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.

Notes on Cleaning bound Protein from RP HPLC columns:

First, a few comments:

• ·         Before proceeding with any column regeneration or cleaning procedures, always refer to the specific advice provided by the column manufacturer. Approved maintenance and cleaning instructions can often be found in the product guide which comes with the new column. Their guidelines supersede these!

• ·         Columns are consumable items. After a suitable amount of use, the time and materials required to regenerate them may cost more than the purchase of a replacement column. Always have a new, spare column on hand.
  

•        Do not overload the column! This is the most common reason for column fouling, flow path contamination and sample carryover issues. In most cases, injection volume should be less than 1% of the column volume (maximum).
  

• ·         Protect your detector. Before washing or cleaning the column, disconnect the column outlet line and direct the column to waste only.

•        Column Storage solutions are not the same as column wash solutions. Never store a column in buffer or ion pairing containing solutions.

For RP supports, if buffers have been used, always start by washing the column down with ultra-high purity water and some organic solvent (e.g. Water/MeOH, 95%/5%) to remove all salts. Use about 10 column volumes to flush these off. Do not wash the column with organic solvents until you have first washed it thoroughly with high-purity filtered water.

Polymeric resins (e.g. PS-DVB) from many manufacturers can effectively be cleaned using 0.1 M Sodium Hydroxide solution or a mobile phase solution containing equal parts of isopropanol (IPA) and 1 to 3 M Guanidine hydrochloride at ~ 50 °C. Optionally, some success has been reported using other solutions such as: 5M Urea (pH 7) buffer solution; 1 M NaCl (pH 7) and even mixtures containing some methylene chloride solvent. Check with the manufacturer first as column damage/plugging may result if their directions are not followed.!

For RP silica based supports (non-SEC), we often start with a series of wash solutions. In most cases, pure water or pure organic solvents such as MeOH or ACN will not remove bound protein (common novice mistakes). An acid, base or even an ion pairing reagent is often needed to clean them. Start simple and monitor.

 

For RP silica based supports (SEC), a high salt buffer solution often releases bound proteins quickly. A mobile phase containing water plus an alcohol (methanol, IPA or ethanol) may also prove effective too.  Optionally, a solution of 0.5 M guanidine hydrochloride may effectively remove bound material.

General Advice: One of the first general wash solutions to start with (especially to remove basic compounds) is a 1% Acetic acid solution in Methanol (50/50). If desired a stronger acid such as 0.1 % Trifluoroacetic acid (TFA) or 0.1 % Formic Acid can be swapped for the acetic acid (where possible, start with a weaker acid). Use a low concentration of acid to achieve a pH of ~ 2.5. This acidic wash can be followed with a neutral solution, or if needed, a later solution where IPA or ACN replaces the MeOH used (50/50).

For extreme cases where the column has been overloaded with protein, a 5 M Urea solution has been proven effective in removing bound protein from silica and polymeric supports too. A word of caution, as the resulting pH of this strong solution may be greater than or equal to pH 9. Many types of silica based RP columns can not withstand strongly basic solutions and the silica inside may dissolve (plugging the column). Start with a lower concentration wash  first. You can always increase it later. Always read the instruction sheet carefully which came with the specific HPLC column to determine if it can be used at these high pH levels. Another salt solution that has shown some promise is 1 M sodium phosphate solution, pH 7.0. Run the salt solutions for about one hour at a moderate flow rate. Follow up all washes with rinses of mixtures of water and MeOH (80/20), then 90% MeOH/Water. 

Please remember that in ALL cases, HPLC columns are consumable items with a limited lifetime. Dispose of them properly when they are damaged or contaminated and replace with a new column. Once you have a fresh clean column to work with, prevent column fouling by developing better quality methods which utilize frequent, properly developed wash methods (using a wash solution which is stronger than your analysis mobile phase), filter all samples and be sure they fully dissolve in solution (100%). *Column fouling is not normal and can be prevented with proper training.

Typical Commercial Strengths of Common Acids and Bases Used in HPLC

CHEMICAL NAME	MOLECULAR WEIGHT	MOLES / LITER	GRAMS / LITER	PERCENT by WEIGHT	SPECIFIC GRAVITY
Acetic Acid	60.05	6.27	376	36	1.045
Acetic Acid, Glacial	60.05	17.4	1045	99.5	1.05
Formic Acid	46.02	23.4	1080	90	1.21
Hydrochloric Acid	36.5	11.6	424	36	1.18
Nitric Acid	63.02	15.99	1008	71	1.42
Perchloric Acid	100.5	11.65	1172	70	1.67
Phosphoric Acid	98	14.7	1445	85	1.70
Sulfuric Acid	98.1	18.0	1766	96	1.84
Ammonia (in H20)	17.0	14.8	252	28	0.898
Potassium Hydroxide	56.1	13.5	757	50	1.52
Sodium Hydroxide	40.0	19.1	763	50	1.53

Data obtained from The Merck Index, 11^th edition (1989).