How to Unravel the Mysteries of Nature: Part Two - Electronspray Mass Spectrometer (ESI-MS)

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Nature is truly magnificent but also unforgiving. Mankind struggled and almost did not make it. The invention of tools helped us to survive and also to strive. In this series I want to talk about some of the most sophisticated tools mankind has ever invented. Tools that help us to unravel the mysteries of nature and to understand our place in this universe.

Man is a tool-using animal. Without tools he is nothing, with tools he is all.    
- Thomas Carlyle

Today I want to present to you an instrument that I worked with for a long time. The electronspray ionization mass spectrometer (ESI-MS). This instrument was vital in my research as it helped me to identify the products of an enzyme-catalyzed reaction. I spent hours in a little room with a very slow computer and little daylight, mixing reaction solutions with ethanol, filling up glass syringes and running the ESI-MS. Mass spectrometers are very important in chemistry and they can be found in every laboratory. Let's get started.

What is ESI-MS good for?

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A mass spectrometer will not tell you the different masses of compounds you found in a sample. Rather, you will get a graph as the one below. The y-axis (vertical) is usually the abundance in %. Abundance is relative, and the biggest peak will always respond to 100%. Thus all other peaks will be relative to the biggest peak. The x-axis (horizontal) is labelled the mass to charge ratio (m/z). If you know the charge of the substances in the sample, you know the molecular mass. Luckily, we knew that our samples had a charge of 1, thus we could just assume that m/z equals molecular mass, making the analysis of our results a lot easier.

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Usually the first step in the analysis of a mass spectrum is to find the peak that corresponds to the molecular mass of the molecule you are most interested in. In the mass spectrum above this is labelled MI+. Very often, your molecule is not exactly equal to its molecular mass, but something in the range of plus or minus 1. That is due to the fact that your molecule may have picked up or lost a hydrogen somewhere on its way through the mass spectrometer. All the other peaks you see in the mass spectrum are probably due to fragmentation. The phenomenon of fragmentation can be curse and blessing. While fragmentation may cause peaks that are more abundant than the peak of your molecule of interest it also creates a more complex spectrum. In some cases a complex spectrum is something you will want to avoid. Sometimes it is exactly what you want as it helps when doing structural analysis of a compound. The fragmentations patterns are a useful tool in determining structure.

So in summary a mass spectrometer can be used to
- Find identity of compound by determining its molecular mass
- Give structural clues about a molecule by using fragmentation
- Verify presence of a molecule by means of finding molecular mass

Fragmentation can be prevented by using so-called soft-ionization sources, like electronspray ionization (ESI). I will explain the mechanism behind ESI next.

Electronspray Ionization (ESI)

There is a very cool and somewhat simple principle at work. I will explain ESI using this illustration below.

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Your sample is diluted in ethanol. Then you fill a syringe with your sample and connect it to the machine. The ESI-MS I used had a pump that would push on the syringe to force sample solution into the ESI compartment at a steady rate (usually around 10 microliter per minute). Once the sample solution reaches the ESI compartment it enters into a needle, which has a high current. The pressure the liquid is pushed into the needle in addition to the current of the needle produces  a stream of small charged droplets (1). After passing through an open space, the stream of charged droplets enters an area of the MS, where the temperature is very high. Here the solution your substrate is contained in evaporates, resulting in smaller droplets (2). Charge is always present at the surface of the charge carrier. Thus the decreasing size of the droplets results in greater charge density at the surface of the droplets. The size of the droplets continues to decrease until the charge density gets to great for the droplet to carry and the droplet explodes. This process repeats itself until you have almost only charged single molecules (3). 

There are many other forms of ionization that can be used in mass spectroscopy. This website gives a good overview on some of the different ionization techniques most commonly used.

Now the charged molecules need to be separated according to their respective mass/charge ratios. While there are several methods, I will talk about the quadrupole mass analyzer. I have used this technique and I think it is very elegant. 

Quadrupole Mass Analyzer

A quadrupole mass analyzer is virtually just an array of four metal tubes that are connected to a electrical source. As can be seen below, there are two tubes that are opposing each other, forming a cross-like arrangement. The opposing rods are connected together. 

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The charged molecules enter the quadrupole mass analyzer. and start moving through the space between the rods. However, a voltage is applied to the rods causing the particles to move in a sinusoidal-like wave through the space in between the rods. Depending on the mass of the molecules and their charge, they are affected by the rods with varying strength. If they are drawn too close to the rods, their trajectory will become very instable, leading to eventual collision with one of the rods and thus, neutralization and exclusion from detection. By varying the voltage applied to the rods, the molecular masses permitted to pass through the quadrupole can be adjusted. This is usually done automatically and does not require any action by the operator. Personally, I think this very design is very elegant. The rods are very rigid and the lay-out is very simple and yet it is sufficient for most laboratory work. 

As I have mentioned, there are several other methods of mass analysis. I included a link to ion trap mass analyzers and time of flight mass analyzers. If you are interested in chemical instrumentation I would recommend reading about both techniques.

I have no background in electrical engineering and I do not feel comfortable to talk about detectors. Here is a link to the website of the University of Bristol that explains a few different types of detectors used in mass spectrometry.

Hopefully, I was able to give you a good insight into mass spectrometry. It is a very important instrument in modern chemistry. I did not use any sources for this work because I really spent this much time working with a mass spectrometer. However, I do want to give you a few things you can read in case you are interested. Just click on the links.

- A video about the quadrupole mass analyzer

- Introduction into mass spectrometry by Michigan State University and an introduction into protein analysis  
 using mass spectrometry by Thermo Fisher

As always, if you have questions please ask! If you liked this article please upvote and share. I am also writing a series on Genius and Madness and some scientific things like the Vitamin C Myth. My goal is to write 1-2 articles about something science related. I really want to help people to better understand science because I think science does not get the recognition it deserves. If you like my work please follow my blog to stay up to date. Thank you very much! 

Cheers @lesshorrible!