Optimizing HPLC Sample Preparation

Optimizing HPLC Sample Preparation

by Caitlin Smith

Careful sample preparation is important prior to high-performance liquid chromatography (HPLC)/ultra high-performance liquid chromatography (UHPLC) analysis for at least three reasons. For one, sample concentration often is needed. Second, the sample may require buffer exchange and/or desalting to place it in the appropriate solution for liquid chromatography. Finally, each HPLC or UHPLC sample will need to undergo at least one filtration step to remove contaminants and particles prior to running it through the liquid chromatography column.

Sample preparation technique

Sample preparation can be achieved using several different methods, and sometimes the best method is dictated by the sample type. However, the most common methods include solid phase extraction (SPE), centrifugation and liquid-liquid extraction. SPE is one of the simplest and most effective methods, and affordable, disposable SPE cartridges that make the technique very user-friendly are available from a variety of manufacturers (Several newer models of SPE cartridges are described below.) Automated SPE systems also are available from a variety of vendors.

Despite the accessibility of user-friendly cartridges and more streamlined protocols for sample preparation, pressure to increase productivity and quality is being felt in the sample-preparation field. “As chromatography has become faster and more sensitive, it has put a lot of onus on getting the sample preparation right,” says Vivek Joshi, senior scientist in the technology development group at EMD Millipore. “In order to get the benefits out of these advances, sample preparation must also become faster and easier to perform. Scientists are more interested in getting their answers and data than spending time preparing samples.” The result is many recent advances in methods to increase throughput in sample preparation—while holding the quality high to keep pace with the increasing demands of UHPLC. Below are some new developments in sample preparation technologies and a few points to consider when choosing products in this developing area.

SPE cartridges and columns for different sample types

To match the myriad types of samples run through HPLC systems, there are a wide range of tools appropriate to different sample types. For example, Waters’ recently released Ostro™ Sample Preparation Plate removes multiple families of phospholipids from biological samples—up to 30 times more phospholipids than traditional removal devices or liquid-liquid extraction methods. In addition, the company’s Oasis® SPE products come in several types of chemistries, depending on the sample (for example, different types of SPE sorbents for bases, acids, strong bases and quaternary amines, strong acids or a mixture of several types). Waters’ Sep-Pak® bonded silica devices also come in a range of chemistries for HPLC and UHPLC preparation, in cartridges, 96-well plates and µElution plates.

Dionex also recently released a range of SPE cartridges that make it easier and faster to remove contaminants from samples before analyzing—hopefully leading to an even clearer chromatogram. The company’s five new types of SolEx™ SPE cartridges can be added online to an HPLC system to help remove contaminants such as pesticides or pharmaceuticals that can be present at low levels in water. These cartridges are designed for use with the Dionex AutoTrace® 280 SPE instrument as well as other SPE instruments.

Importance of filtration in sample prep

Filtering a sample is important for many reasons, foremost among which are clearer chromatographs and cleaner HPLC columns. “For HPLC sample prep, the most important thing is particle-free samples,” says Joshi. “That’s why filtration is a key sample-prep step prior to chromatography. When selecting a filtration device, the following things need to be considered: choice of membrane (based on chemical compatibility with the sample), membrane pore size (based on downstream analysis) and extractables/analyte binding (the lower the better).”

Syringe filters

Researchers who prepare a smaller number of samples per day (fewer than 10, for example) tend to use syringe filters to filter the samples individually. “Scientists preparing high volumes of samples (i.e., more than 100 samples per day) can use multi-well plates and associated robotics to speed up their sample preparation,” notes Joshi. “But for the majority of users (approximately 65% of scientists) who filter somewhere between 10 and 100 samples per day, neither of these solutions is optimal. Filtering dozens of samples sequentially is laborious, and robotics are prohibitively expensive for medium-volume users.”

Vacuum filtration system

EMD Millipore is filling this gap with the Samplicity Filtration System. “This vacuum-based system allows scientists to simultaneously filter up to eight samples directly into standard HPLC vials,” says Joshi. “The system is unique in that it is a vacuum-based filtration system that relieves the manual force and repetitive-motion stress associated with syringe filtration, one of the most common methods of sample filtration prior to HPLC analysis.”

More researchers are embracing UHPLC, and “this means filtration of the sample prior to injection is more critical, as UHPLC systems are less forgiving when solids are present compared to traditional HPLC systems,” says Navin Pathirana, global product manager at GE Healthcare. “Anyone using or switching to such a system should actively consider their filtration step. It is obviously important to choose a filter that functions correctly time after time (for example, without filter bypass) but [researchers] should also consider factors such as ease of use and automation.”

Filtration devices for an HPLC autosampler

GE Healthcare’s Whatman Mini-UniPrep™ is designed for an HPLC autosampler, replacing the syringe, syringe filter, vial, septum and cap. “The Mini-UniPrep essentially consists of two parts, an outer chamber and an inner plunger,” says Pathirana. “The sample is loaded into the outer chamber, and then the plunger is inserted and compressed. The action of compressing the plunger causes the sample to be filtered into the center of the plunger. Once fully compressed, the device can be loaded into the autosampler. Mini-UniPrep is the same size as a standard 2 ml vial. So any autosampler that takes a standard 2 ml vial can take the Mini-UniPrep.”

Syringe filters and filter plates for improved sample prep

Although it is true that well-filtered samples help to prolong the life of HPLC and UHPLC columns by reducing the amount of residue that accumulates within the packing of the column, filtering each sample individually is inefficient when working with large numbers of samples. There are numerous products available to increase the throughput of sample preparation. For example, for small numbers of samples, Pall’s new 0.2 µm Acrodisc® syringe filters were designed especially to preserve UHPLC columns, reducing extractables that can result in otherwise unexplained peaks on chromatograms. The company’s filters for UHPLC sample preparation are available in several membrane types, depending on the chemistries appropriate for the sample.

With larger numbers of samples, you might need to bump up to higher-throughput filtration to save time. Several manufacturers offer filter plates that use the common 96-well plate format to filter 96 samples simultaneously. Offerings include Pall’s AcroPrep 96 Filter Plates. Pall also offers an AcroPrep 24 Filtration System (which filters 24 samples simultaneously) for throughput that falls somewhere between individual samples and 96-well plates.

Automated sample preparation

A wide range of sample-preparation tools are available from Agilent, which recently released its Agilent 7696 Sample Prep Workbench, a new tool designed for automated sample preparation. “The 7696 is unique in that it supports off-line sample preparation for both liquid chromatography and gas chromatography techniques,” says Helmut Schulenberg-Schell, marketing manager for liquid chromatography in the Life Science Group at Agilent Technologies. “In addition, the new 1200 Infinity LC Valve Solutions offer a unique flexibility to configure for online cleanup and enrichment of analytes —unique valving technology suitable for UHPLC.”

Schulenberg-Schell notes that automation makes sample preparation more precise and more productive, saves expensive HPLC-grade solvents and reduces exposure to hazardous reagents. “Sample preparation is fundamental to any analytical laboratory, and over half of the work is still done manually,” he says. It is likely that progressively more sample preparation will become automated to some degree, depending on the throughput. Users who run more than the occasional HPLC sample may have automated sample preparation in their future.

The image at the top of this article is EMD Millipore's Samplicity Filtration System.

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