Online Solid Phase Extraction

Online SPE coupled with HPLC, UHPLC and LC-MS has rapidly been incorporated in a variety of analytical processes. Spark Holland’s SPE systems are a perfect fit in any LC-MS lab, processing multiple batches/assays fast and reliably, with the highest analytical performance, fully unattended. They also have all the tools to perform method development using a systematic approach which generates a working online SPE method in just a few days. These versatile workhorses are a safe investment and allow the user to perform more projects with the highest assay performance in the same time and using the full MS capacity to the lab's economical benefit.

Sample preparation is a critical step in any analytical method, especially in all chromatography techniques where samples have to be homogenous and free of impurities. Today, one of the most widely used sample preparation methods is solid phase extraction (SPE), an extraction method that uses a liquid phase and a solid phase - the same type of stationary phases that are used in liquid chromatography columns - to isolate or concentrate one, or one type, of analyte from a solution.

Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrices, including urine, blood, water, beverages, soil and animal tissue. The stationary phase can come in various forms, such as a packed syringe-shaped cartridge or a 96 well plate, each of which can be mounted on its specific type of extraction manifold. The manifold allows multiple samples to be processed by holding several SPE media in place and allowing for an equal number of samples to pass through them simultaneously. Solid phase extraction cartridges are available with a variety of stationary phases, each of which can separate analytes according to different chemical properties. Most stationary phases are based on silica that has been bonded to a specific functional group. Some of these functional groups include hydrocarbon chains of variable length (for reversed phase SPE), quaternary ammonium or amino groups (for anion exchange), and sulfonic acid or carboxyl groups (for cation exchange).

Concept of on-line SPE

Manual or off-line SPE can often be time-consuming, as well as labour and cost-intensive, compromising productivity. Analyte recovery can be variable from batch to batch causing reproducibility problems. Spark Holland pioneered on-line sample preparation (solid phase extraction, column switching) and introduced on-line sample preparation as an integrated option on an autosampler over 20 years ago.

On-line SPE elution offers several advantages over off-line SPE sample preparation methods. No evaporation or reconstitution steps are required resulting in faster processing and better precision. All of the sample is eluted to the LC or LC-MS system, while maintaining small elution volumes, providing the highest assay sensitivity. No sample preparation is required as sample preparation is carried out during analysis. The use of valve switching ensures all of the samples are in a ‘closed' fully GLP compliant system. During a typical run, an untreated sample will be prepared during the sample LC analysis time, directly transferred to the LC column and analyzed. In a closed on-line system, all data in sequence will be linked only with the prepared sample, ensuring that sample data cannot be mixed up. In practice, with an ‘open' off-line SPE sample preparation system, data mix-up can occur and is impossible to totally eliminate. On-line SPE elution, therefore, is safe both for the sample and importantly, the operator, ensuring error-free analysis and no contact with possible hazardous sample preparation solvents. Lower operation costs can be achieved with no need for additional sample collection vials and the reduction in sample clean up solvents.

Spark high pressure sample processing

Today, online sample preparation using high pressure stream switching is considered the best approach to obtain high assay quality and maximum automation. In addition, online SPE using valve switching provides a closed sample processing system - another plus for sample integrity. High pressure SPE provides sample loading independent of the back pressures on the SPE cartridge. The high-pressure dispenser used for the sample loading goes up to 300 bar and typically the back pressure on the SPE cartridge during the sample preparation steps will be lower than that. High pressure SPE also allows the use of smaller size particles in the on-line SPE cartridges.

Efficient preparation
High pressure SPE provides a constant sample preparation flow rate for all samples. During the sample loading, sample wash and clean-up steps, the solvent flow over the SPE cartridge is constant and not influenced by the back pressures, thus ensuring efficient sample preparation.

Minimal sample pre-treatment
Off-line SPE cartridges require centrifugation or filtration pre-treatment steps due to limited back pressure. High pressure SPE ensures a minimum of sample pre-handling steps and the ability to handle samples having various viscosities.

Precise sample preparation
Smaller size particles act as a disposable ‘mini-LC' column which has about 250 theoretical plates per SPE cartridge, enabling a better and more efficient sample clean up with exactly defined solvent clean up volumes and flow-rates to separate and remove the matrix components from the pre-concentrated sample analytes.

Higher assay sensitivity
Often, with off-line SPE all of the collected elution volumes are not analyzed. During the
on-line elution step, smaller sized particles ensure that the pre-concentrated analytes are eluted in small elution volume when the mobile phase transfers the analyte from the SPE cartridge to the LC column. Maintaining the small elution volumes with the on-line elution of all the analytes which are prepared provides the highest assay sensitivity.

Fast and parallel sample extraction
The on-line elution of SPE cartridges to the LC system is done by valve switching - connecting the SPE cartridge directly to the LC separation column. The flow of the mobile phase transfers the analytes from the SPE cartridge to the LC column. The valve switching is fast and typically, with a LC pump flow of 1 ml/min, all analytes are eluted on-line to the LC column within 30 seconds. After the sample transfer to the LC column the cycle is finished and the automated system prepares the next sample. With this parallel sample preparation and LC analysis, the sample throughput is defined by the LC separation time which is available and the sample preparation of the next sample has to be finished within the same available time. For this reason, high pressure SPE with a constant flow-rate is important and all sample preparation steps will be finished within the available ‘LC separation' time.

Spark on-line SPE systems

Spark Holland offers 20 years experience of on-line SPE, leading the way in the innovative integration of sample preparation functionality into front-end sample
handling systems. New generation high throughput systems offer:
• High pressure SPE with controlled flow rates
• On-line elution to LC and LC/MS systems
• Parallel sample preparation and sample analysis
• Flexibility with two extra high-pressure valves and second extraction cartridge
o Automated method development
o 2D (orthogonal) SPE
o UHPLC with trapping column