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Spectrochimica Acta Part B 53 (1998) 859865

Silicon speciation using reversed phase high performance liquid chromatography-inductively coupled plasma atomic emission spectrometry radial versus axial viewing1
Les Ebdon a,*, Michael Foulkes a, Ken Fredeen b, Chris Hanna b, Karen Sutton2a
a

Department of Environmental Sciences, University of Plymouth, Drake Circus, Plymouth, Devon PL4 8AA, UK b The Perkin-Elmer Corporation, 761 Main Avenue, Norwalk, CT, USA Received 23 January 1998; accepted 18 May 1998

Abstract The speciation of polar silicon compounds by high performance liquid chromatography using both radially viewed and axially viewed inductively coupled plasma (ICP) atomic emission spectrometers, with solid state detection, is described. Optimum operating conditions for the direct aspiration of an organic mobile phase were evaluated for the two modes of viewing and limits of detection, background equivalent concentrations and relative standard deviations of the blank were calculated to compare radially viewed and axially viewed ICPs directly. It was found that no appreciable improvements in detection limits were observed when viewing the plasma axially. This is attributed to the fact that the plasma noise cannot be eliminated for end on viewing through the entire optical path length. Various methods for data acquisition are also described. 1998 Published by Elsevier Science B.V. All rights reserved Keywords: Silicon speciation; HPLC-ICP-AES; Radial vs. axial viewing; Organosilicon compounds

1. Introduction High performance liquid chromatography (HPLC) when coupled with inductively coupled plasma atomic emission spectrometry (ICP-AES) is now well established as a technique for speciation analyses. Using such a coupled technique, both the chemical form of an element and quantitative information may be obtained. The hyphenated technique of HPLC-ICP-AES has been used for the speciation of analytes in areas such as clinical chemistry,
* Corresponding author. Fax: 0044 1752 232 011; e-mail: lebdon@plymouth.ac.uk 1 This paper is published in honor of Professor C.L. Chakrabarti. 2 Current address: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA.

environmental chemistry, food science and toxicology [1]. The speciation of elements such as As, Se, Co, Fe, Pb and Sn has been well documented in the literature and many applications have been described where the inductively coupled plasma has been used as a detector for chromatographic and electrophoretic separation techniques [24]. To date, however, little work has been performed with regard to the speciation of silicon containing compounds. Dorn and Skelly Frame [5] rst described the used of liquid chromatographic techniques for the separation of organosilicon compounds in environmental samples. Both reversed phase and size exclusion chromatography were used to separate the silicon compounds according to their polarity, solubility and size. ICP-AES was shown to

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be a very sensitive method to determine the very low levels of organosilicon species that may be present in environmental samples. Silicon is the second most abundant element on the surface of the earth (28% by weight). It may exist in four major forms; as the elemental form silicon, found in human hair, dental enamel and bones [6]; as the oxidized form silica, SiO 2, a constituent of ceramics and concretes [7]; as silicates, used in analgesics and antacids which are excreted within hours of exposure in urine, or as the organosilicon form, of which the compounds are commonly known as silicones. Polydimethylsiloxanes (PDMS) represent the most versatile and commonly used organosilicon compounds. They are widely used in biomedical applications, principally in plastic and reconstructive surgery owing to the fact that silicon was considered to be chemically, immunologically and physiologically inert in the human body and could pose no ill effects [8]. This has been challenged in a review by Talcott [9], which illustrates the biological hazards of low molecular weight, water soluble breakdown products of PDMS. Three of these compounds are described in this paper. PDMS is manufactured from elemental silicon and is used as the silicon gel lling in breast implants, as well as the rubber implant shell. Causes of silicon bleed through the implant wall have been well documented [10] and, owing to the absorption of body uid components [11,12], the implant may crack or rupture. Silicone may then migrate to sites around the body through the hematogenous and lymphatic systems. It is well known that silicones degrade to lower molecular weight species [13,14] involving siloxane bond redistribution and hydrolysis. Studies are therefore required to identify the actual species present, as this information is essential if the bodys response to these compounds is to be measured. The reversed phase separation of organosilicon compounds requires the use of organic solvents, usually as a component of a binary mobile phase. The direct aspiration of organic solvents into the ICP has traditionally posed difculties [15,16]. Organic solvents tend to cause plasma instability and ultimately extinguish the discharge. Boorn and Browner [17] evaluated the performance of a low

power argon ICP for 30 organic solvents, using the lack of carbon deposition on the injector of the torch as a guide to plasma tolerance. These studies were performed using a radially or side-on viewed ICP and, to date, no investigation has been made as to the effect of organic solvents when viewing the plasma axially or end-on. Although the attainment of lower limits of detection have been well documented for end-on viewing with aqueous systems, an investigation into the performance of axial instruments with organic sample nebulization is required to evaluate if detection limit improvements may be made. Given the abundance of silicon in the environment and human body, studies of the impact of silicones in the human body require sensitive methodology to discriminate between the various forms of silicon which may be present in clinical samples. Spectroscopic techniques, particularly those using ICP-mass spectrometry (MS) and ICP-AES are capable of achieving low limits of detection when coupled to chromatographic separation techniques. However, owing to an isobaric interference, from diatomic nitrogen, with the principal isotope of silicon at mass to charge 28, ICP-AES is a more attractive detector for silicon speciation when compared to ICP-MS. The aim of this work was to make a direct comparison of radial versus axial gures of merit for various silicon species of potential interest in clinical compounds with both a radially and axially viewed ICP-atomic emission spectrometer.

2. Experimental 2.1. Chromatography The separation of three polar silicon containing compounds (inorganic silicon, hexamethyldisiloxane and 1,3-tetramethylsilanediol), using reversed phase chromatography, was performed using a Phenomenex ODS C 18 capped column, 250 2 mm i.d. (Phenomenex, Maccleseld, UK). The column was kept at room temperature throughout the study and samples were eluted with an isocratic mobile phase consisting of 30% methanol in ltered de-ionized water (Milli-Q, Millipore, Harrow, UK). The liquid ow rate was 1.0 ml min 1.

L. Ebdon et al./Spectrochimica Acta Part B 53 (1998) 859865 Table 1 Operating conditions for the radially and axially viewed ICP-reversed phase HPLC system Parameter Frequency/MHz Forward power/W Plasma gas ow rate/l min 1 Auxiliary gas ow rate/l min 1 Nebuliser gas ow rate/l min 1 Solvent Solvent ow rate/ml min 1 Silicon wavelength/nm Nebulizer Spray chamber Radially viewed ICP-AES 40 1150 15 1.0 0.8 MeOH/H _{2}O (30:70%) 1.0 251.611 P.E. Gem Tip cross-flow P.E. Scott Double Pass Axially viewed ICP-AES 40 1450 15 1.0 0.8 MeOH/H _{2}O (30:70%) 1.0 251.611 P.E. Gem Tip cross-flow P.E. Scott Double Pass

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2.2. Instrumentation Two liquid chromatographic spectrometer congurations were used in this comparative study. In the rst instance, a Perkin-Elmer 410 LC pump (PerkinElmer, Beaconseld, UK) was coupled with a radially viewed Perkin-Elmer Optima 3000 ICP optical emission spectrometer (Perkin-Elmer, Norwalk, USA). The Optima 3000 is equipped with a segmented array charged coupled device detector and echelle monochromator [18,19]. The ICP was operated under the conditions listed in Table 1. Optimum power and nebulizer gas ow rate settings were achieved using on-board directed search and exhaustive search algorithms in the Perkin-Elmer Optima 3000 ICP UNIX software, with the signal to noise ratio used as the gure of merit [20,21]. The use of organic solvents in the plasma usually requires more robust operating conditions when compared to the conditions used for conventional aqueous samples. By increasing the RF power and coolant gas ow, a stable plasma discharge may be obtained, which may be operated for an appreciable length of time without carbon deposition on the alumina injector of the plasma torch [17]. In addition, the velocity of sample introduction into the ICP may

be increased by increasing the nebulizer gas ow. A disadvantage of this, however, may be lower analytical signal readings owing to the lower residence time of the analyte in the ICP [16,17]. For comparison, chromatographic separations were also obtained using a Perkin-Elmer 200 LC pump coupled to a Perkin-Elmer Optima 3000 DV ICP emission spectrometer, with axial viewing orientation. The design of the echelle grating and solid state detector was the same as for the radially viewed instrument. Plasma source operating conditions were also optimized for this viewing orientation and are given in Table 1. The introduction of organic solvents into the ICP results in a green plasma plume, characteristic of C 2 emission. The Optima 3000 DV instrument employs a shear gas which cleaves the plasma plume to prevent self-absorption in this region so most C 2 emission can be seen along the central channel of the plasma. Plasma resistance to organic solvents is manifested by an increase in the reected power of the RF generator. The free running RF generator of the Optima 3000 series of instruments brings about adjustment of the variable capacitors via a matching circuit. If the reected power increases dramatically, a safety mechanism operates which extinguishes the plasma to prevent damage to the RF generator. For this reason, the introduction of an organic solvent into the ICP must be brought about by increasing the amount of organic solvent in small increments so that the matching circuits in the RF generator may slowly adjust to compensate for the mismatching of the plasma impedance. However, once the desired amount of organic sample introduced to the sample was achieved, the plasma was stable. In both instruments, a Perkin-Elmer Gem Tip cross-ow nebulizer was used to aspirate the sample into a Perkin-Elmer Ryton Scott double pass spray chamber. A Rheodyne (Rheodyne, Cotati, CA, USA) Model 7125 syringe loading switching valve with 100 ml sample loop was used to inject sample solution into the mobile phase carrier stream. PTFE tubing was used to connect the outlet of the column to the nebulizer. The length was kept to a practical minimum, dictated by the ability to physically move the outlet of the column as close as possible to the nebulizer inlet.

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2.3. Data acquisition Data obtained using the radially viewed instrument were acquired by writing a macro in the UNIX instrument operating software. The data were then converted, using a Perkin-Elmer conversion program, so that it was compatible with Perkin-Elmer Turbochrom 4 chromatographic data acquisition software, which provided integrated chromatographic peak area data. For the axially viewed ICP-AES instrument, newly developed Perkin-Elmer ICP Winlab software enabled the measurement of the transient signals by manipulation of the sample integration time and number of sample replicates measured. The peak area of the spectra was calculated by integrating 3 pixel widths on the corresponding subarray of the solid state detector. All data were stored to a database in Winlab. Chromatograms were then constructed by plotting net emission intensity values as a function of time using Microsoft Excel 5 and Microsoft Query programs. Integrated chromatographic peaks may be easily calculated in Excel 5. By using this method of data manipulation, data density may be improved by a factor of 5 when compared to the previous method employing UNIX software, owing to the increase in data points. 2.4. Silicon standards The silicon standards used in the reversed phase work were (i) hexamethyldisiloxane (Fluka, Gillingham, UK), (ii) a 1000 mg ml 1 commercially prepared inorganic silicon (sodium silicate) standard (BDH, Poole, UK) and (iii) 1,3-tetramethylsilanediol which was synthesized in-house for use as a silanol standard according to a procedure described by Carpenter and Cella [22]. Standards were kept refrigerated until immediately before use owing to their ability to undergo polymerization at room temperature in a matter of hours. It is important, when dealing with such organosilicon compounds, to avoid glassware owing to interactions between the vessel surface. The use of glass asks and bottles may therefore yield erroneous results and, in addition, PDMS lubricants should be eliminated from the sample introduction system and torch assembly [5]. Reagent grade chemicals were used throughout except where otherwise indicated. All standards and

solutions were ltered and degassed with helium prior to use.

3. Results and discussion Previous workers [5] have indicated that organosilicon compounds interact too strongly with silica based packing materials to allow their use for the separation of such compounds. However, a stability study over several days with a 30% capped ODS column did not indicate performance degradation over time. Typical background levels with a standard C 18 ODS column (uncapped) gave high background levels of Si that showed marked daily variation. Sensitivity and detection limit values for the organosilicons degraded with time. Reproducibility (based on peak area) from day to day was poor, typically around 20%. Limits of detection were more than 30 times higher that those attained using the C 18 ODS 30% capped column. The capped column resolved the compounds of interest and permitted the separation of soluble inorganic silicon species. Initially, the three organosilicon compounds were separated using the radially viewed ICP-AES instrument. A typical separation of inorganic silicate, 1,3-tetramethylsilanediol and hexamethyldisiloxane (20 mg ml 1 as silicon) is given in Fig. 1. The retention time for inorganic silicon was 3.8 min, 5.9 min for tetramethylsilanediol and 32.1 min for hexamethyldisiloxane. The overall reproducibility of the separation for each compound was 3.2, 5.4 and 2.3% for inorganic silicon, tetramethylsilanediol and hexamethyldisiloxane respectively (n = 10 replicates). Calibration graphs were linear to 100 mg ml 1 Si for the three compounds of interest. The response, or sensitivity, of the column for each compound differed. The sensitivity was 2601 area units per mg ml 1 Si for inorganic silicon, 2250 area units per mg ml 1 Si for tetramethylsilanediol and 1373 area units per mg ml 1 Si for hexamethyldisiloxane, when n = 10 replicates. The difference in sensitivity was not a result of interactions with the ODS capped column. By removing the column and simply ow injecting the samples into the plasma, a similar difference in sensitivity was observed for the three compounds. The most likely explanation for this difference in sensitivity is that it is caused by the different sample transport efciency

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Fig. 1. Separation of (a) inorganic silicon, (b) 1,3-tetramethylsilanediol and (c) hexamethyldisiloxane by reversed phase HPLC and radially viewed ICP-AES. All compounds present at 20 mg ml 1.

for each silicon compound through the sample introduction system and/or the differences in atomization efciency [23,24]. The higher molecular weight compounds may be transported less efciently into the plasma resulting in lower emission readings for Si and therefore a decrease in sensitivity. The detection limits obtained are given in Table 2 and were 0.1 mg ml 1 Si for inorganic silicon, 0.4 mg ml 1 Si for tetramethylsilanediol and 0.5 mg ml 1 Si for hexamethyldisiloxane. Using the axially viewed ICP-AES instrument, the same separation was performed. A separation of inorganic silicon, tetramethylsilanediol and hexamethyldisiloxane (10 mg ml 1 (as Si)) is shown in Fig. 2. Retention times for the three species were 2.5, 4.5 and 27.9 min respectively with the reproducibility 5.3, 5.7 and 5.9% for the three compounds respectively where

n = 10 replicates. The retention times were slightly different for the separation using the two instruments, as the PTFE transfer line lengths were not the same. The calculated detection limits using the axially viewed ICP-AES are given in Table 2. It can be seen from Table 2 that the radially viewed ICP-AES gave similar detection limits to the axially viewed ICP-AES with improved reproducibility for all three compounds. It should be noted that the integration time of 1 s for the data acquired using the radially viewed plasma was the same as that used for the axial system. It is well known that detection limits are improved dramatically when viewing an inductively coupled plasma axially compared to radial viewing [25]. A general improvement in detection limits of about a factor of ve may be observed when aspirating

Table 2 Comparison of detection limits and reproducibility data for axial and radial HPLC-ICP-AES systems Compound LOD/mg ml 1 Radial Inorganic Si Tetramethylsilanediol Hexamethyldisiloxane 0.10 0.40 0.50 Axial 0.18 0.30 0.48 Reproducibility/% Radial (using 20 mg ml 1 solution) 3.2 5.4 2.3 Axial (using 10 mg ml 1 solution) 5.3 5.7 5.9

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L. Ebdon et al./Spectrochimica Acta Part B 53 (1998) 859865

Fig. 2. Separation of (a) inorganic silicon, (b) 1,3-tetramethylsilanediol and (c) hexamethyldisiloxane by reversed phase HPLC and axially viewed ICP-AES. All compounds present at 10 mg ml 1.

aqueous samples into the axially viewed ICP. This is attributed to viewing the plasma along an increased path length, therefore achieving a higher optical throughput. However, it has been demonstrated [25] that for samples with high levels of matrix constituents, detection limit improvement is markedly reduced. Furthermore, it has been shown by Boorn and Browner [17] that the introduction of organic species into a radially viewed ICP results in poorer limits of detection for a wide range of elements. The separation of these compounds using HPLC
Table 3 Comparison of BEC and RSDB values for radial and axial systems for inorganic Si with a 30% MeOH/H 2O mobile phase Mode of plasma observation Axial Radial BEC/mg ml 1 0.96 0.88 RSDB/% 6.2 3.8

and the radially viewed ICP indicated better reproducibility of chromatographic peaks. This may be attributed to the fact that the optimum viewing height can be found using a radial system where plasma noise, due to nebulization of the organic solvent, is at a minimum. When viewing in axial mode this component of noise cannot be eliminated, therefore the reproducibility of the results is degraded. Table 3 shows a comparison of background equivalent concentration (BEC) and the relative standard deviation of the blank (RSDB) for inorganic silicon where n = 10 replicates. The BEC determines the concentration of the analyte, which is equivalent to the background at the analyte wavelength and is therefore a measure of the sensitivity of the instrument. It can be seen that the BEC values for the two modes of viewing are similar. However, the RSDB is signicantly better when the analytical signal is acquired using the radial mode. The RSDB is the reciprocal of the signal to noise ratio (SNR) where the signal in this case is

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the equivalent of the background signal. An increase in RSDB when viewing in the axial mode indicates an increase in the background noise. This may be attributed to the fact that the optimum viewing height can be found using a radial system where plasma noise, due to nebulization of the organic solvent, is at a minimum. When viewing in axial mode, this component of noise cannot be eliminated, therefore the reproducibility of results is less precise and RSDB values are worse. 4. Conclusions It has been demonstrated that the use of reversed phase HPLC-ICP-AES when viewing the plasma in axial mode offers no signicant improvement in detection limits over viewing the plasma radially. Limits of detection for both modes of viewing are between 0.1 and 0.5 mg ml 1. Although viewing the plasma axially when introducing aqueous samples into the ICP offers a ve- to ten-fold increase in detection limits, no such advantage is seen for samples with a high organic content. The reproducibility of results is less when viewing the plasma axially compared to viewing radially and this is attributed to the fact that the measured analytical signal contains an element of noise that is not as severe when observing the plasma side on. References
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