Comprehensive two-dimensional liquid chromatography (LC

LC) is a relatively new and expanding technology which provides enhanced separation power by combining two LC modes with different separation mechanisms. In this review, practical principles and guidelines for designing LC

LC methods are given. Selected applications from the food, plant, polymer and bioanalytical sectors from the last five years are presented.

In the quest for better separation efficiency, comprehensive two-dimensional liquid chromatography techniques (LC×LC) have been increasing in popularity recently. In 2D-LC, the sample is subjected to two different separation mechanisms, which are practically achieved by using two columns with different stationary phases. In a comprehensive mode, all fractions pass through the whole analysing system. This is the essential difference compared with heart-cutting liquid chromatography (depicted by LC–LC), in which only selected fractions are analysed by the second column. In LC×LC, two separation modes are used to increase the separation efficiency. The instrumentation and operation of LC×LC has been described in recent literature.

  In short, the heart of an LC×LC system is the interface that connects the two columns.

The interface is typically a simple multiport high pressure switching valve, which ensures the collection of the first dimension effluent in aliquots of predefined volumes and allows the transfer of these fractions on the secondary column in an automated way. Low flow-rate employed in the first dimension enables the filling of one sample loop and, at the same time, flushing the second one by fast flow-rate into the second dimension within one valve cycle. Prior to the introduction of a successive fraction onto the secondary column, the analysis of the previous fraction should be finished. Thus, the second dimension analysis time should be equal to (or less than) the duration of a modulation period. This sample cycle, (i.e., the modulation time or sampling frequency) is therefore determined by the analysis time of the second column. To maintain the separation achieved in the first dimension a sufficiently large number of fractions have to be taken from a peak eluting from the first column. Consequently, fast 2D analyses are essential in LC×LC. 

Before coupling, the LC methods in both dimensions should be optimized with respect to the sample characteristics and taking into account all parameters that influence peak capacity, such as orthogonality, sampling frequency and compatibility of both dimensions.  

The optimization should start with selection of the separation mode and the two columns, and then by optimization of the second dimension separation. The design of the LC×LC methodology is summarized in Figure 1. The separation mechanisms used in the two dimensions of a two-dimensional separation process must have different selectivities for the sample components. The sample components should be spread over as broad a separation space as possible in both separation dimensions. The best separation is achieved when there is no relationship between the retention data of the components of the sample in the two separations. Various combinations of reversed-phase (RP) and normal phase (NP), ion-exchange (IEC), hydrophilic interaction liquid chromatography (HILIC) or size-exclusion (SEC) or LC modes can be used in LC×LC. In these combinations, the separation is based on the differences in the molecular structure of separated compounds, such as size, shape and polarity or the specific charge of ionic compounds. 

 Figure 1: Selection of LCÃLC conditions.			

In practice, the separation selectivities in the two LC modes can be more or less correlated because the interaction mechanisms of the analytes with stationary phase and mobile phase are seldom based on a single factor only, and the secondary interaction mechanisms can be correlated in the two separation modes. This must be taken into account when developing real world LC×LC separations. Since the mobile phases used in RPLC×RPLC, IEC×RPLC, RPLC×SEC, NPLC×SEC or HILIC×RPLC separation systems are usually fully miscible and have similar physicochemical properties, these are in practice the most commonly used combinations. 

For example, the combination of IEC and RPLC can be used for the separation of ionic compounds, acids or bases. The combination of SEC with either NPLC or RPLC is useful for the separation of macromolecules, such as synthetic polymers and biopolymers. An NPLC×RPLC combination is more demanding because of eluent incompatibility, and is useful for the separation of samples with two or more types of structural elements that differ in lipophilic and polar properties. For polar compounds, using HILIC for the second dimension separation is a viable option, particularly when stationary phases that are less polar than silica gel (e.g., aminopropyl silica) are used because then mobile phases containing up to 70% water can be applied. Table 1 shows the compatibility of different LC modes. 

 Table 1: The compatibility of different LC modes.			

The selection of column dimensions is crucial in LC×LC. The inner diameter (i.d.) of the second column is typically larger than that of the first column to minimize extra column peak broadening, although it is also possible to use a narrow-bore column with high permeability in the second dimension. The first column is typically long and is operated at lower flow-rates than the second column, which is short and uses a high-speed mobile phase. Here, the speed of the second dimension is a key feature of successful separation and the separation should be as fast as possible, with sufficient resolution.  

Choosing the proper sampling time is essential in designing an LC×LC method.

  The first-dimension separation is maintained more efficiently when the sampling frequency is sufficiently high. To maintain the resolution obtained in the first dimension, a large number of fractions should be transferred along a peak eluting from the first dimension. To achieve sufficient amount of fractions, the first dimension peaks are occasionally broadened by operating the primary column at a lower (than optimal) flow-rate. In this way, a sufficient amount of samplings across the primary peak are obtained. However, the first dimension peak capacity is significantly reduced by operating at a flow-rate below the Van Deemter's optimum. Some guidelines for selection of optimal modulation frequency are available. Three or four samplings across the first dimension peak are often considered sufficient

  and in practice even two sampling periods can usually be tolerated.

  The effect of proper sampling time is demonstrated in Figure 2. Here, selected phenolic compounds were analysed with an RPLC×RPLC combination.

  When three fractions for each peak were sampled [Figure 2(a)], the best overall separation was obtained. With longer modulation time, only 1–2 fractions were samples and the resolution obtained in the first dimension was partly lost. Conversely, when the modulation time was too short, significant wrap-around took place and the peaks were tailing. The wrap-around effect is typical in 2D separations when the modulation time is shorter than the compounds' second dimension retention time and the compounds, consequently, only elute to the detector during the next modulation period and can coelute with the compounds in that fraction. This results in a loss of resolution.

 Figure 2: Effect of modulation time to the overall separation of phenolic compounds in an RPLCÃRPLC system.  (a) Optimal sampling time with three fractions for each first dimension peak; (b) modulation time is too long with 1â2 fractions/1st dimension peak, (c) modulation time is too short with 3â4 fractions/1st dimension peak, resulting in wrap-around. Conditions: First dimension:  C18, 150 Ã 2.1 mm i.d., 3 Âµm, gradient with acetic acid and acetonitrile at 0.1 mL/min, 2nd dimension: + Ultrasphere CN, 75 Ã 4.6 mm i.d., 3 Âµm, eluent 65/35 0.5% (v/v) acetic acid in water/ acetonitrile at 1.9 mL/min. For details, see reference 11.			

After selection of the sampling frequency, the first dimension separation can be optimized, with the flow-rate optimized, taking into consideration the modulation time.  

The inner diameter of the second column should be equal to — or larger than —that of the first column. Short columns packed with small particles (3 µm or less), nonporous, superficially porous or monolithic columns are especially suitable for this purpose. In addition, the column material should withstand high pressure, because high flow-rates are used in the second dimension. Typically, columns of 2–5 cm in length and with an i.d. of 2.1–4.6 mm are used. High-speed separation can be achieved by using monolithic columns, an ultra-high performance liquid chromatography (UHPLC) approach

  or columns packed with partially porous particles. Monolithic columns are also suitable for LC×LC. It should be noted that the use of columns packed with sub-2 µm particles at high flow-rates requires sophisticated instrumentation capable of delivering high pressures (600–1000 bar). The interface valves should also tolerate such high pressures. It is also possible to increase the speed by using elevated temperatures in the second dimension. At elevated temperatures, the viscosity of the mobile phase is reduced and the flow-rate of the eluent can be increased. At 40 °C the viscosity of an eluent is already significantly reduced. In some applications, even higher temperatures have been used, but then the stability of the stationary phase and analytes must be carefully considered.  

The selection of the mobile phases in the first and second dimensions is very important because the whole stationary/mobile phase system controls the retention and the separation selectivity. If possible, the mobile phase used in the first dimension should have low elution strength in the second dimension to focus the fraction transferred from the first dimension in a narrow zone on the top of the second-dimension column before the elution step. This is important, particularly if the transferred fractions are large. Of course, the compatibility of the mobile phases in the first and second dimensions should also be considered — mainly mutual miscibility, solubility of analytes, adsorption of mobile phase components and viscous fingering. In addition, the second-dimension eluent must also be compatible with the detection system. 

In the first dimension separation, gradient elution is generally used to obtain high peak capacity. In the second dimension, isocratic separation is often used because the time necessary for the re-equilibration of the column before the transfer of the next fraction decreases the time available for the separation if repeated gradients are run with each transferred fraction.  

This problem can be avoided in some cases by using parallel gradients spanning over the entire gradient time, both in the first and second dimensions.

  Another area that may be very useful is temperature programming as a replacement for organic solvent gradients, especially in the separation of macromolecular or oligomer compounds. Elevated temperatures in the second dimension separation are also often beneficial because an increase in temperature decreases the viscosity of the eluent and therefore, higher flow-rates can be used.  

It is more difficult to use gradient for the second dimension separation because it can be difficult to get fast and repeatable gradients in a very short time range, typically in a range of only a few minutes. Novel UHPLC systems can also handle fast gradients, when the optimization is done carefully. The use of gradient allows more efficient focusing of the transferred fractions and more efficient separation. 

Table 2 lists contemporary applications of LC×LC that have been published since 2005. As can be seen, a variety of different column combinations have been used in these applications. 

Table 2: Recent applications in LCÃLC			

Food analysis has become very important to determine native and exogenous contaminants and tracing food quality and origin. LC×LC has been used in characterization of wine, orange juice, yoghurt and oils.

  A representative application is the analysis of low-molar-mass organic acids (LMMOAs), which can be used as indicators of food quality. LMMOAs have been analysed by an LC×LC system using a combination of ion chromatography (IC) and RPLC.

  The separation mechanism of IC is based on charge, as well as on the ion size and hydrophobicity, while RP separation is based on the polarity of the analytes. Such column combinations offer nearly orthogonal separation. The correlation coefficient used as an indicator of the orthogonality was 0.0057 (1 indicates fully non-orthogonal and 0 fully orthogonal system). Figure 3 shows that compounds are well scattered across the separation space, demonstrating the orthogonality of the separation in practice. The key feature of the IC×RPLC set-up was the neutralization of the first dimension eluent (KOH) by using a membrane suppressor. Some of the LMMOAs eluted rather late from the second dimension column (~12 min). As a compromise, the modulation time was set to 2 min, which is relatively long, but sufficient separation was still achieved for most of the compounds. Even with this modulation time, some of the lateeluting compounds exhibited wrap around, that is they eluted in the next modulation period. This was acceptable because these compounds did not interfere with other compounds. 

Figure 3: Separation of 24 LMMOAs with modulation time of 2 min. Compound identification in reference 13. Reprinted from reference 13 with permission from Elsevier			

LC×LC has also been successfully used in the analysis of plant extracts, for example, maize,

  Extract of the last mentioned plant was analysed using an LC×LC system that used ultra high pressure liquid chromatography (UHPLC) in the second dimension.

  In the LC×LC set-up polyamine column in NP mode was used in the first dimension and UHPLC C18 reversed-phase column in the second dimension. The sub-2 µm UHPLC column was 3 cm long (i.d. 2.1 mm) and it was maintained at 70 °C. A short modulation time of 20 s could be used at these conditions and 3–12 fractions for each peak eluting from the first dimension could be sampled. The backpressure varied between 805–922 bar depending on the mobile phase gradient composition. Thus, special connections and valves were used in this high pressure set-up. This comprehensive 2D-LC system successfully separated ten glycosides in the extract of 

Separation of synthetic polymers has always been a challenge for chromatographers. Separation of a polymer mixture between two separation dimensions significantly increases the number of resolved individual polymers. LC×LC systems for the separation of polymers have various combinations of columns in the first and the second dimensions: HILIC×RPLC,

  RPLC×SEC (size exclusion column in the second dimension)

  or (temperature rising elution fractionation) TREF×SEC.

  The combination of HILIC and RPLC stationary phases represents really comprehensive separation because the two phases are claimed to have truly opposite separation mechanisms regarding polarity. Another benefit of combining HILIC and RPLC is the use of aqueous–organic mixtures as mobile phases in both dimensions, which makes the two LC systems easily compatible. This type of combination was used for separating non-ionic surfactants, such as amphiphilic polymers.

  These polymers are difficult to separate using one column as they contain hydrophilic and hydrophobic blocks. As an example Figure 4 shows the LC×LC separation of Tween 40 polymer.

Figure 4: LCÃLC separation of Tween 40.27 Reprinted from reference 27 with permission from Elsevier			

Among many proteomics protocols, the (on-line) LC×LC offers an interesting but relatively rarely applied alternative for the analysis of a digested protein mixture. The combination of a cation exchange column in the first dimension and RPLC in the second dimension in connection with ESI-MS was used for elucidation of structural alterations and/or post-translational modifications.

Most LC×LC applications described in the literature are qualitative studies. An example of a quantitative analysis is the determination of organic acids in aerosol particles by LC×LC–TOF-MS.

  The first dimension used a SCX column in an ion exclusion mode and second dimension was RPLC using a short packed C18 column. The quantification was based on the summed peak areas of extracted ion traces. The repeatability of the method was good, with relative standard deviations (RSDs) of the peak areas below 8%. Quantitative LC×LC analysis has also been developed for the characterization and quantification of antioxidant phenolic acids in 

  The optimized LC×LC system used a combination of C18 and cyano columns. In this work, the RSDs for the retention times were better than 0.05% and those for the peak areas ranged from 2–14%. Limits of detection were 18–90 ng/mL. In the same study, LC–MS and LC×LC–MS methodologies were compared in terms of quantification. The results showed that due to the clearly better separation efficiency of LC×LC–MS compared with LC–MS, there was much less matrix effects in the MS detection, and even external calibration gave reliable results in the 2D separation while significant suppression was observed in 1D separation. 

The increased interest in alternative medicine has been reflected in the number of chromatographic applications for analysing traditional Chinese medicine (TCM). For example, root extract of 

 was separated by LC×LC and ESIMS using reversed-phase and amino columns, both tested in two dimensions.

  Seventeen platycosides were identified in the 2D chromatography plot. Another work studied metabolism of 

 administered in rats analysing plasma and striatum samples.

  Over 100 compounds (13 identified) were separated with two LC dimensions that consisted of human serum albumin (HSA) column in the first dimension and RPLC in the second dimension. 

LC×LC is without any doubt technology with a future. It exploits new discoveries in the area of liquid chromatography. The novel developments in fast LC analyses (fusedcore particles, UHPLC systems and monolithic columns) have already created new possibilities for the LC×LC methodology. The main challenge in the field of LC×LC at present is the general lack of robust, commercially available, easy-to-use instruments and software for analysing 2D separation data. Currently, most of the LC×LC applications reported are qualitative in nature. One of the main reasons for this is the lack of suitable software for quantification. Thus, LC×LC definitely needs better tools for data analysis and one viable approach here could be a joint effort in the software development using an open source platform.  

1. I. François, K. Sandra and P. Sandra, 

8. R.E. Murphy, M.R. Schure and J.P. Foley, 

11. M. Kivilompolo and T. Hyötyläinen, 

12. M. Kivilompolo and T. Hyötyläinen, 

14. M. Kivilompolo, V. Obuůrka and T. Hyötyläinen, 

Articles

In this review, practical principles and guidelines for designing LC?LC methods are given.

Comprehensive Two-dimensional Liquid Chromatography (LCxLC): A Review

Clavulanic acid is commonly used in combination with penicillin and cephalosporin antibiotics to overcome bacterial resistance. Clavulanic acid is produced and isolated from 

 with ethyl acetate and then converted to a stable form. One secondary purification approach uses nonaqueous precipitation of potassium clavulanate by adding the potassium salt of 2-ethylhexanoic acid in isopropanol to the primary isolation extract. While 2-ethylhexanoic acid is soluble in the organic solvent, a small fraction may co-precipitate with the potassium clavulanate. 

The United States Pharmacopeia (USP) monograph for potassium clavulanate describes a GC–FID method to determine 2-ethylhexanoic acid in the active pharmaceutical ingredient (API). This contribution describes a simpler approach for determining 2-ethylhexanoic acid in clavulanate with a Reagent-Free Ion Chromatography (RFIC) system. USP clavulanate reference standards were prepared at a 150-fold lower concentration in deionized water than the current USP method, spiked with 2-ethylhexanoic acid, and injected directly without additional sample pretreatment. The target analyte was separated from clavulanate-related peaks using electrolytically generated potassium hydroxide eluent and measured by suppressed conductivity detection. 

Analyses are performed using a Dionex ICS-2100, AS Autosampler and Chromeleon Chromatography Workstation. Separations use an IonPac AG11/AS11 column set (USP L61) with electrolytically generated KOH eluent.

Figure 1 compares 500 µg/mL clavulanate with and without 2-ethylhexanoic acid spiked at the 0.8% acceptance criterion cutoff value specified in the USP monograph. As shown, 2-ethylhexanoic acid is separated in less than 10 min without any interference from minor components in the sample matrix. The method demonstrated good sensitivity with a low limit of detection (LOD) for 2-ethylhexanoic acid of 0.036 µg/mL, which corresponds to a concentration of 0.008% in the API. This represents a 100-fold lower concentration than the 0.8% acceptance criterion cutoff value in clavulanate. Results for linearity, spike recovery, retention time precision and peak area precision determinations show that IC is an accurate and reproducible technique to determine 2-ethylhexanoic acid in clavulanate below the 0.8% acceptance criterion. The lower LOD achieved by this method allows a 150-fold lower API concentration, thereby reducing cost. In addition, this proposed method eliminates the need for two solvent extractions required in the GC–FID method in the current USP monograph, thus significantly simplifying sample preparation, reducing waste, and further improving cost efficiency. 

 Figure 1: Chromatographic comparison of clavulanate without [trace (a)] and with [trace (b)] 4 Âµg/mL (0.8%) 2-ethylhexanoic acid. Peak 1: 2-ethylhexanoic acid (4 Âµg/mL â 0.8%). Eluent: 3 mM KOH from 0â10 min; 3â60 mM KOH from 10â10.1 min; 60 mM KOH from 10.1â20.1 min. Flow-rate: 0.25 mL/min. Temp.: 30 Â°C. Inj. Vol.: 5 ÂµL. Detection: suppressed conductivity, ASRS 300 2 mm, recycle mode,2 mA suppressor current during 3 mM KOH, switch to 38 mA at 10.1 min. 

1. Dionex Corporation. Determination of 2-Ethylhexanoic Acid Impurity in Clavulanate Using a Reagent-Free Ion Chromatography System. Application Note 262 (LPN 2608, Oct., 2010); Sunnyvale, California, USA 

Chromeleon, IonPac, and RFIC are registered trademarks of Dionex Corporation.  

1228 Titan Way, PO Box 3603, Sunnyvale, California 940883603, USA

Departments

Direct Determination of 2-Ethylhexanoic Acid in Clavulanate

Hyaluronic acid is a polymer of disaccharides and can have up to 25000 disaccharide repeat units. The molar mass range is from 5000 Da to 20000000 Da. Hyaluronic acid is mainly used in medical and cosmetic applications. 

GPC/SEC is the method of choice for measuring the molar mass distribution of hyaluronic acid. True molar masses can be obtained when online multi angle light scattering is used in combination with a refractive index detector. The refractive index increment (dn/dc) for light scattering data evaluation can be determined online, measured offline using dedicated instrumentation, or taken from literature. 

Eluent: 0.005 M phosphate buffered saline, 0.069 M NaCl 

Columns: PSS SUPREMA Lux 10 µm 30Å, 10 000Å and 10000Å (8 × 300 mm) + precolumn  

Data acquisition: PSS WinGPC Unity with Compliance Pack 

Detectors: SECcurity GPC1200 RI SECcurity MALLS SLD7000 

High molar mass samples require lower flow-rates and concentrations in GPC/SEC. The GPC/SEC conditions and columns have been optimized for high molar mass hyaluronic acid with respect to loading, flow-rate, column particle size and porosity. For sample preparation the water content of approx. 12% has been taken into account.

 Figure 1: Slice concentration and molar mass obtained with a SLD7000 and an RI detector.

Due to the lack of high molar mass calibration standards the use of light scattering detection is recommended. This allows also to measure true molar masses. 

A refractive index increment (dn/dc) for hyaluronic acid of 0.165 mL/g has been used to evaluate the light scattering data.

  Figure 1 shows the slice concentration measured by the RI detector as well as the online measured molar mass. Sample recovery (compare Conc. Calculation vs Given) was nearly 100% showing that all material eluted from the column.

 Figure 2: Molar mass distribution, molar mass averages and cumulative distribution for a hyaluronic acid obtained using GPC/SEC-MALLS. 

Table 1 shows the molar mass averages of the hyaluronic acid sample obtained using a Pullulan calibration (RI only, apparent molar masses) and using light scattering. The light scattering M

 molar mass is three times lower than that obtained with a pullulan calibration curve.

 Table 1: Comparison molar mass averages: Only light scattering gives absolute molar masses. 

Analytical Ultracentrifugation in Biochemistry and Polymer Science

, Royal Society of Chemistry, Cambridge, 517–531 (1992). 

In der Dalheimer Wiese 5, D-55120 Mainz, Germany

tel. +49 6131 962390  fax +49 6131 96239 11

GPC/SEC–MALLS Analysis of Hyaluronic Acid

The presented ion chromatographic method is used for the simultaneous determination of HF, HNO 

 in acidic texturing baths that are used in the wet chemical etching process of solar cell production. Fluoride, nitrate, sulphate and acetate are determined by conductivity detection after chemical suppression, while the silicon present in the form of hexafluorosilicate is detected spectrophotometrically as molybdosilicic acid after derivatization in the same analysis. The analytical results are validated by titration. 

Energy production from renewable sources such as biomass, biogas, biofuels, water, wind and solar power is becoming increasingly important in our energy-hungry society. Particular interest is given to solar energy, which by human criteria is inexhaustible. Solar cells used in photovoltaic units convert the radiation energy in sunlight directly into electric energy. 

Solar cells are manufactured from ultrapure mono- or polycristalline silicon wafers whose surface is treated in acid etching baths (also known as texturing baths) before being spiked with foreign atoms (P, B). The etching solutions consist of various acids, which act as an oxidizing agent (HNO

), complexing agent (HF), stabilizer and wetting agent (CH

COOH) and determine the surface structure and thus the efficiency of the solar cells. The replenishment of components used up in the etching process extends the bath life and saves costs, though it does require knowledge of the exact composition of the bath, especially the concentration of silicon and hexafluorosilicate. By using titration and ion chromatography (IC), it is possible to determine the key components quickly and precisely.

 Figure 1: 850 Professional IC Anion â MCS and 858 Professional Sample Processor. 

This article describes an ion chromatographic method that separates all relevant components in the bath on an anion-exchange column and identifies them by dual detection in a single run. After suppressed conductivity detection of the acid anions, the undissociated silicic acid reacts in a post-column reaction (PCR) to form molybdosilicic acid, which is determined spectrophotometrically at 410 nm. The concentrations of fluoride and hexafluorosilicate are determined by way of a simple stoichiometric calculation that is performed by the chromatography software. 

850 Professional IC Anion – MCS with post-column reactor

The standard solutions were prepared with CertiPUR standards from Merck (SiO

 in ultrapure water) and the TraceCERT standard from Fluka (acetate solution). All the standard and eluent solutions were prepared with ultrapure water with a specific resistance of more than 18 MΩ·cm. Etching bath samples were provided by a solar cell manufacturer from Germany. 

In the wet chemical etching of silicon surfaces, nitric acid is used to oxidize silicon to form silicon dioxide which is further etched by hydrofluoric acid. 

During the etching process, the concentrations of HF and HNO

 in the etching bath decrease and the concentrations of water and hexafluorosilicate increase. To ensure a constant etching rate and surface properties, the etching bath can be regenerated a few times by subsequent replenishment of spent acids. The increasing concentration of H

, however, limits the number of possible recycling cycles. This requires semi-continuous monitoring of the bath components, which can be achieved conveniently by automated ion chromatography. 

The acid anions present in the etching bath — mainly fluoride and nitrate, though sometimes also sulphate and acetate — are separated under alkaline elution conditions and determined by conductivity detection (Figure 2). In the alkaline eluent, the hexafluorosilicate is converted into orthosilicic acid which is undissociated and therefore "invisible" in the conductivity detector.

Figure 2: Conductivity chromatogram of a simulated etching bath with 25 mg/L fluoride, 20 mg/L acetate and 10 mg/L nitrate. The undissociated orthosilicic acid is not recorded in the conductivity detector. 

The undissociated orthosilicic acid is determined by way of post-column reaction with an acid molybdate solution and subsequent UV/vis detection at 410 nm (Figure 3).

Figure 3: UV/vis chromatogram of a 10 mg/L silicic acid standard. Silicic acid is derivatized to molybdosilicic acid which is then detected spectrophotometrically. 

  produces a fluoride peak in the conductivity detector and a silicate peak in the UV/vis detector. The mass balance derived from the respective peak areas confirms that the concentration of SiF

  results stoichiometrically from the determined fluoride and silicate concentrations, provided there are no other sources of fluoride or silicate present. Thus the concentration of free HF can be calculated as the difference between the total fluoride concentration and the fluoride concentration from the hexafluorosilicate: 

Analysis of the Texturing Baths and Validation

After being diluted at ratios between 1:1000 and 1:5000, four samples from different texturing baths are analysed for their constituents by using the above IC method with dual detection. Figure 4 shows the chromatograms of etching bath sample 1 obtained by conductivity (a) and UV/vis (b) detection.

Figure 4: (a) Conductivity and (b) UV/vis chromatograms of the sample of etching bath 1 with 1:2000 dilution. The chromatographic parameters are the same as those of the prior chromatograms. 

Table 1 provides an overview of the concentrations of the relevant bath components determined by ion chromatography with dual detection. For comparison, the concentrations obtained by titration are also shown. The potentiometric determination of acid concentrations and H

 content was carried out using aqueous acid-base titration with 1 mol/L NaOH solution.

Table 1: Comparison of the concentrations of a few selected bath components determined by ion chromatography and titration. 

Ion chromatography with dual detection allows determining the concentration of all relevant constituents of texturing baths for solar cell production in less than 30 minutes. The acids consumed in the texturing process can subsequently be replenished in a targeted way. This extends the life of the etching baths, guarantees clean and reproducible wafer surfaces, cuts costs and protects the environment. 

3. M. Zimmer et al., 22nd European Photovoltaic Solar Energy Conference and Exhibition, Milan, Italy (2007). 

4. G. Bogenschütz, C. Wilde and C. Hack, Pittcon 2009 (

Ionenstrasse, CH-9101 Herisau, Switzerland

Simultaneous determination of mineral acids, fluoride and silicate in etching baths by ion chromatography with dual detection

Water treatment by disinfection processes is considered to have been the major public health achievement of the twentieth century. Many drinking water utilities have changed their primary disinfectant from chlorine to alternative disinfectants, which reduce regulated organic by-products, such as trihalomethane levels, but at the same time often increase the level of other potentially toxicologically important compounds. The hazardous inorganic oxyhalide by-products are bromate, chlorite and chlorate. The most important of these is bromate, which is formed when source waters containing bromide are ozonated. Bromate is considered a possible human carcinogen. The article is a short review of separations of chlorite, chlorate and bromate in water using ion chromatography (IC) and their determination using various modes of detection.  

In the 19th century, chlorine was introduced to water treatment as a chemical disinfectant, followed by remarkable reductions in cholera, dysentery and typhoid worldwide. The importance of drinking water quality and its influence on human health was well known, but the specific contaminations would not be identified for centuries because the analytical methods available were not appropriate for specific applications. This situation has changed when modern instrumental analytical techniques and methods were introduced into routine analyses. The number of chemicals determined in drinking water has grown exponentially; however, out of hundreds of them, only very few have been studied or have documented proof of their health effects. 

In the 1970s, it was discovered that chlorination of drinking water produced carcinogens, such as trihalomethanes and haloacetic acids. Since 1974, the presence of more than 500 disinfection by-products have been determined in drinking water.

  Since that time, environmental regulatory agencies and drinking water treatment technologists have performed extensive research for alternative disinfection methods that reduce the generation of organic by-products with significant health risks. 

Many drinking water utilities are replacing the most popular chlorine, with alternative disinfectants, such as ozone, chlorine dioxide and chloramines. Ozonation has emerged as one of the most promising alternatives to chlorination. In the early 1980s, it became obvious that the application of ozonization in drinking water treatment not only resulted in the formation of oxygenated compounds but also in the formation of bromide-containing water; brominated organic compounds and bromate as well.  

The identification of new, possibly hazardous compounds in drinking water has become an important task for water suppliers. In an ideal situation, where standards for different intake routes of exposure are fully adjusted to each other, regular monitoring of these compounds should only be necessary when these compounds are carcinogens, or if the relative contribution of drinking water to total exposure or to the tolerable daily intake is high. If formation, toxicity and methods of analysis of metals and selected organic compounds in drinking water were well known, determination of inorganic disinfection by-products at the required low levels was a new challenge for researchers in the end of the 20th century. 

Recently chlorite, chlorate and bromate are the most important inorganic oxyhalide by-products and their concentrations in drinking water have to be controlled. Chlorite is a disinfection by-product that is formed when chlorine dioxide is used for disinfecting drinking water, while chlorate is formed when chlorine dioxide or chloramine is used.

  Chlorination using hypochlorite acid solutions, which contain some ClO

  as a product of HClO disproportionation, may also contribute to ClO

  contamination in disinfected water. Even low levels of chlorite may lead to hemolytic anemia and can have dangerous effects on the nervous system in infants and young children. There is no limit for chlorate because of this limited knowledge about its toxicity; however, the World Health Organization (WHO) recommends minimizing the level of chlorate as much as possible for as long as there is no reliable toxicological data.

 Figure 1: Pretreatment steps for samples, calibration and blank solution according to ISO 15061.10			

The most dangerous inorganic disinfectant by-product — bromate, is formed when water containing bromide is ozonated. Bromate formation can be restricted by careful adaptation of the ozone dosage to disinfectant demand. Other options include lowering of the pH, the use of ozone and hydrogen peroxide, addition of ammonia, removal of bromide before ozonization, and the use of membrane filtration or anaerobic processes.

Figure 2: Separation of F-, ClO2-, BrO3-, Cl-, NO2-, ClO3-, NO3-, PO43- and SO42- in standard solution.			

Bromate has been identified as an animal, and possibly human, carcinogen. The International Agency for Research on Cancer (IARC) has classified bromate in group B-2 (the agent is possibly carcinogenic for humans).

  In 1993, bromate was judged by the WHO as a potential carcinogen, initially at a 25 µg/L level, which was associated with an excessive lifetime cancer risk of 7 × 10

  on account of the limitations in the available analytical and treatment methods. Soon, health effects research indicated that it can be a human carcinogen that poses a potential 10

  risk of cancer after a lifetime exposure in drinking water at a 5.0 µg/L level and a potential 10

The US Environmental Protection Agency (EPA).

  and the Commission of the European Communities

  has recently issued new rules that require public water suppliers to control some of the previously unregulated microorganisms and cancer-causing disinfection by-products in drinking water. According to these regulations, the maximum admissible level (MAL) is 10 µg/L for bromate and 1000 µg/L for chlorite. 

Bromate and chlorite are two of the chemicals parameters included in the EU Directive with a maximum allowed limit of 10 µg/L and 1000  µg/L, respectively. That Directive specifies under Annex III, 2.1: 

"performance characteristics are that the method of analysis used must, as a minimum, be capable of measuring concentrations equal to the parametric value with a trueness, precision and limit of detection specified"

. Both trueness and precision criteria were set at ± 25% of the parametric value. 

Determination of Chlorite, Chlorate and Bromate by using Ion Chromatography

The idealized method for bromate, chlorite and chlorate determinations should meet the following requirements:					

detect the target ions in drinking water with a limit of determination at 25% of the maximum acceptable concentration

The most useful analytical technique to determine inorganic anions and cations seems to be IC, which is a significant addition to the ever-expanding field of chromatographic analysis. IC has seen phenomenal growth in most areas of analytical chemistry since it was introduced in 1975. It has become a versatile and powerful technique for the analysis of a vast number of ions present in the environment and in biological tissues and fluids.

  IC is now considered a well-established, mature technique for the analysis of ionic species. Many organizations, such as the International Standardization Organization (ISO) and the US EPA have standards or regulatory methods of analysis of anions and cations using IC.

In the mid-1990s, the ISO worked on Method 15061 for bromate

  for chlorite, chloride and chlorate determination. The ISO 15061 standard specifies a method for the determination of dissolved bromate in drinking water, raw water, surface water, partially ozonated water and swimming pool water. Measurement of bromate is performed in the range from 0.5–1000 µg/L, with or without sample preconcentration. If preconcentration is necessary, sample is passed through three cation-exchange cartridges, in the Ba

  forms, to reduce the total ionic strength. This standard consists of two parts — a direct method and a preconcentration method, but suffers from the existence of interferences such as chloride and chlorite, which require timeconsuming steps for elimination, clean-up and separation before the instrumental measurements. 

The ISO 10304-4 standard specifies a method for the determination of dissolved chlorite, chloride and chlorate anions in water with low contamination (e.g., drinking water, raw water, swimming pool water). An appropriate sample pretreatment and the use of conductivity, UV or amperometric detectors make the working ranges from 0.03–10 mg/L (chlorate) to 0.01–1000 mg/L (chlorite). 

The methods of bromate, chlorite and chlorate determination using IC can be generally divided into:

direct methods (suppressed conductivity detection)

indirect methods (UV/Vis detection after post-column derivatization)

hyphenated techniques (ICP–MS and MS detection). 

The direct methods are based on the selective separation of ClO

  ions in the presence of other anions in the sample and their detection in the conductivity detector after suppression. 

These methods are relatively simple and inexpensive but the main disadvantage is the proper resolution of ClO

  ions, whose concentrations in real samples differ significantly.  

Better bromate limits of quantification are obtained with indirect methods (derivatization methods) consisting of converting the determined substance, before or after its separation in the analytical column, into derivatives which can then be detected with a UV/vis detector. Reagents such as fuchsine, 

-dianisidine, chlorpromazine, potassium bromide or potassium iodide are applied for the bromate determination with the derivatization methods.

Presently, the technique based on the triiodides formation seems to be the most promising one amongst all the derivatization methods for bromate determination by IC.

  In this connection, in 2009, the Work Group 33 of Technical Committee 147 of the ISO completed the development of the new bromate determination method based on this post-column derivaization reaction with the use of triiodides reaction. It was thoroughly described in 2009 in the ISO standard 11206 entitled 

Water quality - Determination of Dssolved Bromate — Method using Liquid Chromatography of Ions and Post Column Reaction (PCR)

When its verification process within the proficiency testing framework is concluded, it should become the most important standard used for the bromate determination in water. Moreover, it may be expected that it will gradually replace the already used direct method described in the ISO Method 15061. A chromatogram of bromate determination using an indirect method according to reference 15 is shown in Figure 3.

 Figure 3: Chromatogram of ozonated drinking water.			

The best results in terms of bromate detection and quantification limits in water can be obtained with the hyphenated techniques. Among them, the most popular ones are IC–ICP-MS (Ion Chromatography with Inductively Coupled Plasma Mass Spectrometry) and IC–MS (Ion Chromatography with Mass Spectrometry).

  The obvious disadvantages of MS-based detection techniques are that they each add considerable complexity and significant cost to the analysis.  

The use of hyphenated techniques requires an in-depth understanding of the analytical methodologies and knowing the apparatus in minute detail. Such systems are expensive and for this reason, they are currently mainly used for scientific research rather than routine analyses. Additionally, up till now no international IC standard-based ion MS or ICP-MS detection has been declared for the regulatory monitoring of bromate or any other disinfection by-products anions.  

Global and national agencies are continually striving to monitor bromate, chlorite and chlorate levels in drinking water to establish appropriate regulatory limits. Depending on the results of further research, a risk model could indicate a more definitive guideline value for oxyhalides in drinking water.  

Compared with non-ionic chromatographic methods for inorganic disinfection by-products determination, IC is usually faster, more accurate and reliable, less susceptible to sample matrix effects and gives much more information about sample composition. 

Furthermore, for the moment IC is the only accepted standard method for inorganic disinfectant oxyhalide by-product analysis. 

The problem of inorganic oxyhalides also concerns swimming pool waters

  and bottled water which has become a healthier choice than tap water for many people because they believe that bottled water contains fewer contaminants, or dislike the taste of chlorinated tap water.

  The next serious problem which has to be solved is using a KBrO

 in the baking industry and detecting the presence of bromate residues in food samples.

The selection of a proper bromate determination method using IC depends on many factors and is usually affected by the availability of appropriate equipment, the number of analysed samples, the expected analyte content and the required detection and quantification limits.

  Among the three main groups of IC methods recently available (suppressed conductivity, UV/vis and MS detection modes) in the majority of routine laboratories all over the world, methods based on suppressed conductivity detection are used. They are good enough for chlorite and chlorate determination, but are criticized because of costs, and difficulties in bromate determination (< 2.5 µg/L in samples with chloride content over 50 mg/L). 

Hyphenated techniques such as IC–ICP-MS and  IC–MS are still very sophisticated and not popular in routine laboratories. In this connection, derivatization methods for bromate analysis based on UV detection seems to be the most attractive, alternative, reliable, robustness and low operational cost option. 

1. S.D. Richardson, Drinking Water Disinfection By-products,

The Encyclopedia of Environmental Analysis and Remediation

, R.A. Meyers, Ed.; Wiley: New York, 1398–1421 (1998). 

4. IARC. Some Naturally Occurring and Synthetic Food Components, Furocoumarins and Ultraviolet Radiation: Potassium Bromate, 

Monographs on the Evaluation of the Carcinogenic Risk to Human

Stage 1: Disinfectants and Disinfection By-Products Rule. A Quick Reference Guide,

7. Council Directive Concerning the Quality of Water Intended for Human Consumption Directive 98/89/CE

, Commission of the European Union: Brussels. 1998. 

A review of separations of chlorite, chlorate and bromate in water using ion chromatography and their determination.

Analysing Inorganic Disinfection By-products by Ion Chromatography

Biodiesel fuels are part of a rapidly growing alternative fuel industry to replace gasoline with renewable, more efficient and lower emission fuels.

  Biodiesel contains diesel fuel blended with 2–20% (B2–B20) of fatty acid methyl esters (FAME), which are manufactured by transesterification of vegetable or animal oils to generate three molecules of free FAMEs and one molecule of glycerol byproduct. Free and bound glycerol from unconverted triglycerides can cause diesel engine failures. Therefore, ASTM D6751 specifies limits for free glycerol and bound glycerol at 0.02 and 0.24 wt % respectively in biodiesel determined using GC–FID.

  However, this method requires sample derivatization, which increases sample handling time and expense. This application brief describes a high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) method for free and total glycerol in biodiesel that meets the ASTM limit requirements and eliminates the sample derivatization step. 

A Dionex ICS-3000 chromatography system with an electrochemical detector was used. For free glycerol determinations, the biodiesel samples were extracted with water and the aqueous layer was removed for analysis. For total glycerol determinations, biodiesel samples were hydrolysed in 1 M NaOH by refluxing at 95 °C for 1 h, cooled and the aqueous layer removed for analysis.

The method was evaluated by determining the LOD, LOQ and the linearity of response from seven standards (n = 2, 0.05–10 mg/kg glycerol). The method was sensitive, with an LOD (3× 

) of 0.5 and 2.4 µg/kg, respectively. The linearity in the specified range produced a coefficient of determination (r

To evaluate the method for determining free and total glycerol, we analysed ten biodiesel samples. The glycerol peak was well resolved from an unknown peak, as shown in the total glycerol determination of a B100 animal-source biodiesel sample (Figure 1). Free glycerol concentrations ranged from 0.02–11.8 mg/kg (n = 2) while total glycerol concentrations ranged from 0.066–188 mg/kg (n = 2). All glycerol concentrations in the biodiesel samples were within the ASTM specifications of <0.02 (200 mg/kg) and 0.24 wt% (2400 mg/kg), respectively. Recoveries of glycerol added at twice the sample concentrations ranged from 92–100%. 

Figure 1: Comparison of total glycerol in an aqueous solution of a base-hydrolyzed B100 animal sample a) without, and b) with 80 mg/kg added glycerol. Sample preparation: base-hydrolysis at 95 Â°C for 1 h, dilute aqueous layer 1:5, filter 0.2 Âµm. Column: CarboPac MA1 guard and analytical; eluent: 100 mM sodium hydroxide; temperature: 30 Â°C; flow-rate: 0.40 mL/min; injection volume: 5 ÂµL; detection: PAD, Au, Waveform A.1 Peaks: 1) Unknown; 2) glycerol [(a) 0.54 mg/kg, (b) 1.3 mg/kg]. 

LCGC Asia Pacific-06-01-2011

Comprehensive Two-dimensional Liquid Chromatography (LCxLC): A Review

Direct Determination of 2-Ethylhexanoic Acid in Clavulanate

GPC/SEC–MALLS Analysis of Hyaluronic Acid

Simultaneous determination of mineral acids, fluoride and silicate in etching baths by ion chromatography with dual detection

Analysing Inorganic Disinfection By-products by Ion Chromatography

Determination of Free and Total Glycerol in Biodiesel

An Improved SPE-LC–MS–MS Platform for the Simultaneous Quantification of Multiple Amyloid Beta Peptides in Cerebrospinal Fluid for Preclinical or Biomarker Discovery

Valves for Gas Chromatography: Fundamentals

Base Hydrolysis and Accelerated Solvent Extraction (ASE): Determination of Total Fat from Dairy Products

Selectivity in Reversed-Phase LC Separations (Part 2): Solvent-Strength Selectivity