I have been set (or dropped!) the task of setting up an HPLC in our research lab (where I work) from an old system used previously.

I have quite a bit of HPLC experience, but have never set one up by myself before.

Does anyone have any hints/tips about practical considerations in setting one up?

The equipment I have is:

>Perkin Elmer 100 series pump
>Anachem column heater with Rheodne injector
>Perkin Elmer LC90 Sepc detector

I haven't got that much of an idea as to how to start, we haven't got helium into our lab, so I was considering vacuum filtration.

Presumably, as my equipment seems quite "dated" I am not sure whether the valves are pneumatic.

I am hoping to try to interface it through a PE 900 series interface box, to either Turbo-chrom/Totalchrom for data handling.

Hi Lucy

I think you need to be a bit more specific about your problems - do you need advice about data collection or about actually plumbing the system (or both)?

Vacuum filtration is fine for most isocratic applications (gradient assays or use of an ECD may require an on-line degasser or helium sparging).

Ann

Hi Ann,

It's really about where I start, and the practicalities, such as, are there any things I should be looking out for when I set it up, any other bits of equipment I may need (pipes/tubing), if there are extra practicalities, such as "don't put it near a door", etc

Thanks for your reply!

Hi Lucy

Ideally, you should site your HPLC system on a firm, flat surface which is free from vibration, electrical-magnetic disturbances and temperature fluctuations (so don't place it near to a radiator, fan, ventilator or window (it's also a good idea to avoid areas of direct sunlight)).

As for tubing, PEEK (polyetheretherketone) tubing is a good alternative to stainless steel and is chemically inert to almost all HPLC solvents (excluding methylchloride, THF, conc nitric and sulphuric acid). The size you need will depend on the type of columns you intend to use. Most systems using standard bore columns will be plumbed with 1/16" tubing with an ID of 0.007 or 0.010"(if you use microbore columns, you may wish to use tubing with an ID of 0.005"). When you set up your system, remember that tubing lengths should be kept to a minimum to minimise extra-column dead volume and band broadening. It is most convenient to connect the tubing using all-in-one PEEK fingertight fittings rather than separate nuts and ferrules.

I'm not familiar with Perkin Elmer HPLC equipment or the Turbo/Totalchrom software so can't offer any specific advise about data acquisition.

Hope you find the above useful, if you have any other further questions I will do my best to help.

Good luck! :o)

Ann

Should I also be testing the equipment out for leaks as well, before assembly?

You could test your pump out to check that everything is performing well before assembling the system; e.g. is the flow rate accurate? Simple way to test this is to set the pump to your desired flow rate, say 1mL/min, and then collect the waste into a measuring cylinder - in ten minutes time you should have 10mL. You would want to see that the back pressure is stable under load (attach a column to the pump first so that you create a reasonable back pressure.) Any leaks from the pump would be apparent at this point too (in which case the pump may need a service (new pump seals etc.) This is usually simple enough to do yourself (helps if you have a manual for the pump though!)

As for the rest of the system, you will need to assemble it before you can check for leaks (which are usually caused by such simple things as a badly seated Rheodyne loop or inadequately seated or tightened fingertights.)

Leaks from the detector could possibly be something nasty like a broken flow cell so hopefully you won't see any!!

Let us know how you get on! :o)

Ann