Sample loop volume

[Multidimensional]

EK8 wrote: "The problem is I called Waters and the technician said the sample will not be diluted in the loop"; This "person" has no idea what they are talking about or you have misunderstood them. Some are experienced and knowledgeable, some are not. You are being misinformed, but it is not all their fault. Trying to explain the basic fundamentals over the phone (or via email or a web forum) is not realistic. It takes many years of hands-on bench training, with an experienced user available to learn just the BASICS of HPLC.

EK8 wrote: "The problem is I called Waters and the technician said the sample will not be diluted in the loop". One of the fundamentals of HPLC is that we concentrate the sample in as small a volume as possible to load onto the head of the column. This minimizes diffusion and band spreading and is fundamental to obtaining a good separation. The tubing sizes, injection solvent, injection volume, column diameter, mobile phase, temperature and even the flow rate used must be optimized to insure this. *They will be different for different instruments and configurations, different applications etc. If the sample is diluted into the flow path BEFORE it reaches the head of the column, then the peak "slug" will be broad and slowly load onto the column resulting in poor quality chromatography. I strongly recommend you read one of the excellent introduction to HPLC texts to learn these fundamentals. For example, one that we have used for several decades in our classes is: "Practical HPLC Method Development"; by Lloyd R. Snyder, Joseph J. Kirkland, Joseph L. Glajch. Now you can not learn HPLC from reading books (or the web), but you can learn many of the basic fundamental concepts which are needed to use the technique.

Please ask your employer to hire a professional chromatographer (if they are really trying trying to use this technique for something they rely on). Having an experienced industrially trained chromatographer on-site would help move things forward and also provide you with someone to learn from. THE WEB is not a substitute for actual training.

[Hollow]

I don't take it that strict but in the end, neither we nor Waters can tell and guarantee that it will work for your specific case without having it tried.

If the solvent strength of your injected sample is weaker than your initial condition, than your sample will get re-focused at the column head, even if it would have had broadened in the tubing. You may search for Waters "at column dilution" principle for prep-chromatography which essentially makes use of that effect.

If your sample is stronger than your initial eluent, than you may be in trouble and may inject much less than it would be possible under optimized conditions.

And in the end, your peaks will likely end up in volumes between 1000 - 4000 µl on your column (k=2-10), independent how much you have injected, anyhow.
So I wouldn't care too much of small dilution effects from the injector, as long as your compound has a suitable (big enough) retention factor of e.g. >2 under your initial/isocratic condition.

So in the end, you may install the 2 ml loop combined with the 250 µl syringe and inject the 200 µl. The metering device is the syringe and it will only take up what you program and places it in the tubing. Then the needle will be placed back in the flow path and the eluent is then directed through the needle onto your column.

PS: if you want to know how much was effectively injected, then you may weigh your vial before and after the injection. Then you can concentrate your fractions to the same weight (not considering any differences in solvent densities). With 200 µL this should be feasible on analytical balances.

[EK8]

Thank you all for your advice.

I would like to say to Multidimensional - you were correct in what you told me. I called Waters again and had a conversation with a different technician. He said it is very likely that using a 2mL loop will create broad peaks. How broad we can't know unless we actually look at what is happening.

I too thought the sample would be concentrated at the head of the column. The sample solvent is weaker than the initial mobile phase. I will resize the sample loop and see if the time-based fractions harboring bioactivity become more narrow.