Guard column Question

[juddc]

This should be an easy one for most of you, I'd think:

I've recently revised an assay that works quite well on a 100 x 4.6mm C8 column with a 3.5uM particle. Previously, I had used a 150 x 3.9 mm 5uM column (same chemistry), but found some worthwhile gains in resolution and run time with the shift in geometry / particle size. I find that the 3.5uM column doesn't last quite as well as the 5 uM did, however. It's not bad at all- I get at least 800 injections prior to having to think about buying a new column, but I had been getting at least twice that number previously.

I typically don't use guard columns because I'm particular about mobile phase and sample prep and I very rarely run into any column longevity issues, but it's been my experience that smaller particle columns foul a bit more quicly than 5uM ones do and I'd like to have this method as solid as possible prior to validating it and handing it off to QC.

In any case, I'm considering getting a guard column to improve longevity and they're available in two configurations: 10mm x 2.1mm with a 3.5uM particle and 20mm x 3.9mm with a 5uM particle.

Instict tells me to go with the 20mm x 3.9mm with a 5uM particle in order to avoid possible backpressure issues, but I'm not running on experience here. Is there any reason to more closely match particle geometry than column geometry?

Which would you buy and why?

Many thanks!

[DR]

If you have run htrough more than one column at >800 inj. per, I'd skip guard columns.

If you feel the need, however, I'd either match the 3.5µ particle size (the pressure increase will be minimal) or go with a disposable unit that is a reasonably close match to your particle (the male/female looks like a fitting type).

[juddc]

If the method in question was only going to be used in my lab, I wouldn't worry about a guard - and I usually don't. At 800 injections, the peaks are somewhat fronted (T @ ca 0.80 at worst), but separation (Rs of critical pair ca 3.0) and quantification are still OK and I'm at about $0.65 per injection. Nothing to complain about, considering the cost of syringes, filters, solvent, labor, and instrument time.

This method, however, is going over to another lab that demands higher throughput than mine and whose chemists are less experienced with LC, so I'd like to make it as robust as possible. If I can make a relatively small change and extend column life significantly- even from 800 injections - I'll do it

Thanks for the advice!

[danko]

Hi juddc,

What makes you think, the guard column will prolong the analytical column’s life?
If your column wears out due to extensive pressure for instance, then I can’t see the winnings.

Fronting can be attributable to channel/s in the packing material at the head of the column and very high pressure can cause such channels over time.

Or do your sample solutions contain non soluble material? If so, then simple filtration prior to injection is far better solution.

Best Regards

[juddc]

Samples, standards, and mobile phase are always filtered through 0.22 uM nylon and system pressure maximum is roughly 2600 psi on this method with no significant difference seen from old to new columns. 2600 psi should be well within acceptable parameters for this column and system.

The reason I'm considering guard columns is this:

My samples (they're heavy emulsions) require a more hydrophobic extraction solvent to recover the components I'm assaying than my starting MP. I find that 95-100% ACN works best depending upon the sample. My samples are relatively complex with significant hydrophobic material present. As a matter of course, I rinse my columns with 10 system volumes each of buffer free MP and neat ACN after every run, though most of the hydrophobes are not highly soluble in ACN, so not too much gets on column. I would think that this leaves the possibility that over time, I may be fouling the head of the column with small quantities of hydrophobic material and maybe other junk, which might be better caught on the head of a cheaper guard column than on my analytical column.

BTW I would change my rinse solvent to something more excipient friendly, but I only have a four channel system, it's a ternary MP, and the fourth channel is for water for the buffer-free portion of the rinse.

Solvent exchange would be more time consuming than QC would be willing to put up with and I'd worry about both recovery and emulsification, which is pretty easy to do with these samples.

Again, I'm not complaining about column longevity - 800 runs isn't bad, however it's cheap enough to see if a guard column helps or not and if it does, then it should be done.

I really just wanted to know about the appropriate size to purchase -my original question was whether I consider particle size or geometry to be a more important factor, given the column I'm using?

Thanks for your comments and advice!

[Uwe Neue]

Based on my own experience, and that of others, I am a very strong advocate of guard columns. On the other hand, if you buy a lot of columns from Waters, I consider this a worthwhile contribution to my salary .

Anyway, I have demonstrated in the past that you can get 10 000 injections from an analytical column protected by guard columns, and the analytical column looked brand-new afterwards. Guard columns were replaced roughly every 500 injections. A reference analytical column without protection died after less than 2000 inejctions. Of course, there are some limitations to this as well that depend on your mobile phase composition, especially the pH, but this experiment (together with others) demonstrated that guard columns do work...

There is one important aspect to the proper use of guard columns: the material in the guard column should be identical to the packing in the analytical column. If you use a Symmetry C18 analytical column, you should use a Symmetry C18 guard column preferentially packed with the same particle size. You want the guard column to retain the same stuff that would accumulate on your analytical column. If you use a random guard column, this can not be guaranteed.

Otherwise, to all those that do not like to use guard columns: I think you are absolutely right... guard columns are just another pain somewhere that one does not need

[juddc]

I'll assume that the consensus here is that DR's advice is correct and that I should purchase the 2.1 x 10 mm 3.5uM guard rather than the 3.9 x 20 mm 5.0uM guard for my 4.6 x100mm 3.5uM column.

Whether or not I need a guard wasn't really the question. The method is good as it stands. I know its foibles and where I've run contrary to common practice, I've had good reason to do so. Accuracy comes above all and to get there, I bent a sample prep rule, which may have some consequence regarding column longevity...so I use a tough column and am considering a guard.

FWIW, I've seen an unguarded 5uM version of this same column type (Symmetry C8) run >5000 injections under similar LC and sample prep conditions for the same types of samples.

Thanks to all for your input!

[Jumpshooter]

I use a guard column b/c I'm working with finished food products that tend to leave a moderate level of particulate matter in the final organic solvent extract prior to putting in autosampler vials. This even "after" my samples are syringe filtered thru PTFE 0.45 uM ! So far I have about 300 injections on my analytical column. I guess if I did not use a guard I'd be looking to purch a new one by now.

[Jumpshooter]

Oh, and a final point ab out gurad columns---I generally observe that my pump pressure (bars) increase about 1/3 when I use the guard. For example, I analyze samples at a flow rate of 1 ml/min---I noticed that the pump pressure was about 100 prior to installing the guard; after the guard was installed I observe 135 bars. My system is programmed to auto-shut off at 400 bars.

[shaun78]

To throw a slightly different idea out there....

It has been my experience and guard columns go bad without the analyst knowing and it is going to mess up at least one run every time this happens. Analytical guards are also expsneice.

Lets setp back a few years....

Remember those old Rehodyne inline filters that used the stainless steel filter disks. Yeah the ones having a porosity of about 0.5 um.

a filter disk does not have to be matched to a column and it will not alter your chromatography. Anything that makes it through a 0.5 um disk is clearly going to come out the other end of your 3.5 um column...

Cheers!

[juddc]

Jumpshooter - If you still have particulate or emulsified material visible in your analytical samples, you must get that out for the sake of your injector and column. Centrifugation can help prior to filtration. Doubling up filters or changing the diameter (from 13mm-25mm) of the filter can help, too.

Shaun78 - Your frit wil work fine for particulates but not for hydrophobes that may be fully dissolved in the analytical sample, yet sit on the head of a column.

Who'd have thought that a simple question asking only about appropriate guard column geometry would bring such a discussion?

Chris

[Bruce Hamilton]

I almost always use guards, unless a client specifies otherwise. The main reason being that they can protect against mishaps/wear in the injector and unwanted sample gunk.

I tend to use the 3-4 mm long guards ( eg Phenomenex Security Guard ), as they are relatively-cheap, easy to change, and are available in a reasonably-diverse range.

Obviously, it's nice to use the same brand as the column, but several column manufacturers use only the 10-20mm range, which are nice of ion-X and similar protection of column packing against binding junk, but a bit of overkill for my purposes.

It's definitely a good idea to use the same brand and packing for specialised-coating columns. Note, for example, if you look at the Zorbax SB preparative guards ( which are good value ), they use the same guard packing for their Cyano and C8 columns, and another guard packing covers both their C18 and Phenyl columns.

Please keep having fun,

Bruce Hamilton

[Uwe Neue]

Shaun78: I disagree with you. There are many things that make it without difficulty through a filter, but you'll never get it off the analytical column again. Typical examples are samples of biological origin with only partial cleanup. But you can get into the same trouble with samples from a polymer matrix or with polymeric additives, if the polymer is very sticky.

A filter only prevents particulate buildup. If this is all that you expect in your sample or your solvent, than this may be good enough. I like to play it more safely.

The next time you got a column that you do not care about any more, try to make a set of 10 microL injections of milk. It is a fast way to make C18 cheese

[shaun78]

Everyone does have valid points about the use of a frit as your guard.

I, however, would argue that if you are worried about other analytes getting into the column and sticking that will also pass through the frit, then perhaps an inappropriate sample prep/analytical column/mobile phase was initially selected.

My bottom line being I have worked for too many companies performing method development/validation work on anything from exceptionally clean to exceptionally dirty samples. When I used guards myself everything worked fine and I rarely had any problems mainly because I threw out the guards after a perdetermined number of injections (mfg's quality control on guards leaves something to be desired in my mind as I have found somewhat significant variation from lot to lot).

However, when these methods got transfered to the QC labs there was no end of trouble with them. Everytime I investigated the cause(s) of assay failure, it was always determined to be the guard. To me they are more of a pain than they are worth. Hence, if the sample is dirty I use a frit and a carefully chosen sample prep/column/mobile phase.

[juddc]

If the guards worked fine for you during development when you discarded them after a predetermined number of injections, why didn't they work well for QC when they discarded them after the same number of injections? Something doesn't sound right or they weren't following the SOP.

After you determined the number of injections that the guard would tolerate before failure, did you drop that figure by a bit in your SOP to be sure that replacement occurred before failure and QC would be able to avoid such failures altogether?

Sometimes it's simply not possible or economical to purify analytical samples sufficiently to avoid getting something bound to the column. I'm sure you've had samples whose other contents were largely unknown to you (any everyone else) at some point.

Yes, I suppose I could run each sample through an SPE cartridge to hang up hydrophobes in solution, but that would be MUCH more expensive than swapping out a guard column every 500 injections or so - and it's essentially the same thing as hanging a guard column on my system. A frit is dandy for particles but unfortunately won't hang up anything else that could be potentially problematic.

To Uwe - Your milk suggestion brought back memories. Years ago I was charged with the task of finding trace chlorite in raw milk. Sample prep took some doing to get recovery right and keep (expensive) columns from failing quickly. It was actually one of the first methods I ever developed from the start and it was a bit of a trial by fire, but it was fun...and yes, I used a guard column.

Best to all,

Chris