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.

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 Capillary Tubing Connection Volumes:

The length and internal diameter of the HPLC interconnecting tubing used in your system really does matter. The total volume contained in the tubing can dilute your sample or separated peaks. This can effectively undue the work of separating the peak(s) on a column. Extra volume in the tubing can also have the effect of increasing the gradient delay factor for your method (the greater the volume of the tubing from the pump head to the column inlet, the greater the delay in the solvent mixture arriving at the column). In general, keep the the total delay volume as low as possible. This is accomplished by connecting the various modules together using the shortest lengths of tubing possible. For systems which use standard sized HPLC columns (e.g. I.D.'s of 3.0 to 4.6mm and lengths from 100mm to 300mm) the tubing internal diameter should be 0.17mm (0.007"). For systems which use very short, mini or micro bore sized HPLC columns (e.g. I.D.'s of 1.0 to 2.1 mm and lengths from 50mm to 250mm) the tubing internal diameter should be 0.12mm (0.005"). Looked at another way, if the total column volume is less than 750 ul, consider using the smaller internal diameter tubing (0.17mm) to reduce band broadening. 

Here are some tubing volumes to help you evaluate the effect changing the I.D. or length has on the tubing that you use.

I.D. (mm)	I.D. (inches)		ul / cm	ul / inch
0.12	0.005		0.127	0.323
0.17	0.007		0.249	0.632
0.25	0.010		0.507	1.288
0.51	0.020		2.026	5.146
1.02	0.040		8.103	20.581

Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero):

Column Hold-up Volume, Column Dead Time or 'Column Void Volume' (the preferred name) are all different terms we apply to find the internal volume of a packed column  (divided by the flow rate and usually expressed in minutes for the Column Void Time). You must know what this value is BEFORE starting to run an HPLC method or perform liquid chromatography. The value for column void volume changes for different column dimensions and different column support types (e.g. fully porous, superficially porous etc) .

Are you peaks or samples eluting at or near the column void volume? If so, for most modes of chromatography, this implies that no chromatography has taken place and no HPLC method has been developed (SEC/GPC separate based on hydrodynamic volume, so elution at or near the column volume means the sample(s) were excluded from the column). Individuals with little to no chromatography training or experience often make this mistake and create methods which show poor retention. Make sure your methods are designed to retain each sample for a long enough time period on the column (K prime). How do you know how long is long enough? Start by estimating the Column Void Volume (use our table or calculate it for an estimate) then, calculate the K prime value for your sample. The K prime for each peak should be at least 1.5 (>2.0 is the accepted standard for most regulatory authorities) for the method to be useful and selective. *A more accurate value of column void volume will be found by measuring the void volume of your column (please read on).

Knowing the Column Void Volume and the Flow Rate used allows you to calculate the Column Void Time (which is the most useful initial value). Determining  the column void time or T0 ("Tee Zero" as we call it), is necessary to find other important chromatography values such as: the Resolution, Separation Factor and Capacity Factor (K prime aka: "K1") in a chromatography separation. Ideally, it is measured by injecting a sample which is unretained by the column & mobile phase (it passes right through the column support with little to no interaction). It may also be easily estimated for most fully porous, spherical, bare or coated silica supports if you know a few physical specifications of the column and media used. You should first estimate it, then measure it (the two values should be close, +/- 15%). Note: A practical "tip". You can also estimate T0 by noting when the small injector valve pressure peak ('blip') appears on the baseline. It results from the pressure change which occurs from switching the injection valve from the "load" to "inject" positions. Use a low UV wavelength to observe this deflection on the baseline.

Here is short list of typical HPLC column dimensions and their associated estimated void volumes for fully porous silica supports. At a flow rate of 1.000 ml/min these values would also be the same as the void time in minutes.

COLUMN DIMENSIONS (I.D. x Length (mm))                 VOID VOLUME (ml)

                         2.1 x  50                                                                  0.12
                         2.1 x 100                                                                 0.24
                         2.1 x 150                                                                 0.37
                         2.1 x 250                                                                 0.61
                         2.1 x 300                                                                 0.73

                         4.6 x  50                                                                  0.58
                         4.6 x 100                                                                 1.16
                         4.6 x 150                                                                 1.75
                         4.6 x 250                                                                 2.90
                         4.6 x 300                                                                 3.49

                       10.0 x 100                                                                 5.50
                       10.0 x 150                                                                 8.25
                       10.0 x 250                                                               13.75
                       10.0 x 300                                                               16.49

•  Column Void Volume Equation for Std Sized, FULLY Porous Supports:

Column Volume (ul) = (d^2 *Pi * L * 0.7) / 4 ;

•  Column Void Volume Equation for SUPERFICIALLY Porous Supports (e.g. Fused-Core, Core-Shell etc):

Column Volume (ul) = (d^2 *Pi * L * 0.5) / 4 .

   Note: Column Diameter & Length are in mm. Volumes are estimates (always measure to find the actual value).

[Note: All you need is the column's length and ID to estimate it. For most fully porous supports, use a 'Pore Volume' value of 0.70 in the above equation. This is the most commonly measures pore volume found for non-encapped, fully porous spherical bare silica support (please check with the manufacturer for the actual value of your support). For superficially porous supports, use a value of 0.50. Estimating the value will often get you close to the measured value, but due to the unique chemistries used to prepare supports, it is only an approximation.

Always measure the actual void volume of your specific HPLC column with a compound which is unretained by your column. For RP applications which utilize at least 20% organic, Uracil or Thiourea are often used, but some inorganic salts (e.g. sodium nitrite and sodium nitrate) have also been shown to work as well. When determining the "Column Void Volume", you are really measuring the void volume of the column plus any extra-column volume from the injection volume plus all lines connecting the injection to the column and the column to the flow cell. Note: This is very different from the "System Dwell Volume" which includes the volume from the pump (or gradient valve) to the column head.

A more detailed version of this table with other common HPLC Column Sizes and Tubing Volumes for capillary lines are available at the following links (Link #1) or (Link #2).