Adjusting the HPLC Gradient Time For Changes in Column Diameter and/or Length (same particle size)

Changes to the column diameter (to scale the method up or down) can be calculated. For an established HPLC method using the same support type (same exact material and particle size) where the column dimensions and flow rate are known. Note: If only the diameter changes and the lengths remain the same (proper linear flow rates used in both cases), then the resulting gradient times will also be similar. If the column lengths change, then the gradient time will change.

Changes to the Gradient Time (Tg2) used for a second column which has a different diameter, "Dc2" and/or length, "Lc2" can be calculated if you know: 

• Tg1 [Time, of initial Gradient on Column #1];
• Tg2 [Time of second Gradient on Column #2];
  
• Fc1 [Flow Rate of Column 1] ;
• Fc2 [Flow Rate of Column 2];
• Dc1 [Diameter of Column 1]
• Dc2 [Diameter of Column 2];
• Lc1 [Length of Column 1];
• Lc2 [Length of Column 2].

        Tg2 = Tg1 x (Fc1 / Fc2) x (Dc2² / Dc1²) x (Lc2 / Lc1)

 

Example: Initial Method utilizes a 4.6 x 150 mm, 5u column run at 1.00 mL/min with a 10 minute gradient program and we wish to transfer this gradient method over to a column with a 2.1 mm diameter (ID) x 100 mm column run at 200 ul/min.

   Tg2 = 10 x (1 / 0.2) x (2.1² / 4.6²) x (100 /150)

   Tg2 = 10 x (5) x (4.41/21.16) x (0.67) 

   Tg2 =  50 x 0.208 x 0.67

   Tg2 =  6.97 minutes.

The gradient time used on the 2.1 x 100 mm column run at 0.200 mL/min would be ~ 7 minutes (vs 10 minutes on the 4.6 x 150 mm column at 1 mL/min).

 

NOTE: A note about optimized flow rates. If the Column PARTICLE SIZE changes, esp from greater than 3.5 u to less than 3.5 u, then the optimized flow rate may also change too. Please refer to my article; 

• "Speed Up HPLC Analysis Time Using Higher than "Normal" Flow Rates with SMALLER Particles" for more information.

Troubleshooting HPLC Gradient Valve / Proportioning Valve / MCGV GPV Leaks. How to Identify Them.

HPLC pumps which utilize low-pressure mixing VALVES are known by names such as: "Ternary" (3-solvents) or "Quaternary" (4-solvents) pumps.These types of HPLC pump configurations use a single, high-pressure pump head coupled to a multi-port / proportioning valve and represent some of the most popular and versatile pump configurations offered. Featuring random access to multiple solvent bottles (more than two is always better), lower operating costs and less maintenance work provides you with one of the best platforms to develop new methods on. I highly recommend them for most, but not all, HPLC applications (vs. Dual pump, high-pressure "Binary Pumps").

• If your HPLC system utilizes a single, high-pressure pump head coupled to a multi-port valve, then please remember that in addition to pump head maintenance, regular maintenance of the multi-port / proportioning valve is also required.

A few weeks ago I was hired by well known Pharma company to solve a gradient method problem that I was told has stumped their best scientists for almost one year. The client presented me with their validated UHPLC method which suddenly developed a shift in retention time of all peaks. The shift was significant, about 10% of the previous values over a 20 minute run, and had been observed on two different, but similarly configured HPLC systems in their lab. Changing the column to a new one showed no change on either HPLC system. They were out of ideas.

• Before I reveal the cause of the trouble, let us briefly think about what types of changes can result in a small, repeatable shifts of peak retention times. Four common ones that come to mind are: 

(1) Flow Rate changes;

(2) Column Temperature changes;

(3) Column Fouling;

(4) Mobile phase composition changes. 

Start the troubleshooting by ruling out the easy causes first (#1, 2 and 3 above).  

• (1) Flow Rate: When the actual flow rate is in question, start by measuring it manually Never trust the instrument's display screen value or the software's value for flow rate. Measure it. An easy way to measure the flow rate involves timing the amount of liquid that exits the HPLC detector line after a defined period of time. For example: If your flow rate is set at 1.000 ml/minute, then using water, measure the time it takes to fill a 10mL graduated cylinder to the 5 mL line. It must take exactly 5.00 minutes (= 1.00 mL/min). Run this flow test on each pump channel.
  
• (2) Temperature: The HPLC method should be run under controlled column temperature conditions. Verify this. Retention times are a function of temperature (i.e. cooler temps usually result in longer retention times, warmer = shorter). The temperature should be stable (~ 1 or 2 degrees C).
  
• (3) Column Fouling: To prevent fouling, wash the HPLC column with a solution that is STRONGER than the mobile phase after each analysis. Use fresh, clean solutions. Verify that the samples are dissolved in the mobile phase (100% dissolved) and filtered before injection. Verify that the injection volume is less than ~3% of the column volume and the concentration of the sample is not too high (avoid saturating or overloading the column). Solubility is very important for both the sample and any additives used in the mobile phase (to prevent precipitation). Anything that "fouls" the column support will directly effect the retention times and often the peak shape too. Be aware of these causes and take action to avoid them.  *Replacing a suspect column with a new one is often an inexpensive way of troubleshooting a "peak" problem. Always have a NEW spare column on hand for testing. *Columns are consumable items.
  

• (4) Mobile Phase: Changes to the actual amounts of additives, pH or final composition of the mobile phase may impact peak retention times (sometimes, the peak shape too). After all, the final composition used was developed for the purpose of establishing a reliable and reproducible method of analysis. It must be controlled. We must take steps to insure the mobile phase preparation and delivery are accurate. Always prepare fresh solutions each day (esp. all aqueous solutions!). pH values may change after a few days (e.g. even in MeOH / acidic solutions), bacteria/mold/algae grow quickly in many solutions, even in the refrigerator, so only prepare what you need for the day. Evaporation of more volatile solvents (in pre-mixed solutions) can change their actual concentration (always protect them from heat and evaporation).
  

*There is another way that the mobile phase composition can change which often goes unseen. It can change during delivery to the column. The HPLC's low pressure proportioning valve that allows us to easily select and use different solvents can develop small internal leaks, resulting in valve cross-flow leakage. This cross-flow leakage allows liquid (or air, if the line is not connected) to be drawn out of one channel and into another, changing the actual mobile phase composition. This happens because the valve seals, esp if they have been left unused for a long time, can change shape (e.g. shrink) and begin to leak over time. Often the amount of leakage is very small (ul/min), but depending on the method, a small change may result in a significant change to the chromatography.

I reviewed the client's method parameters and concluded that the method met good chromatography fundamentals. Checking the flow rate (using a graduated cylinder) confirmed the flow rate was accurately shown. A review of their mobile phase preparation procedures and methods also appeared OK. Degassing of mobile phase and column temperature were also satisfactory. 

As I looked more closely at the two running HPLC instruments they used, I began to quickly zero in on the most likely problem. 

• A long stream of air bubbles were observed exiting the HPLC pump's gradient valve leading into the high pressure pump head, but no air bubbles were seen exiting the degasser's outlet line (IOW: The vacuum degasser may or may not be the cause, though it is critical to insure the degasser is clean and fully serviced before use. Have the degasser professionally serviced first before proceeding with troubleshooting. Using a damaged degasser will make it difficult to use the pump or run any valid tests as degassed solution is needed). This was observed on several of their HPLC systems, including the two used for this method. The fittings connecting the lines from the degasser module to the valve were correctly connected (as a loose connection would cause air to leak in and must be quickly ruled out). 

The cause was from one or more of the unused gradient valve positions leaking air into the flow path, changing the mobile phase composition. Of four possible mobile phase lines available (A,B,C,D), the client only had two lines connected to mobile phase bottles (A,B) with the remaining two lines left open to the air. The internal valve seals in the unused 'C' and 'D' valve positions had deformed, shrinking in size, sticking,leaking, allowing air to flow into the mobile phase on one of the channels. This resulted in a change of the organic composition % used in the method (due to a cross-flow leak), changing the peak retention times (as the actual mobile phase composition used in their gradient was different). I directed the HPLC pump's outlet line to waste, placed all of the solvent pickup bottle lines (A,B,C,D) in a beaker filled with IPA and allowed the pump to run pure IPA at 1 mL/min across each channel, one-at-a-time (100%), for ~ 20 minutes to re-hydrate the internal gradient valve seals. This was repeated with each valve position, then all of the lines were placed in fresh mobile phase solution, primed and flushed. The system was restarted and the method now ran showing the expected peak retention times. Instructions were provided which included regularly using all of the channels and valve positions plus flushing weekly to maintain valve operation. Use ALL of the lines and flush the valve(s) through all positions, one-at-a-time, on a regular basis. If prolonged flushing with pure IPA does not fix the leak, then it is time to replace the valve. All valves eventually wear out and must be included in maintenance inspections and checks. This is especially true when you purchase your HPLC system at an auction or from an 'equipment' reseller. Never assume that the 10+ year old HPLC valve is OK. Test it first (e.g. Acetone tracer test).

 

Acetone Tracer Test: If you suspect that a cross-flow leak exists on a gradient valve, then one method I use to check for leakage is to mix up a "Tracer" solution of pure organic (often ACN) that has 1% Acetone mixed in (for RP methods). Remove the column and replace with a restriction capillary. Place the tracer solution on the valve position you suspect may be leaking at an appropriate flow rate and set it for 0%. Run one of the other channels with 100% (pure ACN in this example) and monitor the UV (265nm) for the presence of acetone. If the acetone leaks into the channel you are using, it will be easy to observe on the UV trace. So called "bubble" tests (introducing and monitoring the position of a gas bubble into the low pressure solvent line) are not reliable leak detection methods for small leaks. Use a tracer such as acetone to find the leaking channel(s). You can read more about these types of Valve Leakage tests in this article (Click Here).

HPLC to UHPLC Conversion Notes (Gradient Time Program Adjustment)

In an earlier article we discussed how to adjust the flow rate, injection volume and column dimensions when scaling an HPLC method UP or Down. The formula's needed to do this are fairly simple. If we adjust for changes in the column dimensions or flow rate, what types of changes are needed to adjust the gradient time? The formula to make this adjustment is also very simple. Here is the information you need.

Terms Used in Formula:

Time in minutes, Gradient (Initial): Tg1

Time in minutes, Gradient (New):   Tg2

Flow Rate in mL/min, Column (Initial): Fc1

Flow Rate in mL/min, Column (New): Fc2

Column Diameter, mm (Initial): Dc1

Column Diameter, mm (New): Dc2

Column Length, mm (Initial): Lc1

Column Length, mm (New): Lc2

• Tg2 = Tg1 x (Fc1/Fc2) x ((Dc2²) / (Dc1²)) x (Lc2 x Lc1)

Here is an example problem to solve for. 

If we start with a flow rate of 1.000 mL/min (Fc1) on a 4.6 x 250 mm column with 5 micron support (Dc1 & Lc1) and have an initial Gradient Time of 10 minutes (Tg1), then what would the new gradient time be if we switched to a sub 2 micron support in a 2.1 x 50 mm column (Dc2 & Lc2) at 0.200 mL/min (Fc2)? 

To solve the equation we will plug-in the values for each part of the equation separately, then multiply them to obtain the result.

  (Fc1/Fc2):    1.000/0.200 = 5

  (Dc2²) / (Dc1²)  4.41/21.16 = 0.21

   (Lc2 x Lc1) = 50/250 = 0.20

  Tg2 = 10 x 5 x 0.21 x 0.20 

  Tg2 = 2.10 (or 2.10 minutes)

If a 2.1 x 50 mm column was substituted for the 4.6 x 250 mm AND the flow rate was changed from 1.000 mL/min to 0.200 mL/min, then the initial programmed gradient time of 10 minutes would be changed to 2.1 minutes

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.

Gradient Mixing Test For Your HPLC Pump (Step Gradient)

The most popular type of gradient pumping module used to perform HPLC analysis utilizes a low pressure mixing valve in their design. These valves are electronically controlled and proportion the amount of mobile phase from one of several solvent channels into a mixer for introduction to the pump head (*the solenoid valves used for this are sometimes called gradient proportioning valves). They provide random access to multiple solvents (e.g. 4) for method development and column flushing. The mobile phase solutions are mixed at low pressure before entering the high pressure side of the pump head (where they undergo compression). This design requires only one high pressure pumping head and can allow for very high mixing accuracy (often 0.1% per channel) of the mobile phase. This allows for the formation of mobile phase gradients over time which greatly aid in resolving samples apart on the column.

The gradient proportioning valves need to be tested along with the other parts of your HPLC system on a regular basis to insure they are operating within the manufacturer's specifications. They should also be tested anytime you suspect a problem may be present. One quick way to check the operation of two of the valves is to use a tracer compound and STEP gradient to monitor their operation. You can set up a method to perform this test as suggested below.

QUICK GRADIENT COMPOSITION TEST:

Bottle A = 100% DH20;
Bottle B = 0.1 % Acetone in DH20 (*Acetone is the tracer compound);

Flow Rate = 1.000 ml/min;
Column = No column. Install a restriction capillary in place of the column to obtain a backpressure of > 60 Bars;

Detection = 265nm (10 nm bandwidth) UV;

STEP Gradient Program:
    0 to 2.00 min, 0 % B
    2.01 min, 20% B
    4.01 min, 40% B
    6.01 min, 60% B
    8.01 min, 80% B
  10.01 min 100 % B
  12.01 min 20% B
  14.00 min 20% B

Note: If the delay volume (dwell volume) of your system is large, then you may want to adjust the time values shown to LARGER values (i.e. 2 minutes delays are used in this example, but 5 or even 10 minute delays between steps may be more appropriate if your system has > 1 ml dwell volume.

Running the above method should result in a signal trace which shows a step-wise rise to 12.00 minutes (as the acetone concentration increases). The edges of the "steps" should be sharp and the risers should also be close to vertical. The final step change which starts at 10.01 minutes shows a linear gradient change back down to the 20% B level. This line should not have any bumps or dips in it and should transition smoothly back down. The height of the baseline at this point should match the height seen between 2.01 and 4.00 minutes (same 20% B). The height of the proportional steps (e.g. 20, 40, 60, 80) should also be the same. You can use your CDS to measure these height values.

Another useful aspect to view is the S/N ratio at each step. Use your CDS to establish noise windows within each range (e.g. 2.50 to 3.5 minutes). This data is useful when comparing the performance of the pump at different intervals.

If you observe deviations in the height of the proportional steps or dips in the lines, these can be caused by leaking or sticking check valves as well as leaking or sticking gradient proportioning valves. *If you have a quaternary pump, be sure and test all four of the valves used (2x per test).

Lastly, the above example is a generalized method and may or may not be applicable to your specific HPLC pump. Be sure and customize a test method which takes into account the pressure ranges, flow rates, delay volume, mixing volume, and number of low pressure channels used in your pump.