Chromatography Forum

hello everybody,

I am working with my best friend agilent 1200 hplc:)

I have not had any problems with peak purity until now;
I was doing analysis of methyl and aethyl paraben, then i was injecting benzyl alcohol; unfortunately, on alkohol chromatogram i cannot eliminate peaks (small, but they are still at chromatogram) coming from parabens ; I was using option of injection with washing using .... vial; I know that there is an option of writing short program inside the method; it concerns thing like draw ...... from vial, eject from ..... vial and so on but i do not know how to write it properly (the easiest way)

can somebody write me these view lines of program

(i did :
1. draw ..... from vial 100
2.eject .... to vial 99
3.draw ...... from sample, which was my target sequence vial
4.inject

but it did not work very well;
I mean the peak was assymetrical, and sometimes doubled; I want to emphasized that if I was using standart procedure of injection, without the program, peak was symmetrical and single

You may have more than one problem, but here are some ideas:

If you are seeing paraben peaks when injecting benzyl alcohol, you have a "carryover" problem. This is unusual in a 1200, but can happen if you are injecting a high concentration of parabens in the first injection, and low concentration of benzyl alcohol in the second injection.

The "wash vial" procedure, specified in the injector setup, will just clean the "outside" of the sample needle. The needle is dipped in a solvent solution before injection. If you choose the correct solvent, this will remove anything on the outside of the needle. (if you have a well-plate autosampler, there is a "wash station" option, or you can use a vial filled with solvent) The mobile phase flows through the needle after injection, so no parabens would be left inside the needle.

I don't know why you are trying to use the injector program feature. Please explain what you want the injector to do that it is not doing now.

is the needle constantly washed INSIDE or only OUTSIDE during the analysis (when it is inside the port)?

what i was trying to do using injector program was:

in vial number 100 I have only solvent, and vial 99 is empty,so using the injector program I should obtain (below I put commands from the program):

1. draw max volume from vial 100, it means for me, that needle takes 100 ul from vial with clean solvent
2.eject max volume from to vial 99, it means for me, that needle eject everything to this vial
3. draw def volume from sample, it means for me that needle takes 20 ul from vial, which is current in the sequence table
4. inject, it means for me that the volume from the current vial is injected to the column

that it what I put into that program

I hoped that it helped, but still i had some problems with the shape of peaks and their asymmetry;

Mobile phase flows inside needle during analysis, washing process cleans outside part of needle.
Don't forget to left washing vial vithout septum. If washing vial is closed, cleaning is not effective.
When carry-over persists, disconnect needle and needle seal and sonicate in methanol (or water based phases if your mobile phase contains buffer).

Sadilek is correct. I will provide more details.

The Agilent 1200 autosampler is an "in-line" or "integrated" autosampler design. When the sample is "injected" all mobile phase flows through the needle and pushes the sample onto the column. There is no sample left inside the needle. In older "loop" autosamplers, sample could remain in the connecting tubing, causing carryover in the next injection. There is no connecting tubing in your autosampler, so you will never see this problem.

If you are having carryover, it is coming from the outside of the needle. When the needle comes out of the sample vial, there is always a small amount of liquid on the outside of the needle. This liquid contains sample molecules, so the next time the needle goes into a sample vial, those molecules can dissolve off the needle and into the solution, causing carryover. The sample on the outside of the needle can also contaminate the needle seat.

You can minimize this problem by using the "Injection with needle wash" option. Fill a vial with solvent only. Connect a cap but NO septum. Place this vial in the autosampler (I use position 91). In the Injector Setup screen, choose "Injection with needle wash" and enter the location of the vial (e.g., 91). The autosampler will now dip the needle in the solvent before each injection. Replace this solution daily.

Good luck. Write back if you have more questions.

ok i will try it (i tried with the septum so that was maybe a problem);

anyway, what about the injector program option ( i know that I am little too persistent person but i would like to know about that option as well )
are the lines, which I wrote couple days ago, correct? can i use it as 'a second option' to wash the needle?

ok i will try it (i tried with the septum so that was maybe a problem);

anyway, what about the injector program option ( i know that I am little too persistent person but i would like to know about that option as well )
are the lines, which I wrote couple days ago, correct? can i use it as 'a second option' to wash the needle?

Yes, your injector program is correct, but without effect on carry-over. I use injector program for impurity spiking during method development only. I don't recommend to use it for washing, it is a wasting of time only.

Yes, septum on wash vial may be your problem. I show it to newbies in our labs practically with wet needle and empty crimped vial. During puncture is formed a small liquid drop on septum, which is deposited again on needle surface during its removal.

mbicking,
It sounds as if this is resolved, but may I ask why you prefer to wash in a vial instead of using the wash station? I'm just curious because that's one less sample in your tray... but I'm not sure if my washes are lacking somehow.

matthew:
A good question. Answer 1 = "habit." Answer 2 = the vial option is available on all autosampler versions, and it is the only one available for the standard ALS unit. While I do not have a lot of experience with the wash station option, I am always a little suspicious of these designs - the pump has to be working properly (a problem for cheap peristaltic units), and you have to remember to fill the source, and there is one more waste stream to manage. The vial dip is more forgiving to those of us who are busy and/or prone to "senior moments." But if your system is working as is, don't change it.