Instrument Method for automatic AA derivatization

[thhdk]

Hi,

We recently purchased a Thermo Scientific Ultimate 3000 HPLC system to quantitate biogenic amines and aminoacid neurotransmitters. We have problems programming Chromeleon 7.2 to make the autosampler perform derivatization of amino acids using OPA. That is to say we can easily make the autosampler take up 40ul OPA, transfer to 40ul sample, mix 4 times, wait 2 minutes and inject e.g. 15ul. But we get very poor reproducibility with our program (>10% variability). Performing the same derivatization manually using the regular injection routine of the autosampler brings the variability in the range of <1%.

Do any of you have experience in optimizing the chromeleon programming for mixing on the Dionex Ultimate3000 autosampler?

Here's what we have right now:

UdpInjectValve Load
UdpSyringeValve Needle
UdpDraw Air 5ul; GlobalSpeed; GlobalHeight
UdpDraw ReagentAVial: 40ul; GlobalSpeed; GlobalHeight
UdpDispense SampleVial 40ul; GlobalSpeed; GlobalHeight
UdpMix SampleVial; 40ul; 10; GlobalHeight; 4
UdpDispense Drain; 5ul; GlobalSpeed; GlobalHeight
UdpDraw Air; 5ul; GlobalSpeed; GlobalHeight
UdpMixWait 120
UdpDraw SampleVial; Sampler.volume: GlobalSpeed: GlobalHeight
UdpInjectMarker
UdpInjectValve Inject
UdpSyringeValve Waste
UdpMoveSyringeHome GlobalSpeed
UdpMixNeedleWash 50ul

Thanks in advance

Thomas

[Peter Apps]

Hi Thomas

I don't have experience with that particular hardware, but in general to thoroughly mix liquids by sucking into a syringe and expelling again you need to use high eject speeds to squirt the syringe contents into the rest of liquid. With gentle expulsion you get a droplet at the end of the needle that just gets sucked up again with remarkably little mixing with the rest of the fluid. Try it with a dye in one of the liquids so that you can see what is going on.

Peter

[ACrawshaw]

Hi,

As far as I am aware there is not a command "udpMix"? I'm intrigued as to how this command line was added?

If you remove this line and replace it with 4 repeat lines of UdpDraw and UdpDispense, based on the volume you have in the preparation vial, this should help.

Example:
Sampler.UdpDraw SampleVial, 50.000, GlobalSpeed, GlobalHeight
Sampler.UdpDispense SampleVial, 50.000, GlobalSpeed, GlobalHeight
Sampler.UdpDraw SampleVial, 50.000, GlobalSpeed, GlobalHeight
Sampler.UdpDispense SampleVial, 50.000, GlobalSpeed, GlobalHeight
Sampler.UdpDraw SampleVial, 50.000, GlobalSpeed, GlobalHeight
Sampler.UdpDispense SampleVial, 50.000, GlobalSpeed, GlobalHeight

The command at the end of the program also makes little sense as this is used to define the volume of sample that is used the flush the needle. If what I suspect you are tyring to do is a post injection wash, you will need something like the following commands after the MoveSyringeHome command:

Sampler.UdpDraw Wash, 25.000, 8.33, GlobalHeight
Sampler.UdpDispense Drain, 25.000, 8.33, GlobalHeight
Sampler.UdpDraw Wash, 25.000, 8.33, GlobalHeight
Sampler.UdpDispense Drain, 25.000, 8.33, GlobalHeight

Note: With this methodology the mixing is performed within the sample vial, and not within the sample loop. The later can potentially produce poorer mixing effiencies due to the narrow tubing involved.

It may also be worth noting, if your not already, that because you are dealing with 'small' volumes, ideally you want to do this mixing with some sort of insert in you vial. As such you will also need to consider your needle height.


The link below, is for in-needle mixing using Chromeleon 6 commands, but it should at least give you some good pointers.
http://www.dionex.com/en-us/webdocs/110 ... PN2849.pdf

Hope this gives you some ideas.