HPLC Sample Prep: Critical First Steps in LC Analysis
As HPLC systems become faster and employ increasingly exotic stationary phases, sample preparation becomes essential for reliable, reproducible analyses. Biological samples generally require concentration in the target analyte and, in the case of
As HPLC systems become faster and employ increasingly exotic stationary phases, sample preparation becomes essential for reliable, reproducible analyses.
Biological samples generally require concentration in the target analyte and, in the case of proteins, depletion of high-concentration interfering materials. Sample concentration may be carried out through dialysis or with specialized evaporation systems. Porvair Sciences (Leatherhead, UK) and other vendors sell devices that blow heated nitrogen into individual microwells to speed evaporation and sample concentration. Depletion is somewhat harder to achieve, particularly when purifying a low-abundance protein in the presence of highconcentration species.
Depending on the sample type, common prep methods may include solid phase extraction (SPE), centrifugation, or liquid-liquid extraction. SPE is straightforward and available as single disposable cartridges filled by hand or through automated instruments. Protein prep for most LC-MS often involves an additional step, proteolysis, which breaks large molecules into smaller, more manageable peptides. Filtration is the simplest and perhaps most essential LC sample prep technique. Schleicher & Schuell MicroScience (S&S; Dassel, Germany) suggests filtering through a 0.45-micron microporous syringe membrane filter to break up emulsions and retain particulates that clog columns.
S&S recommends regenerated cellulose (RC) membranes, which are hydrophilic, highly resistant to HPLC solvents, and show very low extractables. According to S&S, RC is one of the weakest protein binding membranes, which means it’s suitable for proteomic analysis.
Sample preparation is an oft-overlooked source of error and inconsistency in HPLC analysis. Column and mobile phase conditions may be optimal and steady from run to run, but if samples are prepared differently, they will run differently as well.
That is why Simon Robinson, HPLC product manager at Shimadzu (Columbia, MD), recommends automating sample prep whenever possible. “Otherwise, reaching a point where data becomes untrustworthy is quite likely,” he says. “Automated sample preparation eliminates human error.”
One drawback of automated sample prep, however, is that standards and samples need to be refrigerated, which introduces scheduling issues and somewhat reduces the attractiveness of hands-off operation.
Paul Boguszewski, product manager for sample preparation products at Agilent (Santa Clara, CA), notes that automated sample preparation plays into three significant trends in LC analysis:
- Productivity (or lack thereof), speed of analysis, and throughput, as typified by fast, high-pressure systems (UPLC and fused core technology)
- Sensitivity at ever-lower analyte concentrations, which is the justification for adopting mass detectors (particularly triple-quad)
- Increasing complexity, particularly for biological samples
Agilent has constructed a matrix of preparation methods suitable for small-molecule HPLC analysis. Solid phase extraction is the most complete, versatile technique, removing particulates, proteins, lipids, oligomeric surfactants, and salts. At the other end of the efficiency scale, performing just a tad better than “dilute and shoot,” is liquid extraction, which removes proteins only partially and not much else.
In between are dried matrix spotting, precipitation/filtration, and “smart” titration, which, successively, provide more efficient cleanup.
But as techniques become more efficient, they also raise the cost of sample preparation and complicate the workflow. Many lab managers view this as an absolute negative. This is a mistake, says Mr. Boguszewski, who claims the higher cost is illusory; cleaner samples translate to more robust, reproducible LC analysis, with fewer repeat runs.
Recently Released HPLC Sample Prep Systems
SPE-DEX 4790® Solid Phase Extraction System
- Performs all solid phase extraction steps for aqueous samples directly from their original containers
- Handles 40 mL up to 20 L samples, accommodating SPE disks or cartridges
- Modularity allows for one to as many as eight extractors to be linked and controlled simultaneously
- Compatible with sediment-laden water samples and prevents the formation of emulsions
Horizon Technology
www.horizontechinc.com
Perfinity Workstation
- Powered by Shimadzu hardware components that work in conjunction with five Perfinity optimized columns
- Can be used as a standalone unit for HPLC sample prep and also automates protein separations and mass spec sample preparation for more efficient LC-MS analysis
- Platform is customizable across application areas
Perfinity Biosciences - www.perfinity.com
Shimadzu www.shimadzu.com
Strata™-X-Drug B Solid Phase Extraction (SPE) Sorbent
- Specially designed and quality-control tested for drugs of abuse
- Does not require conditioning, saving time and solvent expense
- Detection for the 11 common drugs of abuse is below the lower SAMHSA cutoff levels
- Does not promote interconversion of norcodeine and normorphine to parent compounds
Phenomenex
www.phenomenex.com
Solid Phase Extraction (SPE) System
MaxiLute™
- Offers the capability to process up to 200ml of sample in one go repeatedly and precisely.
- Each of the 48 wells on the MaxiLute has an individual drain spout ensuring 100 percent sample transfer and zero cross over contamination
- Will not bend or distort because individual SPE cartridges do not have to be repeatedly plugged in and out
Porvair Sciences
www.porvair-sciences.com