HPLC questions?

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I know this isn't a straight EB question, but Fractal hangs out here more than anywhere else AFAIK, and if anyone else can make any positive input I'd be stoked

I'm fascinated with HPLC. OKOK I can't do any chem more complex than making instant coffee, but I seem to have the TLC thing under control from a technical perspective and the spectro ( when it gets used ) is making sense ( thanx burke and morph-man :D ) But one day I'd like to get a 2ndhand HPLC.

Prices in Australia even for used equipment are of course ridiculous. I almost got one on US ebay earlier this year for about a K all up, freight included. Unfortunately upon the rep checking that the unit it turned out the unit wasn't working, it wasn't just the power supply, and that as the unit was no longer supported by the manufacturers it might not be worth shipping here on the off chance the leccy dude here could sort it out for spares. Lucky the rep was most honest and informed me before it was freighted, it weighted about 120kg!

A local 2ndhand price for a prep unit ( which is the sort I'm looking for apparantly, it allows further working with separated compounds after analysis I think? ) is around $15K for a 25 yo unit. No way will I have that kind of money for years unless I can find a client who wants to seriously upscale the technology of the work I do.

But on the US ebay site they always have shitloads of individual HPLC components for SFA. I know I may need to change the power requirements from 110V to 240V, though I have an converter that would handle that for the interim. I know I'd need UV detection as well as standard, so I'd be looking at a variable detector unit.

Can anyone here tell me the feasability of putting together a working prep system from individual components? Is there much hassle connecting stuff of different ages across different manufacturers? And given that I want to do both reverse phase and normal HPLC, what individual components would I be looking for?

What I have so far is... guard column, column, variable detector, pump ( what kind? ) and something that reads things out. Something to interface maybe with the lab PC? Anything else?

I know I'm probably causing qualified and/or better informed ppl than I to squirm in their seats because I'm missing some perfectly straightforward information everyone else takes for granted, so if there's some learning I need to do to comprehend the basics I'd love to hear it

While I'm not chemically minded, I'm a helluva good tech, and once something has a straightforward and functional protocol I can happily work away with it successfully for years

TIA

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Not only might it all work out cheaper, but I'd be able to maybe pick up parts on a piece by piece basis and then so buy individual components when I can afford them :cool:

[Plant Pimp]

Hey! Everyone has to start somewhere.

I'm not a pro at HPLC, but I am an amateur. You seem to have thing pretty much down, but you will need an injector to mix the sample with the mobile phase too and maybe a heater for the column if it requires one (some work at 80C). The only thing I'd be worried about is software compatability between a pump and detector if they both need to be run by software. Having controls directly on the pump and maybe an analogue plotter for a readout would be more compatible?

HPLC usually uses such a small sample size that it's really only good for detection and measurement and doesn't really leave you much separated compounds to work with. If you want to do that, 'column chromatography' would be the way to go. It's alot cheaper than HPLC and can be used in conjunction with TLC. Congrats on getting the technique down, I'd like to get one those kits someday. If you use the same solvent system (polar/nonpolar ratio) on column chromatography as for TLC, you will get the same order of separation. When you get one vial from the column with one isolated spot in TLC, you can change the solvent ratios and see if you can separate it into more spots.

You can find some good deals on auction sites. I almost bought a DNA sequencer for $60!! The shipping turned out to be $400 and they mentioned something about a fork lift, so I passed on that one.

Have you looked into GC? They're pretty simple to use and not too expensive.

Good Luck:)

[ 25. June 2005, 13:57: Message edited by: Plant Pimp ]

[Fractalhead]

Hi DL

I think PP pointed out most of the components i was going to mention. I've got some more specific info for you too but I want to check my notes first. I should write back in the next few days.

I've been looking into the financial feasibility of setting up a semi-preparative HPLC out of old salvaged and bought secondhand bits and pieces. Unfortunately, even thinking about HPLC seems to somehow suck money out of your bank account and i need about $3k to set up a simple system from bits and pieces i already have. That's a lot of school fees to spend on an expensive toy... Any investors out there ????

I've worked out that the best bet is to ask around at old laboratories that have been using hplc systems for a long time and hence have lots of old redundant bits and pieces taking up space. Most old hplc components are cross compatible between brands etc in terms of plumbing. Its just when you want to set up electronic communication between different compononents and a computer that trouble arises.

You mentioned that you would like to set up a preparative system. A true prep system requires a preparative pump that is capable of maintaining high flow rates even against large back pressures. While these offer the ability to separate hundreds of mg or even grams of material at hplc resolution, these are very very expensive and not very commonly available cheaply from secondhand suppliers.

You might be surprised, however, just how much material you can separate with a normal 'analytical' hplc. Quite often you will be able to separate 10s of mg with the right size column and injector configuration. This is quite a bit of material for many applications.

GC is extremely useful aswell but that comes with its own can of worms, again (funnily enough) mostly the type of worms that are easily satisfied with 1000's of $$ Not really any cheaper to set up or run and involves having cylinders of potentially explosive gas sitting around. I would aim for HPLC first just because it is simpler mechanically and electronically, you can more easily assemble a system from lots of different scavenged parts and you might be able to sell the eluents to help pay for the mainenance of the system.

Anyway, I'll dig up some more specific specs for you soon.

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Fractalhead:

I should write back in the next few days....Anyway, I'll dig up some more specific specs for you soon.

Lovely, yes please.

While these offer the ability to separate hundreds of mg or even grams of material at hplc resolution, these are very very expensive and not very commonly available cheaply from secondhand suppliers.

They don't need to be commonly available, they just need to turn up at the right time, like when I can afford the bits Its more a matter of timing.

This is a long term project so if it takes two years to get it together I'm happy with that, cos I have a lot of learning to do on the way there.

GC is extremely useful aswell but that comes with its own can of worms, again (funnily enough) mostly the type of worms that are easily satisfied with 1000's of $$

Damn those expensive worms

Not really any cheaper to set up or run and involves having cylinders of potentially explosive gas sitting around.

Yeah, not into it.

I would aim for HPLC first just because it is simpler mechanically and electronically, you can more easily assemble a system from lots of different scavenged parts and you might be able to sell the eluents to help pay for the mainenance of the system.

My thoughts exactly except for the bit about selling eluents. Hadn't thought that far ahead. Was more into it as an adjunct to assessing best harvesting times for plants and seed and cultures

If you want to do that, 'column chromatography' would be the way to go. It's alot cheaper than HPLC and can be used in conjunction with TLC.

I've heard the same thing said for TLC and HPLC- TLC results can give an excellent guide to protocols for HPLC work- sound true?

If you use the same solvent system (polar/nonpolar ratio) on column chromatography as for TLC, you will get the same order of separation.

You reckon column chromatography is a good intermediate step between TLC and HPLC? I might take that path. I need a column, and something to refill it like a column packer, I need a stand, and of course consumables

I almost bought a DNA sequencer for $60!! The shipping turned out to be $400 and they mentioned something about a fork lift, so I passed on that one.

*longing sigh* if I only knew what to do with a DNA sequencer... there are toys that are way beyond my ken and that will be one of them for a while I fear

Thanks to you both for your excellent replies