John Dolan has been writing "LC Troubleshooting" for 

 for over 25 years. One of the industry's most respected professionals, John is currently a principal instructor for LC Resources, Walnut Creek, California He is also a member of 

How to choose another column to try to solve a separation problem.

In the first two instalments of this series, we've looked at different factors that influence selectivity in reversed-phase liquid chromatography (LC) separations. First, we saw some of the benefits of changing solvent type,

  such as a change from methanol to acetonitrile or tetrahydrofuran. Next, we looked at how a change in the solvent strength

  — the percentage of organic solvent in the mobile phase — was an easier adjustment to make and often had suitable "leverage" to pull apart problem peak pairs. This month's "LC Troubleshooting" will focus on another common technique used to change peak spacing in a chromatogram: changing from one column type to another. 

When I first started using LC, it was thought widely that all C18 columns would give equivalent separations. In fact, the 

(USP) in its column classification scheme classifies C18 columns as "L1 — octadecyl silane chemically bonded to porous silica or ceramic micro-particles, 1.5 to 10 µm in diameter, or a monolithic silica rod".

  Over the past 40 years, however, we've all come to recognize that all C18s are not created equal — many will give dramatically different separations. In the current listing, the L-classification scheme contains 74 different column types,

  including C18, C8, phenyl, cyano and many others. Even columns that have the same label, such as C18 or phenyl, may have significantly different selectivity characteristics. It is not clear how many different reversedphase columns are available, but by some estimates there are at least 1000.

  In our work at LC Resources, we've tested more than 500 different reversed-phase columns. 

So how different are C18 columns from each other? To illustrate this, I've chosen a set of six test compounds that should be fairly well-behaved (see caption of Figure 1 for their identities). That is, at low pH (pH 2.8), they are neutral or un-ionized, so ionic interactions with the column should be minimal. I arbitrarily chose three C18 columns from our database.

  These are from well-known sources and are all type-B, high-purity silica columns. These are simulated separations on 150 mm × 4.6 mm columns packed with 5 µm particles and generating 10000 plates. For simplified comparisons, I've normalized retention to the first peak (anisole). The first column [Figure 1(a)] is our reference column. You can see that peaks 4 and 6 overlap and the resolution is marginal for peaks 2 and 3. If we change to another manufacturer's C18 column [Figure 1(b)], the separation of peaks 2 and 3 is improved, and the separation of the last three peaks has changed, but it is no better than the reference column. A third manufacturer's C18 [Figure 1(c)] is no better — it shows a good separation for peaks 2 and 3, but peaks 4 and 6 still overlap. You can see in Figure 1 that, although the first three peaks are separated by all three columns, the selectivity (relative peak spacing) is different for these peaks. And none of the three columns can separate the last three peaks — even the peak order changes! So you can't arbitrarily swap one C18 for another and expect the same results. But on the other hand, the differences aren't enough that we can make the same change and expect to solve a separation problem. Although there is likely at least one manufacturer's C18 column that will separate all six compounds, changing from one to another in an effort to get the desired separation is not a very good strategy.

 Figure 1: Comparison of simulated chromatograms for C18 columns from three manufacturers. Column dimensions: 150 mm Ã 4.6 mm, 5 Âµm particles; mobile phase: 50:50 acetonitrileâ30 mM phosphate buffer (pH 2.8); temperature: 35 Â°C; column efficiency N: 10000. Retention normalized to peak 1. See Table 1 for column information and text for details. Peaks: 1 = anisole, 2 = n-butylbenzoic acid,  3 = toluene, 4 = mefenamic acid, 5 = ethylbenzene, 6 = trans-chalcone.  

Another technique that has been around as long as I have been doing chromatography is to change the stationary phase. That is, users change from a C18 to a phenyl or cyano or embedded polar phase. We can use the same sample and conditions to see what happens in this case. I've chosen columns from the same manufacturer as the reference column of Figure 1(a). This separation is repeated in Figure 2(a) (with a slightly extended time axis). The sample was then run on the same brand of column from the same manufacturer, but with a phenyl stationary phase for the separation of Figure 2(b). The separation of the phenyl column might be expected to be much different from the C18, because there is aromatic nature to the sample components. The phenyl column separates all the peaks, but not to baseline. We could probably adjust the mobile phase or reduce the particle size (or both) to get baseline separation with this column. Figure 2(c) shows the separation on the same manufacturer's embedded-polar-group (EPG) phase. This column provides a good separation of all the peaks, certainly the best so far. Notice also that peaks 2 and 3 are reversed with the EPG column. The differences between the columns are assumed to be only the stationary phase — the silica particle chemistry should be the same. This technique, of changing to a different stationary phase from the same manufacturer, is one that many people have had success with over the years, but it still involves trial and error, without a certainty of a satisfactory separation.

Figure 2: Comparison of chromatograms for (a) C18, (b) phenyl and (c) embedded-polar-group columns from same manufacturer. Other conditions as in Figure 1;  see text for details. 

Still another way to pick a column that is likely to have different selectivity is to use the column database on the USP website.

  and elsewhere (for example, see reference 8), this database with more than 500 reversed-phase columns can be used to pick similar (equivalent) or different (orthogonal) columns compared to some reference column, such as the one you have already tried. By choosing the "view different columns" option, you can see what columns are most different from the reference column. I tried this out for the present sample containing some acids and neutral compounds at low mobile-phase pH. As my reference column, I chose the same C18 column we used in Figures 1(a) and 2(a), and have repeated the separation as Figure 3(a) (different time axis than the other figures). One of the most orthogonal columns with type-B silica particles (use of the older, low purity, type-A particles is discouraged for new separations) is the one used to obtain the separation shown in Figure 3(b). This is an EPG column from a different manufacturer than the reference column of Figure 3(a). So we've taken advantage of both a change in the stationary phase and a change in the silica source for added selectivity potential. The change is dramatic. Peak 5 has moved forward between 1 and 2, and peak 6 has moved up between 2 and 3. This is definitely a change in selectivity. There is no guarantee that the recommended column will separate all the compounds in your sample, but there is a high probability that the separation will look much different, especially if you take advantage of other selectivity variables. In one collaborative study,

  several pharmaceutical laboratories tested this approach on typical samples that they encounter, and in all 11 of the cases checked, the predicted orthogonal column indeed gave a significantly different separation.

Figure 3: Comparison of separations obtained using (a) a C18 column from one manufacturer with (b) an embedded-polar-group column from a second manufacturer. Other conditions as in Figure 1; see text for details. 

We can make visual comparisons and see that, for the present sample, a change from the column of Figure 1(a) [same as Figures 2(a) and 3(a)] to the column of Figure 2(c) or 3(b) would allow us to separate all the peaks. But which of these separations gives us the largest change? When comparing the selectivity changes when a change in column is made, it is nice to have a quantitative measure of the change. One way to do this is to make a graphic comparison of selectivity. This can be achieved by plotting the retention on one column vs the retention on a second column. An example of this for the columns of Figure 1(a) and 1(b) is shown in Figure 4(a). (Note that plots like this are most useful if we plot the log of the retention factor, 

, for each column.) The scatter about the trend line in the plot is a measure of how different the selectivity is between the two columns. The data cluster tightly about the trend line, and 

 = 0.97 (Table 1) confirms this. Contrast this result to that of Figure 4(b), where the columns of Figure 3(a) and 3(b) are compared. Here, there is much more scatter (

 = 0.49), showing that the selectivity is very different between the two columns.

Figure 4: Plots of log k vs log k for sample of Figure 1. (a) Column of  Figure 1(a) vs 1(b); (b) column of Figure 3(a) vs 3(b). Other conditions as in Figure 1; see text for details. An actual calculation of the change in selectivity, a, is an even better way to compare two columns. Recall that   

 are the retention factors for two peaks. The change in log a between the two columns is related to how far the data points are from the trendline in plots of log 

, as in Figure 4. The larger the average change in log α, |δlog α|

, between two columns, the more different the chromatogram will appear. This measure of change in selectivity, |δlog α|

, is directly related to the standard error (SE) of the plot as 

so it is easy to calculate from plots such as Figure 4. Other work has shown that when selectivity is changed in a reversed-phase separation, whether by a change in column or some other variable (for example, solvent type or solvent strength), a value of |δlog α|

 ≥ 0.1 gives a high probability that the separation will be orthogonal (or significantly different).

  For the present examples, I have made these calculations for the various columns when compared to the reference column [Figures 1(a), 2(a), 3(a)] and summarized them in Table 1. You can see that the column of Figure 2(c) almost reaches the desired change, but that the column of Figure 3(b) has a value of |δlog α|

 that is more than twice as large as the minimum recommendation. This confirms once more our visual observation of the separation that is most different from the original.

Table 1: Comparison of column selectivity. 

  gives still another measure of differences in column selectivity. This is the 

-value calculated from the column characteristics in the database.

  In one study with 67 different compounds of widely varying structure, including acids, bases and neutrals, it was determined that 

  This means that the database can be used to look for columns with large 

-values compared to the current (reference) column, thus giving you confidence that peak-spacing changes will be significant. I have tabulated the 

-values in Table 1 for the six columns we have been examining and the selected sample (neutrals and un-ionized acids). You can see that the column of Figure 3(b) has 

 = 55, whereas all of the other columns have 

s ≤ 16, once more confirming the visual observations that this is the most orthogonal column of the set. 

We have seen that a change in column chemistry can be a powerful tool to change selectivity in a reversed-phase separation. A change from one manufacturer's C18 to one from another manufacturer generally gives marginal gains, as was seen in Figure 1. A more effective way to make the change is to try a different stationary phase. If this change is made within one product line of columns, where the same silica support material is assumed, the change will result from a change in the bonded phase alone. This was shown in Figure 2 for three different bonded phases from the same product line from a single manufacturer. To gain even more leverage in the separation, a change in the base silica along with a change in bonded phase can be made. Although this may be possible within one manufacturer's products, such as by changing to a different bonded phase from a different product line of columns, such a change is more likely if the change is made in both the manufacturer and bonded phase, as shown in Figure 3.  

Using a change in the column to change peak spacing comes with risks of making a poor choice [as in Figure 1 or Figure 2(b)]. Such results also can be costly, because columns are expensive and research time is valuable. A more productive technique is to use some foreknowledge of selectivity differences between specific columns, such as that available in the USP database.

  By using the database, it is easy to identify columns that are most likely to have different selectivity.  

When exploring changes in selectivity, it probably will be more practical to explore solvent-type selectivity

  before column-type selectivity, just because it is less expensive. However, armed with the information we've covered in this discussion, you can make intelligent choices about a change in column, so you can increase the return on the investment in a new column in an effort to change selectivity. When this is taken into account, you may find that screening several columns of different selectivity is a fruitful investment of time early in the separation development process. 

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

 editorial advisory board. Direct correspondence about this column should go to to 

 editor, Alasdair Matheson, at Advanstar Communications, Bridgegate Pavillion, Chester Business Park, Wrexham Road, Chester CH4 9QH, UK, or e-mail 

8. L.R. Snyder, J.W. Dolan and P.W. Carr, 

LC Troubleshooting

Tips on what to consider when choosing another column to try and solve a tricky separation problem.

Selectivity in Reversed-phase LC Separations (Part 3): Column-type Selectivity

The first two articles of this series on green chromatography from the Research Institute for Chromatography (RIC) covered green liquid, gas and supercritical fluid chromatography in practice. Part 3 focuses on sample preparation, potentially the most polluting step of any analytical method because of the high quantities of organic solvents used.   

Great efforts have been made to develop sample preparation techniques that guarantee high recovery and reproducibility and that are also greener, faster, cheaper and easier to automate than older methods. Other green trends are on-site or in-field analysis and the development of sample preparation methods that allow multi-residue determination and/or methods in which several sample preparation processes are combined in one step. Portable and transportable GC and GC–MS systems are now commercially available for on-site analysis. All those methods are contributing to green chemistry because the expensive, energy-consuming transport of samples to laboratories is reduced. On-site sampling also has the advantage that remediation or re-sampling can take place immediately after the first analytical results are obtained.

The advent of advanced mass spectrometric (MS) technologies in recent years had several consequences on our analytical work including the sample preparation step. On the one hand, new and more stringent requirements were formulated by regulatory agencies and more and more samples have to be prepared and analysed. On the other hand, because of the high sensitivity and selectivity of mass spectrometers in GC–MS–MS and LC–MS–MS combinations, sample preparation can be simplified and miniaturized. In the past, enrichment was of vital importance in the sample preparation step because samples were too dilute for direct analysis and needed to undergo a chain of specific treatments to make them compatible with the sensitivity of the analytical techniques used. With present state-of-the-art instrumentation, detectable and quantifiable quantities are in the pico- and femtogramme range and the dictum "the best sample preparation is no sample preparation"

  can be re-considered and re-evaluated. 

 Figure 1: Analysis of pesticides in a spiked surface water by (a) direct LCâMSâMS (b) in-line SPEâLCâMSâMS. Peak numbers (ionization mode, precursor and product ions): (1) Metamitron (+, 203.1, 175.1), (2) Dicamba (â, 221.0, 177.0), (3) Fluroxypyr (â, 253.0, 194.9), (4) 2,4-dinitrophenol (â, 183.0, 108.9), (5) Lenacil (â, 233.1, 150.9), (6) Bentazon (â, 239.0, 132.0), (7) 2,4-D (â, 218.9, 160.9), (8) Bromoxynil (â, 275.9, 78.9), (9) MCPA (â, 199.0, 141.0), (10) DNOC (â, 197.0, 180.1), (11) 2,4-DP (â, 218.9, 160.9), (12) 2,4,5-T (â, 252.9, 194.9), (13) MCPP (â, 213.0, 141.0), (14)  Dimethomorph (+, 388.1, 301.0), (15) MCPB (â, 227.0,141.0), (16) Dimethenamide  (+, 276.1, 244.0), (17) Tebuconazole (+, 308.2,70.0).			

A typical application is the multi-residue determination of pesticides in drinking and surface waters. Figure 1(a) shows the direct analysis of a surface water spiked with 17 target pesticides at 50 ng/L. Before injection, the water sample was acidified with 0.2% formic acid and filtered through a 0.45 µm cellulose filter. The injection volume was 400 µL. The column was a Zorbax Eclipse Plus C18 with 10 cm × 2.1 mm i.d., 3.5 µm particles (Agilent Technologies, Wilmington, Delaware, USA) and a gradient from 10% B to 79% B in 8 min (A = 0.05% formic acid in water, B = acetonitrile) was applied. All spiked pesticides could easily be elucidated and quantified in one run by using alternating positive/negative electrospray ionization with JetStream technology (Agilent Technologies, Santa Clara, California, USA) and multiple reaction monitoring (MRM). Limits of quantification (LOQs) are in the ng/L level for the acidic pesticides (negative mode) and in the subng/L level for the basic pesticides (positive mode). In the 1990s, these sensitivities could only be obtained using labour- and time-consuming sample preparation methods, such as solid-phase extraction (SPE) of a 1 L sample and concentration to 1 mL (enrichment factor 1000), followed by LC-diode array detection (DAD) analysis. Even lower concentrations (ca. tenfold) can be measured by combining SPE placed in-line with LC–MS–MS and using the LC configuration shown in Figure 2. Figure 1(b) shows the analysis of 5 mL of the same water sample spiked at 1/10 compared with Figure 1(a) on the same LC–MS–MS system but equipped with a column switching valve containing up to six Oasis HLB cartridges with 20 mm × 2.1 mm i.d., 25 µm particles (Waters Corporation, Milford, Massachusetts, USA) and an extra pump for loading the cartridges. After sample loading, the cartridge is desorbed using the analytical LC pump and the enriched solutes are analysed. By optimization of LC parameters, peak distortion can be avoided, resulting in very similar resolution to direct water injection, while the sensitivity is increased by a factor of 10 to 12.

Figure 2: Configuration for on-line SPEâLCâMSâMS; Load (SPE): Sample enrichment onto SPE cartridge, Elute (Analysis): Liquid desorption of the SPE cartridge and LCâMSâMS analysis.			

SPE, in its different forms, is nowadays a key component in sample preparation and will be discussed further. It not only removes matrix components that can interfere in MS ion formation (ion suppression) but also selectively enriches the solutes of interest (targets) with a minimum quantity of organic solvent. GC–MS–MS and LC–MS–MS have revolutionized our sample preparation strategies. Systems are now available with complete automated data handling to determine around 600 endocrine disrupting chemicals (EDCs) by GC–MS–MS or more than 300 nonGC amenable pesticides by LC–MS–MS using two transitions and dynamic MRM. We recently described several green sample preparation methods [direct injection, automated off-line SPE and miniaturized liquidliquid extraction (LLE)] that were used in combination with LC–MS–MS for the determination of biogenic amines, mycotoxins, stilbenes, flavonoids, tannins, anthocyanins and phenolic acids in wines and grape juices.

In the framework of this relatively short contribution on green sample preparation methods, it is impossible to review all achievements made in recent years. We refer the readers to some publications dealing with green sample preparation techniques for environmental analysis

  Some new methods will be discussed in depth and illustrated with examples from the laboratories of the authors. A subdivision is made based on the nature of the compounds and the analytical techniques commonly applied for their analysis such as (1) volatile organic compounds (VOCs) that can be analysed by GC via headspace (HS) techniques and (2) semi-volatile organic compounds (SVOCs) that are GC-amenable and nonvolatile (NVOCs) or thermally labile compounds for which LC is the technique of choice.  

Last but not least, a number of fully automated sample preparation techniques are described. Automation is very often accompanied with miniaturization and thus solvent reduction. 

Gas phase sampling is the greenest technique available since inert gases are used as the extracting phase. Moreover, only volatiles are introduced in the GC instrument and column, guaranteeing minimal maintenance costs and long column lives. Different approaches are available for gas phase sampling and the selection is most often dictated by the sensitivity required. Best known are static headspace (SHS), in-tube extraction (ITEX), dynamic headspace (DHS), purge and trap (P&T), solidphase microextraction (SPME) and headspace sorptive extraction (HSSE). Gas phase sampling is intensively used in environmental and food analysis, and recently in metabolomics (breath analysis).

In recent years, the applicability of gas phase extraction has been expanded to the analysis of semi-volatiles by performing the extractions at higher temperatures followed by trapping on a sorbent or a cryotrap to focus the solutes before injection. This solventless technique often called gas phase stripping can be applied, for example, to extract polycyclic aromatic hydrocarbons (PAHs) from dust or soil, residual monomers, antioxidants and stabilizers from plastics, etc. An application developed at RIC concerns the determination of aldehydes, hydrocarbons, free fatty acids, vitamins and sterols in vegetable oils. Fractionation and enrichment of these classes out of the apolar triglyceride matrix is a challenging task.  

The sampling procedure is as follows: 10 mg vegetable oil placed in a glass microvial is introduced in a thermal desorption unit (TDU). Under a carrier gas flow of 100 mL/min helium, the tube is heated from 25 °C to 250 °C at 60 °C/min and maintained at 250 °C for 20 min. The gas phase extract is focused in a programmed temperature vaporization (PTV) injector at -50°C. The cryotrap is then heated at 12 °C/s to 310 °C (5 min) with a split ratio of 1/10 and a helium flow of 1 mL/min. The products are swept in the column and analysed by GC–MS on a HP-5MS 30 m × 0.25 mm i.d., 0.25 µm d

 column (Agilent Technologies, Folsom, California, USA) with a temperature programme from 50 °C to 300 °C at 8 °C/min. Figure 3 shows a typical chromatogram for an extra-virgin olive oil batch. Repeatability of thermal extraction analyses is below 10% relative standard deviation (RSD). 

Figure 3: GCâMS chromatogram of gas phase stripping of olive oil. Peak numbers: (1) Aldehydes, (2) Fatty acids (palmitic and oleic acid), (3) Squalene, (4) Vitamine E,  (5) Sterols.  			

Based on gas phase extraction, we recently described a novel method for the determination of PAHs in ambient air.

  The method is based on active sampling on sorption tubes consisting of polydimethylsiloxane (PDMS) foam, PDMS particles and a TENAX TA bed. After sampling, the solutes are quantitatively recovered by thermal desorption and analysed by capillary GC–MS. The new sampling method has been compared with the classical method using high-volume sampling on a glass fibre filter followed by polyurethane foam (PUF) for 24 h sampling of ambient air. Volumes enriched were 144 L on the mixed bed and 1296 m

  with the classical method. The latter method required high volumes of dichloromethane to recover the PAHs from the glass fibre and PUF filters while the sorptive method is solventless. The concentrations measured using the new method were significantly higher that the values obtained using the classical method, (i.e., a factor 1.2 to 3 higher for the high-molecular-weight PAHs and up to 35 times higher for naphthalene and 23 times for acenaphthene). The total toxicity equivalence (TEQ) value for PAHs was around two times higher compared with the conventional method, illustrating that the concentrations of PAHs in ambient air have been underestimated until now and that miniaturization of the sample preparation step may result in improved accuracy and precision. In the same framework, we recently illustrated that nitroPAHs, which are present in a concentration range of ca. 100 times lower compared with the PAHs in air, can be determined by GC–MS–MS analysis without any further fractionationconcentration step.

  This is due to the unique specificity characteristics of MS–MS.  

Semi-volatile and Non-volatile Organic Compounds

The matrixes in which SVOCs and NVOCs have to be determined include solid and liquid samples. The first step is a solvent extraction step to enrich the target solutes from the matrix. Extraction is commonly followed by a clean-up or fractionation step to selectively remove interferences that disturb the chromatographic or mass spectrometric analysis. 

Well known methods for extraction of solutes from solid matrixes include shake flask extraction, Soxhlet extraction (introduced in 1850!), and its modern forms Soxtec and Soxtherm. The techniques often use hazardous solvents for exhaustive extraction. More environmentally friendly techniques include ultrasonic extraction (UE), supercritical fluid extraction (SFE), pressurized liquid and accelerated solvent extraction (PLE and ASE), microwave assisted solvent extraction (MASE) and matrix solid-phase dispersion (MSPD). 

UE uses mechanical energy in the form of shearing action that is produced by a low-frequency sound wave. The sample is immersed in an ultrasonic bath with a solvent and subjected to ultrasonic radiation for around 15 min. Advantages over other techniques are simplicity, productivity and low solvent consumption. The excellent properties offered by a supercritical fluid, namely tuneable solvating power (selectivity), high diffusivity and low viscosity, have resulted in analytical usage for many years. The green carbon dioxide (CO

) eventually containing some percentages of methanol or acetone, are most often applied. In recent years enthusiasm on SFE has declined as the disadvantages became clearer. The most important disadvantage is the complexity of the extraction procedure which is strongly matrix dependent and needs careful optimization. A procedure developed for one particular matrix does not automatically work for another matrix. PLE encloses a number of extraction techniques that use solvents at elevated temperatures and pressures. Best known is ASE that originates from SFE and uses organic solvents at high temperature and high pressure to leach out the organics from solid matrixes. Compared with extractions at or near room temperature and at atmospheric pressure, ASE delivers enhanced performance by the increased solubility, the improved mass transfer and the disruption of surface adsorption by the conditions applied. Solvent consumption is typically 1/20 of a conventional Soxhlet extraction. Disadvantages of ASE are the lack of selectivity which means that often further clean-up is needed and that the sample is too dilute for direct analysis and concentration is required. 

Recent developments to circumvent these shortcomings are the combination of ASE with SPE and the application of large volume injection (LVI). Another variant of PLE is extraction at high temperatures and pressures with water. Super-heated water extraction (SHWE) including many applications was recently reviewed by Smith.

  Although SHWE is by far the most "green" extraction technique, the main disadvantages of SHWE over ASE are that the solutes are obtained in a dilute aqueous medium and further extraction with an organic solvent is required; that a large number of matrix compounds are extracted as well so that further clean-up is needed; and, last but not least, that the thermal stability of the target solutes under SHWE conditions should be carefully evaluated. MASE uses electromagnetic radiation to desorb organics from their solid matrices. MASE typically operates at 2.45 GHz. In recent years, different systems have become commercially available and they are based on extraction in a closed high pressure vessel with microwave-absorbing solvents, extraction with a non-microwave-absorbing solvent in an open vessel and/or extraction with a non-microwaveabsorbing solvent in a closed vessel applying a micro-wave-energy-absorbing Weflon stir bar (Milestone S.r.l., Sorisole, USA) that heats the solvent. The performance of MASE has been compared with other techniques such as ASE and SFE and similar recoveries were obtained for soil and sediment samples.

  The same disadvantages mentioned for ASE apply to MASE, namely lack of selectivity and the dilution effect. Moreover, care should be taken with solutes that are thermolabile or can rearrange under the influence of electromagnetic radiation.  

  is a powerful technique for disrupting and extraction of solid, semisolid and viscous samples. A small amount of sample is homogenized with bulk-bonded silica sorbent (typical ratio 1 to 4) with a glass pestle in a glass mortar. The shearing forces disrupt the structures of the matrix and disperse the sample over the surface of the sorbent. The homogeneous blend of sorbent and sample is then packed in a syringetype cartridge (like a SPE cartridge) followed by elution of targets and interfering compounds (or viceversa) by consecutive washings with appropriate solvents. A variation of MSPD is dispersive SPE (DSPE) as used in the Quick, Easy, Cheap, Effective, Rugged and Safe (named QuEChERS) multi-residue method for the determination of pesticides in different matrices (see further). 

In the European Community (EC), 1999 will be remembered as the year of the Belgian dioxin crisis. The dioxins (mainly polychlorinated dibenzofurans or PCDFs) originated from animal feed that was contaminated by used transformer oil and many thousands of food samples had to be analysed on polychlorobiphenyls (PCBs). During the crisis, all possible sample preparation techniques were evaluated and a high throughput "green" method based on UE and MSPD was developed. The implementation of the method was accompanied with validation/reproducibility studies and the analysis of certified samples. Samples were homogenised using a blender. From fat samples (chicken or pork fat) 1 g sample was weighed in a 20 mL vial. From eggs, 3 g egg yolk was taken. For animal feed samples or other meat products, a sample size corresponding to 200–500 mg fat was analysed. To the sample, 2 g anhydrous sodium sulphate and 10 mL petroleum ether were added. Tetrachloronaphthalene or octachloronaphthalene may be added as internal standard (IS) to the sample at this stage although external standardization can also be applied. The vial is closed and placed in an ultrasonic bath at 30 °C for 30 min. In this step, the fat and PCBs are transferred from the matrix in the petroleum ether phase, while sodium sulphate adsorbs the water present in the sample.  

After extraction, the sample is allowed to settle. An aliquot (typically 5 mL) is transferred to a test tube and another aliquot (2 mL) is used to gravimetrically determine the fat content of the extract. To the test tube, 2 g of acidic silica gel (44% sulphuric acid) is added and the tube is vortexed for 2 × 30 s. This technique is nowadays called DSPE. In column chromatography and in SPE, the analytes are eluted through a bed and the fat is retained. In DSPE, the fat matrix is allowed to bind on the adsorbent that is mixed with the sample, while the solutes of interest stay in solution. For the fractionation of fat from PCBs, this method works very efficiently. After settlement of the adsorbent or centrifugation, an aliquot of the clear solution is transferred to an autosampler vial. The final volume is not important, because the concentrations are calculated to the initial 10 mL solvent and the sample or fat weight. One technician can easily prepare more than 50 samples a day and the solvent quantity needed is substantially lower (ca. a factor of 20) compared with conventional methods in which, besides extraction, column chromatography has to be performed. The reproducibility and accuracy of the UE–DSPE method was evaluated with the determination of the PCB content in a certified reference material of the EC namely the mackerel oil sample CRM 350 (IRMM, Geel, Belgium). Analyses were performed by four laboratories to which the sample preparation method was transferred and using retention time locked (RTL) GC–MS. The results are summarised in Table 1. 

Table 1: Accuracy and reproducibility test for the mackerel oil sample.			

Extraction of liquid samples (water samples, beverages, biological fluids, organic solvent extracts obtained by extraction of solid samples, etc.) is based on partitioning into an immiscible extracting phase which can be liquidlike in LLE, gum-like in SPME using a PDMS fibre or solid-like in SPE.  

In environmental analysis, LLE is still carried out manually by shaking the water sample with an organic solvent such as dichloromethane or 

-hexane in a separation funnel. The volume of the extract is usually too large for direct injection and in order to obtain sufficient sensitivity, additional evaporation-concentration steps are necessary. Miniaturization of LLE has been the subject of intensive research in the last thirty years. In 1981, W. Jennings presented a rapid liquid-liquid extractor for very small solvent/sample volume ratios (typically 20 µL/40 mL) and applied the technique for analysis of an apple distillate concentrate.

  In fact this can be considered as the first application of single-drop microextraction (SDME) (see further). Micro LLE in the vial of an autosampler has been developed in the beginning of the 90s and illustrated for the extraction of haloforms from drinking water.

  Shortly after the introduction by Pawliszyn et al.

 of SPME (in fact liquidphase microextraction when PDMS is used as fibre coating) numerous micro-LLE methods were introduced. Jeannot and Cantwell,

  described SDME in 1997. The main disadvantage of the method is drop instability and a development trying to overcome this was liquid phase microextraction (LPME) using a membrane between sample and microdrop.

  A similar approach is used in membrane assisted solvent extraction (MESA) developed by Popp et al.

 and commercially available from Gerstel (Mülheim an der Ruhr, Germany) in a fully automated format. Extraction volumes used are typically 100–150 µL.  

In SDME and LPME it can take a relatively long time for equilibration to take place between the aqueous solution and the droplet. This drawback has been addressed by developing a method based on the dispersion of tiny droplets of the extraction liquid within the aqueous solution. The method named dispersive liquid-liquid microextraction (DLLME)

 is in essence close to cloud point extraction (CPE) and can be regarded as multiple-drop microextraction (MDME). In CPE, an involatile surfactant is added while in DLLME a disperser solvent such as diethylether or acetone is used and the extraction medium is a high-density solvent such as tetrachloroethylene. The extraction process can be optimized by using ultrasounds (USA-DLLME or ultrasound- assisted DLLME).

  Notwithstanding the merits of the above techniques, it is very difficult to validate and, moreover, to automate them.  

In SPME using PDMS or polyacrylate (PA), partitioning occurs between the gum-like liquid coated on a fibre and the aqueous solution. The analytes are then desorbed from the fibre in-line with the chromatographic system. SPME is a cheap, fully automated solventless technique that is widely used for preparation of food, environmental and bio samples before capillary GC analysis.

 Stir bar sorptive extraction (SBSE) is based on the same principles as SPME but the quantity of extracting solvent (PDMS) is much higher.

  In a typical experiment, a stir bar coated with 25 mg of PDMS is added to 10 to 250 mL of an aqueous solution containing the compounds of interest and extraction is carried out at 1000 rpm during 30 min. The stir bar is then removed from the sample and the analytes dissolved in the PDMS are released by thermal desorption for GC or liquid desoption for LC analysis. Limits down to the ng/L level can easily be reached. Pre-concentration is based on the log P value and for compounds with low log P values 

 derivatization can be applied. As an example, derivatization was used for the determination of bisphenol-A (BPA) in wash water of polycarbonate (PC) baby bottles. 

derivatization was carried out with acetic acid anhydride. The detection limit was 0.12 ng/L of BPA. PC baby bottles were subjected to simulated every day use and the influence of heating in a water bath and by microwaves has been studied.

SPE is increasingly being applied for isolating or enriching compounds from aqueous samples and is much faster, cheaper and more versatile than most classical techniques. When properly designed, SPE methods need minimum quantities of organic solvents to recover the targets. Moreover, SPE procedures are easily automated using robotics, or can be coupled on-line with chromatographic techniques. The principle of retention (enrichment) is analogous to LC and is suitable for low, intermediate and high polarity solutes. Commonly, SPE media are based on normal phase (NP), reversed phase (RP) or ion exchange chromatography (IEC). Other specific SPE supports include restricted access media (RAM), immunosorbents and molecularly imprinted polymers (MIPs). Giving an overview of the different SPE sorbents and their applications is not within the scope of this paper. Hundreds of SPE cartridges are commercially available and the catalogues of the manufacturers describe their features and applications. The principles and practice of SPE have been described in the book of Simpson (Ed.).

 The syringe type cartridge, packed with particles of 30 µm to 60 µm, is the most common SPE format. Solvent flow through is typically done using a commercially available vacuum manifold. In recent years, because of the performance of modern mass spectrometers in terms of sensitivity, SPE is intensively used to remove matrix compounds rather than to enrich targets. 

A variant of SPE is DSPE. In DSPE the sorbent is added to a solvent extract and unwanted compounds are retained on the sorbent while the target compounds remain in solution. DSPE was intensively used in 1999 in our laboratory for the Belgian dioxin crisis as described before and similar procedures are nowadays used in the QuEChERS multiresidue method.

  Today, the QuEChERS method is by far the best developed multi-residue method for a wide variety of pesticides in several common non-fatty and, since recently, fatty food matrices. DSPE is a key aspect of the method and the type of adsorbents, as well as the pH and polarity of the solvent, can be adjusted for differing matrices and difficult analytes. We recently described the determination of 40 pesticides in baby food using a QuEChERS kit for extraction and DSPE (Agilent Technologies) in combination with UHPLC–MS–MS.

  Detection limits were between 500 ng/kg and 10 ng/kg which is much lower than the maximum residue level (MRL) of 10 µg/kg imposed by the European Union. The green character of QuEChERS has recently been highlighted by the inventors of the technique with about 95% reduction in solvent consumption, 95% in consumable costs and 90% reduction in time

  compared with the traditional multi-residue method used in Europe for nearly 20 years. Automated DSPE clean-up for QuEChERS extracts has been developed and has recently become commercially available.

  One of the disadvantages of exhaustive extraction aimed at in QuEChERS is that the extract may still contain involatile matrix material that can have a detrimental effect on the chromatographic performance. This is especially the case when GC is the analytical method. Contaminated inlet liners lower the response of some pesticides affecting the accuracy and precision of the analysis. The performance can be restored by exchanging the inlet liner and/or cutting the inlet part of the column. An automated solution is provided by using an automated liner exchange system (ALEX).

SBSE is also applied as multi-residue sample preparation method for pesticide analysis.

  Compared with QuEChERS, SBSE has the advantage that the DSPE steps are omitted and that higher sensitivity is obtained (all extracted solutes can be quantitatively transferred to the column using thermal desorption). The limitation is that the log P value of the solutes should be at least 2 for good enrichment. A typical example is shown in Figure 4 for the determination of pesticides in a grape sample. 

Figure 4: SBSEâGCâMS ion extracted chromatogram (IEC) of pesticides in grapes. Peak numbers: (1) Procymidone, (2) Permethrin I, (3) Permethrin II. 			

The initial step can be similar to QuEChERS (i.e., extraction of 10 g homogenized sample with 10 mL acetonitrile). For SBSE, 1 mL of the extract is taken and diluted to 10 mL with water. A stir bar 10 mm long coated with 0.5 mm PDMS is added and the mixture is stirred for 60 min at 1000 rpm. After sampling, the stir bar is taken out with tweezers, dipped in distilled water, placed on lintfree tissue to remove residual droplets and finally put in a liner of a thermal desorption system. Splitless thermal desorption was performed by heating the tube from 40 °C to 280 °C (5 min) at a rate of 60 °C/min. The analytes were cryo-focused in a PTV at –150°C with liquid nitrogen prior to injection. An empty baffled liner was used in the PTV injector. For splitless injection (2 min) the PTV was ramped from –150°C to 280 °C (2 min) at a rate of 600 °C/min. Capillary GC analysis was done on a 30 m × 250 µm i.d., 0.25 µm df HP-5MS column (Agilent Technologies). The oven was programmed from 70 °C (2 min) at 25 °C/min to 150 °C, at 3 °C/min to 200 °C and finally at 8 °C/min to 300 °C. Screening of pesticides was performed using the automatic RTL screener software in combination with the Agilent RTL pesticide library. The RTL screener elucidated by ion extraction at 

= 283 and 183, procymidone (peak 1) and permethrin I and II (peak 2 and 3). The concentration levels were measured at 172 µg/kg (EC norm 5 mg/kg), 20 µg/kg and 83 µg/kg (EC norm for the sum 1 mg/kg), respectively. 

Fully Automated Sample Preparation Stations

Downscaling of sample amounts help tremendously not only in solvent savings but also in opening new perspectives for the development of fully automated sample preparation stations off-line or in-line with the chromatographic system.  

As an example, we described an automated method for the simultaneous determination of six important organotin compounds in water and sediment samples. The method is based on derivatization with sodium tetraethylborate followed by automated HS-SPME combined with GC–MS under RTL conditions. Deuterated organotin analogues were used as internal standards. The method was validated and excellent figures of merit were obtained. LOQs were from 1.3 to 15 ng/L for water samples and from 1.0 to 6.3 µg/kg for sediment samples.

 The method was successfully transferred to a number of environmental laboratories that received accreditation for organotin analysis in environmental samples. Other laboratories, on the other hand, could not implement the method because local regulatory authorities claimed that the used sample preparation technique, namely SPME, was deviating too much from liquid-liquid extraction described in the official method.

  An automated micro LLE-derivatization method was therefore developed for the determination of the two most important organotin compounds, namely tributyltin (TBT) and triphenyltin (TPhT) in water 

All sample preparation steps were automated by using a dual-rail autosampler MPS-2 (Gerstel). Sample and solvent volumes were reduced compared with the official method in order to fulfill the hardware requirements. Figure 5 shows the autosampler equipped with two liquid injection modules (100 µL and 1 mL syringes), two sample trays (2 mL and 20 mL vials), an agitator and two large wash stations for rinsing with ethanol, water and hexane. The GC–MS–MS is equipped with a PTV injector for LVI. 

Figure 5: Dual-rail configuration: (1) 20 mL vial tray, (2) 2 mL vial tray, (3) agitator, (4) large wash station (2x), (5) 100 ÂµL LVI syringe, (6) 1 mL syringe.			

In the reference method, derivatization is performed in 1 L water that is extracted with 20 mL hexane. The hexane layer is then evaporated to 1 mL. In the automated method, 5 mL water sample is pipetted in a 20 mL vial and 5 mL NaOAc/HOAc buffer (pH 5.3) solution is added. The closed vials are then put in the 20 mL vial tray. The programmed steps are presented in Table 2. 

Table 2: Automated sample preparation steps for the analysis of organotin compounds in water			

The robot performs the following basic steps: (1) addition of deuterated internal standards and, in case of calibration addition of native organotin compounds, (2) the 

, (3) LLE with hexane and (4) the transfer of the supernatant to a 1.4 mL sample vial. 50 µL is then injected in the PTV injector. After each addition step, the sample is shaken for a given time and put back into the sample tray. Separation is performed on a HP-5MS capillary column 30 m × 0.25 mm i.d × 0.25 µm d

 (Agilent Technologies, Folsom) in a temperature programmed run from 50 °C to 300 °C at 10 °C/min. TBT and TPhT together with their deuterated internal standards are measured in MRM-mode using the following precursor and product ions: TBT (291.2 to 178.9 and 291.2 to 122.8), TPhT (351.1 to 196.9 and 351.1 to 119.9), TBT-d

 (318.2 to 189.9 and 318.2 to 125.8) and TPhT-d

 (366.2 to 202.0 and 366.2 to 119.9). Figure 6 shows a typical chromatogram of a water sample spiked with the internal standards at 200 ng/L (ppt) and TeBT-d

 (tetrabutyltin) as surrogate at 400 ng/L. The concentrations of TBT and TPhT were 18 and 21 ng/L, respectively.

Figure 6: Automated analysis of organotin (TBT, TPhT) compounds in a water sample.			

 A similar dual-rail autosampler was applied for the fully automated analysis of genotoxic sulphonate esters in pharmaceuticals.

  In this application, the drug substance or reaction mixture is diluted in dimethyl sulphoxide-water. A solution of pentafluorothiophenol is added and the methyl-, ethyl- or isopropyl- pentafluorophenylsulphides are formed that can be analysed by static head space gas chromatography mass spectrometry (SHS–GC–MS). To do this, one robot arm is equipped with a liquid syringe for all liquid handling steps and one robot arm is equipped with a headspace syringe for HS sampling and injection in the GC–MS. Excellent repeatability and high sample throughput are obtained, allowing reaction monitoring and product quality control. 

Techniques that reduce or eliminate solvent consumption in the sample preparation step have been developed in recent years and are gaining in popularity. Implementation in laboratories is, however, not always straightforward and very often hindered by regulatory directives and timeconsuming (re)validation studies to obtain accreditation.  

Nevertheless, implementing these new developments, especially those that can be automated, into routine laboratories is an important step in greening analytical chemistry.  

To conclude, let's compare three methods that are currently used for the determination of PAHs in aqueous samples at the 10 ng/L level namely LLE, SPE and SBSE. In LLE, 500 mL water is extracted with 75 mL dichloromethane, the solvent is evaporated, the residue re-dissolved in 1 mL hexane and 2 µL, corresponding to 10 pg on the column, is analysed by GC–MS. In SPE, 100 mL water is sent over a cartridge packed with octadecylsilica or polystyrenedivinylbenzene particles and the PAHs are recovered in 5 mL acetonitrile or iso-octane. The solvent is evaporated and the residue reconstituted in 0.2 mL hexane and 2 µL, corresponding to 10 pg on the column, is analysed. In SBSE, a PDMS stir bar is added to 5 mL water and then thermally desorbed on-line with the GC–MS system. Quantity introduced is 50 pg on the column. The latter technique not only is solvent free, but (i) more sensitive, (ii) stir bars can be re-used for at least 50 samples and (iii) figures of merit are excellent.  

Article

Discussing the latest developments in sample preparation techniques that reduce environmental impact.

Green Chromatography (Part 3): Sample Preparation Techniques

John V. Hinshaw is senior staff engineer at Serveron Corp., Hillsboro, Oregon, and a member of LCGC's editorial advisory board. Direct correspondence about this column to "GC Connections," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

. For an ongoing discussion of GC issues with John Hinshaw and other chromatographers, visit the Chromatography Forum discussion group at 

Chromatographers have used valves for sample injection and flow redirection since the beginnings of gas chromatography (GC). In this issue, John Hinshaw begins a series of articles on valves for GC.

Analysts have used valves in gas chromatography (GC) since its beginnings. Today, requirements for precise control of gas streams are much more stringent than they were at the onset. From simple gas-sampling valves to electronic pressure control and flow-switched GC×GC modulators, valving and flow switching systems play a crucial role in any modern GC instrument. This "GC Connections" series on valves for gas chromatography will review and examine some of the ways in which valves enable classical and leading-edge applications. The first instalment presents some basic concepts, applications and implementations. 

Chromatographers did not always use the gas-sampling valves that are familiar to modern practitioners. The early pioneers of column-based gas separations — but not necessarily partition GC as we know it today — used instruments that were self-built out of laboratory glass and tubing. Any valves that were used were simple two- or three-way stopcock types that controlled gas direction and flow. In modern elution chromatography a discrete sample is introduced into the column to start a separation, but the early gas separation systems used displacement or frontal elution. A relatively large sample of 1 L or more was adsorbed onto the head of a column and then was displaced with a heavy solvent or with mercury vapour. The requirement to inject a defined sample plug that would generate discrete narrow peaks came with the development of GC — albeit based on retention by adsorption–desorption — in the second half of the 1940s at the University of Innsbruck, Austria, by E. Cremer and F. Prior. Their system used a collection of stopcock valves to introduce a discrete gas sample into a column that then produced recognizable peaks under carrier gas flow. In some other systems of that time a calibrated gas burette provided a measured sample volume; others used crude sample vaporizing inlets. It was not until commercial gas chromatographs became available in the early- to mid-1950s that the first modern gas-sampling valves appeared.

  These were of two types: a rotating tubular valve that has largely disappeared today, and the more familiar concentric rotary valve that now predominates. 

The ability to switch gas streams as well as use interchangeable sample loops to inject small sample volumes with a single movement of a valve actuator ensured the rapid adoption of dedicated gas-sampling valves nearly 60 years ago. A high degree of repeatability along with very low dead volumes and nonexistent sample carryover made the valves ideally suited for the (then) narrower ¼ and ⅛ in. coiled columns. In the time since, rotary valves have undergone significant development in material, construction and application range — including their crossover to liquid chromatography (LC) applications — but they remain fundamentally the same and would easily be recognizable today by the chromatographers of that time. 

Other classes of flow switching devices and applications arose later, in the 1960s and onward. These can be divided into two groups. First came the onset of devices that would lead eventually to what today we take for granted in electronic pressure control (EPC) and related systems. Second was the development of fluidic switching devices, often attributed to David Deans, that have made possible multidimensional and comprehensive GC×GC as well as a number of interesting ancillary flow switching applications. Work in these last areas currently occupies much of the ongoing research in GC; this series will finish with a discussion on the topic. Now let's get started with a look at the basics of rotary valves and their applications.  

Two types of gas stream-switching applications will serve to sketch the capabilities of rotary gas valves. There are many other possibilities for combinations of multiport valves, columns, inlets and detectors. This brief introduction will pave the way for a look at some more involved applications in the next instalment of this series. 

 One of the simplest valved applications is switching the outlet of one column between two detectors. Such a scheme might be used, for example, to change from thermal conductivity detection (TCD) of early, nonhydrocarbon peaks eluted from a porous polymer or molecular sieve column to using more sensitive flame ionization detection (FID) for later hydrocarbon peaks. Figure 1 illustrates a connection diagram for switched detectors. In Figure 1(a) a single pressure controller supplies carrier gas to a column and to a matched restrictor. The column exit is routed to the TCD system and the restrictor is connected to the FID system. The restrictor provides roughly the same flow-rate as the column during isothermal or temperature-programmed operation, which minimizes flow-related detector baseline upsets caused by switching between detectors. An injection device such as a sample loop or heated inlet, located between the carrier gas source and the column, has been omitted to simplify the illustration. 

Figure 1: Schematic diagram of detector selection by flow switching.  

In Figure 1(b) the detector connections have been switched. The column eluant now flows to the FID system and the restrictor to the TCD system. Peaks eluted from the column will be directed to the FID system until the detector flows are switched back to their original positions. So, early peaks such as nitrogen, oxygen or carbon dioxide could be detected by TCD, and later peaks such as methane, ethane and higher hydrocarbons could be detected by FID. Little baseline upset would be expected when switching because the matched restrictor would provide about the same carrier gas flow-rate as the column. Of course, if the detectors were switched midpeak then the signals would change abruptly.   

An implementation of this detector switching scheme can be realized with a multiport rotary valve. Figure 2 shows an exploded view of the inner parts of a simplified six-port rotary valve. The valve consists of a stationary body with small holes on a highly polished surface drilled through to zero-dead-volume connections that are positioned around the periphery of the valve body. A rotating valve core with three cut-away slotted arcs is pressed onto the stationary body and is made to rotate back and forth between two positions, driven by an external actuator shaft (not shown). The holes in the upper surface of the stationary body match up with the ends of the slots so that adjacent pairs of holes are connected by the slots. In one position — as shown in the figure — gas flows from connections 1 to 2, 3 to 4 and 5 to 6. When the core rotates 60°, gas flows from connections 1 to 6, 2 to 3 and 4 to 5. Other common arrangements include four-, eight- and ten-port varieties. 

Figure 2: Beveled rotary valve diagram: (a) a beveled rotating core with slots fits into (b) a stationary valve body with holes. The slots in the rotating core match adjacent pairs of holes in the stationary body. For a six-port valve the core moves back and forth across a 60Â° angle. Low-dead-volume connections are drilled through the stationary valve body into the internal holes.  

Rotary valve actuation is accomplished by a pneumatic or electronic valve actuator. A pneumatic actuator uses air pressure to move a rotor through the degrees of arc required by the particular valve in use. Table 1 lists the degrees of rotation for commonly used rotary valves. Valve actuators have limited degrees of rotation that should match the valve requirements. Although it is possible to use an actuator with a greater degree of rotation than the valve requires, such a configuration will wear out the valve and actuator prematurely due to stress on the actuator as well as on the valve shaft. An electronic actuator substitutes a stepper motor for the pneumatic motor and can provide more precise control as well as positive positional feedback. Electronic actuators can be controlled with encoded binary signals or by serial communications. 

Table 1: Degrees of rotation for rotary valve configurations. Valve actuators must match the valve's degree of rotation.  

The surface-to-surface seal between the rotating valve core and stationary body is critical both in preventing outward leaks from the gas streams to the atmosphere as well as in blocking atmospheric gases from leaking into the internal passageways. Locking out atmospheric gases is particularly important when analysing trace gas levels with extremely sensitive detectors. The sealing surfaces are angled in a way that increases gas pressure upper limits while reducing the force required to rotate the core, unlike a simple planar face-to-face arrangement of the rotating and stationary elements. 

Rotary valves are available in a wide variety of configurations with various pressure and temperature ratings. Typical maximum GC valve working pressures range from 689 kPa up to 2.76 MPa. Maximum temperatures range from 75 °C up to as high as 350 °C, although long-term operation at elevated temperatures will shorten the valve lifetime. Some valves, mostly the types rated for high temperatures, may not work well at subambient temperatures due to hardening of the valve-core material. In general, higher upper pressure limits are associated with reduced temperature ratings. Similar designs are used for LC applications, but with much higher pressure ratings of up to 34.5 MPa for conventional high performance liquid chromatography (HPLC) and as high as 138 MPa for ultrahigh-pressure liquid chromatography (UHPLC). 

Figure 3 illustrates the detector switching application with a four-port rotary valve. In position A the fourport valve connects ports 1 to 2 and 3 to 4. With the column connected to valve port 1 and the TCD system to port 2, the flow from the column is directed to the TCD system while the restrictor flow goes to the FID system. When the valve core is rotated by 90°, the port connections are exchanged. Now, the column flow leaves the valve at port 4 and goes to the FID system, and the restrictor flow leaves the valve at port 2 and goes to the TCD system. Returning the valve to its original position restores the initial flow conditions. 

Figure 3: Detector selection by flow switching: implementation with a four-port rotary valve.  

 Sample loop injection onto the beginning of a column is another common application for stream switching. A six-port rotary valve works quite well for this purpose; Figure 4 shows how this is accomplished. With the valve in position A, ports 1 to 2, 3 to 4 and 5 to 6 are connected. The carrier gas supply and column are attached to ports 1 and 2, respectively, so the carrier gas flows directly into the column in this valve position. A gassampling loop with a volume of about 10 µL to 5 mL is attached to the valve at ports 6 and 3, which then allows a gaseous sample to be introduced into the loop via ports 4 or 5. A gas-sampling syringe, a calibration gas cylinder, or another source of gas can be attached to the valve or to an external fitting that connects to the valve. 

Figure 4: Loop injection onto a single column with a six-port rotary valve.  

When filling the loop it is a good idea to allow at least 10 times the loop volume to flow through to fully purge the loop and connecting tubing of the existing gas content. Backdiffusion of atmospheric gases toward the loop can be mitigated by adding about 30 cm of 0.25 mm i.d. tubing at the sample outlet connection. For the best repeatability allow the loop pressure to decay for a few seconds after filling, so that the loop always reaches atmospheric pressure before injection. In some cases a backpressure regulator can be installed at the sample outlet to create a controlled, elevated loop pressure. Be sure, however, that the loop pressure always is less than the carrier-gas inlet pressure. 

Injection occurs when the six-port valve is moved to the position shown in Figure 4(b). Now the carrier gas flows first through the sample loop and then into the column, flushing the sample loop into the column inlet. The loop contents will be compressed to the carrier-gas inlet pressure, which can help to form a narrower injection profile. The valve must remain in that position for sufficient time to flush the loop completely. At a column flow of 10.0 mL/min with a loop size of 1.0 mL and an inlet pressure of 207 kPa, an injection time of 12 s would provide barely enough carrier-gas volume to flush the loop once. 

Although this time interval represents a carrier-gas volume of 2.0 mL at the column-exit pressure and room temperature, at the inlet pressure it would represent only about half this volume. It could be argued that because the sample is compressed to about half its original volume, the amount of gas needed to flush the loop is also halved, but this point of view ignores the flow of pressurized carrier gas into both ends of the sample loop, from the pressurized column inlet as well as from the gas supply. In most cases it is better to leave the valve in the position shown in Figure 4(b) as long as possible or even until the end of the analysis. 

For the best accuracy, record the atmospheric pressure and then adjust the results to a standard pressure of 101.3 kPa. It isn't necessary to compensate for the loop temperature if the sample loop is positioned inside the GC oven and always at the same temperature. If the sample loop is outside the GC oven and it experiences temperature changes greater than about 10 °C over the course of a day then record the loop temperature and adjust the results to a standard temperature such as 0 °C (273.15 K). The adjustment to standard temperature and pressure conditions (STP) must be done in the same way for calibration as well as sample runs. 

Equation 1 can be used to adjust results for both temperature (

); some data handling systems will perform the adjustments given the correct loop temperature and pressure. Note that the adjustments are applied to the peak area (

) (or height if used) before calculating response factors or concentrations.   

Here, the superscript "0" refers to the standard conditions and the subscript "a" refers to the ambient conditions in the sample loop; pressures must be expressed in the same units and temperatures must be converted to Kelvin. For example, if the local atmospheric pressure is 98.6 kPa and the loop is at 27 °C (300.15 K) then the correction factor to STP conditions would be:   

Using this correction will help reduce variability in calibration and results. In comparison to syringe injection techniques, however, loop injection generally delivers significantly lower relative standard deviations (RSDs). 

Much smaller injection volumes can be accommodated by using the internal volume of the rotary valve itself as the entire sample loop. This technique is applied mainly in valves for LC applications down to as little as 0.1 µL — in the GC realm 10 µL is a practical lower limit for most applications. 

This first series instalment has explored the basics of rotary valves for GC. Detector selection and loop injection are two of the most important applications for gas-stream switching. Modern rotary valves make the setup and use of these configurations simple and robust. The next instalment will address rotary valve configurations for multiple column switching and for samplestream selection. 

 is senior research scientist at Serveron Corp., Hillsboro, Oregon, USA and a member of 

 editorial advisory board. Direct correspondence about this column should go to 

 editor, Alasdair Matheson, Bridgegate Pavillion, Chester Business Park, Wrexham Road, Chester CH4 9QH, UK or e-mail 

Chapters in the Evolution of Chromatography

 (Imperial College Press, London, 2008), pp. 259–290. 

GC Connections

A new series on valves and the pivotal role they play in gas chromatography.

Valves for Gas Chromatography: Fundamentals

New EU GMP Annex 11 Regulations for Computerized Systems

 covering the impact on CSV Policies and Procedures is a two-day educational course held on the 

 in Heidelberg, Germany organized by Concept Heidelberg on behalf of the European Compliance Academy (ECA). 

EU GMP Annex 11 for computerized systems has been an unchanged part of EU GMP since 1992. In 2008, the European Medicines Agency issued a proposed update that was a major change to the regulation. There were also consequential changes to Chapter 4 on documentation that were also issued for industry comment. Within the six month comment period, over 1400 responses from industry were received by the Agency. 

The final version of Annex 11 was issued in January 2011 and becomes effective on 30 June 2011. The structure of the released document has a Principle and 17 clauses but the text is longer than the current version it replaces. The changes to Chapter 4 are no longer consequential but require laboratories to define raw data including electronic records. 

Major changes in Annex 1 mean that applications must be validated and IT infrastructure must be qualified; risk management in both computer validation and change control need to be formalized; the life cycle validation phase has been extensively expanded; the requirements traceability throughout a life cycle moves from a regulatory expectation to a regulatory requirement for the first time; New requirements for data integrity, availability and confidentiality have been introduced, vendor audit reports should be available for inspectors to review; and the use of electronic signatures for signing documents, including records, has now been covered explicitly. 

However, it is the consequential changes to Chapter 4 that update the regulation for documentation that bring major changes to European GMP regulations. There are new requirements for the definition of electronic raw data and the need to keep and manage all electronic records that brings this portion of EU GMP into the 21st century. The new versions of Annex 11 and Chapter 4 combined are Europe's answer to 21 CFR 11 for electronic records and electronic signatures. 

Therefore individuals involved with computerized systems, as well as regulated organizations that operate them, need to be aware of the changes that these updates bring and be prepared for them. Implementation of Annex 11 and Chapter 4 require a good understanding of the changes and how they impact existing computer validation policies and procedures, define and manage electronic records but also how computer systems are validated or revalidated in the future. 

There are three main objectives of this education course, according to the organizers: 					

To review and understand the changes that have been made in the new version of EU GMP Annex 11 before it becomes effective.

To review and understand the impact of the changes to EU GMP Chapter 4 (Documentation) and their impact on the definition of raw data and generation and maintenance of electronic records.

To understand how the requirements of Annex 11 and Chapter 4 interact.

These three objectives aim to allow organizations to plan for the changes to policies and procedures for the validation of computerized systems and management of electronic raw data that the new regulations bring. 

Speakers include Questions of Quality columnist Bob McDowall, UK, and KarlHeinz Menges, European GMP Inspector, Germany. 

6th Symposium on MS and Related Techniques in Advanced Analytical Science

				Hilton Manchester Deansgate, Manchester, UK			

VII International Ion Chromatography Conference

Institute of Environmental Engineering of Polish Academy of Science 

Joint Congress 2011 (ISCC, MSB and GC?GC)

Town and Country Resort and Convention Center, San Diego, California, USA

CASSS - An International Separation Science Society 

LCGC Europe-03-01-2011