QUICKLY UNDERSTAND Liquid Chromatography Mass Spectrometry (LC-MS Simply Explained)
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- Опубликовано: 20 июн 2022
- Liquid chromatography mass spectrometry, what is it, how does it work and why is it useful?
So in the past, we’ve talked quite a lot about different chromatographies, playlist linked by the end of the video, in addition to a few techniques relating to mass spectrometry such as MALDI-TOF. Now, liquid chromatography (LC) is a technique used for separating compounds in a sample based on their affinity to some other molecule and can be paired with mass spectrometry (MS) for further analysis. In other words, it is the merging of these two different techniques! After chromatographic separation, the compounds are desolvated into the gas phase and ionized at an ionization source, and are then introduced into the mass spectrometer for mass analysis.
Liquid chromatography is a good separation technique, especially for larger and non-volatile molecules such as proteins and complex peptides. Combining this with mass spectrometry is especially useful since we can use different types of column chemistries for broad sample coverage. Liquid chromatography is in fact so efficient that it has largely replaced gel electrophoresis for molecular separation. LC is also especially well suited for pairing with MS since it reduces ion suppression which in turn would impede complete ionization needed for MS. An improved version of LC called high performance liquid chromatography also exists and has largely replaced regular liquid chromatography. The four main types of LC are partition chromatography, ion-exchange chromatography, size-exclusion chromatography and affinity chromatography and you can find all four of these chromatographies in my protein purification playlist, linked by the end of this video.
Also, if you have found the video useful so far, please give it a quick like!
In a nutshell, LC workflow is carried out in the following manner: A column, contains a mobile phase as well as a stationary phase. The interaction between our sample and the stationary phase, causes the different compounds in our sample to separate based on some unique difference in quality of these compounds such as size, charge or level of hydrophobicity. When the mobile phase flows out the column, it passes through a detector which “registers” a certain physical or chemical property such as refractive index or light absorption. This is displayed as a signal or “peak”, which corresponds to the amount of the component in the sample. The time at which the analyte is detected is called its RT and compounds of the sample can be confirmed through comparison of their RT to known RTs. However, this is not very reliable and requires secondary confirmation.
Now, the sample can move on to the mass spectrometer. The LC and the MS are coupled through an interface. The solvent is evaporated through the application of heat and analyte molecules are vaporized and ionized. This is crucial because the MS can only measure gas phase ions. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the two most commonly used ionization methods in LC-MS analysis. Then the analyte ions are drawn into the mass spectrometer, where they are subjected to electric fields and/or magnetic fields. The flight paths of the ions are altered by varying the applied fields which ensures their separation from another on the basis of their mass-to-charge (m/z) values. Post-separation, the ions can be collected and detected by a variety of mass detectors, the most common one being the electron-multiplier. The abundances of the ions measured during the analysis of a sample by LC-MS are plotted as a total ion chromatogram (TIC). The plot displays the peak intensities of the analyte ions versus their RT. Further, each point in the chromatogram is associated with the mass spectrum which depicts the ion abundances versus the measured m/z values.
What topic do you want me to cover next? Please let me know by replying to this comment!
manual interpretation of MS spectra
I have covered that topic here I believe: ruclips.net/video/b59u_BHOlnk/видео.html
How to read or interpretation the peptide chromatogram. I run sample but I don't know how to read the result
@@rajashuayb3187 Excellent suggestion, I will put it on my videos-to-do list!
Can you please explain about LC-MS/MS and how it provides a “fingerprint” map of a protein?
😊Excellent explanation! I will recommend your videos to my lab's members.
Thank you!!🙏
Wow such a great channel, keep the good work up man!🌟
Thank you so much!
thank you for the articulate explanation! it is wonderful
That's so sweet of you! Thank you!😇
very good explanation. i will recommend your channel to others
Thank you so much, that means a great deal to me🙏
Helped me for last minute interview preparation. Thank you.
Fantastic to hear! I hope you get the job!
You’re a god send. Thank you
I'm glad that I could help!
Thank you for your explanation
Thank you for your nice words!
I'm studying forensics and in this class we're supposed to be taught the theory behind chromatography
but prof said "research about liquid chromatography all by yourselves and make a presentation explaining all aspects of it including how to use it in forensics, that'll be your final grade" so i was like wtf you're supposed to teach us this. In the online classroom there's barely any info too
in my despair I found your channel with your explanations and visuals and it's a blessing, thank you!!!!
I do have a question about this, you say the "solvent" is evaporated after the HPLC. I'm assuming the solvent is the mobile phase + the sample? Just a little confused on that
Thank you so much for your kind comment. I agree, not very nice of your professor but then again that might be a good way for you to actually remember this stuff!
Currently at work and I don't remember what I said in the video, I'll double check when I can. In general I believe the solvent refers to the substance used for the mobile phase in chromatography.
But I might have made a mistake in the video and referred to both the mobile phase and analyte as the solvent. Let me check😇
Yeah you are right, I'm referring to both the analyte and mobile phase when I talk about the mobile phase/solvent.
However, I believe it would be more correct to say something like "mobile phase which carries the analyte/sample" or "solvent containing our analyte".
I'll try to be clearer in future videos. Thank you for pointing this out👍
Thank you so much
Happy I could help!😇
Amazing❤
Thank you! I appreciate you taking the time to comment!
So is there some sample prep that has to happen prior to LC (like digestion?)? Or is the LC the sample prep?
Depends what you are analyzing. Usually sample prep associated with LC will be required in this case as well. (Digestion is a good example)
hi there Lucas! great video! i was wondering if there is any way of contacting you for further specific questions! thanks
I read all my comments but you can also email me at biotechlucas@gmail.com👍
I need in depth about LC-MS
I'll add it to the list!👍
Will wait for that
I´m so thankfull for your channel!!! Thank you for your effort, keep up the good work! Maybe you can do some videos about Bioreactors. There are not many on yt
Great suggestion Nora, I will add it to my videos ideas list! Thank you🙏
Hi! what is the difference between LC-MS and LC-HRMS?? Thanks
So HR simply stands for high resolution, meaning that it refers to mass spectrometry techniques with higher resolution such as for example FTICR MS i.e. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry which I am currently making a video about😇👍
Please make videos on NMR
Yes, I have be meaning to for a long time! Definitely coming!!
@@LucasLearnz please make it fast my exams are coming you made it so easy just by seeing your video ..i don't have to open my book or do anything
man i go way to far to figure out what the fuck goes down in breaking bad
This is easily the funniest comment I've ever received😂 Thank you!
Kuthollander
ruik ik een nederlander?
Nope, I'm from Finland but my mother tongue is Swedish🙈👍
@@LucasLearnz aahhh that also makes sense hahah
Why can't u explain all R&D equipments
I am sorry, but what do you mean when you say R&D equipment?😇
@@LucasLearnz he means about all the techniques used in biosciences
@@suchetasharma5949 I'll make sure to cover everything I can in the future. Let me know where you want me to start.
who came here from MPMD ?😆
I need to check that out!😇
thankss that was usefull
Excellent to hear!