I have recently acquired my first Shimadzu HPLC system, and I have been trying to put it to use immediately. When recovering a sample using our fraction collector, I have noticed a considerable delay between the obtained signal on screen and the drops coming out of the collector tip. I have determined then (both with a dye and using a bubble) a dead volume of about 560 ul, which gives me a delay of over 1 min at 0.5 ml per minute flux. I am rather disappointed at this delay, as it makes correlating the chromatogram with obtained samples a bit tricky (still learning how to compensate with the collector) and, more importantly, because this means an obtained fraction at the detector will gradually dissolve into this dead volume until it gets to my fraction collector. I am sure this costs me resolution.
Questions:
1) Do you get as much dead volume as this?
2) Did you ever try to reduce this dead volume?
You are correct - 0.5 ml of dead volume is indeed quite a lot and will significantly affect your resolution if you run your chromatography at 0.5 ml/min.
I recommend to check inner diameter (ID) of installed tubings and - if possible - replace them with smaller ID tubing between your detector and fraction collector. For running at 0.5 ml/min you could go down to 150µm ID - but check if that won´t generate too much backpressure (e.g. if running high-viscosity solvents).
Also, to maximize resolution, all installed tubings should be as short as possible and have the minimal ID´s that are still compatible with your intended flow rate. Additionally, any valves, sensors (pH, conductivity, etc) etc, could also be causing dead volumes - if you don´t need them for your analysis take them out of the flow.
I think the best way out will have to be change tubings. I strongly suspect the way the new fraction collector works also increases the demand for long tubes, as there must be a slack to the robot to move. In the past I used to see shorter tubes which were fixed, and the revolving collector with tubes would move... I will discuss this with the responsible provider. Thanks!!
In your case, for example, the volume of a 40 cm long 0.25 mm diameter capillary is 20.4 ul. If you use the common 0.13 mm capillary you are safe, at least concerning the capillary volumes. Be aware of additional capillaries such as a heat exchanger capillary in your detector, or possibly somewhere in your fraction collector that has a volume that you cannot define.
I have an e-book concerning HPLC problems and their solvings, your case is written there... If you need that, send me an email ([email protected])... Good luck
replace (shorten) tubing, the only way...
our shimadzu FRC works with 127 µl vol. tubing length is ofthen a matter of order of the components of the HPLC. Maybe change the order.
Dear Andreas, can you please tell me the model of your FRC system? We have a FRC 10A system, and we have seen that collection tubings are indeed quite thick, however it seems they are the "official" ones, which fit because of an end expansion... Not sure how to change if they are this way.
Dear Eduardo,
we have a 10A system too. Indeed, the tubings are long, but the dead volume is determined by the length tubing from the valve to the drop former. are you sure that you re using the "original Shimadzu" part?
Dear Andreas,
Great! I am getting to the bottom of this. Since you have the very same model system (I just bought mine) could you please have a look at the picture I posted in the link below.
This is exactly my doubt: it seems the tubing I am using is a bit thick and it was installed by the technician, and it seems to have a specific fitting for that position. Is this right?
https://www.researchgate.net/post/Any_one_using_a_HPLC_fraction_collector_system_SHIMADZU_FRC-10A
Also, I have posted a picture of the tubings intalled in the link below. In my system the tubing on the left A was installed at the exit of the detector -- its is quite calibrous. I wanted to changed it for the tubing on the right, however it does not fit exactly.
http://eduardofox.blogspot.com.br/2012/12/temporary-just-to-ask-something-to.html
560 ul dwell volume is quite typical for HPLC system. Indeed some older systems have ~1000 ul dwell volume. Dwell system was decreased by manufacturers gradually to 650 and perhaps ~500 ul in 1990's instruments. Dwell volume of tubing is typically ~100 ul or less, the remaining volume is in the proportioning valves + mixer + degasser. That means you can reduce the dwell volume only little. UHPLC generation of instruments has dwell volume ~100ul of which ~50 ul is mixer. For peptide mapping applications people use larger mixers, so the dwell could be higher. Dwell volume has little to do with the resolution, it just makes analysis longer (because early part of separation happens at isocratic conditions). This is very noticeable for fast gradients, say less than five minutes. I have experience with Waters system, which has volume compensation that could be turned on in sofwtare - injection is delayed to point when the gradient reaches the column. Then the entire separation happens in gradient mode. This could make the method transfer from one instrument toa different type of LC easier.
SOLVED!
The technician had installed a preparative tubing system by mistake, and took quite a long while to realize this (he is quite young). Now he quickly changed it for another one fit for analytic fractionation. I am still going to measure dead volume, but now I am sure it should be fine now, something around 75 ul.
I am very thankful to everyone for the help and attention. My best wishes!
I see this post is five years old, but noticed a great deal of misinformation here which might confuse someone if they find this post in the future. A few comments to try and make some sense of it.
(1) Martin Gilar was on the right track, spotted the error in the terminology and answered correctly that a System DWELL volume of about 0.5 ml is very common and many systems (esp std models) have system dwell volumes of between 30 ul all the way up to 1 or 2 mls. - But this had nothing to do with the original question so let's move on...
(2) Terminology: In this context, "Dead Volume" and "Dwell Volume" are very different things.
- System DWELL Volume refers to the total volume of liquid contained between where the mobile phase is mixed and the head of the column. That includes the injector. Depending on how the system is configured, it could be as little as 30 ul to 2.0 mls! It can be changed too, by re-plumbing the system or manipulating the modes of the A/I.
*For more info: "Determine the HPLC System Dwell Volume (Gradient Delay Volume)"; [ https://hplctips.blogspot.com/2017/02/determine-hplc-system-dwell-volume.html ].
- Dead Volume relates to the internal volume of a part. Usually this is an HPLC column, capillary line or fitting (e.g. union).
**For more info: "Determination of HPLC Column Dead Volume / Dead Time (T zero)"; [ https://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html ].
"Capillary Tubing Connection Volumes"; [ http://www.hplctools.com/capvolume.htm ].
(2) The original question asked if "Is a dead volume of 560 ul in an HPLC too much", but in fact, that was not the question (and the wrong terminology). The real question the poster was trying to ask was IF the dead volume in the tubing which exiting their detector's flow cell (last place you "see" the peak) and led to their fraction collector was too large. It was. This is not related to System Dwell Volume (useful to known and understand for gradient runs), which was what was discussed. Standard (0.17 or 0.25 mm ID) internal diameter tubing is often connected to the waste end of the primary detector if a second detector or fraction collected is plumbed in-series with it . Tubing with an ID of 0.5 mm or greater (1 or 2 mm is common) for use as a waste line. Normally, there is no need to worry about the volume inside the detector's outlet line as it goes to waste. However, if you wish to connect another detector or device (e.g. fraction collector), then it makes sense to reduce this volume. This will reduce the time it takes the liquid (analyte) to get to the device, and far more importantly, reduce the effects of diffusion which will widen and dilute the 'peak' or sample (which would defeat the purpose of separating it so well).
Example: A 'typical' flow cell outlet line, 0.25 mm ID x 50 cm, would have a volume of only ~ 25 ul (for comparison, a larger 0.50 mm ID line x 50 cm would have a volume of ~ 100 ul). *Knowing this volume and the flow rate used would provide you with the estimated time it would take to appear at the fraction collector.
- A word of caution about using narrow ID outlet lines on flow cells. Be sure and only use a flow cell which has a max pressure rating that is safe for use with the restriction connected. Failure to monitor and verify the pressure rating of the cell could result in damage to the flow cell.
I hope this clarifies the issues presented for the next person.
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