Development of a new high temperature/high pressure flow system for the continuous digestion of biological samples Christiane Gra�ber and Harald Berndt* Institut fu�r Spektrochemie und Angewandte Spektroskopie, Bunsen-KirchhoV-Str. 11, 44139 Dortmund, Germany Accepted 22nd January 1999 The development of a new high temperature/high pressure flow system that incorporates resistively heated capillaries for the continuous digestion of various samples is presented. The system allows the continuous digestion of samples at temperatures of up to 260 °C and a pressure of up to 30 MPa (300 bar).Aqueous solutions of simple organic compounds such as glucose, as well as slurries of diVerent biological materials, e.g., Bovine Liver, milk or juice, were digested. The determination of the residual carbon content was performed oV-line by ICP-OES. Compared with other continuous flow digestion systems, unusually low residual carbon contents were obtained. For the majority of biological samples digested, the residual carbon content was 1–13%, corresponding to a digestion eYciency of 87–99%.The potential of the continuous digestion system was illustrated by the digestion of three certified reference materials (Dried Whole Animal Blood, Bovine Liver and Tomato Leaves) and subsequent determination of Mn, Cu, Zn and Fe with ICP-OES. Recoveries between 76 and 109% were found. The RSDs ranged from 2.5 (Mn in Tomato Leaves) to 15% (Fe in Dried Whole Animal Blood).injection analysis and microwave digestion techniques. In an Introduction eVort to shorten digestion times and to eliminate or reduce The determination of elements in solid samples requires the other disadvantages of batch digestion procedures, flow digesdissolution or digestion/manipulation of the solid sample,1,2 if tion systems were developed.15–17 The main advantage of such one refrains from the determination of elements via electrother- flow digestion systems is the possibility for complete automal ablation,3 laser ablation4 or the direct slurry nebulization mation inherent in almost every flow system,18 in addition to approaches5 or direct determination by X-ray spectroscopy.6 others such as high sample throughput, minimized reagent Digestion of solid samples — or aqueous solutions containing consumption, reduced analyte losses and low contamination an organic matrix that can interfere with the analytical determi- and simplified sample handling.Automation is especially nation of an element — can be performed in many diVerent desirable in the analysis of waste water, soil samples, food and ways, e.g., open-vessel hot-plate digestion, and with many biological samples, as well as in process analysis. Several diVerent commercially available digestion systems. In principle, workers have developed and presented diVerent flow digestion high temperatures are essential to achieve as complete a systems in which the determination is carried out oV-line after digestion as possible.7 ‘If the temperature is increased by 10 K the sample has passed the digestion system as well as real the reaction rate is increased by a factor of 2 to 4 on average’.8 on-line systems which are directly coupled to a spec- Convectively heated pressure bomb vessel systems have proved trometer19,20 or a hydride system.21,22 In other systems digesto be the most valuable systems to guarantee complete or tion is carried out in a stopped-flow mode.23–25 There are also almost complete dissolution of solid samples because they several commercially available continuous digestion systems provide elevated digestion temperatures of about 200– (e.g., from CEM, Questron) which can be used for diVerent 230 °C.9,10 One of the most ingenious systems is the high applications.26,27 pressure asher (HPA) developed by Knapp which oVers diges- In all these systems the sample to be digested is transported tion temperatures up to 320 °C.11 Such systems only suVer through a microwave heated capillary consisting of the same from one severe disadvantage: the digestion procedures are, fluoropolymer compounds that are commonly used for batch although complete in most cases, very time consuming.microwave digestion systems, i.e., PTFE or PFA. One excep- Remedial measures concerning time consumption and labour- tion is the system presented by Gluodenis and Tyson.18 Here, intensive manipulation were taken when microwave digestion the PTFE tubing is loosely embedded in a resistive heating systems appeared since they facilitate direct heating of the block.By using PTFE tubing the maximum digestion is sample and therefore generally oVer much shorter times for restricted to less than 250 °C. The limited mechanical strength sample preparation.12–14 Because the power supply is realized of the material merely allows maximum working pressures of by microwave energy the vessel materials have to be micro- up to 35 bar.Since the mechanical strength of the material is wave-transparent. The most commonly used vessel materials decreased with increasing temperature it is not possible to are based on fluoropolymer compounds such as PTFE (poly- apply the maximum admissible temperature of 250 °C and at tetrafluoroethylene) or PFA (perfluoroalkoxy). The general the same time the maximum admissible pressure of 35 bar. problem with these materials is that, due to their limited Therefore, the usual working temperature and pressure are mechanical strength, working temperatures and pressures are much lower than these values (about 200 °C and/or 10–20 restricted to values that are not always suYcient for the bar).The system oVered by CEM, for example, utilizes a complete digestion of certain sample materials. structurally reinforced polymer digestion tube (with a restrictor at the end) that can be operated at 120 °C and 23 bar at the Several workers have tried to combine the merits of flow J.Anal. At. Spectrom., 1999, 14, 683–691 683same time. These conditions are not suYcient for the total principle behind the high temperature/high pressure digestion system required many fundamental studies. The main problem digestion of several types of sample. This can also be concluded from the reports of several workers, who found that after was to find a suitable arrangement for such a system.passing the continuous digestion system there was still particulate matter in the digested solution which had to be filtered.24,26 Experimental A special system was presented by Knapp et al.28 Here, the PTFE tubing is positioned within an autoclave which is filled Instrumentation with nitrogen to a pressure of up to 25 bar. By embedding the A simultaneous ICP-OES instrument (IRIS/AP, Thermo tubing within an autoclave the problem of the limited mechan- Jarrell Ash, Franklin, MA, USA) with axial observation of ical strength of the material is by-passed because the same the plasma and Echelle optics and a CID detector was used pressure exists inside as well as outside the tubing.28 for the determination of elements in the digested solutions.It is obvious that, at present, there exists no material that Instrumental and analytical parameters are given in Table 1. is transparent to microwaves and that is also resistant to high Calibration functions were generated from 0.1–1.0 mg l-1 temperatures and high pressures.Concerning high temperasolutions of the elements. The residual carbon was also deter- tures and high pressures, microwave heated flow digestion mined with this instrument. The calibration function for systems have already reached limitations that can hardly be carbon was generated from 0.1–1 g l-1 solutions. surpassed. However, it is possible to deal with high tempera- A total carbon analyzer (built in-house) was used for the tures and high pressures in a flow system, namely, high temdetermination of the original carbon content of solid samples perature–hydraulic high pressure nebulization (HT-HHPN), and slurried material.where an indirect resistively heated metal capillary is used.29 A standard HPLC pump, a UV-HPLC photometer, a six- A modified arrangement should provide a suitable system port-injection valve and connectors were obtained from Dr. continuous digestion. The high temperature/high pressure flow Knauer, Wissenschaftliche Gera�tebau, Berlin, Germany. system for the continuous digestion of samples that is presented A power supply was built in-house implementing a PID in this work results from a pure nebulization system, viz., (proportional, integral, derivative) controller (Horst, Lorsch, hydraulic high pressure nebulization (HHPN),30 a further Germany). development of which is HT-HHPN.29 In HHPN, a liquid is forced with the aid of an HPLC pump under a high pressure Arrangement of the high temperature/high pressure flow (50–400 bar/5–40 MPa) through a nozzle with a very small digestion system orifice (10–30 mm).In this case, nebulization is realized only by the hydraulic component of the liquid. Apart from pure The arrangement used for the high temperature/high pressure nebulization, HHPN can be considered as an interface between flow digestion system is shown schematically in Fig. 1. An HPLC techniques and atomic spectrometry.Hence, various HPLC pump was used to transport the liquid through the on-line techniques such as matrix separation,31 preconcen- system. The pump head is made of titanium. The valve used tration and trace element speciation analysis are possible.32,33 for sample injection is a six-port injection valve made of PEEK In HT-HHPN, the liquid, which is also transported with the (polyether ether ketone). The connection tubes to the digestion aid of an HPLC pump, is heated to temperatures of up to system consist of PEEK (id=0.75 mm, od=1/16 in).The 360 °C. For nebulization, the superheated liquid is forced sample loop (PEEK, id=0.75 mm, L=5 m, volume=2.2 mL) through a nozzle at pressures of about 200 bar. The aerosol is filled with the aid of a syringe. To connect the PEEK supply eYciency is increased from about 45 to 80% in flame AAS. capillaries (od=1/16 in) to the digestion capillary (od=1/8 in), The essential part of both nebulization systems, viz., HHPN special PEEK connectors with a low dead volume (1/16–1/8 in) and HT-HHPN, is the nebulization nozzle.Its high flow were used. Sealing is guaranteed with PEEK or Teflon ferrules. resistance demands the use of an HPLC pump for the transport The temperature control device consists of a PID controller of a liquid through the flow system. With such a nozzle at the which controls two solid-state relays. Via the solid-state relays end, the system can be considered as a quasi-closed system.the PID controller regulates a variable transformer which is The nozzle can also be considered as a restrictor which connected to a power transformer. The latter is a customary provides a certain back-pressure for the whole flow system. A transformer normally used for halogen bulbs with a maximum liquid can be heated far beyond its atmospheric boiling-point power of 300 W and 12 V. To reach a high temperature within the heated system. Vapour generation is restricted as stability in a simple manner, one of the solid-state relays long as the back-pressure of the nozzle exceeds the vapour delivers a power of 100% while the other via a variable number pressure of the liquid.In HT-HHPN, only a relatively small of resistors delivers a power of about 90%. These resistors are heating zone is necessary (about 12 cm) to reach a temperature connected in series with the variable transformer. If the equilibrium for the liquid and to ensure reproducible nebulization conditions.For the development of a flow digestion Table 1 Instrumental operating parameters for ICP-OES system, it was necessary to scale up the dimensions of the high temperature/high pressure flow system used for nebulization Rf frequency/MHz 27.12 to provide a larger reaction zone and sample residence times Rf power/kW 950 long enough to guarantee complete digestion. Instead of the Argon gas flow rates/L min-1: HHP nozzle a capillary with a small inner diameter can Outer 16 Intermediate 0.5 function as a restrictor.Provided that the flow is Laminar, the Nebulizer 0.6 length of the restrictor capillary necessary to achieve a certain Spray chamber Cyclone type back-pressure at a given flow rate can be calculated using the Sample flow rate/mL min-1 1.4 Hagen–Poiseuilles law. It has to be considered that a deviation Integration time l<360 nm: 30 s l>360 nm: 10 of the nominal inner diameter of 10% leads to a deviation of s the calculate pressure of 46% (p#1/r4).To avoid vaporization Analytical wavelength/nm: C 193.091 inside the capillary, the liquid has to be cooled below its Cu 224.7 atmospheric boiling-point in front of the restrictor capillary. Mn 257.6 In this paper, the development and performance of such a Fe 248.8 high temperature/high pressure system for the continuous Zn 206.2 digestion of biological samples is described and evaluated. The 684 J. Anal. At.Spectrom., 1999, 14, 683–691Fig. 1 Schematic arrangement of the high temperature/high pressure flow digestion system. Upper part: HHP nebulization nozzle as restrictor. Lower part: 50 mm capillary as restrictor. nominal heating value is exceeded by more than 2 °C, the temperatures, high pressures and high acid concentrations as has been shown for the HT-HHPN system.33,34 device ensures that the controller is switched oV completely. This short-term switch-oV is especially important for strongly exothermic digestion reactions.This device ensures the perma- Quartz glass tubing. In the first arrangement a quartz capillary of 2.5 m in length that was specially produced for nent control of energy flux into the sample. Furthermore, it provides the possiblity to heat electric components of low as these experiments was used (Heraeus Quarzglas, Hanau, Germany). The inner diameter of the capillary was 1 mm and well as high resistance by simply changing the power transformer, thus being very flexible.The cables of the power the outer diameter was 5 mm, thus giving a wall thickness of 2 mm. The main problem to be solved was the connection to transformer are connected via strip connectors either directly to the tubing (direct heating) or to a heating wire (indirect the PEEK supply capillaries. Connectors that screw on to the capillary can destroy the material and tend to leak if the heating). The heated zones of the capillaries were insulated with mineral wool to reduce heat losses.system is exposed to high pressure. Therefore, special connectors were fabricated from PEEK that can be glued on the For cooling, either a water/Liebig cooler or an air cooler was used. For the air cooler, two electronic cooling elements quartz capillary with a two component glue that exhibits a particularly high firmness for materials such as glass, metal, (20×4x10 cm; 0.55 kW-1) were screwed together. A channel was drilled through the cooler into which the tubing was ceramics and a variety of plastics (e.g., Aremco-Bond 631; T-E-Klebetechnik, Hannover, Germany). A schematic diagram pressed.The restrictor capillaries used were made of Polysil or PEEKsil (Scientific Glass Engineering, Ringwood, Victoria, of such a connector is shown in Fig. 2. These connectors were able to withstand pressures of more than 20 MPa. Australia). Quartz capillaries with an inner diameter of 50 mm are common in gas chromatography but they are diYcult to Unfortunately, the quartz capillary was destroyed at a pressure of about 18 MPa.A second capillary of 2 m in length was connect to common HPLC capillaries. The Polysil and PEEKsil capillaries have an inner diameter of 50 mm, but they melted together from two 1 m pieces (id 1 mm, od 6 mm, wall thickness 2.5 mm). According to the manufacturer are synthetically coated. The outer diameter is 1/16 in so that the capillaries can easily be connected to normal HPLC (Vogelsberger Quarzglastechnik, Freiensteinau, Germany), the maximum inner overpressure for this capillary is about capillaries with common connectors.Implementation of a restrictor capillary is shown in the upper part of Fig. 1. 6.4 MPa.35 It should be much lower because of the unevenness produced by the melting procedure. However, several experi- The HHPnozzle plate that was used for some experiments consists of Pt–Ir (95+5) and has an orifice of 20 mm (or ments showed that these capillaries were stable beyond 10 MPa at a temperature of 250 °C.The quartz capillary has to be 15 mm). A special nozzle holder made of titanium holds the nozzle. To protect the nozzle from particles that could cause clogging, a titanium filter sieve (d=3 mm) is mounted in front of the nozzle holder. It must be emphasized that this filter is not used to filter any possible particulate residues after digestion. The arrangement implemented with an HHP nozzle as restrictor is shown in the lower part of Fig. 1. Selection of tubing material DiVerent materials were investigated for their suitability as digestion capillaries. The tubing material should withstand high temperatures and high pressures and also be resistant to high acid concentrations. Metal capillaries, e.g., stainless steel, are not suitable as they are partly dissolved under the influence of acids and thus would give high blanks for several elements. Fig. 2 High pressure connector between standard HPLC capillary (1/16 in od) and quartz capillary (6 mm od).An exception are Pt–Ir capillaries. They can be used at high J. Anal. At. Spectrom., 1999, 14, 683–691 685heated indirectly with a heating wire. The heating wire consists electrical isolator. The total length of the tubing is 2.9 m. The volume of the tube is 7.2 mL. The heated zone was coiled of Ni 99.6 (d=1 mm) with a specific electrical resistance of 0,.9 V mm2 m-1 (Isabellenhu� tte, Dillenburg, Germany).A (distance between the coils about 0.5–1 cm; diameter of coiling about 10 cm). The tubing was coiled to obtain a more compact 70 cm length of the quartz capillary was wrapped with 5 m of the wire starting at about 15 cm beyond the inlet. In front of arrangement for ease of handling. The heated part was coated with fibre glass tubing. In the middle of the heated zone a the connection to the restrictor the quartz capillary is cooled with a Liebig cooler (L=20 cm).thermocouple (Ni–CrNi) was fixed which was connected to the PID controller. Finally, the heated zone was insulated with mineral wool to reduce heat losses. The total length of the Glass lined tubing (GLT A). This material consists of a stainless-steel capillary that is coated on the inside with a heated zone is 2.2 m, corresponding to a volume of 5.5 mL. The electrical power required for heating depends on the flow borosilicate glass layer. According to the manufacturer (Scientific Glass Engineering), GLT should withstand strong rate and is shown in Fig. 3. Even at the highest flow rate of 2 mL min-1, the power required to reach a temperature of acids and bases and temperatures of up to 600 °C and briefly up to 800 °C. The material is available in several dimensions, 250 °C was lower than 100 W. The highest voltage necessary was about 5.3 V and the current was 13.1 A. This means that e.g., 1/8 in od, id=1.8 mm or 1/16 in od, id=0.7 mm, standard length up to 1.8 m.The GLT capillary is heated directly. The a relatively small power supply would be suYcient for the eYcient heating of the TL tubing to high temperatures. borosilicate glass layer functions as an electrical isolator so that no electrochemical reaction should take place. After To ensure that the temperature inside the TFE tubing is equal to the temperature measured outside the stainless-steel several digestions it became evident that the borosilicate glass layer was decomposed under high temperature/high pressure tubing, the capillary was heated slowly, without a restrictor at the end of the arrangement, at diVerent flow rates conditions in combination with nitric acid digestion (250 °C, 15 MPa, approximately 6 M nitric acid).Pictures of several (1–4 mL min-1). Additionally, the temperature of the water was measured at the end of the heated part. The diVerence segments of the GLT were taken by a scientific video camera, showing that the glass layer was totally dissolved under the between the temperatures measured outside and inside the capillary was 1–2 °C, thus proving a satisfactory heat transfer digestion conditions stated.Additionally, owing to the strong corrosion of the glass layer, fairly high concentrations of Na, from the stainless-steel capillary to the TFE tubing. Ca and Fe, the main components of the glass layer and the stainless steel, could be detected in the digested solutions.It Pt–Ir capillary. This material can withstand temperatures up to 400 °C and pressures up to 40 MPa. It was tested with was noticed that the glass layer had been destroyed only within the heated part of the capillary. The inlet and outlet of the strong acids and showed good resistance and low blank values for Pt and Ir. For the HT-HHPN system, capillaries of 15 and capillary were still coated. Additionally, the restrictor (nozzle or even the 50 mm restrictor capillary) was blocked frequently 30 cm in length were used.During the manufacturing procedure these capillaries were stretched/drawn. For the continu- by particles that were dissolved from the borosilicate glass layer. The material only seems to be inert up to temperatures ous digestion system, Pt–Ir capillaries of 1.5 m in length were ordered from another manufacturer. Unfortunately, these of about 200 °C. Therefore, GLT is not suitable for a true high temperature/high pressure digestion system.On the other capillaries had hairline cracks, so it was impossible to apply high pressure. These defective capillaries were manufactured hand, this result shows that even glass is dissolved under the stated conditions. Therefore, it is indirect proof of the eYciency by welding. Nevertheless, some orientating experiments were carried out with the 30 cm Pt–Ir capillary which showed that of a high temperature/high pressure flow digestion system. this material should be suitable for incorporation in a high temperature/high pressure flow system.Further investigations Teflon lined tubing (TLT ). Teflon lined tubing (e.g., Alltech Associates, Deerfield, IL, USA) consists of a Teflon (TFE) with this material will follow as soon as another capillary from another manufacturer is available. tube that is mechanically inserted inside a stainless-steel tube. It is available with TFE tubing of, e.g., 3.2 mm od, 1.8 mm id and about 0.19 mm wall thickness. Because most of the results Reagents were obtained with this tubing, its behaviour will be described Analytical-reagent grade water was used for all solutions and in detail.as a carrier stream. Single or multi-element calibration stan- In preliminary experiments the TL (Teflon lined) tubing dards were prepared by dilution of standard solutions contain- was used only for the digestion of glucose solutions. After a ing 1000 mg L-1 of the elements. few months, frequent clogging of the nozzle and restrictor capillary was apparent.Thereupon, the capillary was cut with a silicon carbide saw blade to investigate whether the TFE tubing had been destroyed. It was found that the tubing had snapped and been reduced in diameter and length. It became obvious that during mechanical insertion the TFE tubing must have snapped. Therefore, a second TL tubing was prepared by passing a sturdy PEEK capillary through the TFE tubing to widen it. Furthermore, short pieces of a PEEK capillary (7 cm) were inserted into each end of the TL tubing and glued to the TFE tubing with a special glue (Aremco-Bond 526, T-E-Klebetechnik) appropriate for fluoropolymer materials.This was done to ensure that the liquid is transported only through the TFE tubing and not in the space between the TFE tubing and the stainless steel capillary and to avoid contamination for several trace elements. The TL tubing was connected to the supply capillaries with special connectors (1/16–1/8 in).The TL tubing was heated directly by connecting it to the Fig. 3 Power consumption as a function of flow rate and temperature of the system. power transformer. The inner TFE tubing functions as an 686 J. Anal. At. Spectrom., 1999, 14, 683–691A carbon stock standard solutn was prepared by dissolving residual carbon. The residual carbon can be calculated from eqn. (1): 5.248 g of oxalic acid (C2H2O4·2H2O) in 100 mL of analyticalreagent grade water, thus giving a carbon content of 10 g L-1.RC (%)=RCC×100/Cor (1) All other carbon standards (10, 100, 500, 1000 mg L-1) were freshly prepared by dilution of the 10 g L-1 carbon stock Cor: original carbon content (determined, e.g., with total standard immediately before the measurements. carbon analyzer) in g (carbon) per g (sample) Digestions were carried out using nitric acid (65% m/v, pro RCC: residual carbon content in the digested solution (deteranalysi; Merck, Darmstadt, Germany). mined, e.g., by ICP-OES) refering to original carbon content Cor, in g (carbon) per g (sample) RC (%): residual carbon as percentage of the original carbon Procedure Cor Sample preparation.For the initial digestion experiments DE: digestion eYciency: 100-RC (%). glucose solutions were used since glucose is relatively easy to The original carbon content was determined with a total digest and contains no particulate matter. To prepare these carbon analyzer.Therefore, an amount of the powdered solutions an appropriate amount of glucose was weighed into sample material was weighed with a micro-balance into small a 50 mL (20 mL) flask to which were added about 10 mL Ni containers and transferred into the apparaturs. The material (4 mL) of water followed by diVerent amounts of concentrated was burned in an oxygen stream and the amount of CO2 nitric acid to give a final acid concentration of 10–40% m/v generated was determined by an infrared detector.For the (1.7–7.9 mol L-1). After cooling, the solution was diluted to determination of the original carbon content of milk, the volume with water. sample was diluted by a factor of 20 because of the high As a natural suspension, conventional mixed vegetable juice carbon content. For the determination of the original carbon (a commercial mixture of, e.g., tomato, red beet, onion, celeriac content of vegetable juice, the juice was freeze-dried to reduce and carrot juice) was chosen as an initial sample material that the water content (preconcentration of carbon).The dried contains fibrous particles. To prepare a sample of juice the material was then reacted in the carbon analyzer. bottle originally containing the juice was agitated to provide The eYciency of the digestion was determined via the homogeneity. About 5 g of juice were then weighed into a residual carbon. To obtain the accurate carbon content of the 20 mL calibrated flask and diluted to volume after the addition digested sample by an ICP-OES measurement it was necessary of concentrated nitric acid.to de-gas the sample. If the carbon content was measured To obtain slurries of powdered materials the following without de-gassing the results were found to be rather high. certified reference materials (CRMs) were used: NIST SRM Initially, de-gassing was realized by ultrasonic shaking but 1577 Bovine Liver, NIST SRM 1567 Wheat Flour, IAEA A-2 even after this procedure the carbon contents were still very Dried Whole Animal Blood, NIST SRM 1573 Tomato Leaves high.Hence, the samples were finally de-gassed by passing and Red Beet Powder (Commission of European nitrogen through the digested samples for at least 20 min. The Communities). To prepare slurries of the CRMs, about 0.2 g residual carbon for digested samples (25 g L-1 glucose) that of the sample was weighed (±0.1 mg) into a 20 mL calibrated were de-gassed with nitrogen was ‘0’% and for samples that flask to which were added 5 mL of water followed by the were not de-gassed 37% and if de-gassing was carried out by addition of 1 mL increments of concentrated nitric acid to ultrasonic shaking 29%.These values show that it is necessary obtain a suspension of 20% m/v (3.5 mol L-1) HNO3. Between to de-gas the digested solutions prior to determination of the the addition of each increment of concentrated HNO3 the carbon content by ICP-OES.flask was shaken vigorously to ensure a homogeneous distribution of the solid sample. The slurry was then diluted to Basic investigations volume with water. With some slurry materials frothing was observed; hence, it became necessary to remove the bubbles Most of the following experiments were carried out using a and foam by ultrasonic shaking. TL capillary (od 1/8 in). Because of the large length (3.05 m) and the relatively large inner diameter (1.8 mm) of the capillary, dispersion was expected to be rather large.The dispersion Digestion. About 5 mL of the slurry were drawn into a disposable syringe and injected into a six-port injection valve profile was investigated in two ways. First, by transporting an aqueous solution of Mn through the system and measuring equipped with a sample loop of volume 2.2 mL, corresponding to a sample amount of 22 mg. By turning the valve to the the Mn content in each fractionated sample volume by ICPOES (0.5 mL diluted to 10 mL).Additionally, the solutions injection position the sample was inserted into the aqueous carrier stream and transported towards the heated zone. The contained glucose. The carbon content was also measured by ICP-OES. It was decided to measure the carbon content also sample was digested while passing the heated zone. Subsequently, the sample reached the cooling zone and then because in later experiments the carbon content of the digested solutions acted as an indicator of the eYciency of the sample left the system via the restrictor.The digested sample was collected at the outlet of the system in a single fraction into a digestion procedure. The dispersion profile for an undigested glucose solution containing Mn is shown in Fig. 4. 20 mL flask, thus resulting in a dilution factor of 9.1 (2.2 mL per 20 mL). For a flow rate of, e.g., 1 mL min-1, a sample Additionally, dispersion was monitored by transporting a dye (Eosin Blue) through the system and measuring the absorption volume of 18 mL was collected.Such a comparatively large volume of sample was collected to take into account the large with a UV-HPLC photometer at 252 nm. These measurements led to the same result. For a flow rate of 1 mL min-1, the dispersion within the system. Collection was started 2 min after injection of the sample. To investigate the dispersion maximum of the profile appears at 6–7 min. Theoretically, dilution with the carrier and thus dispersion should be profile, the sample was collected in 0.5 mL fractions and each increment diluted to a final volume of 10 mL.After collection decreased by using capillaries with a smaller inner diameter,36 but the smallest inner diameter available for TL tubing is and dilution, the carbon content and several trace elements were determined oV-line by ICP-OES. 1.8 mm. In low pressure flow systems, dilution with the carrier and The carbon content of the digested solutions was determined by measuring the carbon emission by ICP-OES at 193.091 nm. thus dispersion can be avoided by deliberately embedding the sample in small volumes of air (segmented flow technique).The residual carbon content expressed as a percentage of the original carbon content of the undigested sample yields the For a high pressure system, a relatively large volume of air J. Anal. At. Spectrom., 1999, 14, 683–691 687indicating that temperatures of at least 210 °C are necessary for the eYcient digestion of a glucose solution.Further elevation of the temperature (220–240 °C) does not seem to increase the oxidation rate of carbon significantly and thus the digestion eYciency any further. For an acid concentration of 20% HNO3, the carbon content is already decreased at a temperature of 190 °C. After evaluation of the variation of the carbon profile with increasing temperature, solutions containing the same amount of glucose (25 g L-1) were digested and the residual carbon (RC%) was determined quantitatively.For this digestion, an acid concentration of 20% HNO3 was used. Again, the temperature was varied from 25 to 250 °C (25, 130, 150, 170, 190, 210, 230, 250 °C). For a temperature of 130 °C, the residual carbon was 68% (RSD: 1%); for 150 °C, 23.5% (RSD: 9%); for 170 °C, 6.5% (RSD: 6%); for 190 °C, 1.6% (RSD: 15%); Fig. 4 Dispersion profile of undigested glucose solution with Mn for 210 °C, 0.5% (RSD: 20%); and for temperatures of 230 as indicator. and 250 °C, no carbon could be detected in the degassed solutions (n=5) (detection limit for carbon: 1.5 mg L-1).has to be introduced as the air becomes compressed under In further experiments the amount of glucose to be digested high pressure. An initial volume of 2.2 mL of air was injected was elevated (25, 50, 75, 100, 150, 200, 250 g l-1). Whenever that should result in a buVer volume of 10 mL after com- digestion at 250 °C was found to be incomplete (RC >2%) pression.With an inner diameter of 1.8 mm of the capillary, the acid concentration was increased (10, 20, 30, 40% m/v). the length of the compressed volume under a pressure of Fig. 6 shows the residual carbon (RC%) as a percentage of 20 MPa should be 4 mm, thus providing a satisfactory distance the original carbon content of the digested sample for diVerent between sample and carrier. However, obviously, the air is initial glucose contents and acid concentrations. For small compressed and probably becomes completely dissolved in the amounts of glucose (25, 50 g L-1), a nitric acid concentration carrier stream.After introduction of the air, the pressure of up to 20% is suYcient to reach RC values of less than 2%. suddenly drops considerably which can be observed by follow- For higher amounts of glucose (75–250 g L-1), higher acid ing the pressure display of the HPLC pump. Pressure is rebuilt concentrations are necessary (30–40% m/v).It can be seen only very slowly as the air becomes compressed and dissolved that even a 25% (250 g L-1) glucose solution can be digested in the carrier stream. Owing to the inevitable pressure drop with 40% HNO3, resulting in a residual carbon content of after injection of a discrete volume of air, the segmented flow about 2%. The relative standard deviation is 3–16% for the technique, often used in low pressure flow systems, cannot be determination of the RC%.For original glucose contents transferred to high pressure flow systems. Furthermore, the higher than 150 g L-1, the relative standard deviation is less pressure drop within the system leads to an undesirable vapour than 10% (n=6). generation along the heated zone because boiling of the liquid occurs. Residual carbon (digestion of slurries) The first suspension to be digested was mixed vegetable juice Results (5 g juice per 20 mL digestion suspension). Digestion of the vegetable juice was carried out using a TL capillary with a Residual carbon (digestion of glucose solutions) restrictor capillary at the end of the digestion system.With an Solutions containing 25 g l-1 glucose were digested with 10% acid content of only 10% HNO3 and temperatures between HNO3. The digested sample was collected in 0.5 mL fractions 200 and 230 °C, frequent clogging of the restrictor capillary into 10 mL flasks as mentioned above. The carbon content of occurred.At a temperature of 240 °C and an acid concentration each fraction was measured by ICP-OES without any of 20%, a series of digestions were carried out without any de-gassing because only the variation of the carbon content should be observed. In a series of experiments the digestion temperature was increased from 190 to 240 °C. The resulting profiles are shown in Fig. 5. At temperatures below 210 °C, no digestion takes place. Starting at a temperature of 210 °C the carbon content of the digested solution suddenly decreases, 10 RC (%) 9 25 g L–1, 10% HNO3 25 g L–1, 20% HNO3 50 g L–1, 10% HNO3 50 g L–1, 20% HNO3 75 g L–1, 10% HNO3 75 g L–1, 20% HNO3 75 g L–1, 30% HNO3 100 g L–1, 30% HNO3 150 g L–1, 30% HNO3 200 g L–1, 30% HNO3 200 g L–1, 40% HNO3 250 g L–1, 40% HNO3 8 6 7 5 4 3 2 1 0 Fig. 6 Residual carbon as a function of the original glucose content Fig. 5 Residual carbon profile at increasing digestion temperatures. and of the concentration of the digestion acid. 688 J.Anal. At. Spectrom., 1999, 14, 683–691clogging. Even samples containing higher amounts of vegetable means at a 95% probability level. Table 3 additionally contains the calculated texper, the critical t-value, tcritical, and the result juice (e.g., 10 g juice per 20 mL) could be digested without any problem. These initial experiments with vegetable juice as of the t-test. the sample showed that, in general, the system is suitable for the digestion of samples that contain particulate matter.Discussion Thereupon, various pulverized CRMs (see under Procedure) The aim of this work was to develop a new system for the were selected for digestion. Arbitrarily, a slurry content of continuous digestion of diVerent sample materials and to prove 10 g L-1 (1% m/v) was chosen. Since digestion of vegetable the validity of the principle and the feasibility of a high juice with only 10% HNO3 led to frequent clogging of the temperature/high pressure flow digestion system.Heating of restrictor and thus incomplete mineralization of particulate the tubing was accomplished by direct or indirect resistive matter, for the digestion of the pulverized material an acid heating. DiVerent tubing materials were tested such as quartz concentration of 20% HNO3 was provided. After optimization, capillaries, glass lined tubing and Teflon lined tubing. a digestion temperature of 250 °C was found to be suitable. Under these conditions each of the listed sample materials (see Development of the arrangement/selection of tubing material under Procedure) could be digested.The digested solutions were in all cases clear and colourless. Furthermore, no eventu- The most practical material and the least expensive was Teflon ally undigested particles led to blockage of the restrictor. The lined tubing. The majority of the presented results were only material that gave rise to complications was Pine Needles obtained with this material.From a diVerent point of view the (NIST CRM 1575). The particles within the slurry were too TL tubing can be regarded as a pressure bomb stretched in large and stringy so that not even injection into the sampling length (‘long tubing bomb’) as, usually, pressure bomb vessels valve was possible. This problem can be overcome by drilling such as the ‘classical’ pressure bomb described by To� lg consist holes in the injection valve. With an injection valve containing of a stainless-steel vessel in which is embedded a PTFE liner.holes of a larger inner diameter (e.g., 1/8 in HPLC sample However, the best tubing material for a continuous digestion introduction valve) and by the use of a larger sample injection system should be Pt–Ir capillaries as they allow temperatures coil this problem can be overcome. To date, no erosion of the up to 300 or 400 °C. However, these capillaries have to be valve components was observed.In addition, slurries of Pine produced to order. Yet it seems to be important that the Needles can be injected and digested if they are left standing capillaries are manufactured by stretching/drawing. for at least 1 d after preparation of the slurry. Concerning safety it can be stated that, in general, the The carbon content measured in the digested solutions of handling of high pressure is substantially safer in metalvarious sample materials is listed in Table 2. In addition to covered tubes of a small diameter than in bombs or in the relative standard deviation, the original carbon content unprotected PTFE tubing.Temperature is controlled by a PID (Cor), the residual carbon content (RCC) and the residual controller that ensures total shut-down of energy flux in the carbon (RC%) are listed. It can be seen that the residual event of highly exothermic reactions. Pressure is controlled by carbon is rather low. For the majority of digested samples it the aid of an HPLC pump.If clogging should eventually is less than 10%. Only for Bovine Liver and Dried Whole occur, the mp immediately stops if the maximum pressure Animal Blood is the value above 10%. This may be attributed is reached. By using a TFE tube that is surrounded by a to the comparatively high original carbon content and to the stainless-steel capillary, pressure is no longer a limiting paramfact that the slurry consisted of rather large clots that may eter in digestions.not have been totally digested. Applicability/application of the system Elemental recoveries After the development of the system it had to be tested concerning its applicability to the digestion of diVerent sample For three CRMs, viz., Tomato Leaves, Dried Whole Animal Blood and Bovine Liver, the content of some selected elements materials. Initially, only glucose solutions were used as they can be digested relatively easily and contain no particles. The in the digested solutions was determined oV-line by ICP-OES.The results were compared with the certified values. In Table 3 residual carbon was evaluated as a measure of the eYciency of the digestion system. These preliminary experiments showed the analytical results and the certified values are listed. In general, the results are in the range of the certified values. that a considerable amount of carbon was present in the solutions after digestion in the form of CO2 that falsifies the Only the value for Fe in Dried Whole Animal Blood (76% recovery) is significantly lower than the certified value.To actual digestion eYciency. Complete de-gassing was realized by passing a stream of nitrogen through the digested solutions date, no explanation can be given for this behaviour. In Table 3 the relative standard deviations and the recovery are before measurement of the carbon content by ICP-OES. Highly concentrated glucose solutions up to 250 g L-1 can be also listed.A significance test was performed for the resulting Table 2 Digestion eYciency for diVerent biological materials Originala Residual Residualc Digestion carbon content, Carbonb RSD carbon content, Carbon (%) eYciency (%) Material Cor/g g-1 content/mg L-1 (%) RCC/g g-1 (RC%) (100-RC%) Tomato Leaves 0.34 25±5 20 0.023 6.7 93.2 Wheat Flour 0.38 14±3 21 0.013 3.4 96.6 Red Beet Powder 0.33 20±3 15 0.018 5.5 94.5 Bovine Liver 0.44 53±8 15 0.048 11 89 Whole Animal Blood 0.52d 71±25 35 0.065 12.5 87.5 Cocoa Powder 0.38 5±1 20 0.005 1.2 98.8 Vegetable Juice 0.02 8±1 13 0.0003 1.4 98.6 Milk 0.07 12±1 8 0.0011 1.6 98.4 aMeasured with total carbon analyzer.bCarbon content measured in digested solution by ICP-OES. cResidual carbon calculated as percentage of original carbon. dValue from the literature.37 J. Anal. At. Spectrom., 1999, 14, 683–691 689Table 3 Trace determinations in digested slurries of CRMs Concentration RSD Certified Recovery Significant measureda/mg g-1 (%) value/mg g-1 (%) texper tcritical diVerence NIST SRM 1573 Tomato Leaves— Cu 11±1 9.1 11±1 100 0 2.26 No Mn 244±6 2.5 238±7 103 2.13 2.23 No Fe 593±35 5.9 690±25 86 6.4 2.36 Yes Zn 67±3 4.5 62±6 108 2.92 2.09 Yes IAEA-A2 Dried Whole Animal Blood— Cu 45±3 6.7 45±4 100 0 2.18 No Fe 2.6±0.4 15 3.41±0.26 76 4.7 2.36 Yes Mn 114±11 9.6 123±21 93 1.46 2.10 No Zn 77±5 6.5 89±9 87 4.4 2.11 Yes NIST SRM 1577 Bovine Liver— Cu 207±11 5.3 193±10 107 2.84 2.31 Yes Zn 128±8 6.3 130±13 98 0.48 2.13 No Fe 292±10 3.4 268±8 109 5.5 2.36 Yes aMean and standard deviation (n=6).digested with a nitric acid concentration of 40%. In further hydride system to obtain on-line digestion and determination. Initial experiments showed that it is possible to connect the experiments, natural suspensions and slurries of powdered CRMs were digested. Even these suspensions containing proposed digestion system directly to an ICP-OES instrument. In these experiments the outlet of the restrictor capillary was diVerent types of particulate matter could be digested under the influence of high temperature and high pressure, resulting coupled to the Meinhard nebulizer of the spectrometer.Problems arose due to the dissolved CO2 and NOx present in in high digestion eYciencies between 87 and 99% depending on the respective material. The rather low amounts of residual the digested sample. To prevent the plasma from being extinguished, only small sample amounts can be digested and directly carbon should even make possible the subsequent determination of elements by electrochemical detection methods.In led into the spectrometer. The problem of extinguishing is not as serious when coupling the digestion system directly to a general, it was proved that temperatures of 250 °C are suYcient for the almost complete digestion of a variety of biological flame atomic absorption spectrometer. Working with a nozzle as restrictor it should also be possible to use the HHP nozzle and botanical materials.Because of the rather large inner diameter of the TL tubing, dispersion leads to the dilution of for direct nebulization of the digested samples into the plasma or even an HT-HHP nozzle. In both cases for ICP-OES, the sample in the carrier. In spite of the large dilution a sample volume of 2.2 mL was suYcient for the digestion of biological desolvation of the aerosol of the digested sample solution is necessary because of the high aerosol yield provided by this slurries and the subsequent determination of minor, major and trace elements.Carry-over from one sample to the next nebulization technique. A further possibility of direct nebulization of the sample into a spectrometer can include the use of was not observed as the elements under investigation are present in the biological materials at rather low concentration the restrictor capillary. As mentioned above, the digested solution is cooled below its atmospheric boiling-point before levels.Additionally, the system is cleaned continuously by the carrier. It is not yet known whether carry-over would occur leaving the system via the restrictor capillary. However, if the liquid were not cooled it would reach the restrictor capillary for the subsequent digestion of samples which contain the elements at considerably diVerent concentrations. at rather high temperatures. This should result in thermospray eVects that can be used for direct nebulization. If suYcient sample material is available, e.g., urine, waste water or juices, larger sample volumes (10–20 mL) can be Furthermore, the system can be automated to achieve computer-controlled digestion procedures.Another area for injected and a larger segment of the sample can be collected after digestion, rejecting the diluted front and end (tailing) of investigation is the use of alternative reagents for the digestion of diVerent materials, e.g., mixtures of HNO3 and HCl, the sample.peroxodisulfate and H2O2. Further developments In a further development of the digestion system the use of a Conclusion Pt–Ir capillary is intended. This capillary is available in a A new continuous flow digestion system using resistively much smaller inner diameter, thus reducing the dilution of the heated metal-covered tubing has been presented that works sample in the carrier stream. Since this material can be heated under real high temperature/high pressure conditions (260 °C, to temperatures up to 300 or even 400 °C it is expected that >200 bar).The feasibility of the continuous digestion of the digestion eYciency can be increased further. Higher digesvarious sample materials, liquid as well as slurrry samples, has tion temperatures should allow higher flow rates and shorter been demonstrated. The eYciency of the oxidation of the residence times and a reduction of the digestion time as well organic matrix was about 90–99% depending on the digested as the use of a shorter capillary and thus a more compact sample material.Several minor, major and trace elements were system for ease of handling. In the near future further investidetermined with recoveries of about 87–109%. This work has gations will be carried out with a system incorporating a Pt–Ir shown that the hitherto existing limitations concerning tem- capillary, especially for the digestion of waste waters.This perature and pressure can be overcome. It can be expected sample material can contain rather large amounts of silicates that in future still higher temperatures can be reached. that are hard to digest. However, with temperatures up to 400 °C, even the digestion of such a complex sample material should be possible. Acknowledgement In addition, it is intended to connect the continuous digestion system directly to an ICP optical emission spectrometer, This work was carried out with the financial support of the Bundesministerium fu� r Forschung und Technologie as well as a flame atomic absorption spectrometer, a photometer or a 690 J.Anal. At. Spectrom., 1999, 14, 683–69117 H. Matusiewicz and R. E. Sturgeon, Fresenius’ J. Anal. 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