LC Trapping - An Introduction by Optimize Technologies

O P T I M I Z E

T R A P P I N G

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 inline, 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.

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W W W. O P T I M I Z E T E C H . C O M

Trap Cartridges as Sample Pre-concentrators Often, samples arrive for LCMS analysis in concentrations that are undesirably low. For instance, picograms or even femptograms of a protein of interest may be present in a few milliliters of solvent, resulting in a sample concentration that is below the detection limit of the instrumentation being used. In addition, the matrix itself may be less than optimal for injection onto an LCMS system (non-volatile, buffered, etc.). In these situations, it is of course desirable to increase the concentration of the target analyte in the sample. The most practical way to achieve that goal is to reduce the volume of the sample matrix without losing any of the target analyte dissolved within it. By using a trap cartridge as a sample pre-concentrator either on or off-line, this can be achieved conveniently, and with a minimal amount of manual sample handling.

OPTI-LYNX™

Off-line Sample Pre-concentration This simple method allows for manual off-line pre-concentration of samples without requiring

additional valve-switching capability. A trap cartridge chemistry is selected with affinity for the target

SYRINGE

analyte, and the sample is manually driven across the

AUX PUMP

trap bed with a syringe, or a small pump (syringe, peristaltic, etc.) Flow should be steady, and within

fig. 1a

the range of typical LC flow rate for the trap bed diameter being used – slower is generally better. As the sample passes through the trap, the target analyte is retained on the trap bed while the sample OPTI-LYNX™

matrix is sent to waste (fig 1a). Once the sample has passed through the trap, the target analyte can be eluted in a small volume of stronger solvent (fig. 1b). If salts were present in the sample matrix, a quick rinse step with a non-

buffered solution prior to the elution step would be advantageous. Elution could also take place

SYRINGE

AUX PUMP

fig. 1b

via manual delivery of solvent, but it would also be easy to remove the trap cartridge from the manual loading rig and install it into a holder already in-line OPTI-LYNX™

upstream from an analytical column or within an injection loop (fig. 1c). In these situations, it is of course desirable to increase the concentration of the target analyte in the sample. The most practical way to achieve that

OPTI-LYNX™

goal is to reduce the volume of the sample matrix without losing any of the target analyte dissolved within it. By using a trap cartridge as a sample pre-concentrator either on or off-line, this can be achieved conveniently, and with a minimal amount of manual sample handling.

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fig. 1c

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W W W. O P T I M I Z E T E C H . C O M

On-Line Sample Pre-concentration

GRADIENT PUMP

For a more convenient and automated means of pre-concentrating samples, a trap cartridge can be placed in-line in the loop of an injection or switching valve, providing a means of partial or complete

WASTE

automation. The injection valve is plumbed in a way that allows solvents from two different sources to

AUX PUMP

flow through the trap depending on the position of the valve (fig. 2a).

DILUTE

During the loading phase, the sample solution is pumped across the trap bed using an auxiliary pump (peristaltic, piston or syringe pump would be fine). Sample matrix is flushed to waste. Again, it may also be desirable to wash the trap bed with a salt-free solution after loading, to ensure that any buffer salts

fig. 2a

have been rinsed away. LC COLUMN

Once the entire sample has been pumped through the trap cartridge, the elution step can commence. Prior to elution, it might be a good idea to follow the sample-loading step with a flushing solvent to ensure

GRADIENT PUMP

that any sample remaining in tubing leading to the trap cartridge makes it across the packing material. The trapped and concentrated sample can now be eluted from the trap bed using a small volume of suitably strong organic solvent (fig. 2b). Eluent from

WASTE

the trap can be sent directly to a mass spectrometer, or on to an analytical column for further separation.

AUX PUMP

It might even be desirable to follow the preconcentration step with a 2DLC scheme if the analyte contains a complex mixture of proteins and peptides.

fig. 2b

CONCENTRATE LC COLUMN

For more applications such as sample loops, fitting kits and packed capillary columns, please contact Optimize for more information.

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TEL: 503-557-9994

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W W W. O P T I M I Z E T E C H . C O M

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 six main products with many variations – low-volume capillary products suitable for direct installation into injection valve ports and columns, variable volume quickconnect products that can be connected in-line anywhere on your system, low-volume ultra high pressure traps, and variable volume ultra high pressure hand-tight cartridge traps.

Advantages: • Manual, In-Line and Direct Connect

• Bed volumes range from .12µL to 100µL

holder configurations

• More than 30 packing material and phase options

• Hand-tight holders offering hand-tight

• Auto adjusting (ZDV) port connections

cartridge replacement

• Low-volume, low-dispersion cartridges

• Medium, high and ultra high pressure

• Custom configurations available

(20,000 + PSI) products

OPTI-TRAP™

OPTI-LYNX™

TRAP COLUMN

MICRO TRAP

TRAP COLUMN

MEDIUM PRESSURE

HIGH PRESSURE

1,500 PSI / 100bar

6,000 PSI / 400bar

EXP®2

EXP®

NANO TRAP

STEM TRAP

TRAP COLUMN

ULTRA HIGH PRESSURE

20,000+ PSI / 1400bar

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W W W. O P T I M I Z E T E C H . C O M

TEL: 503-557-9994

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