Ultra High Pressure Column Packing

Ultra-high pressure HPLC can be one method to combat the high sample complexity of shot gun proteomics mixtures. During the years there have been an o lot of effort in optimizing the hardware setup. For example using solid core particles or longer columns.

The Coon Lab tried to reduce the van Deemter A-Term by creating an really tight and homogeneous packing bed of C18 particle utilizing pressures of over 2000bar for column packing. The packed columns turned out of display reduced backpressure and improved sequence coverage of +23%.

How this has been achieved?

Well, here a special type of pump was used. An air driven liquid pump based on pneumatic principle from heskel. This company usually operates in the hydrogen-gas fueled car industry but there multi head pump system which are able to create outlet pressures of up to 7000bar shown to be beneficial for column loading. The fittings and values used have been manufactured by HiP. Especially the connection from the slurry reservoir, having a ID of 1mm to the capillary with and OD of 0.3mm seems to be crucial to me (an modified femal to male fitting was used).

Source: https://pubs.acs.org/doi/ipdf/10.1021/acs.analchem.8b02766

EvoSep One nanoLC - combines low pressure sample loading and offline gradient formation for reproducible proteomics measurements

Usually reproducibility is a huge problem in nanoLC MS using an conventional 2-column setup utilizing sub-2µm solid core particles.

Due to the high pressure that is applied to achieve best efficiency, mechanical parts have reduced life time, undergo faster service intervals and obtain performance variation (pressure fluctuation).These phenomena led to an fluctuation of retention times and resolution from run to run.

Although nanoSpray displays better sensitivity researchers have been going back to microflow application because it offers a good comprise because sensitivity and reproducibility.

The new Evosep one nanoLC introduced a few concepts, which are going to overcome these limitations. The system has 4 low pressure pumps and a single high pressure pump linked by the sample loop.

The Evosep one pre-forms a gradient at low pressure and high flow and stores it within an sample loop. While sitting together with sample no mixing occurs.

Before loading into the sample loop the sample is cleaned up with an zip-tip like tip, also at low pressure. Since the loop is directly located behind the tip hydrophilic peptides won’t get lost and will be transferred entirely to the analytical column.

Since the gradient is pre-mixed only a single pump is required for eluent delivery. This is way better for reproducibility than two separately working pumps for each of the mobile phases.

Further, this unique system provides less carry over and an optimized duty cycle with less overhead time (-35% time saving compared to EASY1200 nano).

All in all this system provides great features and I am pretty sure it will become the method of choice for short gradient clinical proteomics.

Source: https://www.biorxiv.org/content/biorxiv/early/2018/05/15/323048.full.pdf

The company evosep has got a pretty cool homepage and a youtube channel, which definitely should be checked out.

FUNPET - half ion funnel, half ion carpet

FUNPET is a hybrid ion funnel ion carpet MS interface. It combines the advantages on an classical stacked ring RF-only driven ion funnel and an ion carpet.
The ion carpet consists of multiple planar (isolated) metal rings with decreasing diameter stacked together. In the center there is orifice.

To each of the rings there is an RF and  DC applied. The RF (180 degrees out of phase between the rings) keeps the ions away from colliding with the rings and confines them in front of the ion carpet and its centric orifice. The DC is decreasing the closer it gets to get orifice, providing a electric gradient field for focusing the ions into the orifice and transmitting them into the next pressure stage.

Interestingly, the design includes an jet disruptor which causes dissipation of neutral gas molecules and therefore reduces the gas load for the next vacuum stage and increases ion transmission.

More information about the FUNPET and the ion carpet technology can be found at

https://link.springer.com/article/10.1007%2Fs13361-018-2038-3

or under http://www.massspecpro.com/technology/ion-optics/ion-carpet