Bio-Inert LC?

[Karen01]

Could someone explain for types of compounds/work is a bio-inert LC important and why?

Thanks,
- karen

[Gerhard Kratz]

Some proteins can absorb to the surface of stainless steel. That's the reason why. Titanium frits, PEEK tubing etc. can help to avoid that. But most Proteins can be separated on a standard HPLC system.

[Karen01]

Thanks... I would assume that would matter most for aqueous SEC or IEX. But would it be likely to matter for RP? The organic (ACN typically) would mostly likely prevent sticking, no?

- Karen

[dorota]

Bio-inert LC would be my first choice for separation of proteins by ion exchange chromatografy. A mobile phase for IEC usually contains high concentration of NaCl. Look here for typical conditions: http://www.mnstate.edu/provost/IonExchangeProtocol.pdf
or here: http://www.seaviewsci.com/vydac/vydacpubs/vhpionex.pdf

[Andy Alpert]

These are two different concerns:
1) Getting the proteins to elute from the column (per Gerhard's response);
2) Preventing attack of the mobile phase components (especially Cl-) on the stainless steel of an HPLC system (per Dorota's response).
Making all the components out of titanium, glass, PEEK etc. solves the second problem but doesn't always solve the first. There can still be "hot" sites in titanium frits that adsorb proteins (titanium frits are actually made from an alloy that contains a significant amount of aluminum). The result can be elution ~ 10% later than usual and tailing peaks. In cases where either 1 or 2 is a problem, we instruct our customers to elute their column overnight at a low flow rate with 40 mM EDTA.2Na. This passivates metal surfaces in both the HPLC system and in the column. It eliminates most interaction of proteins with metal components of the system and also renders stainless steel resistant to chloride ion attack for about 3 weeks. Consequently, we feel that there's no need to buy a "bioinert" system. Such systems often feature compromises in pressure limits and tend to cost more.
I might note that it's essential to use a column that's designed to be compatible with intact proteins. That means a silica-based material with a thick, hydrophilic coating (unless you're performing hydrophobic interaction chromatography) or a polymeric material with a hydrophilic surface, cf. the VHP materials that Dorota posted the link to [NOTE: these are no longer in commercial production]. Don't put a protein on a polystyrene column and expect to see it coming out the other end in a well-shaped peak.

[Gerhard Kratz]

I agree with Andy. RP with ACN means denaturating conditions for Proteins. If you use Titan frits and PEEK tubing it will help a little bit. Some manufacturers can pack your RP material also in PEEK tubing. If you do the neccessary maintenance of your standard HPLC system, it should be not a problem.

[Karen01]

Thanks all...

It sounds like it a bio-inert system usually is not needed but sometimes is...

BTW I'm talking about analytical , not prep use. Right now it looks like it will be primarily for proteins and peptides, but some small molecule work will likely be needed as well.

I expect the system will be primarily used for RP but I think there is a significant chance it will be used for some IEX and SEC.

I don't think I'll need to be doing any NP ... but you never know.

So let me ask this, are there any downsides (besides price) to having a bio-inert systems for general use? Are there limitations in mobile phase? I suspect there might be for some NP solvents.

Thanks,
- Karen

[DSP007]

Hi
Looking out of our village . Bio-Inert LC is stupid marketing moves.
Better to see with my own eyes, what material was been done.
Example
http://www.icct.ru/Practicality/Papers/ ... 03-Big.jpg
- MiLiChrome-1 (model1968-1983 year, apparently the world's first UPLC (column 2x64 mm) . Architecture is very open - to the modern computer can be connected through the staff connector) . Pumps (syringe 2.5 ml , 5MPa 0.003-1ml/min) - glass, tantalum, Teflon, Teflon-communication, cell - quartz, Teflon (holder - steel), column- stainless steel or glass, silicagel or C18RP-silicagel (Lachema ChSFR). The concept of exclusion sorbent 40 years ago was not known, although (but) the "KCK" ( silicagel for draining gas, pores not normalized 300-1000 A) is normal exclusion sorbent.

There are more nuances, more than sorbtion.
Denaturation of proteins under the influence of components mobile phase , such as TFA. Fortunately the current sorbents sufficient quality and can not be taken into account.
Modern DAD detector use a powerful light beam, which can lead to protein denaturation.
From this point of view , Milichrom monochromator "jumping the mirror"very interesting.
In general .Principles of inert LC systems was been known for half a century, but then was not advertising idea.