HPLC System Dead (Dwell) Volume. Is It Static or Can It Change During a Method? Autoinjectors and Gradients.

I recently read a post on a popular LinkedIn chromatography group where a user asked "if it is possible for the total system volume of their HPLC system to change during a method? Would it effect sample retention times? If so, how? If not, why?" Almost all of the group members who responded to the question said that it was impossible for the system volume to change once the HPLC system was installed! Note, we are referring to the HPLC "System" volume, not the column volume in this question. Column volume is fixed, but the total system volume is not fixed. Another reason why you should not believe everything you read on the web! The question tests your practical knowledge of how HPLC systems operate (specifically, how HPLC injectors operate).

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.

Troubleshooting HPLC Injectors (Manual and Automated)

Sample injectors are a critical component of a chromatography system. Understanding how they operate as well as the proper techniques to use and maintain them are fundamental skills needed to operated an HPLC system. Lets briefly discuss some of these fundamentals as applied to a standard manually operated HPLC injection valve and also in an automated mode as found in an autosampler. *Note: You should always refer to the specific manufacturer's product specifications, operation, servicing documentation or support personnel before servicing any injector.

MANUAL INJECTION VALVE Notes:

These valves allow you to use a high precision syringe to manually fill a fitted "loop" with a sample and then, by turning a valve handle, introduce the sample to the high pressure flow stream directed toward the column inlet. Sample loops are available in a wide range of volumes and take just minutes to install. The injector valve has very tiny openings inside which are moved between two different positions (LOAD and INJECT). The LOAD positions allow the valve to seal off the internal high pressure flow from the loop to allow it to be safely filled with sample at atmospheric pressure. The INJECT position introduces the liquid contained inside the loop to the main flow path (under high pressure). The parts must be clean and seal well to insure proper function. Leaks from all areas (except the vent) are not acceptable and indicate a problem. Here are a few tips regarding the use of manual injectors in HPLC.

• Use the correct type of sample syringe. Usually these are high precision glass syringes with Teflon plungers. The needle tip style is the most critical item! Most injectors are designed to only work with a needle which has a squared off tip (NOT a point as is commonly used in GC!). The most common gauge used is #22. Always check with the valve manufacturer to determine the correct style and gauge of needle before use.
• Leave the valve in the INJECT position during the entire run to flush it clean of sample and stay equilibrated with your method. Switch it back to LOAD only when you are ready to load a new sample.
• For high reproducibility and accuracy within one HPLC system, fill the loop with at least three times the volume of the loop with sample to insure that the entire path is full of sample. This is known as the complete or over-filled loop method. *Choose your loop volume size with this in mind. Loading the same volume as the loop will often result in poor accuracy.
• Loops often do not contain the exact volume stated on them. They can be off by ~25% so consider this when injecting partial volumes and not using the standard over-filled loop method.
• Types of common leaks: (1) Leaks at the needle port (needle seal worn); (2) Leaks behind the valve stator (worn rotor seal, buffer crystals dried inside, over pressured, scratch on rotor); (3) Leaks at the vent (liquid should be expelled from the vent when filling the loop only. Other leaks indicate a problem). Note: Rotor seal damage can cause sample carry-over problems so valves should be inspected at regular intervals (~ 6 months).

AUTOMATED INJECTION VALVE (auto-injectors) Notes:

These valves use a high precision syringe or high pressure pump to fill a fitted "loop" with a sample and introduce the sample to the high pressure flow stream, all automatically. Most function exactly the same as described above, though some are based on true high performance liquid chromatography pumps so have no "syringe" at all (e.g. Agilent 1100/1200 designs) Here are a few tips regarding the use of automated injectors in HPLC.

• Regular maintenance is even more critical with auto-injectors since you often can not see what is going on during the injection cycle. Leaks, if present can be harder to find so make it a habit to visually check all of the areas around the injector regularly.
• Needle and Needle Seats are normal wear items on these instruments. As such, they require routine checking for leaks or damage and replacement when worn.
• Vial Caps: If you make multiple injections from one vial (or large volume injections) with a tightly sealed vial cap, a vacuum can form inside the vial causing volumetric errors to occur in your samples (resulting in you injecting less sample each time). Leave the caps slightly loose to avoid this problem. Multiple injections into the same vial can also cause the needle hole to become enlarge over time allowing the sample or solvent to evaporate over time, changing the concentration of the sample (more concentrated). Replace the cap and seal with a new one if used multiple times. Always leave the cap slightly loose.
• Loop volume: Autoinjectors often incorporate one large loop to handle a wide range of sample volumes. This is a trade off of accuracy for convienence. Accuracy is often poorest at the very low end of the range and best near the middle to high end. Always verify the reproducibility of the injector to inject a specific volume through statistical analysis of repetitive injections.
• Types of common leaks: (1) Leaks at the needle seat (needle seat worn); (2) Leaks behind the valve stator (worn rotor seal, buffer crystals dried inside, over pressured, scratch on rotor); (3) Leaks at the vent (liquid should be expelled from the vent when filling the loop only. Other leaks indicate a problem). Note: Rotor seal damage can cause sample carry-over problems so valves should be inspected at regular intervals (~ 6 months).

These are just a few tips related to HPLC injectors. Please consult the service documentation for your specific instrument to better understand how the system works and what areas you should be monitoring. Understanding HOW these systems operate (and can fail) is one of the most important skills you can learn as a chromatographer. Take the time to understand the complete flow path of your system before using it.