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.

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

HPLC Maintenance & Repair Parts To Have on Hand for HPLC Systems

HPLC (UHPLC) systems are complex instruments which require periodic inspection, cleaning and maintenance. These tasks are critical to maintain the performance, reliability and accuracy of the instrument. If you have not done so already, I strongly recommend that you create formal standard operating procedures (SOP's) which address: (1) The frequency of when routine and non-routine maintenance procedures should be performed; (2) The types of maintenance and/or repair procedures used (e.g. piston seal replacement, A/I rotary valve seal replacement); (3) The exact step-by-step procedure to follow in performing these tasks and (4) The Performance Verification or Qualification steps and procedures which are to be performed to verify that any repairs made have been done correctly. *An instrument log book should be employed to document these procedures over time.

Periodic "General Maintenance" of the HPLC is one type of service procedure which should be scheduled at a set frequency (Example: Every 6 months) and will serve to provide a time to clean, inspect and repair/replace any parts which are worn due to normal use. Such routine HPLC maintenance is often referred to as a basic "Preventative Maintenance" service (or "PM Service"). Spare parts common to your HPLC system(s) should be on hand to perform these scheduled maintenance procedures as part of a normal PM service.

Here is a list of common parts that should be on hand for a "typical" HPLC system used in a pharmaceutical laboratory. Please consult the appropriate manufacture's product literature to determine the correct parts needed for your own HPLC system. This list is presented as a general guideline only:

• Capillary tubing, fittings (nuts and ferrules): Assorted fittings, usually made of 316 Stainless Steel, but could be made of polymeric materials. Always have spare precut and polished chromatography tubing of appropriate I.D. and lengths for use with your HPLC available at all times. Insure that the nuts and ferrules used are appropriate for your brand of HPLC system and the columns used as different manufacturers have different specifications for their fittings and ferrules. Many types are not interchangeable.

• Detector Lamps: At least one spare bulb of a type designed for your specific detector should be on hand. Note that some detectors use multiple lamps so you may need to have more than one type available for each detector. Some lamp bulb types (e.g. tungsten) can be safely stored and last for several years while other types, such as Deuterium bulbs, loose substantial energy after as little as 6 months. If you have several detectors of the exact same design, then there is often no need to stock multiple replacement bulbs for each one. Instead, stock enough bulbs to service one detector as it is unlikely you would see failure of more than one detector on the same day (an exception to this guideline is if you perform PM services on all of the instruments at the same time, then you may want to have multiple bulbs available).

• Pump Pistons: One set of spare new pistons should be kept on hand for each pump module. As with lamp bulbs, if you have several identical pumps, then there is often no need to stock multiple sets of pistons for each one. Stock only as many as you expect to use in one year. Clean and inspect the pistons during each PM for any signs of scratches or surface abrasions. Under routine use, pistons should only require general cleaning and last a long time before replacement is required (> 1 year). Mobile phases which contain high concentrations of salt buffers often accelerate this wear requiring more frequent replacement. *Always install new piston seals when replacing pistons.

• Pump Piston Seals: At least one set of spare new piston seals should be on hand for each pump module. Seals wear out more frequently than pistons. You should go through two or more sets of piston seals before you need to replace the pistons. If the piston seals leak, inspect the pistons for wear (replace with new ones or clean and reuse) and install new piston seals. Mobile phases which contain high concentrations of salt buffers often accelerate this wear.

• Solvent Pickup Filters: These are the large particle filters which sit inside your solvent or mobile phase bottles. They are often made from stainless steel or sintered glass with porous inlets (~10 to 30 micron) and can clog or become fouled over time (esp. when used with aqueous buffers). In some cases these can be cleaned using sonication (not sintered glass filters, only steel or polymeric!). Note: Sometimes it is most cost effective to replace them with new filters then clean and re-use them.

• Inline Frits/Filters: You may have an inline filter placed after your PUMP head, but before the column inlet to collect any remaining particulate matter. These filters can extend the lifetime of the entire HPLC system (esp. the A/S, A/I and Column), but will only do so if changed on a regular basis. Some manufacturers incorporate this type of filter into the design of their pump modules. An example of this can be found on the HP/Agilent brand model 1050, 1100 and/or 1200-series pumps. These have an inexpensive 10 micron PTFE frit installed in the outlet valve of the pump. This filter catches all of the normally occurring piston seal debris and larger mobile phase particles and should be changed every month. Other pre-filters are installed in cartridges just before the column inlet. These often overlooked pre-filters filters must be replaced about once each month to do their job properly. Keep plenty of spare filters on hand.

• Auto-injector Rotary Valve Seals: If you have an auto-injector, then a high pressure valve is probably used to switch the sample into the flow path for analysis. This valve will have one or more parts which require regular inspection, cleaning and periodic replacement. Mobile phases which contain high concentrations of salt buffers often accelerate this wear. The valve rotor seal is the most common part which requires replacement.

• Auto-Sampler Needle: A needle should last a very long time, but depending on the frequency of use and type of vial septa encountered it can require replacement at regular intervals. A good general guideline would be to keep one spare needle on hand for every 2-4 systems.

• Auto-Sampler Needle Seat: The needle seat often requires more frequent replacement than the needle due to repeated mechanical wear. A good general guideline would be to keep one spare needle seat on hand for each system.

• UV/VIS Detector Flow Cell: While not actually a required PM spare part, this one is worthwhile to have. If you employ a UV/VIS flow cell, then I always suggest you keep one dedicated spare flow cell on hand which matches the size and volume of the type you use in your instrument. A spare flow cell can prove to be very valuable as a troubleshooting tool if you believe that you have contaminated or clogged your current flow cell. A quick swap can answer the question and get you back to work quickly saving hours or days of lost time. *Note: This extra flow cell should be kept separate from all instruments for use as a tested spare only and not used for regular analysis.

If you have suggestions for other types of common HPLC spares to add to the list or to have on hand, then please let me know.

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.