Methanol (MeOH) and acetonitrile (ACN) are the two most popular organic solvents to use in the mobile phase for reversed-phase (RP) liquid chromatography (LC) separations. Tetrahydrofuran (THF) is also used to a lesser extent. Why are these solvents the most popular ones? Are there alternative solvents that should be tried? This month's 

 examines these and some other aspects of solvent selection for LC.

In the 1970s, Rohrschneider made an extensive study of the properties of various solvents.

  Among other things, he showed that, in some cases, when two solvents were mixed, properties of a third solvent could be obtained.

This suggested that some subset of all possible solvents could be used for chemical purposes. Around the same time, Lloyd Snyder was interested in the various solvent properties that were important in LC separations, especially reversed-phase.

 From other studies, he knew that three properties were of particular significance in terms of selectivity: the acidic, basic and dipolar nature of the solvents. Snyder recalculated Rohrschneider's data based on these three properties and plotted them in what is now known as the solvent selectivity triangle. A subset of these data is shown in Figure 1, where each solvent is plotted according to its proportion of acidic, basic and dipolar properties. The heavy dots show the central tendency of several solvent classes, some of which I have labelled. 

In principle, three solvents — one with 100% acidity, one with 100% basicity and one with 100% dipolarity — would be located at the extreme corners of the triangle. If these solvents existed, we should be able to blend them in different proportions and get the properties of solvents lying within the boundries of the triangle. Of course, such solvents do not exist. Most solvents have a mixture of acidic, basic and dipolar properties, so they lie away from the corner and usually away from the edges of the triangle. Since the desired solvents don't exist at the corners of Figure 1, we might consider other solvents that are close to the corners — solvents that exhibited primarily acidic, basic or dipolar properties. For example, carboxylic acids (RCOOH in Figure 1) are quite acidic, amines (for example triethylamine) are basic, and the chlorinated solvents (for example dichloromethane, MeCl

) have dipole properties. If we were to blend these three solvents together, we might expect to get intermediate properties. But again, we are foiled, because the chlorinated solvents don't mix with the others and two phases result — not a desirable mobile phase characteristic! We can continue working our way toward the centre of the triangle, searching for solvents that are miscible and have a dominance of one of the desired properties. For example, alcohols (such as MeOH) have some acidic properties, ethers (for example THF) have basic properties, and nitriles (for example ACN) have dipole properties. These three solvents are mutually miscible and form a subset (shaded area) of the total triangle. It would be nice to conclude that because of Snyder's work, these three solvents were selected as the solvents of choice for reversed-phase separations, but this would be a bit presumptuous. By the time this work was completed, these three solvents were already the most popular solvents, but now we know why they had been found to be most effective. The enhanced acidic, basic and dipolar properties of one solvent can assist certain chemical interactions better than another mobile phase solvent, so that difficult-to-separate solutes can now be separated. 

Figure 1: Classification of solvent properties according to their acidic, basic, and dipolar nature. Adapted from references 2 and 3. 			

One popular application of the solvent selectivity triangle is based on a set of seven experiments using various solvent blends. This strategy formed the basis of the Sentinal system introduced by DuPont when they were in the LC business and was adapted as the PerkinElmer Solvent Optimization System (PESOS). These products enabled automated or semi-automated selection of the best solvent to separate a particular sample. The concept of this approach is shown in Figure 2. The MeOH–ACN–THF solvent triangle of Figure 1 is adjusted to an equilateral triangle for convenience. One solvent is chosen to start with and its concentration in water or buffer is adjusted to get retention in the desired range in an effort to achieve a separation, for example, 50:50 MeOH/buffer. Next, a second solvent is adjusted to give similar overall retention, for example, a 40:60 ACN/buffer gives similar retention. Then the third solvent is blended with buffer to get similar retention. For example, a 30:70 THF/buffer in the present situation. By appropriate adjustment of the organic–buffer ratio, the three mobile phases should give very similar retention times when the entire separation is considered — individual retention times are expected to vary between solvent systems. These are experiments 1, 2 and 3 at the corners of the triangle in Figure 2. Next, 1:1 blends are made of the mobile phases from each corner to generate the runs 4, 5 and 6 on the edges of the triangle. Finally a 1:1:1 blend formed the central experiment, 7, to complete the sevenexperiment set. Retention data from each of these experiments could be entered into the optimization programme and results for conditions between the experimental points could be calculated to find the best possible separation, often a quaternary blend of MeOH, ACN, THF and buffer. 

Figure 2: The solvent-selectivity triangle approach to method development. 			

Alternatively, the seven chromatograms could be printed and set out in the same triangle pattern with visual interpolation to identify the best separation conditions. An example of this approach is shown with the simulated chromatograms of Figure 3. The MeOH/buffer run (#1) separates only four peaks of the fivecomponent mixture — the last two peaks are merged. The ACN/buffer run (#2) nearly separates the last two peaks, but the second two have merged and reversed. The THF/buffer run (#3) has its problems, too — the last two peaks are nicely separated, but the second two are still a problem. It is reasonable to assume that intermediate mobile phase conditions will give intermediate separations when a mobile phase condition is varied. So, if we were making these runs one at a time and examining the results before proceeding, we might skip run #5, because it is obvious from #1 and #2 that an intermediate separation of the last two peaks will be no better than for the binary mobile phases. The same prediction could be made for the second two peaks in run #6 based on #2 and #3. Run #4, a 50:50 blend of #1 and #3 is able to separate all five peaks to baseline by giving up part of the excess separation of the first three peaks observed in #1 and part of the excess separation of the last two peaks from #3. The quaternary blend of 1:1:1 of #1, #2 and #3 to form run #7 is unsatisfactory too.  

Figure 3: Simulated partial chromatograms for various mixtures of MeOH, ACN and/or THF with buffer. Chromatograms positioned to correspond to Figure 2. 			

This solvent triangle approach of method development was quite popular in the 1970s and 1980s. It was fairly intuitive to use and generated useful methods. It was equally good for developing isocratic and gradient methods. However, because the mobile phases often comprised all three solvents plus water, they tended to be complex and the more components contained in a mobile phase, the more things can go wrong. This, coupled with the less reproducible nature of the columns of the day, resulted in methods that often needed constant tweaking to keep running. As we'll see in next month's 

, further understanding of reversed-phase retention behaviour introduced the solvent-strength optimization technique that largely supplanted the solvent selectivity triangle as the most popular approach to method development. But just because it isn't as popular today doesn't mean that we should discard the solvent triangle approach — it is a powerful tool that can help to solve difficult problems, especially after simpler techniques have failed. 

We've seen how the solvent selectivity triangle helps us understand why ACN, MeOH and THF have become complementary and preferred solvents for reversed-phase LC mobile phases and how the triangle can serve as an organizational tool for using solvent type to adjust peak spacing in a chromatogram. There are other uses for the solvent triangle as well. 

We can use the solvent triangle to help us make wise choices for the selection of additional solvents to test. For example, if we find that MeOH in the mobile phase does not give us the peak spacing we need, we might be tempted to try another alcohol — perhaps ethanol or propanol. But the properties of all of these alcohols cause them to be located in the lower left corner of the triangle. This suggests that it is unlikely that we'll find dramatically different chromatographic properties by changing to a different alcohol. We'd be much better moving to an entirely different part of the triangle if our goal is to use solvent selectivity to move peaks around in the chromatogram. On the other hand, there may be other reasons why we might switch from one alcohol to another. For example, larger alcohols, such as propanol, tend to be less denaturing to biomolecules than methanol — it would be reasonable to expect similar, but not identical, chromatographic properties when making this change. As another example, we might find that the basic properties of THF are favourable for a particular separation, but its other properties, such as relative instability and high UV absorbance, make it less than ideal for some applications. It is possible to successfully substitute another ether, such as methyl tertiary butyl ether (MTBE), for THF in some cases. (MTBE, however, is not fully miscible with water, so may require a co-solvent in the mobile phase.) 

Still another application of the solvent triangle is to identify alternate mobile phase solvents if the supply of solvent is limited. In the alcohol example above, we saw that the properties of the different alcohols were sufficiently similar that a change in alcohol type is unlikely to be an advantage when trying to change selectivity. Conversely, this suggests that we could change from one alcohol to another if necessary for other reasons. For example, if the availability of HPLCgrade methanol became scarce for some reason, we might reasonably switch to ethanol with the hope of obtaining a similar separation. For various economic, political and practical reasons (see reference 4) HPLC-grade acetonitrile has become quite expensive in the last few years. It would be nice to find another solvent that had similar properties to use as an alternative. Unfortunately, there aren't any readily available solvents in the dipole corner of the triangle that fulfill this requirement, so we are stuck with acetonitrile. If ACN is necessary to get the desired separation and no alternative solvents are available, we have to approach the problem from a different angle. We could change from using a 150 × 4.6 mm i.d. column to a 150 × 2.1 mm column. The reduction in column diameter will allow us to lower the flow rate by a factor of 5 and get the same retention time, for example, from 1 mL/min to 0.2 mL/min. This would reduce the ACN consumption by fivefold, as well. Or we could switch from a 150 × 4.6 mm column packed with 5 µm particles to a 100 × 4.6 mm column filled with 3 µm particles. Both of these columns will generate approximately 10000 plates with real samples, so the 100 mm column should reduce the run time by one third — a 15 min run would drop to 10 min, with a corresponding saving of solvent. Or we could combine the two changes and switch from the 150 × 4.6 mm, 5 µm column to a 100 × 2.1 mm, 3 µm column and reduce the solvent consumption by 5 × 3 = 15-fold. Of course, if these changes were made, we might have to make some other adjustments, such as reducing the amount of injected sample or adjusting the system plumbing to reduce extracolumn volume.  

The solvent selectivity triangle helps to simplify the number of solvent choices that are likely to be beneficial to explore during LC method development. It also gives us an organizational framework when exploring solvent selectivity as a means of resolving difficult-to-separate peaks. Finally, we can get some insight into the pros and cons of alternate solvents if we want to make a change. Next month's 

 will consider an alternative to solvent-type selectivity: solvent-strength selectivity. 

 is vice president of LC Resources, Walnut Creek, California. He is also a member of 

 editorial advisory board. Direct correspondence about this column should go to "LC Troubleshooting", Bridgegate Pavillion, Chester Business Park, Wrexham Road, Chester, CH4 9QH, UK, or e-mail the editor, Alasdair Matheson, at 

Articles

LC Troubleshooting

Examining the properties of the three most commonly used solvents and how to select the most suitable mobile phase composition.

Selectivity in reversed-phase LC Separations (Part 1): Solvent-type Selectivity

A simple, rapid and sensitive method based on solid-phase microextraction (SPME) and gas chromatography–mass spectroscopy (GC–MS) was developed to determine five chlorophenols and their two major metabolites in aqueous samples. To improve the peak shape, silylation was performed on the SPME fibre to get less polar forms, which are more favourable for GC–MS analysis. All parameters influencing SPME–GC–MS performance, including pH, salinity, extraction time, derivatization time, as well as desorption time, were carefully optimized. The method was successfully applied to the analysis of chlorophenols and their metabolites, excluding tetrachlorohydroquinone in pond water with recovery percentages ranging from 80.4% to 91.4%.

Chlorophenols (CPs), are known to contaminate the environment, and are highly toxic, poorly biodegradable and potentially carcinogenic.

  Within the list of 11 phenolic compounds considered as major pollutants, drawn up by the US Environmental Protection Agency (EPA), CPs are among the most toxic and carcinogenic.

  They are produced commercially for use as preservative agents, pesticides, antiseptics, disinfectants and are also used as intermediates in many industries including the pharmaceutical industry.

  In addition, some chlorophenols, especially 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP) and 2,4,6-trichlorophenol (2,4,6-TCP), could be produced when water is disinfected with chlorine, resulting in unpleasant and persistent organoleptic properties in the disinfected water.

CPs enter the environment either directly from industrial waste or indirectly as important metabolites of some other chlorinated pesticides, such as PCBs.

  As well as CPs, their two major metabolites, dichlorohydroquinone and tetrachlorohydroquinone, also show high toxicities. They were reported to be more toxic because they could cause DNA damage by breaking a single strand of DNA chain.

  This makes the simultaneous determination of CPs and their metabolites very important when accessing their environmental risks. However, until now, most publications have put the emphasis on the analysis of one specific CP, or a group of CPs, but have ignored their metabolites. 

Different techniques have been used to determine chlorophenols. Most of them used chromatographic-based instrumentation such as gas chromatography (GC)

  and high performance liquid chromatography (HPLC), particularly reversed-phase liquid chromatography (RPLC)

				 in combination with ultraviolet detection (UV),

  Apart from when coupled with fluorescence or an MS detector, HPLC coupled with other detection methods suffers from interference from matrix compounds such as humic substances naturally occurring in environmental samples. GC is therefore preferable because of its high resolution, high sensitivity and selectivity when coupled with a versatile, selective detector such as an electron-capture detector (ECD), atomic emission detector (AED) or mass spectrometer

  During the GC separation process, peaks obtained from free chlorophenols show strong tailings. In this case, derivatization methods were suggested to get less polar forms, which could improve the peak shape.

  Silylation is the technique of choice because phenolic functional groups can be readily derivatized, and the reaction mixture can be directly injected into the GC without further sample pretreatment. This technique, however, requires prior separation of the analytes from the sample to avoid quenching of the silylating agent by the large amount of water or ethanol present in the matrix. Therefore, two of the major drawbacks of these technologies are laborious sample pretreatment and poor recoveries because of the volatile nature of the derivatives. 

Due to the low concentration levels of CPs and the complicated matrices of environmental samples, different preconcentration methods have been used prior to the analysis. Liquid-liquid extraction (LLE)

				 have been widely used. However, these traditional methods have the well-known disadvantages of being solvent- and sample consuming, time- and labour consuming and also have the risk of analyte losses. Therefore, liquid phase microextraction (LPME),

				 and head-space solid phase microextraction (HS-SPME)

  have been proposed as alternative methods to avoid the use of large volumes of sample and organic solvent. However, the precision of LPME suffers from the instability of the droplet when using single droplet LPME as a sample preparation method. Recently, research concerning the determination of chlorophenols by SPME has focused on the evaluation of new fibre coatings.

  These research projects have broadened the range of SPME applications. However, the new fibre coatings are not commercially available and are instable from fibre to fibre, which is the main problem to be solved.  

The aim of this paper is to develop a simple, rapid and sensitive method for the determination of chlorophenols, as well as their degradation metabolites, in aqueous samples. To achieve this, SPME in combination with GC–MS was used. In addition, the silylation method was used to improve the peak shape. This is different from the traditional silylation procedure. The silylation in this paper was carried out on the SPME fibre based on head-space concept (i.e., the fibre was inserted into the headspace of silylating agent), after the extraction of chlorophenols from aqueous matrix. On-fibre silylation has the advantages of SPME but also makes up for the drawbacks of traditional silylation because the analytes have been separated by the fibre from the matrix.

The small volume of water remaining on the fibre can react with the excess vapour of the silylating agent, whereas the silylating agent would be quenched by the large amount of water or ethanol present in the matrix during the traditional silylation procedure. Moreover, the derivatives generated on-fibre could be introduced directly and immediately into GC, which avoids the losses of analytes. 

 The SPME manual fibre holder was from Supelco Inc. (Bellefonte, Pennsylvania, USA). Due to the polar property of CPs and their metabolites, polyacrylate (PA) fibre (Supelco) with 85 µm thickness was used for their extraction. The SPME fibres were conditioned under helium for 1–2 h in hot GC injection port before extraction. The SPME sampling stand and the heat/stir plate used in extraction were also from Supelco. The magnetic stirring bars (10 × 3 mm) were from Aldrich (St Louis, Missouri, USA). 

The standards of 2,6-dichlorophenol (2,6-DCP, 99%), 2,4,6-trichlorophenol (2,4,6-TCP, 98%), 2,5-dichlorohydroquinone (2,5-DiCH, 98%), tetrachlorohydroquinone (TeCH, 99%) and Bis(trimethylsilyl)trifluoroacetamide (BSTFA) were purchased from Acros Organics (Geel, Belgium), whereas pentachlorophenol (PCP, 98%), 2,3,4,5-Tetrachlorophenol (2,3,4,5-TeCP, 10 ng/µL) and 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP, 10 ng/µL) were from Sigma-Aldrich (St Louis, Missouri, USA). 

The individual stock standard solution of 1 g/L 2,6-DCP, 2,4,6-TCP, 2,5-DiCH, TeCH and PCP were prepared by dissolving 0.0250 g of each standard in acetonitrile in a 25 mL volumetric flask. The 10 mg/L stock mix-standard of the above five compounds were prepared by adding 0.1 mL of each stock standard in a 10 mL volumetric flask and diluted with acetonitrile. The solutions were protected from light at 4 °C. The series of working standard solutions were freshly prepared by diluting proper volume of stock mix-standard solution, 2,3,4,5-TeCP and 2,3,4,6-TeCP with acidified Milli-Q water (pH 3). Three millilitres of each working standard was put into 4 mL sample vials, and then sealed with hole-caps and Teflon-faced silicone septa (both purchased from Supelco). The derivatizing reagent was also prepared by adding 100 µL BSTFA into the 4 mL sample vial, and then sealed with hole-caps and Teflon-faced silicone septa. All chemicals and reagents used in this study were analytical grade. 

 The sampling was performed by immersing a SPME fibre directly into the 3 mL standard solution or sample for 60 min at ambient temperature under magnetic stirring for the adsorption of analytes onto the fibre coating. After extraction, the fibre was then inserted into the headspace of BSTFA. The analytes on the fibre were derivatized for 5 min to ensure the completion of silylation. The GC programme started as soon as the fibre was immediately inserted into the GC injector. Desorption time was set for 3 min with the temperature 270 °C. 

Instrumentation and Operating Conditions:

 A Hewlett-Packard (Palo Alto, California, USA) 6890 gas chromatograph equipped with 5973 mass spectrometer and the MS Chemstation with Window Operational System (1701AA series) software was used for analysis. A 30 m × 0.25 mm i.d. × 0.25 µm HP-5MS fused-silica capillary column, coated with 5% biphenyl and 95% dimethylpolysiloxane (from HP Inc.) was used The column temperature programme was set as follows: 100 °C held for 1 min; 15 °C/min to 160 °C; 5 °C/min to 300 °C; held for 7 min. The total run time of the programme was 40 min. The GC injector port was used in the 'splitless' mode and held isothermally at 270 °C for SPME injections for the duration of the run. Helium was used as the carrier gas, at a flow rate of 1 mL/min by using electronic pressure control. The GC–MS interface temperature was maintained at 280 °C. The MS was operated in the electron impact (EI) ionization mode with electron energy of 70 eV, and mass-to-charge ratio (

) scan ranged from 50 to 550 amu to determine appropriate masses for selected ion monitoring (SIM). The MS ion source and mass filter (quad) temperatures were held at 230 °C and 150 °C, respectively. 

Development of SPME-Silylation Procedure and GC–MS Analysis:

 In this study, the post-derivatization with silylation was performed on the fibre of SPME following extraction by using BSTFA gaseous phase. Generally, the silylation combined with SPME is difficult because the silylmethylated reagents and silylmethylated derivatives will hydrolyse in aqueous solutions. To overcome this problem, a method based on the silylation in the BSTFA gaseous phase after SPME was proposed. In this method, the fibre after extraction was then pierced immediately into the Teflon-faced silicone septa on the headspace of BSTFA, in which the adsorbed chlorophenols were derivatized with BSTFA. 

Figure 1: (a) TIC chromatogram of silylmethylated derivatives in mix-standard solution; (b) SIM chromatogram of silylmethylated derivatives in pond water [extracting individual quantification ions for each derivative]. 			

Because CPs and their metabolites are polar compounds, commercially available polar SPME fibre coating, namely 85 µm polyacrylate fibre, was used for extraction. As CPs and their metabolites showed weak acidic properties, we primarily adjusted the pH value of standard solutions to 3 in order to get neutral forms of the target compounds for better extraction by SPME fibre. Figure 1(a) shows the chromatogram of derivatives (10 µg/L) obtained after SPME extraction and BSTFA silylation. All compounds were well separated under the above temperature programme. To increase the sensitivity, MS was used to quantitatively analyse CPs and their metabolites by selected ion monitoring (SIM) mode. The molecular weight, retention time, molecular ion and qualifier ions for each derivative are listed in Table 1. To minimize the baseline shifting after a derivatizing reagent peak, the signal was turned off as soon as the reagent appeared and turned on again after the reagent eluted. 

Table 1: Molecular weight of parent compounds, retention time, molecular ion and qualifier ions of the derivatives of the analysed compounds. 			

 The optimum desorption time was studied by varying desorption time of polyacrylate fibre in the GC injector at 270 °C with 10 µg/L mix-standard solution (pH 3). The results showed that the desorbed amount of derivatives of target compounds on the fibre increased at 3 min. The carryover test was run to measure the amount of derivatives retained on the fibre after 3 min exposure. The fibre was placed back to the GC injector (270 °C) for another period of 3 min. None of the derivatives were found during the carryover test, which indicated that 3 min desorption was completed for target derivatives. Thus, 3 min was chosen for the subsequent experiments. 

 In derivatization, the silylation efficiency of target compounds after SPME was determined by the reaction time of BSTFA. In this work, the effect of reaction time on the silylation was studied by varying the exposure time to BSTFA from 1 min to 60 min at room temperature. According to the results shown in Figure 2, the efficiency of silylation for most selected compounds reached the maximum at 5 min, afterwards, the responses decreased with the increase of reaction time. The reason might be that long derivatization time could promote the release and the degradation of the derivatized chemicals from the fibre. Because the derivatized analytes were more volatile than their mother compounds. Long derivatization time might cause their release from fibre, thus influence the final results. Moreover, the silylated analytes might get desilylation when the derivatization time was too long. Thus, the optimum derivatization time was finally selected to be 5 min. 

Figure 2: Effect of derivatization time on peak areas of silylmethylated derivatives by SPMEâGCâMS. 			

The standard solution of target compounds (10 µg/L, pH 3) was used to investigate the optimum conditions for SPME procedure. The equilibrium time for SPME depends on the rate of mass transfer in the aqueous phase. The optimum extraction time was determined by varying the extraction time from 20 min to 90 min. All the extractions were carried out at room temperature and a constant rapid stirring was used to increase the rate of mass transfer in aqueous phase. As shown in Figure 3, the amount of most target compounds extracted, except PCP, increased with the increase of extraction time, and was in equilibrium at about 60 min. Based on the consideration of shorter pretreatment time, the optimum extraction time was set at 60 min. 

Figure 3: Effect of extraction time on peak areas of silylmethylated derivatives by SPMEâGCâMS. 			

Generally, to increase the extraction efficiency in sample solution, favourable conditions for forming neutral forms of target compounds should be selected. Since selected compounds show weak acidic property, pH values of 3.0 and 7.0 were investigated. During the extraction procedure, extraction was kept at room temperature for 60 min and derivatized for 5 min, with 3 min desorption followed. Figure 4 illustrates the responses of the pH optimization. The results indicated that a higher sorption capacity was obtained at pH 3.0 than at pH 7.0. Thus, pH 3.0 was chosen as final experimental condition in this work. 

Figure 4: Effect of pH on peak area of silylmethylated derivatives by SPMEâGCâMS. 			

 The salt content in the sample solution affects hydration of ionizable compounds, changes their solubility in aqueous phase and thus affects the extraction efficiency by the fibre. The salinities of the sample solution were studied by adding different amounts of NaCl into the sample solution. Three concentrations of NaCl, 0%, 15% and 30%, were selected for salinities study. Figure 5 shows the effects of salinities on the response of the target compounds. During this procedure, adsorption was kept at room temperature for 60 min with sample pH at 3.0, and the derivatization time was 5 min. It was observed that, for 2,6-DCP and DiCH, the responses increased with the increase of ion strength, while for the rest of the compounds, responses reached the maximum when the concentration of NaCl was 15%. The possible reason for this phenomenon where extraction efficiency initially increased with the increase of salt content and then decreased with the further increase could be a "salt-out" effect and a reverse "salt-in" effect. Normally, salt content influences the extraction efficiency by this so-called ''salt-out effect''; salt would decrease the solubility of target compound in aqueous phase and thus increase the partition coefficient between fibre coating and aqueous phase. However, in some cases, the solubility of the compounds might not change and the addition of salt may decrease the amount extracted by decreasing the activity coefficients of the analytes.

				 Considering the overall responses of the target compounds, a concentration level of 15% NaCl was selected. 

Figure 5: Effect of salinities on peak area of silylmethylated derivatives by SPMEâGCâMS. 			

Calibration Curve, Precision and Detection Limit:

 The SPME and post-silylation procedures were performed according to the optimized conditions. The electron impact (EI) ionization and selected ion monitoring (SIM) mode were used to determine the detection limit of selected compounds in aqueous solution. The concentrations of working solutions were selected to be 0.01, 0.1, 1 and 10 µg/ L respectively. The limits of detection (LOD) and the limit of quantification (LOQ) of selected compounds were calculated based on the 3σ and 10σ criteria, respectively. The precision was determined by performing three replicates from the aqueous solution, containing 1.0 µg/L of target compounds. The results are shown in Table 2. The linear ranges of target compounds by SPME procedure spanned over three orders of magnitude, with correlation coefficients ranging from 0.9963 to 0.9999. The relative standard deviation (RSD) of target compounds ranged from 3.9% to 10.4%, showing good reproducibility for all target compounds. The LOD and the LOQ of target compounds ranged from 2.3 × 10

Table 2: Linearity, detection and quantification limits of target compounds in pure water obtained by SPMEâGCâMS. 			

Determination of CPs and their Metabolites in Pond Water:

 Pond water was selected as a real sample for evaluation of the developed method. Under the optimized conditions, the concentrations of seven selected compounds determined by SPME are shown in Table 3 and the SIM chromatogram is shown in Figure 1(b). Only three compounds, 2,3,4,5-TeCP, 2,3,4,6-TeCP and PCP, were detected by the developed method. To further evaluate the influence of matrix on direct determination of CPs and their metabolites by this method, the recovery test was performed by adding 1.0 µg/L mix-standards of selected compounds into pond water. Recoveries of most compounds were satisfying, ranging from 80.4% to 91.4%, except 2,3,5,6-TeCH (Table 3). Based on the fact that 2,3,5,6-TeCH has similar structure with 2,5-DiCH, the matrix would effect the extraction of 2,3,5,6-TeCH in the same way with 2,5-DiCH. The possible reason for bad recovery of 2,3,5,6-TeCH might be that some soluble enzymes produced by some microorganism in pond water made the dechloronation of 2,3,5,6-TeCH very fast.

  In this case, standard-addition should be applied to get the accurate concentration for 2,3,5,6-TeCH. 

Table 3: Determination of pondwater by SPME and GCâMS. 			

A rapid and simple method for the determination of five CPs and their two major metabolites in an aqueous sample has been developed using gaseous phase on-fibre silylation following SPME. It is sensitive and selective. An 85 µm of polyacrylate-coated fibre could be used to have the limit of detection (LOD) and the limit of quantification (LOQ) of target compounds range from 2.3 ×10

  µg/L respectively. This method has been shown to exhibit adequate precision and linearity. The method has been used to analyse CPs and their metabolites in pond water. The recoveries were satisfactory except for 2,3,5,6-TeCH.  

Financial support from the National Natural Science Foundation of China (NSFC, No. 20977116 and 40601090), Natural Scientific Foundation of Guangdong Province (No. 9151027501000114) and Program for New Century Excellent Talents in University are gratefully acknowledged. 

is associate professor at the Hygiene Detection Centre, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, China.  

is associate professor, School of Life Sciences, Sun Yatsen University, Guangzhou, China.  

is associate professor at the School of Marine Sciences, Sun Yat-sen University, Guangzhou, China. 

 is a lecturer at the School of Marine Sciences, Sun Yat-sen University, Guangzhou, China. 

is a bachelor of science student at the School of Life Sciences, Sun Yat-sen University, Guangzhou, China. 

 is a professor at the School of Life Sciences, School of Marine Sciences, Sun Yat-sen University, Guangzhou, China. 

Environmental Analytical Chemistry, Comprehensive Analytical Chemistry

, vol. XXXII, Elsevier, Amsterdam, 1999, pp.537. 

, October 26, 1984. Environmental Protection Agency, part VIII, 40 CFR part 136, 1981, p.58. 

Article

A method based on SPME and GC–MS was developed to analyse chlorophenols and their metabolites in aqueous samples.

Determination of Chlorophenols and their Degradation Metabolites in Environmental Samples by SPME and on-fibre Silylation with GC–MS

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For those seeking to develop new skills or learn more about chromatographic techniques, an attractive feature of Pittcon is the informative short courses. There are about 61 short courses associated with liquid chromatography and gas chromatography. Several of these short courses are new to Pittcon this year and include topics such as 

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New Concepts of TOF-MS for Fast GC and GC

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The Conference itself includes 53 invited symposia which showcase nationally and internationally recognized scientists. Some of the symposia of interest include: 					

Quality by Design in HPLC: The Balance Between Chromatography and Chemometrics

 presented by Loren Wrisley, Mike McBrien, Imre Molnar, Moheb Nasran and John Dolan will focus on the QbD steps related to chromatographic separations. It will include examples of the application of QbD, such as determining the most robust method, deciding on the best selectivity and transferring methods.

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s by Susan Richardson, Helene Budzinski, Brian Buckley, Rebecca Trenholm and Rebeca Pinhancos will discuss where and how pharmaceuticals and their metabolites/degradants enter the environment and how the newly introduced high resolution liquid chromatography, such as UHPLC, with high resolution mass spectrometers (Q-TOF and Orbitrap) and fast-scanning mass spectrometers (Q-TOF and Q-TRAP) allow the detection of pharmaceuticals present at trace levels in water. 

Advances in Microscale Liquid Chromatography

 by Barry Karger, Stephen Weber, J Michael Ramsey, Gert Desmet and Mary Wirth will cover recent advances in the application, theory and instrumentation for miniaturized liquid chromatography systems including capillary, microfluidics and monolithic columns.

New Enantiomeric Separation Approaches for the Pharmaceutical Industry

 by Daniel Armstrong, Christopher Welch, Antal Peter, Kyung Gahm and JT Lee will describe the newest chromatographic separation methods, including new chiral stationary phases, screening procedures, preparative separations and the separation of unusual chiral entities (such as nonprotenic amino acids).

As well as the invited symposia, there are an additional 200 papers that are related to chromatography in the organized contributed and poster sessions and workshops. The advance registration fee of $105 allows unlimited access to all parts of the technical programme as well as the exhibition. Pittcon also offers free one-day registration on 

 attracts nearly 20000 attendees from industry, academia and government from 90 countries worldwide. Each year thousands of scientists take advantage of the opportunity to see the latest in instrumentation all on one exhibition floor. At this year's Exposition, 1?000 exhibiting companies, including leaders in chromatography instrumentation and accessories, will be present at the Georgia World Congress Center from 

A complete list of exhibitors is available on the Pittcon website at 

Rhone Conference Centre, Amsterdam, The Netherlands

Dubai International  Convention and Exhibition Centre, Dubai, UAE

Budapest Congress and World Trade Centre, Budapest, Hungary

Departments

Event News

A simple, highly-precise non-invasive near-infrared spectroscopy (NIRS) method to characterize a wide range of stationary phases is described. This new approach produces results in a few seconds and offers significant advantages over conventional approaches.

The design of novel stationary phases — with enhanced selectivity, specificity and speed — is a constant challenge in chromatographic and electrophoretic separation science. Therefore, next to the calculation of chromatographic parameters, the characterization of chemical and physical properties of stationary phases is essential. Conventionally, column performance is checked by a test run which only allows users to prove efficiency column by column. Chemical parameters, including surface coverage, are normally analysed by burning combustion or frontal analysis. Physical parameters, including pore size, pore volume, surface area and particle size, are usually analysed by scanning electron microscopy (SEM), mercury intrusion porosimetry (MIP) and Brunauer Emmett Teller (BET). All these methods are extremely time consuming, invasive and require special equipment and specially trained lab staff. 

To achieve fast high-throughput control of the chemical and physical parameters of stationary phases we introduced near-infrared spectroscopy (NIRS, 4000–10000 cm

 ) as a non-invasive, easy-to-handle technology that achieves simultaneous analysis within only a few seconds at higher precision than traditional methods. This new approach is applicable to porous/non-porous materials, silica gel, polymer beads, carbon-based nanomaterials (e.g., fullerenes), monoliths for solid-phase extraction (SPE), liquid chromatography (LC) micro-liquid chromatography (µ-LC) materials and substances used for material enhanced laser desorption ionization (MELDI) and coated capillaries for capillary electrophoresis (CE) and capillary electrochromatography (CEC).  

The established NIRS technique not only enables identification of the crude carrier material but also the ability to control the success of any derivatization procedure — including reversed- phase, normal phase, ion exchanger, immobilized affinity chromatography (IMAC), affinity chromatography and endcapping methods — by applying principal component analysis (PCA) to the spectra recorded.  

Partial least square regression (PLSR) enables the determination of physical parameters, including porosity, total porosity, pore size, pore volume, particle size and surface area, with one single measurement in an established quantitative model. For a detailed investigation, NIRS imaging, which benefits from the combination of a NIR spectrometer with a microscope, allows the analysis of materials of interest with a lateral resolution down to 4 µm. 

For the optimized design of polymeric phases, real-time 

 monitoring to control the polymerization progress can be extremely helpful. In this article, the advantages of this novel NIRS-based method for the fast high-throughput quality control of stationary phases is described. 

In the late 1940s, near infrared spectroscopy (NIRS) became important in commercial applications in various industrial fields for analytical characterization and material analysis.

  The method became more popular in the eighties because of the availability of stand-alone NIR instruments offering enhanced reproducibility, new instrument accessories and statistical software. Improvements were made in imaging systems, light-fibre optics, optimized detectors and sample desk probes, alongside progress in miniaturization and the use of chemometrics to correlate the spectral data with physical and chemical properties of the sample.

  In- and on-line methods could be developed to perform kinetic studies and to control and monitor automated processes, including chemical

   NIRS spectroscopy is considered superior to mid-IR (MIR) for industrial applications because in the 4000–10000 cm

  range overtone and combination vibrations appear that are less intense than fundamental vibrations in mid-IR. This allows the use of much larger sample thicknesses. In the field of chemical and polymer research, NIR is mainly applied to control the polymerization and chemical processes of products with defined patterns and characteristics.

  Parameters such as porosity, anisotropy, composition, crystallinity, surface area, molecular weight, particle diameter, monomer content, intra-molecular interactions, etc., are investigated by characteristic absorptions, reflections and scattering effects in the NIR region. 

Silica gel derivatized with alkyl chains of different lengths (C4–C30) is the most frequently applied phase for thin layer chromatography (TLC), SPE and all kinds of LC in different disciplines, including proteomics, metabolomics and phytomics.

Nanomaterials based on carbon not only offer an excellent starting material for further derivatization but also offer a high ratio of surface to size and an enhanced capacity as a result. MELDI is a technique where human serum samples are incubated onto chemically (the type of derivatization) and physically (particle size, porosity, surface size) well-defined carrier materials. Following an optimized washing step, nonbound peptides are eluted and the remaining incubated material is put under investigation using matrix assisted laser desporption/ionization time-of-flight mass spectrometry (MALDI–TOF-MS). The resulting mass spectrum is used as a fingerprint to distinguish between different forms and/or stages of any illness, for example, prostate or breast cancer, using bioinformatics. 

In many cases polymeric materials are helpful in SPE

  but they have also attracted attention as a starting material in immobilized affinity chromatographic (IMAC)-based proteomics (MELDI) as well as as a reactor for enzymatic digestion.

Figure 1:  Investigated stationary phases and their application. 			

Monolithic capillary columns synthesized by copolymerizing bis(vinylphenylethane) and methylstyrene have been shown to be highly suitable for the separation of even low- (phenolic compounds, flavonoids) and high-molecular-weight analytes (peptides, proteins, oligonucleotides) by µ-LC.

  An overview of the different kinds of synthesized, stationary phases investigated in our laboratory and their application is provided in Figure 1. 			

In the following section the benefits of NIRS for the fast high-throughput characterization of chemical and physical parameters is discussed. 

Figure 2: PCR model for the classification of silica and different kinds of derivatization. 			

Silica is the main material used in all chromatographic-based separations

  and NIRS is an important technique to investigate its properties. A qualitative model based on principal component regression (PCR) allows clustering of materials with differences in particle size and/or surface area, pore structure and derivatization in a threedimensional factor plot. The basis for differentiation is found in corresponding overtone and combination stretching and deformation vibrations of C–H, N–H and O–H groups in the near-IR region (spectra not shown). As a selected example the cluster depicted in Figure 2 allows the classification of pure silica, silica-C18 and aminosilica.

  The sensitivity to differentiate between different particle size at consistent porosity and the sensitivity to differentiate between different porosity at consistent particle size was verified successfully in a detailed study described in reference 19. NIRS in combination with principal component analysis (PCA) was introduced to control success of endcapping procedures.

Figure 3: Predicted (NIRS) versus reference property for the determination of: (a) particle diameter; (b) specific surface area. 			

A quantitative approach enables the determination of surface coverage

  as well as particle size, pore structure and specific surface area and simultaneously.

  Particle size is routinely determined by electron microscopy, light scattering or the Coulter-Counter (CC) method. Recording 230 spectra of 23 different silica and silica-based phases allowed the establishment of a partial least square (PLS) model with higher precision compared with conventional applied methods. The CC method shows relative standard deviation of +15% to –12% and the novel NIR method is only 8.6%. The same is valid for the determination of surface area: BET analysis results in RSD% of ± 7.8% and NIRS of < 6.8%. Description of pore structure includes the determination of pore diameter and volume, which are routinely determined by size exclusion chromatography. Figure 3 shows the linear NIRS-based regression models for the quantitative determination of particle size and surface area. An impression upon the high efficiency of the novel NIRS- based method for the quantitative analysis of the parameters surface coverage, particle size, surface area and pore volume can be gathered from Table 1 which provides data for 18 selected silica phases. 

Table 1: NIR results on physical properties of investigated silica phases: Particle size, pore diameter, specific surface (18 selected examples). 			

Carbon-based Nanomaterials: C60-fullerenes

The suitability of NIRS as a derivatization control tool of modified nanomaterials was checked for C60fullerene samples. C60-fullerenes were oxidized to the corresponding epoxyfullerene and further covalently attached to amino-derivatized silica. The material proved to be highly efficient in desalting cell lysate samples and for the highly selective SPE of low-molecular weight plant ingredients.

  Each sample was scanned ten times in a wavenumber range from 4000–10000 cm

  — the final spectra is presented as an average. Figure 4 shows the final recorded NIR spectrum including an assignment of the most relevant vibrations identified. 

Figure 4: NIR spectrum of C60-fullerene covalently attached to amino-silica for SPE. 			

Polymer Beads: Poly(glycidyl methacrylate-co-divinylbenzene)

The potential to monitor on-line polymerization between glycidyl methacrylate (GMA) and divinylbenzene (DVB) by NIRS in diffuse reflection mode was successfully verified. In the first step, band assignment of glycidyl methacrylate, divinylbenzene and its copolymerized form will act as the basis for all further investigations. In this case, the most abundant vibration could be assigned to the C–H- and C=O-stretching vibrations at 4368 and 4524 cm

  originating from GMA, followed by a C–H stretching vibration at 8748 cm

  from DVB. In the following steps these vibrations were used to establish a qualitative cluster plot for the control of the monomer to crosslinker ratio in the resulting polymeric bead. In a quantitative model the monitoring of the on-line polymerization control was accomplished by measuring the signal intensities of the vibrations discussed above using light fibre optics outside of the reaction flask (Figure 5). Starting the reaction after 40 min, it took a further 140 min until the DVB core was formed. Addition of GMA after 180 min caused an immediate formation of poly(glycidyl methacrylate-co-divinylbenzene) particles, with increasing weight starting to sag to the ground of the reaction flask. After this, yield is increased and dispersion of particles from the ground caused an increased absorption. This model can also be used for the on-line measurement of other parameters including, for example, particle size and surface area (data not shown). The well characterized material is successfully applied in our laboratory for MELDI as well as a reactor for enzymatic digestion.

Figure 5: On-line monitored control of glycidyl methacrylate divinylbenzene copolymerisation. 			

Monoliths Copolymerized from Methylstyrene and Bis(vinylphenyl)ethane

Imaging spectroscopy with a resolution down to 4 µm can be successfully applied for the quality control (QC) of poly[methylstyrene-co-bis(vinylphenyl)ethane] monoliths in capillaries with an inner diameter of 200 µm.

  In our laboratory this technique was installed to control the ratio between monomer and crosslinker, which appeared to be extremely useful because the measurement is performed non-invasively and the same monolithic capillary can be used for LC separations. Recording spectra (average of 10 scans) in reflection mode over a wavenumber range from 4000–10000 cm

  of 1 = 39%, 2 = 40%, 3 = 41%, 4 = 42%, 5 = 35%, 6 = 43%, 7 = 38%, 8 = 36%, 9 = 37% monomer + crosslinker amount allows us to also calculate the ratio in a quantitative model. Simultaneosuly, the ratio of macro-, meso- and micro-pores can be determined by using mercury intrusion porosimetry (MIP) as a reference method (Figure 6) for the establishment of the highly selective model.

Figure 6: Resulting pore volume and pore area distributions by gradually increasing the MS/BVPE content. 			

Quality control of latex and fullerenol coated capillaries (inner diameter < 75 µm, thickness of coating < 0.1 µm) can be achieved by NIR imaging combined with multivariate data analysis in a similar approach as described in the previous chapter. Coating of the capillaries' inner wall is helpful in preventing irreversible protein adsorption during electrophoretic separation process.

  The interpretation of the resultant model gives information about the homogeneity of the applied chemical coating. Figure 7 shows a two dimensional factor plot for the differentiation between bare fused silica, latex and fullerenol derivatized. The corresponding image is depicted in the small insert. 

Figure 7: Two-dimensional factor plot for the identification of bare fuesed silica (FS), fullerenol (FU) and latex derivatized (LD) silica. The small insert shows the original recorded image. 			

The suitability of NIRS to simultaneously control chemical and physical properties of stationary phases including monoliths, silica particles, polymeric beads, coated capillaries and C60fullerene bound silica, was described. As measurement is extremely fast, this method has the potential for use in high-throughput control and should help to ensure quality. 

				The authors want to thank the Leopold-Franzens University, Innsbruck, Austria for financial support (Young Scientist Award 2007).			

 is associate professor at the Institute of Analytical Chemistry and Radiochemistry, University Innsbruck, Austria. The institute is headed by Professor Dr. Günther Bonn and has extensive experience in the design of novel stationary phases. In 1997, Christian Huck established a research group focusing on infraredspectroscopy (IR), which merges these two disciplines in a synergistic manner. The group has produced nine patents and more than 120 scientific contributions have been published in the fields of chromatography and/or spectroscopy, respectively. 

A non-invasive near-infrared spectroscopy method to characterize a wide range of stationary phases.

Fast High-Throughput Chemical and Physical Characterization of Stationary Phases by NIRS

In mass spectrometry (MS) the laser, together with electrospray ionization (ESI), has become an essential tool to advance our analytical pursuits in the sciences. The 50th anniversary of its invention marks an appropriate time to examine the device's importance in MS practice.

History credits the Hughes Research Lab's Theodore Maiman as the first to successfully operate a laser, in 1960. Since then, according to the LaserFest website, the US Patent office has granted more than 55000 patents involving the laser. 

The laser has become an important asset in mass spectrometry (MS). Indeed, its usefulness nearly rivals that of electrospray ionization (ESI) in the ability to examine the molecular underpinnings of biological systems. So it is fitting that we consider the laser from a perspective of MS practice, and speculate on its future. 

The Optical Society of America (OSA) and the American Physical Society (APS) are jointly promoting LaserFest (

), which they describe as "a multi-year celebration designed to commemorate the 50th anniversary of the invention of the laser." Toward that end, the partnering societies plan a range of activities including public outreach events, lectures, symposia and educational demonstrations. Many of the proceedings will recognize and honour the accomplishments of those who discovered, developed and applied laser technology. Among those memorialized will be Charles Townes, who figures prominently among the pioneers of laser technology. Townes developed the forerunner to the laser — the "maser" — and published, with Bell Laboratories' Arthur Schawlow, a key theoretical paper in 1958 that led to the laser's commercial development. In 1960, Townes and Schawlow were jointly awarded the first laser patent. 

The four types of lasers are gas, solid, liquid, and semiconductor. 

Low-cost, helium-neon (HeNe) lasers are ubiquitous in the educational setting and emit energy at a variety of wavelengths. The typical low-cost units operate at 633 nm and are common in education. The HeNe mixture — the gain medium — is pressurized so that helium = 1 Torr and neon = 0.1 Torr. The gas mixtures contained within a glass tube of 1 cm diameter and 0.25–1 m length. Two electrodes connect to a high-voltage DC source that generates a discharge inside the tube acting like a "pump". Two parallel mirrors are placed around the gain medium, one in front of the other, so that only mirror 1 shows the complete reflection. Mirror 2 shows a partial reflection. When electric current passes through, a continuous light wave flows inside the tube with constant frequency, developing "coherent" light waves that emanate from mirror 2. 

MS systems often trigger ionization by means of a nitrogen laser. Others, though, like the solid-state diode YAG lasers (neodymium-doped ytterbium aluminum garnet), are also used. A chemical matrix protects the molecule of interest from destruction by direct laser energy and enables vaporization and ionization. Essentially a chromophore, the matrix absorbs laser energy, which, liberated from the surface (where both matrix and analyte have been applied), then transfers or assists ionization of the analyte. 

Other examples of gas lasers include higher efficiency, carbon dioxide units that emit high-power beams at about 10 µm. Such devices are often are used in industry for cutting and welding. Argon-ion lasers emit light in the 351–528.7 nm range. Depending upon the associated optics and type of laser tube, a variable number of lines are usable. Nevertheless, the most frequently used lines are 458 nm, 488 nm and 514.5 nm. Metal-ion lasers such as helium-silver (HeAg, 224 nm) and neon-copper (NeCu, 248 nm) generate deep, ultraviolet wavelengths. The very narrow oscillation line-widths of such lasers make them useful for fluorescence-suppressed Raman spectroscopy. 

In solid lasers, a ruby-like cylindrical crystal serves as the gain medium, which is surrounded by a helical xenon flash lamp that acts as a "pump". Mirrors are arranged similar to those of gas lasers. A red laser beam results on the discharge of electric current. 

Liquid lasers use organic dyes as their gain medium. In semiconductor lasers, known as injection laser diodes (ILD), current causes visible light modulation from the ILD. These lasers are often used in electronic equipment. 

Karas and Hillenkamp introduced matrix-assisted laser desorption ionization (MALDI) in 1985.

  Along with electrospray ionization, MALDI has dramatically affected the way we investigate biomolecules, particularly proteins. Initially used to analyse intact proteins, MALDI is now widely used to analyse peptides that result from protein digestion. (Enzymatic protein digestion, a standard procedure in proteomics, is a means for identifying proteins by reducing the larger molecule into significant subsets.) MALDI's production of singly charged ions, which simplifies spectral interpretation, is also a prominent advantage for synthetic polymer analysis. 

MALDI is well represented at the annual American Society for Mass Spectrometry (ASMS) each year, where about 6000 attendees bring about 3000 talks and posters. In 2010, there were 499 focused on MALDI. The numbers were slightly larger in past years (748 in 2004 or 30% of all abstracts submitted to ASMS that year), but MALDI clearly remains a significant identifiable technique. 

A "soft" ionization technique (relative to electron ionization, for instance), MALDI especially benefits analyses of biomolecules and biopolymers (proteins, peptides and sugars) and other large organic molecules that tend to fragment when desorbed and ionized by conventional ionization methods. MALDI is similar to ESI in that both are soft techniques that are less likely to damage a molecule of interest. Both ESI and MALDI conserve charge and display good sensitivity. Yet MALDI yields many fewer and less abundant multiply charged ions; and electrospray, considered the more quantitative of the two techniques, produces more fragmentation from the multiply charged ions when appropriate activation is supplied after the ionization step.

Three of MALDI's shortcomings become evident when the technique is applied to intact proteins and protein complexes. Because of possible source and analyser combinations for ESI and MALDI sources, the peaks of singly charged ions produced by MALDI are broader than the multiply charged, equivalent ions produced by ESI. Most limiting for the protein analysis using high performance mass spectrometers equipped with powerful analysers, however, is the mass range limitations: singly charged protein ions are simply not detected. Fragmenting singly charged ions is also more problematic, limiting structure determination from enzymatically digested proteins (or small proteins within the mass range of the instrument). Broad peaks make accurate mass determination difficult, and important subsequent steps like database searching yield only limited success. MALDI's other shortcoming is that a practitioner must find a suitable sample preparation method (Figure 1). Although MALDI generally requires little, if any, exotic sample preparation, it is sometimes difficult to find a matrix with properties that yield ions from solution complexes. Often, a peak for an intact, specific complex is observable only for the first application onto a fresh sample spot. Though a topic of some debate, it seems that if the same area is irradiated again, only dissociation products or nonspecific clusters are observed.

 Figure 1: The matrix-assisted laser desorption ionization (MALDI) traditional solvent-based process as developed by Karas and Hillenkamp, with possible limitations at each step. (Courtesy S. Trimpin, Wayne State University.)			

  has shown that, contrary to conventional wisdom, using standard MALDI conditions can produce multiply charged ions similar to those obtained using ESI. A few references in the literature describe multiply charged ions similar to those created by ESI using a commercial, atmospheric pressure (AP-MALDI) infrared (IR) neodymium:yttrium aluminum garnet (Nd:YAG) laser to ablate an acidiﬁed glycerol matrix.

David Muddiman's group (North Carolina State University, Raleigh, North Carolina, USA) developed a technique referred to as liquid matrix-assisted laser desorption electrospray ionization (liq-MALDESI), which generates multiply charged ions from liquid samples deposited onto a stainless steel sample target biased at a high potential. Muddiman says: "We have observed a singly charged radical cation of an electrochemically active species indicating oxidation occurs for analytes and therefore water; the latter would play a key role in the mechanism of ionization".

Trimpin showed that UV nitrogen laser ablation of standard MALDI matrixes at atmosphere could produce multiply charged ions similar to ESI, with high sensitivity without an applied voltage. Calling the method laserspray ionization (LSI) distinguishes it from AP-MALDI, in which highly charged ions are not observable from solid matrices. In the initial work, the laser is shot through the sample holder in transmission mode (Figure 2). The matrix–analyte was placed in solution onto a glass microscope slide using the "dried droplet" method described by Karras

  in which the sample is allowed to dry and is then ablated using transmission geometry, in which the laser beam impinges the matrix from the bottom after passing through a glass microscope slide. Recent work shows the method can be applied using conventional reflective geometry on a commercial AP-MALDI source design as well.

  A major difference between LSI and other methods of producing multiply charged ions is that laser ablation occurs directly from a solid matrix and no voltage is required. 

Figure 2: Laserspray ionization (LSI) schematic. In transmission geometry, laser ablation of the solid matrix/analyte sample occurs from the backside without the use of a voltage. In reflective geometry, the laser hits from the front. The transfer capillary, the region in which the matrix is removed from the highly charged matrix/analyte cluster, is crucial to produce ESI-like multiply charged LSI ions. (Courtesy S. Trimpin, Wayne State University.)			

Forward momentum of charged matrix–analyte molten droplets is generated by the deposition of energy into the matrix by the laser pulse. The result propels the highly charged droplets sufficiently close to the MS ion entrance where they come under the influence of the vacuum and are transported with the flow. Just as in ESI, evaporation of the solvent (matrix) in the charged droplets is necessary to produce highly charged analyte ions. 

According to McEwen, "The charge state can be switched to singly charged ions nearly instantaneously by changing the voltage applied to the MALDI target plate." Under normal, atmospheric MALDI operating conditions, a voltage is applied to the target plate, resulting in the observance of singly charged ions. The authors found that "At or near zero volts, highly charged ions are observed for peptides and proteins. Thus, switching between singly and multiply charged ions requires only the manipulation of a single voltage."  

One of the great benefits of ESI as a technique — multiple charging that allows direct measurement of analytes well beyond the usual single-charge acquisition limits of the hardware — is thus achievable using the LSI source not achievable with AP-MALDI. 

Trimpin focuses on resolving the shortcomings of MS laser applications: "Over the years, mass spectrometry has been a powerful analytical tool and a breakthrough in biological research. MS has been less successful in characterizing materials with restricted solubility and materials not easily ionized, whether due to complex composition or the desire to detect unadulterated or unmodified compounds." 

When coupled with recent hybrid techniques like ion mobility, the LSI approach makes even more sense because it adds enhanced spatial resolution to the mix. Apart from its specific advantage of allowing a practitioner to choose between charge states (single versus multiple), LSI offers MALDI-like advantages: laser for high-spatial resolution, ability to work solvent-free for insoluble materials and speed. It also brings ESI-like advantages: working at atmospheric pressure, extended mass range of high performance mass spectrometers, and improved fragmentation (for example, electron-transfer dissociation, or ETD).

The concluding part of the series on the role of system suitability tests in the pharmaceutical industry.

LCGC Europe-12-01-2010

Selectivity in reversed-phase LC Separations (Part 1): Solvent-type Selectivity

Determination of Chlorophenols and their Degradation Metabolites in Environmental Samples by SPME and on-fibre Silylation with GC–MS

Event News

Fast High-Throughput Chemical and Physical Characterization of Stationary Phases by NIRS

The Year of the Laser

Why System Suitability Tests Are Not A Substitute for Analytical Instrument Qualification or Calibration (Part 2)