of molecules like DNA & proteins which are invisible to our naked eye, as well as molecules whose presence we spy see we want to put numbers to (quantify) Don’t have a lot of sample, never you fear, with the help of the NanoDrop you can still use Beer!
The Beer Lambert law (Beer’s law) allows you to convert between concentration (c) of a dissolved thing (solute) & absorbance (A) of light – if you know the extinction coefficient (how well the solute absorbs (steals) that light) & the path length (how far the light travels through the solution, which influences how many chances it will have to collide with solute & get absorbed).
Light – and other forms of electromagnetic radiation (EMR) is made up of little packets of energy (photons) traveling in waves.
All wavelengths of light travel at the same speed in the linear direction, but different wavelengths of light have photons with different energies – and the more energy the photon has, the more it wants to move – but since it has to go at the same speed as its less-energetic neighbors it resorts to “bouncing up and down more” – the wave peaks come at a higher frequency (closer together).
If a photon has just the right amount of energy for a certain molecule, and its bouncy path is on a collision course with that molecule, that molecule can “steal it” – absorb a photon. If not, the light just passes through – a little off-track but the same strength.
When a molecule absorbs a photon from the part of the light spectrum we can’t see, we don’t even know anything changed (but the spectrophotometer can so we can measure DNA, protein, & RNA molecules which absorb UV light). But if a photon gets absorbed from the visible part of the spectrum it’s like it steals a piece of the rainbow, leaving you with colored light – the color of which depends on what color was “stolen”
Because different molecules have different makeups they absorb different wavelengths of light to different amounts, giving them a unique absorbance spectrum – it will absorb most strongly at certain wavelength(s) but can usually absorb more weakly elsewhere.
To get a full absorbance spectrum, you shine light of all wavelengths (well, at least all visible & some ultraviolet (UV)) & measure what goes through (is transmitted). Whatever doesn’t go through is assumed to be absorbed (or abducted by aliens…). So absorbance = 1-transmission.
We can characterize how much a molecule absorbs light at any wavelength (we usually choose its “favorite” – peak absorption) using its extinction coefficient.
In addition to using this to measure concentrations of “boring” solutions like pure DNA or protein you just need to know the concentration of to use for something else, you can use it to measure more “exciting” solutions where molecules are actually changing (though honestly I think the boring stuff’s pretty exciting too!)
If molecules react to form new molecules with new makeups, those new molecules may absorb light differently. We can measure this absorbance and use it to track a reaction’s progress or see how the reaction conditions (e.g. how much you start with, temperature, time) affect things. These types of experiments are often referred to as “colorimetric assays” because you’re measuring color as your read-out.
So how does it work? We take whatever solution we want to measure and put it in a plastic or glass holder called a cuvette which has a window for light to shine through & stick it in a spectrophotometer which actually shines the light through one side & measures what comes out the other. Different cuvettes can have different path distances the light has to travel, and the Beer Lambert law takes this into account – the longer the path, the more molecules light’s likely to hit (regardless of the concentration) – and the more molecules it hits, the more chances there are to be absorbed. Our calculation of concentration is based on how much light gets absorbed so we need to account for this distance.
It’s also important to have a blank – this is just the liquid you have your samples in and all the “constant” components (e.g. salts, etc. that are in each reaction) – its made up of molecules too so it’ll have a characteristic absorbance that will always be there, whether or not the reaction we’re looking for actually occurs. And some of its absorbance spectrum might overlap with our products’. So we want to subtract it out so we don’t confuse it for our signal.
The equation is: A = εcl
A = absorbance
ε = extinction coefficient (aka molar absorptivity coefficient) – specific for particular molecule & particular wavelength; units of L mol-1cm-1
c = concentration (in mol/L) – this is molarity – a mole is just a chemist’s “baker’s dozen” – it’s Avogadro’s number (6.022 x 10^23) of something – solute molecules or donuts, it’s just a number http://bit.ly/2r4RnrX
l = path length (in cm)
Cuvettes are great for things where you have “large” samples. But if you don’t have much sample though, you’ll want something smaller-scale.
The NanoDrop spectrophotometer has a little pedestal you put a drop of liquid on (a really tiny drop, like 1-2μL “μL” stands for microliter and it’s a millionth-of a liter, or a thousandth of a milliliter. Then you lower the arm -> it contacts the liquid then pulls up a little bit and, when it does, it pulls on the liquid. It does this thanks to surface tension. Surface tension occurs because the molecules of the liquid like each other more than they like the air – so they try to stay together & maximize the liquid-liquid interactions while minimizing their combined air exposure. more here: http://bit.ly/2AeB1B6
When you put the drop on, surface tension causes it to remain drop-like. But when you lower the arm & squish it down, some of the water molecules stick to the top surface. And when the arm pulls back up, these molecules get lifted – and the other water molecules don’t want to leave their friends behind -> as a result a column of liquid forms
This column is just like the column of liquid in the cuvette except it’s much smaller and it’s held up by surface tension instead of being barricaded by glass/plastic. And it’s “sideways” – the light travels through from above and is recorded below
Another difference about this column is that the path length’s adjustable (the column can be pulled up higher or squished) – so it can adjust for different concentrations (e.g. if your sample’s too concentrated it’ll see this squish down to shorten the path length so the light doesn’t meet as many, or if it’s too dilute it can pull up to make sure lots of molecules get hit by light). The NanoDrop software then has to correct for this when it uses Beer’s Law to get to concentrations
A couple things we commonly use it to measure are concentrations of nucleic acids (DNA or RNA) and proteins. It’s easier to get columns to form for nucleic acids than proteins because the proteins often weaken the surface tension – not all parts of proteins like water, so it’s harder to get continuous water networks to stay as you pull up the column —> column breaks. But there’s strength in numbers, so, when loading protein, I usually load 2uL, but I load 1 or 1.2 for nucleic acids.
Another time we’re interested in extinction coefficients is with fluorophores – when most molecules absorb light, they just let off the extra energy they got as heat. But fluorophores are molecules that give back energy as light – but of a lower energy (longer wavelength, lower frequency) because the process isn’t completely efficient.
Just like with normal chromophores, the extinction coefficient tells you how well the fluorophore will absorb light of a specific wavelength. The higher the extinction coefficient, the more light will get absorbed and the more light will be emitted. more here: http://bit.ly/31vcK5G