Does anyone develop methods using 250mm columns?

[mikea]

Hi all,

I’ve had sensitivity issues with a mixture of bases and hydrophobic compounds, specifically in that my early eluting bases had poor S/N. This is mainly because the diluent needed to dissolve both classes of compounds is 50:50 ACN:water (plus 0.1%TFA) and the point in which the bases elutes from in the gradient is ~39% ACN. I’ve used alkysulfonates to try to retain the bases longer so they will elute closer to 50% ACN in the gradient, however the baseline becomes much noisier at the DL level, which defeats the purpose of trying to retain the peak longer. pH has also been looked at up to 10.5, but tailing was quite high (2.2)

I’ve found that if I switch to a 250mm column (from a 150mm column of the same brand/manufacturer), the bases are retained longer (keeping a simple mobile phase of ACN:water:0.1% TFA) and elute at ~45%ACN, which helps a lot with the S/N and peak tailing. Also, my overall run time is only increased by 2 minutes.

My question is, does anyone develop methods using 250mm length columns anymore? Although my chromatography is a bit better using a longer column (and larger particle size) I’m wondering what I’m overlooking in moving to a longer column.

Thanks for your input.

[Anthony Crawshaw]

Do you really need to use acetonitrile?

Have you consider or tried methanol?

This would give a slightly different selectivity and potentially could help with the retention issue.

[Mark Tracy]

Not as often as I used to. I usually try a 3µ 4.6x150mm column first. But there are times when more plates is the only answer and I go for 5µ 4.6x250mm. Dionex's new Explosives columns are only sold in 250mm length because shorter columns don't quite make the resolution.

[Sallybeetle]

I also start with a 3-µm 150x4.6 mm column. Rarely do I have to go up in length. What stationary phases have you tried?

[Bruce Hamilton]

I mainly use 250mm x 5um , because I have a diverse range, and instrument time isn't a major issue. The speed and sensitivity advantages of 150 x 3um make them very attractive for most people.

My question would be " why has your instrument got a baseline noise problem during the gradient that affects your detection limit?. "

If time and sensistivity/resolution are important, it may be worthwhile to investigate the issue, otherwise, just use what works...

Please keep having fun,

Bruce Hamilton

[Bryan Evans]

Yes, there are a lot of people using 250mm, 3um columns.
We sell them all the time for people who need more efficiency
for complex samples.

[mikea]

Thanks so far for the comments. I've had great success in separating about 13 peaks in a combination of bases and more hydrophobic small molecules using mainly C18 stationary phases and the following setup.

Sample diluent (50:50:0.1 v/v/v) ACN:water:TFA
Mobile phase A: Water +0.1%TFA
Mobile phase B: ACN + 0.1%TFA
gradient: 30%B to 70%B in 20 minutes
injection volume: 15ul
column: ACE 3 C18 150 x 4.6, 3um

However, the molar absorptivity of the bases is about 20-fold lower than the more hydrophobic species in the mixture at the same concentration. To maximize the s/n of the earlier eluting bases, I’ve been trying to keep them on the column long enough so the organic strength will better match the organic strength at the time they elute. If I attempt to increase the concentration or increase the injection volume to 30ul, peak tailing of the earlier eluting bases becomes an issue for the response factors (ie greater than 1.7), which just looks terrible.

I’ve found that I can use a ACE 5 C18 250mm x 4.6, 5um, get the same selectivity using the same gradient and increase the injection volume to 40ul and obtain a tailing value of ~1.5-1.6 for the RF standards. The increased injection volume allows for better sensitivity and the longer column keeps my bases on the column slightly longer for better compatibility with the mobile phase.

It’s been a while since I’ve had to use a 250mm column. It just looks…archaic to me, as if I’m doing something wrong. 

[Mark Tracy]

You are not doing anything wrong at all. Long columns are more tolerant of sample overload than short columns. You found a satisfactory solution to your problem.

There is a lot of talk about moving to short/fast columns these days (I have done my share of it) and that is great if time or solvent are in short supply and chromatographic resolution is in surplus. But resolution and quantification are the essential requirements of any method, and you do what you must to get them. The only time that using a 250mm column would be a bad idea is if you develop a method that has far more resolution than necessary and don't take the time to find out if a faster method would be satisfactory, then stick your QA lab with an unnecessarily low-throughput method.