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 Peak Splitting. Common Reasons For It

True "Split" HPLC peaks, not resulting from co-elution of another peak, can be caused by a number of chromatography problems. Here are a few examples and their solutions:

1. Sample overload. Sample overloading is one of the most common reasons for observing peak "splitting". Reduce the sample concentration by factors of ten to see if the peak shape improves. 
2.  A poor quality HPLC method. Poor quality methods which do not use mobile phase solutions which are at an appropriate pH (*If the pH of the mobile phase is close to the pKa of the sample, then split peaks may result); which does not dissolve the sample in (should be fully soluble) or are unstable, show sample or mobile phase precipitation can cause this effect. Always check solubility before starting.
3. A partially plugged or fouled column. A dirty or fouled column (from not washing down properly with a solution which is STRONGER than the mobile phase). Analysis methods should be followed by separate wash methods to remove all bound material and any late eluters,
4. Wrong injection solution. Peak splitting may be the result of dissolving and injecting your sample in a solution that is stronger than your mobile phase. Dissolve and inject samples in the mobile phase or in a solution which is a slightly weaker solution (not stronger).
5. A poorly packed column, void at column inlet, a dirty frit or poor mechanical connection (i.e. improperly swaged fitting). These types of structural or mechanical defects can each result in peak "splitting" (all of these are less common today than in the past using modern HPLC columns). When present, a dirty inlet frit can be replaced with a new one, or the column can sometimes be backflushed to remove any accumulated material. Connections should always be double checked.
6. Detector data rate set too low. Too few peaks collected over time may result in integration errors and inaccurate peak symmetry problems. Read more about how to determine the best data collection rate at this link.

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.

HPLC K Prime. Also known as: Retention Factor, Capacity Factor): One of the Single Most Important HPLC Parameters of All

The role of Capacity Factor / Ratio (K prime) in liquid chromatography is to provide a calculation or ratio which defines how much interaction the solute (sample peak) has with the stationary phase material relative to the mobile phase (IOW: the relative time the sample spends interacting with the support vs. the mobile phase). If this interaction is too short (i.e. K prime less than 1.0), then no chromatography has taken place and you have just developed a "flow-injection" method (the same as if no column was used) instead of a chromatography method. The method fails all validation and is NOT fit for purpose. Sample Retention must be long enough to demonstrate that the method developed is specific to the sample and shows good selectivity (retention) for the sample analyzed. This is true for most, but not all modes of liquid chromatography (3). 

• New and ever 'experienced' users lacking training in HPLC often make this error, developing methods where the sample has little to no retention on the column. We routinely review methods where the actual K prime of the key sample is measured to be at or near 0, failing to show any chromatography took place in the method (*The Journals are filled with thousands of examples of invalid HPLC methods of analysis). This mistake is made because the author(s) have no formal training in liquid chromatography and do not understand the basics of the technique. 
• Note: Slowing down the flow rate to "show" a later elution time DOES NOT increase K prime (a very common novice mistake) !!! 
  

Observance of the fundamentals of chromatography are key to developing high quality HPLC methods. For most modes of HPLC, highest on this list of basic fundamentals is that the sample(s) be retained on the HPLC column used and not eluted out at or near the column's void volume (we often refer to this time in minutes as, "T-zero" or "t0"). Many chromatography methods fail this simple test of retention and are invalid as written. Knowing what a compound's retention or capacity factor is allows us to be confident that it has been retained and eluted past this critical point, but to first calculate K prime, we first need to know the HPLC column's void volume. 

Calculation and/or measurement of the Column Void Volume should be one of the very first chromatography method development tasks you learn to perform. Knowing the column void volume allows you to determine the retention time of an unretained sample and the resulting retention factor (K prime) of each sample eluted after it. To do this, you must calculate the column void volume AND inject a sample which will not be retained by the column to determine what time an unretained sample will be eluted off the column. This establishes the 'T' zero time, or T(0). The time it takes an unretained compound to elute off the column is critical to know. If your HPLC method does not retain the sample on the column long enough past this time, then you are not allowing any chromatography to occur. Once you have this T(0) value, you can then determine the retention factor (the "K Prime") of your actual sample(s) using the simple ratio formula below. Your final method should baseline separate all compounds apart and, if properly developed, each sample peak will often have K Prime values between 2.0 and 10.0. K prime values of greater than 10 are acceptable, but often show minimal improvements to resolution. Try and insure that the earliest eluting peak in your sample has a K Prime of  >1.5. Do not develop methods which only result in K Primes of less than 1.5 (an indication of poor quality chromatography). 

Note 1: Many regulatory agencies (e.g. The USA FDA) requires that K prime values for HPLC separations be equal to or greater than 2.0 to meet Specificity acceptance criteria (System Suitability/Method Validation). After all, if it elutes at or near the void volume, then your method is not specific for anything. Besides being unscientific in design, your method will fail System Suitability and fail validation. IOW: It does not meet this basic requirement.

• K Prime, K1 (Capacity Factor or Retention Factor) Formula:

       k1 = [T(R) - T(0)] / T(0)

• (where T(R) equals the retention time of the peak in minutes and T(0) is
  the retention time of an unretained peak). 
• *The 'K Prime' of your sample must be > 1.00. A value greater than 1.5 should be your goal.

Example #1: 
 T(0) found to be 2.90 minutes and the sample elutes at 5.80 minutes. k1 = 5.80 - 2.90 / 2.90. k1 = 1.00.

Example #2:
 T(0) found to be 2.90 minutes and the sample elutes at 9.10 minutes. k1 = 9.10 - 2.90 / 2.90. k1 = 2.13.

Example #3:
 T(0) found to be 1.75 minutes and the sample elutes at 1.74 minutes. k1 = 0. No retention and no chromatography have taken place at all. The method is invalid.


Note 2: I see and read published HPLC methods (including "Validated Methods" !) every week which ignore this fundamental requirement and present data showing little to no retention of the primary sample on the column. Most are RP methods run on popular C18 columns and show the main peak of interest eluting out as a nice sharp peak right at the void volume.  These methods often describe the sample analyzed as "100% pure" and are fully validated (because the person doing the work may not have had any HPLC experience or training)! A mixture will always look like a single peak by HPLC when no 'chromatography' is employed to separate out all of the possible components. The sample must be retained on the column for a period of time before we can conclude anything about its purity by the method employed.

Note 3: In some cases, when other modes of chromatography are utilized (e.g. ion exchange, size exclusion chromatography (SEC / GPC), K prime is not as relevant. The mode of chromatography can affect the interpretation. For example: This is because size exclusion chromatography relies on the sample's interaction with well defined pores inside the support (inclusion/exclusion) to separate based on molar size. A variety of pore sizes can be used to "filter" the sample. So a large K prime value might be normal for a molecule that is low in molecular weight and spends a lot of time working its way through the column. A high molecular weight sample might just "shoot" through the column due to little or no interaction with the pores. You still need to have retention on the column, but now it is determined by how long it takes the sample to find its way out of the column. SEC columns are bracketed by Pore Size (e.g Mw. Excluding all samples that do not "fit"). With size exclusion columns, determine the Retention and Exclusion times, not the K prime). This article is specific to thr more common cases where traditional HPLC NP or RP modes are used. In these cases, low K prime values indicate no retention took place and the method fails all claims of specificity for the sample (selectivity is absent or poor). HPLC methods with little to no selectivity fail scientifically as no chromatography has taken place.