How to calibrate an autosampler loop[

[ziyads]

Dear:
Can any one tell me how to calibrate an autosampler loop or how to verivy its volume?

[shaun78]

I would hope that there are better ways of doing this and that someone will be along to post.

But, if not...

I had to do this before on a TOC instrument. How we did it was essentially disconnect the loop from where ever to leads into. We had the loop draw water and took note of the temperature in the lab. The loop would then expel the water into a tared container. Using the density of water, we knew what our water weight should be for xx mL's.

I have never seen this done on LC. I would imagine that it would be next to impossible as you would be dealing with microliters. I have always verified injector/loop accuracy by linearity using caffiene and a capillary restrictor. If the injector is not injecting the proper amounts, then your linearily is not going to pass...

I hope this helps you out some.

[JA]

Discussed previously:

http://www.sepsci.com/chromforum/viewtopic.php?t=5275

[ljc]

Water weighs a mg per microliter. Just evacuate the loop with air (blowing or pulling through it). Then tare the loop on a 4 place balance. Then, draw water into the loop very carefully taking care to keep drops off the outside of it and not to lose any water from inside of it. Then weigh the loop again and the weight in mg is the volume in microliters. Repeat a couple of times until you get "good at it" and take an average. No matter what or how you measure something, there's stats involved. Ironically I've found this easiest to do with narrow tubing (10 micron i.d.) because the water tends to not drip out so easily. Another way to do it is to use a long length of tubing to get a "microliter per unit length" then just cut off the appropriate length to get the volume you want. Somewhere there must be a table showing lengths of tubing of certain diameters to get desired volumes, or it can be calculated (volume = pi(R**2)*length) ? But measuring seems the surefire way...

[DR]

^ method will tell you the volume of the loop but it still fails miserably in capturing the volumes associated with bulkhead fittings, channels in valves etc. that all do contribute to your injection volume, especially in an overfill mode.

In the above linked thread, there is a good suggestion that you use the r², RSD and intercept for a series of standard injections to assess the precision and accuracy of the entire injection/sampling system.

[ljc]

I'm not sure I get DR's concern about the extraloop volumes in the plumbing. Unless your loop size is very small (1 uL), the extra volume inside the valve and rotor seal should be negligable, shouldn't it ? In a typical 6-port valve like a Rheodyne 7010, the only extra volume outside the loop that actually gets injected is in the stator face and rotor seal on the exit side, which should be really tiny. Any sample that is overfilled goes to waste.

[mcbart]

We miss many info. First if you have a general interest in understand loop volume verification or if you are experiencing some problem with your autosampler/loop. In the 2nd case these info must be collcetd:

1) loop volume and brand
2) are you doing full-loop or partial-loop injection
3) your syringe volume

[DR]

I'm not sure I get DR's concern about the extraloop volumes in the plumbing. Unless your loop size is very small (1 uL), the extra volume inside the valve and rotor seal should be negligable, shouldn't it ? In a typical 6-port valve like a Rheodyne 7010, the only extra volume outside the loop that actually gets injected is in the stator face and rotor seal on the exit side, which should be really tiny. Any sample that is overfilled goes to waste.

In an overfill, you get the channel volume from entry & exit sides. The Rheodyne spec I once heard for channel volumes was ±17%, fill volume for each channel is at least a microliter or two (can't remember). ZDVU volumes (aren't really zero and) vary from manufacturer to manufacturer - add volumes of all of these fittings and channels to a small (<20µL) loop and you may well see some variation from system to system and probably more from vendor to vendor.

[ljc]

I guess I'm not seeing it still DR; you have the loop itself, the "dead volume" between the end of the loop and the channel inside the valve face, and the small volume formed by the rotor seal. Any other sample liquid should be vented to waste on the next fill, shouldn't it ? Don't want this to turn into a dissertation, but just want to see where the variation can come from.