How Reversed-Phase Liquid Chromatography Works Mark R

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January 2014 Volume 27 Number 1 www.chromatographyonline.com

Expand Your Horizons A look at emerging sample preparation technologies

LC TROUBLESHOOTING GC CONNECTIONS PITTCON PREVIEW What is the point of the column The origins of GC carrier gases A look to Chicago 2014 dead time?

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Volume 27 Number 1 COVER STORY 37 SAMPLE PREPARATION PERSPECTIVES Electrical Potential as a Driving Force in Sample Preparation Ronald E. Majors Entirely new sample preparation technologies continue to be introduced, mainly in the academic sector. Some of these technologies will undoubtedly stay in the academic laboratory. However, some new technologies may “cross the chasm” and eventually become a standard laboratory procedure. This instalment will examine some of those methods.

Features 8 Determination of Sulphur-based Odourants in Commercially Available Natural Gas with Flow Modulated Comprehensive Two-Dimensional Gas Chromatography Taylor Hayward, Ronda Gras, and Jim Luong Pulsed-ﬂ ow-modulated comprehensive two-dimensional gas chromatography with ﬂ ame ionization detection was used to separate and identify all common sulphur compounds found in natural gas.

18 How Reversed-Phase Liquid Chromatography Works Mark R. Schure, Jake L. Rafferty, Ling Zhang, and J. Ilja Siepmann This tutorial on reversed-phase LC explains the role of solvent, chain conformation, solute position, and retention dynamics.

43 Pittcon 2014 Preview A look at what’s in store for chromatographers at Pittcon 2014, which will be held at McCormick Place South, Chicago, USA, from 2–6 March 2014. Columns 28 LC TROUBLESHOOTING Column Dead Time as a Diagnostic Tool John W. Dolan What good is that big, ugly peak at the beginning of the chromatogram?

33 GC CONNECTIONS The Origins of GC Carrier Gases: Putting a Genie in the Bottle John V. Hinshaw This month’s instalment tracks the progress of helium, hydrogen, and nitrogen carrier gases as they start their journey through a GC system.

50 THE ESSENTIALS Optimizing LC–MS and LC–MS–MS Methods Secondary parameters in the interface and mass analyzer can often Editorial Policy: have a major impact on sensitivity and reproducibility. This column All articles submitted to LC•GC Europe considers how and when to consider optimizing these parameters are subject to a peer-review process in association through a study of the working principles of LC–MS analysis. with the magazine’s Editorial Advisory Board.

Cover: Departments Original materials courtesy: Westend61 45 Products 48 Events

4 LC•GC Europe January 2014

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Taylor Hayward, Ronda Gras, and Jim Luong, Dow Chemical Canada, Analytical Technology Center, Alberta, Canada.

Alkyl mercaptans, alkyl sulphides, and cyclic sulphides are added in various blends to commercial natural gas as a safety precaution to identify its presence by smell. Although several different analytical methods exist to measure these compounds, few are all-encompassing techniques that can measure all the sulphur compounds simultaneously at appropriate levels. Pulsed-flow- modulated comprehensive two-dimensional gas chromatography with flame ionization detection was used to separate and identify all common sulphur compounds found in natural gas. The column set used in this method consisted of a polydimethylsiloxane-like stationary-phase selectivity in the first dimension with a low-bleed wax stationary phase in the second dimension. This set was optimized to separate key sulphur compounds found in natural gas from this hydrocarbon matrix. Detection limits of this method were determined to be 0.1 ppm (v/v), and a linear calibration range spanning from 0.1 ppm to 30 ppm (v/v) was used to perform quantitative analysis on a residential natural gas supply. Reproducibility of peak volume of seven successive injections on these test analytes averaged 3.7%.

Natural gas is highly flammable, colourless, and require an odourant to be detectable by smell when odourless; therefore malodorous compounds are added natural gas concentrations reach one-fifth of the lower as a method to identify the presence of this gas. Most explosive limit, which represents a level of about 1.25% in commonly, these compounds consist of a blend of alkyl air (Federal Regulation, 49 CFR, 192.625). Most sulphur mercaptans, alkyl sulphides, or cyclic sulphides (1,2). odourants have an odour threshold of approximately These structures are prominent because of their desirable 1 ppb (3), and therefore are minimally required to be physical and chemical characteristics. For example, at concentrations of 100 ppb in natural gas. Generally, the blends need to be odorous, volatile, flammable commercially available residential natural gas contains liquids that have a low odour threshold and will not oxidize metal pipelines (1,2). The most common sulphur compounds used as odourants are tert-butyl mercaptan, tetrahydrothiophene, methylethyl sulphide, dimethyl sulphide, isopropyl mercaptan, n-propyl mercaptan, and KEY POINTS sec-butyl mercaptan. Two or more of these compounds • A method to determine key odourants in natural gas are added to natural gas before it is delivered to the was developed. consumer (1,2), and depending on environmental or • GC×GC with pneumatically controlled modulation was economical factors, they will be blended in various used to separate sulphur compounds from each other combinations and percentages. as well as from a carbon matrix. The ability to monitor these odourant blends in all their • The use of a modulator increased signal detectability variant forms is beneficial to natural gas providers to and detection levels as low as 100 ppb for each certify their presence at detectable levels by smell and sulphur compound were achieved. ensure consumer safety. North American regulations

8 LC•GC Europe January 2014

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detection (FPD) (7–10), sulphur chemiluminescence Figure 1: Single-dimensional chromatogram of a 1-mL natural detection (SCD) (11–13), mass spectrometry (MS) (14), or gas injection (blue trace) overlaid with sulphur standard mix ion mobility spectrometry (15). The use of FPD requires (red trace). The analytical conditions are described in the text. the sulphur compounds of interest to be well separated Modulation was deactivated. Standard peaks: 1 = dimethyl from the hydrocarbons in the matrix to reduce the effects sulphide (7.22 ppm [v/v]), 2 = isopropyl mercaptan (5.67 ppm of quenching (16–18), which involves multiple switching [v/v]), 3 = tert-butyl mercaptan (4.71 ppm [v/v]), 4 = n-propyl valves. SCD affords great selectivity of sulphur response mercaptan (5.86 ppm [v/v]), 5 = methylethyl sulphide over carbon with low detection limits, but has a higher (5.87 ppm [v/v]), 6 = sec-butyl mercaptan (4.88 ppm [v/v]), cost of ownership. Furthermore, this necessitates 7 = tetrahydrothiophene (6.02 ppm [v/v]). equipment specifically dedicated to sulphur detection. 50 MS in selected ion monitoring mode can also be used; however, if there is insufficient separation between 40 7 the analyte of interest and the interference there is a 30 possibility of a false positive. For example, in selected ion monitoring mode an abundance of ions can cause peak 20 6 Signal (PA) 4 broadening because of electrostatic repulsions within a 3 5 10 1 2 cloud of similarly charged ions (19). Ultimately, this could

0 offset the centroid of the sulphur species of interest or 0 5 10 15 20 25 these coeluted hydrocarbons may overlap and create a Time (min) false positive sulphur response (19). Comprehensive two-dimensional gas chromatography (GC×GC) is an emerging technique that uses the Figure 2: Raw chromatogram of modulated sulphur standard separation power of two columns with different selectivity gas mix. The inset shows a magnified view of the tert-butyl (20–22). All analytes in this method undergo separation mercaptan peak. The chromatographic conditions are by both columns, and the process is facilitated by a described in the text. Standard peaks: 1 = dimethyl sulphide rapid re-injection from a modulator. Flow modulation was (0.90 ppm [v/v]), 2 = isopropyl mercaptan (0.71 ppm [v/v]), first described by Seeley (23) and Amirav (24). Basic 3 = tert-butyl mercaptan (0.59 ppm [v/v]), 4 = n-propyl operation of modulation includes effluent from the first mercaptan (0.73 ppm [v/v]), 5 = methylethyl sulphide column filling a collection channel within the plate, and (0.73 ppm [v/v]), 6 = sec-butyl mercaptan (0.61 ppm [v/v]), then that effluent is rapidly injected into the second 7 = tetrahydrothiophene (0.75 ppm [v/v]). column by switching flow through the channel creating a pulsed flow. Not only does the separation power increase 18 in GC×GC, but signals are enhanced as modulation creates sharp narrow peaks that elute off the second

6 column. GC×GC has been coupled to selective detection 10 4 5 3 1 2 systems such as FPD and SCD to analyze sulphur

Signal (PA) species in complex matrices (25–27). Here, GC×GC is used to measure prominent sulphur odourants found in natural gas in one method. Utilization of this technique 0 10 12 14 16 18 20 can be beneficial to separate sulphur compounds of Time (min) interest from the hydrocarbon matrix as well as to identify the species added to a natural gas sample. Furthermore, signal enhancement from modulation allows detection sulphur odourants with 0.5–10 ppm levels. At these at low levels meaning that these sulphur species can be levels, natural gas can be readily detected by smell at monitored with flame ionization detection (FID) over an concentrations well below the lower explosive limit. It is appropriate range. also necessary to ensure that these odourants are not overdosed, which can lead to further expense to the Experimental natural gas supplier. Excessive additions in natural gas Chromatographic Conditions: A model 7890 gas can cost companies millions of dollars in unnecessary chromatograph (Agilent Technologies) was used for chemical cost as well as investigations because of false development of this method and was equipped with positive alarms reported by consumers. Higher levels of two split–splitless inlets, two flame ionization detectors, sulphur species within the distribution pipelines may also and a CFT GC×GC flow modulator. Flow was delivered corrode these pipes and create potentially hazardous to the modulator by a pneumatically controlled module leaks. with the system running in constant flow mode. Raw Currently, odourants in natural gas are monitored chromatograms were collected using Chemstation by various sensors such as olfactory detection (ASTM software version B.04.03 (Agilent) and were then D6273-08) and lead acetate strip tests (4–6). Gas deconvoluted to produce the 3D colour plots using GC chromatography (GC) has also been demonstrated Image software version 2.2 (Zoex Corporation). to detect these compounds, which usually involves a A 1-mL gas sample was injected manually into the selective detection method such as flame photometric split–splitless inlet at 275 °C. The system was operated

10 LC•GC Europe January 2014

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Table 1: Limit of detection for the prominent sulphur compounds in natural gas with and without modulation. Compound 2 mL/min 4 mL/min

Concentration (ppb [v/v]) Signal/Noise(p-p) Concentration (ppb [v/v]) Signal/Noise(p-p) Dimethyl sulphide 113 11.4 726 2.5

Isopropyl mercaptan 89 10.8 570 2.4

tert-butyl mercaptan 74 15.3 473 3.3

n-propyl mercaptan 92 13.6 589 3.2

Methylethyl sulphide 92 22.6 590 3.3

sec-butyl mercaptan 77 13.3 491 3.1

Tetrahydrothiophene 95 27.4 605 3.9

Table 2: Reproducibility of peak volume, first- and Figure 3: Colour plot of sulphur standard gas mixture second-dimension retention time on seven successive approaching detection limits. The concentrations are as injections of a standard mixture of the prominent sulphur described in Table 1, and the chromatographic conditions are compounds in natural gas. as described in the text.

Compound % Relative Standard Deviation 2 Concentration (ppb [v/v]) Peak Retention Time Tetrahydrothiophene Volume n First Second 1.5 -Propyl mercaptan Dimethyl sulphide Dimension Dimension Methyl ethyl sulphide Time (s) Time Dimethyl sulphide 726 2.4 4.2 × 10-4 0.67 sec-Butyl mercaptan 1 Isopropyl mercaptan 570 4.1 0.0 0.49 Isopropyl mercaptan tert-Butyl mercaptan tert-butyl mercaptan 473 3.7 4.5 × 10-4 0.73 6 8 10 12 14 16 18 20 n-propyl mercaptan 589 1.7 4.0 × 10-4 0.51 Time (min)

Methylethyl sulphide 590 3.4 3.9 × 10-4 4.3 × 10-4 methylethyl sulphide, and tetrahydrothiophene, which sec -butyl mercaptan 491 5.0 0.0 0.63 were obtained from Sigma-Aldrich. Natural gas samples Tetrahydrothiophene 605 5.4 2.3 × 10-4 0.22 were supplied by Direct Energy.

Results in split mode with a split ratio of 2:1. The inlet liner used GC×GC Conditions and Optimization was an ultra-inert liner (Agilent) in an effort to maintain Column Set Selection: Many methods used to analyze minimum activity of the sulphur analyte with the liner sulphur compounds in natural gas suppress matrix surface. Hydrogen (Praxair) was used as the carrier gas interferences by means of selective detection. With at a rate of 1 mL/min through the first column, controlled GC×GC, separation is maximized by the use of two by electronic pressure control, and 22 mL/min through columns with orthogonal selectivity. Column selection the second column, delivered from the pneumatically is a vital component in optimizing the separation controlled module. An oven temperature programme capabilities, which in this method is used to separate the was used that began at an initial temperature of 40 °C sulphur compounds of interest in the first dimension and for 2 min, then programmed to 250 °C at a rate of 5 °C/ then separate these compounds from the matrix by the min. The FID system was maintained at 250 °C with an second-dimension column. additional 20 mL/min of hydrogen to the carrier flow rate, To separate the sulphur analytes in the first-dimension 350 mL/min of air (Praxair), and 30 mL/min of nitrogen column, a nonpolar, low beta ratio (β) stationary phase makeup gas (Praxair). The modulation period was was chosen because it offers advantages of inertness, optimized to be 4.0 s, with a fill time of 3.70 s and an good separation of low retention factor (k) solutes, and inject time of 0.30 s. high diffusivity. Recently, in collaboration with Restek, a The first-dimension (1D) column had dimensions of low β value, inert stationary phase with selectivity similar 30 m × 0.250 mm with a film thickness of approximately to PDMS was developed. In using a flow modulator it is 3.0 µm. The stationary phase was a base-deactivated advised to use a maximum column internal diameter of 100% polydimethylsiloxane (PDMS) (Restek). The 0.250 mm to avoid excessive pressure pulsing. Thus, the second-dimension (2D) column was a 5 m × 0.250 mm, first-dimension column was made to a β equal to 21 with d 1.0-µm f Agilent VF-WAXms column. a film thickness of approximately 3.0 µm and an internal Chemicals: The sulphur test analytes used in this study diameter of 0.250 mm. were dimethyl sulphide, isopropyl mercaptan, n-propyl The first-dimension column is utilized to separate the mercaptan, tert-butyl mercaptan, sec-butyl mercaptan, sulphur compounds of interest. The analytes spend

12 LC•GC Europe January 2014

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Figure 4: Sulphur standard gas mixture spiked into Figure 5: Natural gas sample taken from a residential a natural gas matrix. Concentrations of each sulphur source with identified sulphur components labeled. component: Dimethyl sulphide (0.90 ppm [v/v]), isopropyl mercaptan (0.71 ppm [v/v]), tert-butyl mercaptan (0.59 ppm [v/v]), n-propyl mercaptan (0.73 ppm 2 [v/v]), methylethyl sulphide (0.73 ppm [v/v]), sec-butyl mercaptan (0.61 ppm [v/v]), and tetrahydrothiophene 1.5 (0.75 ppm [v/v]). The chromatographic conditions are Methyl ethyl sulphide Time (s) Time described in the text. tert-Butyl mercaptan

1 Methane Ethane n-Propane n-Butane n-Pentanen-Hexane 2 4 8 12 16 20 24 Time (min) Tetrahydrothiophene n-Propyl mercaptan Methyl ethyl sulphide 1.5 Dimethyl sulphide sec-Butyl mercaptan

Time (s) Time as the peaks eluted out of the second-dimension column Isopropyl mercaptan tert-Butyl mercaptan Methane are compressed with the higher carrier velocities. 1 Ethane The modulation period was developed and optimized to n-Propane n n-Hexane n-Butane -Pentane be 4.0 s, where the modulation number was at least three, 2 4 8 12 16 20 24 Time (min) meaning that the peak eluted from the first-dimension column was modulated at least three times. This results in accurate representation of the peak, while effectively the most time interacting with this phase, therefore maximizing the signal intensity (30,31). A sample of the it will have the most influence in the separation. raw chromatographic sulphur peaks are displayed in Single-dimension separations of these sulphur Figure 2. Flush and fill times of the modulation chamber compounds with this column produce chromatograms were adjusted to obtain Gaussian peaks eluted from with Gaussian peaks that are well resolved; among the second-dimension column, which are essential for a hydrocarbon matrix such as natural gas, however, accurate volume determination in the colour plots that are coelutions occur that make detection and identification used for quantitation. of these sulphur compounds difficult. Figure 1 shows Figures of Merit: Calibration was performed using single-dimension chromatograms of natural gas overlaid test analytes of each of the sulphur compounds. Linearity with a gas mixture of the sulphur compounds ranging is observed over a range of 0.1–30 ppm (v/v), which is at approximately 5 ppm (v/v). These concentrations representative for sulphur compounds typically found represent upper limits of these species that would be in natural gas (1,2). Correlation coefficients of each added to natural gas. While compounds like isopropyl test analyte are all above 0.99. Recoveries of these mercaptan and tetrahydrothiophene are separated from sulphur compounds spiked into a natural gas matrix at hydrocarbons in natural gas, the remaining sulphur approximately 0.7 ppm (v/v) were all above 95%. compounds show either full or partial coelution. To Detection limits were obtained by measuring the signal analyze these compounds in a conventional GC provided by the tallest peak in the raw chromatographic method, selective detection is required. However, data and then ratioed to the peak-to-peak (p-p) noise. sulphur-selective detectors only provide information At the 100 ppb (v/v) level, the signal-to-noise ratio on the sulphur species present, and as previously (p-p) was calculated to be approximately 10–27 for mentioned, the coelutions present could pose a problem each sulphur compound. Table 1 displays the minimum for quantitation with MS in selective ion monitoring detectable concentration for each of the sulphur mode. However, in GC×GC, the second dimension is compounds tested. A colour plot of this mixture is shown utilized to separate the sulphur analytes from the matrix in Figure 3. The well formed peaks seen in this colour plot and detection is provided by FID; therefore all species were achieved by deconvoluting the raw chromatograms present can be separated, monitored, and quantified. (Figure 2), after which volumes can be determined for The second-dimension stationary phase was quantitation. Also shown in Table 1 are the minimum determined to be a wax phase with a film thickness of detectable concentrations and signal-to-noise ratios 1.0 µm (β = 63). Previous work demonstrated selectivity obtained without modulation demonstrating minimum that retains methyl mercaptan, which is eluted near detectable concentrations approximately six times heptane (28). Thus, the selectivity of the wax stationary larger at lower signal-to-noise values. These results phase will have more retention of the sulphur analytes demonstrate the improved detectability when using over the hydrocarbon matrix, effectively providing the modulation, which leads to the possibility of detecting necessary separation in the second dimension. sulphur species at the lower limits required by federal Modulation Optimization: The column flow rates regulations. through the first and second dimension were 1 mL/min Reproducibility of seven successive injections at and 22 mL/min, respectively. These values are typical concentrations ranging from 475 ppb to 730 ppb (v/v) using a modulator to achieve optimally modulated peaks is displayed in Table 2. As shown, the percent relative of the effluent coming off of the first dimension (23,29). standard deviation (%RSD) spans from 1.7% to 5.4%, with Furthermore, these rates achieve a signal enhancement an overall average of 3.7%. Table 2 shows %RSD values

14 LC•GC Europe January 2014

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determined on retention times in the first and second column used in this method and the members of the dimension, which demonstrate excellent reproducibility Analytical Technology Center, particularly Vicki Carter, with %RSD values less than 1% in all analytes tested over for their help and support. Funding for this project was a period of four weeks. Overall, the chromatographic made possible from contributions by Alberta Innovates conditions are reliable and robust and retention Technology Futures and the Dow Analytical Technology time marking can be used to identify these sulphur Center Technology Renewal and Development 2012 compounds when present in natural gas. Programme. Figure 4 shows these sulphur compounds spiked into a natural gas matrix at concentrations of about References 0.6 ppm (v/v). Target analytes are well separated from (1) M.J. Usher, in Odour Fade – Possible Causes and Remedies the hydrocarbon matrix where coelutions occurred in (Elf Atochem North America, Inc., Philadelphia, Pennsylvania, USA, 1999), pp. 1–12. single-dimension analysis (Figure 1). Natural gas contains (2) D. Tenkrat, T. Hlincik, and O. Prokes, in Natural Gas, P. Potocnik, hydrocarbons beyond hexane, and the first sulphur Ed. (Sciyo/InTech, Rijeka, Croatia, 2010), pp. 87–103. species of interest are eluted near pentane. This method (3) F.A. Fazzalari, Ed. Compilation of Odour and Taste Threshold may then be extended to analyze odourants found Data (ASTM Data Series DS 48A, West Conshohocken, Pennsylvania, USA, 1978), pp. 1–508. in propane that is distributed and used in residential (4) P.D. Wehnert, in Determination of Proper Odourization of Natural households, as propane contains similar odourants used Gas (Proceedings of the International School of Hydrocarbon for identification of its presence. Measurement, 2002), pp. 747–750. (5) A.V. Litvinov, P.O. Unchenko, and I.N. Nikolaev, Meas. Tech. 50, Sulphur Compounds in Natural Gas: A residential 548–550 (2007). sample of natural gas was obtained by sampling a (6) K.-H. Kim, Sensors 11, 1405–1417 (2011). natural gas line into a Tedlar bag and injecting it into (7) R.L. Firor and B.D. Quimby, Hydrocarbon Process. 82, 79–81 × (2003). the GC GC system. The resulting chromatogram is (8) L. Huber and H. Obbens, J. Chromatogr. 349, 465–468 shown in Figure 5. Retention time identifies a blend of (1985). tert-butyl mercaptan and methylethyl sulphide in natural (9) J. Macak, J. Kubat, V. Dobal, and J. Mizera, J. Chromatogr. 286, gas. The concentrations of the species (based on their 69–78 (1984). (10) D.C. Pearson, J. Chromatogr. Sci. 14, 1521–1528 (1976). peak volumes) were found to be 2 ppm and 0.7 ppm (11) R. Shearer, D. O’Neal, R. Rois, and M. Baker, J. Chromatogr. (v/v), respectively by external calibration. This represents Sci. 28, 24–28 (1990). a blend of approximately 75:25 tert-butyl mercaptan to (12) H.P. Tuan, H.-G. Janssen, and C.A. Cramers, J. High Resol. Chromatogr. 18, 333–342 (1995). methylethyl sulphide, which is a common combination (13) H.P. Tuan, H.-G. Janssen, E.M. Kuiper-van Loo, and H. Vlap, J. (1,2). High Resol. Chromatogr. 18, 525–534 (1995). The ease of determining the sulphur compounds in (14) S.-W. Myung, S. Huh, J. Kim, Y. Kim, M. Kim, Y. Kim, W. Kim, this matrix should be further noted. The sample was and B. Kim, J. Chromatogr. A 791, 367–370, (1997). (15) J. Luong, R. Gras, R. Van Meulebroeck, F. Sutherland, and H. taken right from the natural gas line and directly injected Cortes, J. Chromatogr. Sci. 44, 276–282 (2006). into the GC×GC system, and the use of retention time (16) L. Kalontarov, H. Jing, A. Amirav, and S. Cheskis, J. templates allowed for the rapid identification of the Chromatogr. A 696, 245–256 (1995). (17) W.A. Aue and X.-Y. Sun, J. Chromatogr. A 641, 291–299 (1993). sulphur compounds present. This method can be used (18) M. Dressler, J. Chromatogr. A 270, 145–150 (1983). to analyze sulphur compounds in natural gas from any (19) R.E. Pedder, Application Note RA_2011A, Extrel CMS L.P., provider as it simultaneously detects all prominent Pittsburgh, Pennsylvania, USA (2002). (20) P.J. Marriott, S.-T. Chin, B. Maikhunthod, H.G. Schmarr, and S. sulphur species. Furthermore, not only does this Bieri, TrAC 34, 1–21 (2012). technique allow for detection of the sulphur compounds (21) M. Adahchour, J. Beens, R.J.J. Vreuls, and U.A.Th. Brinkman, present, but also all of the hydrocarbons in the same TrAC 25, 438–454 (2006). analysis. This method may be very beneficial as a (22) M. Adahchour, J. Beens, R.J.J. Vreuls, and U.A.Th. Brinkman, TrAC 25, 726–741 (2006). complementary technique to using a selective detection (23) J.V. Seeley, N.J. Micyuc, J.D. McCurry, and S.K. Seeley, method such as SCD or MS. American Laboratory 38, 24–26 (2006). (24) M. Poliak, M, Kochman, and A. Amirav, J. Chromatogr. A 1186, 189–195 (2008). Conclusion (25) S.-T. Chin, Z.-Y. Wu, P.D. Morrison, and P.J. Marriott, Anal. A method has been developed to analyze key odourants Methods 2, 243–253 (2010). found in commercially available natural gas by GC×GC (26) J. Blomberg, T. Riemersma, M. van Zuijlen, and H. Chaasbani, with pneumatically controlled modulation. This technique J. Chromatogr. A 1050, 77–84 (2004). (27) R. Hua, J. Wang, H. Kong, J. Liu, X. Lu, and G. Xu, J. Sep. Sci. uses the separation power of GC×GC to separate 27, 691–698 (2004). sulphur compounds from each other as well as from (28) R. Gras, J. Luong, V. Carter, L. Sieben, and H. Cortes, J. a hydrocarbon matrix. Furthermore, because this Chromatogr. A 1216, 2776–2782 (2009). (29) P.McA. Harvey and R.A. Shellie, J. Chromatogr. A 1218, technique uses a modulator, increased signal 3153–3158 (2011). detectability is achieved that allows for quantitation (30) W. Khummueng, J. Harynuk, and P.J. Marriott, Anal. Chem. 78, at levels as low as 100 ppb (v/v) of each sulphur 4578–4587 (2006). (31) P.J. Marriott and W. Khummueng, LCGC Europe 22, 1–12 compound. This method allows for direct injection of a (2009). natural gas sample for identification and quantitation of odourants added. Taylor Hayward, Ronda Gras, and Jim Luong are Acknowledgements with Dow Chemical Canada, in the Analytical Technology The authors would like to thank Jaap de Zeeuw (Restek) Center in Fort Saskatchewan, Alberta, Canada. Direct for his contribution of the low beta ratio, first-dimension correspondence should go to: [email protected]

16 LC•GC Europe January 2014

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magentablackcyanyellow ES374224_LCE0114_017_FP.pgs 01.08.2014 23:35 ADV How Reversed-Phase Liquid Chromatography Works

Mark R. Schure1, Jake L. Rafferty2,3, Ling Zhang2, and J. Ilja Siepmann2,4, 1Superon and Theoretical Separation Science Laboratory, Kroungold Analytical, Inc., Blue Bell, Pennsylvania, USA; 2Department of Chemistry and Chemical Theory Center, University of Minnesota, Minneapolis, Minnesota, USA; 3Department of Chemistry, North Hennepin Community College, Brooklyn Park, Minnesota, USA; 4Department of Chemical Engineering and Materials Science, University of Minnesota, USA.

The keys to understanding reversed-phase liquid chromatography (LC) are provided at the molecular mechanism level as determined by high accuracy molecular simulation. The essential features of C18 stationary-phase chains in contact with methanol–water and acetonitrile–water mixtures are discussed in the context of bonded-chain geometry, spatial distribution of alkane and alcohol solutes, retention mechanism, and retention thermodynamics. This tutorial is intended to be applicable to a wide audience ranging from occasional users of liquid chromatography to separation scientists.

The technique referred to as reversed-phase liquid magnetic resonance (NMR), infrared (IR), and fluorescence chromatography (LC) is the workhorse of all LC techniques. spectroscopy detect bonded‑phase chain conformations Continued improvements in columns, instrumentation, and and solute associations for a large number of molecules application methodologies have led to reversed‑phase simultaneously; these conformations and interactions are LC being used in more laboratories and delivering results complex, the signals are difficult to decompose into single faster, with higher resolution. We confine the scope of our molecule information, and widely diverse signals lead to discussion of reversed‑phase LC to column materials that spectroscopic broadening and loss of resolution. Hence, contain a bonded‑phase layer of a hydrophobic material, reversed‑phase LC retention mechanisms have not been usually dimethyl octadecylsilane (C18), bound to a porous silica revealed by these techniques. Some spectroscopic techniques support. The C18 chains function as the retentive material, have given outstanding structural information, for example, yet details of chain conformation, how the solutes of interest determining the distribution and types of silanol groups on interact with the chains, where the solvent lies in proximity to silica and revealing the silica bonding details of derivatizing the chains, and the thermodynamics of this process have been agents by solid‑state NMR spectroscopy (1). lacking for many years. It is ironic that with the high popularity We have learned a great deal about the reversed‑phase of this technique, the details of the retention mechanism LC system through the use of high‑accuracy molecular have been so difficult to obtain. The purpose of this tutorial simulations (2–4). Simulation offers a unique methodology to is to enlighten readers about the detailed inner workings of understand disordered and microheterogeneous systems and reversed‑phase LC. this has been the primary technique used for many years in the For many years researchers believed they could figure study of liquids (5,6). Simulation is necessary because of the out the mechanism of reversed‑phase LC by examining the complexity from the large number of simultaneous interactions pattern of retention times produced by injecting different that take place (the so‑called “n‑body problem” of liquids). but chemically related solutes into columns with different These types of problems cannot be understood or formulated stationary‑phase chemistries using a range of mobile‑phase mathematically unless drastic simplifications are made. These compositions. This is a fallacy because retention time simplifications render such theories of limited use. With the experiments cannot give a microscopic picture of the advent of high‑speed computers and advances in simulation bonded‑phase chains and inference of the structure of these chains is largely circumstantial. The retention time experiment is thermodynamic in nature — it is determined by the distribution of the solute between the mobile‑phase solvent system and the stationary phase. This distribution is KEY POINTS the subject of what is often referred to as “phase equilibria.” • High accuracy molecular simulation was used to No microscopic mechanism can be gleaned from data examine the mechanism of reversed‑phase liquid of this type (that is, thermodynamic data). In essence, chromatography. thermodynamics is just a bookkeeping system, nothing more • High amounts of organic modifier in the solvent lead to and nothing less. swelling of the chains, but its effect is subtle. Spectroscopic investigations have also been inconclusive • Further insights into solvent chain confirmation, solute and have not revealed details of the retention mechanism. position, and retention thermodynamics are described. This is because spectroscopic techniques like nuclear

18 LC•GC Europe January 2014

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Figure 1: Left: Snapshots from simulations in different Figure 2: Snapshots illustrating the structural effects of

solvents, C18 chains (grey), and alkane and alcohol solutes grafting density. The stationary phase is depicted as tubes (large spheres with green, red, and white indicating CHx, with CHx groups in grey, hydrogen in white, oxygen in hydroxyl oxygen, and hydrogen, respectively). Middle and orange, and silicon in yellow. Solvent molecules are shown

right: Density proﬁles of C18 chains, methanol, water, and in the ball and stick representation with oxygen in red, CH3 acetonitrile for pure water (W), pure methanol (M), pure groups in blue, and hydrogen in white. The silicon substrate acetonitrile (A), and solvent mixtures given as mole percent. atoms deﬁne the position z = 0 Å. The average end‑to‑ T = 323 K, grafting density of 2.9 µmol/m2. Adapted, in part, end distance of the chains, as a function of coverage, is from references 4 and 14. shown in grey on the side of each molecular representation as a level plot. The methanol mole fraction is 0.5 (0.69 v:v 1.2 W Total methanol:water) and T = 323 K. Adapted from reference 15. 0.8 C18 Acetonitrile W Methanol 30 0.4 Water 20 zGDS 0 33M 33A 10 z (Å) 0.8 0 33M 0.4 1.6 µmol/m2 2.3 µmol/m2 2.9 µmol/m2 3.5 µmol/m2 4.2 µmol/m2

0 67M 67A

p ( z ) (g/mL) 0.8 (7). Alternatively, this effect has been described by Walter and 67M 0.4 colleagues (8) as a dewetting process. Partial dewetting has been confirmed by molecular simulation (9), in which it was 0 M A shown that the chains do not collapse on the surface when run 0.8 under pure water conditions. We have found, in fact, that the M 0.4 probability of collapse of octadecane molecules in pure water is very small and the solvent composition does little to affect 0 0 10 20 100 20 30 the conformation of C chains (10). 0 10 20 30 18 z (Å) z (Å) z (Å) The density profiles of C18 chains bound to silica are shown graphically in Figure 1, for seven solvent systems. These are labelled as pure water (W), pure methanol (M), pure methodology, this method of investigation has become acetonitrile (A), and the systems 33% methanol in water (33M), more practical and has now evolved to the point where 67% methanol in water (67M), 33% acetonitrile in water (33A), simulation can reproduce the energetics of reversed‑phase and 67% acetonitrile in water (67A). In the four cases of the LC accurately. Since simulation gives mechanical details mixed solvent systems, these percentages are given as the of reversed‑phase LC on an atomic scale (that is, chain mole percent of the organic modifier, as opposed to the usual conformation, solute and solvent locations) the mechanism volume‑to‑volume designation. We prefer mole percent or the of the reversed‑phase LC process can be revealed using mole fraction because it is independent of temperature and simulation methodology. As we will show, the comparison pressure. For these simulations at 323 K, the 33M solvent of simulation with experiment is impressive and provides is 53% methanol by volume. The 67M is 82% methanol by an assurance that these computational results for model volume, the 33A solvent is 60% acetonitrile by volume, and the systems carry a sufficient amount of realism to learn about real 67A solvent is 87% acetonitrile by volume. chromatographic systems. On the left in Figure 1, snapshots of C18 chains are shown In this tutorial we will guide readers through a number of our for various methanol concentrations. The results for acetonitrile simulation findings that give the microscopic details needed are similar. The independent variable z shows the distance in to understand reversed‑phase LC. This is not only useful for angstroms from the site of chain attachment at the outermost chromatographers in understanding practical separations, but it silicon atom of the silica surface (at z = 0 Å) and perpendicular may also prove useful towards designing new stationary phases to the surface. We will use this distance repeatedly throughout for liquid‑based separations other than reversed‑phase LC. A this tutorial. In all of these cases, the chain conformation detailed description of the simulation methodology, the bonding is highly disordered but with a preference to stand roughly procedure, and further details are given in two review articles perpendicular to the surface, as will be shown in the following (3,4). We refer readers to these references and others given here figures. For the model systems shown here the silica support is for the detailed workings of this methodology. assumed to be a planar substrate; simulations for a cylindrical pore yield behaviour in good qualitative agreement with the Structure of the Bonded-Phase Chains and Solvent planar substrate (11). Location As shown on the right of Figure 1, the solvent penetration The starting point for discussing reversed‑phase LC is the into the bonded‑phase region is not extensive. Water and chain conformation. This point has been contentious because methanol are found hydrogen‑bonded to free silanol groups of the observation that when reversed‑phase LC columns are at the silica surface (peaks at z = 3 and 4 Å for water and run in 100% aqueous solvent and subsequently depressurized, methanol, respectively). From both of the snapshots on the retention is diminished drastically and this has been thought to left and from the seven average density graphs, it is shown be caused by chains collapsing and lying flat on the surface that very little water is found where the carbon density of

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the C chains is high. For the pure water simulation (W), it Figure 3: Local methanol mole fraction (x ) relative to 18 local appears that the water does not reach its liquid density until the bulk solvent mole fraction (x ), as a function of the bulk ≈3 Å (that is, about one molecular diameter) away from the distance from the silica substrate. This graph shows the ends of the C chains. Looking at the red curves (the water enhanced methanol concentration within the chain region for 18 density) and the black curves (the C chain density) it can different surface coverages. Conditions are the same as for 18 be seen for all of the solvent systems where water is present data in Figure 2. The dashed vertical lines are the GDS. The that there is a small distance between the chain ends and the methanol mole fraction is 0.5 (69% methanol by volume) and z value where bulk water density is established. This space T = 323 K. Adapted from reference 15. is caused by the lack of hydrogen bonding capability when water is near a hydrophobic interface. This space represents 3 1.6 µmol/m2 a low water density region characteristic of the lack of surface wetting. In all cases shown in Figure 1, the penetration of 2.3 µmol/m2 organic modifier into the chain region increases as the solvent 2 2.9 µmol/m concentration of organic modifier increases. At low organic 3.5 µmol/m2 modifier concentrations (33M and 33A), there is a significant 2 4.2 µmol/m2 enrichment in the organic modifier concentration at the interface between bulk solvent and the chain region. We will

bulk address this effect in more detail below, noting this excess has / x

local been measured experimentally (12,13). x In Figure 1, the grey zone indicates the width of the 1 chain‑solvent interface that covers 80% (from 10% to 90%) of the change in solvent density. The orange dotted line is the Gibbs dividing surface (GDS), a plane that represents the location of the chain‑solvent interface where the solvent density is half‑way between the bulk and interior solvent density, as determined by an interpolation procedure we have used 0 0 10 20 30 extensively (3,4). The GDS, as used here, represents a simple z (Å) (but artificial) plane defining the border between mobile and stationary phases.

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Figure 4: Distribution coefﬁcient proﬁles, K(z), for n‑butane Figure 5: Incremental retention free energies of the (left) and 1‑propanol (right) solutes in various mobile phase methylene and hydroxyl groups as a function of location for T = solvents. The stationary phase consists of C18 chains with different solvent compositions. Conditions are 323 K, surface coverage of 2.9 µmol/m2 and T = 323 K. The dashed surface coverage of 2.9 µmol/m2. Energies are given in joules orange lines are the GDS and the shaded grey regions give (1 kcal = 4.184 kJ). Adapted from references 2 and 14. the 10–90% liquid region. Adapted from reference 14. 2.5 20 W 2400 120 33A W 67A 0 A Butane 0 33M (kJ/mol) (kJ/mol)

1600 80 Propanol 2 67M OH CH -2.5 67M Z ∆ G GDS ∆ G 800 40 -5 -20 0 10 20 30 0 10 20 30 0 6 z (Å) z (Å) 33A 33M

60 4 envisioned the bonded chains to stick out from the surface. As 30 2 shown previously through simulation (15), the chain geometry is dependent on surface coverage as illustrated in Figure 2. 0 0 For relatively low coverage, the chains can lean over but K ( z ) 67A 67M K ( z ) are generally oriented away from the surface. As coverage 10 2 increases, the chain extension is driven by excluded volume and the chain backbone, for parts close to the surface, 5 1 orients approximately perpendicular to the surface. At all surface coverages, the chain ends show a relatively random 0 0 A M arrangement. At medium coverage, the C18 chains at carbon numbers 2 2 above about half of the chain length orient rather randomly. However, at the highest coverage shown in Figure 2, 1 1 the chains are more oriented towards an approximately perpendicular configuration. 0 0 0 10 20 0 10 20 30 The average end‑to‑end distances (the distance from z z (Å) (Å) the first carbon to the last carbon in the C18 chain) for these surface coverages are 10.2 Å, 11.6 Å, 11.6 Å, 12.6 Å, and 14.9 Å, respectively. These results indicate that the chain As can be seen from the graphs in Figure 1, the location of the orientation and average end‑to‑end distance are highly GDS depends only weakly on solvent composition, although the dependent on coverage. These numbers compare favourably GDS is slightly closer to the silica surface under pure methanol with the value of 17 ± 3 Å obtained by neutron scattering (16) composition. The width of the chain‑solvent interface is larger in pure methanol and with C18 that was bonded to silica under as the organic modifier concentration increases. This is slightly maximum coverage conditions (16). more pronounced for methanol than for acetonitrile. Overall, this Notice in Figure 2 that at the lowest coverage the solvent has behaviour shows that the organic modifier concentration is more much easier access to the surface silanols than at the highest diffuse within the chain‑solution interfacial region as the organic chain coverage. In all cases, a gap between the solvent modifier concentration is increased. and the chains exists. This is a consequence of the limited The chain conformation, as implied from these density solubility of water and the organic modifier in the C18 chains. plots, shows rather subtle effects from changes in the solvent This interfacial dewetting behaviour is rather universal where environment. This is shown in Figure 1 in which the density hydrophobic chains meet hydrophilic liquids. of the C18 chains, as a function of distance from the silica As mentioned previously, for smaller organic modifier surface, extends further from the silica surface with increasing concentrations, an excess of the organic modifier is found organic modifier concentration. This suggests that the chains in the interfacial region between bulk solvent and the C18 stretch or swell with higher modifier concentration although the chains. This is illustrated in Figure 3 where the excess in the effect is subtle. relative modifier concentration is reached progressively farther Overall, it appears from Figure 1 that very little solvent is away from the silica surface for higher chain densities. For present in the interior of the chain system for highly aqueous the highest chain density, almost twice the concentration of mobile phases and there are simply no solvent molecules methanol is obtained in the chain region as compared to the to displace the solute. The solute, therefore, has little to no bulk solvent composition. competition with the solvent for interaction sites within the chain interior region. Solute Molecule Spatial Location One of the many questions that have been asked about Chain Coverage, Tilt, and End-to-End Distance reversed‑phase LC is whether solute molecules adsorb Many researchers have speculated that the chains are on or partition into the chain system. In Figure 4 we show folded on the surface of the support material and others have the distance‑dependent distribution coefficient, K(z), for

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Figure 6: Schematic of the thermodynamic cycle used to It appears that n‑butane exhibits both a partitioning decompose the contributions to retention. behaviour (the first K[z] peak at 7.5 Å) and an adsorption behaviour where the solute lies near the chain‑solution Gas phase interface. The orientation of the molecules can be determined using metrics such as the orientational order parameter -∆G ∆G mobile Stationary (3,4) which shows that the n‑butane solute within the chain interior region orients preferentially perpendicular to the n silica surface, whereas the ‑butane above the C18 chains orients preferentially parallel to the surface in an adsorbed Mobile phase Stationary phase configuration near the chain‑solution interface. Hence, G ∆ retention hydrophobic solutes act with both partitioning (within the chains) and adsorption (on the chain’s outer surface) retention mechanisms. As shown in Figure 4, higher organic modifier n‑butane and 1‑propanol solutes in seven solvent systems. concentrations push the location of the adsorbed n‑butane to K(z) is the ratio of the average number density of solute higher z locations within the more diffuse interfacial region. The molecules in the stationary phase to the average number mean location of the partitioning n‑butane peak increases only density of solute molecules in the mobile phase. Higher K(z) slightly with increasing organic modifier concentration. values indicate regions of higher solute retention. Averages The preferred location of the 1‑propanol solute clearly are important here because this number varies as molecules indicates adsorption at the solvent–C18 interface for the solvent move between the mobile and stationary phase. The conditions of pure water and for small concentrations of the simulation technique we use (3,4) to compute these results organic modifier. As the concentration of organic modifier is gives these numbers directly. increased, the location of the 1‑propanol solute becomes more As can be seen from Figure 4, there are two maxima for diffuse and part of this is because of the increased penetration of K(z) of n‑butane for all of the solvent systems investigated the organic modifier into the chains. For the mixed solvent cases, here. These maxima occur within the chain interior (z ≈ 7.5 Å) the preferred location of the 1‑propanol solute is shifted to larger and below the GDS. We use n‑butane as a prototypical z values, that is, towards the region where enrichment of the hydrophobic alkane solute with no hydrophilic groups and organic component is found at the chain–solvent interface. 1‑propanol as a model solute with a hydrophilic group (the For the pure acetonitrile solvent, significant amounts of alcohol group) and some hydrophobic character. 1‑propanol are found near the substrate. This is because of

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to surface silanols or to solvent molecules that are bound to Figure 7: Computed and experimental incremental transfer the silanols. The second region (z ≈16 Å) is found only for free energies of a methylene group (top) and a hydroxyl pure water where the solute can form a hydrogen bond to group (bottom) for methanol‑water and acetonitrile‑water the partially dewetted solvent. Note that the magnitude of the solvent mixtures. The retentive phases include dimethyl incremental free energies is much larger for hydroxyl groups octadecyl silane (ODS or C ) bound to silica and bulk liquid 18 that can form hydrogen‑bonds than the solute methylene group n‑hexadecane (C ). The C surface coverage is 2.9 µmol/ 16 18 that favours lipophilic interactions with C chains. Also note m2, T = 323 K. The simulation data are from our laboratory 18 that the interactions of hydroxyl groups with chain elements is and are described in a number of papers (2–4,18). The quite thermodynamically unfavourable, as denoted by the large experimental data are taken from Carr and colleagues (19), positive free energies in Figure 5. For C stationary phases and Barman (20), and Alvarez‑Zepeda (21). 18 those formed by longer ligands, both adsorption and partition play a role for nonpolar solutes, whereas adsorption is always W 1 the major mechanism for solute molecules with polar groups.

Vapour 0 Infuence of the Mobile-Phase Composition on

(g/mL) 33M

2 -1 33A Retention Thermodynamics CH 67M 67A

∆ G Few topics in reversed‑phase LC have caused more -2 M A confusion than the retention mechanism and the subsequent ODS ODS ODS ODS ODS ODS C16 -3 C16 ODS C16 C16 C16 C16 C16 thermodynamic analysis. Early attempts at a quantitative description of reversed‑phase LC retention, such as the Vapour 0 solvophobic theory of Horváth and colleagues (17), failed for C16 C16 C16 C16 C16 C16 C16 the same reason that a quantitative theory of liquids failed: The

(g/mL) -10 2 ODS complexity of the liquid state prevents a simple description of

OH ODS ODS ODS ODS ODS A

∆ G liquid chromatography. -20 67A M ODS 67M 33A 33M Simulation For the thermodynamic cycle used to analyze the retention Carr et al. W -30 Barman et al. Alvarez-Zepeda et al. mechanism, an ideal gas reference state is included that is assigned a free energy of transfer equal to zero because there are no intermolecular interactions in this state. This cycle is direct hydrogen bonding of 1‑propanol to unreacted silanols; given schematically in Figure 6. the split into two peaks in Figure 4 reflects different orientations The solute can be in any one of the three states: In the mobile of the 1‑propanol solute because of the solute being either phase, the stationary phase, or in the reference (gas phase) the acceptor or donor in the hydrogen bond formed with a state. To measure the contribution of the solvent one considers residual silanol and due to different extents of steric crowding moving the solute from the ideal gas reference state to the from the surrounding methyl side chains of the C18 ligands. mobile phase. To measure the contribution of the C18 chains, The hydroxyl group on 1‑propanol is a much more effective the surface silanols, and the embedded solvent, one considers hydrogen bond former than acetonitrile, which is only a weak moving the solute from the ideal gas reference state to the hydrogen bond acceptor. It should be noted that for pure stationary phase. The retention process is then described by methanol solvent, most of the available silanols form hydrogen ΔG = ΔG − ΔG bonds to the solvent and, hence, there is no peak near the retention stationary mobile [1] substrate for 1‑propanol. The K(z) plots for a homologous series can be converted This equation and the thermodynamic cycle indicate that the to an incremental free energy diagram, as shown in Figure 5 net free energy of solute retention, that is, moving a solute (2–4). The incremental free energy of a methylene group and from the mobile phase to the stationary phase is the difference a hydroxyl group are free energy metrics that facilitate easy between the free energy of insertion from the gas phase to the comparison with experiment. This is because the phase ratio, mobile phase and the insertion from the gas phase into the the ratio of mobile‑phase volume to stationary phase volume, stationary phase. is most difficult to accurately measure in an experiment but is The complete thermodynamic cycle is shown in Figure 7 with not needed for comparison of incremental free energies. free energies expressed as the incremental free energies of As can be seen from Figure 5, the methylene groups exhibit methylene and hydroxyl groups, as described and used above. n the most favourable interactions within the C18 interior region; that We also include a bulk retentive phase of liquid ‑hexadecane is, negative free energies indicate more “favourable” interactions as a purely liquid–liquid partitioning process for comparison. than with the bulk solvent (and higher probability of occurrence). Here is how to interpret the data illustrated in Figure 7. As Incremental free energies of zero indicate locations that do discussed with Figure 5, any solute movement towards more not contribute to retention and free energies greater than zero negative free energies is referred to as a favourable transfer. (referred to as “unfavourable”) show a depletion of solute groups However, as with any thermodynamic event, the time scale and are less likely to occur. At z ≈ 12 Å, where adsorption occurs, of transfer is not specified. In all of these cases, only the the methylene groups are slightly less energetically retained transfer of a methylene group from an ideal gas to pure water and this is likely because of the higher solvent density which is unfavourable; free energy has to be expended to put a permeates the chains at the interfacial region. methylene group into water. In this one case the transfer is For the hydroxyl group, the incremental free energies are deemed solvophobic. In all other cases the methylene group negative in two regions: The first at low z values where the prefers the mobile phase and the favourability increases solute hydroxyl group forms a hydrogen‑bond either directly from 33% methanol–water and 33% acetonitrile–water (all by

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mole fraction) to pure methanol and pure acetonitrile. These Solvent: interactions are deemed solvophilic. • Highly water‑rich solvent mixtures form a nonwetting gap For all solvent compositions studied, lipophilic interactions between the chains and the bulk fluid. • of methylene groups with the C18 (ODS) stationary phase drive Water, methanol, and, to a lesser extent, acetonitrile occupy the retention. This is indicated by the arrow going downward the residual silanols. (showing the favourable nature of the interaction) that connects • The organic component is enriched in the interfacial the solute group in the solvent phase to the solute group in the region between chains and the bulk fluid. This enrichment ODS phase. is dependent on organic modifier concentration (more Note that the free energy of transfer of a methylene group enrichment at lower organic modifier) and chain density from the gas phase to the retentive phase does not vary much (higher enrichment at higher chain density). with mobile‑phase composition. However, the decrease in retention as the organic modifier concentration is increased Chain Conformation: is driven by an increase in the solvophilic interactions with the • Chains are conformationally disordered with segments solvent. Although the free energy transfer of a methylene group closer to the chain termini showing more disorder. With into ODS (that is, C18) and C16 are very similar in magnitude, increasing surface coverage, the disorder is reduced this is somewhat coincidental because the mechanisms and the chains align more perpendicular to the surface. are different (4). Again, this highlights the danger of using • Higher amounts of organic modifier in the solvent lead to thermodynamics to imply a retention mechanism. swelling of the chains, but the effect is subtle. For the transfer of a hydroxyl group into the mobile and stationary phases from the gas phase, it is found in Figure 7 that Solute Position: all transfers are favourable. In all cases, however, the transfer • Alkane‑like solutes can be found within the chain system of the hydroxyl group from the mobile phase into the stationary (partitioning) and in the interfacial region of the chains phase is unfavourable (that is, the arrows in Figure 7 are upward (adsorption) for both acetonitrile–water and methanol– facing). As shown in Figure 7, it is energetically more favourable water mixtures. These solutes are found predominately to transfer a hydroxyl group into the mobile phase than the perpendicular to the surface deep within the C18 chains stationary phase. This point is echoed in Figure 5 where the and lying preferentially parallel to the surface within the incremental free energy, as a function of distance, shows that interfacial region. the free energy of a hydroxyl group is only favourable (that is, • Solutes with hydroxyl groups (such as alcohols) are ΔG negative OH) close to the substrate because of hydrogen preferentially located closer to the interface of the C18 bonding with silanol groups, whereas it is unfavourable for most chains and the solvent. This allows for enhanced hydrogen of the C18 chain region. The magnitudes of these free energies bonding to occur with solvent systems containing water. are such that addition of a hydroxyl group reduces retention by a larger amount than addition of a methylene group increases Retention Thermodynamics: • retention in C18. For alkanes, the driving force for retention is lipophilic. As shown in Figure 7, simulation and experiment give very Solvophobic interactions are only pertinent for solvent systems similar results. This is a tribute to the simulation methodology with very high water concentration. However, the decrease in (2–4), which has evolved to be the leading simulation retention upon increase in the organic modifier concentration methodology for research in phase equilibria studies for some is mostly because of an increase of the solvophilic interactions. time (22). For any simulation to be believed it must agree • For hydroxyl groups, the strongest interaction is solvophilic, with experiment. Simulation has enabled a profound, and that is, it is more favourable for a hydroxyl group to be in the in many cases surprising, picture into the inner‑workings of solvent region than in the stationary phase. The difference in reversed‑phase LC that was not available by other methods. the favourable interactions with the solvent and the stationary We have studied other stationary phases and solutes than phase decreases with increase of the organic modifier those discussed here, for example, embedded polar groups concentration. (23), studies with bare silica (24) used in hydrophilic interaction • The magnitude of the hydroxyl incremental free energy is chromatography (HILIC), and retention of polycyclic aromatic much larger than for the methylene group — it takes many hydrocarbons (PAH) (25) amongst others. In addition, we methylene groups to balance the effect of one hydroxyl group. have studied the effects of pressure, chain length, and solute length (11) and a host of other variables pertinent to liquid We welcome any feedback from readers that will help us chromatography. Other research groups have used molecular clarify, focus, and expand this work. dynamics simulations to probe structure and dynamics of related chromatography systems including chiral stationary phases Acknowledgements (26), adsorption of acridine orange at the reversed‑phase LC We are grateful to Pete Carr for many stimulating chain‑solvent interface (27), and solvent mobility at bare silica discussions and to Jack Kirkland for his helpful comments surfaces (28). One of the many lessons learned is that retention on this article. Financial support from the National Science is highly dependent on the chemical nature of the solute and Foundation (CHE‑0718383 and CHE‑1152998) is gratefully this makes reversed‑phase LC both useful and fascinating at the acknowledged. Part of the computer resources were provided same time. by the Minnesota Supercomputing Institute.

Conclusions References (1) D.W. Sindorf and G.E. Maciel, J. Am. Chem. Soc. 105, 3767–3776 (1983). The key insights for the reversed‑phase LC system of C18 chains (2) J.L. Rafferty, L. Zhang, J.I. Siepmann, and M.R. Schure, Anal. Chem. bound to silica as determined by molecular simulation include: 79, 6551–6558 (2007). 26 LC•GC Europe January 2014

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(3) J.L. Rafferty, J.I. Siepmann, and M.R. Schure, Adv. in Mixtures in Reversed‑Phase Liquid Chromatography,” PhD thesis, Chromatography, E. Grushka and N. Grinberg, Eds. (CRC Press, Georgetown University, 1991. Boca Raton, Florida, USA, Vol. 48, 2009), pp. 1–53. (22) J.I. Siepmann, S. Karaborni, and B. Smit, Nature 365, 330–332 (4) R.K. Lindsey, J.L. Rafferty, B.L. Eggimann, J.I. Siepmann, and M.R. (1993). Schure, J. Chromatogr. A 1287, 60–82 (2013). (23) J.L. Rafferty, J.I. Siepmann, and M.R. Schure, Anal. Chem. 80, (5) M.P. Allen and D.J. Tildesley, Molecular Simulation of Liquids (Oxford 6214–6221 (2008). University Press, Oxford, England, 1989). (24) J.L. Rafferty, J.I. Siepmann, and M.R. Schure, J. Chromatogr. A 1223, (6) D. Frenkel and B. Smit, Understanding Molecular Simulation, 2nd Ed. 24–34 (2012). (Academic Press, New York, USA, 2001). (25) J.L. Rafferty, J.I. Siepmann, and M.R. Schure, J. Chromatogr. A 1218, (7) M. Przybyciel and R.E. Majors, LCGC North Am. 20(6), 516–523 9183–9193 (2011). (2002). (26) R. Arjumand, I.I. Ebralidze, M. Ashtari, J. Stryuk, and N.M. Cann, J. (8) T.H. Walter, P. Iraneta, and M. Capparella, J. Chromatogr. A 1075, Phys. Chem C 117, 4131–4140 (2013). 177–183 (2005). (27) A. Fouqueau, M. Meuwly, and R.J. Bemish, J. Phys. Chem. B 111, (9) L. Zhang, L. Sun, J.I. Siepmann, and M.R. Schure, J. Chromatogr. A 10208–10216 (2007). 1079, 127–135 (2005). (28) S.M. Melnikov, A. Hötzel, A. Seidel‑Morgenstern, and U. Tallarek, J. (10) L. Sun, J.I. Siepmann, and M.R. Schure, J. Phys. Chem. B 110, Phys. Chem. C 117, 6620–6631 (2013). 10519–10525 (2006). (11) J.L. Rafferty, J.I. Siepmann, and M.R. Schure, J. Chromatogr. A 1216, Mark R. Schure is with Superon and the Theoretical 2320–2331 (2009). Separation Science Laboratory at Kroungold Analytical, Inc., in (12) D. Westerlund and A. Theodorsen, J. Chromatogr. 144, 27–37 (1977). (13) F. Gritti, Y.V. Kazakevich, and G. Guiochon, J. Chromatogr. A 1169, Blue Bell, Pennsylvania, USA. 111–124 (2007). Jake L. Rafferty was a graduate student and postdoctoral (14) J.L. Rafferty, J.I. Siepmann, and M.R. Schure, J. Chromatogr. A 1218, researcher with the Department of Chemistry and Chemical 2203–2213 (2011). Theory Center at the University of Minnesota in Minneapolis, (15) J.L. Rafferty, J.I. Siepmann, and M.R. Schure, J. Chromatogr. A 1204, 11–19 (2008). Minnesota, USA, and is with the Department of Chemistry (16) L.C. Sander, C.J. Glinka, and S.A. Wise, Anal. Chem. 62, 1099–1101 at North Hennepin Community College in Brooklyn Park, (1990). Minnesota, USA. (17) C. Horváth, W. Melander, and I. Molnár, J. Chromatogr. 125, 129–156 (1976). Ling Zhang was a graduate student with the Department of (18) J.L. Rafferty, L. Sun, J.I. Siepmann, and M.R. Schure, Fluid Phase Chemistry and Chemical Theory Center at the University of Equilib. 290, 25–35 (2010). Minnesota, USA. (19) R.P.J. Ratatunga and P.W. Carr, Anal. Chem. 72, 5679–5692 (2000). J. Ilja Siepmann is with the Department of Chemistry and (20) B.N. Barman, “A Thermodynamic Investigation of Retention and Selectivity in Reversed‑Phase Liquid Chromatographic Systems,” Chemical Theory Center and the Department of Chemical PhD thesis, Georgetown University, 1985. Engineering and Materials Science at the University of (21) A. Alvarez‑Zapeda, “A Thermodynamic Study of Acetonitrile–Water Minnesota, USA.

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Column Dead Time as a Diagnostic Tool

John W. Dolan, Walnut Creek, LC Resources, California, USA.

What good is that big, ugly peak at the beginning of the chromatogram?

Often considered a necessary evil, large, off-scale peak (Figure 1[b]). 150 mm = 1.5 mL. For columns of the first peak in a chromatogram Although there are more exact other internal diameters, you can use t can be a useful diagnostic measurement techniques for 0, V ≈ Ld 2 tool for troubleshooting liquid such as injection of D2O, most of M 0.5 c /1000 [2] chromatographic (LC) separations. us just use the retention time of the d Most people I encounter refer to peak. I prefer to pick a measurement where c is the column internal this as the column dead time peak, that is easy to reproduce, because diameter in millimetres. For a 50 mm t t × V × × abbreviated 0. However, it has a most of the time an estimate of 0 2.1 mm column, M ≈ 0.5 50 wide variety of other names: Junk is sufficient. For Figure 1(a), this is 2.12/1000 = 0.11 mL. Either of peak, garbage peak, solvent front, the point the disturbance crosses these estimates is good to within t or hold-up time, with M as the most the baseline, noted by the arrow. approximately ±10% for columns common alternative abbreviation. Because a large unretained peak packed with totally porous particles. This represents the time it takes usually is off scale so that the top These estimates are based on the V something to go through the LC of the peak may be inconvenient to assumption that M represents ~65% column that does not interact with locate, I usually pick the point where of the volume of an empty column the column. A corresponding dead the peak rises from the baseline and that about half of this volume V volume (or hold-up volume), M, is (arrow in Figure 1[b]). Of course is inside the particles and half is the volume of mobile phase inside the retention time reported by the between the particles. the column. This volume comprises data system is another convenient The column dead time is simply the both the volume of mobile phase measurement of the dead time. column volume divided by the flow between the packing particles (the rate, F (in millilitres per minute): interstitial volume) and the volume Often considered a t = V F within the particles (the pore volume). 0 M/ [3] t necessary evil, the ﬁ rst We’ll see that 0 can be a useful diagnostic tool to identify potential peak in a chromatogram Using t0 as a Diagnostic Tool t problems with an LC method. can be a useful diagnostic I regularly use 0 to help diagnose tool for troubleshooting problems submitted to me by Measuring t0 readers. Here are some of the ways If we want to use the column dead liquid chromatographic this can be useful. time as a tool, we need to be able (LC) separations. Verify the Unretained Peak: I find to identify it. Most LC detectors that it is useful to check to be sure t t will generate a peak at 0, the To confirm a measured value of that the presumed 0 peak is in the t most obvious exception being 0 or to determine it if there is no right place. For this, simply calculate t the mass spectrometric detector corresponding disturbance in the 0 using equation 1 or 2 and 3, then (liquid chromatography–mass baseline, as with LC–MS, we can compare this to the observed peak spectrometry [LC–MS]). Therefore, estimate the column dead volume, in the chromatogram. For example, if V t the chromatogram usually has a M, and convert it to 0. If you are the chromatogram of Figure 1(b) was V × peak similar to the first baseline using a 4.6-mm i.d. column, M can obtained with a 150 mm 4.6 mm disturbance in Figure 1. If the be estimated as follows: column operated at 2 mL/min, t = sample is very clean and has 0 ≈ 1.5 mL/2 mL/min 0.75 min. V ≈ L minimal unretained material, a M 0.01 [1] This agrees with the observed peak small baseline disturbance as at approximately the same retention V L shown in Figure 1(a) may appear. where M is in millilitres and is time. If the calculated and measured t More commonly, there is sufficient in millimetres. Thus, for a 150 mm values of 0 differ by more than × V = × unretained material to generate a 4.6 mm column, M 0.01 ~20%, it is advisable to try to figure

28 LC•GC Europe January 2014

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t of this can be found in an earlier “LC Figure 1: Examples of 0 peaks (arrows). (a) Chromatogram with little unretained t Troubleshooting” column (1). The material; (b) large peak normally observed at 0. outlet check valves can also leak if they become contaminated. If you (a) (b) have a pump with outlet check valves, sonicating them may help. If you choose to sonicate the check valves, be careful that you know how they are assembled, in case they come apart in the process. Worn pump seals can also leak, resulting in a lower 1 2 3 4 2 4 than expected flow rate. Check the Time (min) Time (min) maintenance log for the pump. If the seals haven’t been replaced in the past year, I would suggest replacing them. If the seals are newer, you rate set properly? It should also be can inspect the pump more closely Figure 2: Illustration of exclusion of a obvious that historical retention data for possible signs of leaking. Most charged molecule from packing pores. should be consulted to be sure an pumps have a hole or drain tube (a) No ion pairing reagent present, abnormality in t really exists. below the pump head behind the charged base is poorly retained, acid 0 Assuming that the flow rate is set check valves, where any leakage is retained; (b) with ion pairing reagent, correctly, the most likely causes from the seals will exit. Look for signs charge on particle surface attracts of a flow rate problem are leaks, of leakage, such as visible liquid base, increasing its retention, but air bubbles, and problems with or white deposits of buffer residue. repels the acid, excluding it from the the check valves or pump seals. A Replace the pump seals if there is any pores. See text for details. Adapted secondary symptom may be low question of its integrity. from reference 3. pressure, depending on how far off t0 Smaller Than Expected: If t t 0 is. If leaks are not obvious, I would the observed 0 peak comes out (a) (b) earlier than expected, one of two Base Acid Peaks that are eluted possibilities exists. The easier to Neutral Neutral before the expected address is a mistake in the flow Acid retention t time are most rate setting so that the flow rate is 0 too high. Although I suppose it is likely excluded from the possible, I have never heard of a pores of the column. pump or controller software failure Base that resulted in excessive flow rates, open the pump purge valve and run so operator error is the most likely 5 mL or so of solvent to waste from source of a flow-related problem. each flow channel in use. Thus, if you With the most common forms of LC are using a two-pump high-pressure (reversed phase, normal phase, ion t mixing system, purge both pumps; exchange, ion pairing, and so forth), 0 if it is a low-pressure mixing system, should be the first disturbance in the purge each solvent line. This should chromatogram. If something is eluted 0 5 10 0 5 10 remove any bubbles from the system. earlier than the expected retention of t If the problem persists, carefully 0, the compounds may be excluded check each fitting in the pressurized from the pores of the packing out why. Several possibilities are flow stream for leaks. Sometimes the material. The exception to this is discussed below. fine point of a twisted laboratory wipe in size-exclusion chromatography, t t 0 Larger Than Expected: If 0 is larger or facial tissue can be used to probe where everything should come out t than expected, the most likely cause the fittings for possible leaks. If leaks before 0. If a molecule is restricted is a flow-related problem. For isocratic in the flow stream are not found, a from entering the packing pores, it methods, the retention time of retained pump problem is most likely. only has access to the volume of peaks should change by the same If you are using acetonitrile as one column between the particles (the t proportion as 0 when the flow rate is of the solvents and the pump does interstitial volume), which I mentioned changed. If, for example, the above not have active check valves, it is at the beginning was approximately t case had an observed 0 of 1.0 min, possible for the inlet check valves half of the total solvent volume, or the retained peaks should increase by to stick. This is from the formation 30–35% of the column volume if 1.0/0.75 = 1.33-fold. If this is confirmed, of polymers on the surface of the the total dead volume is ~65%. I’ve check for flow-related problems. Larger check-valve seat, and usually can seen the interstitial volume quoted as t than expected values of 0 indicate a be corrected by sonicating the 40% of the column volume (2), but drop in the flow rate. Always check the check valves for a few minutes in in either case, a significant portion most obvious case first — is the flow methanol. A more detailed discussion of the dead volume is inside the

30 LC•GC Europe January 2014

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particles. If sample molecules are in situ ion-exchange surface with a factor can be estimated by using t0 restricted from entering the pores of negative charge that is used to retain as the unit of measure for retention the packing, they will be eluted before the positively charged adrenaline. and measuring retention beginning t0. Such peaks may be confused with However, although the pH of the at the observed value of t0. For the the true t0 peak, because we are mobile phase was not changed, chromatogram of Figure 1(b), this is used to assigning the first peak in the you can see that the naphthalene done by dividing the baseline up in chromatogram as t0. sulphonate is now unretained. This units of t0 instead of minutes. The first Two common causes of restricted is because the pores now contain peak is eluted a little over 1 t0 unit access to the pores exist. The a net negative charge that repels past t0, so it has a k value of a little most obvious is that the molecules the negatively charged naphthalene more than 1. Similarly, the second are too large to enter the pores. sulphonate. You can imagine a and third peaks have k values of a A rule of thumb is that the pore similar situation where a pore with bit more than 2 and 3, respectively. diameter should be 3–4 times the a net charge would repel a sample Although equation 4 does not apply to hydrodynamic radius of the molecule. molecule of opposite charge, resulting gradient elution, the general principle Most analytical columns have pores in exclusion from the particles and of keeping the first peak away from t0 in the 8–12 nm (80–120 Å) range, elution before the column dead to avoid interferences still holds. From which will accommodate molecules time. This phenomenon, called ion this standpoint, I like to see the first of up to ~10,000 Da. Above this exclusion, is occasionally observed in peak of interest in a gradient come off size, for example proteins, ion-exchange chromatography. Any the column at least 1 t0 unit past t0. larger-pore columns, with 30–40 nm chemical change in the pore surface pores, are used. Thus, if your that repels sample molecules will Conclusions sample is a pharmaceutical product, have the same result. Although at first glance, the solvent although the analyte of interest peak at the beginning of the may be <1000 Da, the formulation chromatogram has no value, it can may contain polymers or other When the first peak be a useful tool to help diagnose excipients in excess of 10,000 Da comes out after the problems with the chromatogram. that may be excluded from the expected retention time, We can compare column dead pores. In some cases, dimers the problem is usually time estimates with the observed or other aggregations of sample retention time of the t0 peak and molecules can result in a flow-related and most get an idea of what might be going material that is stable enough to commonly caused by a wrong. When the first peak comes chromatograph, but too large to leak. out after the expected retention time, enter the pores. Any of these large the problem is usually flow-related molecules may be eluted before the and most commonly caused by a column dead volume. Using t0 to Check for “Good” leak. Peaks that are eluted before the A second cause of restricted Chromatography expected retention t0 time are most access to the pores is that chemical Another use I make of t0 is to check likely excluded from the pores of the repulsion between a sample molecule for the quality of the separation, column, because they are too large or and the pore may exist. An example especially when readers submit are repelled from the pores. So we can of this is illustrated in Figure 2 (3). In problem chromatograms for me to see that nothing (t0) really is a useful Figure 2(a), a sample of adrenaline diagnose. For isocratic separations diagnostic tool. (base), benzyl alcohol (neutral), (those with a constant mobile phase and naphthalene sulphonate (acid) composition), the retention factor, k, References is separated on a C18 column with is calculated as: (1) J.W. Dolan, LCGC North Am. 26(6), 532–538 (2008). a methanol–buffer mobile phase (2) U.D. Neue, HPLC Columns (Wiley-VCH = at pH 6. Adrenaline has a pKa of k (tR – t0)/t0 [4] New York, USA, 1997), p. 53. 8.55, so it will be fully ionized under (3) J.H. Knox and R.A. Hartwick, J. 204 these conditions, and naphthalene where t is the retention time of the Chromatogr. , 3–21 (1981). R (4) J.W. Dolan, LCGC North Am. 25(7), sulphonate has a pKa of <1, so it peak of interest. The retention factor 704–709 (2007). will also be ionized. Adrenaline is is a measure of the distribution of unretained because in its charged the sample between the stationary John W. Dolan is vice president form it is very polar and not retained. phase and the mobile phase. As an of LC Resources, Walnut Creek, On the other hand, naphthalene indicator of chromatographic quality, California, USA. He is also a member sulphate has sufficient nonpolar I like to see 1 < k < 20, or better of LC•GC Europe’s editorial advisory nature that it is well retained, even 2 < k < 10, as has been discussed in board. Direct correspondence though it is ionized. With the addition past “LC Troubleshooting” columns about this column should go to “LC of 14 mM octane sulphate as an (for example, reference 4). If k < 1 is Troubleshooting” LC•GC Europe, ion-pairing reagent, the results of observed, the peak is likely to have 4A Bridgegate Pavilion, Chester Figure 2(b) are obtained. Here, the poor retention-time reproducibility Business Park, Wrexham Road, ion-pairing reagent is assumed to and more likely than strongly retained Chester, CH4 9QH, UK, or email the be immobilized on the surface of the compounds to have interferences from editor-in-chief, Alasdair Matheson, at C18 stationary phase, creating an the tail of the t0 peak. The retention [email protected]

32 LC•GC Europe January 2014

magentablackcyan ES374287_LCE0114_032.pgs 01.09.2014 00:18 ADV GC CONNECTIONS The Origins of GC Carrier Gases: Putting a Genie in the Bottle

John V. Hinshaw, Serveron Corporation, Beaverton, Oregon, USA.

Have you wondered how the gas chromatography (GC) carrier gases helium, hydrogen, argon, and nitrogen are transformed from their natural conditions or precursors into highly purif ed compressed states inside laboratory gas cylinders or generators? Here, we track the genesis of the top four carrier gases before they start their journey through a GC system.

Unlike liquid chromatographers, This month’s instalment asks the xenon are and will remain too expensive gas chromatographers have only a following question: Where exactly do to be taken seriously as GC carrier few mobile phases to apply to their the various GC carrier gases come gases, but it is possible that desperate separations. The mobile phase in from? The obvious and wrong answer is, gas chromatographers with no helium liquid chromatography (LC) plays “from a gas cylinder”. Instead, consider on hand may have tried one of the an active role in determining solutes’ the elemental or natural states of the noble gases if they could find a cylinder. retention characteristics, and it does primary carrier gases helium, nitrogen, Neon seems like a logical candidate. so in relation to the chemical nature hydrogen, and argon. Some of these If helium prices continue to increase of the stationary phase. To that end, are quite common in nature; others are then perhaps neon, which costs more liquid chromatographers employ rare. How are the carrier gases obtained than five times the price of helium, will various solvents, solvent mixtures and from nature and purified to better than become another helium replacement gradients, buffers, and other additives 99.9999%? like hydrogen. in combination with a wide array of Beyond simple cost considerations, stationary phase modes and types that Natural Occurrence there are performance reasons to give them degrees of control over their Table 1 lists gases that have been prefer certain carrier gases. Hydrogen, separations that the rest of us might used — or at least could be used for example, is sometimes preferred dream of. — for GC mobile-phase duty. The because of its low viscosity and high In gas chromatography (GC) the second column of Table 1 lists their diffusivity. The low viscosity means situation is much more limited. The role average concentrations in the natural that less pressure is required to attain of the GC mobile phase is reduced to atmosphere at sea level. Nitrogen is, of any particular flow rate with hydrogen, simply carrying solute molecules along course, the most abundant gas with a which translates to better performance the column while they are not dissolved 78.1% portion. It is followed by oxygen at higher flows and with narrower-bore in or adsorbed on the stationary phase. at 20.9%, although pure oxygen is columns. Higher diffusivity produces All of a GC separation’s selectivity omitted from the table since it makes a more efficient separations around the can be attributed to the stationary decidedly poor and chemically active optimum carrier gas velocity region. phase alone. The GC mobile-phase carrier gas. Purified air, however, has Neon, on the other hand, has high job description of “inert” and “carrier” been used as a carrier gas in certain viscosity and low diffusivity, which make gas sounds about as dull as it can limited situations such as with some it less attractive. be. And also unlike in LC, GC carrier portable gas chromatographs and at Helium, a nearly nonrenewable gases come with some nonintuitive limited low column-oven temperatures. natural resource, is light enough that, behaviours that can be daunting to Argon is the next most abundant along with hydrogen, it attains escape understand at first, such as the effects atmospheric carrier gas, followed by velocity after it is released into the of gas compressibility on flow and carbon dioxide as used for SFC. The atmosphere and simply leaks off into separation. These side effects of a carbon dioxide level in Table 1 is the space where it is swept away by solar gaseous mobile phase have been dealt reported monthly mean value at Mauna wind. Not that these elements are with in innumerable publications and Loa in Hawaii as of October 2013 (1). scarce — by some accounts hydrogen discussions, and I won’t go into them The trace gases neon, helium, krypton, comprises 73.9% of the known baryonic any further in this article. Also, for space hydrogen, and xenon, which taken matter in the universe, followed by reasons the use of carbon dioxide for together account for about 25 ppm of helium at 24%. Both are replaced in supercritical fluid chromatography (SFC) the atmosphere’s content, are listed the Earth’s atmosphere at slow rates; will not be addressed in any detail. below carbon dioxide. Krypton and the balance between replenishment

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Table 1: Properties of carrier gases. Gas Abundance in Dry Boiling Point (K) at 101 kPa Gas Viscosity Relative Diffusivity† Air (by volume) (Pa.s × 10-6) at 0 °C Nitrogen 78.1% 77.4 16.7 0.30 Argon 0.93% 87.3 21.0 0.37 Carbon Dioxide 394 ppm 217 K at 523 kPa (triple point) 13.7 0.23 Neon* 18 ppm 27.1 29.4 0.43 Helium 5.2 ppm 4.2 18.7 1.0 Krypton* 1.1 ppm 120 23.3 0.2 Hydrogen 0.6 ppm 20.3 8.40 1.2 Xenon* 0.1 ppm 166.6 21.2 — *Not commonly used in chromatography. †Calculated for n-octane at 130 °C from the Fuller-Schettler-Giddings equation (2) as cited in reference 3.

and loss accounts for their average along the route to pure nitrogen and the incoming air flow is switched to a natural concentrations in air shown in argon carrier gases. second filter bed, while the pressure Table 1. A chromatography laboratory Cryogenic Fractionation: In in the first is reduced to release with helium carrier gas in use would be cryogenic fractionation for recovery of the trapped oxygen and impurities above average — aren’t they all — with nitrogen, argon, and oxygen, ambient into an exhaust air stream. The a considerably higher background air is first filtered and pressurized. oxygen-enriched air can be recovered helium level. It’s the higher levels of Condensed water is removed, and — for example, for use in portable hydrogen in the laboratory that are then the air stream is further dried and oxygen supplies. The two filter beds are of real concern and that drive the hydrocarbons plus carbon dioxide are used alternately to give a continuous installation of hydrogen detectors and removed by active adsorbent beds flow of purified nitrogen. The final better laboratory ventilation. such as molecular sieves. The purified nitrogen purity is controlled by flow rate, air is cooled until it liquefies, and then the size of the adsorbent beds, the use Sourcing, Separation, and the oxygen and argon are fractionally of multiple PSA stages, and filtration (4). Purifcation distilled from the nitrogen. Neon, PSA can produce nitrogen output with Most carrier gases come from natural krypton, and xenon can be separated as a purity as high as 99.9995%, which is sources. Hydrogen is the exception. The well by applying additional fractionation suitable for many GC applications. hydrogen supplied in pressurized tanks steps. The resulting purified gas streams Membrane Separation: If moderately is most often obtained as the by-product are stored in a liquid state or in a pure nitrogen in the range of 95% of other commercial reactions and gaseous state as industrial-purity bulk to 99.5% pure is suitable for an processes. Laboratory generation of gases. application then purification by hydrogen uses electrolysis of water, Typically, the initial cryogenic selective membrane permeation may and hydrogen is the only carrier gas fractionation process yields nitrogen be an option. Filtered pressurized air that is truly generated in the laboratory. purities around 99.95%. Further is fed into one or more hollow-tube A laboratory nitrogen generator does purification is required to bring the membrane separators, from which not actually generate nitrogen, it just gas up to ultrahigh purity or research purified nitrogen emerges in the main separates and purifies nitrogen from grade for service as a GC carrier gas flow stream and the impurities — an incoming air stream. Helium, as the or for other specialty gas services. including oxygen, water, and carbon reader must know, is a story unto itself. Additional fractionation steps, as well as dioxide — are taken off in a separate Helium is a natural resource that is not pressure-swing adsorption, can be used. stream. In a nitrogen separator the replenished at anything near its rate Pressure-Swing Adsorption: membrane has permeation and of consumption — it is irreplaceable. Pressure-swing adsorption (PSA) is a mechanical characteristics that Helium and hydrogen are discussed in smaller scale air-separation technique selectively permit impurities to pass their own sections below. that is used at the industrial level through while blocking nitrogen. The Nitrogen and argon are also obtained when the size and cost of a cryogenic ultimate nitrogen purity depends on the from natural sources for industrial and plant is not practical or necessary. number and type of membranes used. laboratory applications, but the available PSA purification is also found in Generally a membrane separator alone quantities are not in any danger of some laboratory nitrogen generators. will not yield sufficiently pure nitrogen depletion. They are both produced in PSA works by passing air through for GC carrier gas purposes. large quantity by separation from air and initial filters that remove particles, Helium: Helium has become expensive then further purified until the desired hydrocarbons, and the bulk of the and difficult to obtain in recent years. GC grade is obtained. Commercial air water. Next, the cleaned air passes Anyone who has had to purchase separation plants also produce oxygen through a carbon-sieve bed at elevated and manage helium supplies has and can yield neon, krypton, and xenon. pressure, where oxygen plus residual experienced this first-hand. The There are three types of air separation water and carbon dioxide are adsorbed reasons for this change include generally in use today. Cryogenic while purified nitrogen flows through. increasing demand for applications fractionation is the most familiar first step As the bed approaches saturation, such as superconducting magnets for

34 LC•GC Europe January 2014

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magnetic resonance imaging (MRI) Helium is separated from natural gas increase (6). A significant portion of as well as for military applications. At by liquefaction of the other gases the hydrogen carrier is lost through the the same time the available supply including nitrogen, methane, and column walls, especially for smaller of helium in the United States has carbon dioxide, followed by fractional diameter columns. The actual average shrunk, because of difficulties with distillation of the still-gaseous helium. velocity has to be measured by distribution and the imminent closure Like other industrial gases, helium is timing an unretained peak — simply of the diminishing National Helium further purified to reach laboratory reading the electronic pressure control Reserve. Today, however, it appears research purities suitable for GC. (EPC) system reported value won’t that the shutdown is delayed. Other Hydrogen: Naturally occurring be accurate because EPC does not factors make helium’s future look hydrogen molecules are found only at compensate for this effect. bleak, including the slow shift to shale low concentrations in the atmosphere natural gas sources that contain no — free hydrogen escapes the same Conclusion helium, and a lack of incentive for way as helium. Fortunately, and unlike GC carrier gases traverse varied and natural gas producers to bring new helium, hydrogen is easily synthesized complex paths on their way to the gas helium separation facilities on line. by industrial steam reformation of natural tanks and generators that sometimes There is some hope for a loosening gas: are taken for granted in the laboratory. of the worldwide helium supply with Some carrier gases, such as nitrogen + → + the opening this year of significant CH4 H2O CO 3H2 and argon, are sourced from the + → + additional helium production facilities in CO H2O CO2 H2 [2] atmosphere, whereas helium is a Qatar and the continuing demand from nonrenewable natural resource found existing supplies in Africa and China. Other industrial reaction pathways primarily in natural gas. Hydrogen is Helium is generated naturally in the to hydrogen also exist. Hydrogen has the only carrier gas that is synthesized, earth’s crust primarily as the by-product received considerably more attention either on-demand in laboratory gas of uranium and thorium radioactive in recent years because of a popular generators or as a by-product of alpha decay processes that start with resurgence as a transportation industrial chemical processes. All of the stable naturally occurring isotopes fuel. Perhaps some day gas the carrier gases must be purified 238U (half-life 4.5 × 109 years) and 232Th chromatographers will be able to tap substantially and multiple times to attain (half-life 1.1 × 1010 years): into their automobiles for a field supply the levels required for GC. of carrier gas. The next “GC Connections” 238U → 234Th + He2+ In the laboratory, hydrogen is instalment will address the storage 232Th → 228Ra + He2+ [1] produced via electrolysis of water: and use of both carrier and other GC gases, and the multiple steps by which → + Additional decay processes yield 2H2O 2H2 O2 [3] pressures and flows are controlled on more alpha and other nuclear particles the way to a GC inlet. until the decay chains end at lead. Water-to-hydrogen oxidation– The alpha particles (He2+) recombine reduction can be carried out in a References with electrons and form helium upon number of ways. Modern laboratory (1) U.S. Commerce, National Oceanic and Atmospheric Administration, Earth System impacting nearby minerals. Clearly, hydrogen generators use a Research Laboratory, Global Monitoring in-house generation of helium is not proton-exchange membrane (PEM) that Division, “Trends in Atmospheric Carbon a possibility, although some have passes protons (H+) for recombination Dioxide,” retrieved November 2013 from quipped that it might be possible with into hydrogen molecules while being http://www.esrl.noaa.gov/gmd/ccgg/ trends/#mlo. a self-sustaining fusion reactor; clearly impermeable to the gaseous hydrogen (2) E.N. Fuller, P.F. Schettler, and J.C. not an imminent source. It is possible to and oxygen reaction products. The Giddings, Ind. Eng. Chem. 58, 19–27 recover small amounts of helium from hydrogen is then passed through a (1966). (3) L.S. Ettre and J.V. Hinshaw, Basic cryogenic air separation processes, platinum membrane that blocks all Relationships of Gas Chromatography but the cost is considerably more than other molecules and attains a hydrogen (Advanstar, Cleveland, Ohio, USA, 1993), obtaining helium from natural gas. The purity of six nines or better. The oxygen pp. 44 – 47. total amount of helium that could be is vented. (4) S. Ivanova and R. Lewis, CEP Magazine, 38–41 (June 2012). recovered by atmospheric fractionation Gas chromatographers who have (5) M.A. Cook, Nature 179(4552), 213 (1959). would not make much of an impact on run their fused-silica columns with (6) J.E. Cahill and D.H. Tracy, J. High the worldwide demand. hydrogen at elevated temperatures Resolut. Chromatogr. 21, 531–538 (1998). The total amount of terrestrial helium have, perhaps unwittingly, performed John V. Hinshaw is a senior scientist generated by natural radioactive membrane purification of their carrier at Serveron Corporation in Beaverton, decay has been estimated at about gas right through the column walls. Oregon, USA, and is a member of 3000 metric tons annually (5). The Fused silica is hydrogen-permeable at the LC•GC Europe editorial advisory helium thus formed remains entrained the high end of GC oven temperatures, board. Direct correspondence about in impervious rock, but it is released above about 320 °C. It is not difficult this column should be addressed to through fissured rock to collect in gas to observe that the net flow rate “GC Connections”, LC•GC Europe, 4A pockets along with other natural gases. through the column becomes less Bridgegate Pavilion, Chester Business The resulting helium concentrations than the mass flow rate entering the Park, Chester, CH4 9QH, UK, or email in natural gas can range from a few column by measuring the average the editor-in-chief, Alasdair Matheson, parts-per-million up to as high as 7%. carrier gas velocity as temperatures at [email protected]

36 LC•GC Europe January 2014

magentablack ES374284_LCE0114_036.pgs 01.09.2014 00:18 ADV SAMPLE PREPARATION PERSPECTIVES

Electrical Potential as a Driving Force in Sample Preparation

Ronald E. Majors, Sample Preparation Perspectives Editor

To achieve lower detection limits and better selectivity, researchers are always exploring new ways to enhance sample preparation and separation technology. The addition of electrical potential to existing technologies or for entirely new approaches is relatively unexplored but can create another dimension of selectivity in sample preparation. In addition, speed and sensitivity are added benef ts. This instalment looks at the use of electroenhancement to solid-phase extraction, solid-phase microextraction, and membrane and liquid extraction.

We are all familiar with the role compounds encountered are EE, the immiscible phases that are of electromigration in enhancing charged or could be made charged. electrically conductive are kept separations in capillary and between electrodes, and upon gel electrophoresis and related Electroextraction addition of an electric field, charged techniques and in capillary Most of us are familiar with liquid– particles travel from one phase to electrochromatography. In addition, liquid extraction (LLE) because another, thereby separating anions electrical potential has been used in it is still one of the most popular and cations, as depicted in Figure 1. electrochemical and other forms of techniques used for sample Three‑phase systems are also chromatographic detection. The use cleanup. In LLE, two immiscible used where anions and cations are of electrical potential to enhance and liquids are used to partition attracted into the two outer phases add another dimension of selectivity analytes from a sample into one leaving uncharged particles in the to sample preparation is still a of the two phases, an organic middle phase. Generally, organic relatively new, noncommercialized solvents need to contain a small technique. The purpose of this Sample preparation using amount of water so that they can be instalment is to discuss how made conductive. In two‑phase EE, electrically driven enhancement has electrolytic potential in a rapid electromigration takes place already proven to offer some major bioanalysis is particularly because ions in the organic phase advantages to the preparation of attractive since many are subjected to very high electric samples whose analytes of interest field strength resulting from low are ionized or can become ionized of the water-soluble conductivity. Because the electric by the appropriate adjustment of pH compounds encountered field of the aqueous acceptor phase to the separation media. We explore is much lower, ions in the organic five sample preparation methods are charged or could be phase will migrate at high velocity where the application of a charge made charged. to be concentrated just beyond the across barriers provides enhanced liquid–liquid interface. selectivity, often leaving undesired phase and an aqueous phase. The apparatus used to conduct matrix and impurities behind, thereby After shaking the phases together electroextraction consists of a vial accomplishing the goals of sample in a separatory funnel, analytes with a conical bottom, a bottom preparation. that prefer the organic medium grounded electrode in contact The main purpose of sample are partitioned into it while those with the lower aqueous layer and preparation is to transport the analytes that prefer the aqueous a capillary (injection port) to inject analytes of interest into a medium media, usually polar compounds or the sample solution, and an upper that is compatible with the analysis ionized compounds, are partitioned electrode in contact with the upper method, to remove interferences, and into it. Electroextraction (EE) is organic phase. The EE experiment to concentrate the analytes so that a sample enrichment technique can also be performed in an they can be more easily detected that focuses charged analytes electrophoresis‑like capillary and and quantitated. Sample preparation from a large volume of one phase this version is referred to as capillary using electrolytic potential in into a small volume of aqueous electroextraction (cEE). Lindenburg bioanalysis is particularly attractive phase through the application of and colleagues (1) used a wide‑bore since many of the water‑soluble an electric current. In two‑phase capillary connected to a two‑way,

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Figure 1: Diagram showing the Figure 2: Setup for three‑phase electroextraction. principle of electroextraction.

−

Organic phase

DC supply Conductive pipette tip

Organic flter phase Aqueous acceptor phase

Aqueous acceptor phase

Aqueous phase

+ phase by applying an electric field analyte increases, the extraction between the donor and acceptor. rate increases, with the partition In principle, the experiment works coefficient across the aqueous– like electromembrane extraction organic filter interface being the (EME) (see next section), but the limiting factor. Large molecules 10‑port switching valve interfaced intermediate organic phase isn’t like proteins will not be transported to a liquid chromatography–mass immobilized in a membrane. The into the acceptor phase through spectrometry (LC–MS) system. extraction occurs when the electric the organic filter phase. Thus, This system allowed up to 100 µL field is applied between the aqueous the three‑phase electroextraction of sample to be extracted, and donor and the aqueous acceptor technique could be quite useful for they showed improved detection phase (shown as the green drop bioanalysis. of peptides in an unspiked plasma suspended in the organic filter phase In their recent paper, Raterink sample. For these real samples, in Figure 2). In this experiment, no and coworkers (3) demonstrated limits of detection values were in stirring is required, as is the case that model compounds (carnitines) the 10–50 nM range for several for single‑drop microextraction, could be extracted from a 50‑µL angiotensin peptides, which and a rapid, complete extraction sample into a small 2‑µL acceptor indicates the potential for cEE as is accomplished. A fairly large droplet in only 3 min. Samples of an on‑line sample concentrating donor volume can be used and a acylcarnitines were spiked into technique. In another application, small acceptor volume (microlitre to a human blood plasma sample this same laboratory applied cEE– submicrolitre) is possible, thereby and were found to be effectively LC–MS to urine metabolites including providing large enrichment factors extracted, but enrichment factors amino acids and acylcarnitines (2). and allowing direct injection into a were slightly lower compared to nano electrospray ionization (ESI) MS model analytes, presumably because Three-Phase Electroextraction source. By choosing an optimized of protein binding. Calibration A recent variation of electroextraction organic filter phase, selectivity of curves for the spiked carnitines in termed three-phase electroextraction the extraction can be improved. human plasma showed linearity also uses the principles of EE (3). Enrichment factors were found to be over two orders of magnitude. A In this technique, charged analytes related to the polarity of the organic proof of principle for the on‑line migrate from an aqueous donor filter phase with relative standard integration in an automated phase through an immiscible organic deviation (RSD) values less than nanoESI‑direct infusion‑MS platform filter phase into an aqueous acceptor 15%. Also, as the polarity of the was demonstrated. These preliminary

38 LC•GC Europe January 2014

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Five Keys to Successful GC Methods

A FORMAT FOR EVERYONE This new e-book is available for the iPad or for a standard computer web browser

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John Hinshaw, Specifi c topics covered include: renowned expert in gas ® Peak Problems: How to handle partially chromatography and resolved or distorted peaks that yield poor quantitation the longtime author of the “GC Connections” ® System Operation: The steps to follow for restoring an idle GC column to operating column in LCGC, provides condition invaluable advice about ® Air Leaks: What happens when air leaks how to handle some of the into the carrier-gas line, and what to do most common diffi culties about it faced by users of gas ® Preventive Maintenance: How to avoid chromatography as well as crises through periodic maintenance of your GC system with best practices to avoid ® problems in the fi rst place. Upgrading GC: Guidelines for upgrading your GC laboratory to use high speed GC and generate your own gases

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liquid–liquid–liquid membrane Figure 3: Diagram of (a) the EME setup and (b) principle of operation using extraction with an added electrical pethidine as a model compound (5). potential. The setup was used to (a) (b) isolate and concentrate a series of basic drugs in an acidified solution Negative electrode Positive electrode of water, human plasma, and human urine. Enrichment factors were in the range of 7.0–7.9. At the time, Power they speculated that this approach supply could become “an interesting tool + for future isolation within chemical analysis”. Since then, they and other CH3 N+ research groups have applied this priniciple to not only flat supported liquid membranes (SLMs), but + CH3 O O also to inexpensive, supported hollow‑fibre membranes that make it experimentally more convenient for + + Fibre wall impregnated with small volumes. organic solvent In 2010, Astrid Gjelstad from Acceptor phase (pH ~2) the above‑mentioned University of Donor phase (pH ~2) Oslo group published a paper in LCGC (5) demonstrating the use of such a system for the enrichment of pethidine, a basic drug, in aqueous solution. The EME setup that she Figure 4: Experimental setup for electroextraction–SPME. used is illustrated in Figure 3. The setup and procedure is very SPME holder similar to hollow‑fibre liquid‑phase microextraction (HF‑LPME) (6,7), + but in EME electrodes are inserted into the sample and the acceptor solution, respectively, and the DC supply electrodes are connected to a direct current electrical power – supply. In EME, the driving force for the extraction is the electrical Pt electrode potential, and this parameter must be optimized. Normally, extraction recoveries increase with increasing Fibre voltage up to a certain level, after Sample solution which there is no further gain in recovery versus voltage (5). The optimal voltage must be established by experimental optimization, as this Stirring bar voltage is dependent on both the analytes and the composition of the Magnetic stirrer SLM. In EME, an electrical potential is applied over the electrodes of typically 5–100 V, creating an electrical field over the SLM. For EME of basic analytes, the anode is results showed that three‑phase EE 300‑V electrical potential imposed located in the donor sample solution, may prove to be an automatable, across a thin (200 µm) supported whereas the cathode is placed in the novel sample pretreatment for liquid membrane for 5 min assisted acceptor solution. Both the donor high‑throughput bioanalysis. the electrokinetic migration of and acceptor solutions should be drug substances from a 300‑µL acidic (dilute hydrochloric or formic Electromembrane Extraction donor compartment to 30 µL of an acid). The sample must be acidified A number of years ago, acceptor solution (4). The membrane to make sure that the basic analytes S. Pedersen‑Bjergaard and K.E. was impregnated with 2‑nitrophenyl are ionized. Thus, the basic analytes Rasmussen of the University of octyl ether immobilized in the pores, are extracted as protonated species Oslo demonstrated that the use of a making the experiment a form of from the sample, through the SLM,

40 LC•GC Europe January 2014

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SPME fibres (13). The technique Figure 5: Modified SPE cartridge for studies of electric field‑assisted SPE. Shown has been called electroenhanced is an expanded view; the final assembly is tightly sandwiched to eliminate dead solid-phase microextraction volume. (Courtesy of Professor Susanne Roth, University of Campinas, Brazil.) (EE‑SPME). In this approach, the fibres were used with the direct immersion mode in aqueous samples using an applied potential to extract methamphetamine. The experimental setup is depicted in Top electric contact Figure 4. When used with an applied voltage of 12 V, the fibre coated with carboxen‑polydimethylsiloxane (CAR‑PDMS) was more efficient for methamphetamine than those Top electrode constructed of polyacrylate, PDMS, or PDMS‑divinylbenzene. The PTFE frits CAR‑PDMS gave sixfold greater sensitivity than the polyacrylate and 1.5‑fold greater sensitivity Sorbent than the other two fibres. After this optimum fibre was established, the workers went on to optimize the PTFE frits solution pH (pH 7), applied voltage (12 V), extraction time (20 min), and Thermistor stirring speed. Overall, the EE‑SPME Bottom electrode technique gave a more efficient PTFE frits Although commercially the area of

Bottom electric contact electroenhancements to sample preparation protocols is relatively undeveloped, the use A recent innovation has nitrobenzene are typically used for of electric potential the SLM (5). For extraction of more been the application of polar substances, these solvents can create additional an electric potential to are normally added to an ion‑pair selectivity, sensitivity, and commercial SPME fbres. reagent or another modifier to speed. facilitate the mass transfer of analyte and into the acceptor solution. The across the SLM. Typical examples extraction for methamphetamine acceptor solution is also acidic to are di‑(2‑ethylhexyl) phosphate and compared to the use of regular SPME support the electro‑kinetic transfer tris‑(2‑ethylhexyl) phosphate (5). — a 159‑fold higher enrichment and to avoid back‑extraction into Acidic compounds have only been factor. The final analysis was the SLM. For acidic analytes, the extracted a few times by EME, and in performed using thermal desorption direction of the electrical field is these cases 1‑octanol has been used and gas chromatography–mass reversed, and alkaline conditions as supported liquid. Several reviews spectrometry (GC–MS). The (dilute sodium hydroxide or ammonia have been published summarizing calibration plot under the best solution) are used in the sample and applications of EME (8–12). selected parameters was linear in the the acceptor solutions to maintain range of 0.5–15 ng/mL (r = 0.9948). the analytes in their charged Electroenhanced Solid-Phase For the analysis of methamphetamine configuration. EME is more rapid Microextraction in human urine samples, the limit of than HF‑LPME because the driving Solid‑phase microextraction (SPME) detection (LOD) was 0.25 ng/mL with force is an electrical potential is a popular extraction technique a satisfactory RSD of 6.12% (n = 3). rather than a pH gradient. The EME used in gas, liquid, and headspace Recovery of methamphetamine in the experiment can often be completed sampling and analysis. Research spiked urine samples was 86.9%. in under 5 min. groups have been investigating ways For EME of basic substances, to make the technique even more Electric Field-Assisted immobilized solvents like selective and sensitive. A recent Solid-Phase Extraction 2‑nitrophenyl octyl ether, 1‑ethyl‑ innovation has been the application Solid‑phase extraction (SPE) 2‑nitrobenzene, and 1‑isopropyl‑4‑ of an electric potential to commercial is one of the most popular

www.chromatographyonline.com 41 magentablackcyanyellow ES374300_LCE0114_041.pgs 01.09.2014 00:18 ADV SAMPLE PREPARATION PERSPECTIVES

sample preparation techniques around some of these problems application, especially when the used before chromatographic by optimizing the design of the feasibility of automation has been analysis. It relies on similar electrodes to minimize bubble demonstrated. There are other areas principles of retention‑elution formation. In a buffered system, where electrical potential can aid that are characteristic of liquid there were no observed pH changes sample preparation, such as the chromatography so a number of during the E‑SPE experiment. For focusing of analytes before capillary mechanisms (that is, reversed unbuffered wash systems, they electrophoresis and electrofiltration. phase, normal phase, anion and came up with a clever bidirectional These techniques may be discussed cation exchange, affinity, and so on) flow modification (not shown here), in future “Sample Preparation are available to the chemist. The that negated the pH changes that Perspectives” instalments. application of electric potential as resulted from hydronium formation an additional force for the SPE of during electrolysis by introducing References ionizable or ionic compounds was solvent into the cartridge at a (1) P.W. Lindenburg, F.W.A. Tempels, investigated by Brazilian workers higher flow rate than the solution U.R. Tjaden, J. van der Greef, and T. Hankemeier, J. Chromatogr. A 1249, (14). The modified syringe‑type leaving the cartridge (15). This 17–24 (2012). SPE cartridge used in their studies solvent neutralized and diluted the (2) P.W. Lindenburg, U.R. Tjaden, J. is shown in Figure 5. In the final ions generated during electrolysis. van der Greef, and T. Hankemeier,

assembly, the various elements Temperature increases inside the Electrophoresis 33, 19–20 (2012). (3) R.‑J. Raterink, P.W. Lindenburg, R.J. were closely sandwiched together. cartridge were no more than 8 °C. Vreeken, and T. Hankmeier, Anal. They termed the technique E-SPE. They were able to reverse the Chem. 85, 7762–7768 (2013). In their investigations of E‑SPE, polarity in real time and minimize (4) S. Pedersen‑Bjergaard and K.E. Rasmussen, J. Chromatogr. A 1109, they studied the sorption or elution the effects of electro‑osmotic flow. 183–190 (2006). Using a flow extraction system that (5) A. Gjelstad, LCGC North Am. 28(2), they developed, they were able to 92–112 (2010). The electrical (6) S. Pedersen‑Bjergaard and K.E. control the flow rate, electric current, enhancement techniques Rasmussen, Anal. Chem. 71, electrical potential, and temperature 2650–2656 (1999). are especially applicable inside of the cartridge. (7) S. Pedersen‑Bjergaard and K.E.

The authors compared the Rasmussen, J. Chromatogr. A 1184, to situations where the 132–142 (2008). recovery with and without the (8) M. Balchen, L. Reubsaet, and S. analytes of interest application of electrical potential Pedersen‑Bjergaard, J. Chromatogr. A are ionic or ionizable across the packed bed. For the 1194, 143–149 (2008). (9) M. Balchen, T.G. Halvorsen, L. model compound marbofloxacin, in dilute solution, Reubsaet, and S. Pedersen‑ recovery was improved by 2.3 times Bjergaard, J. Chromatogr. A 1213, such as might be or reduced 4.2 times in comparison 14–17 (2009). to conventional SPE when the (10) C. Basheer, S.H. Tan, and H.K. Lee, J. encountered for drugs Chromatogr. A 1213, 14–18 (2008). and their metabolites top electrode was used as the (11) J. Lee, F. Khalilian, H. Bagheri, and cathode or anode (by reversing H.K. Lee, J. Chromatogr. A 1216, in biological fluids. polarity), respectively. The results 7687–7693 (2009). (12) L. Xu, P.C. Hauser, and H.K. Lee, J. demonstrated that the electric Chromatogr. A 1214, 17–22 (2008). behaviour of a model compound, the field acted as an additional driving (13) T. Yin Tan, C. Basheer, M.J. Yan Ang, antimicrobial marbofloxacin, using force and contributed to enhance and H.K. Lee, J. Chromatogr. A 1297, 12–16 (2013). both conventional C18 SPE and extraction efficiency and selectivity (14) R. M. Orlando, J. J. Rodrigues‑ electric field‑assisted C18 SPE. The in the SPE procedures. Rohwedder and S. Rath, electrical potential was applied only Chromatographia On‑Line Publication during the wash step, but this step Conclusion DOI 10.1007/s10337‑013‑2565‑9, 28 September 2013. was considered the most important Although commercially the area of (15) http://biq.iqm.unicamp.br/arquivos/ step to obtain adequate cleanup and electroenhancements to sample teses/ficha94196.htm. extraction efficiencies. preparation protocols is relatively The application of electric potential undeveloped, the use of electric “Sample Preparation Perspectives” across an SPE cartridge wasn’t as potential can create additional Editor Ronald E. Majors is an straightforward as they expected. selectivity, sensitivity, and speed analytical consultant and is a Similar to the development of any as was shown here using a few member of LC•GC Europe’s new technique, they uncovered published examples. The electrical editorial advisory board. Direct issues that had to be addressed in enhancement techniques are correspondence about this the final design. Bubble formation especially applicable to situations column should be addressed to at the electrodes and pH changes where the analytes of interest are “Sample Preparation Perspectives”, because of electrolysis of water, ionic or ionizable in dilute solution, LC•GC Europe, 4A Bridgegate temperature increases, and such as might be encountered Pavilion, Chester Business Park, electro‑osmotic flow affected by for drugs and their metabolites in Wrexham Road, Chester, CH4 silanol content of the SPE sorbent biological fluids. The technique 9QH, UK, or e‑mail the editor‑ were a few of the problems they of three‑phase electroextraction in‑chief, Alasdair Matheson, at encountered. They were able to work looks particularly promising for this [email protected]

42 LC•GC Europe January 2014

blackcyanyellow ES374619_LCE0114_042.pgs 01.09.2014 17:04 ADV Pittcon 2014 Preview

Marian Nardozzi, The Pittsburgh Conference, Pittsburgh, Pennsylvania, USA.

Pittcon 2014 will offer the opportunity to engage with new products and services from instrument vendors, participate in a diverse educational technical programme, and engage with colleagues from around the world in the one place. The conference will be held from 2–6 March 2014 at McCormick Place South in Chicago, USA.

Pittcon is held annually and offers attendees the chance promise a new wave of GC instruments of reduced size, to explore, evaluate, and compare the latest analytical improved speed, and improved resolution. and ancillary equipment from leading companies. It is • Ion Mobility Separations in Proteomics and Structural estimated that approximately 50% of exhibitors launch Biology, presented by Alexandre A. Shvartsburg, Pacific new products and technologies at the event, giving Northwest National Laboratory (Washington, USA), will visitors a first‑hand opportunity to find out about the latest focus on how ion mobility separations (IMS) hybridized developments. Technical experts will be on hand to give to MS have emerged as a powerful analytical approach. advice on critical issues and solutions, as well as offering • Method Development Strategies for Two-Dimensional live product demonstrations. There will be an opportunity Liquid Chromatography, by Dwight Stoll, Gustavus to attend exhibitor seminars that provide valuable insight Adolphus College (Minnesota, USA), will bring together into functionality and applications. Several speciality several experts in 2D LC from a variety of backgrounds areas will be open on the floor including: The New to present their perspective on best practices for Exhibitor and Laboratory Information Management method development in 2D LC. Systems (LIMSs) sections along with the Japanese and • Imaging Mass Spectrometry of Biological Tissues and German pavilions. Cell Cultures, by Amanda B. Hummon, University of Notre Dame (Indiana, USA), will show how imaging Technical Programme mass spectrometry is a growing area of biological A key feature of the conference is the diverse technical research, with new developments in ionization and programme. Beginning on Sunday 2 March, the sample preparation. programme features symposia, contributed and oral • Advances in Mass Spectrometry Based on Ultrashort sessions, workshops, awards, and posters presented Pulse Laser Technology, by Martin E. Fermann, by world‑renowned speakers. The opening session IMRA America Inc. (Michigan, USA), will offer a will feature the Wallace H. Coulter Plenary Lecture, state‑of‑the‑art account of the various prevalent “Quantitative Proteomics in Biology, Chemistry, and directions in ultrashort pulse mass spectrometry, Medicine”, presented by Steven A. Carr, director of comprising improved methods for desorption, proteomics at the Broad Institute of MIT and Harvard vaporization, and ionization using fs and ps pulses. (Massachusetts, USA). Carr will discuss the new era • Capillary Liquid Chromatography — A Powerful Tool in of quantitative biology that has been enabled by mass Analytical Chemistry, by Stephen G. Weber, University spectrometry (MS)‑based proteomic technologies. Other of Pittsburgh (Pennsylvania, USA), will discuss how highlights of interest are: using capillary columns allows one to minimize waste • New Wave of Gas Chromatography, by Milton L. Lee, and concentration sensitivity, interface with the Brigham Young University (Utah, USA), will present dominant detectors, easily control temperature, and new developments in gas chromatography (GC) that accommodate novel stationary phase structures. ©Cesar Russ Photography. www.chromatographyonline.com 43

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• Liquid Chromatography in Microfluidics: A • Getting the Most out of Capillary Gas Chromatography: Workhorse Tool is Going Small Scale, by Adam T. A two‑day advanced level course that will be taught Woolley, Brigham Young University (Utah, USA), by Matthew S. Klee, an independent consultant and will provide information on recent innovations of recognized authority in the area of GC analysis and microfluidic systems that combine multiple on‑chip instrumentation. sample preparation processes with subsequent • Optimizing the Performance of Your Gas Delivery electrophoretic separation. These miniaturized System to Obtain Best and Consistent Results While systems are automated and use small volumes of Reducing your Gas Costs: A half‑day class that will be samples and reagents, offering excellent potential for taught at beginner level by Frank Kandl who has over rapid biomarker analysis. 30 years of experience in design and manufacturing of • Clinical Analysis: The Next Frontier in Mass gas delivery systems. Spectrometry, by Timothy J Garrett, University of Florida • Practical Gas Chromatography: Presented by Eugene (Florida, USA), will assemble scientists from both the F. Barry, a professor of chemistry at the University of research and clinical fields to discuss new and old Massachusetts Lowell, USA, the course will span two mass spectrometry tools being used in clinical analysis days and will be taught at an intermediate level. and offers attendees the opportunity to discuss the potential for MS in patient‑oriented analyses. Networking Sessions The ability to network face‑to‑face has always been one Pittcon Honours Scientific Achievement of the major benefits of investing the time and cost of An important feature of Pittcon is to recognize and travel to attend Pittcon. Conferee networking sessions honour the achievements of scientists who have made are one of the ways that attendees with similar interests outstanding contributions to analytical chemistry and are brought together to discuss new techniques, resolve applied spectroscopy. The Chromatography Forum of problems, and brainstorm new ideas. These 2 h sessions are included in the cost of registration and cover topics Approximately 50% of exhibitors will such as analytical tools, laboratory management, and launch new products and technologies at techniques used in different applications. Pittcon 2014 Stay Connected At first glance, Pittcon can seem a bit overwhelming. The the Delaware Valley Dal Nogare Award will be presented free Pittcon 2014 mobile application will help iOS and to Mary J. Wirth, Purdue University (Indiana, USA). An Android device users to organize schedules, technical awardee is chosen on the basis of his or her contributions sessions, networking sessions, short courses, search to the fundamental understanding of the chromatographic exhibitor listings, and find Chicago restaurants and process. Featured within this particular prize presentation entertainment. The tool is available to use before, during, session will be the following presentations: and after the event. • Packing Capillary LC Columns with Sub-2 Micron This year’s conference and exposition will attract Particles, by James Jorgenson, Justin Godinho, Edward members of the scientific community from industry, Franklin, and James Grinias, University of North academia, and government — all to one place — for one Carolina at Chapel Hill (North Carolina, USA). exciting, productive week filled with discovery, innovation, • The Changing Relationship Between the Column and and education. Registration is $150 before 10 February the Instrument in Modern HPLC/UHPLC, by Ronald E. ($300 after this date) and includes unlimited, week‑long Majors, LC•GC Magazine access to all events and facilities. • Super-Resolution Spectroscopy Reveals Molecular-Scale Detail in Ion-Exchange Protein Contact: Marian Nardozzi Separations, by Christy Landes, William Marsh Rice E-mail: [email protected] University (Texas, USA). Location: McCormick Place, Chicago, Illinois, USA • Fluorescence Imaging of Single-Molecule Retention Tel: +1 412 825 3220 ext: 203 Trajectories in Reversed-Phase Chromatographic Fax: +1 412 825 3224 Particles, by Joel Harris, University of Utah (Utah, Registration: www.pittcon.org/register USA).

Professional Development Another important aspect of Pittcon 2014 is the short Stay Connected with LC•GC course programme. Short courses are affordable and are suitable for the seasoned professional, the recent The LC•GC team will be attending Pittcon 2014, bringing graduate, or the researcher interested in learning a new you the latest news and updates from around the event. skill set. More than 70 courses are available covering a Tweet us via @LC_GC or follow us on Facebook LCGC range of subjects relevant to chromatographers. Taught Magazine to keep up-to-date or to send us your personal by leading field experts, there are courses ranging from conference highlights. beginner to advanced levels in half‑, one‑, or two‑day LC•GC — Solutions for Separation Scientists sessions. The following is a sample of the short courses www.chromatographyonline.com on offer:

44 LC•GC Europe January 2014

magentablackcyan ES374317_LCE0114_044.pgs 01.09.2014 00:19 ADV PRODUCTS Mass spectrometer olfactory port The Impact HD system is a new PerkinElmer has addition to the UHR-QqTOF mass introduced the GC spectrometry product line from Bruker SNFR olfactory port Daltonics. The company report that it accessory to capture has more than 40,000 full-sensitivity human sensory data resolution (FSR), making it ideal for and correlate it with applications where trace analysis from analytical data when complex, high-background matrices using PerkinElmer is a challenge — such as biomarker GC–MS systems. The accessory is reported by the research, identif cation of impurities, or company to be able to measure and characterize complex residue screening. aromas for a complete, clear aroma prof le of food, www.bruker.com beverage, f avour, and fragrance samples. Bruker daltonics, Massachusetts, www.perkinelmer.com usA. PerkinElmer, Massachusetts, usA.

HPLc columns Infrared spectrometer UCT, LLC, has introduced a line of Selectra PFPP (pentaf uorophenyl Scientists monitoring propyl phase) HPLC columns greenhouse gases, plant that offer increased selectivity ecology, carbon sequestration, and reproducibility for difficult and volcanic emissions, compounds. The company among other CO2 analysis report that the range is ideal for applications, can now perform the analysis of charged bases, isotope ratio analysis at electronegative compounds, the sources of samples. halogenated, isomeric, and The Thermo Scientif c Delta Ray Isotope Ratio Infrared amine-containing compounds. Spectrometer is a transportable category of analyzer for The columns use multiple the continuous measurement of isotope ratio values from selectivity mechanisms including dipole-dipole, hydrogen CO 2 in ambient air. bonding, aromatic, and hydrophobic interactions. www.thermoscientif c.com/deltaray www.unitedchem.com thermo Fisher scientif c, Massachusetts, usA. uct, LLc, Pennsylvania, usA.

96-well plate Emulation technology The TELOS Agilent Technologies has MicroPlate is a introduced the third version of 96-well plate for Intelligent System Emulation the extraction of Technology (ISET) for the 1290 drug molecules Inf nity Binary LC or 1290 Inf nity from small-volume Quaternary LC systems. According biological f uid to the company, ISET enables samples. It is the transfer of existing HPLC available as either amd UHPLC methods between loose wells or LC systems via a mouse click, populated plates, in 5- and 10-mg sorbent masses, and allows regardless of the brand, and delivers unchanged retention analyte elution in as little as 50 µL. The full range of TELOS neo time and peak resolution. Generic SPE sorbents (PRP, PCX, WCX, PAX, and WAX) are www.agilent.com available in this novel format. Agilent technologies, california, usA. www.kinesis.co.uk Kinesis, st Neots, uK.

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High temperature GPC Lc–Ms–Ms Tosoh Bioscience has Shimadzu has announced the launch introduced of the EcoSEC-HT, a the LCMS- compact high temperature 8050 triple (HT) GPC/SEC system. quadrupole The system is reported LC–MS–MS to provide stable instrument for thermostatization up to trace-level 220 °C. Autoinjector, quantitation in clinical research and other markets. The pumps, and a dual-fow instrument incorporates proprietary ultrafast technologies RI detector are integrated as well as a newly developed ion source and collision cell into a compact design. The technology. optional sample processing unit is reported to process up to 24 www.shimadzu.eu samples. TSKgel GMHHR- HT2 GPC columns were specifcally Shimadzu Europa GmbH, Duisburg, Germany. developed for use with the EcoSEC-HT. www.ecosec.eu Tosoh Bioscience GmbH, Stuttgart, Germany.

sEc–MALs uPLc system Wyatt has launched the miniDAWN The Acquity UPLC I-Class system from TREOS, a SEC–MALS Waters reportedly produces the accurate detector that can and reproducible separations for reportedly be coupled analyzing compounds that are limited in to any HPLC system amount or availability, providing the most or used off-line in a information possible while accelerating micro-batch mode for laboratory results. Pairing this ultra-low determining absolute dispersion system with Cortecs UPLC molecular weights and sizes of proteins and polymers directly. columns is said to deliver exceptional A dynamic light scattering (DLS) option enables it to determine levels of efficiency, performance, and the size of very small molecules (<8 nm) eluting from each throughput, resulting in narrower peaks slice of a chromatograph. The detector has been designed for and higher peak capacity. “plug-and-play” functionality. www.waters.com www.wyatt.com Waters corporation, Massachusetts, usA. Wyatt technology, california, usA.

Automated fltration HPLc material The Filtration option AkzoNobel has presented for the Gerstel its latest preparative HPLC MultiPurpose Sampler product, Kromasil EternityXT. (MPS) enables efficient An extension of the Eternity automated clean-up grafted technology platform, of up to 98 samples it is reported to have or extracts combined an extended lifetime for with other sample preparative applications. preparation steps or Based on a 10 µm Kromasil with introduction to 100 Å silica matrix, with an LC–MS–MS or GC–MS system. Filtration can improve the high mechanical stability, the company report that the reliability of the analysis and of the analysis system. Liquid EternityXT can be used at pH from 1 to 12, giving fexibility transfer is reported by the company to be performed with exact in large-scale applications optimization. control of fow and volume for highly reproducible results. www.kromasil.com www.gerstel.com Akzonobel, Bohus, sweden. Gerstel GmbH & Co.KG, Mülheim an der Ruhr, Germany.

46 LC•GC Europe January 2014

magentablackcyanyellow ES374316_LCE0114_046.pgs 01.09.2014 00:19 ADV Products

Ion chromatography Gas generators The modular 940 Peak Scientifc has introduced Professional IC Vario the Peak Precision Series, a system from Metrohm is modular gas generator system reported to be ideal for designed to be tailored to any research applications gas chromatography laboratory’s and routine use. The needs. The system eliminates the system is self-monitoring inconvenience of gas cylinders, – all system and while allowing separation and method parameters are analysis of samples and complex permanently checked mixtures with reportedly high against the respective limits and standards. Results can be accuracy in the most spatially traced back to every single step of the analysis including sample economic laboratories. preparation, making users prepared for even the toughest www.peakscientifc.com audits. The system enables in-line preparation of eluents of any Peak scientifc Instruments Ltd, Inchinnan, scotland. composition and concentration. metrohm.com Metrohm, Herisau, switzerland.

Lc columns Raptor LC columns from Restek are reported to combine the speed of superfcially porous particles with the resolution of highly selective USLC technology. This new species of column more easily achieves peak separation and faster analyses without expensive UHPLC instrumentation. The frst phase released — biphenyl — is reportedly ideal for bioanalytical work, and more phases will be released soon. www.restek.com/raptor restek corporation, Pennsylvania, usA.

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magentablackcyanyellow ES374613_LCE0114_047.pgs 01.09.2014 17:04 ADV EVENT NEWS

20th International Symposium on Separation Sciences 18–23 May 2014 (ISSS 2014) 38th International Symposium on Capillary Chromatography and 11th The jubilee 20th International Symposium on Separation Sciences (ISSS GC×GC Symposium 2014) will take place at the Hotel DAP in Prague, Czech Republic, from Congress Centre, Riva del Garda, Italy 30 August to 2 September 2014. The symposium will bring together specialists Organizers: Luigi Mondello, from all areas of separation science from around the world to partake in the Chromaleont, University of Messina, exchange of new ideas from both academia and industry. Italy The scientific programme will include plenary and keynote lectures presented by Tel: +39 334 3612788 leading scientists, contributed oral presentations, and poster sessions. The topics E-mail: [email protected] to be featured and discussed include: Miniaturized capillary and chip-based Website: www.chromaleont.it/iscc fluidic separation media; hyphenated and multi-dimensional techniques; method development; chemometric approaches; quality assessment; ultra-fast high-flow and high-temperature gas and liquid chromatography; and sample pre-treatment 9 July 2014 and preparation. In addition, advances in column technology will be addressed Recent Advances in focusing on micro- and nano-particle, core–shell, and monolithic technologies. the Applications of Other topics that will be covered range from new stationary phase chemistries for Chromatography-Mass high hydrophilic interaction liquid chromatography (HILIC) and chiral separations, Spectrometry to Environmental to molecularly-imprinted media and stationary phases providing a dual retention Matrix Analysis mechanism, with an emphasis on novel application methods, especially in food, British American Tobacco Group clinical, and environmental analysis. Special emphasis will be put on novel Research and Development, application methods, especially in food, biomedical, and environmental analysis. Southampton, UK The following eminent scientists have been confirmed as presenters at Organizers: Environmental Mass the symposium: D. Berek; T. Bolanca; B. Buszewski; V. Coman; D. Corradini; Spectrometry Special Interest Group F. Foret; M. Gertsiuk; M. Gilar; M. Holčapek; V. Kašicka; I. Klebovich; J. Lehotay; of the BMSS W. Lindner; M. Macka; L. Mondello; Mx. Novotný; P. Sandra; P. Schoenmakers; Tel: +44 (0)161 973 7032 F. Švec; and I. Vovk. E-mail: [email protected] The 20th ISSS symposium will be a unique opportunity for exhibitors to Website: www.bmss.org.uk display their products and services to the participants. A poster competition will be organized for young scientists and students. Last but not least, a rich social programme will be organized, including a symposium dinner, boat trip, and a Send any event news to Bethany sight-seeing walk around Prague. Degg: [email protected] E-mail: [email protected]; Website: www.isss2014.cz

LCGC ON-LINE HIGHLIGHTS

Q&A: Marine Pollution Reflecting on Innovation LCGC Blog: The Heat is On! Chris Reddy gave LCGC the run-down It may be a new year but there is still LCGC blogger Tony Taylor gave his on the ongoing time to review insights on how analysis of the the instrumental to develop a Deepwater Horizon innovations of 2013 more logical oil spill, and how with this selective aproach to GC comprehensive review from The temperature GC×GC works in Column. programme practice. bit.ly/1jXp1G3 development. bit.ly/1gbdKyJ bit.ly/19aLoWD

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48 LC•GC Europe January 2014

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magentablackcyanyellow ES374190_LCE0114_049_FP.pgs 01.08.2014 23:33 ADV THE ESSENTIALS Optimizing LC–MS and LC–MS–MS Methods An excerpt from LCGC’s e-learning tutorial on optimizing LC–MS and LC–MS–MS methods.

Many parameters within liquid and it will change with the analyte analyte in solution (eluent pH higher K chromatography–mass spectrometry type, eluent system, and flow rate. This than analyte p a for acids and lower (LC–MS) methods are “locked and left” parameter is perhaps the most overlooked for bases). This can result in orders of — that is, they are optimized using a few variable in terms of improving instrument magnitude improvement in instrument methods when new, then only changed response and one should consider sensitivity; however, it may also require if absolutely necessary because of optimizing to improve not only sensitivity an adjustment in high performance poor analytical performance. This is fine but also reproducibility. At a higher liquid chromatography (HPLC) operating when methods are performing as they applied potential, one should be cautious conditions to properly retain the ionized should be and producing data which is to assess quantitative reproducibility analyte or reoptimize the separation fit for purpose, but the following question because there are various nonideal spray selectivity. The use of ion-pairing reagents should also be asked: Do you know when modes that will give rise to a signal but will such as trifluoroacetic acid should be your data aren’t as good as they could result in variable ionization efficiency. avoided and volatile buffers chosen so K be? Here we consider some parameters The efficiency of ion sampling from that the buffer p a is within ±1 pH unit of in LC–MS that can be optimized to within the electrospray will vary widely the eluent system pH. improve sensitivity and reproducibility in with analyte type, applied voltage, solvent Ion suppression or enhancement LC–MS. system, drying gas parameters, and eluent effects occur when there are species that The most significant choice in many flow rate. The position of the sprayer strongly compete with, or are suppressed LC–MS methods is the mode of ionization. relative to the sampling orifice (both axially by, the presence of your analyte. When The generally accepted rule is that and laterally) should be optimized when quantitative sensitivity is poor or the electrospray ionization (ESI) works best the highest sensitivity is required. analyte signal is highly irreproducible, for higher-molecular-weight compounds Nebulizing gas flow rate and heating one should investigate the effects of the that are more polar or ionizable, and requirements will change with the nature eluent system and matrix on the instrument atmospheric pressure chemical ionization and flow rate of the eluent, and, in response. These effects can often be (APCI) is best for lower-molecular-weight, general, smaller droplets are preferred overcome by altering the eluent system to less-polar compounds. Atmospheric within ESI experiments to improve reduce the ionic strength or change the pressure photoionization (APPI) was the efficiency of the droplet charging buffer type, or by more studious sample originally designed to work with less-polar process. When changing eluent systems preparation, designed to isolate the analyte analytes, however the careful choice (especially the amount or nature of the from the interfering matrix component. of dopant or the use of direct ionization organic component) or flow rate, consider The vast majority of LC–MS instruments sources can significantly extend the optimizing the nebulizing gas settings. are capable of analyte declustering, capability of APPI techniques. In all API Similarly, drying gas requirements will especially with respect to water molecule source types there are a multitude of change with the changing nature of the clusters. The application of an accelerating interrelated factors that affect the degree eluent system (including during gradient voltage in the first stage of the mass K of analyte ionization, including analyte p a analyses where the range of organic spectrometer to cause low energy value, electronegativity, relative proton composition alters greatly [large ΔΦ]) collisions with background molecules can affinity, ionization energy, and volatility. and should be optimized especially often lead to an improvement in instrument “New” analytes should be screened when eluent systems are highly aqueous. sensitivity, primarily because of a reduction using all available techniques to optimize Electrospray efficiency is often improved in noise around the analyte signal. instrument response. Furthermore, by adding “dopants” such as isopropanol When performing MS–MS analysis, although the “polarity” of the interface to allow a reduction in droplet surface it is important when seeking optimum may seem obvious when screening lower- tension; however, note that the sprayer sensitivity and quantitative accuracy to molecular-weight acids or bases, one position, nebulizing gas, and drying optimize the collision energy as well as can often be surprised at which polarity gas settings may need to be altered for the “dwell” time between measuring ion mode gives the optimum signal with more- optimum signal response. transitions to avoid “cross-talk” effects complex molecules. Be sure to screen In ESI, analytes may be charged that can significantly affect quantitative analytes in both polarity modes to ensure through processes such as charge measurements. optimum response, especially when transfer or triboelectric charging at More Online: the analyte is not ionizable, and avoid the capillary tip. Although this may “guessing” which mode might be best. produce a satisfactory response, one Get the full tutorial at

The capillary (sprayer) voltage can have should consider adjusting the eluent www.CHROMacademy.com/Essentials (free until 20 February). a major effect on efficiency of ionization, system to give the ionized form of the

50 LC•GC Europe January 2014

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