Is there any advantage in premixing ACN/water (0.1% TFA in both) in gradient RPHPLC. Is there any benefit in using 5%ACN in water instead of 100% water as component A, and 5% of water in ACN instead of 100% ACN as B? From my prospective this may only contribute to the human error and waste of time not talking about the changes in mobile phase composition with time if He purging is involved. Does anybody have a different opinion?

Some (most?) low pressure-mixing gradient LC systems will have problems (eg., outgassing) if you ask them to mix 100% water and 100% ACN. I think MeOH is even worse.

Your point about changes in mp composition with sparging is well taken. If you are analyzing proteins & peptides, for example, an enormous shift in Rt is observed for a very small alteration in mp ACN content. However, if you arrange to sparge only for a minute or so, then blanket the solution with He, you can get very good results. For small peptides (insulin digest fragments), we have obtained 0.2-0.4% rsd on the retention times over 18 runs (including reprep. of the mp). (Thanks to Vydac for this tip.)

I don't pretend to understand the physics of mixing, but know from experience that for the absolutely quietest baselines (i.e., maximum sensitivity), premixed components will mix better than pure ones. Otherwise, for most applications it doesn't matter if you premix or not, and it is a bother.

As long as you don't excessively sparge, you shouldn't have retention drift problems. Only 1 volume of helium is required to degass an equal volume of mobile phase -- that is one liter of gas for 1 liter of liquid. Doesn't take much...

One way to minimize variations in premixed mobile phases is to weigh the components, calculating the required weight from the density of the liquid, instead of measuring them in a graduated cylinder. (This was suggested at a seminar I attended several years ago.) I agree with JD that composition change from sparging should be minimal if you don't overdo it.

Do you need to pre-mix? It depends on the instrumentation that you are using. Newer systems have improved solvent mixing capabilities making pre-mixing unnessary. If you want more info go to the Waters web page (www.waters.com) go to the application libary. In the search query enter wpp203 or wpp214 These 2 pdf documents are a good source of info on this topic.

If you are using older technologies, I agree with previous messages, pre-mix and sparge lightly.

A.A.

We find very little need to pre-mix when using the Waters Alliance system using an in-line vac degasser but for most solvents we use (ACN, MeOH, THF etc)a quick blast in a sonic bath under vac before putting them on the system seems to be needed.

I agree with JD (see above). I have experienced much quieter baselines when there is a small amount of solvent "premixing", even if it is only 2% of a solvent premix.

Beside the point if premixing is needed an other thing is importanted. You should not put a reversed phase column in 100% aqueous -- the bonded phase tends to collapse.

If you are using Alliance or HP 1100 binary then there is no problem with mixing by a pump.

Janusz

Of course, there are advantages to premixing of ACN and water for your gradient. Just as there was an improvement with older systems, the improvement of premixing is also present on newer models with degassers.Just not as great a degree of improvement.

It is very important, especally in method development phase to acuratly measure the volumes you are mixing. Volumetric flask , I find are the best and most reproducible. 500.0ml organic from a 500.0 vol. lask plus 500.0ml water from a 500.0 vol flask for a 50/50 mix. NOT 500.0 organic and qs to volume in a 1000.0 vol flask.

He sparging is also benifecial even with the in-line vacumn degasser. The degasser functions more effieciently with He as the desolved gas.Don't belive me? Give it a try on your instrument.

But as everyone has told you ,don't over sparge. I seen countless techs drop a spagre line in the mix and walk away! They just don't understand why their RT shift! I even stopped of who was spaging and used a black rubber stopper to weight the spage line to the bottom of the mobile phase(for an impurities method!!)


Also be aware that in-line degassers usally work by kicking in when the vacumn drops below a set point. This may cause a long term baseline cycling to occur. Particulaly in normal phase. The solution to this is to switch to continuas degassing( if possible) or not use the degasser at all.

You say human error makes the pre mix "not worth the time". That same human error will inadvertantly run your column at 100% water!

The real answer is "It depends on your method."
The best thing is to be ultra conservative in the method devlopment phase, and then test ruggedness and Robustness. It may be that for a assay with a small number of good response components. Its not a realvent issue. But for a low level impurities or trace analysis, It may be critical.

Do the work only you can answer this question on your instrument with your assay!
;-)

Great post and sound advice!

-- Tom Jupille / LC Resources