The analytical pyrolysis technique hyphenated to gas chromatography–mass spectrometry (GC–MS) has extended the range of possible tools for the characterization of synthetic polymers and copolymers. Pyrolysis involves thermal fragmentation of the analytical sample at temperatures of 500–1400 °C. In the presence of an inert gas, reproducible decomposition products characteristic for the original polymer or copolymer sample are formed. The pyrolysis products are chromatographically separated using a fused-silica capillary column and are subsequently identified by interpretation of the obtained mass spectra or by using mass spectra libraries. The analytical technique eliminates the need for pretreatment by performing analyses directly on the solid or liquid polymer sample. In this article, application examples of analytical pyrolysis hyphenated to GC–MS for the identification of different polymeric materials in the plastic and automotive industry, dentistry, and occupational safety are demonstrated. For the first time, results of identification of commercial light-curing dental filling material and a car wrapping foil by pyrolysis–GC–MS are presented.

Structural analysis and the study of degradation properties are important to understand and improve performance characteristics of synthetic polymers and copolymers in many industrial applications. Traditional analytical techniques used for characterization of polymers and copolymers such as thermal analysis and Fourier transform infrared (FT–IR) spectroscopy have limitations or are not sufficiently sensitive (1). Pyrolysis techniques hyphenated to gas chromatography–mass spectrometry (GC–MS) have extended the range of possible tools for the characterization of synthetic polymers and copolymers. Under controlled conditions, at elevated temperatures (500–1400 °C) in the presence of an inert gas, reproducible decomposition products characteristic for the original polymer or copolymer sample are formed. The pyrolysis products are chromatographically separated using a fused-silica capillary column and subsequently identified by interpretation of the obtained mass spectra or by using mass spectra libraries (such as the National Institute of Standards and Technology [NIST] or Wiley). Pyrolysis methods eliminate the need for pretreatment by performing analyses directly on the solid polymer or copolymer sample (1). (Please note that this article was presented at the XVII European Conference on Analytical Chemistry, which was held in Warsaw, Poland, on 25–29 August 2013).  

Most of the thermal degradation results from free radical reactions initiated by bond breaking and depends on the relative strengths of the bonds that hold the molecules together. A large molecule will break apart and rearrange in a characteristic way (2–4). If the energy transfer to the sample is controlled by temperature, heating rate, and time, the fragmentation pattern is reproducible and characteristic for the original polymer or copolymer. Another sample of the same composition, heated at the same rate to the same temperature for the same period of time, will produce the same decomposition products. Therefore, the essential requirements of the apparatus in analytical pyrolysis are reproducibility of the final pyrolysis temperature, rapid temperature rise, and accurate temperature control. Depending on the heating mechanism, pyrolysis systems have been classified into two groups: the continuous-mode pyrolyzer (furnace pyrolyzer) and pulse-mode pyrolyzer (flash pyrolyzer, such as the heated filament, Curie-point, and laser pyrolyzer). The pyrolysis unit is directly connected to the injector port of a gas chromatograph. A flow of an inert carrier gas, such as helium, flushes the pyrolyzates into the fused-silica capillary column. Figure 1 shows the schematic view of the furnace pyrolyzer used in our investigation. The detection technique of the separated compounds is typically MS, but other GC detectors have also been used depending on the intentions of the analysis (1,4).  

Figure 1: Schematic view of the furnace pyrolyzer used in this study.			

The applications of analytical pyrolysis–GC–MS range from research and development of new materials, quality control, characterization and competitor product evaluation, medicine, biology and biotechnology, geology, airspace, and environmental analysis to forensic purposes or conservation and restoration of cultural heritage. These applications cover analysis and identification of polymers, copolymers, and additives in components of automobiles, tyres, packaging materials, textile fibres, coatings, half-finished products for electronics, paints or varnishes, lacquers, leather, paper or wood products, food, pharmaceuticals, surfactants, and fragrances.  

Our earlier publications (1,5–12) presented the analysis and identification of degradation products of commercially available synthetic polymers and copolymers by using analytical pyrolysis hyphenated to gas chromatography with flame ionization detection (GC–FID) and GC–MS. In this work, new examples of applications of this analytical technique for the identification of different polymeric materials are demonstrated. 

: Plastic particles from industrial filter fins, a car wrapping foil, unknown fibres, and commercial light-curing dental filling material were used in the investigation. 

Instrumentation and Analytical Conditions

: Approximately 100–200 µg of solid sample was cut out with a scalpel and inserted without any further preparation into the bore of the pyrolysis solids-injector and then placed with the plunger on the quartz wool of the quartz tube of the furnace pyrolyzer Pyrojector II (SGE Analytical Science). Three spots on each sample were analyzed in duplicate. The pyrolyzer was operated at a constant temperature of 550, 600, 700, or 900 °C. The pressure of helium carrier gas at the inlet to the furnace was 95 kPa.  

Pyrolysis–GC–MS measurements were made using two apparatus. In the first apparatus (1), the pyrolyzer was connected to a Trace 2000 gas chromatograph (ThermoQuest, CE Instruments) with a quadrupole mass spectrometer Voyager (ThermoQuest, Finnigan, MassLab Group) operated in electron ionization (EI) mode. A 60 m × 0.25 mm, 0.25-µm Elite-5ms fused-silica GC capillary column (PerkinElmer Instruments) was used. The GC conditions were as follows: programmed column temperature: 60 °C for 1 min, then 60–100 °C at 2.5 °C/min, 100–280 °C at 10 °C/min (20-min hold at 280 °C). The temperature of the split–splitless injector was 250 °C and the split flow was 50 cm

/min. Helium, grade 5.0 (Westfalen AG), was used as a carrier gas. The helium programmed pressure was 70 kPa for 1 min, then 70–110 kPa at 1 kPa/min (hold at 110 kPa to the end of analysis) was used. The transfer line temperature was 280 °C. The MS EI ion source temperature was kept at 250 °C. The ionization occurred with a kinetic energy of the impacting electrons of 70 eV. The current emission of the rhenium filament was 150 µA. The MS detector voltage was 350 V. Mass spectra and reconstructed chromatograms (total ion current [TIC]) were obtained by automatic scanning in the mass range

35–450 u. Pyrolysis–GC–MS data were processed with the Xcalibur software (ThermoQuest) and the NIST 05 mass spectra library. 

In the second apparatus (2), the pyrolyzer was connected to a 7890A gas chromatograph with a series 5975C quadrupole mass spectrometer (Agilent Technologies Inc.) operated in EI mode. A 59 m × 0.25 mm, 0.25-µm 

 DB-5ms fused-silica GC capillary column (J&W Scientific) was used. Helium, grade 5.0 (Westfalen AG), was used as a carrier gas. The GC conditions were as follows: programmed column temperature: 60 °C for 1 min, then 60–280 °C at 7 °C/min (hold at 280 °C to the end of analysis); programmed helium pressure: 122.2 kPa for 1 min, then 122.2–212.9 kPa at 7 kPa/min (hold at 212.9 kPa to the end of analysis). Second set of GC conditions: programmed column temperature: 75 °C for 1 min, then 75–280 °C at 7 °C/min (hold at 280 °C to the end of analysis); programmed helium pressure: 122.2 kPa for 1 min, then 122.2–212.9 kPa at 7 kPa/min (hold at 212.9 kPa to the end of analysis). 

The temperature of the split–splitless injector was 250 °C and the split ratio was 50:1. The transfer line temperature was 280 °C. The MS EI ion source temperature was kept at 230 °C. The ionization occurred with a kinetic energy of the impacting electrons of 70 eV. The quadrupole temperature was 150 °C. Mass spectra and reconstructed chromatograms (total ion current) were obtained by automatic scanning in the mass range 

 35–750 u. Pyrolysis–GC–MS data were processed with the ChemStation software (Agilent Technologies) and the NIST 05 mass spectra library.  

Figure 2: PyrolysisâGCâMS chromatogram of plastic particles from industrial filter fins at 700 Â°C obtained with apparatus 1. Fused-silica GC capillary column: 60 m Ã 0.25 mm, 0.25-Âµm df Elite-5ms. GC conditions: programmed column temperature: 60 Â°C for 1 min, then 60â100 Â°C at 2.5 Â°C/min and then 100â280 Â°C at 10 Â°C/min (20 min hold at 280 Â°C); splitâsplitless injector temperature: 250 Â°C; split flow: 50 cm3/min; helium programmed pressure: 70 kPa for 1 min, then 70â110 kPa at 1 kPa/min (hold at 110 kPa to the end of analysis). For peak identification, see Table 1. 			

Pyrolysis–GC–MS of Plastic Particles from Industrial Filter Fins

: A sample of plastic particles from industrial filter fins was pyrolyzed at 700 °C to identify its composition. Figure 2 shows the obtained pyrolysis–GC–MS chromatogram of the sample. Based on the decomposition products summarized in Table 1, the plastic particles were identified as a mixture of poly(hexamethylene adipamide) (nylon 6-6) and polypropylene glycol with a small amount of styrene–butadiene rubber (SBR). The peaks of propylene and acetone indicate the presence of polypropylene glycol. The main decomposition product of nylon 6-6 is cyclopentanone (retention time [

] = 10.51 min). Other peaks in Figure 2, like butene/1,3-butadiene (

 = 14.39 min), are typical pyrolysis products of SBR (1,2,5,6,18). The small peak of 4-isopropylphenol (

 = 28.15 min) may be a clue to the presence of polycarbonate or bisphenol A epoxy resin (5,6). All of the pyrolysis products and the materials identified from pyrolysis products in filter fins are summarized in Table 1.  

Table 1: Pyrolysis products and identified materials in plastic particles from industrial filter fins.			

Pyrolysis–GC–MS of a Car Wrapping Foil

: The next object of identification was a car wrapping foil pyrolyzed at 600 °C. Figure 3 shows the obtained pyrolysis–GC–MS chromatogram of the car wrapping foil. Based on the decomposition products summarized in Table 2, the plastic material was identified as a mixture of flexible poly(vinyl chloride) (PVC) with bis(2-ethylhexyl) phthalate (BEHP) plasticizer and poly(hexamethylene adipamide) (nylon 6-6). The chromatogram in Figure 3 shows the typical pyrolysis products of PVC, like hydrogen chloride (

 = 25.93 min). This is the result of the formation of double bonds by the elimination of hydrogen chloride from the poly(vinyl chloride) macromolecules, followed by the breaking of the carbon chain with or without cyclization reaction (2). The detected cyclopentanone (

 = 10.46 min) is generally known as a characteristic pyrolysis product of nylon 6-6 (2,3,6). Methyl methacrylate (

 = 8.20 min) identified in pyrolyzate is formed from poly(methyl methacrylate) (6) and most likely comes from the adhesive film. Thus, the identified 3,3-diphenylacrylonitrile (

 = 36.69 min) may be from the adhesive layer of the foil. 

Figure 3: PyrolysisâGCâMS chromatogram of a car wrapping material at 600 Â°C obtained with apparatus 1. Fused-silica GC capillary column 60 m Ã 0.25 mm, 0.25-Âµm df Elite-5ms. GC conditions: programmed column temperature: 60 Â°C for 1 min, then 60â100 Â°C at 2.5 Â°C/min and then 100â280 Â°C at 10 Â°C/min (20 min hold at 280 Â°C); splitâsplitless injector temperature: 250 Â°C; split flow: 50 cm3/min; helium programmed pressure: 70 kPa for 1 min, then 70â110 kPa at 1 kPa/min (hold at 110 kPa to the end of analysis). For peak identification, see Table 2.			

The thermal decomposition of the plasticizer bis(2-ethylhexyl) phthalate identified in car wrapping foil leads to the formation at 600 °C of 2-ethyl-1-hexene (

 = 28.63 min) (1,7). In the car wrapping material, the rest of the tetrahydrofuran solvent (

 = 6.99 min) was also detected. Table 2 shows the identified ingredients of the pyrolyzed car wrapping foil.  

Table 2: Pyrolysis products and identified materials in car wrapping foil.			

Identification of Unknown Plastic Fibres: 

A sample of unknown plastic fibres was pyrolyzed at 700 °C and 900 °C, respectively, to identify its composition. Figure 4 shows the pyrolysis–GC–MS chromatogram of the sample pyrolyzed at 900 °C. Based on the decomposition products summarized in Table 3, the fibres were identified as polyaramid [poly(

-phenylene terephthalamide)] (Figure 5). The main identified degradation products of polyaramid at 900 °C are benzene (

 = 11.81 min). Currently, polyaramid fibres have only been characterized in a few publications using thermal analysis (thermogravimetry, derivative thermogravimetry, and differential thermal analysis), infrared spectroscopy techniques (13–15), and pyrolysis–GC–MS (2,16–19).  

Figure 4: PyrolysisâGCâMS chromatogram of polyaramid fibers at 900 Â°C obtained with apparatus 2. Fused-silica GC capillary column: 59 m Ã 0.25 mm, 0.25-Âµm df DB-5ms. GC conditions: programmed column temperature: 75 Â°C for 1 min, then 75â280 Â°C at 7 Â°C/min (hold to the end of analysis); programmed pressure of helium carrier gas: 122.2 kPa for 1 min, then 122.2â212.9 kPa at 7 kPa/min (hold at 212.9 kPa to the end of analysis). For peak identification, see Table 3. 			

Polyaramid fibres are a class of heat-resistant, strong synthetic fibres. They are used in aerospace and military applications for ballistic-rated body armour, fabric, ballistic composites, and fire fighters protective clothing as well as in bicycle tyres and as an asbestos substitute. 

Table 3: Pyrolysis products of polyaramid fibres. 			

Identification of Commercial Light-Curing Dental Filling Material

: A number of dental filling materials are presently available for tooth restorations. The four main groups of these materials, which dentists have used for about 35 years, are the conventional glass-ionomer cements, resin-based composites, resin-modified glass-ionomer cements, and polyacid-modified resinous composites (20). Light-curing glass-ionomer cements contain polyacrylic acid, chemically or photo-curing monomers (multifunctional methacrylates, like triethylene glycol dimethacrylate or 2-hydroxyethyl methacrylate), an ion-leaching glass, and additives (initiators, inhibitors, stabilizers, and others) (20). Resin-modified glass-ionomer cements are now widely used in dentistry as direct filling materials, liners, bases, luting cements, and fissure sealants (21). These materials mainly consist of polymer matrix and glass-ionomer parts. The polymer matrix is based on a monomer system and different multifunctional methacrylates with additives (21). Methacrylic monomers, such as bisphenol A glycidyl methacrylate (Bis-GMA), urethane dimethacrylate (UDMA), triethylene glycol dimethacrylate (TEGDMA), and 2-hydroxyethyl methacrylate (HEMA), are the main components of resin-based dental filling materials. The presence of additives such as initiators, activators, inhibitors, and plasticizers in uncured dental material mixture is necessary (21).   

Figure 5: Chemical structure of poly(p-phenylene terephthalamide) (polyaramid).			

Figure 6 shows the total ion current pyrolysis–GC–MS chromatogram of commercial light-curing dental filling material pyrolyzed at 550 °C. The pyrolysis products identified by using mass spectra library NIST 05 are summarized in Table 4. The carbon dioxide (

 = 6.85 min) identified in pyrolyzate is formed from polyacrylic acid (2,18). The identified substances 2-hydroxyethyl methacrylate (HEMA) (

 = 13.65 min), ethylene glycol dimethacrylate (EGDMA) (

 = 19.48 min), and triethylene glycol dimethacrylate (TEDMA) (

 = 28.72 min) are known as standard composites of dental filling materials (1). Other compounds in Table 4, such as bisphenol A (

 = 33.10 min) or bisphenol A diglycidyl ether (

 = 42.42 min), are probably formed by thermal degradation of bisphenol A diglycidyl mono- or dimethacrylates. The presence of the additives, such as the antioxidant 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT) (

 = 23.17 min) or the UV-absorber drometrizol (

 = 31.95 min) was also confirmed. The triphenylantimony (

 = 34.55 min) identified in pyrolyzate is used as catalyst in the UV-induced polymerization (1). 

Figure 6: PyrolysisâGCâMS chromatogram of commercial light-curing dental filling material at 550 Â°C obtained with apparatus 2. Fused-silica GC capillary column: 59 m Ã 0.25 mm, 0.25-Âµm df DB-5ms. GC conditions: programmed column temperature: 60 Â°C for 1 min, then 60â280 Â°C at 7 Â°C/min (hold at 280 Â°C to the end of analysis); programmed helium pressure: 122.2 kPa for 1 min, then 122.2â212.9 kPa at 7 kPa/min (hold at 212.9 kPa to the end of analysis). For peak identification, see Table 4.			

Analytical pyrolysis–GC–MS has been proven as a valuable technique for the analysis and identification of organic polymeric materials in the plastic and rubber industry. For the first time pyrolysis–GC–MS was used for the identification of commercial light-curing dental filling material and for the identification of a car wrapping foil. This technique allows the direct analysis of very small sample amounts (5–200 µg) without the need for time-consuming sample preparation. 

Table 4: Pyrolysis products of commercial light-curing dental filling material. 			

Peter Kusch, Gerd Knupp, Wolfgang Fink, Dorothee Schroeder-Obst, and Johannes Steinhaus

 are with Hochschule Bonn-Rhein-Sieg, University of Applied Sciences in the Department of Applied Natural Sciences, in Rheinbach, Germany. 

 is with Dr. Obst Technische Werkstoffe GmbH, in Rheinbach, Germany.  

Advanced Gas Chromatography – Progress in Agricultural, Biomedical and Industrial Applications

, M.A. Mohd, Ed. (InTech, Rijeka, Croatia, 2012), pp. 343–362.  

Analytical Pyrolysis of Synthetic Organic Polymers

 (Elsevier, Amsterdam, The Netherlands, 2005). 

 (CRC Press, Boca Raton, Florida, USA, 2007). 

(4) K.L. Sobeih, M. Baron, and J. Gonzales-Rodrigues, 

(6) P. Kusch, G. Knupp, and A. Morrisson, in 

, R.K. Bregg, Ed. (Nova Science Publishers, New York, New York, USA, 2005), pp. 141–191. 

(8) P. Kusch, V. Obst, D. Schroeder-Obst, W. Fink, G. Knupp, and J. Steinhaus, 

(10) P. Kusch, V. Obst, D. Schroeder-Obst, G. Knupp, and W. Fink, 

(12) P. Kusch, W. Fink, D. Schroeder-Obst, and V. Obst, 

(13) S. Villar-Rodil, A. Martínez-Alonso, and J.M.D. Tascón, 

(14) F. Suárez-García, A. Martínez-Alonso, and J.M.D. Tascón,

Articles

Features

The pyrolysis–GC–MS method enables direct analysis of solid or liquid polymers without sample pretreatment, as illustrated here for various materials, including a dental filling material and a car wrapping foil.

Application of Pyrolysis–Gas Chromatography–Mass Spectrometry for the Identification of Polymeric Materials

Sample preparation has often been viewed as the bottleneck in analytical procedures. Surveys have shown that time is typically the most frequent problem area for sample preparation procedures and that analysts can easily spend a majority of the total analysis time on sample preparation. While newly developed extraction techniques address time, modern chromatography advances are also moving towards faster separations. Based on these considerations, what is high-throughput sample preparation? Do modern extraction methods adequately address the issue of time? How can we address the analytical need for speed?

The self-proclaimed world's greatest rock and roll band famously sang, "You can't always get what you want." It's easy to apply the Rolling Stones admonition to analytical sample preparation because surveys (1–3) have consistently shown that time, along with cost and solvent use, is among the most significant desires of analysts. Traditional sample preparation methods are often the rate-limiting step in the overall sample analysis process. We can envision three approaches for addressing the desire for high-throughput sample preparation: parallel sample processing, automation, and improvements in the process kinetics. 

Evolution of High-Throughput Analysis from Microplates

Over the past several years, we've heard increasing calls for high-throughput sample preparation. But what is high-throughput sample preparation? It many cases, high-throughput stems from the high-throughput screening approach to combinatorial chemistry. This approach is the most established, based on the 96-well plate format. These microplates have been established for two to three decades. Key considerations for the acceptance of this approach are standardization of microplate dimensions and attributes by the Society for Biomolecular Screening and development of ancillary devices like repeating pipettes, vacuum manifolds, and more. Countless vendors are involved. Sample preparation and analysis using microplates includes liquid–liquid extraction (LLE), protein precipitation, solid-phase extraction (SPE), fluorescence, matrix-assisted laser desorption–ionization mass spectrometry (MALDI–MS), and separations. 

Wells (4) presented a list of some of the uses of filtration microplates in pharmaceutical development, including: 					

clarification of acid, base, or organic digests of plant materials in medicinal chemistry,

solution-phase synthesis in combinational chemistry using resin scavenger media,

membrane-based proteolytic digestion before MALDI–MS,

filtration of plasma or serum samples in combination with direct injection techniques,

filtration of reconstituted extracts, and

solid-supported LLE, SPE, or exclusion chromatography.

As we can see, the development of 96-well plates and the major applications of this approach are in bioanalysis of liquid samples. This is because the transfer of liquid samples from analytical operation to another is somewhat straightforward. Recently, Scoffin (5) reviewed liquid transfer in high-throughput systems. These liquid handling systems operate either by air displacement or positive displacement and can reliably work with microlitre volumes of volatile or viscous liquids. Other combinations of liquid handling and sorbent-based extraction, including solid-phase microextraction (SPME), automated disposable pipette extraction, and microextraction by packed sorbent have also been used in high-throughput approaches (6,7). By combining these solvent-free, sorbent-based extractions with syringe-needle or pipette configurations, direct extraction and liquid transfer is accommodated. 

Turbulent Flow Chromatography in Sample Preparation

In recent years, turbulent flow chromatography (TFC) has emerged as a high-speed extraction alternative for liquid samples. When flow rates increase significantly, the flow pattern shifts from a laminar, bullet-shaped profile to a turbulent profile. An example of this flat, turbulent flow profile is shown in Figure 1. Turbulent flow minimizes zone spreading and, most importantly for sample preparation purposes, facilitates radial diffusion (that is, mass transfer) to the stationary phase. Two years ago, sample preparation applications of TFC were discussed in this column (8). To achieve turbulent flow conditions, large, porous particles are combined with high flow rates. In general, 30–60 μm particles packed in 0.5–1.0 mm i.d. columns with flow rates greater than 1 mL/min and up to 6 mL/min achieve these conditions. Dramatic reductions in time are observed, while maintaining accuracy, precision, detection limits, and other figures of merit. Low-molecular-weight compounds readily diffuse into the porous particles, whereas larger compounds are less retained and are eluted to waste. Proteins are reported to be more selectively removed from biosamples using TFC than restricted-access media (RAM) or solid-phase extraction, as illustrated in Figure 2 (9). Thus, small molecule analytes in samples like whole blood, serum, plasma, or urine can be delivered to the injection port when coupled to high performance liquid chromatography (HPLC) in an on-line manner using column switching. Although minimal sample handling is necessary, resulting in fast analysis, the high flow rates necessary to achieve turbulent flow result in a large solvent consumption. Observed matrix effects require the use of standard addition. Recent reviews (10–13) have illustrated the use of TFC for biofluids, foods, and environmental samples.  

Figure 1: Example of turbulent flow profile.			

So far, we've focused the discussion of high-throughput sample preparation on liquid samples. The reasons are two-fold, as previously mentioned. Liquid handling is easier than manipulation of solids and the microplate technology drove the development of high-throughput methods. How can we make the analysis of solid samples high-throughput? Do the more recently developed extraction techniques take us in that direction?  

Figure 2: Comparison of turbulent flow chromatography (TFC), restricted-access media (RAM), and solid-phase extraction (SPE) cleanup columns as a function of the molecular weight excluded during sample cleanup. Adapted from reference 9.			

During the time when 30–60 min chromatography runs were standard, Soxhlet extractions took 6–48 h. This bottleneck was even further exacerbated when spectroscopy or other rapid analytical procedures were used. During the past 20 years or so, several automated techniques (including supercritical fluid extraction [SFE], microwave-assisted extraction [MAE], and pressured liquid extraction [PLE]) have been developed, which provide the hope for high-throughput sample analysis. In addition to automation of the extraction procedure, these methods are characterized by very rapid extraction times compared with the traditional methods. However, simultaneously to the development of these techniques, advances in sensors, mass spectrometry, and chromatography have rendered even these modern technologies to remain the rate-limiting part of an analytical procedure. Sample preparation that takes 20 min, while several-fold faster than Soxhlet or shake-flask methods, are still slow compared with 5 min analyses. In the days of 6–48 h Soxhlet extraction and 30–60 min chromatographic analysis, the ratio of sample preparation time to chromatography run time was 6–96:1. However, it is common to perform, for example, 12 Soxhlet extractions simultaneously, so the ratio becomes 0.5–8:1. SFE, PLE, and MAE extractions commonly take 20 min, but have we really gained anything? It is now routine to perform chromatography with 10–30 min run times or faster. So the ratio remains 0.5–6:1. 

Figure 3: Relative percent of oil extracted from 725-Î¼m coffee particles as a function of time (min) using various extraction processes. Adapted from reference 15.			

The fundamental difference between extracting solid samples and liquid samples is the role of diffusion. Diffusion in liquids at room temperature is on the order of 10

/s, while diffusion in solids at the same temperature is on the order of 10

/s. But what about those solid extraction methods that operate at elevated temperatures? We have conducted a systematic comparison of SFE, MAE, PLE, and sonication extractions to examine the rate-limiting role of diffusion in these extraction methods. We extracted the oils from ground, roast coffee using these methods and determined the diffusion coefficients using the hot-ball model (14). Extractions were performed using dichloromethane at 100 °C under sub-optimal conditions, which would allow the determination of the diffusivity. Soxhlet extraction is performed at some elevated temperature near, but below, the boiling point of 39.8 °C, while the modified Soxhlet approach includes leaching in boiling solvent as the main portion of the extraction. Ultrasonic extractions are performed in open-vessels, so the bulk solvent cannot exceed the boiling point, but on a molecular level temperatures during cavitation can reach 5000 K. PLE and MAE in this work were performed at 100 °C. Figure 3 displays the kinetic curves from these extractions (15). When the hot-ball model is applied to this data, the diffusion coefficients shown in Table 1 are obtained. The results show that analyte diffusion, under similar extraction conditions applied across each method, were within the same order of magnitude. It seems unlikely that temperatures greater than those used in these techniques will be routinely applied for analytical extractions. Thus, when developing high-throughput extraction systems, the kinetics of extraction will not differentiate these alternative extraction procedures. PLE, MAE, and SFE seem to be the preferred approaches for developing high-throughput processing, but advantages with each technique will arise because of instrumental configurations. 

Table 1: Average diffusion coefficients obtained from the extraction of oils from ground, roast coffee.			

Is High-Throughput Sample Preparation Necessary?

As discussed, the major approaches to achieve high-throughput sample preparation stem from parallel sample processing, automation, and the kinetics of the sample processing procedure. The success observed in the high-throughput sample preparation of liquids comes, in part, from automation and parallel processing. The microplates process 96 or more samples simultaneously. The instrumental approaches to extracting solids are also configured for automation and parallel processing. The kinetics of extraction is fundamentally limited by practical considerations. So perhaps the best approaches for high-throughput analysis will result from the integration of methods. Just as turbulent flow chromatography is coupled to liquid chromatography (LC), thermal desorption is coupled with gas chromatography (GC), and 96-well microplates are coupled to MALDI–MS, similar methodologies combining rapid sample and processing parallel approaches will begin to address this need. However, the analyst must also keep in mind what is truly desired. High-throughput sample preparation is not the ultimate goal, but rather high-throughput analysis. Chemometrics and other statistical approaches can glean the desired information from minimal analysis, and dilute-and-shoot or direct analysis MS eliminate the need for sample preparation entirely, which is perhaps the fastest method of all.  

is an Associate Research Professor at South Dakota State University. His research interests include green chemistry, alternative solvents, sample preparation, high resolution chromatography, and bioprocessing in supercritical fluids. He earned his PhD in 1990 at Brigham Young University under the direction of Milton L. Lee. 

 is an analytical consultant and is a member of 

 editorial advisory board. Direct correspondence about this column should be addressed to "Sample Prep Perspectives", 

, 4A Bridgegate Pavilion, Chester Business Park, Chester, CH4 9QH, UK, or e-mail the editor-in-chief, Alasdair Matheson, at 

High Throughput Sample Preparation Methods and Automation Strategies

, (Elsevier, Amsterdam, The Netherlands, 2003) pp. 199–254. 

(6) J. Pereira, C.L. Silva, R. Perestrelo, J. Goncalves, V. Alves, and J.S. Camara, 

(8) J.L. Herman, T. Edge, and R.E. Majors, 

(9) O. Nunez, H. Gallart-Ayala, C.P.B. Martins, and P. Lucci, 

(10) J. Pan, C. Zhang, Z. Zhang, and G. Li, 

(11) O. Nunez, H. Gallart-Ayala, C.P.B. Martins, P. Lucci, and R. Busquets, 

(14) K.D. Bartle, A.A. Clifford, S.B. Hawthorne, J.J. Langenfeld, D.J. Miller, and R. Robinson, 

(15) J.L. Driver and D.E. Raynie, in preparation (2014). 

Cover Story

Sample preparation has often been viewed as the bottleneck in analytical procedures. Surveys have shown that time is typically the most frequent problem area for sample preparation procedures. While newly developed extraction techniques address time, modern chromatography advances are also moving towards faster separations. Based on these considerations, what is high-throughput sample preparation? Do modern extraction methods adequately address the issue of time? How can we address the analytical need for speed?

Exploring the Possibilities of High-Throughput Sample Preparation

Columns packed with superficially porous particles (SPPs) have created considerable excitement over the last few years. Indeed, this column technology manifests the advantages of fully porous material (loading capacity, retention) and some beneficial properties of nonporous particles (kinetic performance). This review provides an updated overview of the theory behind the success of SPP technology, trends, benefits, and limitations. It also summarizes the latest developments of sub-2-µm SPPs and instrumental constraints associated with their use. Finally, it describes several applications to illustrate the performance and the universal applicability of these newly engineered particles.

Superficially porous particles (SPPs) (also called core–shell, fused-core shell, partially porous, pellicular, or solid-core) are made of a solid, nonporous silica core surrounded by a porous shell layer with similar properties to those of the fully porous materials used in conventional high performance liquid chromatography (HPLC) columns. The "fused-core" terminology was originally introduced by Jack Kirkland to describe the manufacturing procedure that "fuses" a porous silica layer onto a solid silica particle (1). 

The very high efficiency achieved on columns packed with sub-3-µm SPPs, combined with convenient operating conditions (modest back pressures and the ability to use conventional HPLC instruments), has generated significant interest in the chromatographic community and widespread applications in many fields (2). Columns packed with sub-3-µm SPPs rival the efficiency of columns packed with sub-2-µm fully porous particles, but the former generate only half the back pressure. As a result, practitioners can use such columns on regular HPLC equipment, leading to the initial interest in these materials and their successful application. Moreover, further performance improvement is possible with very fine SPPs (1.3–1.7 µm), though the use of ultralow-dispersion ultrahigh-pressure liquid chromatography (UHPLC) systems (for example, extracolumn peak variance σ

) is mandatory. Faster analysis and higher efficiency is always desirable in liquid chromatography (LC), particularly to pharmaceutical scientists or researchers in life science wishing to attain higher productivity in the laboratory or more accurate analysis (higher resolution) of very complex samples. Reducing analysis time while maintaining resolution requires high kinetic performance (more separation power per unit time) using smaller particles, better particle morphology, or both. 

The initial intent of applying SPPs was to enhance kinetic performance in the analysis of large biomolecules such as therapeutic proteins. The rationale behind this concept was to improve column efficiency by shortening the diffusion path that molecules have to travel and, thus, improve their mass transfer kinetics (3,4). Shell-type (pellicular) particles were first developed by Horváth and colleagues in the late 1960s for the analysis of large molecules in ion-exchange mode (5). Shortly afterward, Kirkland demonstrated that superficially porous particles (pellicular materials) with 30–40 µm diameters could provide much better separations than totally porous ones (6). In 2001, a new column for fast protein or peptide analysis was introduced that was packed with a 5-µm SPP with a shell thickness of 0.25 µm.  

In 2007, a revolution started with the commercialization of a new generation of sub-3-µm SPPs adapted for separation of small and large molecules (7) by Advanced Materials Technology. This material possesses a 1.7-µm solid core covered by a 0.5-µm-thick shell of porous silica. It combined the advantages of both fully porous and nonporous particles. In particular, this improved particle design solved the problem of the low loading capacity of early pellicular particles because an approximately 75% volume fraction of these particles is still porous. Since then, many providers commercialized SPPs with particle sizes ranging between 1.3- and 5-µm. Table 1 provides a current list of commercially available columns packed with SPP materials, their pertinent properties, and available bonded phases.  

Table 1: A current list of HPLC columns made with commercially available SPP materials including the newest sub-2-Âµm SPPs.			

Column Performance: The Impact of Shell Thickness

In 1956, the famous van Deemter equation was proposed to describe column performance (plate height) as a function of linear velocity (8). Since then, several plate height and rate models have been derived for LC by numerous researchers. Knox suggested a three-term equation to describe the dependency of the theoretical plate height (

) of a column as a function of linear velocity (

 are constants, determined by the magnitude of band broadening because of eddy dispersion, longitudinal diffusion, and mass transfer resistance, respectively. In practice, the 

 term is the sum of contributions from resistance to both trans-particle mass transfer (

The application of reduced parameters is common in engineering to compare the performance of columns in normalized units. It is useful to convert 

 to a dimensionless parameter such as reduced plate height (

 is particle diameter. The advantage of this approach is the ability to compare the performance of columns packed with particles of different sizes or structures (

). With the use of the reduced parameters, equations similar to equation 1 can be written. Therefore, the total reduced plate height (

) can be written as the sum of at least four different contributions: 

are the reduced plate height contributions from eddy dispersion, longitudinal diffusion, trans-particle mass transfer, and external film mass transfer, respectively. 

The initial idea of preparing SPPs was to increase column efficiency by reducing mass transfer resistance across particles (

). However, it turned out that trans-particle mass transfer resistance is far from being the main contributor to band broadening in LC for small molecules (especially for modern columns packed with small particles) (3). The enhanced performance of SPP materials clearly lies in contributions from other factors.  

(the diffusion speed inside the particle pores) of an SPP depends on the ratio of the diameter of the solid core to that of the SPP (

). As this ratio (ρ) increases, the mass transfer kinetics becomes faster through the particles. Similarly, the external mass transfer (

) process depends on the particle's structure. It has been demonstrated that mass transfer kinetics is mainly affected by the external film mass transfer resistance across the thin layer of the mobile phase surrounding the external surface area of the particles (3). The trans-particle mass transfer resistance is important for large molecules when the diffusion time inside the particles is much longer, but not so critical for small molecules. As far as diffusion time is concerned, the 2.7-µm Halo (Advanced Materials Technology) and Poroshell 120 (Agilent Technologies) phases are equivalent to a 1.8-µm fully porous packing material, while the 2.6-µm Kinetex material (Phenomenex) is equivalent to a 1.3-µm fully porous stationary phase (10). 

Additionally, the presence of a solid core within an SPP has a direct impact on longitudinal diffusion (the B term of the van Deemter equation). It decreases the B term contribution to the plate height by about 20% when the ratio of the core to the particle diameter (ρ) is 0.63 (for example, Halo [Advanced Materials Technology], Ascentis Express [Sigma-Aldrich], BrownLee SPP [PerkinElmer]) (3,4). However, the improvement in overall efficiency is not only because of the reduced internal particle porosity of SPPs: It was experimentally determined that the solid core reduced longitudinal diffusion by about 30% in comparison with fully porous particles (11). This reduction in longitudinal diffusion produces only a ~10% increase in total overall column efficiency compared with columns packed with fully porous particles.  

Figure 1(a) shows the theoretical reduction in reduced plate heights (

) on a relative scale for trans-particle mass transfer (

) as a function of particle structure (ρ) (4). Theoretically, on average about ~40–60% lower 

 values are expected of current SPPs compared to fully porous materials of the same diameters. Please note that Figure 1(a) does not say anything about the relative contribution of the three different dispersive phenomena to the total 

Figure 1: The theoretically expected reduction of (a) reduced plate height contributions and (b) the volume fraction of the porous shell as a function of particle structure (Ï). The light blue zones indicate the Ï range of commercially available SPP materials.			

Finally, according to several experimental results, eddy dispersion (the 

 term in the van Deemter equation) in SPP columns is significantly smaller (~30–40%) than that of the columns packed with fully porous particles of similar size (12–14). This is unexpected because particle structure should not affect the multipath dispersion of solutes when travelling through the column. When modern SPP columns are operated at their optimum linear velocity, 50% of the band dispersion results from long-range packing heterogeneity (12–14). However, it is still unclear whether this significant improvement in efficiency is because of narrower particle size distribution (PSD) of SPPs or their higher density, which enables them to form very homogeneous, efficient packed beds. Some recent studies have indeed indicated that SPPs displaying a very narrow PSD can lead to unprecedented low minimal plate heights (3,4). Another explanation proposed was that some SPPs appear to have rougher surfaces than fully porous particles. This might also have a positive influence on packing quality, apart from the narrowness of the PSD.  

Based on Figure 1(a), a thinner shell is advantageous for efficiency, but it also reduces sample loading capacity and analyte retention. Therefore, the optimum shell thickness is a compromise between efficiency, sample loading capacity, and analyte retention. The sample loading capacity and expected retention factor of a given solute are proportional to the stationary phase volume. Therefore, sample loading and retention are expected to be somewhat lower on SPPs than on fully porous particles. Figure 1(b) shows the volume fraction of the porous layer of some typical SPP structures. Please note that nominal particle size and shell thickness values were considered for Figure 1; however, in practice, the average and nominal values could be slightly different. 

As can be seen in Figure 1(b), the commercially available SPP materials present a huge variety in terms of volume fraction of porous material, between 27% and 75%. SPPs with low volume fractions correspond to relatively large wide-pore particles (for example, 3.6-µm Aeris WP [Phenomenex] or 5-µm Poroshell 300 [Agilent Technologies]) developed for large molecules. In practice, the loadability of most commercially available sub-3-µm SPP materials seems to be similar to that of fully porous particles of the same diameter (15,16). This result may be explained partly by the relatively high surface area of the silica in the porous shell. It is also conceivable that sample molecules do not penetrate appreciably into the centre of totally porous particles (15). Similar to loadability, it appears that the retention characteristic of sub-3-µm SPP materials may not be significantly different from that of their totally porous counterparts when operating under identical conditions (16).  

Latest Developments in SPP Technology: Decreasing the Particle Size

The last decade has seen significant enhancements in LC performance brought about by the development of new columns packed with sub-2-µm fully porous particles and improved UHPLC instrumentation. This new level of performance originates from the achievement of lower height equivalent of a theoretical plate (HETP) values, which is manifested in practice as narrower peaks eluted in a very short time and with superior resolution.  

As described above, the exceptional performance of columns packed with SPPs with low-molecular-weight analytes is related to a reduction of eddy dispersion and longitudinal diffusion. Because of these unexpected but beneficial features, SPPs are likely to have better morphology for further performance optimization via particle miniaturization (17). Therefore, column providers have introduced very fine sub-2-µm SPPs. In 2009, 1.7-µm particles with ρ = 0.73 appeared on the market and showed quite impressive kinetic performance. Minimal HETP values between 2.6 and 4.3 µm were observed with low-molecular-weight solutes, resulting in some very efficient separations of peptides (18). 

The real breakthroughs in column performance came in 2013, when two providers introduced 1.3- and 1.6-µm SPP materials. The 1.3-µm packing (ρ = 0.69) provided exceptionally low minimal plate heights of ~2 µm, corresponding to a plate count of up to 500,000 plates/m (19,20). It was shown that the 1.3-µm SPP material provides the shortest analysis time among existing stationary phases for efficiencies below 30,000 plates. Despite its excellent chromatographic performance, it is evident that the efficiency of this column is limited by current instrumentation in terms of upper pressure limit and extracolumn band broadening. Even at 1000 bar, it is hard to reach optimal linear velocity, thanks to its low permeability (

) (19). The effect of extra-column band broadening of current UHPLC equipment still has a major impact on the apparent kinetic performance when using short narrow-bore columns (50 mm × 2.1 mm) packed with a 1.3-µm SPP material. In spite of these limitations, this 1.3-µm material seems very promising for ultrafast separation of small molecules and peptides in both isocratic and gradient elution modes (19,21).  

Very recently, a column packed with 1.6-µm SPPs (ρ = 0.70) also appeared on the market. The kinetic performance of 1.3-, 1.6-, and 1.7-µm materials was compared in a recent study (22). It was found that the 1.3-µm phase outperforms the two comparators in ultrafast separations. Conversely, the 1.6-µm packing seemed to be the better size for high-resolution chromatography, in isocratic and gradient modes for small molecules and peptides. This exceptional performance was attributed to its more favorable permeability (

) and somewhat higher mechanical stability of the columns (Δ

of 1200 bar). Figure 2 shows the comparison of these three packings using gradient kinetic plot representation obtained from experimental data for small molecules and peptides (21). When constructing these plots, the maximum achievable peak capacity values (

) at any flow rate were considered for a given gradient time (

) to estimate the highest achievable performance for a defined gradient time (21). For these calculations, a generic scouting gradient run from 5% B to 95% B (Δφ = 0.9) was assumed. The ratio of gradient time and achievable maximum peak capacity (

) is plotted against the maximum peak capacity (

). In this representation, the minimal analysis time to achieve a certain peak capacity can be discussed and compared. As shown here, the 1.3-µm material is most useful for ultrafast separations requiring a peak capacity of less than 250. If higher efficiency is required, the best choice is the 1.6-µm material. The 1.6-µm material outperforms the 1.3-µm packing for small molecule separations when 

 > 300. Please note that these plots show the maximum achievable performance when operating the columns at their maximum pressure capability (kinetic performance limit). 

Figure 2: Gradient kinetic plot comparison of columns packed with 1.3-, 1.6-, and 1.7-Âµm SPPs for the separation of (a) small molecules and (b) peptides. Maximum operating pressure: ÎPmax = 1200 bar for Cortecs and ÎPmax = 1000 bar for Kinetex columns, ÎÎ¦ = 0.9 (5â95% B), S = 6 for butylparaben and S = 14.8 for decapeptide. Adapted from reference 22 with permission.			

These sub-2-µm SPP materials were also successfully applied to the separation of tryptic digests of proteins generating 0.5–2 kDa peptides (21,22). With 50 mm × 2.1 mm columns, peak capacities between 200 and 300 were reached within 10 min (depending on the operating conditions). 

Instrumental Considerations for Using Columns Packed with SPPs

: Commercially available SPP materials can be classified into three groups based on their particle sizes and operating pressure requirements: Relatively large particles of 3.6, 4, 4.6, and 5 µm; sub-3-µm particles (2.5, 2.6, and 2.7 µm); and sub-2-µm materials (1.3, 1.6, and 1.7 µm). The columns packed with 3.6–5 µm SPPs can be operated in the same pressure range as conventional 3–5 µm fully porous particles; therefore these materials can be operated on any conventional HPLC systems.  

The 2.5–2.7 µm SPP packings can operate at one-half or one-third of the pressure compared to fully porous sub-2-µm particles; therefore it is theoretically possible to use these columns on conventional HPLC instruments. In generic conditions, when using an acetonitrile–water mobile phase, there is little need to go beyond 400 bar when using shorter columns packed with 2.5–2.7 µm particles. Indeed, the maximum viscosity of acetonitrile-water mixture is around 1 cP at 25 °C, while the viscosity of methanol–water and isopropanol–water mixtures can reach 1.6 and 2.9 cP at 25 °C, respectively. The nominal mechanical stability of some 2.5–2.7 µm SPP columns is 600 bar, whereas the pressure capability of conventional HPLC systems is limited to 400 bar. The pressure capability of conventional HPLC systems seems to be appropriate for columns packed with 2.5–2.7 µm SPPs, but the full benefits cannot be attained with columns of standard dimensions. Indeed, operating these columns at a pressure higher than 400 bar not only provides faster separations, but also allows for the use of alternative (more viscous) solvents to adjust selectivity. 

Sub-2-µm SPP materials can be operated optimally only on UHPLC equipment, since they require operating pressures higher than 400 bar. Such columns are generally stable up to 1000–1300 bar, and their real benefits can be observed only beyond 800 bar. Because of their low permeability, very high pressure is required to operate these columns at reasonable flow rates for fast separations (19). 

: The currently available commercial HPLC instruments can be classified into three groups according to their system dispersion: Optimized UHPLC systems for fast separations with very low dispersion (σ

); hybrid HPLC or UHPLC systems recommended by vendors for both fast and conventional separations (σ

); and conventional HPLC systems with extracolumn variances over 50 µL

  (23). The extracolumn peak variances of several commercially available instruments were reported in the literature. As expected, the impact of system variance on apparent column efficiency could be critical, particularly for shorter columns in 2.1-mm i.d. format. Some model calculations were performed to illustrate the importance of system dispersion, based on experimental efficiency data observed on columns packed with SPPs of different dimensions. Figure 3 shows the effect of extracolumn variance on the percentage of remaining (observed) column efficiency for 1.3-µm (50 mm × 2.1 mm), 2.6-µm (50 mm × 2.1 mm and 50 mm × 3 mm), and 5-µm (150 mm × 4.6 mm) SPP columns (assuming an analyte with a moderate retention factor of 

Figure 3: Effect of the extracolumn variance on the remaining (observed) column efficiency for columns packed with 1.3-, 2.6-, and 5-Âµm SPPs (50 mm Ã 2.1 mm, 50 mm Ã 3 mm, and 150 mm Ã 4.6 mm). A retention factor of k = 5 was assumed for the model calculations. The yellow area corresponds to 

Perspectives in Modern HPLC

This review provides an updated overview of the theory behind the success of SPP technology, trends, benefits, and limitations. It also summarizes the latest developments of sub-2-?m SPPs and instrumental constraints associated with their use.

Superficially Porous Particles: Perspectives, Practices, and Trends

Organic polymer monoliths are mainly used to separate macromolecules in gradient elution liquid chromatography (LC) because of their favourable porous structure. The main reason for the poor behaviour of organic polymer monoliths when separating small molecules isocratically was attributed to the lack of small pores with a stagnant mobile phase, and the resulting low surface area. Recent efforts have improved the separation power of organic polymer monoliths for small molecules, offering column efficiencies up to 50,000–80,000 plates/m. This review describes recent developments in the preparation of organic polymer monoliths suitable for the separation of small molecules in the isocratic mode, and discusses the main factors affecting the column efficiency.

It has been over 20 years since Hjertén published a paper describing the development and application of a "continuous polymer bed" (1). The "macroporous polymers membranes" described by Svec and Tennikova (2) followed and "continuous rods" were later introduced by Svec and Fréchet (3). These continuous materials initiated extensive research of the novel types of stationary phases that consisted of one piece of porous material filling a whole volume of a cylindrical column, later named "monolithic stationary phases" (4). Since then, monolithic stationary phases have been established as useful members of a family of stationary phases with inorganic silica-based (5) and organic polymer-based matrices (6–8).  

Photo Credit: GÃ¼nay Mutlu/Getty Images			

The morphology of monolithic materials with an interconnected network of micrometre-sized flow-through pores enables high flow of the mobile phase at moderate back-pressures and fast separations. Silica-based monoliths are suitable for the separation of small molecules (5,9) while polymer monoliths have been successfully used for fast gradient separations of synthetic and natural polymers (10). The high temperature and chemical stability of organic polymer monoliths in the separation of low molecular compounds triggered significant efforts in tailored preparations of polymer-based, highly efficient monolithic stationary phases suitable for fast and efficient analysis of low molecular compounds. This topic has been recently reviewed by Svec (11) and Nischang (12).  

This article attempts to summarize current progress in the preparation of organic polymer monoliths suitable for the separation of small molecules. Numerous protocols including adjusting the polymerization mixture composition, controlling the polymerization time and temperature, and post-polymerization surface modifications are discussed.  

Organic polymer monoliths are usually — but not always (13) — prepared by radical polymerization of monovinyl monomer and crosslinking divinyl monomer in the presence of pore forming solvents, typically alcohols (6). The polymerization reaction begins by decomposition of a radical initiator triggered by an external input, such as elevated temperature or UV light. At an early stage of the reaction, polymerization nuclei are formed and (usually) a divinyl crosslinking monomer is incorporated (7,14). At a later stage of polymerization, the nuclei grow and a highly interconnected moiety is formed. Finally, the heterogeneous, "cauliflower-like" polymeric material fills the whole fused-silica capillary space. At the end, the monolithic block is flushed to remove all unreacted components of the polymerization mixture. Figure 1 shows several examples of monomers and crosslinkers usually used for the preparation of organic polymer monoliths.  

Figure 1: Examples of functional and crosslinking monomers usually used for the preparation of porous polymer monoliths. (1) butyl methacrylate, (2) lauryl methacrylate, (3) ethylene dimethacrylate, (4) N,N-dimethyl-N-methacryloxyethyl-N-(3-sulphopropyl)ammonium betaine, (5) styrene, (6) vinylbenzyl chloride, (7) divinylbenzene.			

The concentration of monomers in the polymerization mixture has a significant impact on the density and efficiency of the prepared columns. Eeltink et al. improved the column efficiency from 13,000 plates/m to 67,000 plates/m by decreasing the concentration of all monomers in the polymerization mixture from 40% to 20% (15). By keeping the crosslinker concentration constant, and gradually increasing the concentration of the monomer in the polymerization mixture to 25%, the column efficiency of 30,000 plates/m was achieved at elevated temperatures (16).  

Previously, the possibility of isocratic separation of low molecular alkylbenzenes has been tested on butyl methacrylate-co-ethylene dimethacrylate capillary monolithic columns (17). By increasing the 1-propanol concentration in the pore-forming mixture from 60% to 62% and then to 64% the column efficiency was improved from 5000-plates/m to 35,000 plates/m. The effect of 1-propanol on the internal structure of prepared monolithic stationary phases is demonstrated in Figure 2. At the same time, Coufal et al. used butyl methacrylate as a functional monomer and ethylene dimethacrylate as a crosslinker to prepare a monolithic column with an efficiency of 37,000 plates/m (18).  

Figure 2: Effect of 1-propanol concentration on internal structure of (poly)methacrylate monolithic stationary phase. (a) 37.2% of 1-propanol in polymerization mixture, (b) 38.4% of 1-propanol in polymerization mixture. Adapted and reproduced with permission from D. MoravcovÃ¡, P. Jandera, J. Urban, and J. Planeta, (2003), J. Sep. Sci. 26, 1005â1016. Â© John Wiley and Sons.			

Different alkylmethacrylate monomers with functional groups including C2, C4, C6, C8, C12, C16, C18, and isobornyl were used to prepare monolithic stationary phases differing in hydrophobicities (16,19–22), with an efficiency in the range of 30,000–40,000 plates/m. From among the monomers, lauryl methacrylate (C12) usually showed the highest efficiency (16,23,24), which might be the result of specific interactions inside the polymerization mixture. 

Horváth et al. prepared a poly(styrene-co-divinylbenzene) monolithic capillary column with a reported efficiency of 43,000 plates/m for non-retained dimethylsulphoxide (25). Another report described the efficiency of 91,000 plates/m for non-retained uracil on a poly(styrene-co-divinylbenzene) column prepared from 20% styrene, 20% divinylbenzene, 40% 1-propanol, and 20% formamide (26). 

The addition of methacrylic acid into the polymerization mixture of styrene, divinylbenzene, toluene, and isooctane significantly changes the separation power of capillary columns (27). A column without methacrylic acid in the polymerization mixture showed the surface area of 0.1 m

/g and could not separate alkylbenzenes at all; however, after the addition of methacrylic acid (in the ratio of 1:1:2 of styrene, methacrylic acid, and divinylbenzene) the column had a surface area of 261 m

/g and provided a baseline separation of alkylbenzenes with the efficiency of 28,000 plates/m (Figure 3). The methacrylic acid obviously contributes to the formation of a significant number of small pores in the monolithic stationary phase (11,27).  

Figure 3: Examples of small molecules isocratic separations on monolithic poly(styrene-co-methacrylic acid-co-divinylbenzene) capillary column. Mobile phase: 65% aqueous acetonitrile; flow rate: 4 Î¼L/min; column length: 170 mm; UV detection: 241 nm. Analytes: (1) thiourea, (2) phenol, (3) aniline, (4) benzene, (5) toluene, (6) ethylbenzene, (7) propylbenzene, and (8) butylbenzene. Adapted and reproduced with permission from A. SvobodovÃ¡, T. KrÃ­Å¾ek, J. Å irc, P. Å Ã¡lek, E. Tesarova, P. Coufal, and K. Å tulÃ­k, (2011), J. Chromatogr. A 1218, 1544â1547. Â© Elsevier.			

The addition of 2-hydroxyetlyl methacrylate in the polymerization mixture containing divinylbenzene and ethyl vinylbenzene was shown to reduce the plate height of the columns for small molecules from 160–490 μm to 40–76 μm, which was attributed mainly to the decrease in the domain size of the monoliths, leading to lower eddy dispersion and mass transfer resistance contribution to the band broadening in the column. Similar to methacrylic acid, the addition of 2-hydroxyetlyl methacrylate in the polymerization mixture enhanced the formation of small pores and significantly increased the surface area of prepared materials up to 500 m

The addition of polystyrene to porogen solvents was another way of increasing the efficiency of glycerol dimethacrylate-based monolithic columns for the analysis of small molecules (29). Efficiency increased from 5500 plates/m to 18,500 plates/m; and to 34,000 plates/m with the use of a low-molecular-weight polystyrene (50,000), a medium-molecular-weight polystyrene (600,000), and an ultrahigh-molecular-weight polystyrene (3,840,000) solution in chlorobenzene, respectively, in glycerol dimethacrylate-based monolith (29). 

The efficiency of polymethacrylate monolithic stationary phases can also be increased by employing alkyl dimethacrylate crosslinkers with various alkyl chain lengths between the two methacrylate units (30). Hydrophobic stearyl methacrylate has been polymerized in the presence of tert-butanol and 1,4-butanediol with dimethacrylate crosslinkers including ethylene dimethacrylate, butanediol dimethacrylate, hexanediol dimethacrylate, neopentyl glycol dimethacrylate, methyl octanediol dimethacrylate, and nonanediol dimethacrylate. Even though these materials have low surface areas of only several m

/g, they allow surprisingly efficient separations of low molecular alkylbenzenes (30). Methyl octanediol dimethacrylate yielded the highest separation efficiency of 83,000 plates/m and 52,000 plates/m for unretained thiourea and retained butylbenzene, respectively. 

A single monomer with crosslinking functionalities in pore-forming solvents can be used to synthesize monolithic stationary phases suitable for the separation of small molecules. Initial reports used divinylbenzene (31,32), however, it usually contains significant concentrations of styrene monomer (11). Several others monomers were used for this purpose, including glycerol dimethacrylate (29,33–36), methylene-bis-acrylamide (37), and poly(ethylene) diacrylate (38).  

Tetrakis(4-vinylbenzyl)silane thermally polymerized in 1-dodecanol and toluene forms a monolithic material (39) with the surface area of 79 m

/g for 20% monomer concentration in the polymerization mixture. The column permeability decreased with higher concentration of the monomer. The monolithic columns prepared in this way were used for gradient elution separations of small molecules, including carboxylic acids, phenols, and amines. An efficiency slightly higher than 20,000 plates/m was achieved for isocratic separation of alkylbenzenes.  

Developments in the preparation of organic polymer monoliths suitable for the separation of small molecules in the isocratic mode are described and the main factors affecting column efficiency are identified.

LCGC Europe-06-01-2014

Application of Pyrolysis–Gas Chromatography–Mass Spectrometry for the Identification of Polymeric Materials

Exploring the Possibilities of High-Throughput Sample Preparation

Superficially Porous Particles: Perspectives, Practices, and Trends

Developments in the Preparation of Organic Polymer Monoliths for the Separation of Small Molecules

UV Detector Problems

Vol 27 No 6 LCGC Europe June 2014 Regular Issue PDF