The present invention relates to analytical chemistry, and in particular relates to sample preparation for molecular analysis.
In order to carry out molecular analysis (the task of identifying one or more compounds in a sample) of any product, a sample of the product must be in such a form that it can be easily analyzed by chromatography, spectroscopy, mass spectroscopy and/or nuclear magnetic resonance instrumentation
Because these analytical instruments require substantially pure isolated analytes, some intermediate slops, generally referred to as “sample preparation”, must be carried out to isolate the compounds of interest from the sample matrix in which they might be found and prepare them for analysis by instrumentation.
The task of identifying one or more compounds in a sample—presents an enormously larger set of possibilities and challenges related to sample preparation. The number of “naturally occurring” compounds (those produced by plants or animals) is immeasurably large, and the capabilities of modern organic and inorganic synthesis have generated—figuratively or literally—a similar number of synthetic compounds.
There is tremendous interest in identification or quantitative measurement for compounds of interest as it relates to industrial processes, and for environmental testing for contaminants in waste water, soil, and air.
Even a small group of recognizable representative samples would include pesticides in food, other synthetic chemicals in food (antibiotics, hormones, steroids), synthetic compositions (benzene, toluene, refined hydrocarbons) in soil, and undesired compositions in everyday items (e.g., Bisphenol-A (“BPA”) in polycarbonate bottles and other plastic food packaging.
In general extraction has been a main form of sample preparation; i.e., drawing one or more compounds of interest from a sample by mixing the sample with a solvent into which the desired compound(s) will be extracted from the sample so that it can be measured by an analytical technique.
For several generations (and continuing to date), sample preparation in the form of extraction has been carried out by the well-understood Soxhlet method which was invented in the 19th century. In the Soxhlet technique, a single portion of solvent circulates repeatedly through a sample matrix until extraction is complete. To the extent, the Soxhlet method has an advantage, it allows an extraction to continue on its own accord for as long as the boiling flask is heated and the condenser is cold.
This method of extraction can take hours to completely extract the compounds of interest. Other concerns of safely from flammable solvents, hazardous waste and breakable glassware are significant drawbacks to this method.
Another commonly known extraction method uses ultra-sonication; i.e., the irradiation of a liquid sample with ultrasonic (>20 kHz) waves resulting in agitation. Although ultra-sonication has an advantage of speeding up the extraction process the disadvantages are that it is a labor intensive, manual process and uses large amounts of solvents.
In more recent years analytical scale microwave-assisted extraction (MAE) has been utilized. MAE uses microwave energy to heat solvents in contact with a sample in order to partition analytes from the sample matrix into the solvent. The main advantage of MAE is the ability to rapidly heat the sample solvent mixture. When using closed pressurized-vessels the extraction can be performed at elevated temperatures that accelerate the extraction of the compounds of interest from the sample matrix. MAE accelerates the extraction process, but has its disadvantages as well. In the microwave healing process typically a polar solvent is needed to provide dipole rotation and ionic conduction through reversals of dipoles and displacement of charged ions present in the solute and the solvent, limiting non-polar solvent use. MAE uses expensive, high-pressure vessels that do not provide a means of filtering the extract, and they must be cooled before pressure can be released.
In the 1990's automated apparatuses for the extract ion of analytes were developed. These apparatuses incorporated solvent extraction in pressurized cells under elevated temperatures and pressures and are referred to as “Pressurized Fluid Extraction” (“PFE”) or “Accelerated Solvent Extraction” (“ASE”). PFE has shown to be similar to Soxhlet extraction, except that, the solvents are at elevated temperatures where they exhibit high extraction properties. This procedure was first developed by Dionex (Richter DE et al., Anal Chem 1996, 68, 1033). One such PFE automated extraction system (Dionex ASE) is commercially available.
PFE was initially used for environmental contaminants (EPA Method 3545, herbicides, pesticides, hydrocarbons) in soil, sediments and animal tissues but has expanded to use in foods, pharmaceutical products and other biological samples.
PFE provides an efficient extraction, but still has not overcome the major bottlenecks associated with the many steps necessary to prepare a sample for analysis. PFE utilizes multiple-component cells and many steps. The cells are tightly packed with the sample and other packing material to eliminate any void areas in the cell, enhance separation, and avoid channeling. Preparing a cell for analysis can typically take 15 minutes. The cells are pre-pressurized at pressures up to 1500 psi and heated up to 200° C. prior to adding the solvent. Extraction is based on chromatographic principles to force the hot solvent through the column. Cycle times can take up to 20 minutes and the requirements of high pressure lead to secondary disadvantages with respect to cost and maintenance.
Newer PSE or ASE techniques attempt to address some of these difficulties, but still require that the cells be tightly packed, adding to the complexity and overall time required for each extraction.
Sample preparation, although having developed over the years, nevertheless remains the major bottleneck in molecular analysis. Accordingly, although the Soxhlet, Ultrasonication, MAE and PFE techniques have their advantages, each remains relatively time-consuming. As a result, when multiple samples are required or desired to provide necessary or desired information, the time required to carry out any given extraction-based molecular preparation step reduces the number of samples that can be prepared in any given amount of time, thus reducing the amount of information available in any given time interval. To the extent that, measurements are helpful or necessary in a continuous process, this represents a longer gap between samples or before an anomalous or troublesome result can be identified.
In recent decades, advances in liquid chromatography have led to analogous uses of packed columns in a technique referred to as solid phase extraction (“SPE”). Originally, chromatography was used to separate fractions in mixed samples for analytical purposes, and indeed it still serves this purpose very very well.
In SPE, the chromatography technique is modified to extract an anal vie from a matrix. Nevertheless, SPE fundamentally remains liquid chromatography technique in which molecules spread out (travel at different speeds) within a column based on their polarity, the particle size and polarity of the packed column (stationary phase), the polarity of the flowing liquid (mobile phase), the size (length and diameter) of the column and specific factors such as “hold-up volume,” “linear velocity,” and “flow rate.” See, e.g.. Arsenault, J. C. 2012. Beginner's Guide to SPE. Milford Mass.: Waters Corporation. (Arsenault 2012).
Although SPE is useful, it has limiting characteristics, some of which include the following factors. First, a proper description of SPE is “liquid-solid phase extraction” because the sample matrix that holds the analyte is almost always a liquid.
Second, because SPE is essentially a liquid chromatography technique, it requires either column packing steps or a new column for each test, along with a potential pre-swelling step depending upon the material selected or required for the stationary phase. SPE typically requires different methods and manipulative steps for different analytes.
Third a more deliberate (slower) flow through the packed column tends to produce belter separation among the fractions. Thus, in a very real sense slower SPE is better than faster SPE.
Finally, if an additional driving force (i.e., in addition to simple gravity flow) is required to move solvent through the SPE column, an external liquid or gas pump, or a centrifuge, or a vacuum pull must be incorporated, which in turn increases, to some lesser or greater amount, the complexity of the technique and any supporting systems.
More recently, a dispersive solid phase extraction (“dSPE”) method referred to as “Quechers” or “QuEChERS” (“quick-easy-cheap-effective-rugged-safe”) has become a standard for extraction preparation of molecular samples. Dispersive SPE addresses some of the disadvantages of SPE, but still requires an extraction step, the adjustment of pH with an appropriate ionic salt, is labor-intensive (even if advantageous compared to other methods), and requires two separate centrifuge steps.
Quechers is in many ways less complex than Soxhlet extraction, but still requires a multi-step process. In the literature, this is sometimes called a “three step process”(e.g., Paragraph 0153 of U.S. Patent Application Publication No. 20160370357), but in reality Quechers requires at least the following: homogenization of the matrix that contains the analyte of interest; adding extraction solvent; band agitation; buffering; a second agitation step; a centrifuge separation step; decanting; dispersive solid phase extraction (“dSPE”) clean up; a second centrifuge separation step; and decanting the supernatant liquid following the centrifuge step.
In addition to the multi-step handling and transfer of the solvent, the sample, and the various mixtures, each of the centrifuge steps takes a recommended five minutes; so that the full Quechers sample preparation takes at least about 15-20 minutes.
Accordingly, although the Soxhlet, SPE, and Quechers (dSPE) methods have their advantages, each remains relatively time-consuming. As a result, when multiple samples are required or desired to provide necessary or desired information, the time required to carry out any given extraction-based molecular preparation step reduces the number of samples that can be prepared in any given amount of time, thus reducing the amount of information available in any given time interval. To the extent that measurements are helpful or necessary in a continuous process, this represents a longer gap between samples or before an anomalous or troublesome result can be identified.
In summary, among other disadvantages current sample preparation techniques are slow, require a large number of separate steps, use excess solvent, are difficult to automate, and operate under high liquid pressure.
Accordingly, a need continues to exist for efficient rapid extraction-based molecular preparation techniques.