HPLC system care and troubleshooting is an important activity in any analytical chemistry lab. Liquid chromatography systems have become real workhorses for laboratory chemical analysis, but scientists have developed a love-hate relationship with these systems. The wide range of applications delights users, while varied complexity, robustness, and performance frustrate them. Unlike other laboratory tools, these systems are not a “plug and play” solution. There are several brands and models, but the basic operation is similar across the board. Lab technicians need some training and experience to get LC systems to perform well. This article will cover the basics of maintaining and troubleshooting your HPLC system.
All liquid chromatography systems have the same four basic components – Injectors, Pumps, Columns, and Detectors. There can be other components added before, after, or inline with these items. For example:
Mass Spectrometer (after) – essentially another detector, but there’s enough here to cover in a separate article
Most pump-related issues are due to a failure to prime the system properly. Upon startup, users should always prime the pump and verify that the flow is constant on all channels. Pump-priming issues can be detrimental to chromatography results in several ways, including no flow, erratic flow, mixing problems, incorrect gradients, bubble formation, and more.
Isocratic Pump – This is the simplest pump type. It uses a single pump to deliver a single solvent for the mobile phase.
Binary Pump – This pump uses two pumps and can mix up to two different solvents in varying proportions over the course of a run for gradient mobile phases.
Quaternary Pump – This pump uses a single pump, but has a special proportioning valve to mix up to four different solvents for complex mobile phase gradients.
What’s Wrong with my HPLC?!?!? … Troubleshooting the Chromatogram
Injector Failure – Sample is not introduced to the flow path. Check injector. Clean if it’s clogged. Repair if malfunctioning. If you’re using an autosampler, check to see if it’s aspirating sample successfully – bypass the autosampler and manually inject sample to see if it’s the problem.
Pump Failure – Mobile phase not flowing. Check/repair pump.
Column Issues – Sample is binding to the column and not eluting – change separation column and/or guard column type.
Solvent Issues – Sample is incompatible with mobile phase, or mobile phase is not compatible with column – change mobile phase solvent.
Solvent Issues (evaporation/concentration changing, atmospheric gas absorption/pH changing, leaks) – De-gas and sparge solvent with an inert gas. Seal solvent containers. Check for and repair leaks (tubing, fittings).
Gradient Issues (one solvent absorbs/retains more of the sample than other) – Change one or both mobile phase solvents. Employ baseline subtraction.
Contaminated Column – Flush column with solvent. Change column type to avoid build up and slow release.
Pressure Issues – Filter mobile phase and samples. Reduce sample or solvent viscosity. Use larger tubing.
Temperature Issues – Look for changing temperature over the run. Control temperature in the detector flow cell.
Temperature Issues – Stabilize environmental control. Check for effects from the HVAC system or other nearby equipment that throws off heat or cooling. Control temperature in the detector flow cell.
Bubbles / Mixing Issues (in the pump) – Flush and prime pump. De-gas mobile phase. Increase system volume.
Clogs (partial) – Remove clog. Replace tubing. Flush with solvent.
Pump Issues – Repair or replace the pump.
Electrical Noise – Look for and remove sources of interference with a cyclical pattern. Change circuit. Install line conditioner.
Bubbles (in the mobile phase, column, or detector) – Check for and repair leaks (tubing, fittings). De-gas mobile phase. Flush or backpressure detector cell or column to removed trapped bubbles.
Contaminated Column – Flush column with solvent. Change the column.
Electrical Noise – Check connections. Look for and remove sources of interference. Clean contacts. Shield cables. Change circuit. Install line conditioner.
Detector Issues – Flush detector cell using manufacturer’s cleaning protocol. Adjust sensitivity/gain. Replace the lamp.
Bubbles – Check for and repair leaks (tubing, fittings). De-gas mobile phase.
Electrical Wiring Issues – Check wiring, leads, and connectors. Repair or replace broken wiring or connectors. Clean connectors and leads.
Electrical Noise – Check connections. Look for and remove sources of interference with a sharp, intermittent pattern (valves, compressors, lighting). Clean contacts. Shield cables. Change the power circuit. Install line conditioner.
Detector Issues – Check and replace the lamp.
Bubbles / Mixing Issues (in the pump) – Flush and prime pump. De-gas mobile phase. Increase system volume.
Pump Issues – Repair or replace the check valve. Repair or replace the pump plunger or seals. Check for and repair leaks.
Electrical Noise – Look for and remove sources of interference with a cyclical pattern. Change circuit. Install line conditioner.
Bubbles / Mixing Issues – Check for and repair leaks (tubing, fittings). De-gas mobile phase. Flush or backpressure detector cell or column to removed trapped bubbles. Increase system volume.
Clogs (partial) – Remove clog. Replace tubing. Flush with solvent.
Electrical Noise – Check connections. Look for and remove sources of interference with an intermittent pattern. Clean contacts. Shield cables. Change circuit. Install line conditioner.
Positive and Negative Peaks or Only Negative Peaks
No Issues – Negative peaks may be normal if you’re using a refractive index detector – no changes.
Solvent Issues – Filter solvent. Change to different solvent.
Bubbles – De-gas mobile phase.
Only Negative Peaks – Change detector polarity.
Injectors transfer your sample from the source vial or plate into the LC system flow path. Once introduced, the sample flows through the column to the detector(s) and out to the waste. Once again, improper priming can be the main cause of issues relating to the injector. Any air present in the system can affect the injection volume and cleanliness. Priming the system properly removes air bubbles from the sample and wash syringes. This will improve the system reproducibility and reduce sample carryover.
Maintaining your separation column is critical for your LC system. This is the heart of the system. This component enables the separation of constituents in your sample. Problems with your column will translate directly to your results. Refer to your column’s documentation for appropriate wash and storage solvents to use. Always flush the column after runs with a compatible organic solvent.
Failure to clean your column will impact column life and performance. This is especially important if you plan to store the column for an extended period. Replace your column if the packing material has become contaminated or ineffective. Run a test standard through the system regularly to track changing column performance.
There are many detector types and models available for LC systems. But, detector maintenance is a simple procedure that’s common across types. You need to know the back pressure your detector flow cell generates at a known flow rate for a solvent. This will create a baseline for you when troubleshooting the system. You can use this value to gauge system readiness.
To do this:
Record the pressure while flushing solvent through the detector (D).
Remove the inlet line to the detector and record the pressure again while flushing (C).
Subtract C from D.
This is important because the most common issue with detectors is a clogged flow cell. If the flow cell is partially plugged, the pressure will be much greater at the detector inlet. The flow cell can rupture if completely plugged, resulting in an expensive repair. To maintain the flow cell, always flush with a clean solvent that is at least 20% organic. This will help maintain the flow cell and keep it clean and free of blockages.
Fittings & Tubing
Improper connection of tubing in fittings can also have an impact on results. A common error is to leave a void at the end of the tubing inside of the fitting. It’s important to seat the tubing flush to the end of the fitting.
LC tubing size can have a tremendous impact on chromatographic results. When replacing tubing, make sure to use the correct sizes. If the tubing internal diameter (ID) is too big it will cause peak shape issues. Peaks will become wider (and shorter with larger tubing). If the tubing is too long or too narrow, it will generate higher pressures and can also cause peak issues.
Mobile Phase (The Lifeblood of the System)
The mobile phase is the solvent that carries your sample through the HPLC system. This can be water, an aqueous buffer solution, or an organic solvent or mixture. You can use an isocratic mobile phase – a single solvent at a constant concentration. Or you can use a gradient mobile phase – two or more solvents at changing ratios over time. The majority of HPLC issues originate from mobile phase problems.
To fix these issues, follow these suggestions:
Start with fresh HPLC grade solvents. If using water, get it from an HPLC-grade purification system.
Filter the mobile phase using a 0.2-micron filter.
De-gas the solvents well before use.
Never add more solvent to open bottles; replace them when empty.
Bacteria can grow in pure water; be careful to use fresh, filtered water.
Use a 10-20% organic solvent/water mixture to inhibit bacteria growth for storage.
Maintaining Your Equipment – Shameless Plugs
Of all of the types of lab equipment, HPLC systems tend to be the most DIY-friendly for maintenance – and their owners tend to be pretty skilled as well. It comes with the territory. Not every problem is easy to solve and regular preventative maintenance service is important for continued performance. Atlantic Lab Equipment would be happy to work with you to help with your yearly PM services. Also, if you have equipment that’s no longer used, we’ll buy your surplus lab equipment. Of course, if you’re looking to expand, or get a backup 0f your favorite system, we provide high-quality refurbished equipment.
Please feel free to check out what we have to offer:
Are you considering whether to enter the world of integrated laboratory automation? Many labs begin with the end in mind and focus on the detection technology – e.g. DNA sequencing, HPLC, mass spectrometry, imaging. Lab automation is often not considered until much later. Sample preparation is frequently a manual activity. Automation is commonly seen only as an answer to sample volume issues. Yet, until it reaches a critical point, increased sample load is often addressed by adding “bodies.”
Lab space and budgets are both limited at many labs – even at some large companies. So how can you get the benefits of integration for this scenario? If your protocols need a few or smaller peripherals, you could take a workstation-based integration approach. If you need more capacity or have larger devices to integrate, you can take a distributed integration approach. For this a collaborative robot arm, like the PreciseFlex PF400, is perfect. In both approaches, you can save significant costs by working with a premium refurbished equipment provider, like Atlantic Lab Equipment. It’s not uncommon to save about half the cost of purchasing new equipment.
Why Use Integrated Laboratory Automation?
The benefits of integrated lab automation go beyond addressing high sample volumes. It’s best to think about your plan for automation early in developing your lab processes to take advantage of what lab automation has to offer:
Improved process control – repeatability, consistency, error reduction
Enhanced sample and material tracking – error reduction, and helpful for troubleshooting
Increased lab safety – less repetitive stress injuries
Better resource utilization – deploy scientists for value-added activities instead of tedious tasks
Adding personnel might seem like the path of least resistance to handle increased sample load or extra methodologies. But there are significant costs associated with “manual automation.” Besides the direct costs of salary, benefits, and training, laboratory space costs can be an important factor.
In biotech hot spots, like Cambridge, MA, lab rent can approach $80 per square foot per month. The typical lab technician is going to occupy about 20-30 linear feet of bench space. This is about 90-150 square feet, which translates to about $7-$11k/month for the lab space. Don’t forget to add taxes, insurance, and maintenance to that triple net lease, and you’re now over $100/sf. Of course, other places are less expensive – you might only be paying $15-$20/sf in RTP for lab space.
A common lab scenario involves adding instrumentation and automation in a piece-wise approach. Add a fluorometer for plate reading; add a centrifuge for spin-downs; add a washer for, well, washing; add a liquid handler for pipetting; and so on. Each of these instruments operates on their own, connected by “sneaker net.” The lab personnel moves the material from one piece of equipment to another. This is non-integrated automation … YOU are the automation!
When we start to connect each of the components needed to perform a complete protocol, at least physically, and perhaps also informatically, we have integrated automation. The liquid handling system is usually at the center of this type of setup. It’s performing the lion’s share of the work. The other devices are “peripherals.”
The two ways to provide integrated automation for lab protocols are:
Workstation-based integrated automation: The liquid handling system is the “hub” that provides the pipetting and the plate movement. Peripherals integrate directly to the liquid handling robot.
Distributed integrated automation: A separate robot arm is the “hub” and provides the connection between the liquid handling system and the other peripherals.
The most cost-effective and space-efficient solution handles the task with the least hardware. Liquid handling workstations are powerful pieces of equipment for the lab. Complex protocols can be performed on many samples in a tight space. Do you have a limited number of protocols that can use a couple of smaller peripherals? Then a workstation-centered integration approach may be your best option.
Early liquid handlers only focused on pipetting. Other operations happened off the robot. Modern liquid handling robots offer much more capability for integrated applications. Of course, liquid handlers can perform pipetting tasks like sample transfers, reagent additions, dilutions, cherry picking, and mixing. But, the ability to mount multiple arms on the workstation enables integrated automation. This allows you to have (depending on the manufacturer):
an 8-tip arm for reformatting between different types of labware, or individualized operations like normalization or liquid level sensing for varying sample volumes,
a 96- or 384- channel head for plate-based operations like reagent additions or mother-daughter transfers, and
a plate manipulator arm for moving plates around the deck and to/from peripheral devices.
That last point is the kicker – a plate manipulator is the key to integration. If your protocols only involve liquid transfers, you may not need any integration. But most protocols need some other devices. Common peripherals include thermal cyclers, shakers, heat blocks, incubators, plate washers, plate readers, magnetic bead plates, or storage.
A plate manipulator can take care of this. This arm includes a gripper to grasp the microplate to move it around the deck. Some arms can also rotate and use a cantilever (sideways) gripping configuration. The Tecan RoMa arm and Hamilton iSWAP arm are two good examples. Other plate manipulators are overhead (vertical) and grab the plate from the top. These manipulators are typically attached to the 96- or 384-tip heads.
Vertical plate manipulators are best when plates only need to move within the main deck area. They can access some peripherals, but only ones that provide plenty of room in the “landing area” for the plate. These devices need to be within the robot envelope, taking up valuable deck space.
Cantilevered plate manipulators are the most flexible. They can access peripherals with tighter entry points; reach outside of the main envelope, which saves deck space; and rotate to access peripherals in different orientations. And, some can reach below the deck to access peripherals under the robot or handle tubes instead of plates.
What does the typical vision of a distributed system conjure up? Do we see big, industrial-looking robot arms with an array of instruments circled around a big table behind a tough safety enclosure? This is one way to go, but this is an article about integrating on a budget and in small spaces. How can we talk about distributed automation?
Enter the collaborative robot (a.k.a. cobot). A collaborative robot is a robot arm that is so safe that it needs no safety enclosure. Lab personnel and the robot arm can occupy the same space without risk of injury. This drastically reduces the space needed to operate. These robots can be placed next to the liquid handler and the peripherals to create a complete distributed system on a bench top.
The leader in this category is Precise Automation with the PreciseFlex PF400 sample handling robot. The PF400 is a 4-axis SCARA robot that can live on the bench top. It marshals plates, tip boxes, reagent troughs and other consumables between devices. The arm has a reach of almost 2 ft, a z-height of 16”, and a rotation of 347°. With this, the PF400 can easily access the liquid handler, external storage, and a host of additional devices. The extended reach version can access up about 30”, and an extended height version extended the z-direction to almost 30”. This provides an extensive working volume in a small footprint.
How can adding a robot arm help you to keep costs down and minimize space usage?
Smaller, simpler liquid handler – since the liquid handler has fewer responsibilities, you can use a smaller workstation. For example, in the workstation-based approach, you may need a Tecan Freedom EVO 150 or 200 with 2 or 3 arms (one of them a RoMa arm). With a cobot-based approach, you may only need a Tecan Freedom Evo 100 or 150 with 1 or 2 pipetting arms.
Multi-tasking – with the extra envelope capacity of the cobot, you may be able to configure one system for multiple protocols, using different methodologies. One liquid handler (usually the bigger investment) and many peripherals. Where with the workstation-based approach you are limited to a narrow range of protocols for the limited peripherals. Automating other, different protocols would require a second workstation-based configuration.
Workstation-based approach – double the cost, double the space.
Cobot-based approach – less than double the cost (the cost of the extra peripherals), and about the same space (think vertical stacking).
There are two methods to control and coordinate the activities of the robot and devices:
Scheduling software, like Genera from RetiSoft, will manage the protocols and help to determine the material flow. In addition, the software can interface with your LIMS system for advanced sample tracking. This software is tightly coupled with the PreciseFlex robots and has a long design history.
Direct programming can also be used to statically schedule the order of operations between devices. The PreciseFlex robots include an embedded programming environment that is very similar to Visual Basic. This is a good low-cost option for one or two instrument protocols that do not require dynamic scheduling.
Your liquid handler is a marvel of modern engineering. These helpful systems can relieve lab employees of most sample processing tasks. Benefits include increased productivity, fewer errors, reduced injuries, and less boredom. This all leads to lower turnover for lab personnel and reduced costs for the lab. These improvements can be so significant that these robots become the focus of the lab. The love is so strong that many labs even give their liquid handlers names and personalities! (At one company, liquid handling systems were named after the divas – Whitney, Celine, Christina, and Brittney! At another, names were Lord of the Rings themed.)
We appreciate these machines for the work and support they offer our laboratories. But, made of metal and plastic, it’s easy to forget they need proper care, much like their human counterparts. We see the same story time after time:
a new robot enters the lab
production ramps up due to the increased capacity
weeks turn into months and everyone is happy
then … the liquid handler performance drops
What can you do to set up your system for success?
Loss of pipetting performance (lower accuracy and precision) is bad enough. But in extreme cases, performance issues can completely shut down a lab. You can avoid this terrible situation. Nobody wants to be “down.” Regular preventative care (by you) and annual service (by the pros) will keep your robot in great shape. Proper care and feeding boil down to a few common-sense tactics:
Establish a maintenance plan (and follow it)
Keep your system clean (inside and out)
Make sure your connections are air-tight
Use the correct / high-quality materials – liquids / reagents, consumables, and parts
Have an annual preventative maintenance service
Care of the liquid system – the heart of the liquid handler
Some very advanced technology goes into modern lab automation systems, including:
Precision motion control
Air and liquid-based dispensing
Liquid level sensing
Device integration and communications
Although, a simple view of a liquid handler is: pumps, moved by motors, connected by tubing to pipette tips. Each of these basic elements has their own potential for failure. Knowledge of how they function will help us prevent problems and keep the system running. Most liquid handling systems have at least two sets of pumps: a fast pump and syringes.
The large volume, fast wash pump
The fast pump also called a wash pump, is for moving large amounts of liquid. The fast pump can be a piston pump, diaphragm pump, or peristaltic pump. The fast/wash pump generally supplies water for washing the system out. These pumps can also supply buffer or other liquids for filling large volumes. This happens with lower precision than syringe pumps, but much faster.
To pass the original manufacturer’s testing, the fast pump must be able to meet a specific flow rate. This is a large volume in a set amount of time; 1600 µL per second for example. In most cases, there is a large margin of safety in this specification. A good pump and tubing should have no problem moving 150% of this required rate. If the source lines become clogged, worn, or leaky, the flow rate can plummet. You can flush the tubing lines with distilled water, and even replace the tubing if it becomes too fouled. You may be able to clean the tubing with a cleaning agent. The manufacturer may have recommendations for solutions and procedures to use for cleaning. But, if the pump is the source of the problem, then replacing the entire pump assembly is more cost effective than repairing it.
The precision pipetting system – syringes
The syringe pumps are for delivering more precise volumes. Syringes are smaller piston pumps that can dispense volumes from <1 µL to 10 mL or so. These pumps are best utilized in steps requiring smaller or more controlled volumes. Also, syringes can aspirate liquid from one container and dispense into another, while fast pumps are usually one-way – only dispensing liquids. The accuracy and precision requirements for syringes depends on their capacity. A smaller syringe, say 500 µL, will have a stricter accuracy and precision specification than a larger syringe, like 10 mL. The manufacturer will factory test the syringes using either colorimetric or gravimetric methods. As with the fast pump unit, it’s most effective to replace the entire syringe if one becomes faulty.
The best maintenance task for fast pumps and syringes is a distilled water system flush. Liberal flushing of the system will rinse away corrosive liquids and solid particles. There is no danger in doing this often. It’s recommended to perform a flush each time the system has completed handling biological fluids or reagents. Also, if the system has been sitting unused for a while, a system flush will help to keep it from deteriorating.
Consistency is key with all maintenance procedures. Many labs create log books to document these steps with signatures, times, and dates. Most active labs will keep the liquid handling robots busy, and regular use is good for these systems. But, intermittent robot users should be extra vigilant about keeping the system clean with regular flushes.
Leaks and Clogs
Obstructions and leaks are the #1 problem to avoid on pipetting systems. Leaks can be problematic because they may not be visible on liquid handlers. We tend to think of leaks as dripping liquid OUT of the system. This can and does happen, but you may be more likely to encounter leaks that let air INTO the system. Leaks and clogs affect the pipetting pressures, which kills accuracy and precision. Manufacturers design their systems to resist leaks and clogs, but they are common.
Leaks – running an (air-)tight ship
While very rare in a properly assembled liquid handler, leaks can happen and are most common at the connections. The pressure inside a system’s tubing changes during operation. This expansion and contraction can loosen connections over time. You should be especially concerned about the “finger-tight” connections between diluters and valves. These can loosen over time. While a puddle is the most obvious sign, visible bubbles in any of the system’s lines is also a good sign that there is a leak. Even tiny leaks can have a large effect on the accuracy and precision of the liquid handler. The pipetting action depends on the liquid column to provide a capillary force. Trace the flow of liquid from the source and look for where the bubbles originate. Then ensure all connections from there are tight. This should fix the problem. But the tubing will need replacement if leaks continue to appear.
Clogs – keeping the openings clear
The likeliest place for a clog to occur is in the smallest openings: a diluter, a syringe, or pipette tip itself. Diluters are valves that switch liquid flow between the pump and the syringes. Regular flushes are your go-to maintenance tactic. But, the next best way to keep these openings clear is to use only approved materials for your system. This includes reagents, systems liquids, consumables, and parts. Using incorrect or low-quality materials can cause buildup in or degradation of the system’s liquid path.
For example, most of the tubing on the liquid handler is durable PVC. This is an excellent tubing material for most applications. But it’s incompatible with certain reagents. For example, using a system liquid like DMSO will quickly degrade the tubing and cause leaks. Planning with a trained application engineer can help reduce the risk of this. In some cases, you can use an alternate tubing material like PEEK, that is resistant to the solvent. In other cases, you can try to avoid contact of the materials with the incompatible parts of the system.
The end of the line – leaks or clogs at the tips
Leaks or clogs will occur with both fixed and disposable tip systems. Fixed, washable tips are often for procedures that call for low volume transfers. Since these tips are thin metal tubes, they can become bent (obstructed) or cracked (leak) if damaged in a crash. Proper software scripting and consideration during protocol development can reduce this risk.
Disposable tips are less likely to clog, but leaks can form if the tips are not seated well on the tip adapter. Improper setup of the system or the use of low-quality tips can cause this leak. Both conditions are preventable. Professional service during installation or maintenance can solve the setup problem. And careful selection and validation of tip vendors will avoid the seating issue.
One other source of leaks is where the fixed tip or disposable tip adapter inserts into the tubing. Both fixed and disposable pipette tips depend on this connection to be air-tight. This is not a common point of failure, but the improper initial setup of the system can cause leakage over time.
The hard(ware) part of the system – easy to maintain
We’ve spent most of the time discussing how to avoid or solve problems with the pipetting part of the system. With good reason, this is the critical part that affects results most. We can’t ignore the rest of the system, though. In general, the liquid handling part of the system is the most error-prone. But, the rest of the system hardware requires some TLC as well to keep it in tip-top shape. Movement is an important part of the system. Most liquid handling robots have arms controlled by motors mounted on wheels/bearings. This arm usually runs along a track or rail covered in lubricating grease. This grease can gather dust and debris which will cause performance issues. If the wheels/bearings become fouled, they could bind or skip and cause motor errors or inaccurate movements. You can care for this yourself, as follows:
wipe away the contaminated residue with an alcohol solution
replace the lubricant with a small amount of clean (manufacturer qualified) grease
The Annual Preventive Maintenance Service
Yearly maintenance by a trained service engineer is important for a healthy liquid handler. A typical annual preventative maintenance (PM) service will include at least the following:
replacement of all the system liquid tubing for syringes and fast wash pumps
inspection/replacement of worn syringes and diluter valves
inspection/replacement of worn pipettes tips and tip adapters
inspection/cleaning/lubrication of moving parts
calibration of movement
PM visits are available as pay-as-you-go services or can be parts of a yearly service contract. Annual service contracts usually also include emergency service coverage. You’ll need to determine the best approach for your lab based on your usage of your lab automation system. If it’s a critical part of keeping your lab running, then you may want to consider a service contract. The cost of lost productivity from downtime can far outweigh the price of a service contract.
Atlantic Lab Equipment is ready to help you find a system that is best suited for your lab’s needs. We can also provide ongoing support for your newest team member! With proper installation and annual maintenance by a service professional, and weekly and monthly maintenance by your lab staff, a liquid handling robot will provide a very real return on investment for many years.