The numerous and incorrect responses posted to the initial question made me realize that this would be an excellent job interview question for chromatographers seeking employment. The question certainly tests the users practical knowledge of liquid chromatography hardware and systems. An intermediate or advanced level user with a few years of experience should have the practical knowledge of the HPLC system flow path and how it effects sample retention times and method development to know the answer. A novice user would not be expected to have this same level of practical knowledge and answer incorrectly. Additionally, most chromatography books only address concepts and fundamentals, but to be a good chromatographer you also need a great deal of practical hands-on knowledge about the how the chromatography hardware operates. This information is obtained through receiving proper training and practical hands-on experience running a wide variety of methods with real samples to solve complex problems. This is a very 'hands-on' technique.
To get back to the original question posed, "if it is possible for the system volume of their HPLC system to change during a method?" Knowledge about column void volume, system swept volume (system dwell volume), gradient composition delays and most importantly of all, how the flow path is manipulated in an autoinjector (or a manual injection valve) to inject a sample into the flow path are all needed to formulate an answer. Which parts of an HPLC system contribute to the total system dwell volume? The total volume of liquid contained in the system from the inside of the pump head to the column and detector inlet or flow cell contribute to the total system volume. These parts are pre-plumbed. The mobile phase mixer and/or pulse dampener are two parts (e.g. ~300 ul) which may contribute a significant percentage of the volume up to the column head. However, of more concern in this case and also a significant contributor of total delay volume in an HPLC system is the injection loop (usually ~100 ul). For manual injection and auto-injector valves, this loop is of a fixed volume, but allows for partial filling (though the loops used are not really accurately measured as the metering device is responsible for most of the volume accuracy). For both types of valves, the loop volume should be at least as large as the largest volume needed (e.g. 100 ul size is common). If the loop size is 100 ul and you only inject 1 ul of sample into a std loop of 100 ul, then you are placing your 1 ul sample up against a slug of 99 ul of mobile phase. While this dilutes the sample and allows some diffusion to take place, spreading out the sample (not ideal), when injected into a typical 4.6 x 250 mm, 5u column (which has a volume of ~ 2.90 mls), it normally has very little negative effect on the chromatography seen. The effect can be dramatically different when using a tiny column with a small volume (e.g. 2.1 x 50 mm, 3u). The diffusion effect can result in very wide peak widths resulting in poor loading and resolution. A physically smaller volume loop is needed to improve the performance.
However, when we run a gradient analysis another effect is introduced, gradient delay. The mobile phase composition is mixed at the pump head outlets or in a mixer after the pump(s). It takes a specific amount of time for this mixture to reach the head of the column. This time delay is known as the gradient delay. The flow rate and the volume of liquid contained in the tubing from where the liquid is mixed to the head of column determines how long this delay lasts. Since the flow rate normally remains fixed during a method, the total volume of liquid between these two points is the critical value we are interested in. The larger the volume, the longer the delay before the mobile phase composition reaches the column head.
- Gradient Delay Example: Flow rate = 1.00 ml/min; Volume between pump and head of column is 0.300 mls. Delay volume is 300 ul and the Gradient Delay Time would be 0.3 minutes. So the mobile phase composition that we programmed into the pump does not actually reach the column until 0.3 minutes after we programmed it to occur.
Depending on the value of this volume, the delay from the time the gradient program starts until the gradient reaches the head of the column will vary. This is a critical concept to understand when developing gradient methods and especially when transferring gradient methods to other HPLC systems (as different systems have different dwell volumes). This poses a minor inconvenience to method development and we need to take it into account so we program composition changes with enough time in between them to allow the changes we programmed to have time to take place and cause the desired effect.
How do we change the volume of the Autoinjector (or manual injector) without re-plumbing the system? One of the most common methods used to reduce the total flow path volume of an autoinjector is to program the injector to switch the injection loop (which has a large volume) out of the flow path immediately after the injection, instead of leaving it directly in the flow path for the remainder of the method. Remove the loop and you subtract the loop volume from the total dwell volume. This will reduce the total system volume (dwell volume) at the start of the method which will also reduce the total gradient delay observed. The newly mixed solvent composition will arrive at the column head sooner. *Using the previous example of a system with a 300 ul gradient delay volume, toggling the injection valve to switch out the 100 ul loop from the flow path would reduce the total delay volume by one third, from 300 ul to 200 ul. So this illustrates a well known technique to change the total system dead volume (dwell volume) of an HPLC system without manually re-plumbing it. Most autosamplers (autoinjectors) provide this loop "toggle" feature as standard in their software menus for exactly this purpose. It can also be time-programmed into most injector's (if no "feature" or menu option is available) and can also be employed with manual injection valves too by placing them back in the "Load" position after injection.
Summary: Can the HPLC system swept volume be changed during a run? YES it can.
How? One of the easiest ways is by switching the injection loop out of the flow path during the analysis.
I think I saw that LinkedIn post on one of the chromatography groups. It is an excellent question which really does test the persons skills and experience. In our lab, no one knew the correct answer, just as you said. They also admitted that they do not understand the flow path of our HPLC systems either. We have never had anyone train us in HPLC. What we know has come from trial and error and our managers do not seem to understand that we need training to do good job.
ReplyDeletePlease keep up these great and free trainings. Everyone in our lab reads and discusses them. We have leared a lot.