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).  

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-Î¼m 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.			

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.  

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

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.  

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-Î¼m 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.			

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.  

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

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.  

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-Î¼m 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.			

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).  

Table 3: Pyrolysis products of polyaramid fibres. 			

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.  

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

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 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-Î¼m 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.			

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).  

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

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. 

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

Feature

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

Have you wondered how your gas chromatography (GC) system sets and controls gas pressures, flows, and carrier gas velocities electronically? Here, we describe the requirements for and the operation of electronic gas control systems for GC columns and detectors.

Computerized or electronic pneumatic control (EPC) systems for carrier gas, split flow control, and detector gases abound in modern gas chromatographs (GC), as well as in headspace samplers and column switching systems. The accuracy and repeatability of EPC are superior to that of manual adjustment, and the improved control of instrument parameters greatly reduces the possibility for making gas-related mistakes. Computerized pneumatics excel at controlling column pressure drop or detector gas flow rates. An EPC system generally relieves operators from having to make repetitive adjustments and measurements with a flow meter and stopwatch. However, running an EPC system blindfolded, so to speak, by never cross-checking actual gas behaviour with selected set-points, only invites trouble. Like any computer system, the results can only be as good as the column and gas parameters that a user enters. Thus, a good working understanding of how an EPC system works and what goals are to be accomplished is essential for obtaining the best possible results. 

In an EPC system, gas flows from the gas supply input, through a metering valve, into a pressure or flow transducer, and then out to the device — inlet, detector, or other GC component — that consumes the gas. The GC system sends a set-point value to the EPC controller, which returns the measured flow or pressure value from its transducer. The set-point and actual values are compared in the EPC system, which adjusts the metering valve as required to maintain the desired set point. The EPC controller incorporates column and carrier gas characteristics to determine the necessary pressure drop at any given moment. Atmospheric and gas supply pressures plus controller temperatures are included as required to compensate for drift and instabilities. 

In the simplest configurations an EPC channel acts as a carrier gas flow controller for a packed column or controls a detector gas, such as air or hydrogen, for flame ionization detection (FID). Two controllers — one pressure and one flow — can provide the split flow and inlet pressure for a capillary column inlet splitter. Other more complex applications include pressure or flow controllers for auxiliary devices such as purge-and-trap or headspace samplers, or pressure-switching controllers for multidimensional column systems. 

Computer-controlled pneumatics cannot prevent operators from selecting inappropriate column pressures or split flow rates. An operator may easily establish incorrect conditions and become misled as to the reasons for a problem. Because of their complexity and flexibility, computerized pneumatic systems offer analysts more opportunities for errors. 

One of the most common applications for EPC is the control of capillary (open-tubular) column carrier gas. The column flow rate and the carrier gas linear velocity are complex functions of the column dimensions, oven temperature, and type of carrier gas. The mathematical relationships between GC column dimensions, temperature, and flow, pressure, and linear velocity are well understood. Fortunately for today's GC users, all of this is incorporated into the EPC system, which acts as a kind of gas calculator. 

A wide-bore 30 m × 530 μm column is a good example to help understand the ins and outs of EPC control. At 50 °C with helium carrier gas this column will require about 4.1 psig of inlet pressure to maintain a flow of 5.6 mL/min or an average carrier gas linear velocity of 40.0 cm/s. I know this because I used the EPC "calculator" in my GC system to compute the pressure, flow, and velocity by first entering the column dimensions, carrier gas type, oven temperature, and desired velocity of 40 cm/s. Because I am using a split inlet, I entered the split ratio as well. With this column, a desired split ratio of 20:1 results in a split-flow set point of 112 mL/min. 

An alert reader will realize at this point that they could have entered an incorrect column length or internal diameter, or perhaps failed to correct an existing set of parameters from the previously installed column. This type of error would, for the most part, go unnoticed by the GC system itself unless a required pressure or flow could not be attained by the controller. Suppose that the column internal diameter was mistakenly set at 320 μm. Entering a velocity of 40 cm/s would result in a pressure drop of 20.7 psig. This is achievable by the EPC controller, which upon increasing the pressure to that level would cause the actual 530-μm column flow to increase to 41.9 mL/min with an average linear velocity of 190 cm/s! The split ratio of 20:1 would still call for a split flow rate of 112 mL/min, and so the actual split ratio would be more like 2.67:1.  

These erroneous operating parameters are within the capabilities of the split inlet pressure and flow controllers, so the GC system would not report an error even though the column was operating very far from its desired set point. A subsequent injection might alert the operator to a problem; the peaks would be eluted too rapidly and they would be much larger than expected. If not identified immediately, this type of problem could cause serious difficulties later on. Of course, if the retention times were known under the correct conditions then this situation would be evident after one injection.  

Rather than wait until after a run is recorded to discover such errors, the operator can double-check flow rates or unretained peak times after a column has been installed and the preliminary setup completed. Time an unretained peak or measure the flow at the column outlet and compare that to what the EPC system says they should be. Bear in mind that direct flow measurement is more difficult with smaller internal diameter columns, and if necessary use an electronic flow meter that is rated for low flow rates. 

Small deviations in the actual column internal diameter or length will give rise to relatively small errors in actual velocities or flows, as discussed in more detail in another "GC Connections" instalment (1). For the best accuracy, it's a good idea to include the stationary phase film thickness, if greater than about 1 μm, with the column dimensions entered into the GC system. 

The pressure drop that's required for a particular column flow depends on the oven temperature as well as the column dimensions and carrier gas, so the operator must specify the temperature at which the desired flow is to be achieved — this is nearly always the same as the initial oven temperature. So far so good, but what happens when the column temperature increases during oven temperature programming? The column flow rate depends on the oven temperature, as well as many other factors, so the flow and velocity will change during temperature programming. The EPC split inlet controller maintains a set pressure level and does not directly control flow or velocity — it must instead calculate the pressure required to establish a desired flow or velocity. What are the effects of choosing these different pneumatic control modes? 

 As temperatures increase so does the carrier gas viscosity, which causes the flow and velocity to decrease at higher temperatures if the pressure is kept constant. Figure 1 shows the relationships of temperature and gas viscosity for three common GC carrier gases. EPC systems use these relationships to calculate carrier-gas viscosity dynamically as the oven temperature changes. Figure 1 gives a good idea of how large this viscosity effect can be. Going from 50 °C to 250 °C, for example, causes helium viscosity to increase by about 40%. The other carrier gases undergo viscosity changes of a similar magnitude.  

Figure 1: Carrier gas viscosity as a function of temperature (adapted from reference 2). The dots correspond to published measurements; the solid lines represent interpolated data and the dashed lines extrapolated data.			

With a constant inlet pressure drop, the column flow decreases during temperature programming. Figure 2 illustrates the effect on carrier gas flow and average linear velocity of changing the oven temperature while holding the pressure drop constant, using our example wide-bore column with a constant 4.1 psig of helium carrier gas. The column flow rate of 5.6 mL/min at 50 °C decreases to 2.5 mL/min at 250 °C, a 55 % loss. Across the same temperature range, the average linear velocity decreases from 40 cm/s at 50 °C to 28 cm/s at 250 °C, a 30% loss.  

Figure 2: Theoretical effect of column temperature on (a) corrected carrier gas flow and (b) average linear velocity with constant pressure drop. Column dimensions: 30 m Ã 0.530 mm; pressure drop: 4.1 psig; carrier gas: helium; column outlet pressure: 1 atm.			

When using a constant inlet pressure with temperature programming, the reduction in flow rate at elevated temperatures can cause unwanted peak broadening as the carrier gas velocity departs from optimum, and it also can extend elution times unnecessarily. Usually in this situation, choosing a different pneumatic operating mode will produce better results. 

An EPC system can be programmed to increase the column pressure drop sufficiently to maintain a constant carrier-gas flow rate as the temperature increases. Figure 3 shows how this choice affects the pressure drop and the average linear velocity. The EPC controller maintains a constant column flow of 6.0 mL/min by increasing the pressure drop from 4.5 psig at 50 °C to 9.1 psig at 250 °C. Note that the average linear velocity increases as the oven temperature goes up, which perhaps runs counter to intuition. But with a constant flow rate the linear velocity can remain in a more efficient region somewhat higher than optimum, which will largely avoid column efficiency losses. Peaks will be eluted sooner and at lower temperatures, reducing the total run time compared to constant pressure operation.  

Figure 3: Theoretical effect of column temperature on (a) carrier gas pressure drop and (b) average linear velocity while maintaining constant corrected column flow. Column dimensions: 30 m Ã 0.530 mm; column flow: 6.0 mL/min; carrier gas: helium; column outlet pressure: 1 atm.			

 Large shifts in column flow rate can affect detector operation. Constant column flow is desirable for consistent detector function during temperature programming. Mass spectrometry (MS) detection solute ionization fragmentation patterns depend somewhat on the source pressure, which in turn depends on the incoming flow rate, especially with higher column flows. Granted, the 530-μm column example discussed here is not the best choice for direct interfacing to a bench-top mass spectrometer, but the benefits can be significant in terms of consistent spectra and library searches for those narrower i.d. columns that can be connected directly.  

When FID is used with hydrogen carrier, it's a good idea to keep the total hydrogen flow through the detector constant. This can be accomplished in two different ways, either by maintaining a constant column flow rate or by programming the detector hydrogen flow to compensate for changes in the column flow, that is, by keeping the sum of the column and detector hydrogen flows constant. 

Ultimately, the most important consideration may be peak resolution and not necessarily detector performance, especially in situations where the separation is marginal. Deciding whether constant pressure or constant flow will yield a better temperature-programmed separation, let alone the effects of changing the temperature programme itself, is a more complex consideration that doesn't give itself over very well to purely theoretical modelling. Relative peak positions in a chromatogram depend heavily on the thermodynamic relationships between the stationary phase and a solute's chemical characteristics. As a result, individual peaks move in different ways relative to each other as the temperature changes. 

In general, the more the chemical natures of a pair of peaks differ, the greater the effect of changing the column temperature or temperature programme will be. At higher flows peaks will be eluted earlier in a temperature programme, and therefore at somewhat lower column temperatures. But this change of elution temperature may or may not improve the separation of a pair of peaks if both are not affected in the same way. This effect is illustrated in the series of test chromatograms shown in Figure 4. Here, a test mixture was injected with either constant pressure or constant flow programmed pneumatics, with the initial pressure drop the same in either case. The programme rate was doubled in each of four consecutive runs, from 3 °C/min to 6 °C/min, 12 °C/min, and finally 24 °C/min.  

Figure 4: Series of chromatograms at increasing oven temperature programme rates with constant pressure or constant flow controlled pneumatics. Temperature programming from 50 Â°C, hold 2 min, then to 250 Â°C at: (a) 3 Â°C/min; (b) 6 Â°C/min; (c) 12 Â°C/min; (d) 24 Â°C/min. Constant pressure at 4.1 psig (P) or constant flow at 6.0 mL/min (F). Column: 30 m Ã 0.530 mm, 1.3-Î¼m  df EC-Wax. 1.0 Î¼L manual injections split 20:1 at 200 Â°C. Peaks: 1 = n-C10, 2 = n-C11, 3 = n-C12, 4 = n-C13, 5 = 2-octanone, 6 = 1-octanol, 7 = 2,6-dimethylaniline, 8 = 2,4-dimethylphenol.			

The overall effects of constant flow compared to constant pressure operation are clear in these chromatograms: The peaks are all eluted sooner with constant flow. The last peak in the run, 2,4-dimethylphenol (DMP), is eluted at 24.85 min with constant pressure and at 22.79 min with constant flow at 6 °C/min. Similar reductions in retention times hold for the other peaks when the flow rate is kept constant, across all of the selected programming rates. 

The effects on retention times of increasing the temperature programme rate far outweigh the differences observed between constant pressure and constant flow pneumatic modes. The DMP peak moves in from 40.66 min at 3 °C/min (elution temperature = 166 °C) to 11.1 min at 24 °C/min (elution temperature = 250 °C). Again, there are no surprises here — the temperature effects are quite strong. 

The overall effect on peak resolution, however, is somewhat unexpected. The two closest-eluted peak pairs, 

-decane–2-octanone and 2,6-dimethlyaniline (DMA)–DMP, show opposite trends. Resolution increases with temperature programme rate for the first pair but decreases for the second pair, regardless of the pneumatic programming mode. Interestingly, the constant pressure mode gives slightly better resolution than the constant flow mode for the first pair, but makes little difference for the later peak pair. Figure 5 plots the resolution of these two peak pairs as a function of the temperature programming rate and the pneumatic mode.  

Figure 5: Peak resolution (RS) as a function of temperature programming rate and pneumatic mode for (a) n-C13 and 2-octanone, and (b) 2,6-dimethylaniline and 2,4-dimethylphenol. P = constant pressure and F = constant flow. Conditions and chromatograms from Figure 4.			

Although the resolution of DMA–DMP is high in all cases — there is no reason to be concerned with this peak pair itself — if there were other peaks between these two in a "real" sample, then it would be interesting to find an optimum set of conditions for the overall chromatogram instead of choosing the best case for the first peak pair. A temperature programming rate of 12 °C/min might be a good choice because it keeps the resolution of the first pair while still utilizing a good portion of the available resolving power between the second pair.  

A better choice might be to use a two-ramp temperature programme instead of a compromise single-ramp programme. At the time that the first peak pair is being eluted, the later-eluted DMA–DMP pair has not started to move along the column much, so cutting the temperature programming rate back to 6 °C/min or even 3 °C/min halfway through the run would make some sense. That way, the later-eluted pair would behave in much the same way as found by running the entire chromatogram at the lower rate, while the earlier pair would get the full advantage of the maximum available resolution. Of course, other solutions might include changing to a narrow-bore column or investigating the effects of a different stationary phase composition. 

Overall, these examples demonstrate that there can be significant differences in how peaks behave relative to each other as the column pneumatic and thermal conditions are varied. Clearly, such changes can affect resolution in ways that may not be anticipated. Chromatographers should always validate and confirm that required peak resolution and quantitation performance levels are met after making any changes to the chromatography. 

Computerized pneumatic control adds a very capable multipurpose tool to the chromatographer's tool belt. Such systems make setting up a GC system easier by automating some tasks. They can give more repeatable results from run to run and laboratory to laboratory. Some detector performance improvements can be obtained with constant carrier-gas flow control. And computerized pneumatics excel in facilitating advanced separations techniques. But when it comes to chromatographic performance gains in solute resolution, the choice of constant flow or constant pressure control can produce significant differences for multiple peak pairs in the same analysis. 

is a senior scientist at Serveron Corporation in Beaverton, Oregon, USA, and is a member of the 

 editorial advisory board. Direct correspondence about this column should be addressed to "GC Connections", 

, Honeycomb West, Chester Business Park, Chester, CH4 9QH, UK, or e-mail the editor-in-chief, Alasdair Matheson, at 

Introduction to Open-Tubular Column Gas Chromatography

GC Connections

Have you wondered how your GC system sets and controls gas pressures, flows, and carrier gas velocities electronically? Here, we describe the requirements for and the operation of electronic gas control systems for GC columns and detectors.

Electronic Control of Carrier Gas Pressure, Flow, and Velocity

Although it has been happening to some degree for centuries, food adulteration is increasingly becoming a worldwide epidemic, as evidenced by the melamine scandal of 2008 and recent meat and fish substitutions at major food chains. Analysts are applying more sophisticated chromatographic, spectroscopic, and enzymatic analytical techniques to monitor and measure food adulterants, which are often economically motivated. In this instalment, guest authors Jeff Hurst and Kendra Pfeifer from Hershey Foods explore high performance liquid chromatography (HPLC), ultrahigh-pressure liquid chromatography (UHPLC), and mass spectrometry (MS) approaches being adopted to keep ahead of the food adulteration game. 

Food adulteration, sometimes called food contamination, is an intriguing topic that can sometimes seem daunting because it is in everyday commerce and not just the analytical laboratory. First, let's discuss a few general thoughts on food adulteration.  

The focus of this instalment is on detecting food adulteration with high performance liquid chromatography (HPLC), but a veritable arsenal of techniques are currently being used. These include HPLC coupled with a variety of detection methods ranging from UV to mass spectrometry (MS) and other nonchromatographic techniques such as immunoassay and mid-infrared (IR), near infrared (NIR), and Raman spectroscopy.  

Another interesting point of contention is that there are some who perceive scientists that are involved in food adulteration from the contaminations side as second-rate scientists. However, that seems to be far from the truth because those scientists exhibit not only a certain level of scientific expertise, but also ingenuity. One obviously doesn't condone these food adulteration activities, but we should be aware.  

Food fraud is seemingly large and growing, with articles on the topic appearing in publications ranging from 

published an article titled "A la Cartel", indicating that organized crime is diversifying into food and alcoholic beverages (1). Several examples are given in the article, including the horsemeat scandal in 2013 and another case in which nearly 2500 jars of honey were filled with sugar syrup. There was also a report on the seizure of 17,000 L of fake vodka worth $1.7 million (around €1.3 million). Finally, this article contained information from Europol that in the United Kingdom crooks have switched from drugs to food since everyone buys food and drink. 

Despite the recent news coverage, food fraud is nothing new (2). The Romans had laws that focused on the adulteration of wines because wine back then tended to become bad rather rapidly and a number of items were added to improve the flavour. One of the compounds happened to be lead salts, which sweetened the wine and likely added to the lead load in the Roman population. In the early 19th century, Frederick Accum wrote a book titled 

Treatise on the Adulteration of Food and Culinary Poisons Exhibiting the Fraudulent Sophistications of Bread, Beer, Wine, Spirituous Liquors, Tea, Coffee, Crème. Confectionary, Vinegar, Mustard, Pepper, Cheese, Olive Oils, Pickles and other Articles Employed in Human Commerce

 (3). In that time period, used tea and coffee grounds could be purchased inexpensively. The tea grounds were then boiled with sheep dung and ferrous sulphate and coloured with a mixture of tannin, copper acetate, and ferrrocyanide while coffee grounds were mixed with other roasted bean, gravel, sand, and chicory. Burnt sugar was used to add colour to coffee. In the case of confectionary, lead, copper, and mercury salts were used to make bright colours that were eye catching for children, but toxic. Green vitrol, alum, and salt were added to give beer a good head because beer was sometimes diluted.  

The most likely event that brought the topic of food adulteration to the forefront today was the melamine incident in 2008, which killed six Chinese infants and sickened more than 30,000. Another recent incident occurred in the past month, in which meats used by two very visible restaurant chains was found to be tainted — meat more than a year old was mixed with fresh meat (5,6).  

, the top five foods targeted for adulteration are milk, olive oil, honey, saffron, and seafood (4). The adulterants can be divided into three categories: targeted, nontargeted, and economically motivated (EMA). This division recognizes that there is a crossover effect and that HPLC plays a key role in the detection of adulterants. Although there is no exact definition for economically motivated adulteration, the United States Food and Drug Administration (FDA) adopted a working definition for EMA as the "Fraudulent, intentional substitution or addition of a substance in a product for the purpose of increasing the apparent value of the product or reducing the cost of its production, that is, for economic gain" (7). Common types of EMAs include substitution or dilution of an authentic ingredient with a cheaper product (for example, replacing extra virgin olive oil with a cheaper oil), flavour or colour enhancement using illicit or unapproved substances (such as unapproved dyes), and substitution of one species with another (such as fish species fraud). 

Figure 1: Chromatogram of the separation of cyanuric acid and melamine and their C13 analogues on a 50 mm Ã 2.1 mm, 2.6-Î¼m dp 100-Ã  Kinetex HILIC LC column.			

In the case of milk, many individuals tend to focus on melamine as the "poster compound" for milk adulteration. Figure 1 shows a chromatogram of a melamine-contaminated milk with the compounds cyanuric acid and melamine identified by MS using an isocratic mobile phase consisting of acetonitrile and 100 mM ammonium acetate. Peaks 1 and 2 are cyanuric acid and its 

C analogue and peaks 3 and 4 are melamine and its 

C analogue. While melamine is a high visibility target, it is being monitored and anecdotal information indicates that it is now being replaced by urea and even amino acids because they are more difficult to identify. In addition to melamine, soy protein, corn syrup, whey, leather, and even shampoo have been reported as potential adulterants in milk. When leather is added to milk, it can be hydrolyzed to improve its solubility. The use of this material can be detected by the determination of the amino acid hydroxyproline from the hydrolysis of leather protein that is not seen in milk protein (8). There is an active group at the United States Pharmacopeia called the Skim Milk Advisory Group that is investigating potential adulterants in skim milk, one of which is soy. Figure 2 shows an example chromatogram of various samples ranging from skim milk powder to soy (9). Other potential contaminants could be different types of milk such as goat's milk, which can be detected at the 1% level by the determination of beta-lactoglobulin.  

Figure 2: UHPLC chromatograms in Masslynx (Waters) format: Red = pure skim milk powder (SMP), black = 99:1 (w/w) SMPâsoy protein isolate (SPI), purple = 90:10 (w/w) SMPâSPI, and green = pure SPI. Detection: UV absorbance at 215 nm.			

 The contamination of olive oil with other oils including corn, sunflower, safflower, and sesame requires continual testing to verify  it is pure olive oil with both polyphenols and triglycerides used as marker compounds. Furthermore, olive oil contains a higher concentration of oleic acid than other oils, but less linoleic and linolenic.  

In a similar vein, wines are being adulterated by the addition of polyphenols. Other things that have been added to wine include pigments and glycerol to give a wine "body". In a paper published in 

(10), the authors described an HPLC method for the anthocyanins in red wine, in which elderberry extracts were added to improve the colour. The method determined that wine adulterated with the elderberry contained an extra peak attributed to cyanidin-3-bubioside-5-glucoside. 

 The third food on the top five list from 

 was honey. The dilution of honey with less expensive materials, such as corn syrup, has been around for decades. The initial work on this topic was done by White of the United States Department of Agriculture using nuclear magnetic resonance (NMR) spectroscopy (11), but as technology evolved there have been a number of HPLC applications to monitor this phenomenon. In addition to the EMA activities, there is also concern about honey being contaminated with the antibiotic chloramphenicol used by beekeepers to treat their hives against the crippling foulbrood disease. Figure 3 provides a chromatogram from a liquid chromatography–mass spectrometry (LC–MS) method developed for this application (12) with either a 100 mm × 4.6 mm RP-18e column (EMD Millipore) or a 250 mm × 4.6 mm, 5-μm 

 Zorbax XDB C18 (Agilent Technologies). For both columns, the mobile phase was 45:55 (v/v) methanol–0.2% aqueous ammonia acetate at a flow rate of 1 mL/min.  

Figure 3: (a) Total ion chromatogram (TIC) and (b) extracted ion chromatogram (EIC) obtained from chloramphenicol with detection by MS in negative ESI mode.			

 Because of its cost, saffron is fourth on the list (according to 

), but other literature seems to be more inclusive by indicating that spices, in general, are targets for adulteration. 

 indicated seven spices of concern: cayenne pepper, cumin, coriander, pepper, saffron, turmeric, and salt (13). Unscrupulous suppliers can adulterate cayenne pepper with sawdust and colours, cumin with sawdust, turmeric with sawdust and yellow colours, and saffron with corn silk. Figure 4 provides a chromatogram of Sudan Red I, II, III, IIB, and IV dyes corresponding to peaks 1–5, respectively, which can be used to adulterate spices by enhancing colour. The separation was performed on a 150 mm × 4.6 mm, 4-μm Synergi Polar-RP 80A LC column (Phenomenex) using a 65:20:15 (v/v/v) methanol–acetonitrile–water mobile phase. Detection was UV absorbance at 480 nm.  

Figure 4: Chromatogram of sudan dyes (see text for details).			

 As was indicated in the introduction, earlier this year there was an incident in China where tainted meat was mixed with fresh meat and provided to unsuspecting organizations including KFC and McDonald's (6). Last year in Europe, it was determined that horsemeat was mixed with beef. In addition, there is a lot of data indicating that a large percentage of fish is mislabelled, leading to a cheaper fish being sold as a more expensive one (14). This does not include the issue with farmed and wild caught salmon. Although DNA-based techniques have been widely used, a number of HPLC techniques including proteomics, or "foodomics" as it is now called, have been used to help solve this quandary. Furthermore, information about fish fraud indicates that a substantial amount of fish sold in outlets ranging from sushi bars to the local fish market is not as advertised. A recent report found that fish samples purchased at grocery stores, restaurants, and sushi bars in major cities were often mislabelled, including red snapper (actually tilefish); white tuna and butterfish (actually escolar); wild Alaskan salmon (actually farmed Atlantic salmon); caviar (actually catfish roe); and monkfish (actually puffer fish) (14). 

A paper by Giaretta (15) described an ultrahigh-pressure liquid chromatography (UHPLC) method using myoglobin as a marker for meat adulteration with an example given on detecting pork in beef. Figure 5 is an example chromatogram of a variety of meat types. This method used a Protein-Pak Hi Res Q column (Waters) with photodiode-array detection and a mobile-phase system consisting of three buffers in a discontinuous gradient. A final example can be seen in a recent study in which Chou and coworkers (16) used HPLC with electrochemical detection using copper nanoparticle-plated electrodes to differentiate meat from 15 animal species.  

Figure 5: Chromatogram of myoglobin from different sources.			

This column instalment has provided a modest snapshot on the use of HPLC techniques to address challenges in EMA of the food supply. This issue will continue to be a moving target with a need to be vigilant to monitor developments of EMA in parallel with the food industry, instrument vendors, and government labs. There is still the need for simpler sample preparation protocols and it would seem that an expansion of the various "ambient" LC–MS techniques in this area could be helpful. One of the techniques that is being touted by HPLC–MS vendors is the application of exact-mass LC–MS, but, in our opinion, one must ensure that peak identifications are appropriate since sometimes standards are not available.  

Finally, as the adulterers become more sophisticated, it seems like we will need more sophisticated HPLC-based techniques such as foodomics paired with other instrumental techniques like immunoassay and Fourier transform infrared (FTIR), NIR, and Raman spectroscopy. 

is a manager of regulatory affairs in the quality and regulatory affairs department at the Hershey Company. She has a bachelor's degree in chemistry and a master's degree in food science. Kendra has a wide variety of experiences within the corporation with a tenure in the analytical research and services organization before joining the regulatory affairs group. She was active in the Association of Analytical Communities (AOAC) methods committee and implemented robotics in the lab environment.  

is a principal scientist with the Hershey Company. He is the author of a substantial number of papers on HPLC and food analysis and is a member of numerous scientific organizations including the American Chemical Society (ACS), the Institute of Food Technologists (IFT), the American Society for Mass Spectrometry (ASMS), and the American Association for Integrative Medicine (AAIM). He indicates that Hershey was his first "real job". 

 is an analytical consultant and is a member of 

's editorial advisory board. Direct correspondence about this column should be addressed to "Column Watch", 

, Honeycomb West, Chester Business Park, Wrexham Road, Chester, CH4 9QH, UK, or e-mail the editor-in-chief, Alasdair Matheson, at 

Swindled: The Dark History of Food Fraud, from Poisoned Candy to Counterfeit Coffee

 (Princeton University Press, Princeton, New Jersey, USA, 2008). 

Treatise on the Adulteration of Food and Culinary Poisons Exhibiting the Fraudulent Sophistications of Bread, Beer, Wine, Spirituous Liquors, Tea, Coffee, Crème. Confectionary, Vinegar, Mustard, Pepper, Cheese, Olive Oils, Pickles and other Articles Employed in Human Commerce 

(Longman, Hurst, Rees, Orme and Brown, London, UK, 1820). 

http://www.nbcnews.com/id/28787126/ns/world_news-asia_pacific/t/face-execution-over-china-poison-milk-scandal/

http://money.cnn.com/2014/07/21/news/companies/kfc-mcdonalds-china/

http://foodfraud.msu.edu/wp-content/uploads/2014/01/CRS-Food-Fraud-and-EMA-2014-R43358.pdf

(8) "Dionex Solutions: Methods for Detecting Leather Protein Adulteration in Milk," Dionex Corporation, 

http://www.dionex.com/en-us/markets/food-beverage/news-articles/lp-110606.html

(9) J.E. Jablonski, C. Pardo, L.S. Jackson, B. Rohrback, J. Moore, and M. Han, "Chemometrics and UPLC-UV to Detect Adulteration of Skim Milk Powder with Soy Protein Isolate," poster from United States Pharmacopeia Skim Milk Powder Advisory Group.  

Column Watch

In this instalment, guest authors Jeff Hurst and Kendra Pfeifer from Hershey Foods explore high performance liquid chromatography (HPLC), ultrahigh-pressure liquid chromatography (UHPLC), and mass spectrometry (MS) approaches being adopted to keep ahead of the food adulteration game.

When Bad Things Happen to Good Food: Applications of HPLC to Detect Food Adulteration

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LCGC Asia Pacific-11-01-2014

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

Electronic Control of Carrier Gas Pressure, Flow, and Velocity

When Bad Things Happen to Good Food: Applications of HPLC to Detect Food Adulteration

Vol 17 No 4 LCGC Asia Pacific November 2014 Regular Issue PDF