This article is the second part of the series on method development for drug impurity profiling. The first part described the first two steps: the selection of a set of dissimilar chromatographic columns (i.e., columns with different selectivities), and the selection of a suitable chromatographic column and optimization of the mobile phase pH. This second part will discuss the last two steps: optimization of the organic modifier composition and optimization of the gradient slope and the temperature.

An impurity is defined as 'any component of the new drug substance that is not the chemical entity defined as the new drug substance',

  and can be induced during drug synthesis, manufacturing and/or storage.

 To develop chromatographic impurity profiles by means of reversed-phase high-performance liquid chromatography (RPLC), different sequential steps can be considered, determined by the influence of the factors on the selectivity; (1) selection of a set of dissimilar chromatographic columns, (2) selection of a suitable chromatographic column from the above set and optimization of the mobile phase pH, (3) optimization of the organic modifier composition and (4) optimization of the gradient slope and the temperature. 

In Part 1, the first two steps were discussed.

  After selection of a set of dissimilar chromatographic columns, the drug impurities mixture is screened on these four or five dissimilar columns at, for example, four different pH values, to select the most suitable column and the optimal pH value.  

In Part 2, we will focus on the following two steps: (3) optimization of the organic modifier composition and (4) optimization of the gradient slope and the temperature. The best column and pH, selected in step (2), will be used to screen the mixture at different mobile phase compositions (different organic modifier compositions).  

As already mentioned in the previous paper,

  it is not suitable to model either selectivity factors or resolutions (responses representing the separation degree or quality) directly as a function of the organic modifier fractions. Thus, for each compound (impurity) a model is built, relating its retention time (or factor) to the organic modifiers fractions. This allows predictions of the retention time (or factor) of each compound at intermediate organic modifiers compositions. Then, for each composition, the predicted retention times (or factors) and corresponding peak widths (considered constant in gradient elution and modeled for isocratic elution) are sorted from high to low, enabling the calculation of the selectivity factors or resolutions between consecutive peaks. The optimal organic modifiers composition is that where the minimal selectivity factor or the minimal resolution, such as the selectivity factor or the resolution of the worst separated peak pair, is the highest.   

Optimization of the Organic Modifier Composition

The third step in developing drug impurity profiles consists of the optimization of the organic modifier composition. The chromatographic column and the mobile phase pH, selected from the second step, are used to screen the drug substance and its impurities at several isoeluotropic organic modifier compositions.  

Figure 1: Solvents triangle, with x1, for instance, representing an ACN/H2O mixture, x2 MeOH/H2O, and x3 THF/H2O. Water content is determined by solvent strength. Numbers 1â10 (see Table 1). 

In general, for a three-component mixture, for example with modifiers methanol (MeOH), acetonitrile (ACN) and tetrahydrofurane (THF), all possible compositions can be represented in a triangle (Figure 1). In Table 1 the compositions forming some mixture designs are shown. Each vertex of the triangle represents a condition where the organic part of the mobile phase only consists of one modifier. Each side represents binary mixtures of organic modifiers and inside the triangle ternary mixtures can be found. Additionally, each condition contains a given amount of water to make all compositions isoeluotropic. Any composition can be considered as a mixture with given fractions of x

, each ranging between 0 and 1. When applying a gradient elution, isoleluotropy of the beginning and end conditions often is ignored. In fact in a gradient elution one moves from one triangle to another, from one with a larger water content to one with a smaller.  

Table 1: Three-component mixture designs: (3,1) simplex lattice design with 3 (points 1 till 3) experiments; (3,2) simplex lattice design with 6 (points 1 till 6) experiments; (3,3) simplex lattice â centroid design with 7 (points 1 till 7) experiments; or augmented simplex lattice â centroid design with 10 (points 1 till 10) experiments. 

A choice can be made between the optimization of either binary organic modifier mixtures alone, or binary and ternary organic modifier mixtures jointly. To limit the complexity of the mobile phase, one may initially chose to only use binary mixtures of organic modifiers when the vertex compositions do not result in baseline-separated chromatograms. In such situations, only one side of the triangle is explored (e.g., side 2 to 3 in Figure 1). Two different retention models can be constructed depending on whether the model is based on two or three measurements (Figure 2). The model for retention based on two measurements, [such as each vertex of one side of the triangle (points 2 and 3 in our example)], is a linear model (Equation 1). One based on three measurements, [such as each vertex of one side of the triangle (points 2 and 3) and a given point on that side of the triangle (e.g., point 6 in Figures 1 and 2)], is a quadratic polynomial model (Equation 2).  

where k is the retention factor, x the fraction of one of both modifiers (between 0 and 100% or between 0 and 1) and b

 are the regression coefficients of the models.  

Figure 2: Optimization of a binary mixture of organic modifiers using (a) a linear model, and (b) a quadratic polynomial model to model retention. Si = substance i from mixture. 

To optimize a binary mixture of organic modifiers, the drug mixture is analysed at two or three conditions and the model (Equations 1 or 2) is built for each impurity. In Figure 2, this is represented for four substances. For each intermediate mixture of organic modifiers, the retention is predicted for all impurities. At each composition, the retentions are then sorted and the selectivity factor α or the resolution Rs is calculated for each pair of consecutive peaks. To determine the optimal conditions, the minimal selectivity factor α

 is plotted as a function of the composition of the mobile phase. The plot does not have a smooth behavior because the plotted α

 values at different conditions may originate from different peak pairs. That composition where α

 is maximal is then selected as the optimum, for example X = 40% x

 in Figure 2(a), meaning that 2/5 of composition x

To explore the two other sides of the triangle, the above procedure is repeated for the sides 1 to 2 and 1 to 3. For the optimization of binary and/or ternary mixtures of organic modifiers (if you wanted to consider compositions in the entire triangle, for example), either three (points 1, 2 and 3 in Figure 1), six (points 1 to 6 in Figure 1), or seven (points 1 to 7 in Figure 1) experiments are executed. 

The three-experiments (3,1) simplex lattice design (Figure 1 and Table 1, exp 1 to 3) is used to calculate the coefficients of the model.  

The six-experiments (3,2) simplex lattice design (Figure 1 and Table 1, exp 1 to 6) is used to calculate the coefficients of the model. 

The seven-experiments (3,3) simplex lattice–centroid mixture design (Figure 1 and Table 1, exp 1 to 7) is used to calculate the coefficients of the reduced cubic model.  

In Equations (3–5), k is the retention factor, t

 are the fractions of the first, second, and third organic modifier, respectively, and b

 are the model coefficients. Most applied when considering the entire triangle, is the latter situation based on seven experiments. Its model (Equation 5) is equivalent to the quadratic model we have been discussing earlier in the data handling column about response surface designs.

  Here, the model also represents a response surface.  

The model coefficients of Equation 5 can simply be estimated from one or some measured results (Table 1).  

Experiments 8 to 10 (Figure 1 and Table 1) together with experiments 1 to 7 form another mixture design, for which the model of Equation 5 can be built. However, software is needed here to estimate the coefficients.  

Figure 3: Visualization of the grid over the triangle. 

To optimize a ternary mixture of organic modifiers, again a model is built for each impurity. Similar to the optimization of binary mixtures, for each intermediate composition of organic modifiers retention is predicted for all impurities. The intermediate compositions are determined by creating a grid over the triangle (Figure 3). At each composition, the retentions are then sorted and Rs or α is calculated for each pair of consecutive peaks. To determine the optimal conditions, α

 is plotted or visualized as a function of the composition of the mobile phase (Figure 4). That composition where α

 is maximal is then again selected as the optimum.  

Figure 4: Visualization of the predicted minimal resolutions for the 66 grid points of Figure 3. 

Optimization of the Gradient Slope and the Temperature

The fourth step in developing drug impurity profiles consists of the optimization of the gradient slope and the temperature. It concerns factors with less influence on the selectivity. This step often can be considered optional or rather as a fine-tuning of the method. It can be done using response surface designs. The types and properties of these designs were already discussed in reference 4. However, concerning the data analysis to determine the best conditions, again a similar approach, as in Part 1 for pH and here for organic modifier composition, is applied. Therefore, this approach will not be further elaborated here. 

In general, the optimized conditions for two examined factors, x

 (e.g., gradient slope and temperature), to separate a mixture of compounds, is derived from a plot of Rs

 grid conditions, as shown in Figure 5. Similar plots are obtained for the triangle (mixture domain) when plotting Rs

 values as a function of the organic modifier composition.  

Figure 5: Plot of Rsmin values for the different grid conditions considered during the optimization of factors x1 and x2. 

In this data handling column, step (3) optimization of the organic modifier composition and step (4) optimization of the gradient slope and the temperature, of the development of chromatographic impurity profiles were discussed in more detail. 

 is a professor at the Vrije Universiteit Brussel, Belgium, department of Analytical Chemistry and Pharmaceutical Technology, and heads a research group on chemometrics and separation science.  

 is a former PhD student from the same department [funded by a specialization grant awarded by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT)], and is currently working as a postdoctoral fellow at the Umea University, Umea, Sweden. 

 is a postdoctoral fellow of the Fund for Scientific Research (FWO, Vlaanderen, Belgium) working at the same department on experimental designs and their applications in method development and validation, and on the development and data analysis of herbal fingerprints.  

1. International Conference on Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH) guideline Q3A(R2), Impurities in new drug substances, 2006, 

3. B. Dejaegher, M. Dumarey and Y. Vander Heyden, 

Articles

Practical Data Handling

A systematic approach to developing drug impurity profiling methods.

Method Development for Drug Impurity Profiling (Part 2)

Now that several years have passed since the introduction of ultrahigh-pressure liquid chromatography (UHPLC), I think we all can agree that it is a technique that is here to stay. I'm not convinced that UHPLC will be replacing all LC methods in the near future. However, UHPLC has moved beyond the research and development environment into more routine use in many companies. Some companies use UHPLC for the development of analytical methods and then covert them to conventional LC methods for routine use; others use UHPLC in all areas for new applications; others are still staying with the traditional LC methods and equipment. 

Through contacts in my teaching and consulting activities, attendance at scientific meetings and e-mail with readers like you, I have collected a number of tips and techniques that  are used in practical applications to get the most out of UHPLC equipment. It is this subject that I would like to explore a bit in this month's "LC Troubleshooting". I will add more to this in the future, so if you have found particular tricks that you would like to share with other readers, drop me an e-mail (

) and include "UHPLC" in the subject line. For the present discussion, I will make a binary division. LC (or as many call it, high performance LC or HPLC) will refer to equipment that operates up to the conventional pressure limits of 6000 psi (400 bar); UHPLC will refer to all equipment that operates at higher pressures. 

As is summarized in Table 1, the passageways through the typical UHPLC system are smaller than those for conventional LC systems. To minimize dispersion problems when 5 µm diameter particles are used as a column packing, connecting tubing should be no larger than 0.175 mm i.d. in the areas where the sample travels: between the autosampler and column and between the column and detector. If 3 µm particles are used, 0.125 mm tubing is required. Even this latter tubing can be a challenge. I remember when Hewlett-Packard first introduced their 1190 system, which was one of the early low-dispersion LC systems. Originally it was plumbed with 0.125 mm tubing, but after numerous problems with tubing becoming blocked in routine operation, they had their service engineers replace the 0.125 mm tubing with 0.175 mm i.d. tubing, which is less prone to blockage. The further challenges of UHPLC with sub 2 µm particles require even less extracolumn peak broadening, which means that even smaller tubing diameters are used; typically 0.00625 mm i.d., which is even more prone to blockage.

 Table 1: LC versus UHPLC particulate challenges			

One of the complaints I commonly hear about 3 µm particle diameter columns is that they become blocked much more easily than 5 µm columns. The primary reason for this is that 5 µm columns use 2 µm porosity frits to hold the particles in the column. This frit size is not sufficiently small to retain all the particles in many 3 µm particle columns, so they typically use 0.5 µm porosity frits on the outlet and either 0.5 µm or 2 µm frits on the column inlet. With sub-2 µm particles, the 0.5 µm frit is not satisfactory, so 0.2 µm frits are commonly used. Those of you who have had a microbiology class along the way will remember that 0.22 µm porosity filters are used to remove bacteria from solution. Ever see an analytical chemistry laboratory that is bacteria-free? 

So how do these items affect the practical use of UHPLC in the laboratory? With 3 µm or 5 µm particles, sample filtration often can be avoided by simply placing the samples in a benchtop centrifuge, turning the speed up to the maximum for 5 min to settle any particles, and transferring the supernatant to the injection vial. As a safety measure, I strongly recommend using an 0.5 µm porosity in-line filter between the autosampler and guard column or analytical column to catch the occasional sample particle that was missed in the centrifugation step. With sub-2 µm columns and 0.2 µm frits, the centrifugation step might not be sufficient. If this is the case, every sample must be filtered through a 0.2 µm porosity frit — a very slow process. Whether centrifugation or filtration is used, a 0.2 µm porosity in-line filter is strongly recommended as a safety precaution.  

Another source of particulate matter that can block the column is the mobile phase. Conventional LC applications with 3 µm or 5 µm particle columns are fairly tolerant to minor levels of mobilephase contamination. For example, if the buffer or other aqueous mobile phase is filtered through a 0.5 µm porosity solvent filter, it can be used for at least a week in most laboratories, and many laboratories extend this to two weeks. For UHPLC applications, many workers observe sufficient microbial growth in aqueous mobile phases that fresh buffers must be made daily and filtration through a 0.2 µm porosity filter is required. In either case, the reservoir should be replaced with a clean one each time the mobile phase is replenished, so that a minor microbial contamination does not amplify itself in the next batch of mobile phase.  

Still another source of particulate matter is the wear of piston seals and injection valve rotors. I recently saw a UHPLC presentation that discussed the use of a continuous seal wash to help keep the piston cool so that the heat generated by the seal gripping the piston did not cause the seal to melt. The data also showed a reduction of piston seal lifetimes of fourfold at the highest pressures tested. Similarly, the increased pressure of the UHPLC system requires that the rotor seal and stator of the injection valve be pushed together more tightly, increasing friction and reducing the seal lifetimes. So it is imperative to pay close attention to system software that counts piston and injection cycles so that seals can be changed on a timely basis to minimize particles from these sources. 

What does all this tell us? As one of my friends terms it, "chromatographic hygiene" must be at a much more stringent level for UHPLC than for LC applications. But many workers take care to keep particulate matter out of the system and can use UHPLC reliably in routine analysis with great results. 

When most of us think about the increased pressure that is observed with a UHPLC system, it seems logical that it results from smaller-particle columns and higher linear velocities. We get faster separations at higher pressure and don't think much else about it as long as the equipment can handle the increased pressure. However, it isn't quite so simple. It has been known for more than 40 years

  that pressure can change the molar volume of solutes; this can result in changes in selectivity, or relative peak spacing in the chromatogram. I know that this came as a surprise to me when we were testing one of the first UHPLC systems. We observed that the peak spacing for a closely eluted triplet changed dramatically as we changed the pressure from 5400 psi (370 bar) to 12800 psi (880 bar). 

With the right compounds, changes in peak spacing can be very dramatic. In the example of Figure 1,

  a change in column pressure was achieved by adding a restriction capillary between the column and detector; the same flow rate and other conditions were used. You can see that as the pressure was increased from 43 bar (625 psi) to 739 bar (10700 psi), there are two peak reversals. Peaks 3 and 4 (diphenhydramine and acetophenone) reversed retention order, as did peaks 5 and 6 (protriptyline and nitrobenzene). These changes are attributed primarily to a differential change in the molar volume of the solutes.

Figure 1: Pressure selectivity effects: Separation observed at (a) 43 bar and at (b) 739 bar by the addition of a restriction capillary after the column. Column: C18; mobile phase: acetonitrileâphosphate. Peaks: 1 = thiourea, 2 = propranolol, 3 = diphenhydramine, 4 = acetophenone, 5 = protriptyline, 6 = nitrobenzene. Adapted from reference 3.			

What does this mean in practical terms? With the relatively small changes in pressure encountered in conventional LC systems (for example, a factor of two when the flow rate is doubled), most of us don't ever notice this effect. However, one of the common deployments of UHPLC technology in the pharmaceutical industry takes advantage of the speed of UHPLC to develop new methods in the research laboratory more quickly than could be done with conventional LC equipment. But there also is a realization that many methods are performed in a production environment where the equipment, skills and environmental conditions might not be as favourable. So to improve the chances of success, the UHPLC methods are converted to conventional LC methods on 3 µm or 5 µm particle columns that can be operated on existing, lower-pressure equipment. This can save in training, equipment costs and method downtime. However, when pressure selectivity is taken into account, scaling a method from UHPLC to LC might not be as simple as initially thought. This is of special concern for methods that must be capable of detecting many small peaks, such as stability-indicating or impurity methods. This does not mean that the UHPLC development and LC application strategy cannot work, but it does mean that special care should be taken to check for possible changes in peak spacing when the system pressure is changed. For a nice discussion of other effects of pressure in LC applications, see the introduction to reference 3. 

When liquid passes through a bed of particles, the friction between the two phases creates heat (sometimes called viscous heating). As with pressure effects, with the 3 µm or 5 µm particles and 1–2 mL/min flow rates used with conventional LC methods, we seldom notice this effect. However, when the particle sizes are reduced to < 2 µm and flow rates (or more properly, linear velocities) are increased, a significant amount of heat can be generated. As has been discussed here before (for example, in reference 4), a change in column temperature can have a dramatic effect on the separation. In fact, peak reversal can occur from changes in column temperature. The use of temperature can be a powerful tool to help optimize a separation, but it also can be the source of problems in a separation if it is not carefully controlled. The effect of temperature on peak shape is shown in the illustrations of Figure 2. In Figure 2(a), a fully temperature-equilibrated column is shown. The incoming mobile phase, column bed and column oven are all at 70 °C. The uniform temperature distribution across the column means that all the molecules in a single band travel at the same speed, no matter where they are relative to the column diameter and a relatively narrow peak results. 

Figure 2: Effect of uneven column heating: (a) column and mobile phase fully equilibrated, (b) cold incoming mobile phase, (c) frictional heating within column.			

If solvent preheating is ignored, the incoming mobile phase can be at a significantly lower temperature than the rest of the system, as shown in Figure 2(b). Here, the solvent at the centre of the column is at a lower temperature than that at the edges of the column, where the column oven has its strongest influence. As a result, molecules in the centre of the column travel more slowly than their warmer siblings closer to the column wall. This causes peak broadening. The opposite can happen if heat is created within the column, as is illustrated in Figure 2(b). Here, the centre of the column is warmer than the edges, where heat is dissipated into the column wall and oven, so the molecules in the centre travel more quickly than the ones at the edge. Once again, peak broadening results. 

The combination of these temperature effects can be interesting. In one case, we noted that a sub-2 µm column generated the narrowest peaks (highest plate number) at 65 °C. Upon further investigation, we found that the oven was not performing very well, so that most of the column heating was provided not by the column oven, but by the preheated solvent (the oven was, in fact, acting as a radiator to cool the column). So under these conditions, the solvent coming into the column was warmer than that leaving (and at the column walls). On the other hand, the small particle size and high linear velocity created heat within the column, so the temperature at the column outlet was higher than at the inlet. This also caused peak broadening. But at 65 °C, the cooling effect of a poorly performing oven and the heating effect of frictional heating balanced each other and the column temperature was uniform, resulting in the best performance. Although this was an interesting observation, it certainly is not conducive to easily transferred methods. It is very important to preheat the mobile phase for methods operating above ≈40 °C, to have a column oven that works well, and to pay attention to possible influences of frictional heating. 

UHPLC can be a great tool to speed method development and routine analysis. However, it is not without potential problems. In this discussion, we saw that cleanliness is extremely important to reliable operation. Solvents should be filtered and changed more often than with conventional LC systems. More attention may need to be given to preventive maintenance of pump and injection rotor seals. Care should be taken to minimize particulate matter in samples either through centrifugation or filtering. Additional protection of the column should be undertaken by installing a 0.2 µm in-line filter just downstream from the autosampler in every system.  

We also reviewed two phenomena that can be a surprise when using UHPLC. Pressure selectivity is seldom observed with conventional LC methods, whereas it might be more prevalent in UHPLC, especially when UHPLC methods are scaled to lower pressures for routine operation. Similarly, frictional heating is not a common problem under conventional LC conditions with 3 µm or 5 µm particles, but can potentially change selectivity and column efficiency with UHPLC. 

As my mother always told me when I was learning to drive as a teenager, "Be careful. Pay attention to what you are doing." This still is good advice to her chromatographer son decades later. 

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

 editorial advisory board. Direct correspondence about this column should go to "LC Troubleshooting", Poplar House, Advanstar Communications, Park West, Sealand Road, Chester, CH1 4RN, UK, or e-mail the editor Alasdair Matheson at 

LC Troubleshooting

An essential guide to getting the most from your UHPLC system.

UHPLC Tips and Tricks

				Department of Chemistry, University of York, York, UK,

 National Centre for Atmospheric Science, University of York, UK,

 Energy and Resources Research Institute, University of Leeds, Leeds, UK,

 The Dolomite Centre, Unit 1, Anglian Business Park, Hertfordshire, UK,

 National Physical Laboratory, Middlesex, UK.  

Capillary gas chromatography is a powerful and versatile technique that has revolutionized organic chemical separations and analyses, and is now one of the most widely used methods in analytical chemistry. It is reasonably fast, reproducible and has a broad scope of application. In combination with a flame ionization detector (FID) or a mass spectrometer (MS), it forms one of the most powerful tools for environmental analysis and has been used in many studies to measure volatile organic compounds (VOCs).

While modern GC systems are highly sophisticated analytical tools, they remain relatively bulky and power intensive and are generally not field portable. The heating of capillary GC columns has evolved little over the past few decades and is primarily based on the turbulent fan oven, which remains an excellent means to achieve even heating of the column. The nature of GC and the size of such ovens make it a difficult technique to use in remote or off-grid locations for environmental research. Therefore, there is great potential in this area for microfabricated GC systems that are compact, robust and with low-power demands. Devices where all subcomponents of the GC system (injector, column, detector) are formed in a single planar fabrication have particular attraction, including potentially improved robustness, lack of interconnections and a structural geometry that is much easier to heat using planar devices. 

Figure 1: (a) The most simple Chip 1 comprising 7.5 m and  1.4 m columns with 320 Âµm internal diameters. (b) The improved Chip 2 comprising connected 7.5 m and 1.4 m columns with 250 Âµm internal diameters, as well as having an edge connector and etched preconcentrator and modulator channels.			

The first miniaturized GC, created by Terry et al. at Stanford University in 1975, was fabricated on a silicon wafer using photolithography and chemical-etching techniques.

  The microGC included an injection valve and used an internally mounted thermal conductivity detector. The 1.5 m long, 40 µm deep, 200 µm wide column had a rectangular cross-section. Difficulty in evenly coating rectangular channels and in miniaturizing the other GC components meant that the resolving power of the column was poor compared with standard columns of the day. 

Figure 2: (a) Shows a cross-section of the glass device taken using an Alicona G4 Infinite Focus microscope, (b) the same glass section using a Wild Heerbrugg M400 microscope. Measurements of three bores give internal column diameters of 242 Âµm, 250 Âµm and 248 Âµm with a manufacture target of 250 Âµm. (b) Shows how the use of an asymmetric pair of glass thicknesses places the GC channels within a few hundred microns of one surface of the device for direct heating.26			

Since then, chip-based gas chromatography has received relatively limited attention compared with silicon-fabricated on-chip electrophoresis and liquid chromatography systems. However, various versions of miniaturized and microfabricated GCs have been reported. While many of these devices encompass all instrument components on a smaller scale, a number merely combine conventional drawn capillary narrow bore columns with miniaturized detection and data handling technologies.

  The µChemLab developed by Sandia National Laboratories is, however, an example of a hand-held miniaturized GC instrument for VOC measurement that incorporates a microfabricated etched column.

  It comprised a preconcentrator consisting of a thin silicon nitride membrane supporting a patterned metal film-heating element, a 1 m long 100 × 400 µm high-aspect ratio GC column on a silicon wafer and an array of surface acoustic wave sensors.  

 While silicon has been the favoured substrate to date for microfabricated GC columns,

  other materials have been used in their construction, including porous silicon,

  In addition, a variety of fabrication techniques have been used including DRIE, DRIE polymer vapour deposition, stereolithography, silicon bulk micromachining and Lithographie, Galvanoformung, Abformung (LiGA). 

Figure 3(a): Separation using the 7.5 m long, 320 Âµm internal diameter planar glass column with 2 Âµm OV-101 stationary phase, placed in a GC fan oven and with a FID detector. Blue trace is an injection of the BTEX standard mixture and red trace is a mixture of 2 ppm nonane, alpha-pinene and 3-carene with a 500 ÂµL s ample loop.26			

Of the miniaturized devices previously reported, a common feature has been square or rectangular-sided channels to form the columns. These designs often result in a "pooling" of stationary phase at the column corners and can produce an uneven coating of stationary phase film. As analytes spend a longer time in these areas of thicker coating, peak broadening and general degradation of the separation performance may result.

  A limited number of circular channelled microfabricated devices have been reported, notably by Potkay et al. who reported columns of 1 m length and 90 µm diameter with a semi-circular cross-section

  and Pai et al. reporting the use of circular cross-sectional silicon serpentine columns with a 250 µm internal diameter.

  Circular cross-sectional channels are able to perform better over a larger range of flow than other reported column shapes, resulting in increased system design flexibility. 

Figure 3(b): Separation of gasoline vapour using 7.5 m GC column programmed from 3 to 100 Â°C at 10 Â°C/min heated using Peltier and resistive elements. Detection is by low-power photoionization detector.			

The planar microfabricated GC system reported here uses acid-etched borosilicate glass as the working substrate. The use of larger glass wafer areas, which may be micromanufactured at reasonable costs when compared with silicon, results in capillary channels that are circular in cross-section with column dimensions analogous to those used in laboratory GCs. 

 The glass devices reported here were fabricated by the Dolomite Centre, UK, a specialized manufacturer of micro-scaled glass components. The design used two glass (Schott B270, Mainz, Germany) wafers (0.5 mm and 2.5 mm thick) with etched channels bonded together to form a single "chip". The chemical etching involved the surface of each half of the glass chip being covered with a chrome and then a photoresist layer. The photoresist layer is exposed to UV light through a mask outlining the channels. This causes it to become soluble to the photoresist developer compared with the unexposed photoresist which remains insoluble. On removal of the exposed photoresist an engraved pattern of the channels remains which is then used to etch the chrome layer. Finally, the glass is wet-etched with hydrofluoric acid solution along these preformed channels. The HF etching process is controlled to ensure symmetrical, hemi-spherical channels. After the photoresist and chrome layers are fully removed the two halves are brought into contact with the channels aligned. The flatness of the glass surfaces results in strong bonding through van der Waals forces. No heating is required and chips fabricated using this process can withstand pressures up to 50 bar. 

Figure 3(c): 2D separation using the 7.5 m and 1.4 m long, 250 Âµm internal diameter columns coated with OV-101 and OV-17 respectively. Individual peaks have been rescaled to give common peak maxima intensity.			

Two different chip designs have been used in this work. Figure 1(a) shows Chip 1, while Figure 1(b) shows Chip 2 — an improved version of the first. Improvements seen on Chip 2 include the presence of an edge connector which provides an easy means of sample introduction and gas pressure modulation in a single connector unit, and connection of the channels between the primary and secondary columns, modulator and detector which allows comprehensive GC separation. Figures 2(a) and (b) show a cross-section of a portion of Chip 2. Both figures clearly show the uniformity of the column dimension, the accurate alignment of the two halves of glass and that the profile is very close to circular. Measurements of three bores at the chip edge gave internal column diameters of 242 µm, 250 µm and 248 µm, with the target internal diameter being 250 µm. Figure 2(b) shows how the use of an asymmetric pair of glass thicknesses places the GC channels within a few hundred microns of one surface of the device for direct heating. 

 There are two main methods of capillary GC column coating — static and dynamic. Static coating is widely used to deposit a highly uniform film of stationary phase onto column walls. This can be difficult to produce by the dynamic method which is based on pumping solution through the column. We used a static procedure here to deposit a film of non-polar DPMS stationary phase (OV–101, Restek, Bellefonte, Pennsylvania, USA) on the inside of the Chip 1 primary column. The column was filled with a solution containing the stationary phase prior to controlled evaporation of the solvent. A final weighing confirmed the mass of stationary phase deposited and calculation showed that the mass of 11 mg was equivalent to a uniform layer 2 µm thick. 

Figure 3(d): 2D separation using the chip heated by planar manifold with commercial Agilent FID. Only the raw detector signal is shown to illustrate the peak widths achievable using the on-chip modulator when an optimized commercial FID is used rather than the low-cost PID.			

One-Dimensional Separation Performance in a Laboratory GC with FID:

 The coated chip was initially tested using the fan oven, temperature control, injection and flow control systems of a laboratory GC (Varian 3800, Santa Clara, USA) This enabled the separation capabilities of the coated glass column to be characterized with an established FID. Using high-purity helium (BIP Air Products, Allentown, Pennsylvania, USA) as carrier gas, a standard BTEX mixture containing 10 µmol/mol (molar ppm) of benzene, toluene, ethylbenzene, 

-xylene was injected via a 500 µL gas sample loop. A second ppm gas mixture of nonane, α-pinene and 3-carene was injected separately. Automated valve changes, as part of the Varian 3800 GC system, were used. The oven was programmed to run at 30 °C for 0.2 min, then ramp at 20 °C/min to 100 °C. The switching valve was heated to 170 °C. The results are shown in Figure 3(a), which shows good separation between all of the injected components with symmetrical peak shape and elution in 4 minutes. Peak skew is less than 1.8 for all peaks (the majority less than 1.3). An isothermal chromatogram indicated around 2000 theoretical plates as measured for toluene. 

Figure 4: (a) Profile view showing the layout of components in the temperature-controlled stack used for heating and cooling the glass GC. (b) Plan view layout of the temperature-controlled stacks. Stack 1 is under the primary 7.5 m long column. Stacks 2 and 3 are positioned under the secondary 1.4 m column and the injection region, respectively.26			

Thermal Control in Constructed GC Manifold:

 Uniform heating of a GC column is central to its performance, requiring accurate and reproducible column temperatures and with minimal spatial temperature gradients. For the device reported here, combinations of direct heating approaches were investigated. These included the direct heating of the glass wafer with metal heating elements placed above and below the device and a more complex arrangement involving a stack of thin film-resistive elements and Peltier devices shown in Figure 4(a) in profile view.  

Since glass is a relatively poor heat conductor, the separation of the zones of the device allows the differential heating of the injector from the columns and other regions. This was tested using three different control thermal stacks and temperature zones shown in Figure 4(b). The heater consisted of a polyimide-encapsulated heater and Peltier devices bonded directly to a finned aluminium heatsink using thermally conductive epoxy adhesive. The heatsink was equipped with a centrifugal blower for rapid cooling of the metal fins. Temperature monitoring of the stack was achieved using a PT100 polyimide-encapsulated sensor (Jumo GmbH, Fulda, Germany) placed between the heater and the glass chip. A configurable CompactRIO module supplied by National Instruments (Austin, Texas, USA) running Labview Realtime operating system was used to control the temperature of the stack. The temperature profile across the chip was determined using a matrix of 16 thick film platinum resistance temperature sensors (Labfacility DM503, Bognor Regis, UK), which were attached across the chip surface in an X pattern using thermally conductive epoxy adhesive. The temperature data collected was displayed in the form of a colour map overlaid on a two dimensional image of the glass chip using Labview software from National Instruments. Figure 5(a) shows the temperature profile across the surface of the chip when stack 1 (7.5 m column) is heated to 100 °C and all other stacks are left at ambient, while Figure 5(b) shows the measured temperature profile when stack 1 is cooled to 10 °C, with stack 2 (1.4 m column) held at 100 °C and unheated stack 3 (preconcentrator) remaining at ambient temperature. The 2D images reveal that the divided thermal stack arrangement is capable of independently controlling the temperature of at least three discrete regions of the glass GC device. The column temperature range is between 0–200 °C, with linear ramp rates of between 5 and 20 °C/min. Typical power consumption was 25 W mean or 30 kJ per analysis, two orders of magnitude less than conventional turbulent fan ovens. 

Figure 5: (a) Measured temperature profile with stack 1 heated to 100 Â°C, all other stacks left at ambient.  (b) Measured temperature profile when stack 1 was cooled to 10 Â°C whilst holding the temperature of stack 2 at 100 Â°C. The temperature remained uniform (Â±2 Â°C) over stack 1 and the unheated stack 3 remained at ambient temperature.26			

One-Dimensional Separation Performance with Direct Resistive Heating and PID:

 Following characterization of performance in the standard lab GC, the glass column was tested with direct heating using manual loop injections. A low-cost and low-power miniaturized photoionization detector (PID) (Alphasense, Great Notley, UK) operating with a lamp having an ionization potential of 10.6 eV was attached to the column outlet. The PID has a linear dynamic range of 5 ppb to 50 ppm (isobutylene). A two-stage regulator was used to provide a constant head pressure of helium carrier and was connected to a 6-port valve (VICI, Houston, Texas, USA) and operated manually. A 250 µL sample loop made from treated stainless steel was fitted to the valve in a configuration to allow sample filling in one position and sample injection in the other. The 6-port valve was connected to the inlet of the chip by a 10 cm long 1/16" Silcosteel tube and a 20 cm long 0.32 mm inner diameter fused-silica capillary connected by a reducing union (Thames Restek, Saunderton, UK). The effluent from the 7.5 m long column was connected to the PID by 0.32 mm inner diameter uncoated fused-silica capillary. The detector was integrated into the unit and to a data acquisition system. The carrier gas head pressure was set to 12 psi. Gasoline vapour was injected onto the column at a starting temperature of 3 °C. A temperature ramp of 10 °C/min up to 100 °C was applied. As seen in Figure 3(b), reasonable chromatographic separation of the highly volatile C5 – C8 fraction of the complex hydrocarbon sample was achieved in less than 3 mins. 

Figure 6: Schematic of the in-line fluidic differential flow modulator. 			

Two-Dimensional Separation Performance in GC Manifold with PID:

 Chip 2 was used to investigate comprehensive separations, with the primary column coupled to the secondary column via an on-chip fluidic differential flow modulator. The modulation setup can be seen in Figure 6. Primary column coating was conducted as previously described. The modulation valve was tested with gas phase samples and narrow peaks were obtained by fine tuning the carrier and modulator gas pressures. The secondary 1.4 m column was coated with a film of 50%-phenylmethylpolysiloxane stationary phase (OV17, Sigma Aldrich, St Louis, Missouri, USA). A dynamic technique was used for these preliminary results for ease and speed of coating. The coated chip was then tested using the PID and chip heating manifold. The overall system has a total peak power consumption of ~80 W (mean ~30 W) and is approximately briefcase sized with the outer frame dimensions being 33 cm (w) × 41 cm (l) × 14 cm (h).  

A 6-port automatic injection valve was used fitted with a 250 µL sample loop made from treated stainless steel. The valve was connected to the chip's edge connector by a short length of peek tubing. The effluent emerging from the 1.4 m secondary column was connected to the PID by a 0.25 mm inner diameter uncoated fused-silica capillary. The carrier and modulator gas pressures were set to 15 psi and 14.5 psi respectively. The modulation period was set to 2 s, with filling set to 1.5 and flushing set to 0.5 s. A gas mixture comprising pentane, hexane, cyclohexane, toluene, ethylbenzene, 

-xylene and nonane was injected onto the column at a starting temperature of 30 °C. A temperature ramp of 20 °C/min up to 100 °C was applied. Figure 3(c) shows an early experiment using 2D separation on the glass chip. While first dimension separation is successful, second dimension separation is not ideal. The response of the low cost PID was not sufficiently fast to allow modulated peaks to fully return to baseline. We tested the true separation performance of the device by replacing the PID with a fast commercial FID from an Agilent GC system (Agilent 7890A, Santa Clara, California, USA) by means of a ~50 cm long transfer line. The resulting raw FID data, shown in Figure 3(d), shows much improved comprehensive GC peak shape and that the chip-based columns and modulator are roughly equivalent in performance to other systems reported in the literature. A key element of our future research is therefore to connect a compact, low power, but fast-responding PID to our chip-based GC system. Development in fabrication capabilities now allows for channels of differing widths to be etched on the same glass wafer. We are currently working on redesign of the column geometries to allow for a longer second column than used here in combination with a narrower bore primary column. 

A planar 2-dimensional GC chip with fully circular channel profiles has been microfabricated from glass using acidetching techniques. Relatively long column lengths and standard internal diameters have been achieved. Lowpower direct resistive heating and cooling of the glass chip produced a uniform heating profile across the columns when held both above and below ambient temperatures and multiple temperature zones have been achieved within the same chip. The one-dimensional performance of the device was proven using a standard GC oven for injection, heating and FID detection. Coupling of the directly heated column to a low cost, low-power PID showed reasonable separation of gasoline vapour. Comprehensive separation has shown some promising preliminary results from the separation of ppm gas mixtures of a set of VOCs when coupled with commercial GC detectors. 

The approach described here offers substantial potential as the basis for a field-portable GC and is a useful alternative to typically square-channelled silicon devices of restricted physical size and higher material cost. The ability to directly cool this device using the Peltier effect may offer substantial advantages for the analysis of very volatile species over typical cryogenic cooling of drawn capillaries in standard GC ovens. 

 graduated from Limerick Institute of Technology with a degree in Pharmaceutical and Forensic Analysis and is currently a PhD student in the Department of Chemistry at the University of York. 

 is professor of atmospheric science at the University of York, a research director of the National Centre for Atmospheric Science and technology advisor to the Natural Environment Research Council. He completed his BSc and PhD at the University of Leeds and was a lecturer there between 1999 and 2003. 

 has a BSc from the University of Strathclyde, and a PhD in chemistry from University of Leeds. She received the Desty Memorial Prize for separation science in 2009 and is currently a lecturer in chemistry at the University of York. 

 obtained a BSc and PhD from University of York. He has worked as an mass spectrometry instrumentation specialist at the Central Science Laboratory and also for manufacturers VG and Thermo, before returning as a research fellow at the University of York in 2008. 

 has been a researcher of chromatographic techniques for more than 40 years and has published in excess of 400 articles on these and related topics. He is currently an Emeritus professor at the University of Leeds. 

 is the head of engineering at The Dolomite Centre, a specialist manufacturer of microfluidic devices. 

 obtained a combined degree in physics and chemistry at the University of Edinburgh. He worked in the Gas Metrology group at NPL for 4 years, and has recently completed a Masters in Science Policy at the University of Edinburgh. 

 obtained a BSc at Regent Street Polytechnic in 1967. He designed control and protection instrumentation for submarine nuclear reactors for over 25 years before joining NPL where he creates specialized laboratory facilities, including patented gas dilutors. 

 obtained a MChem and PhD from the University of Manchester. After working as a postdoctorate fellow there for 2 years she moved to NPL in 2008, where she has been leading their work on VOCs in the Gas Metrology group. 

 has a MChem from the University of Southampton and a PhD in chemistry from Imperial College London. He is currently a Senior Research Scientist in the Analytical Science Division at the National Physical Laboratory. 

 has more than 30 years experience in gas chromatography and gas analysis. He has worked at NPL for 8 years, prior to that he worked at the National Office of Measures in Hungary. 

 is a Fellow of the National Physical Laboratory. He has a degree in physics from Oxford University and a PhD in laser physics from Southampton University. His research concerns the development of accurate standards and measurement methods for gases and their standardization internationally.  

Regular Article

The development of a microfabricated comprehensive GC that can be used as a portable measurement device.

Lab-on-a-Chip GC for Environmental Research

The Fifth International Symposium on the Separation and Characterization of Natural and Synthetic Macromolecules (SCM–5

) will be held in Amsterdam, The Netherlands from 

. The SCM series of meetings have gained a reputation as a premier platform for obtaining and exchanging information on characterization methods for "large" molecules. As the title of the meeting suggests, SCM is very much concerned with separations. Almost all macromolecules occur in complex mixtures. Therefore their detailed molecular characterization inevitably relies on separation techniques. Among these, liquid-chromatographic (LC) techniques are arguably the most prolific.  

There will be a great deal of attention for studies into the understanding and application of size-exclusion chromatography (SEC), which is the workhorse of polymer separations. Various advanced detection techniques for SEC with be intensively discussed. Other isocratic ("critical") and gradient LC separations will be addressed in many presentations, as well as comprehensive two-dimensional liquid chromatography (LC×LC), which is an immensely useful technique for characterizing complex mixtures of polymers.  

Field-flow fractionation (FFF) techniques were conceived by the formidable J. Calvin Giddings more than 30 years ago, but a strong resurgence became apparent during the previous SCM meeting in 2009. This year a great deal of attention will again be devoted to FFF techniques and a one-day course will be offered just before the 

 meeting to bring newcomers up to speed with the exciting possibilities of FFF.  

The FFF course will be presented by two of the greatest experts in the world: Kim Williams of the Colorado School of Mines (Golden, Colorado, USA) and Meyung Hee Moon of Yonsei University in Seoul, South Korea. This course will allow scientists to decide whether — and how — to apply FFF for their specific applications. It also provides a perfect platform for the state-of-the-art presentations during the SCM–5 conference. 

 Another hot area in separation science is mass spectrometry (MS). The application of MS for the analysis of macromolecules has been — and still is — making amazing progress. The SCM–5 meeting will take place in the Rhône Conference Centre, which can be easily and rapidly reached from central Amsterdam. There will be a large exhibition, which will be integrated with posters sessions, luncheons and coffee breaks. 

There is also an opportunity for exhibitors to present new developments in the form of vendor seminars. 

 promises to be every bit as informative, challenging and enjoyable as the previous meetings in this series. Details of the conference programme and further information can be found on the conference website at 

3rd International Conference on Polyolefin Characterization (ICPC)

			Montreux Music and Convention Centre, Montreux, Switzerland  

www.selectbiosciences.com/conferences/EBS2010

Departments

Event News

Office chromatography (OC) aims to boost the performance of ultrathin-layer chromatography (UTLC). What obstacles were you aiming to overcome with this technique?

Firstly, miniaturized plate formats are very difficult to handle and manage with the thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) equipment currently available. This lack in instrumentation combined with the reduced surface activity of monolithic UTLC layers has hampered its progress for almost a decade. It is also not easy to find out how to use UTLC plates correctly. For example, when analysts transfer a particular HPTLC method to UTLC they get different chromatographic results. But for a successful transfer, the elution strength of the mobile phase has to be reduced significantly and nanolitre volumes of a more concentrated solution need to be applied to generate real sharp start zones, which is crucial. 

Planar chromatography is the only chromatographic method that can synergetically benefit from print and media technologies because of its open planar format of the stationary phase. Office chromatography (OC), which aims to combine print and media technologies with chromatography, will lead to the development of instrumentation that will visibly improve the results that can be achieved on such small plates. The print of sharp start zones offers a good start for OC. Secondly, for UTLC plates particularly, the continuous print of the mobile phase is possible because the highly reduced layer thickness scales the relevant supply volume down; separations can be performed in 1 min

  with mobile phase consumption only in the microlitre range. Different types of UTLC plates have been reported to accelerate the chromatographic separation.

  Finally, this minor mobile phase volume can be evaporated by infrared (IR) bulb scans of a flatbed scanner positioned beneath the plate. Electronic documentation of the dry plate by different light bulbs, which also fit in the flatbed scanner, allows a high-resolution image of the small-scale plate dimension. All in all, OC will offer the first real chance for a one-click planar chromatographic system because all steps (application, development and evaluation) are integrated in one system. Of course, such a system will lower the degree of flexibility, but an online system would be convenient to many analysts because it increases the free time available for the analyst compared with TLC or HPTLC. OC is online and the whole proces can be  started with one click. 

How did you come up with the idea of using office equipment to solve this problem?

In 1997, Professor Nyiredy applied for a patent coupling a liquid jet with TLC.

  However, neither the proof of principle nor any results were ever reported. Just one paper was published in 2001 that used a flatbed scanner for documentation and evaluation of TLC plates.

  Later, another one for HPTLC plates was published in 2006.

In 2005, we started printing on TLC/HPTLC plates. We published the homogeneous printing of derivatization reagents with experimental proof.

  For the international symposium on HPTLC in Berlin in 2006, we printed the name badges and the social evening menu list on a TLC plate, called the 'magic dinner card' as the menu list was not visible. The magic dinner card revealed the delicious menu list just to skilled analytical scientists who found out that a derivatization reaction was essential. First by heating the plate on a heating plate, which was placed beside the buffet, the menu list printed with the sweetener sucralose on a ß -naphthol impregnated plate was colourized. 

Figure 1: Milestones of planar chromatography.1			

 bacteria through the printer's nozzles onto the TLC plate.

  Later I noticed, just few months earlier,  Paul Calvert published a paper in 

Working with miniaturized plate formats was always a technical challenge and we started thinking about printing not only derivatization reagents or bacteria, but also start zones on the plates. The experimental proof was successfully demonstrated in a recent paper in 

  Then we discovered that printing the mobile phase is also possible and even the generation of a minor forced flow. Evaporation of the mobile phase is not an issue due to the microlitre-load of the plate. Finally, the potential for a planar online system was judged to be realistic. 

Figure 2: Sample application on ultrathin layers with a piezoelectric nanodispenser (PipeJet P9, BioFluidex) (left) and an inkjet printer (Pixma iP 3000, Canon) (right); sample zones 3-fold enlarged. 			

What benefits does this approach offer the chromatographer over existing techniques? 

Having the idea of OC does not mean we have a working system on the bench. The approach is in its infancy, but already shows potential. The benchspace will be significantly reduced compared with step-based TLC/HPTLC techniques. An online planar analysis will be possible. All steps will be conveniently integrated in a one-click system. The analysis time will be shortened due to the reduced migration distance of UTLC plates. 

The disadvantages are that fewer types of UTLC layers are available at the moment and that the result must be available in the form of an image (i.e., it cannot cover the whole UV-range or record UV/Vis spectra like classical TLC/HPTLC scanners do). 

Figure 3: Potential chromatographic one-click system in printer size.			

Benefits compared with HPLC/GC include high-parallel sample throughput and high matrix-tolerance, which often allows a reduced sample preparation. Compounds left at the origin of the adsorbent are visible. A disadvantage is the lower separation power, but this can be mitigated by a selective derivatization or by the use of electronic filters. 

Figure 4: Separation of food dyes E131 (blue) and E124 (red) on a nanostructured UTLC plate in 40 s parallel for 20 samples; plate size 8 cm x 1 cm, band length 3 mm. 			

Are there any particular applications where you see this technique becoming popular?

It is intended to be used in a wide range of applications. For example, in pharmacies, chemical education, chemistry departments and on-site. One major application area could be monitoring the absence or presence of analytes in process control, quality control, cleaning validation or the assay of samples. For authenticity proof of medicines it could ideally be used because the planar image allows the direct comparison of many samples in parallel analysed under identical conditions. High-throughput analysis is another application field because the sample throughput capacity of OC is good. Plate sizes of 20 × 2 cm could analyse 50 samples in parallel. 

Figure 5: Digital evaluation of the UTLC separation of food dyes E124 (red), E131 (blue) and E 100 (yellow) using electronic filters. 			

How do you intend to develop this research?

As I mentioned before there is still a lack of different types of plate. Besides the development of a commercially available system, new miniaturized plate structures offer an immense potential regarding separation speed and phase design for solving specialized analytical tasks. This is the focus of our cooperation with Professor Michael Brett (

). Recently, interesting UTLC approaches have been suggested, such as electrospun,

  Hard-core particles are of interest in planar chromatography. Further goals will be the improvement of digital image evaluation tools and creation of intuitive software making the one-click analyses convenient. An opportunity to see first hand the progress in HPTLC and OC is by attending the 

International Symposium on High-Performance ThinLayer Chromatography

 received her doctorial degree in Chemistry under the supervision of Professor H. Jork and Professor H. Engelhardt in Saarbrücken, Germany. She has worked in industry as head of the CAMAG laboratory and as a senior consultant. She is currently associate professor at the Institute of Food Chemistry at the University of Hohenheim in Stuttgart, Germany. In 2009 she received the honourable Kurt-Täufel Award of the Young Research Scientist of the Society of Food Chemistry, the biggest division of the German Chemical Society. She was appointed co-chair of the International Symposium on HPTLC and editor of the CAMAG Bibliography Service CBS, in which the database CCBS has over 10000 abstracts with TLC/HPTLC-specific information listed. 

12. G. Morlock, C. Stiefel and W. Schwack, 

Alasdair Matheson spoke to Gerda Morlock about her radical approach to boost the performance of ultrathin-layer chromatography.

Q&A: Out Of Office Reply

The previous installment in this series looked at how moderate peak tailing affects resolution and degree of peak overlap. When peaks tail, even moderately, extra time between them is required to attain the same resolution as for nontailing peaks due to the overall increase in peak widths caused by tailing. Even when the same resolution is attained, however, the degree of area overlap between tailing peaks is greater than for nontailing peaks, and more so for the amount of excursion of the first peak into the second. Thus, additional resolution is required for tailing peaks to achieve the same degree of peak area overlap as a pair of nontailing symmetrical peaks. The amount of area overlap of one peak into another can be measured as the percentage of a peak's area that lies underneath the adjacent peak, where the peaks are separated with a specific degree of resolution, as in the previous two installments in this series.

  In that case, where the peaks were of equal size, resolution of 2.0 was needed to bring the degree of overlap of a first tailing peak into the second down to about the same level seen with symmetrical peaks at resolution of 1.5, at around 0.5%. This was despite the moderate increase in peak separation required to keep the measured resolution the same for tailing peaks as for nontailing ones. 

The degree of peak overlap can be calculated easily enough with the theoretical peak model being used here, but it isn't a realistic measurement of the effects that lessthan-ideal peak separation has on actual area measurement of peaks that are co-eluted in a chromatogram. With nonselective detectors, it isn't possible to measure the two peaks independently, as was done here to obtain the degree of overlap. Even with a mass spectrometer or other selective detector, there often isn't sufficient selectivity to obtain 100% discrimination between overlapping peaks, so questions of the effects of peak overlap on quantitative measurements are not answered by overlap calculations. 

In some cases, mathematical peak deconvolution can be used to tease apart two partially merged peaks, but practically speaking, for routine applications, we have to fall back on the integration parameters available in chromatography data processing systems. To test how well merged tailing peak areas are estimated, I exported a series of synthesized chromatograms with moderately tailing peaks using asymmetries (

) of 1.4 in which the peak resolution was set at 1.0, 1.5 and 2.0. Three common peak integration methods were selected for the estimation of peak areas: a vertical drop from the valley point between the peaks down to the baseline; a tangential skim drawn from the valley out to the down-sloping side of the second peak; and an exponential skim underneath the second peak. Somewhat unrealistically, no noise was added to the generated peaks, but in this case, I was more interested in the measurements themselves and not the degree of uncertainty across multiple measurements. These results show how one particular data processing system performed. Others would vary, but I expect they might produce very similar results. 

Table 1 enumerates the results of this tailing peak faceoff, and Figure 1 shows what the separations looked like at resolution of 1.0. As was the case with symmetrical peaks,

The concluding part of the series focuses on testing quantitative area measurment.

LCGC Europe-11-01-2010

Method Development for Drug Impurity Profiling (Part 2)

UHPLC Tips and Tricks

Lab-on-a-Chip GC for Environmental Research

Event News

Q&A: Out Of Office Reply

When Peaks Collide (Part 3)

Analysing Metal Ions by CE