Trapping - What is it? What are trap cartridges and how are they used?

Trapping: The selective retention and subsequent elution of analytes within a sample. Trapping is a chromatographic technique, but typical phase boundary effects like partitioning are not being exploited. Many trapping applications could be described as "digital", or on/off chromatography, because solvent conditions are selected to ensure that there are only two retention states - one where solvent strength is weak enough to allow the analyte to bind to the stationary phase without eluting, and one where solvent strength is sufficient to cause immediate and complete sample elution. The term trap cartridge refers to a packed bed with suitable capacity to completely retain a given amount of target analyte within a sample. Trapping is often used in conjunction with mass spectrometry - the trap cartridge serves to clean up (and/or pre-concentrate) the analyte, while the MS handles detection and identification. The MS can also make up for low separation resolution and column efficiency that would not be acceptable with other modes of detection. In return for the trade-off in resolution, trap cartridges offer greatly reduced analysis time (a benefit common to all short/fast bed formats.)

Example 1: Sample Cleanup ("Trap and Dump"), Desalting

The analyst has a simple mixture - a compound of interest dissolved in a medium not suitable for injection onto an MS (highly aqueous, salt-containing, etc.). For instance, protein samples often arrive for analysis in buffered solutions. This might be for stability reasons, or the salts might be present due to a preparation step such as a digest. This salt has to be removed prior to mass spectrometry analysis, as they will foul the MS interface. A trap cartridge can be placed within the loop of an injection valve to conveniently capture the analyte. Once the buffer salts and impurities have been washed away, mobile phase strength can be increased and the compound eluted onto an analytical column, or directly onto the MS.

Example 2: LC Separations on the MS Timescale

A big problem with LC-MS is that the LC part takes a lot longer than the MS part. With MS, it is not always necessary to achieve a high level of resolution, but a trap cartridge can be used to provide a small amount of chromatographic separation. Using switching valves, two trap cartridges could even be used in a tandem/parallel set-up, reducing the LC cycle time by 50%. These two factors can be combined to produce an automated fast LC-MS system that works at the pace of the MS, not at that of the LC.

Example 3: Separation of A Mixture of Proteins

Traps are often used in protein separations. You can actually use two or more cartridges in a row to assist with separation of mixtures of charged and neutral proteins. For instance, an ion exchange cartridge could be used in-line, followed by a C18 cartridge. Additional switching and injection valves would be used to offer increased solvent routing flexibility, as follows:

Gradient pump → Injection Valve → Ion Exchange Trap → Switching Valve → C18 Trap → Mass Spectrometer

The protein or peptide mixture would first be injected inline ahead of the ion exchanger. Use of a weak mobile phase solvent would cause charged proteins to be retained by the ion exchanger, while neutral proteins would pass through to the C18 cartridge and be retained there. A switching valve in between the exchanger and the C18 bed allows switching of the solvent being delivered to just the C18 trap. The neutral proteins can be eluted from this trap onto the MS by increasing solvent strength. Then, charged proteins could be selectively eluted from the exchanger onto the C18 column via salt gradient (the valve downstream from the C18 trap would switch so that salts were sent to waste, not to the MS). A second subsequent elution off the C18 trap (in conjunction with a solvent switch) sends these charged proteins onto the MS, free of salts and contaminants.

Optimize Products for Trapping

Optimize offers a wide selection of novel packed-bed products, all of which can be used effectively in a sample trapping or on-line purification application. The following products are well-suited for use as trap cartridges, but we encourage you to call us to discuss your particular requirements - we have numerous bed formats in a variety of dimensions, and many options for standard or custom-packed stationary phases that can be tailored to your application. Our trap cartridge offering is centered around four main offerings with many variations - a low-volume capillary product suitable for direct installation into injection valve ports and columns, a variable volume quick-connect product that can be connected in-line anywhere on your system, a low-volume ultra high pressure stem trap, and a variable volume ultra high pressure hand-tight cartridge trap.

OPTI-PAK® Capillary Traps

This product is perfect for direct connection to injection and switching valves, and offers a very low-volume bed format combined with auto-adjusting ZDV connection into any 10-32 port. Use for very small sample volumes and low flow rates. This product is rated to 6,000 psi. 

OPTI-LYNX™ Trap Cartridges

These cartridges offer quick-connect convenience and a larger bed for greater sample capacity and higher flow rates.

EXP® Stem Traps

The slim architecture allows it to easily fit into crowded instrument compartments or to connect directly to tightly-spaced injection ports. When tightened by hand it seals to 8,700+ psi. When tightened by wrench on the incorporated wrench flats, it seals to 20,000+ psi. The low volume, low dispersion cartridges are perfect for your UHPLC volume-critical applications.

EXP® Trap Cartridges

The Hand-Tight EXP Trap Column is rated for use at 20,000+ psi and can connect to any injection valve or column with 10-32 threads or can be connected in-line with 1/16” stainless tubing. For your UHPLC applications that require a larger packed bet, these cartridges are the best choice on the market today.

When Do I need to use a Guard Column?

Guard columns perform two primary functions:

• First: they capture irreversibly retained compounds - substances that would otherwise permanently bind to and foul the stationary phase of the analytical column.  They guard the analytical column from premature failure.
• Second: they serve a sacrificial role - when mobile phase pH is extreme enough to degrade the column packing, a guard column with similar phase characteristics can "take the hit", and prolong the life of the analytical column. Guard columns also function as pre-column filters, protecting the analytical column from particulate contamination. However, if the mobile phase is of moderate pH and samples are free of irreversibly retained compounds and other contaminants, particulate contamination can be handled without a guard column, using only a precolumn filter.

Selecting a Guard Column: Factors to Consider

Liquid chromatography has become an increasingly sophisticated separatory technique over the last two decades, as advances in instrument technology have led the way to lower detection limits, increased automation and more powerful data-handling capabilities. In similar fashion, improvements in packing material and column design have led to more efficient, selective and reproducible separations. Even with these substantial design improvements, however, the analytical column continues to be a problematic link in the HPLC component chain. With the cost of quality analytical columns continually increasing, and with the price of some specialty analytical columns reaching into the thousands of dollars, effective pre-column protection is a wise precaution in today's cost-conscious HPLC laboratory.

Choosing a Guard: When choosing a guard column, there are several factors that are important to consider. The fundamental role of a guard column is to trap particulates, chemical contaminants and other foreign material in order to prevent them from fouling the analytical column. A guard column containing the same type of packing material as the analytical column will provide the best protection. Chemical contaminants and other substances with a propensity for fouling octadecyl stationary phases would be best trapped by an octadecyl guard column; contaminants that cause deterioration of a particular packing material would first attack the stationary phase in the sacrificial guard, protecting the analytical column for as long as the capacity of the guard column will allow.

A second factor to consider is a guard column's potential for contributing dispersion through introduction of extra-column volume. The installation of a guard column requires that additional connections, and often extra tubing, be incorporated in a section of the chromatograph where excessive dead volume can make or break an assay. A well-designed guard column will minimize the amount of extra tubing required for connection to the analytical column, thus minimizing extra-column dispersion.

When selecting optimal precolumn protection for your HPLC, you should also consider potential ways in which the guard column will affect separation efficiency. To use a guard column is to extend the length of the packing bed by a finite distance. A well-designed guard column will impart minimal influence on the efficiency of the analytical separation. A guard column that affects efficiency to any great degree will cause problems when validation time arrives, or when the guard itself must be replaced.

Optimal Protection by Design:  If a prospective guard column is to provide optimal performance and protection for your analytical column, it must address the physical and chemical constraints previously mentioned. Guard Columns from Optimize have been designed to offer reliable low-dispersion column protection, while eliminating any chance of dead-volume introduction in the precolumn region. Optimize Guard Columns offer tool-free connection with an automatically adjusting tube stem for perfect zero-dead-volume connections, regardless of the analytical column manufacturer's tube stop depth. This patented design guarantees that the tube stem will automatically bottom out securely against the tube-stop of any 10-32 female port. Some guard columns incorporate molded one-piece couplers or swaged-on ferrules that establish a fixed tube-stop depth for connection to the analytical column. Guards with fixed tube stems will introduce either dead-volume or leaks at the connection with the analytical column when they are switched from one port to another. This situation can occur even when a fixed-stem guard column is switched between different 10-32 ports from the same manufacturer. Because tube-stop depth will vary from port to port due to normal manufacturing tolerances, a guard column that utilizes a fixed tube stem will often leak or introduce dead volume when it is switched between different female 10-32 ports.

A guard column should perform its primary function of column protection in a manner that is as chemically transparent as possible to the chromatographic separation. When selecting a guard column, choose high quality packing materials that are a good match for the stationary phase of your analytical column. Also, be sure that the guard column you select is designed to minimize extra-column effects. Guards that avoid fixed or pre-swaged tube stems, and connect directly to the analytical column without the need for extra tubing, are the optimal choice.

Filtration: A Systemic Approach to Protecting Your System

Obtaining reliable, consistent results from your HPLC system requires continuous consideration of chemical and mechanical factors that can negatively impact system performance. For your laboratory to remain competitive, instrument down-time must be kept to an absolute minimum. Many common instrument problems are caused by the presence of particulate contamination introduced by the mobile phase or as a by-product of system operation. Particulates can cause a variety of maintenance headaches, including check valve failure, excessive wear of the piston and seal, and high back-pressure due to frit blockage. A consistent, holistic approach to filtration is an effective means of minimizing particulates in your flow path. It is also a vital precaution that will keep your chromatograph working for you (and not the other way around).


Mobile Phase / Solvent Filtration: The first and perhaps easiest place to prevent particulate contamination is in the mobile phase. Buffer salts, improperly cleaned glassware, microbial contamination and pre-filtering procedures are all potential sources of particulates. Solvent manufacturers adequately filter HPLC-grade solvents prior to bottling; ideally, there should be no need for additional pre-filtering in your laboratory if your mobile phase contains only HPLC-grade solvents. Mobile phases prepared with buffer salts or other solid reagents should be filtered prior to use, however. A 0.2 or 0.45 µm membrane filter is recommended for this filtration step.  It is also a good idea to discard the first few milliliters of solvent that pass through the filter.  Solvent reservoir filters are a useful filtration accessory, providing a first line of protection as the solvent enters the system. OPTI-SOLV® Solvent Reservoir Filters are an excellent choice for this application; OPTI-SOLV secures your inlet tubing at the bottom of the solvent reservoir without the use of tools or fittings, and is machined from PTFE and Titanium. This well-swept filter ensures access to all but the last few milliliters of solvent, and will not trap bubbles or leach extractable contaminants. Solvent reservoir filters are available with a choice of frit porosities. For analytical HPLC applications, a 2 µm frit is the best choice, while a larger porosity frit such as 10 µm should be selected for flow rates exceeding 10mL/min.

Piston Seals:  Piston seals are a common source of particulate contamination in the flow stream. As the piston moves back and forth through the piston seal at high pressure, seal fragments can slough off from the mating surface of the seal. The size and shape the particles will vary depending upon the nature and quality of the material from which the seal is made. These variables are important to consider, as they directly impact the ease with which shedding particles can be removed from the flow path. Particles too small to be trapped by a 2 µm filter, or large enough to be trapped within the frit but below the surface, will cause the most trouble.  The exclusive polymer blends used in our ITB™ and OPTI-SEAL®  piston seals are specifically formulated to shed particles large enough to be trapped at the surface of a 2 µm frit. Because particulates in the pump head can cause increased friction and abrasion between the piston and seal, excessive seal wear is both a source and a symptom of particulate contamination.


In-Line Filtration:  The buildup of precipitated salts and other particulates within the pump can be minimized by regular flushing with non-buffered mobile phase. Some pumps include a piston flushing utility that allows the piston to be rinsed on the low pressure side of the seal while the pump is in use. Daily piston flushing can help prevent salt precipitation on the surface of the piston and can prolong the life of both the piston and piston seal. For most effective protection from seal shedding particulates, we recommend the use of an in-line filter installed downstream from the pressure transducer but prior to the injection valve or autosampler. OPTI-SOLV In-Line Filters are a convenient and cost-effective solution for most in-line filtering applications, providing rapid access to replaceable frits without requiring disconnection of attached tubing.

Check Valves with Filters are a bad idea:  Some instrument manufacturers supply check valve assemblies equipped with a filter element on the outlet side. While a filter frit in this location may seem to be a better option than no filter at all, this arrangement is problematic. First, the use of an in-line filter upstream of the pressure transducer complicates pressure-based diagnosis of instrument performance. Second, placement of a frit this close to the outlet check valve causes particulates to build up in a location where they are likely to cause problems. Check valve failure is often brought on by the adherence of contaminants to the surface of the ball, causing it to stick or seal improperly. In-line filtration of seal debris and other particulates downstream from the pressure transducer will minimize the chance of check valve contamination and will not complicate system diagnostics.


Pre-Column Filtration:  Two more potential sources of particulate contamination can cause trouble in the region beyond the injection device but before the analytical column. First, the injection device can introduce particles resulting from rotor seal wear or the coring of vial septa. Second, contamination may be introduced by the sample matrix. The analytical column can be damaged by irreversibly retained contaminants in the sample matrix, or by salts that precipitate due to incompatibilities between the sample matrix and mobile phase. Where practical, most of these types of contamination can be addressed in the sample preparation procedure. In cases where additional filtration is required between the injector and column, a low-volume, low-dispersion filter and/or guard column must be employed. OPTI-SOLV Mini Filters and OPTI-GUARD® Guard Columns work extremely well for applications requiring pre-column protection. Our patented, automatically adjusting tube stem design assures zero-dead-volume connections and low dispersion in an area where excessive dead volume can make or break your assay.

What Is All This Stuff About Dead Volume, Swept Volume, and Void Volumes?

This is definitely an issue of semantics. Often, two chromatographers will be speaking about the same concept, but using different terms.  Even more confusing, the word one uses will also be in the other's lexicon, but it will have a completely different meaning. To try and clear the waters, let us define some concepts and how you may hear them referred to.

Dead Volume: As a difficult term to define, dead volume often refers to the amount of volume in the flow path. However, if this definition is used, then there is no way to define the spaces in the flow path that give rise to non-linear flows. Therefore, we prefer to use the term swept volume to refer to the volume in the flow path, while ascribing the term dead volume to areas outside of the flow path that can contribute to bleeding, carryover, and cross contamination.  

What is meant by outside the flow path? Often gaps will be introduced into the LC system causing Laminar flows to no longer exist. These most commonly are the spaces that occur in connecting tubing: if tubing is not bottomed out in the tube-stop, a small space, or dead volume, is created. These small spaces are hiding areas for sample and provide a great place for the remixing of analytes to occur. This is why all Optimize products are designed to minimize dead volume. The floating stem design automatically adjusts to virtually any tube stop, making a flush zero-dead-volume (ZDV) connection; and the OPTI-LYNX™ quick-connect hardware system that only needs the tubing connection to be made only once, with all subsequent OPTI-LYNX connections being identical.

This is not to say that minimizing swept volume is not important. Any where swept volume occurs is a place where mixing can happen. So, if you want to minimize mixing--which you probably do--you should also minimize the excess contributions to swept volume. The most obvious, and easiest, way to do this is by using the minimal amount of tubing to make connections. This is especially critical in the region between the column and the detector where excessive volume can result in a degradation of the detector signal.

There are a few more volumes we should consider. There is the total system volume, which we have defined as swept volume, and the column volume. For column volume, there are a few definitions to examine. First, there is the amount of space in the column not taken up by packing material, which is referred to as void volume or interstitial volume. We really don't think that either is more correct, though we prefer to use interstitial volume because it is more definitive. The interstitial volume is commonly expressed temporally by the use of terms like void time, column dead-time, t0, or tm. All of these are measurements of the time before an unretained component is detected, usually uracil. While these are the common ways to express the void volume or interstitial volume, it should be noted that these measure the contribution not only from the column, but any extra volume from the injector through to the detector. Furthermore, they are expressed in units of time, not volume. To get volume, you must perform the favorite scientific pastime of dimensional analysis (this may excite some more than others-probably physicists).

Lastly, we should say a few words about void, or column void. This is a great example of the over use of a word, void, that leads to a great deal of confusion. When void is used by itself, it is referring to a void, or space, in the actual column where there is no packing material. The presence of a void in the column is absolutely debilitating, and often spells the end of its useful life (at least as a column; it still has a very utilitarian life as a paperweight).

We hope this cleared up any confusion, both of our usage and the usage at large. It should be noted, though, that these are our definitions and you or others could use the terms above to refer to completely different things. The best way to avoid confusion in any LC conversation is to be sure that these common terms have the same meaning to all involved.

As with most analytical instruments, HPLC systems require some amount of preventative maintenance if they are to perform optimally. By proactively replacing key components within their expected lifetime, unexpected and costly instrument downtime can be minimized for mission-critical systems.

Service Contracts

They are certainly a convenient, if overpriced, means of handling instrument upkeep, but most routine preventative maintenance tasks can be handled quickly and easily without a visit from a service representative, even if you don’t have the benefit of an internal metrology department.  By selecting easily replaced maintenance components and following proper installation procedures, you can implement your own effective preventative maintenance program.  Outlined below are recommended maintenance schedules for commonly replaced HPLC system components.  These schedules can be used as general guidelines to assist you in implementing a preventative maintenance schedule for your HPLC system, but can be adjusted based on your experience with maintenance cycles and typical component lifetimes in your laboratory, in order to more closely match your operational conditions.

Piston Seals

No other wetted component within a typical HPLC system experiences as much direct wear as the piston seal.  The average lifetime of a piston seal will vary depending upon several parameters, such as the seal material, the composition of the mobile phase, the presence of particulates or precipitated salts around the seal, and the condition of the piston itself.  Most laboratory preventative maintenance (PM) schedules call for replacement of the piston seal on a quarterly or semi-annual basis, or every three to six months.  In general, the piston seal should be replaced at least once every six months, providing there is no excessive and unusual wear.  Where conditions are less than optimal, more frequent replacement will likely be required, such as a highly corrosive solvent or extreme operating conditions.

You can maximize seal lifetime and performance by following a few simple guidelines. First, try to pick the best seal for the mobile phase you will be using (view our seal-solvent compatibility chart for more information, or contact us for a specific recommendation for your application.) Second, ensure that your piston is in good condition, is free of adhered particulates and salts, and shows no sign of wear. Finally, minimize the chance of seal damage during installation by presoaking the seal in methanol or isopropanol, and by using a seal forming tool when available.

Check Valves

As with piston seals, the lifetime of a check valve will vary depending upon chromatographic conditions. The ball and seat are subject to wear, and the ball may also become coated with particulates or other matter, causing sealing problems. The majority of laboratories routinely replace check valves every six months. Once again, longer or shorter lifetimes may be the norm in your laboratory, and the frequency of replacement may require some adjustment.  However, with an Optimize Technologies OPTI-MAX® Check Valve, you should get a year or more of reliable use.

When selecting a replacement check valve for your HPLC or UHPLC, you will usually have two options. For older pump models, you can use complete assemblies (or rebuild existing assemblies with multi-component rebuild kits), and for newer models you can use a cartridge-based check valve system. Replacing whole assemblies means that you throw out the entire check valve housing and internal components and replace the assembly and install a new one. This is convenient, but possibly expensive and certainly wasteful. Rebuild kits are more economical, but the procedure is time-consuming and troublesome, and there is a risk of improper assembly and contamination. Also, some rebuild kits and assemblies incorporate a filter element within the outlet check valve. While in-line filtration is a worthwhile addition to any HPLC, putting a filter within the outlet check valve assembly has two major drawbacks. First, a filter above the outlet ball will concentrate particulates in a region where they can potentially cause check valve failure. Second, the presence of an in-line filter element upstream from the pressure transducer can complicate pressure-based instrument diagnostics.

Cartridge-based systems are the preferable choice, due to greater convenience and reliability, greater economy, less waste, and reduced risk of contamination during installation. When selecting cartridge check valve products, be sure to focus on manufacturing quality and product performance, and beware of dubious marketing claims. For instance, several manufacturers claim that their check valve cartridges are “self-priming.” This carries little importance or credence, as check valves are by design a passive flow restriction device – they are self-priming in the same way that rivers are self-flowing. Positive hydrostatic pressure causes mobile phase to flow through ball & seat type check valves – as long as there is minimal resistance to flow in the forward direction, a check valve won’t get in the way of the mobile phase. The specifications that really matter in terms of check valve performance include ball/seat material, finish quality, leak rating, and amount of allowed ball travel.

Pistons

The pistons inside an HPLC pump do not require routine replacement, but they too are subject to wear, and should be inspected as part of the PM schedule.  Pistons should be examined at least once a year and whenever the seal is replaced. Close examination of the surface finish can reveal potential problems such as flat spots, out-of-roundness, adhered particulates or buffer salts, scratches and cracks.  Light microscopes can be useful for inspecting the condition of a piston.

Particular attention should be paid to the piston when UHMW-PE seals are to be installed in place of PTFE-based seals. UHMW-PE provides considerably longer seal lifetimes when used with mostly aqueous mobile phases, but the PE material is less forgiving than the softer PTFE blend.  A worn or damaged piston that performs adequately with a PTFE seal may cause problems with UHMW-PE. 

When switching to UHMW-PE seals after using PTFE, it is a good idea to replace the piston at the same time.

UV Detectors

For UV detectors, source lamps are the most common consumable item requiring routine replacement.  The frequency of replacement will vary depending upon the amount of usage, but it can be estimated based upon the frequency of use and the anticipated lifetime of the lamp.  Typically, source lamps will require replacement annually or semi-annually.  Some types of lamps come pre-aligned in holders, ready for immediate installation. If pre-aligned lamps are not available for your detector, alignment will be required as part of the installation process. This can be a somewhat involved procedure, and you may wish to consult with qualified personnel if you have not successfully performed an alignment before.

Autosampler or Injection Valve

For most sample injection devices, the only components that require regular replacement are rotor seals and liquid seals.  This can usually be performed on an annual basis.

Conclusion

In a productive lab, the cost of HPLC downtime can cost thousands dollars for each hour the system is down. Implementing an effective preventative maintenance program for your HPLC can help prevent minor maintenance issues from becoming a major drain on your time and budget.