Determine the HPLC System Dwell Volume (Gradient Delay Volume)

Note: The total HPLC gradient system dwell volume is different than the HPLC column’s void volume. Two different terms for two very different measurements.

When we perform gradient HPLC analysis, the mobile phase composition is changed over a period of time. The mobile phase is mixed in real time by the pump(s), mixer and/or valves, then transported to the injector and finally, on to the head of the HPLC column. The total volume of liquid contained between where the mobile phase is mixed and the head of the column helps us determine when the newly mixed solution arrives at the column head (it is not instantaneous). This delay is often referred to as the gradient delay time (or delay volume) and its value will vary for different HPLC systems due mainly to differences in tubing dimensions used, pumping system type and the design of the flow path. 

For example: If the system dwell volume is found to be 1 ml and the flow rate used is 1.000 ml/min, then the gradient delay time is one minute. 

So how do we know what the system dwell volume or gradient delay volume is? Well, we measure it of course!

Measure the ‘System Dwell Volume’ (aka: Gradient Delay Volume)*:

(1) REMOVE any HPLC column(s) and install a Zero Dead Volume Union (*ZDV) or a restriction capillary of know volume in its place.

(2) Prepare Two Different Mobile phase solutions:

Bottle ‘A’: HPLC grade Methanol (MeOH).

Bottle ‘B’: HPLC grade Methanol with 0.1% acetone added (v/v).

(3) Set your UV/VIS detector to 265 nm (8 nm Bandwidth, Reference OFF).

(4) Program a suitable system flow rate and create a simple Gradient Method (linear change) which starts at 0.0 minutes with 100% ‘A’ (HPLC grade Methanol) and 0% B (HPLC grade Methanol with 0.1% acetone added) and runs to 0% ‘A’ and 100% ‘B’ for about 10.0 minutes (actual times used will depend on your selected flow rate).

(5) Flush and degas both solutions, ‘B’ first, then ‘A’ through the system until you get a nice clean, flat baseline. Make sure their is enough backpressure on the pump (>40 bars) to obtain a stable signal (use a restrictor or back-pressure regulator if needed).

(6) No injection should occur during this method.

(7) Start the method (RUN) and observe the 265 nm signal over time. At some point you should observe the signal begin to rise. When you see this signal change occur, the acetone has finally made it from the pump head to the detector’s flow cell. Make note of the time this occurs. 

Using the known flow rate and observed signal change time, you can now estimate the total system dwell volume. 

Example: If you observe the signal start to rise steeply at 2.00 minutes and your flow rate was 1.000 ml/min. Your system dwell volume would be 2.000 mls. 

A more accurate system dwell volume value can be obtained by next running the same method with an injection of acetone (e.g. 1 ul) and noting the time at which the injection peak is first seen. That will give you the time it takes the sample (and therefore the volume needed) to go from the injector to the flow cell. If you subtract this time off the system dwell time you recorded in the last test, you will have the actual measured time from the pump head (or proportioning valve) to the head of the column (vs the flow cell). Normally the volume contained in this tubing and flow cell are very small relative to the volume in the rest of the system, so we can ignore them. However, when using some of the very low volume columns (e.g. 2.1 x 50 mm), the volume contained in these areas can become significant so when appropriate, we need to be aware of them.

Failure to take into account changes in HPLC system dwell volumes can result in methods which no longer work or provide different results. This is because the gradient rate change you program in your method may not allow enough time for the new mobile phase composition to reach and flow all the way through the column in the time that you have programmed. A common mistake we see is when users forget to adjust the gradient profile when changing column dimensions or program changes using too fast a time.

BTW: One common trick we use to improve compatibility between systems which have different dwell volumes is to include an initial (time 0.0)  isocratic hold-time into the start of each method. If all systems used have system delay volumes under 3 mls, then add a 3 minute isocratic hold time at the start of each method (if 1.000 ml/min flow rates are used), before any gradient starts. While not the best way to deal with the issue, this type of “cheat” can make it possible to quickly adapt a method for use on several different system types.

*Note: This is a generic method to determine the system dwell volume or gradient delay volume. Detector signal buffering and flow cell volume also adds to the delay and in some cases, must also be accounted for too. There are many other methods which can be used for this determination as well. This proposed example serves to illustrate the concept only.

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.

Appropriate Mixer Volume for HPLC and UHPLC Applications

For gradient analysis, most analytical scale HPLC (UHPLC) systems incorporate a solvent mixer which is designed to balance the requirements of moderate dwell volume, low noise and good mixing efficiency. Depending on the method run, the ideal mixer's volume may in fact be completely different than the one installed in your chromatography system. A high-pressure mixing Binary pump can often work well with a slightly lower volume mixer than a low-pressure mixing ternary or quaternary pumping system (because the high pressure mixing gives you a head start), but both pump types benefit from additional mixing.

• Be sure to also consider the volume of any pulse dampener used too as these often have large internal volumes and act as mixers. Some pulse dampeners also incorporate the pressure transducer and/or mixer. These types of combination modules may limit the types of modifications which can be made to optimize the mixing and reduce the dwell volume.

• Don't forget to address the dwell volume contribution of the autosampler, injector loop, interconnecting tubing (extra column volume) and detector flow cell too when optimizing the flow path of your HPLC system.

Here are some general guidelines to help you determine the appropriate mixer volume for your own HPLC system. Note: Since many types of mixer designs exist (static, dynamic, shear...), these are guidelines only. There are some commercially available, high efficiency, low-volume mixers available which can reduce the need for a large volume mixer. Your specific application should be taken into account to determine which size is best.

HPLC System Mixer Volume Choices - Size Matters ("Mixer Volume")

SMALL: Fast or ultrahigh speed separations using low volume, small particle columns. These types of applications depend on a low dwell volume mixer for gradient analysis. To achieve this, your HPLC system should be plumbed with narrow bore capillary tubing (example: 0.005" ID; 0.12mm ID) and include a gradient mixer with a volume of less than 100 ul for low flow rates (example: ~35 ul is rather common size). 

LARGE: High Sensitivity Analysis: Gradient analysis where sensitivity is key, benefit from larger volume mixers to minimize contributions of any UV absorbing additives (e.g. TFA) and turbulence in the flow. Traditional 300 to 750 ul mixers often work well in these applications, provided that the column volumes are also large. Smaller column volumes will require smaller mixer volumes to reduce the added dwell effect.

MEDIUM: Routine HPLC Analysis: Typical analytical separations using 3 to 5 mm ID columns (x 100 mm or longer) usually benefit from modest sized mixers within a range of 200 to 400 ul volume. For these applications, I often start with a recommendation to use a mixer which has 10% of the columns volume as a starting point. For a typical 4.6 x 250 mm, 5 micron porous support column, which has about 3 mLs of internal volume, a 300 ul volume mixer usually provides enough mixing volume for routine gradient analysis.  


Additional Info:

Back in the 1980's we often related mixer volume to intended flow rate/column dimensions. For example: A mixer size of 25 ul was suggested for 50 ul/min flow rates (commonly used with 1 mm ID columns). A mixer size of 200 ul was suggested for 200 ul/min flow rates (commonly used with 2.1 mm ID columns) and 350 ul mixer volume for 1.000 ml/min flow rates (commonly used with 4.6 mm ID columns). Note: Mixers such as these, with large volumes relative to the column volume contributed to large gradient delay times, but this was, and still is, of less concern for isocratic methods.

As mentioned before, the type of mixer, column volume, flow rate and mobile phase characteristics will help suggest the most applicable volume for your application. When in doubt, select a larger mixer volume for isocratic analysis (less baseline noise, better for gradients) and a smaller one if reducing gradient analysis delay volume is critical.

HPLC Flow Cell Volume & Path Length:

Modern UV/VIS detectors offer several different flow cell options. The option(s) you select can make a big difference in the level of signal sensitivity, sample dispersion and response you obtain. If you fail to note which type of HPLC flow cell you use in a particular system, then you may discover some problems when transferring a method to a different instrument. Always record the flow cell volume and path length used as part of your method description. 

Flow Cells Usually Differ In Three Ways:

(1) Maximum Rated Back-pressure;

(2) Flow Cell Volume and

(3) Flow Cell Path length. 

Let’s take a look at these in more detail.

• Maximum Rated Back-pressure: Unless the detector is in series with another detector, column or has a back-pressure regulator on it, the expected back-pressure on a typical flow cell’s outlet is just about one bar as it usually is directed to an open waste line. *This topic will be discussed in more detail in the future as part of another “hint and tip” topic. Today we are more concerned about the remaining two options:

• Flow Cell Volume: Analytical flow cells are commonly offered in nl to ul sizes. Depending on your instrument setup, column and sample(s), one flow cell volume may make more sense than another. After you have spent time separating and concentrating the peak of interest into a tiny volume you do not want to elute it off the column and mix it with another peak because the cell volume is too large. Ideal cell volume is a compromise between sample dispersion and sensitivity. The best choice will be determined mostly by the actual peak volume of your separated sample. The general rule is that your flow cell volume should be no larger than 10% of your peak volume and ideally ~ 2.5% (a 1:40 ratio), but there are some exceptions to this rule. When in doubt, experiment with different cells and do not forget to consider the total volume of all the connecting tubing and valves in your system as these contribute to many issues when the column volume decreases (such as when using mini or narrow bore columns are used). Some common analytical cell volumes offered by various manufacturers are 2 ul, 6 ul and 13 ul. For narrow bore columns (~ 2.1mm ID) a smaller cell volume (~ 2 ul) will result in less sample dispersion, while a larger cell volume may increase overall sensitivity (esp. when used with a longer path length). Mid-bore or Mid-Size columns (2.1 to 4.6mm ID) often are best suited to cell volumes around 6 ul to minimize dispersion and still provide good sensitivity. Larger flow cells such as the common 13ul size often have longer path lengths which can be used to enhance sensitivity. Standard 4.6mm ID columns often benefit from a 13ul volume cell to provide maximum sensitivity with less concern for dispersion effects when larger columns are used (e.g. 4.6 x 250mm). Keep in mind that these are general guidelines only. Most samples contain many peaks of varying width & volume, so you will need to select the cell volume that is optimized to most of the peaks found in your sample.

• Flow Cell Path Length: The flow cell’s path length affects the intensity of light reaching the detector (Beer-Lambert law). For the same volume of sample, the apparent concentration of the sample will appear to be higher if the path length is longer. There is no established standard for ‘path length’ so it is important that you always known what the path length of each flow cell is in your detector (10 mm is very common). Just as volumes vary, manufacturer’s offer different flow cells with varying path lengths. Even identical detectors can use flow cells with identical volumes, but have different path lengths. When comparing the analysis results obtained from two different instruments, always make note of the flow cell dimensions used in each instrument. If the method is to be accurately reproduced on a second system, then the flow cells used should have the same geometry (volume and path length). One way that the difference in path length can be used to enhance sensitivity of an existing method is to use a flow cell with a longer optical path length. For example, if your current flow cell has a path length of 6 mm you could replace it with one having a longer path length of 10 mm. This would increase the sample peak response (as more light would be absorbed) in your method. *This fact can be useful to squeeze out additional sensitivity in a method and often does not require any change of column or conditions.