Chromatography Forum

Good morning all,

We have this autosampler in our lab.

One of my colleagues has been using it for quantitative work and found that he wasn't seeing any peaks at a certain injection volume.

I ran a check to see if the AS was consistent in terms of the volume taken from the sample vial. I filled a vial with water and after each injection (e.g. 100uL) I weighed to see if there was a loss of 100mg. Perhaps there is air trapped somewhere in the system, as the apparent mass taken up was much less than expected. It also varies depending on the position of the vial.

Is this a sensible way to assess the performance of the AS? This is quite an old design (pull-to-fill) and I'm no expert on them, but perhaps this check isn't suitable?

Any experts on here that can help?

many thanks,
Chrom

A pull-to-fill design actually has to withdram *more* than the nominal volume in order to account for the amount left behind in the needle and transfer line, so something is seriously wrong. Possibly a blockage somewhere or a bad seal on the syringe.

One of my colleagues has been using it for quantitative work and found that he wasn't seeing any peaks at a certain injection volume.

is this "certain" volume on the high or low end?

before doing some tests, make sure your AS is purged properly with inital mobile phase. As for the "purge inj" make sure you have a column installed, cause it needs some backpressure for success.

If your detector is working fine, you could do a linearity check as well.
Inject different volumes of a solution of (eg.) caffein dissolved in mobile phase. Plot Area vs. Vinj
-> how does the curve looks like? Linear fit should give r2 >= 0.999.

Second check would be for reproducibility: do six replicates of a certain volume of the caffein solution and calculate %RSD of the peak areas. This should be <1%

As for the accuracy, the Waters OQ test is similar to the one you did, but they do six replicates of 50µl of water (in series). The average volume withrawed should be 50 +/- 0.5µL = mg (if I remeber correct). The balance should read at least 0.1 mg

The 717plus has the "needle in line", today also know as "FTN: Flow-through-needle" design, so it just takes up the volume specified. Afterwards the needle is just switched into the flow path and all of the sample goes to the column.

Possible sources of your errors:
- be aware that the point of inlet is on the side of the needle, about 4-5mm above the tip. Fill your vial with enough sample: if you make a lot of replicates of large volumes, maybe use several vials for not to fall below this needle inlet level.

- if you have limited sample volumes, use "total recovery vials" (insert style), but make sure the small part is filled with your sample and not with trapped air.

- edit: valve #3 is defective (->call Waters) (not valve4)
-- does the "compression check" pass or fail?

- as Tom suggested: syringe is not sealing properly (plunger tip worn out?)

It also varies depending on the position of the vial.

very strange! No idea for this...

- were the vials filled with different levels?

- is the carousel working correct eg. does it move to the correct vial?
--> place several groups of 3 vials around the carousel, then specify injections from the middle vial of each group. Are all (and only) the correct vials punctured, none of the left or right ones?

thanks for your responses.

ok, I was playing with this all day and think I've discovered the problem, but first of all nothing to do with vial position. forget I said that!

we actually have two of these.... so I switched syringes to see if that was the issue. it wasn't, as the other AS functions perfectly.

NOTE: I forgot to mention this earlier, but extremely important: When the syringe was drawing liquid during the sample run set-up, I saw that air bubbles were going into the syringe, then disappearing once the waste valve (valve 3) opened (you hear a little click). This air bubble issue doesn't appear when you do purges (or indeed manual purges), the reason being that valve 3 is already open, and continues to be. I tested and retested and if the valve is closed, then air gets drawn into the syringe.

The really interesting thing is that on the functioning autosampler, the valve is open when the syringe is drawing sample out of the vial, in preparation for injection. And in this case everything works as intended.

how is it possible that the valve configuration is wrong in the case of the non-functioning AS? is this something that is configured when installing an AS for the first time? I'll be going through the manual but please come back with any thoughts based on my observations so far

thanks
C

when drawing the sample from vial valve 3 must be closed, otherwise the sample wouldn't be taken up. (and if it is not properly sealing, the inj accurracy would be poor)

I don't think the sequence of the valve operation could be changed without removing and reprogramming of ROM-chips. So it must be another explanation for these errors and observations (maybe the display just shows the status with some delay)


But how about the compressions check? Does it pass or fail?

As for the normal purge, the pump must be running with some flow (e.g. 1 ml/min) and a column must be installed to provide sufficient backpressure.
Otherwise the whole purge cylce won't work properly.

I would to 2-3 purge cycles with MeOH before doing the compression check.

If compression check fails repeatedly, some of the valves (V3), seal pack or maybe some of the tubing connetctions are defective/leaking.

-> adjust/calibrate seal pack
-> check tubing connections
-> call Waters, if Valve is faulty

The valve was gone

strike!

thank you for response.

A pull-to-fill design actually has to withdram *more* than the nominal volume in order to account for the amount left behind in the needle and transfer line, so something is seriously wrong. Possibly a blockage somewhere or a bad seal on the syringe.

Unique issue in Waters Alliance just like Waters 717 Autoosampler is that the needle at STREAM never leaks once we closes V3,V2 manually and then we checked it at WET and SEAL positions too. This means Valve 1 is okay
However when we test V1 by the procedure it hangs on with Restrictor Missing. with small amount of leak probably Needle Wash solution on vial found.
Seal Pack ADJUST test also hampered with Restrictor Missing message.

Same happens in V2 test . The Waste Valve 3 open upon PURGE mode but again when it performs compressibility test hangs on with restrictor failure error.

I cant believe all my Waters Alliance have same issue and 1 717 autosampler. Something is really wrong.

I noted and did the battery replacement too.

Plz guide

Thanks

One of my colleagues has been using it for quantitative work and found that he wasn't seeing any peaks at a certain injection volume.

is this "certain" volume on the high or low end?

before doing some tests, make sure your AS is purged properly with inital mobile phase. As for the "purge inj" make sure you have a column installed, cause it needs some backpressure for success.

If your detector is working fine, you could do a linearity check as well.
Inject different volumes of a solution of (eg.) caffein dissolved in mobile phase. Plot Area vs. Vinj
-> how does the curve looks like? Linear fit should give r2 >= 0.999.

Second check would be for reproducibility: do six replicates of a certain volume of the caffein solution and calculate %RSD of the peak areas. This should be <1%

As for the accuracy, the Waters OQ test is similar to the one you did, but they do six replicates of 50µl of water (in series). The average volume withrawed should be 50 +/- 0.5µL = mg (if I remeber correct). The balance should read at least 0.1 mg

The 717plus has the "needle in line", today also know as "FTN: Flow-through-needle" design, so it just takes up the volume specified. Afterwards the needle is just switched into the flow path and all of the sample goes to the column.

Possible sources of your errors:
- be aware that the point of inlet is on the side of the needle, about 4-5mm above the tip. Fill your vial with enough sample: if you make a lot of replicates of large volumes, maybe use several vials for not to fall below this needle inlet level.

- if you have limited sample volumes, use "total recovery vials" (insert style), but make sure the small part is filled with your sample and not with trapped air.

- edit: valve #3 is defective (->call Waters) (not valve4)
-- does the "compression check" pass or fail?

- as Tom suggested: syringe is not sealing properly (plunger tip worn out?)

It also varies depending on the position of the vial.

very strange! No idea for this...

- were the vials filled with different levels?

- is the carousel working correct eg. does it move to the correct vial?
--> place several groups of 3 vials around the carousel, then specify injections from the middle vial of each group. Are all (and only) the correct vials punctured, none of the left or right ones?

Unique issue in Waters Alliance just like Waters 717 Autoosampler is that the needle at STREAM never leaks once we closes V3,V2 manually and then we checked it at WET and SEAL positions too. This means Valve 1 is okay
However when we test V1 by the procedure it hangs on with Restrictor Missing. with small amount of leak probably Needle Wash solution on vial found.
Seal Pack ADJUST test also hampered with Restrictor Missing message.

Same happens in V2 test . The Waste Valve 3 open upon PURGE mode but again when it performs compressibility test hangs on with restrictor failure error.

I cant believe all my Waters Alliance have same issue and 1 717 autosampler. Something is really wrong.

I noted and did the battery replacement too.

Plz guide

Thanks