Hello

We have recently identified a problem in our lab where our analyte is adsorbing to the HPLC vials resulting in poor reproducibility. We are using normal glass 2mL vials. The adsorption occurs if silanized vials are used and was worse when polypropylene vials were evaluated. The diluent is nearly 100% aqueous. If the diluent is altered to contain 10% ACN, the adsorption does not occur. Ideally, we would like to avoid changing the diluent. Are there other types of inert vials or vial cleaning procedures that might help aleviate this problem. Any advice will be greatly appreciated.

Thanks

without any clue as to what is absorbing, and from what concentration, it would be a bit speculative to suggest a solution for your problem. You did not provide data on what would seem to be a logical next step in the evaluation. do you get adsorption on just plain glass vials, ie no nonpolar phase to absorb in?

We experienced a similar problem som years ago related to our vials (and it turns out HPLC system too), where the diluent was 100% de-ionized water. Our investigation revealed:

- Using bottled HPLC grade water did not cause the problem
- Adding NaCl (to add an inorganic polar agent) to the solution (made with lab supply deionised water) bought the results back to the expected level
- Changing to a reputable branded vial did not cause the problem (we used vials from Agilent for this product after that).
- The autosampler we were using also contributed to the low results - we had been using TSP Spectra System AS1000 and when it was run using an Agilent 1050 the result were higher.

The final part in particular should indicate you may need to consider other aspects of you analysis too.

Since you indicate that silanized vials are unacceptable for this application as well as polypropylene vials, it's possible that you are experiencing problems associated with the general tendency of glass to acquire "reversed phase" properties when exposed to the atmosphere. While I don't believe its well-known exactly what causes the effect, you can demonstrate the effect by taking a glass microscope slide from a brand-new package and demonstrating that the slide has a very low contact angle when a drop of water applied (a drop of water will not form a "bead" on the surface of the glass but rather spread quite diffusely over the surface). However, if you leave this microscope slide open to your laboratory atmosphere for somewhere between three and seven days and repeat the application of a drop of water you will see a much higher contact angle with the drop of water generally staying tightly confined to a single hemisphere was relatively little spreading. You can restore the original hydrophilic properties of this very same microscope slide by a number of methods including: exposure to powerful oxidizing agents, exposure to UV/ozone or treatment with alcoholic KOH (generally a 5-10% solution in ethanol is used). It seems possible that you are observing problems caused by this general increase in the hydrophobicity of glass over time. I would try cleaning your vials by one of the above methods, checking before and after to make sure the contact angle has been significantly decreased and then see if this helps eliminate the need to add acetonitrile to your sample.

By the way, presenting this problem from occurring in the first place can be accomplished by careful control of storage conditions. For example, don't open your package of vials until just before you need to use them (assuming they are sufficiently clean in the first place). Another option, is to store them submerged in ultrapure water after cleaning to prevent contact with air.

Thanks for the information. Based on the results we have seen thus far, we definately need a more hydrophillic surface, however, I had no idea how to accomplish this. I will evaluate the cleaning procedures you discussed above.

Chromacol sell inert glass autosampler vials (they call these 'gold grade' for some reason), might be worth trying these?

Good luck

Why not just using plain glass? Silanized glass is hydrophobic (just like a C18 column), and polypropylene is obviously even more hydrophobic.