Quickly Understand High Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography, or HPLC, is the single biggest chromatography technique to most laboratories worldwide.
If you have watched any of my videos on column chromatography, the main idea is the same. That is to say HPLC has 4 aspects of it in common with other chromatography techniques:
1. It separates mixtures of substances into their constituent components
2. It has a stationary phase and a mobile phase
3. The mobile phase flows through the stationary phase and carries the sample with it
4. Different components in the sample will travel at different rates, due to how they interact with the stationary phase, thereby separating them
However, HPLC is a highly improved form of column chromatography. A pump forces the solvent through the column under pressures of up to 400 atmospheres. The stationary phase is usually made of solid particles such as silica or polymers. This pressure makes the technique much faster than normal types of column chromatography which in turn allows for much smaller particles for the column packing material, which in turn leads to more particle surface area for interaction between the mobile and stationary phase. Ultimately, this leads to much better separation! HPLC is in addition highly automated and extremely sensitive.
Let us look at how this device operates in greater detail:
- A mobile phase is connected to a delivery pump which speeds up the overall process.
- Then the liquid sample is added to the setup, usually through a different tube.
- Then the sample gets carried with the mobile phase through the column and the stationary phase.
- At this point, the sample gets separated into their constituent parts.
- Then a detector converts the amount of each component into an electrical signal.
If you feel like this was a clear explanation, please show some quick love by liking the video!
The two most common types of HPLC are normal-phase HPLC and reversed-phase HPLC.
Normal-phase HPLC is less common of the two and uses a non-polar solvent. In addition it is filled with tiny silica particles which are polar. These polar silica particles interact more with polar compounds, making compounds stick longer the more polar they are. This results in polar compounds moving slower through the column while non-polar compounds move more quickly through it.
Reversed-phase HPLC is the more common and works in the exact same manner EXCEPT that the silica particles have been modified in order to make them non-polar AND a polar solvent is used. This causes the exact opposite effect, making the more non-polar compounds move slower and the more polar compounds move faster!

[@biotechlucas4126]

What topic would YOU like me to cover?

[@sakasan100]

Could you do a video on the topics "'Carbon - 13 NMR spectroscopy" and ''UPLC - Ultra-performance liquid chromatographic method'' ?

[@biotechlucas4126]

@@sakasan100 Here's the Carbon - 13 NMR Spectroscopy video: czcams.com/video/UKm0MtSLLNw/video.htmlsi=xFSv7EibQOSAOful
I'll add the UPLC video to my to do list!

[@sakasan100]

@@biotechlucas4126 Great, thank you 👍🏻❤

[@ludvahve]

thank you very much. Can you give a lecture on the analysis of different real human chromatogram data (sanger) with a longer video?

[@dr.shajijohn1251]

Excellent, Short and crisp

[@biotechlucas4126]

Thank you so much!

[@sebastianhernandezrodrigue8390]

Thank you for making this way more easier ! 🤩

[@biotechlucas4126]

I'm glad to hear I could help!😊👍

[@abass4311]

Thank you very much. Very wonderful

[@biotechlucas4126]

Thank you!

[@SunilKumar-vs8ny]

Need a lengthy video of HPLC and gas chromatography

[@biotechlucas4126]

Will do!👍 Anything in particular you want me to include?

[@supunkahawaththa6812]

Short and sweet

[@biotechlucas4126]

Thank you🙏

[@patrycjapajdak2491]

a very useful video, thank you! :))

[@biotechlucas4126]

I'm grateful to hear you found it helpful!👍

[@bafreeen.m]

really appreciate it thank you ❤️

[@biotechlucas4126]

I'm happy you found it helpful!🙏

[@BaidaA-qj7gx]

Synthesis of methylbenzoate, including (. (TLC) and GC. thanks

[@biotechlucas4126]

Thank you for the suggestion😇👍

[@theunknownsamurai7426]

As a laymen how do i read a HPLC Report?

[@willlawrie2644]

is the separation method of HPLC better than normal LC?

[@biotechlucas4126]

Yes, their resolving power is better!

[@kagithelvayim]

2:13 liked it 👍👍👍

[@biotechlucas4126]

Wow, thank you so much for watching so many of my videos and commenting on them!! I'm super grateful!

[@abhishekkumarsaurav9760]

Include image, so that it becomes easy to understand

[@biotechlucas4126]

Thank you for the feedback!

[@jothidharshinichannel5225]

Thin layer chromatography

[@biotechlucas4126]

Already got it!😄
czcams.com/video/DLxpYBWzOAw/video.html

[@kylitrixgames4980]

Kuthollander