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).

Vespel, Tefzel or PEEK Valve Rotor Seals ?

One of the most common HPLC preventative maintenance parts is the injector valve rotor seal. Worldwide, the majority of these chromatography parts are produced by Rheodyne (IDEX) or Valco Instruments (VICI) and used by the major instrument manufacturers in their products. These valve seals are critical to maintaining a leak free, high pressure seal inside the injector. They are subjected to a lot of wear and chemical exposure with use. They have a finite lifetime which may be very short, in some applications (a few months) or last for many years in others. *The most common reason for rotor seal damage is a lack of flushing when buffers are used. Regular flushing down of the flow path (without the buffer) is required to maintain a clean flow path. Buffer deposits and crystals scratch and damage the rotor surfaces. Depending on the specific use and application, rotor seals are often replaced as a preventative measure once every 6 or 12 months. They should also be replaced whenever they are scratched, heavily worn, no longer sealing well, leak or become contaminated. Failure to maintain your injector's parts may lead to HPLC carry-over contamination problems.

The choice of rotor seal material should be based on: (1) the types of chemicals it will come into contact with; (2) the working pH range; (3) the temperature range.

• Note: When choosing a valve rotor seal material, please refer to the valve manufacturer's information, compatibility and advice. Blends and properties may vary between vendors so always verify compatibility with them before use.

 
Common HPLC Rotor Seal Material Types:

 Vespel ^®: Chemically, this is a polyimide blend (DuPont). One of the most widely used materials for HPLC valve rotor seals. It has excellent chemical compatibility with most HPLC mobile phases, excellent temperature stability and a pH limit of 10. An excellent choice for most applications.

Tefzel ^®: Chemical name, ethylene-tetrafluoroethylene (aka, "ETFE"). It has excellent chemical compatibility with most HPLC mobile phases and a higher pH limit of 14. Tefzel's preferred applications are where very high (>9) or very low (<3) pH solutions or mobile phases are used. Not compatible with some chlorinated solvents and in most forms it has a temperature limit of 50°C.

PEEK: Chemical name, polyether-ether ketone. Known for applications where biocompatibility and / or high temperatures are of concern. Like Tefzel, it has excellent chemical compatibility at room temperature with most HPLC mobile phases. Most vendors report a working pH range between 1 and 14. Unlike Tefzel, it has a much higher temperature limit (i.e. 100°C or higher), but its resistance to some chemicals appear to degrade with increasing temperature. Contraindicated where it will come into contact with solutions of THF, methylene chloride, DMSO or concentrated acids (i.e. nitric or sulfuric). Some sources have observed problems with chloroform as well, especially with PEEK tubing, so it may not be recommended for those applications.

 

For more information on troubleshooting HPLC injector valves, please refer to this linked article, "Troubleshooting HPLC Injectors (Manual and Automated)". 

Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems

"Carry-over" is a term used to describe a type of sample contamination which causes sample peaks to re-appear in later runs which do not actually contain the sample (e.g. blank runs). The contamination can last for several sequential runs, often decreasing in amount after each injection (which is a key observation when troubleshooting). When proper instrument training has been provided, modern HPLC system designs make carryover extremely rare, but when it does appear, the contamination can be due to: (1) A lack of HPLC maintenance; (2) Overloading samples which foul the column; (3) Poor Wash Vial Usage and/or Sample Vial Selection; (4) Inadequate operator training in how to set-up and use the chromatography system. *Note: Proper operator training greatly reduces the chances of contamination and is the most overlooked reason for the problem.

The Types of HPLC Carry-Over Contamination; Why They Occur and How To Reduce Them:

(1) A Lack of HPLC Maintenance: Most auto-injector valves rely on a rotary seal to move the sample from the needle loop to the flow path of the system. The components within these valves wear out and should be inspected at least every 6 months and replaced when needed. Also, always check the needle seat and needle for signs of wear or leaking. Note: Look for signs of leaks by the injector. Leaks always indicate a problem and should be fixed immediately. Don't run samples when you have leaks. Your method and data obtained will be invalid. Any worn parts should be replaced and the system performance tested. One of the most common causes of carry-over is due to a worn sample injector valve rotary seal. A worn seal can allow sample to be retained in the worn grooves, in-between injections, resulting in sample peaks appearing in subsequent runs. Additionally, buffer salts can lodge between the seals causing leaks or carryover. Routine HPLC service and, if applicable, flushing of all buffers/salts every day can eliminate these issues.

(2) Column Fouling / Overloading of Sample: If you inject too high a concentration of sample and overload your column with material, then it is possible that your column will continue to bleed sample long after the analysis is over. This also happens when the sample has a high affinity for the support you have chosen too. Failure to regularly flush and clean your HPLC column on a regular basis can also result in a similar problem as retained material is released from the column over time. Avoid this problem by performing a loading study to determine how much material can be effectively loaded on to the column. Next, create a wash method which utilizes a stronger solvent than your method (often utilizing a gradient) which will wash away any strongly retained material in between runs. This is critical if you are running an isocratic method as material will be retained on the column and must be washed off at frequent intervals using a stronger wash solution. *When using only isocratic methods, people often do not initially observe carry-over problems (because the sample just sticks to the column and accumulates over time). When the solvent strength is changed or the method is revised to a gradient, then the problems start... Avoid the problem by selecting the right column (which retains, then elutes ALL of the sample), not overloading the column (do a loading study) and washing the column down with a stronger solution that fully dissolves (not precipitates out) any remaining material off the column after each run.

(3) Wash Vial Usage and/or Sample Vial Selection: If you are using a modern high-pressure, "Flow-Through" design autoinjector (e.g. Agilent 1100, 1200, 1260, 1290), then carryover is rarely an issue as these modern injectors use a high pressure pump to aspirate and inject the samples directly into the flow path, reducing the need for any wash stage. The entire HPLC's injection flow path is continuously washed with mobile phase during the analysis run. This dramatically reduces the chances of any sample re-appearing in later runs. The need for a separate wash vial is nearly eliminated in this way as the needle, needle seat, loop, injector pump and valve are all flushed clean during each method. Many older auto-injector designs utilize either a low pressure injector (glass syringe) or injector pump which is not part of the main flow path. These injectors benefit from a separate wash vial as they are not continuously cleaned. Effective cleaning requires that a wash vial be employed in these cases. It should be filled with mobile phase or a solution which will dissolve any remaining material which might still be in the system.

When sticky sample solutions are used, separate Wash Vials can be used to reduce contamination with either older or newer injector designs . Sometimes these sticky samples can adhere to the outside of the needle while it is being withdrawn from a vial which has a septa which has been punctured many times. High puncture rates tend to open up the hole resulting in a lack of "wiping' of the needle surface upon withdrawal. *For vials that are punctured many times, it is critical to replace the septa OR use septa materials which seal for a long enough time frame to reduce this effect. Septa needle wiping eliminates some of this contamination. Two types of contamination can occur from this problem. (a) When the needle is dipped into a vial (same or different one) which also has a large septa opening, it can carry some of the sample with it and deposit it into the new vial (or onto the septa of the vial). (b) The contamination can also run down the needle itself and drip onto the needle seat at the time of injection resulting in contamination of the seat or sample.

One of the easiest solutions to reduce external needle contamination involves incorporating a wash vial which contains a solution which is optimized to quickly dissolve the sample into solution. This sounds simple, but many chromatographer's choose wash solutions which do not enhance the cleaning aspect of the needle at all. For example: Mobile phase, which is normally ideal, but does not work in some cases. Samples such as peptides, proteins, fats, oils and/or lipids can be troublesome as their solubility can be at odds with the mobile phase chosen. For the wash vial to be effective, it must quickly dissolve the material. The needle can be first "dunked" (dipped) into one vial containing the solution and withdrawn, followed by an aspiration and wash in a second wash vial. If needed, you take this cleaning one step further and use additional aspiration steps to serially dilute any remaining material. These wash vials must be changed frequently (easily done by having several wash vial positions programmed in the system). Additionally, the caps should be left OFF the wash vials to reduce pickup contamination each time they are used (this step is critical).

Lastly, if you are analyzing sticky materials which are known to interact with metals found in chromatography systems, consider using a system which incorporates bio-compatible materials such as titanium, tantalum and/or polymers such as PEAK. You can also utilize plastic sample vials (e.g. PP) or plastic vial inserts too.

(4) Inadequate Operator Training: Good chromatography requires a complete understanding of the hardware used and the fundamentals of HPLC. You must be able to troubleshoot the complete flow path of the system and understand the concepts of chromatography as used in method development. This is not a technique best learned by trial and error, but rather through mentoring using logical steps. Depending on your skill set, troubleshooting a "carry-over" problem in an HPLC system can take minutes to months to diagnose and solve. We learn these skills through hands-on experience and training. Reading many of the better books and articles on the subject matter helps too. Get as much practical hands-on training as you can. Ask your supervisor or manager(s) to invest in you by purchasing professional training for you in this field so you can learn on your own systems. You will learn far faster this way and spend less time troubleshooting problems and more time running samples, accurately in less overall time. Training also costs just a fraction of what the instrumentation and your salary are. If you have acquired the fundamental skills, a skilled teacher can impart about one years worth of practical knowledge to you in as little as one week of training.

Summary: The two most common reasons for sample carry-over contamination in an HPLC or LC/MS system are due to: lack of operator training and/or lack of system maintenance (most commonly manifested as a worn injector rotor seal).

 Note: This article specifically addresses carry-over contamination as it relates to the most commonly used HPLC, UHPLC and LC-MS autoinjectors (or autosampler modules).

You may wish to read a related article on "Troubleshooting HPLC Injectors (Manual and Automated)" found at this link: http://hplctips.blogspot.com/2013/06/troubleshooting-hplc-injectors-manual.html

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.

Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)

Few things in chromatography are more frustrating than dealing with large pressure fluctuations (>1% ripple). If the pump pressure is unstable, and fluctuating up and down, then it will negatively impact your ability to analyze, measure and integrate sample peaks in a reliable manner. A smooth, flat baseline is needed to run and develop methods, collect the data (peaks), integrate and report the results which are reproducible. Baseline instability during an analysis may lead to the entire analysis being declared invalid.

So what causes the HPLC pressure to sometimes fluctuate in a wild manner up and down on your HPLC system? Unfortunately, many things... Most result from poor training, incorrect operation techniques, but some are maintenance related so be sure and keep your chromatograph in excellent condition. Maintain a logbook for each instrument and record what types of maintenance and service have been performed over-time, with the date and list of parts used/replaced. Additionally, maintain a preventative maintenance schedule (e.g. every six months) to inspect and clean the entire HPLC system to check condition, verify operation and minimize unproductive down time. 

HPLC Pump or System Pressure Fluctuation Causes and Solutions:

• Air / gas In the Liquid or Mobile phase (Failure to Degas Mobile phase OR loose fittings) --- Air gets into the system due to a leak or from gas trapped in the mobile phase. Find and correct the cause of the leak and/or degas the mobile phase (use continuous Vacuum degassing or a Helium sparging system only). Leaks are the most common cause of instability, but insufficiently degassed solution is a close second. Make sure your degasser is working 100% correctly (they require professional servicing every 5 years). HPLC pumps require degassed mobile phase for reliable operation.

• Loss of Prime. Improper Priming of the System --- Failure to flush ALL of the lines with freshly degassed mobile phase, before use (every day), will often result in all kinds of instability problems until all of the old gas-filled mobile phase has bee purged from the system. *This could take many column volumes of liquid. Make sure you account for any vacuum chamber volume too. Properly prime the pump heads before use.

• Sticking Check Valve(s) --- If air is exiting the pump outlet, the pump will not function properly. Both Inlet and Outlet valves should be inspected. Remove and clean the check valve(s). Be sure the pump is fully primed with liquid as the check valve might just have an air bubble in it (common on Waters, Thermo and Shimadzu systems). Sometimes sonication of the valve for ten minutes in a beaker containing warm solvent does the trick (e.g. MeOH or IPA/Water). Though very rare, ACN has a bad reputation for polymerizing in solution. If the system has sat unused for a long time OR was not properly flushed out when last used, it is possible that particulate matter may clog the flow path. Small sticky particles may form (ACN polymerization) and cause the check valve to stick inside the housing (use fresh, filtered solvents only to prevent these problems). Clean and inspect any suspect valve first. Replacement of the check valve may be needed in some cases to restore operation. Note, this problem of "sticking" check valves is most likely to be an issue in HPLC pumps with mechanical (gravity or spring) check valves with ruby balls, not modern style active inlet check valves ("AIV") which are electromechanical (solenoid valves) and are very reliable, much less susceptible to these problems. In any case, verify operation of all valves while under pressure (backpressure is needed for them to function correctly).
  

• Worn Pump Piston Seals --- Commonly observed as rapid up/down spiking on all channels and an inability to maintain or produce backpressure (the pump will often prime with no problem, as this is done at low-pressure). Run a formal pump high-pressure leak test at max pressure to confirm (remove the column and replace with a calibrated backpressure restriction line for all testing). Clean pistons and replace piston seals to repair (you should have spare pistons and seals on hand). *Seals are a maintenance item so expect them to wear out and need regular replacement.

• Flow rate too low (may be inappropriate for system). Running at a flow rate that is below the optimum range of the specific instrument (i.e. System rated for 200 to 2,000 uL/min, but run at 100 to 200 uL/min or at the limit of the range) may result in an unstable baseline. The cause may be due to pump cavitation, loss of prime, non-optimized piston stroke volume.

• HPLC System Back-pressure too low to maintain prime in system. Most types of analytical HPLC systems require a minimum system back-pressure of 40 or more bars to maintain enough pressure (mechanical compression) on the component parts to run in a reliable fashion (*Water's Article number: 32564 states the back-pressure must be at least 1000 psi for their Alliance systems). Too low a pressure often results in a loss of prime, cavitation, mixing problems, turbulence and poor reproducibility. Correct sizing of column, particle size, flow rate and mobile phase composition should all take into account achieving enough back-pressure on the system to maintain a stable baseline throughout the entire analysis. Monitor the system back-pressure at all times for stability. High quality research grade HPLC systems are often capable of maintaining stable isocratic flows with less than 1% ripple and 0.2% ripple common ("ripple" is a term we often use to describe the pump's pressure output over time relative to the baseline (S/N)).

• Mixing Problem (gradient or isocratic online mixing) --- If your active mixer or proportioning valve (AKA: Gradient valve) is defective or dirty, then one or more of your mobile phase channels may not be getting to the pump. Air would most likely be mixing with the mobile phase causing the unstable flow. Clean or replace the valve. Note: Always try flushing the gradient valve with pure IPA, then DH20 for about twenty minutes. This sometimes restores operation by wetting and flushing the internal seals (which may dry out).

• Wrong Pump Solvent Compressibility Settings --- In HPLC we routinely subject different liquids to very high pressures which can result in measurable liquid compression. The degree of actual compression for each liquid varies, but the modern HPLC pump can compensate for this to improve the accuracy of the mixing and flow delivery.  Most pumps provide for user adjustable solvent compressibility values. If the value input varies a great deal from the actual liquid in the system, then it can result in pressure fluctuations. Example: Water has a value of 46, but Methanol 120. Using the wrong value can cause instability.  

• Poor Solubility, Mobile Phase  --- Sometimes the mobile phase which has been prepared (or mixed online) is not 100% soluble. This could be due to an inorganic salt additive which has not gone into solution or failure to fully mix and filter the mobile phase before use. Ultrasonication, a bit of heat and stirring for 20 minutes can help to get everything dissolved. 

• Dirty inline filter --- A fouled or partially plugged filter can disrupt the normally smooth flow into a turbulent one. Some are installed as part of the pump (i.e. HP/Agilent brand pumps) and should be changed out every month (Yes, for the PTFE frit, replace it once a month with a new one). Other systems use these pre-filters downstream of the pump before the injector. Clean or replace all filters frequently. If used in your system, these are regular maintenance items and should be part of a general 'PM' program.

• Dirty Solvent Pickup Inlet Filters: These can become obstructed or fouled over time (esp. if used with aqueous solutions!). Just as with any built-in filter, the multiple solvent inlet pickup filters should be cleaned or replaced on a regular basis to prevent particulate or any material which may contaminate or restrict the flow path from entering the system. Mobile phase pickup filters are often 10 to 20 um and connect to the bottom of the low pressure (e.g. Teflon) solvent lines in each bottle. If you use 316 Stainless steel filter (recommended for organic solvents), they should be removed, cleaned in an ultrasonic bath, rinsed and replaced monthly. If you use sintered glass or other disposable type filters (often used with aqueous solutions), they should be disposed of on a regular basis and replaced with new ones (replacement, not cleaning is recommended because sintered glass can not be sonicated and should be disposed of to prevent bacterial, mold or fungal contamination). A quick way to check if one filter is causing the pressure to fluctuate is to remove the filter from the one line, then re-test the system. If the problem goes away, then returns when you re-install the filter back on the line, the filter may be obstructed (replace it),

The above list includes some of the most common reasons for unstable baselines. Other non-pump related causes would include a bad / old detector lamp(s) or contaminated mobile phase. To find the cause, test and verify the operation of each component part of the HPLC. Troubleshooting Advice: Test one part at-a-time, before moving to the next part. Never assume anything, test, re-test and verify or prove at each step.

Method Development Hint: Use your HPLC Diode Array Detector (DAD or PDA) as a Spectrophotometer

One of the many useful features of a UV/VIS scanning diode array detector is that it can be employed in flow injection mode to scan a sample and provide you with some useful data about the absorbance characteristics of the sample (which probably contains a mixture of components). Unlike a spectrophotometer, you only need about 1 ul of sample instead of a 1ml cuvette and only 15-20 seconds of time to gather the data.

Why do this? I use this feature often when I receive a new and unfamiliar sample for method development. I set up the detector to scan and store all wavelengths, in steps of 2nm, from 210nm to 450nm and inject the sample in flow injection mode (that means no-column is present and I easily do this using the By-Pass position on my column selector). In a very short amount of time I can view the resulting spectra of the sample which aids me in selecting the initial discreet wavelengths to monitor. For example: If I notice that the sample shows some absorbance at 410nm using the flow injection run, then notice while developing the analysis method that none of the peaks seen show absorbance near 410nm, then I can assume that I may still have some components retained on the column.

Setup Hints:
(1) For this to work well, you should have a high performance, low volume switching valve or automated column selection system (e.g. The LC Spiderling Column Selection System) installed so you can easily by-pass your column (otherwise, remove your column and place a high pressure, low volume union in its place).
(2) Set the diode array detector to a high sampling rate because the sample is going to fly through the flow cell quickly. Use a sampling rate that is faster than you would use if a column was there to disperse the sample and slow down the peaks.
(3) Choose a wide range of wavelengths to scan and store. If the sample appears colorless to the eye in solution and I am running in a UV transparent solvent such as acetonitrile, then I often use a range of 210 to 450nm.

Troubleshooting HPLC Injectors (Manual and Automated)

Sample injectors are a critical component of a chromatography system. Understanding how they operate as well as the proper techniques to use and maintain them are fundamental skills needed to operated an HPLC system. Lets briefly discuss some of these fundamentals as applied to a standard manually operated HPLC injection valve and also in an automated mode as found in an autosampler. *Note: You should always refer to the specific manufacturer's product specifications, operation, servicing documentation or support personnel before servicing any injector.

MANUAL INJECTION VALVE Notes:

These valves allow you to use a high precision syringe to manually fill a fitted "loop" with a sample and then, by turning a valve handle, introduce the sample to the high pressure flow stream directed toward the column inlet. Sample loops are available in a wide range of volumes and take just minutes to install. The injector valve has very tiny openings inside which are moved between two different positions (LOAD and INJECT). The LOAD positions allow the valve to seal off the internal high pressure flow from the loop to allow it to be safely filled with sample at atmospheric pressure. The INJECT position introduces the liquid contained inside the loop to the main flow path (under high pressure). The parts must be clean and seal well to insure proper function. Leaks from all areas (except the vent) are not acceptable and indicate a problem. Here are a few tips regarding the use of manual injectors in HPLC.

• Use the correct type of sample syringe. Usually these are high precision glass syringes with Teflon plungers. The needle tip style is the most critical item! Most injectors are designed to only work with a needle which has a squared off tip (NOT a point as is commonly used in GC!). The most common gauge used is #22. Always check with the valve manufacturer to determine the correct style and gauge of needle before use.
• Leave the valve in the INJECT position during the entire run to flush it clean of sample and stay equilibrated with your method. Switch it back to LOAD only when you are ready to load a new sample.
• For high reproducibility and accuracy within one HPLC system, fill the loop with at least three times the volume of the loop with sample to insure that the entire path is full of sample. This is known as the complete or over-filled loop method. *Choose your loop volume size with this in mind. Loading the same volume as the loop will often result in poor accuracy.
• Loops often do not contain the exact volume stated on them. They can be off by ~25% so consider this when injecting partial volumes and not using the standard over-filled loop method.
• Types of common leaks: (1) Leaks at the needle port (needle seal worn); (2) Leaks behind the valve stator (worn rotor seal, buffer crystals dried inside, over pressured, scratch on rotor); (3) Leaks at the vent (liquid should be expelled from the vent when filling the loop only. Other leaks indicate a problem). Note: Rotor seal damage can cause sample carry-over problems so valves should be inspected at regular intervals (~ 6 months).

AUTOMATED INJECTION VALVE (auto-injectors) Notes:

These valves use a high precision syringe or high pressure pump to fill a fitted "loop" with a sample and introduce the sample to the high pressure flow stream, all automatically. Most function exactly the same as described above, though some are based on true high performance liquid chromatography pumps so have no "syringe" at all (e.g. Agilent 1100/1200 designs) Here are a few tips regarding the use of automated injectors in HPLC.

• Regular maintenance is even more critical with auto-injectors since you often can not see what is going on during the injection cycle. Leaks, if present can be harder to find so make it a habit to visually check all of the areas around the injector regularly.
• Needle and Needle Seats are normal wear items on these instruments. As such, they require routine checking for leaks or damage and replacement when worn.
• Vial Caps: If you make multiple injections from one vial (or large volume injections) with a tightly sealed vial cap, a vacuum can form inside the vial causing volumetric errors to occur in your samples (resulting in you injecting less sample each time). Leave the caps slightly loose to avoid this problem. Multiple injections into the same vial can also cause the needle hole to become enlarge over time allowing the sample or solvent to evaporate over time, changing the concentration of the sample (more concentrated). Replace the cap and seal with a new one if used multiple times. Always leave the cap slightly loose.
• Loop volume: Autoinjectors often incorporate one large loop to handle a wide range of sample volumes. This is a trade off of accuracy for convienence. Accuracy is often poorest at the very low end of the range and best near the middle to high end. Always verify the reproducibility of the injector to inject a specific volume through statistical analysis of repetitive injections.
• Types of common leaks: (1) Leaks at the needle seat (needle seat worn); (2) Leaks behind the valve stator (worn rotor seal, buffer crystals dried inside, over pressured, scratch on rotor); (3) Leaks at the vent (liquid should be expelled from the vent when filling the loop only. Other leaks indicate a problem). Note: Rotor seal damage can cause sample carry-over problems so valves should be inspected at regular intervals (~ 6 months).

These are just a few tips related to HPLC injectors. Please consult the service documentation for your specific instrument to better understand how the system works and what areas you should be monitoring. Understanding HOW these systems operate (and can fail) is one of the most important skills you can learn as a chromatographer. Take the time to understand the complete flow path of your system before using it.