DSpace at VNU: A new CE with contactless conductivity detection method for the determination of complex cationic compositions: Application to the analysis of pen inks

Alkali and alkali earth ions always excluding Fr+, Be2+ and Ra2+, and less frequently excluding Rb+ and Cs+ are the most commonly studied metal cations by CE-C4D because some of them, su

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A NEW CE WITH CONTACTLESS CONDUCTIVITY DETECTION METHOD FOR THE DETERMINATION OF COMPLEX CATIONIC COMPOSITIONS APPLICATION TO THE ANALYSIS OF PEN INKS

Jorge Sáiz1*, Carlos Martín-Alberca1, Thanh Duc Mai2,3, Carmen García-Ruiz1

1 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, and University Institute of Research in Police Sciences (IUICP) - University of Alcalá, Ctra Madrid-Barcelona Km 33.6, Alcalá de Henares 28871, Madrid, Spain

Keywords: capillary electrophoresis; contactless conductivity detection; metals; cations; pen inks

1 Introduction

CE with capacitively-coupled contactless conductivity detection (C4D) has been proven to be

a feasible and suitable technique for the separation and determination of inorganic anions and cations Some well-known reasons are that CE is a clean, cheap and fast technique, with a simple design and separation principle, that uses very little sample volumes and

consumables Since the detection of analytes in C4D depends on the difference of their conductivities from that of the separation medium, C4D is a good detection system for the determination of inorganic ions The particular case of the separation and determination of metal cations by CE-C4D has attracted the interest of several researchers in the last decade Many different kinds of samples have been studied, including: waters [1-4], table salts [5], fertilisers [6], plant exudates [7], virgin olive oils [8], post-blast explosive residues [9, 10] or fireworks [11] among others Alkali and alkali earth ions (always excluding Fr+, Be2+ and

Ra2+, and less frequently excluding Rb+ and Cs+) are the most commonly studied metal cations by CE-C4D because some of them, such as Na+, Ca2+, Mg2+ and K+ are widely present in a number of diverse samples Very often, BGEs based on 2-(N-morpholino)ethanesulfonic acid/histidine (MES/His) (usually at equimolar concentrations) have been employed for the separation of these metal cations by CE-C4D [6,10-17], because of the high ionic strength, low conductivity and suitable migration velocities of the co-ions and counter-ions [18] However, these BGEs are suitable only for the analysis of samples in which Na+, Ca2+ and Mg2+ are in molar ratios close to one, since they tend to co-migrate when they are present at different ratios [18] Another drawback is that Ni2+ migrates very close to the electroosmotic flow (EOF) signal in this BGE [6,12] According to our experiences in the laboratory, this behaviour may produce the partial or absolute loss of this peak after small changes in the EOF intensity that naturally occur inside the capillary When MES/His buffers are used, Zn2+, Co2+, Cu2+ and Ni2+ are shown as negative-going peaks because of their low conductivity, which is attributed to individual degrees of complexation of these cations with the BGE components [16,19] This should not be inherently considered as

a drawback if the detectability is still good enough for these cations However, detection sensibilities for some heavy metals may be poor when their conductivities are too close to the conductivity of the MES/His BGE More specifically, Pb2+, Cd2+, Zn2+, Co2+, Cu2+ and Fe3+show reduced sensitivities in comparison to lighter metal ions when using these buffers [16,19]

For the reasons mentioned above, alternative BGEs have been investigated in recent years

in order to find more suitable electrolytes for the determination of metal cations BGEs composed of acetic acid and His have been mainly used for the analysis of samples containing light metals [1,9,20,21], the overlapping of Na+ and Mg2+ still being a problem in certain cases [21] Citric acid has been used as additive in electrolytes to form complexes with double charged cations and to reduce their mobility [7] However, according to our experience, the addition of citric acid may result in the loss of certain double charged cations, such as Ca2+, Mg2+ or Sr2+, because of their strong complexation with the acid BGEs based on lactic acid (Lac) and His have also been recently investigated [22] Lac-based buffers have become promising electrolytes for the separation and determination of metal cations by CE-C4D The reason is that Lac also forms complexes with certain double charged cations, improving the resolution between Na+, Ca2+ and Mg2+ and extending their linear dynamic ranges above those obtained with a MES/His BGE [5,8,22]

CE, in many of its variations and configurations, has been used for the analysis of inks Different inks, such as fountain pen inks [23], ball point pen inks [24,25], gel inks [26], water-based inks [27], stamp inks [28] or inkjet inks [29,30] have been analysed by CE Most of these studies have focused on the comparison of different electrophoretic profiles in order to study similarities between the studied inks [23, 26, 29, 30], while others have focused on the separation and determination of the dyes used [24,25,27] However, to the best of our knowledge there have been no studies providing qualitative and quantitative data of metals

in inks by CE Certain inks may contain certain types of metals, which may be useful, for example, for their identification and classification Moreover, some metals in inks produce environmental issues and cause negative health effects when the ink is accidentally swallowed In this context, CE-C4D might be seen as a cheap and fast methodology for the study on the metal composition in inks

This work aims to develop a new methodology for the study of complex cationic compositions A new Lac-based BGE was investigated for the separation and determination

of 17 metal cations (Cs+, Rb+, K+, Ca2+, Na+, Mg2+, Mn2+, Sr2+, Li+, Ba2+, Fe2+, Pb2+, Cd2+,

Zn2+, Co2+, Cu2+, Ni2+) and ammonium by CE-C4D and compared to other previously published procedures The developed method was applied to the analysis of 11 water-based inks, in order to provide qualitative and quantitative information on their metal composition

2 Experimental

2.1 Reagents, standards and samples

All chemicals used were of analytical grade Standard solutions of analytes (100 mM) were prepared from nitrate or chloride salts MES, Lac, His, β-alanine (β-Ala), α-hydroxyisobutyric acid (HIBA), 18-Crown-6 and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich (St Louis, MO, USA) Ultrapure water (resistivity of 18.2 MΩ x cm at 25°C) was obtained from a Millipore Milli-Q water system (Bedford, MA, USA) Different pens with water-based inks from different manufacturers and of different colours (blue, red, black, purple, and pink) were purchased in a local store (Madrid, Spain)

2.2 Procedures and instrumentation

Different BGEs were prepared fresh daily by dissolving the corresponding amount of acid and amino acid in ultrapure water and then adding the corresponding concentration of additives, which were prepared in 100 mM stock solutions The pH-values of the BGEs were experimentally measured with a pH-meter model Crison GPL-21 (Crison Instruments, Barcelona, Spain) Eleven pens with water-based inks of five different colours (black, purple,

pink, blue and red), named with letters from A to G, representing different manufacturers, were opened and their inks were transferred to 1.5 mL Eppendorf tubes Inks were diluted in ultrapure water before their injection, in such a way that the minimum dilution (maximum concentration) needed was 1:50 (v:v) while the maximum dilution was 1:8000 (v:v) The dilutions were done in order to obtain concentration values for the analytes that fitted in the calibration curve

Experiments were performed on a CE PA-800 system from Beckman Coulter (Fullerton, CA, USA) The instrument was fitted with a ER225 C4D (eDAQ, Deninstone East, NSW, Australia) The detector excitation frequency was set to 1200 kHz and the amplitude to 100% for all the BGEs used Bare-fused silica capillaries of 50 μm I.D and 365 μm O.D (Polymicro Technologies, Phoenix, AZ, USA) with a total length of 90 cm and an effective length of 68 cm were employed The capillaries were conditioned prior to their first use by flushing at 20 psi with 1 M NaOH for 40 min, then water for 5 min, and finally BGE for 30 min Injections were carried out at 0.5 psi for 10 s Between runs, the capillary was rinsed with running BGE for 2 min at 50 psi to obtain good precision in the analysis The capillary temperature during the electrophoretic separations was kept at 25°C The applied voltage was 30 kV and a voltage ramp from 0 to 30 kV of 0.1 min was set at the beginning of the separation

The software Chart v.5.5.6 from eDAQ (Deninstone East, NSW, Australia) was used for data recording The software Peakmaster v.5.3 [31] was used for the calculation of ionic strengths, conductivities, and buffer capacities of the BGEs as well as to collect pKa values

of the BGEs constituents

3 Results and discussion

3.1 Optimisation of the CE-C 4 D method

Three different BGEs were assessed in order to know their appropriateness for the separation of the 17 metal cations and NH4+ The first BGE studied was the commonly used electrolyte in CE-C4D composed of 20 mM MES/His The ionic strength of this buffer is 10.2

mM with the conductivity of 4.7 x 10-2 S/m and the buffer capacity of 23.1 mM The second BGE was composed of 20 mM Lac/His, which has recently been used by our group because

it greatly improves the resolution between Na+, Ca2+ and Mg2+ [5] The ionic strength of this BGE (18.7 mM) and the conductivity (9.9 x 10-2 S/m) are greater than those of the MES/His BGE whereas the buffer capacity is smaller (5.8 mM) The last BGE, which has never been used for cations analysis by CE, was composed of 20 mM Lac/β-Ala Its ionic strength (8.1 mM) is lower than that of the MES/His BGE The buffer capacity (22.6 mM) is similar, whereas its conductivity is slightly higher (6.1 x 10-2 S/m) than that of the MES/His BGE The pKa values of Lac and β-Ala are very similar (3.9 and 3.4, respectively), similar to the case

of MES and His (6.1 and 6.0, respectively) According to these values, this is, presumably, a good buffer for CE-C4D

Electropherograms for the separations of different cationic species are shown in Fig 1 2

mM 18-Crown-6 was added to each buffer to allow the separation of NH4+ and K+ for being a regularly used concentration In the electropherogram shown in Fig 1A for the separation with the BGE composed of MES/His, it is remarkable that there is a good separation with good detectabilities of the first 10 cations within 300 s However, the method showed less sensitivity for the following analytes and the last four analytes appeared in the electropherogram as inverted peaks According to de Carvalho et al [6], Ni2+ migrated very close to the EOF and in certain analyses they co-migrated together, probably due to small changes in the EOF intensity, leading to the partial or complete peak loss Cu2+ and Co2+also co-migrated under these conditions as well as Fe2+ and Pb2+ The detectability was very

low for both Fe2+ and Pb2+, especially for the latter, which was over 150 µM Only when the concentration of Pb2+ was intensively increased a tailed peak appeared with a small peak of

Fe2+ co-migrating together (data not shown) In the electropherogram for the separation with the BGE composed of Lac/His in Fig 1B, Na+ migrated before both Ca2+ and Mg2+, due to the complexations of the double-charge cations with Lac This is the reason why the linear dynamic ranges of Na+, Ca2+ and Mg2+ are increased with this buffer [5] Interestingly, Mn2+and Sr2+ also changed their migration order as well as Pb2+ and Cd2+ Under these conditions, and due to the decreased EOF magnitude produced by the lowered pH compared to that of the MES/His BGE, the migration of Ni2+ was delayed to more than 800 s The buffer did not allow to detect Fe2+ and Pb2+ at the concentrations used (150 µM) and more concentrated solutions were needed in order to detect these peaks The detectability for Zn2+ and Co2+ was also much decreased, while the baseline noise was also increased due to the higher conductivity of this BGE The electrolyte composed of Lac/β-Ala (Fig 1C) provided the best detection for most of the cations, compared to the other two BGEs studied

17 out of the 18 analytes were shown as positive-going peaks Interestingly, although the Lac/β-Ala BGE had the lowest pH value (pH 3.62) among the 3 studied buffers, the separation of the 18 cations was faster than with the other two electrolytes This could happen as a result of the degree of complexation between His and certain double-charged cations, such as Ni2+, Zn2+ and Co2+, which lengthen the separation when His is used Fe2+and Pb2+, with very poor sensitivities when the previous BGEs were used, showed now good detectabilities (see LODs in Table 1) The noise of the baseline (noise intensity) was comparable to that obtained with the MES/His electrolyte due to their similar conductivities, while the baseline produced by the Lac/His buffer was noisier The BGE based on Lac/β-Ala was thus considered as the most suitable electrolyte for the separation of the metallic analytes Further investigations were carried out on this Lac/β-Ala BGE to improve the separation of co-migrating peaks, such as Fe2+ and Cd2+ or Mn2+ and Sr2+

As it is shown in Fig 2, four different concentrations (10, 20, 30 and 40 mM) of Lac/β-Ala with 2 mM 18-Crown-6 were used to study the effect of the BGE concentration in the separation of the analytes The increase in the BGE concentration led to the prolongation of the migration time of the analytes When 10 mM Lac/β-Ala was used, peaks were broadened and many of them co-migrated due to the electrodispersion produced in this BGE When the concentration was increased up to 40 mM, the baseline became extremely noisy, affecting the detection sensitivity The time for each separation in this case was increased to more than 15 minutes Moreover, the peaks of Li+, Ba2+, Fe2+, Mn2+ and Sr2+ were overlapped under these conditions For these reasons, concentrations higher than 40 mM were not studied The BGE composed of 30 mM Lac/β-Ala behaved similarly to the one composed of

40 mM Lac/β-Ala and many analytes co-migrated, such Na+ and Ca2+; Mg2+ and Sr2+; Co2+,

Ba2+ and Li+; and Fe2+ and Cd2+ The buffer composed of 20 mM Lac/β-Ala also produced the co-migration of Fe2+ and Cd2+ as well as certain overlapping of Sr2+ and Mg2+ peaks However, the separation of other peaks, such as Co2+, Ba2+ and Li+ was excellent under these conditions 20 mM Lac/β-Ala with 2 mM 18-Crown-6 was therefore selected as BGE for the following experiments It is interesting to mention that higher concentration of the BGE constituents produced changes in the migration order of certain double charged cations, such as Ca2+, Sr2+, Cd2+ and Co2+ This was attributed to an increase of complexation degrees between Lac and those analytes, produced when the concentration of Lac was increased

The addition of HIBA to the running buffer was proven to modify the mobilities and migration order of certain anions and cations [1] The addition of HIBA to the BGE was studied in order

to improve the separation of the selected analytes As can be seen in Fig 2, an increase in the concentration of HIBA showed an improvement in the separation of Fe2+ and Cd2+, which were baseline separated at HIBA concentrations of 5 mM and higher Baseline separation was also obtained with the same concentration of HIBA for the peaks of Sr2+ and Mg2+ The

peaks of Na+ and Ca2+ overlapped when 10 mM HIBA was used Thus, 5 mM HIBA was chosen as the optimal concentration to achieve the separation of the highest number of cations (Fig 2)

Finally, Fig 3A shows the electropherogram for the separation of the 18 cations selected in this study under the final conditions As a note, it is possible to improve the resolution between Cs+ and Rb+ by adding 1-10% (v/v) ethanol to the BGE However, this negatively affects the resolution of Na+ and Ca2+, since they started to co-migrate with the addition of 1% (v/v) ethanol

3.2 Performance assessments

The performance of the developed CE-C4D method was evaluated in terms of linearity, sensitivity and intermediate precision of migration times and peak areas for each of the 18 selected cations in this study Corresponding data are shown in Table 1 Two different series

of standard solutions were prepared for the study of the linear working ranges, with each series containing alternated analytes to avoid peak overlapping The working ranges depended on the species and they were studied over three ranges of magnitude, which were well covered by each analyte without losing the linearity Five-point calibration curves were created for quantitative analysis Excellent correlation coefficients were obtained in all cases, with R2 values of 1.0000 for Pb2+ (see Table 1) Good LODs were obtained, which were in the lower micromolar range for most of the analytes Due to the similar conductivities shown

by this cation and the BGE, the LOD for Cu2+ was 75 μM Although the selectivity for this cation was excellent under the optimised conditions, the method should not be used if sensitive detection of Cu2+ is required In this specific case, the employment of the BGE composed of 20 mM Lac/His is recommended because it showed the best LOD value for

Cu2+ (16 μM) The intermediate precision of peak areas and migration times were determined over 10 consecutive injections in the same day and 5 injections in different non-consecutive days The RSD values for intraday migration times were excellent (RSD < 0.5

%) and the RSD for peak areas (< 10.0 %) and inter-day migration times (< 4 %) and areas (<18 %) were also acceptable

3.3 Application to the analysis of pen inks

The electrophoretic method was applied to the analysis of water-based inks from 11 different pens in order to provide information on their metallic compositions Table 2 shows the concentrations of the determined analytes in each ink analysed Ammonium was found at different concentrations in all the inks, probably due to the use of ammonium hydroxide in water-based inks in order to maintain an alkaline pH [32] Calcium was also found almost in all the samples analysed, at lower concentrations than those of ammonium Calcium carbonate is used in inks as an extender, which is a white, transparent, or semi-transparent component pursuing to reduce the cost of the ink by increasing the area covered [33] Sodium was found at higher concentrations than those of calcium in all the inks analysed The presence of sodium may be explained by the use of certain surfactants to impart lubricity to the inks, enhancing the penetration of the ink into the writing paper [34] Ammonium, sodium and potassium (potassium being found only in 3 out of the 11 inks), could also come from other inorganic salts that are commonly added to water-based inks Li+was found in pen A-blue at 17.6 mM while Ni2+ was only determined in the ink of the pen A-black (Fig 3B) at a lower concentration (1.2 mM) The results revealed that analysed pen inks differed in their composition, not only depending on the colour, but also depending on the manufacturer The developed electrophoretic method could be used for qualitative and quantitative purposes, such as the comparison or differentiation of inks, for quality control and industrial statistics for pen inks The analysis of written paper might be also possible