Chromatography Forum

After injecting the sample in injection port and subsequently loading, many people take out the syringe out of port, while other keep it in port itself even during sample run.

Is there any serious concern for either practice?

It's been over a decade since I've injected HPLC samples manually, as autosamplers have better precision as well as the obvious time-savings benefits. We always left the syringe in the loading port, and always used a loop size that we could fill completely, so we could absolutely rule out over-filling.

There are two steps to making a manual injection.

1-Loading athe loop with the injector handle in the "load" position.
2-Injecting the sample by moving the handle to the "inject" position.

The syringe should never be removed when the handle is in the "load" position. Doing so will suck some of the sample back into the syringe guide. Once the sample has been injected, the sample loop is inline with the column and the syringe may be safely removed.

Same as the post above, and you might consider completely filling the syringe with deionized water after injecting until the next sample is injected. This reduces carry-over by the syringe quite a bit (it works well even in one digit ppb ranges to use the same syringe for several days with rather clean samples)

Mat,
one does not only reduce, but eliminate syringe carrover by taking the piston out, washing it and drawing solvent through the syringe body with vacuum.

Dear All,

Your comments are all valid. I just want to add a few words I have found useful regarding manual injection.

Manual and automated injection can be equally precise if you overfill the sample loop (7 volumes at least), this eliminates the laminar flow problems you have when displacing a liquid with another. The extra sample volume is obviously wasted, but if sample is plentiful there is no problem at all.

It is true, is better to leave the syringe in the valve while doing the injection. Carryover can be best handled doing a solvent flush, between injections, with the use of a sample port cleaning adapter. This is a small plastic piece that fits the valve filling port and is attached to a syringe. Doing this flushing, with a suitable solvent, cleans the chanel between the port and the valve core and the rest of the lines.

Good luck,

josebenjamin

This washing of the injector doesn´t remove carryover due to material getting in between stator and rotor (or dissolving in rotor?). As an example, we had to take Rheodynes apart and wash the parts to get them cleaned. For substances that have a tendency to get stuck there we inject directly into the loop (injector removed).

H W Mueller,

Can you please explain how you do this?

This washing of the injector doesn´t remove carryover due to material getting in between stator and rotor (or dissolving in rotor?). As an example, we had to take Rheodynes apart and wash the parts to get them cleaned. For substances that have a tendency to get stuck there we inject directly into the loop (injector removed).

HW Muller,

You must have had some very special case of compound that sticks to everything. My comment about washing the injection port lines said..."with a suitable solvent". If the cleaning solvent is a good one for the samples I dont see how it can not be removed. If it reacts or adsorb on the valve´s core then a better choice of material would solve the problem. Tefzel is usually better than Vespel.

Good Luck,

josebenjamin

Tefzel or Vespel, it doesn´t matter, the largest part (or all) of the problem was diffusion in between rotor and stator. This has been discussed before, others have seen this, some thought it was a solvation. What I saw can be explained by diffusion. Whether you see this or not has in large part to do with the desired (required) sensitivity, you do not necessarily need outlandish substanes.

HW Muller,

Now I understand your comments better. I must say that I have never seen such problem in over two decades of HPLC work. If there is some very small difussion of sample solution in the extremelly narrow space you describe, I would expect the problem to be very small or none at all if there is some sort of washing between injections.

I have tried to determine the extent of carryover many times and in most cases the problem was too small to worry about it. I must say as well that I have seen only one autosampler (Shimadzu) that copnsistently gave zero carryover no matter how we tried to see it.

Good Luck

josebenjamin

A few more points should be made: It appears that the analytes are not only diffusing in there but that mechanical action transports them there. The amount reaches a plateau (best seen if you have an internal standard present or unknowns of similar concentration in all samples). Injecting blanks, as well as moving the injector (Inject >>> Load >>> inject) without an injection, or even the presence of a canulae, then shows carryover which is diminished very slowly. Washing was hardly better, if at all, than a blank injection. Any sliding parts/areas will do this, syringes are particularily notorius. I don´t understand why some syringes come with these guides which make removal of the piston a chore (the first thing I do when I get such a syringe is removing all that crab).
The reason I believe that this is mostly due to accumulation between the rotor and stator is the ease with which it is removed by simple washing of the parts. Contrarily, when I was working with a Siemens GC with automatic injection, using a plunger that transported the sample through teflon/glass gaskets at 280°, fatty acids certainly dissolved in the teflon: They could not be washed out, the gasket had to be replaced after too short a time. Bum design.

Forgot, a repeat is also in order: We have fair evidence that ouabain has been labeled a hormone, because of Rheodyne and/or syringe carryover into samples derived from human body fluids. Some people never bother injecting a blank.

Dear All,
Besides all important point one has to
-clean ur syringe with diluent for atleast five six times minimum before moving to sample
-remove bubles from the syringe before injection, for that just keep filled syringe upward down, tap to allow bubble to settle up near needle and push slowly to remove it.
-if u want to inject 10 ul inject at least 100 ul. (7 to 10 times) to get reproducible result
-everytime make a habit to inject smoothly by kepping same time, flow.
-check for viscosity of solvent and inject accoridngly. Take care using normal phase chromatography.
-do not remove syringe before inection.
-check the tubing coming out from the injector port, if syringe is not properly fixed in seal or likage is there than u may not get reproducible result.
-if u have problem with syringe clogging clean with proper solvent and if applicable finaly use plenty of water to clean.
-never use ultrasonic for cleaning syringe.

skunked_once,
sorry, I just saw your question today, probably on injecting into the capillary?
For that we just plumb in a large inner diameter tubing (0.8 mm, if I remember correctly) ahead of the column, stop flow, separate this tube on one end to insert the syringe cannulae (we first placed a cut off silicon rubber stopper on the cannulae so as to provide a seal of syringe to fitting of the tube) loosen the fitting on the other end of the tube to be able to fill it, close everything, restart flow. One has to do everything in the right (obvious) sequence and positioning to prevent air from getting in. For convenience, we use VALCO bulk fittings which are mounted with the tube (or loop. if you wish) on an aluminum angle piece.

nawazsheikh,
as stated above, it is about impossible to get all material out of a syringe unless you take the piston (plunger) out, etc. (especially if you remove the air bubble at the piston (there is about 1µL volume in the cannulae of a standard 10µL syringe, also some stuff gets in between the piston and syringe...).
How much volume one injects depends also on the loop volume and whether you inject against or with the flow (has to do with laminar flow as mentioned).
Never had occasion to need this, but if your syringe can be taken apart (cannulae removed, etc.) what would be the reason not to use ultrasound on the parts?

On the two waste connections: If you connect long (plastic is common) tubes to them, bending (hanging) down from the injector and filled with waste solution, you can draw air into your system via a syphoning action.