John Dolan has been writing "LC Troubleshooting" for 

 for over 20 years. One of the industry's most respected professionals, John is currently the Vice President of BASi Northwest Laboratory, McMinnville, Oregon and a principal instructor for LC Resources, Walnut Creek, California. He is also a member of 

We will discover how to find the “sweet spot” in terms of retention for a liquid chromatographic separation as well as how much retention change can be expected for a selected change in organic mobile-phase percentage or column temperature.

	This is the second instalment in a series about how to use chromatography fundamentals to estimate the impact of various parameter changes on liquid chromatography (LC) separations. In the first discussion (1) we looked at the influence of the column plate number (N) and saw that for most situations, starting method development with a 10,000-plate column makes the most sense. This is a good compromise between separation power, pressure, and
	run time. The effect of further changes in column length (L) or particle diameter (d

) can be easily estimated without doing the actual experiments.

	This month, we’ll continue the discussion with an emphasis on the influence of the retention factor (k) on the separation, expressed as resolution (R

). We’ll consider the general case, specific cases, and how to estimate the influence of a change in mobile phase percent organic (%B) or temperature on retention. As with the prior instalment of this series, we’ll limit ourselves to isocratic separations and reversedâphase conditions to simplify the discussion, but much of this instalment will apply to gradients and other separation modes.

	Once again, we’ll use the fundamental resolution equation to guide the discussion:

	            (i)      (ii)       (iii)

	where α is the selectivity between two peaks with k-values of k

), it is usually simpler to use the retention factor in discussions like the present one. Recall that the retention factor is calculated from t

	From equation 1, we can see that resolution is a function of k/(1 + k) (term iii). Thus, R

will increase with k, but not linearly. For example, if k = 1, k/(1 + k) = 0.5, whereas if k = 100, k/(1 + k) ≈ 1.0. I find that this relationship is easier to understand in a graphic representation, such as in Figure 1, where retention, expressed as k, is plotted against resolution, expressed as k/(1 + k). Initially resolution increases rapidly with increases in k, then flattens out as k exceeds ~5.

 shown in Figure 1 can help us to decide in advance what is the desired range of k-values we would like to aim for in a separation. Let’s look at three different aspects of this: the improvement in R

 we get for some increase in k, the cost of an increase in k to increase R

, and the stability of the separation as it relates to k.

 The plot of Figure 1 shows us that for small values of k, R

 increases rapidly with increased k. For example, a change of k from 0.5 to 1.0 (black dots in Figure 1) increases the relative resolution by 50% from 0.35 to 0.53. A further doubling of k from 1.0 to 2.0 gives only a 33% increase in resolution. A fivefold increase of k from 2 to 10 increases resolution by another third, but a change of k from 10 to 20 only changes resolution by 5%. This tells us that if we want to use k as a lever to improve R

, it will be most effective at small values of k.

Another way to look at the data of Figure 1 is to consider how much it costs for an increase in k and compare this to the gain in resolution we might get for that cost. Because most LC methods are run in an automated mode, the cost of analysis time is usually not significant as long as a batch of samples can be run in a reasonable time, such as a 14-h (840âmin) overnight run. As an example, let’s consider a method run on a 150 mm × 4.6 mm column at 2.0 mL/min and a sample batch size (comprising both samples and calibration standards) of 100 injections. Under these conditions, t

 ≈  0.75 min; we can rearrange equation 3 and solve for retention time:

	If we use the same examples as above with k = 0.5, 1, 2, 10, and 20 (black dots on Figure 1), we can use equation 4 to calculate retention times of 1.3, 1.5, 2.3, 8.3, and 16 min, respectively. (I’ve rounded my numbers here, so your results may vary a bit if you repeat these calculations.) These translate to batch run times 100 times larger of 113, 150, 225, 825, and 1575 min. You can see that methods where the last peak is eluted with k > 10 would exceed the allotted 14-h window.

	Another potential cost of adjusting k has to do with detection limits for a method. Longer retention times mean broader peaks (peak width is directly proportional to retention time under isocratic conditions). If peak area is assumed constant, peak height will be inversely proportional to peak width (or retention time). This relationship means that doubling retention, as when k is increased from 10 to 20, will halve the peak height. For trace analysis, peak height, not peak area, is the limiting factor for detection limits. Clearly smaller k-values are desired for taller peaks.

	A final cost-related factor is that of solvent costs. The purchase price, preparation costs, and disposal costs all add up. Shorter run times (smaller k-values) will reduce solvent costs.

 The general curve shape for Figure 1 is one we often see in chemistry, such as when tracking reaction completion. It is common to see a rapid rise in the dependent variable with a small change in the independent variable, then with continuing increases in the independent variable, the change in the dependent one levels off. The same happens here for changes in resolution as k is increased. For values of k > 5, there is only a small increase in resolution for each successive increase in k, whereas for k < 2, the changes are quite dramatic. Values of k can change because of normal laboratory variation. For example, as discussed below (Rule of Three), a 1% change in %B (the organic content of the mobile phase) can be expected to change k by approximately 10–15% for a 500-Da sample. A 1% error in mobile-phase preparation is certainly within the normal expectations of laboratory variation. Because the curve of Figure 1 is quite steep at low k-values compared to higher ones, small k-value samples will be more susceptible to significant changes in resolution caused by normal fluctuations in mobile-phase composition. For this reason, it is best to avoid conditions that give k < 2 for maximum method robustness.

After considering the above discussion, it is clear that selection of an ideal value of k has tradeoffs-robustness, resolution, run time, detection limits, solvent expense, and so forth. Another complicating factor is that the typical sample contains components that vary somewhat in retention time, so we would like to fit the sample into a range so that the k-value of the first peak is not too small and the k-value of the last one is not too large. Based on the above discussion, a range of 2 ≤ k ≤ 10 is ideal, being a good compromise between robustness and run time. However, some or many samples, depending on the application, have too broad a polarity range to fit within this k-range. In such cases, extending the range to 1 ≤ k ≤ 20 is reasonable. When the ratio of the k-values of the first and last peaks exceeds 20–40, a gradient method will usually be favoured over isocratic.

	The data in Figure 1 implies that an increase of k will always increase resolution, but this conclusion is a rather broad generality that tends to fail in the case of specific samples. To understand this relationship better, we can classify samples as “regular” and “irregular” (see discussion of reference 2 for more details). It has been shown (3,4) that the relationship between k and the mobile-phase percent organic (%B, expressed as Φ, where Φ = %B × 0.01):

	Where k0 is the k-value of a solute at 0% B and S is the slope of the plot, and is a characteristic of the solute. S can be estimated as follows:

	where MW is the molecular weight in daltons. For some types of samples, plots of equation 5 appear as in Figure 2. Here it can be seen that the log k versus S plots are parallel or tend to fan out from each other as %B is reduced. In a case like this, the chromatogram would spread out, with increasing resolution at smaller values of %B, as might be expected from the behaviour observed in Figure 1. This kind of sample has been referred to as a “regular” sample. Regular samples tend to be those closely related in structure, such as homologs or isomers. The sample of Figure 2 is a group of structurally related triazine herbicides.

Many, if not most, samples include components that are not as closely related as those just discussed. Such samples vary in core structure as well as in the type and number of functional groups. It is common for plots of equation 5 for these samples to appear more like that of Figure 3. In Figure 3, compounds represented by the black, blue, and green lines behave much like those of Figure 2 in that they are roughly parallel. However, the compound represented by the red line moves relative to the others. At ~45% B, the red and black lines cross, so the two compounds would be coeluted. This is also the crossover point for the red and black compounds; at %B > 40%, the red compound comes out first and at %B < 40% B, it comes out after the black one. In contrast to the expected behaviour of Figure 2, the compounds represented by the black and blue lines converge as %B is reduced, so their resolution gets worse, not better, with increased k. Samples in which the slopes of the log k versus %B plots differ significantly are referred to as “irregular” samples.

The information we gather from Figures 1 and 3 can be valuable guides for LC method development. Figure 1 tells us that we’ll get the “best” chromatographic behaviour with 2 ≤ k ≤ 10 (or 1 ≤ k ≤ 20 if the sample won’t fit within this restricted retention range). This is a good general guideline. However, samples that behave as the one in Figure 3 are common enough that we can’t assume that larger k-values (longer retention) will always improve the separation. Sometimes α (equation 2) will remain relatively constant as %B is changed and sometimes it will vary significantly. This tells us that fine-tuning k can be a powerful tool to fine-tune the resolution.

	Another useful tool that we can use to save experimental runs is to estimate the change in retention (expressed as k) for a selected change in %B. We can do this estimation quite accurately based on two experimental runs that allow us to make plots as in Figures 2 and 3. For example, chromatograms run at 30% B and 50% B would allow us to measure retention for each compound, convert it to k and plot the results as in Figure 2 or 3. Alternatively, we could use equations 5 and 6, along with a single experimental run and knowledge (or guess) of the analyte molecular weights to construct similar plots with a little less accuracy (depending on how many assumptions we make). Equation 6 tells us that a 400-Da compound will have an S-value of approximately 5, which is a convenient number to use as an average or typical S-value for small molecule samples (for example, <1000 Da). From equation 5, we can construct an estimate of the change in k (âk) for a given change in %B (â%B, where â%B = 10% is equivalent to âΦ = 0.1):

	Thus for S = 5 and â%B = 10%, we get âk = 10(

) ≈ 3. This is what is often referred to as “The Rule of Three”, where a 10% change in the organic solvent concentration of the mobile phase is expected to change k by approximately threefold. This is a very handy tool for estimating how much retention will change when the mobile phase is modified. I used a similar estimate for the earlier discussion of the effect of a 1% change in %B due to normal variation in the laboratory.

	Although I won’t go into detail here (see discussion in Section 2.3.2.2 of reference 2 for more information), we can get plots similar to Figure 2 or 3 if we plot log k versus 1/T, where T is temperature in kelvins. From such data, we can generate a rule of thumb that an increase in temperature of 1 °C will decrease k by 1–2%. This rule of thumb can be useful to help estimate the effect of a change in column temperature on retention and also alerts us to the importance of column temperature control if we want stable retention times.

	We have examined the influence of the retention factor, k, on resolution. After we considered the risks (costs) and benefits of various values of k, using Figure 1 as a guide, we concluded that we are likely to get the “best” chromatography and a good balance of costs (time, solvent costs, resolution, and so forth) if we can adjust the chromatographic conditions to achieve 2 ≤ k ≤ 10, or if the sample won’t fit into this retention range, 1 ≤ k ≤ 20. When k < 1, retention tends to be less stable, resolution is very sensitive to small changes in k, and there is more chance of interferences from the unretained materials at t0. When k > 20, run times become excessive and peak heights suffer. When samples won’t fit in a retention range of 1 ≤ k ≤ 20, a gradient is likely a better choice.

	We also saw that while it is a general pattern that resolution increases with an increase in k (as in Figure 1 or 2), there are many cases in which the sample components vary enough that changes in relative retention and even peak reversals can be seen with either increases or decreases in %B. This means that even if the sample is eluted in the 1 ≤ k ≤ 20 range, fine-tuning %B can be a useful tool to move peaks around relative to each other.

	Finally, we saw that we can further save the cost of experimental runs by estimating the change in retention or k for a specific change in %B or temperature. If two experimental runs are made, such as at 30% B and 50% B for Figures 2 and 3, quite accurate estimates can be made for the change in retention that can be expected with a specific change
	in %B.

	Often if we start method development with a column that generates N ≈ 10,000 plates (1), find conditions that generate 1 ≤ k ≤ 20 for our sample components, and then fine-tune %B for the best retention, we will obtain a satisfactory separation. If this is the case, method development can be a fairly simple process. If we have optimized these two factors (terms i and iii of equation 1) and are not successful, it is time to look for help in term ii of equation 1, as will be discussed in the next instalment of this series. 

		L.R. Snyder, J.J. Kirkland, and J.W. Dolan, 

Introduction to Modern Liquid Chromatography

, 3rd edition (Wiley, 2010), section 2.5.2.1.	

		K. Valkó, L.R. Snyder, and J.L. Glajch, 

		L.R. Snyder, J.W. Dolan, and J.R. Gant, 

		T. Braumann, G. Weber, and L.H. Grimme, 

		N.S. Wilson, M.D. Nelson, J.W. Dolan, L.R. Snyder, R.G. Wolcott, and P.W. Carr, 

		N.S. Wilson, M.D. Nelson, J.W. Dolan, L.R. Snyder, and P.W. Carr, 

 has been writing “LC Troubleshooting” for LCGC for more than 30 years. One of the industry’s most respected professionals, John is currently a principal instructor for LC Resources in McMinnville, Oregon, USA. He is also a member of LCGC Europe’s editorial advisory board. Direct correspondence about this column via e-mail to 

LC Troubleshooting

Count the Cost, Part 2: Increasing Resolution by Increasing Retention

Our annual review of new liquid chromatography (LC) columns and accessories introduced at Pittcon and throughout the previous year.

	In accordance with recent tradition, this article is intended to cover chromatography columns and accessories commercially released after Pittcon 2016 through this year’s conference held in Chicago 5–9 March 2017. LCGC once again sent out a survey in early 2017 asking vendors to supply them with any products launched after Pittcon 2016. Instrumentation is covered in this issue of LCGC (1), and gas chromatography and sample preparation will be covered in the May issue. Because information for this instalment of “Column Watch” is obtained sporadically over the course of many months, it is very possible that some information has been missed. Readers are encouraged to check with specific vendor sites for additional products as well as more detailed information regarding what is covered here.

	The vendors that responded to the survey with high performance liquid chromatography (HPLC) and ultrahigh-pressure liquid chromatography (UHPLC) columns and accessories are listed in Table 1. The list contains very wellâknown vendors such as Waters and Agilent Technologies, but also several lesser-known vendors. The product range is also highly varied from traditional reversed-phase columns using fully porous materials to specialty columns. As in years past, with such a varied list, further discussion requires some breakdown of the different offerings. Similar to 2016, one differentiation can be made based on offerings that target small or large molecules. Within the small-molecule realm three subcategories could be established: reversed-phase, hydrophilic interaction liquid chromatography (HILIC), and chiral offerings. Each of these will be discussed separately. For large-molecule products, subareas involving reversedâphase mode and size-exclusion chromatography are identified and discussed. Columns commercialized using ion-exchange chromatography for both small and large molecules will be discussed separately. Furthermore, there were products launched for supercritical fluid chromatography (SFC) that deserve some discussion. An interesting and potentially game-changing microchip chromatography device based on pillar array technology is also featured. Lastly, several accessories relating to HPLC are noted.

The product offerings assigned to the small-molecule, reversed-phase category are listed in Table 2. A total of 22 new surface chemistries are noted from the list. Continuing recent trends, many of the new columns are intended to round out selectivity or particle size offerings within existing lines. Of the 22 columns listed, nine are manufactured using superficially porous particles (SPP), indicating that the particle architecture continues to garner attention. Enhancements of fully porous particles (FPP) are also noteworthy. For example, Phenomenex’s Luna Omega phase reportedly includes a thermal modification that provides improved performance, inertness, and robustness as compared to conventional FPP products. Another theme consistent with 2016 is the continuing effort to increase column stability for wider pH range utility. Agilent Technologies (HPHâC18), ChromaNik Technologies (SunShell C18), and Imtakt Corporation (DZ-C18) all launched phases with enhanced pH stability. Separation Methods Technologies commercialized an interesting set of short-chain reversed-phase chemistries (C1, C2, and C4). The company contends that their proprietary bonding strategy provides “proper deactivation” of silanols as well as resistance to acid hydrolysis that plagues many short-chain surface chemistries. Another interesting trend from this year’s offerings is the presence of several “polar C18” chemistries. Phenomenex launched a polar C18 stationary phase in both the Kinetex and Luna Omega lines and Waters added its T3 chemistry to the Cortecs brand. In each case the surface modification is noted to impart a balanced retention for hydrophilic and hydrophobic molecules. Phenomenex also came out with a chemistry denoted as PS C18 which is reported to include a positively charged modification to the surface that aids in the retention of acidic analytes. More complete, full line offerings were launched by Agilent (InfinityLab Poroshell) and Restek (Force Performance).

 HILIC, which is often employed for the retention and separation of very polar compounds, continues to show growth. Table 3 lists product offerings in the HILIC category. The seven new entries in this category match the number of new columns reported in 2016. It is interesting to note that three of the seven entries are built on polymeric phases. HILICON introduced a polymeric version in its iHILICâFusion line. Shodex commercialized two polymerically based HILIC phases. The VN-50 2D diol phase is designed for oligosaccharide analysis, and the VC-50 2D carboxyl chemistry is reportedly applicable to monoamine type neurotransmitters and oral anti-diabetic drugs. The company also notes that the PEEK housing reduces undesired interactions with the column hardware. MicroSolv Technology continues to fill out its line of silica hydride based phases with a new amide chemistry. Fully porous– and superficially porous–based HILIC phases continue to be promoted. Advanced Chromatography Technologies added an amino chemistry to its FPP ACE offering, and both Agilent Technologies (bare silica) and ChromaNik Technologies (amide) added to their respective SPP lines.

 Eight new chiral phases were introduced after Pittcon 2016. This number is a significant increase over the same time period from Pittcon 2015 through the “New Chromatography Columns and Accessories for 2016” column instalment where only three entries were noted (2). Cellulose- and amylose-based chiral phases dominate the list provided in Table 4. Chiral Technologies extended its line of meta-immobilized 5-µm phases through the introduction of Chiralpak IG. The company also introduced amylosic and cellulosic phases on 2-µm FPP particles designed for UHPLC. ES Industries continued to fill out its ChromegaChiral line with two new chemical modifications of amylose that provide what the company refers to as unique enantiomeric separation behaviour. Phenomenex introduced an immobilized cellulosic phase, Lux i-Cellulose-5, which is designed to be more robust for strong organic solvents than coated polysaccharide phases. The company also launched the Lux-AMP phase. The proprietary chemistry is specifically designed for the enantiomeric separation of amphetamine and methamphetamine. Separation Methods Technologies reported the development of a teicoplaninâbased chiral stationary phase. The company reports that the column is useful for performing a wide variety of chiral separations with amino acids, carboxylic acids, and neutral compounds. Like 2016, it is clear from these new product introductions that amylose and cellulose phases based on FPP particles continue to dominate the field. New this year is the introduction of sub-2-µm chiral columns (Chiral Technologies); however, SPP technologies have still not entered this realm.

	The separation of large molecules such as peptides, proteins, and oligonucleotides often requires larger pore sizes than are typically used in small molecule applications. Full characterization of these complex molecules often requires multiple modes of separation, including reversed-phase chromatography, size-exclusion chromatography (SEC), ion-exchange chromatography, and hydrophobic interaction chromatography (HIC). 

 In last year’s report, columns intended for reversed-phase analysis of large molecules was dominated by chemical modification of SPP particles. That trend continues this year. However, a new line of columns from ES Industries (MacroSep BIOâGold) demonstrates that new lines based on fully porous particles are still being developed. New columns intended for reversed-phase chromatography of biomolecules are presented in Table 5. Also included in the table is a new set of offerings from PolyLC, Inc. for HIC. 

	Advanced Materials Technologies introduced a 1000-Å C4 column intended for very large molecules such as monoclonal antibodies (mAbs). The larger pore size as compared to many “large-molecule” columns (typically 160–400 Å) provides less restriction into the pores and thus can provide higher resolution of minor variants of mAbs. The column is based on SPP technology. Fortis Technologies reports the expansion of its SPP portfolio with SpeedCore BIO Peptide (160 Å) and SpeedCore BIO Protein (300 Å) columns. Also based on wider pore SPP technology, these offerings are intended for improved separations of peptides and proteins, respectively. Oligonucleotides also fall into this category of large molecules and offer their own set of chromatographic challenges. Phenomenex recently launched Clarity Oligo-XT, an organoâsilica SPP phase intended for the reversed-phase characterization and purification of synthetic oligonucleotides. The column is reported to be stable at high pH, which is often used in oligonucleotide separations.

	HIC likely deserves its own category within the large-molecule realm. However, for the sake of space it is lumped in here with the reversedâphase offerings. HIC is often a valuable tool used to separate polar variants of proteins. HIC, however, traditionally uses high concentrations of nonvolatile buffers, rendering it incompatible with mass spectrometry (MS) detection. A presentation by Andrew Alpert of PolyLC, Inc., at HPLC 2016 described efforts to render HIC MS friendly (3). Using moreâhydrophobic analogs of a poly(propyl aspartamide) stationary phase, Alpert demonstrated that proteins could be denatured in MS-friendly(er) mobile phases that may lead to a possible marriage of the two techniques (4). Since that time, PolyLC, Inc., has launched three versions of the polyaspartamide phases discussed at HPLC 2016 (pentyl, hexyl, and heptyl) that permit MS-compatible HIC for proteomics and quality control of biotherapeutics.

Size-Exclusion Chromatography– Gel Permeation Chromatography:

 SEC continues to witness resurgence largely as a result of the focus on large-molecule therapeutics. SEC resolves analytes based on molecular size and is often used as a complementary technique to reversed-phase analysis of protein. Gel permeation chromatography (GPC) uses the same process of separation by molecular size. Separation by molecular size in aqueous mobile phases is commonly referred to as SEC, whereas separation in organic solvents such as tetrahydrofuran is normally referred to as GPC. Columns used in SEC and GPC are often characterized by strict control of pore size and by inert surface chemistry. Control of the pore size dictates how molecules of various sizes can diffuse into the pores, while the inertness of the surface limits secondary interactions that may interfere with molecular size separation and determination.

	Table 6 lists the SEC and GPC column offerings launched since Pittcon 2016. The number of new offerings is similar to those reported last year. Jordi Labs released a divinylbenzene GPC phase in both 3- and 13-µm particle sizes that promises high efficiency, high capacity, and lower back pressure.  Shodex continued to expand its offering with the release of two new chemistries on polymeric supports. The HK-404L phase is intended for rapid GPC analysis, and the LW-803 product is designed for antibody and antibody aggregate separations. Smaller-particle designs continue to infiltrate the SEC–GPC market in an attempt to enhance resolution and speed. Phenomenex extended its line of sub-2-µm SEC columns with the release of the Yarra 1.8µ SEC-X300 column. The column’s 300-Å pore size is intended for the characterization of mAb aggregates, antibody–drug conjugates (ADCs) and biosimilars with a separation range of 10–700 kDa. Similarly, YMC Co. Ltd. launched a 2-µm diol phase that reportedly exhibits greater resolution than standard 3- and 5-µm materials.

 Ion-exchange chromatography is a separation technique that exploits strong interactions between opposite charges of a surface and an analyte. Various surface supports may be modified to carry permanent (strong cation and anion exchange) or variable (weak cation and anion exchange) charge that can be used to interact with and separate analytes with the opposite charge. As with SEC, ion-exchange chromatography is often used as a complementary tool for the analysis of large molecules. As shown in Table 7, however, the product launches this year are dominated by columns intended for small-molecule analysis. ES Industries released the Epic SCX column, which the company claims provides superior cation loading and stability that results from a high density bonding technology. Thermo Fisher Scientific also released two new chemistries in its IonPac line. According to the company, the AS23 phase provides advanced capabilities to detect oxyhalides among other inorganic anions in various water matrices of interest and the CS20 product provides enhanced performance for the analysis of inorganic cations and amines. The lone “large-molecule” ion-exchange product release came from Phenomenex. The Clarity OligoâSAX phase is interestingly built on a nonporous, 5-µm particle and is intended for the high-resolution analysis of synthetic oligonucleotides.

SFC continues to receive interest because of improvements in instrument design, the desire to perform greener separations, and the high resolution and speed the technique often provides. The lone line of new SFC phases reported since Pittcon 2016 comes from Waters Corporation. Several surface modifications, including 2-picolamine, diethylamine, diol, and 1-aminoanthracene built on Water’s bridged ethyl hybrid (BEH) particle technology were released under the brand name Torus. The company claims that the particle is designed especially for SFC and is enhanced by a two-stage bonding process that provides stable retention and unique selectivity.

	An interesting and potentially game-changing separation device was featured at Pittcon 2017. PharmaFluidics launched the first microchip chromatography device that is manufactured using lithographic techniques. The device features a perfectly ordered backbone that is formed by etching interstitial volume out of a silicon wafer. The company reports that the device produces highly reliable and robust reversed-phase, nanoâLC separations with unprecedented peak capacity. It is certainly possible that this development may provide a revolutionary advance for liquid chromatography.

	Although most of the attention in this series of column instalments goes to the development of new columns, accessories play a vital role in enabling liquid separations. Table 8 lists several LC-related accessories that have been recently developed. Analytical Sales and Service, Inc., reported the release of the MonoSLEEVE column heater device, which features advanced “plug-andâplay” technology. Mott Corporation launched HPLC–UHPLC static mixers intended to supply better sensitivity by greatly reducing baseline noise and improving lower limits of detection. According to the company, the mixers are easy to install, are pressure rated up to 20,000 psi, and can maintain high efficiency over a wide range of flow rates. Optimize Technologies released a series of trap columns under the brand name EXP. On-line trapping facilitates automation for high-throughput analysis and can be used for multidimensional chromatography processes. High-pressure capillary connections often frustrate the practitioner. Prolab Instruments has developed a novel capillary fittings system called Zircofit that is designed to allow users to assemble connections within minutes. The company reports the achievement of 20,000-psi pressure ratings with this design. VICI AG International launched a new back-pressure regulator, JR-BPR3, which reportedly extends the application range of the company’s existing line. The company claims the new regulator is manually adjustable between 90 bar and 300 bar and can be mounted into any column oven that maintains temperatures up to 80 °C. Lastly, ghost peaks in gradient LC can not only be a nuisance, but may also interfere with detection and quantitation of target analytes. The source of ghost peaks is often impurities in solvents used in mobile phases. Welch Materials has developed what it calls the Ghost-Buster column to help alleviate the issue. The column, packed with proprietary material, is installed between the gradient mixer and the sample injector and is intended to remove contaminants from the mobile phase and thus reduce or eliminate undesired responses in gradient analyses.

	Overall, the products released over the past year represent evolutionary developments. The greatest number of new columns falls into the category of line extensions or “gap-filling” new product lines for individual companies based on existing accepted technologies. New known chemical modifications of both SPPs and FPPs of various particle sizes continue to be developed, and these modern particles continue to find utility in niche separation modes such as HILIC and chiral. Several reported treatments of FPP surfaces may lead to more revolutionary products, and several new chemistries, especially hydrophobic–hydrophilic balanced phases, may offer unique and powerful selectivities.

	As opposed to last year, there seems to be less emphasis on large-molecule separations in terms of the number of phases that have been launched. That said, several developments in largeâmolecule separations could play a key role in the future of HPLC. An emphasis on even larger-pore-size particles may, for example, enable improved analysis of mAbs and other biotherapeutics. Techniques complementary to reversed-phase chromatography such as SEC–GPC and ion-exchange chromatography continue to grow at a steady pace. It is noteworthy, however, that no affinity chromatography columns were reported to be released over the past year. Is it possible that the emphasis on large-molecule separations is slowing?

	The commercialization of pillarâarray nano-LC devices may be the most revolutionary development over the past year. It will be interesting to see if the device leads us to more widespread acceptance of chip-based separations.

	Product reviews would not be possible without the contributions and cooperation of the manufacturers that have responded to the LCGC survey. Their effort is greatly appreciated. Although LCGC has made every attempt to include every submission in the series of review articles, it is possible that some have been missed. If there have been omissions or if you want to be sure to be included in the 2018 review series, please contact Laura Bush, Editorial Director, LCGC Europe, at 

	M.W. Dong, LCGC Europe 30(4), 208–218 (2017).

	A. Alpert, A Series of New Materials for Direct HIC-MS Analysis of Proteins in Top-Down Proteomics, presented at the 44th International Symposium of High performance Liquid Phase Separations and Related Techniques (HPLC 2016), San Francisco, California, USA, 2016.

	D.S. Bell, LCGC Europe 29(9), 506–515 (2016).

	D.S. Bell, LCGC Europe 29(4), 214–224 (2016).

 is senior manager of separations technologies and workflow development at MilliporeSigma (formerly SigmaâAldrich/Supelco). With a B.S. degree from SUNY Plattsburgh and a Ph.D. in analytical chemistry from The Pennsylvania State University, Dave spent the first decade of his career within the pharmaceutical industry performing analytical method development using various forms of chromatography and electrophoresis. During the past 15 years, working directly in the chromatography industry, Dave has focused his efforts on the design, development, and application of stationary phases for use in HPLC and hyphenated techniques. In his current role at MilliporeSigma, Dr. Bell’s main focus has been to research, publish, and present on the topic of molecular interactions that contribute to retention and selectivity in an array of chromatographic processes.
	
	Direct correspondence to: 

Column Watch

New Chromatography Columns and Accessories for 2017

This instalment highlights new high performance liquid chromatography (HPLC) systems, modules, and software that were introduced at Pittcon 2017 and during the year prior, and describes their innovative features and significant benefits. 

	This annual review of new high performance liquid chromatography systems and related products covers products introduced during the last 12 months. The majority of the products were launched at Pittcon 2017. 

	The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) is one of the world’s largest conferences on laboratory science. The 69th Pittcon was held at McCormick Place in Chicago, the largest conference centre in the United States, from 5–9 March 2017. The last time Pittcon was held in Chicago was in 2014 (and 2011 prior), and it will be there again in 2020. Note that Chicago, Orlando, Philadelphia, New Orleans, and Atlanta are the cities selected for hosting Pittcon with Chicago in the rotation every 3 years and the others about every 6 years. This frequency makes sense since Chicago is the third largest city in the U.S. and is also an industrial, agricultural, financial, transportation, and communication centre. We had great weather this year at the conference with many sunny days and spring-like temperature in the 50s.

	Pittcon is impressive with ~2000 presentations, 100+ short courses, and a huge exposition with over 800 exhibitors, including over 120 new manufacturers displaying for the first time. Although the number of attendees has been dropping in recent years, registration hit 13,300 after three days. With open admission on Thursday, a total of above 14,100 was expected, which is very good news for the conference and an indication of a healthier economy and positive outlook for instrumentation. This year’s conference featured a plenary lecture by Dr. Karl Deisseroth, a professor of bioengineering at Stanford University, on insights on optogenetic proteins using electron microscopy and mass spectrometry (MS). Once again, Pittcon 2017 provided a “mecca” for analytical chemists and a premier venue for instrument manufacturers.

	The perfect marriage of ultrahighâpressure liquid chromatography (UHPLC) and MS instruments continues to be the megatrend in chromatography in recent years (1–5), even though this is another off year for new UHPLC system introductions. With many secondâgeneration UHPLCs debuted by all major manufacturers in prior years (2,3), this year we are seeing mostly line extensions, applicationâspecific systems, modules (particularly new MS or MS add-ons), and software products.

	Table 1 lists the new product introductions (arranged alphabetically by vendor name), followed by more detailed descriptions or commentaries. 

New HPLC and UHPLC Systems and Line Extensions

	New HPLC, UHPLC, supercritical fluid chromatography (SFC), ion chromatography (IC), modules, chromatography data systems (CDS), and related software are summarized in Table 1 and described with more details below.

	Agilent 1260 and 1220 Infinity II systems-Two years ago Agilent introduced the 1290 Infinity II LC systems. This year, Agilent completed the InfinityLab LC Series with the 1260 and 1220 Infinity II systems, all to deliver full method compatibility. Innovative 1290 Infinity II LC technology is leveraged with the 1260 Infinity II Multisampler with dual-needle sampling and Vialsampler. A larger column oven, which fits 30-cm columns and can accommodate four columns with a single switching valve, and a range of detectors are common to both systems. The 1260 Infinity II pumps have a 600-bar pressure limit and are available in either stainless steel or bioinert format. The 1220 Infinity II system remains an integrated 600âbar system available with an isocratic or a dual-channel gradient pump that can be paired with a manual injector or an autosampler, a variable-wavelength UV detector or a diodeâarray detector, and external special detectors including a massâselective detector. All systems can be operated with Agilent OpenLab CDS Software as well as third-party software with the universal Agilent Instrument Control Framework.

	Bruker Elute LC series-Bruker introduces the Elute LC series systems for LC–MS analysis: SP (700 bar), UHPLC (1300 bar for ultrafast analysis), OLE (for on-line extraction), and HT (high-throughput with the new PAL3 autosampler).

	Cromite portable HPLC-Cromite introduced a handheld, portable HPLC system that consists of an isocratic pump with a 275-bar or 4000-psi pressure limit and a max flow rate of 4 mL/min, and a UV–vis detector with a light-emitting diode (LED) source that provides up to four customizable wavelengths. The system is designed with novel approaches to mobile-phase storage and sample introduction and preparation, and it includes a single-ended leak-proof column. The system weighs 2.5 lb (1 kg) and has overall dimensions of 6.8 × 3.2 × 3.4 in. (173 × 81 × 86 mm). The device can be controlled from a PC, smartphone, or tablet via Bluetooth and can be powered off a 12 VDC power supply or external battery. The company is targeting applications in education, environmental, and production QC testing with this offering. Figure 1 shows an example chromatogram obtained using the Cromite portable HPLC system.

Knauer Azura Analytical and SemiâPrep HPLC-Knauer offers the Azura as a system solution for analytical to preparative HPLC. The analytical systems are available with binary or quaternary pumps (stainless steel at 700 and 1000 bar as well as a biocompatible, ceramic version at 400 bar). The semipreparative 200-bar systems are capable of flow rates as high as 50 mL/min and are available in both stainless and biocompatible formats. These systems can be configured with appropriate autosamplers, column ovens, and UV–vis, refractive-index, and diodeâarray detectors. The systems can be controlled by a controller, a mobile tablet, or a chromatography data system (CDS) such as OpenLAB EZChrom, ClarityChrom, and Chromeleon.

	Prolab Instruments ZirconiumHT-The Zirconium High-Throughput UHPLC system is a micro-UHPLC system with a novel trapping column configuration for loading and analyzing samples at the same time using only one binary pump. Suggested applications are 2D micro and nano-LC analyses.

	Shimadzu Cannabis analyzer-The Shimadzu Cannabis analyzer is a HPLC–UV system designed specifically for quantitative determination of cannabinoid content for potency with three method packages optimized for highâthroughput, high sensitivity, and high resolution. Figure 2 shows an example chromatogram for potency testing of 11 cannabinoid standards on the system.

	Sotax Xtend On-Line Dissolution with Waters Acquity H-Class UPLC-For those who have been waiting for a modernized replacement of the Alliance-based 2695D automated dissolution analyzer, the wait is over. Waters, in conjunction with Sotax, now offers an automated UHPLC system based on the Acquity H-Class UPLC system and Sotax’s dissolution testing apparatus with Sotax’s sample manager (SAM) to allow sample withdrawals, fractions collection, and autoinjections from USP dissolution systems.

	Bonna-Agela OCTOPUS purification system-Bonna-Agela Technologies (acquired by Danaher last year) introduced a flexible and easy-to-use modular purification system for both flash and preparative HPLC with a family of four pumps (1–200 mL/min and pressure limits of 14–200 bar [200–3000 psi]) that can be equipped with automatic fraction collection, an autosampler, a column oven/switcher, and UV–vis, photodiodeâarray, evaporative light scattering, refractiveâindex, or MS detection.

	Agilent 1260 Infinity II SFC-The Agilent 1260 Infinity II SFC system now offers an improved Multisampler autosampler, which uses an innovative feed injection principle that allows 0.1–90 µL of injection volume range with the higher sample capacity of 432 2-mL vials. The 1260 Infinity II SFC binary pump can provide pressures of 600 bar at up to 5 mL/min. The Infinity II SFC control module now offers full flow into evaporative light scattering or MS detection systems. A novel low dispersion nozzle with variable back-pressure settings enables outstanding peak shapes in this case.

	Metrohm Eco IC-The Eco IC ion chromatograph is designed for routine analysis of anions, cations, and polar compounds in environmental testing and education applications. The system is complete with a suppressor, a conductivity detector, software, and an optional autosampler with up to 36 samples.


	
	Cecil Instruments Electrochemical Detection Adept HPLC system-Cecil Instruments offers a modular HPLC system with electrochemical detection that supports DC-amperometric, scanning, and pulsed modes.

	FF Runge Mikron detector series-These very compact photometric, fluorimetric, and conductivity HPLC detectors are based on LED technology.

	Thermo Scientific Vanquish Flex binary pump-The Vanquish Flex binary system now features a binary highâpressure gradient pump with 2 × 3 solvent channels (1000 bar or 15,000 psi) and low gradient delay volume capable of delivering flow rates of up to 8 mL/min. The entire system provides a biocompatible flow path.

	Wyatt ViscoStar III-The Wyatt ViscoStar III viscometer detector offers measurements of intrinsic viscosity for sensitive macromolecular size and conformation for polymer branching analysis. The detector uses MarkâHouwink-Sakurada parameters and hydrodynamic radii intrinsic viscosity calculations using Wyatt’s ASTRA software package in size-exclusion chromatography.


	
	JASCO HPLC–MS and SFC–MS-JASCO offers the Advion Expression compact single-quadrupole MS system for its HPLC–MS (LC-4000 Series) and SFC–MS (SFC-400 Series SFC) systems with mass-triggered fraction collection and integrated system control using the ChromNAV CDS.

	PerkinElmer QSight MS/MS system-The QSight tripleâquadrupole system is a highly sensitive dual source self-cleaning LC–MS/MS analyzer that enables laboratories to test complex samples for food contaminants (such as pesticides) for regulatory compliance. QSight provides a complete pesticide solution from sample preparation and extraction to analysis and reporting.

	Sciex X500B QTOF-Sciex launches the compact X500B QTOF system designed to bring simplicity, performance, and robustness for biotherapeutic analyses. The new SCIEX OS software interface and BioPharmaView 2.0 processing software provide
	an integrated LC–MS solution for experts or
	novices performing standard biotherapeutic characterization. 

	Sciex QTRAP 6500+ LC–MS/MS-This combined quadrupole–ion trap system includes the highly sensitive triple-quadrupole 6500+ MS/MS system for high-throughput LC–MS/MS analysis for both small- and largeâmolecule quantitation. The MS system is equipped with the multicomponent IonDrive technology for enhancing sensitivity and is capable of six orders of magnitude of linearity, up to 20,000-u/s scan rates, and 5-ms polarity switching.

	Shimadzu LCMS-8045-Shimadzu’s LC-MS/MS-8045 system is a mid-range triple-quadrupole MS system that performs routine quantitative analyses in food safety and environmental testing. The instrument features a scan speed of 30,000 u/s without the loss of mass accuracy and a polarity switching time of 5 ms. It is operated using Shimadzu’s LabSolutions software. A dual electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) source is available.

	Shimadzu CLAM-2000 clinical laboratory automation module for LC–MS-The CLAM-2000 system automatically performs all of the sample preparation procedures necessary for analyzing blood (from blood collection tubes) and other biological samples by LC–MS/MS for therapeutic drug monitoring and testing of abused drugs.

Thermo Scientific Q Exactive HF-This compact quadrupole-orbitrap hybrid LC–MS/MS system designed for rapid quantitation of proteins, peptides, lipids, metabolites, and contaminants is capable of scan rates up to 18 Hz and a maximum resolution of 240,000 FWHM.


	
	Agilent 1290 Infinity II preparative fraction collector-This automated open-bed fraction collector allows efficient and flexible automated collection of up to 432 fractions or volumes up to 5.9 L triggered by time-, peak-, or mass-based mode.

	Analytical Sales and Services Sentinel-The Sentinel is a fluid-level indicator for monitoring up to three bottles of waste or mobile-phase containers and is equipped with status display and alarm functions.

	Biotech AB DEGASi Compact and Prep+ degassers-Two new degassers were introduced: The DEGASi Compact degasser with a small internal volume and the DEGASi Prep+ degasser that supports preparative HPLC, flash chromatography, and highâthroughput applications.

	ECOM ECF 2096 fraction collector-The ECF 2096 automated fraction collector collects up to 48 tubes of 8-mL volumes. 

	JM Science Sonic reservoir system-A monitoring system for up to three HPLC reservoirs and waste containers using sonic detection and an LED display.

Chromatography Data System and Other HPLC-Related Software


	
	Agilent OpenLAB CDS 2.1-This newest version of OpenLAB CDS provides a networked or standalone environment that combines ease of use with rigorous regulatory compliance. This latest platform is an LC-, GC-, LC–MS-, and GC–MS-capable CDS for the QC laboratory with strong data integrity and audit trail review capabilities. Version 2.1 contains many new features including an extended support for non-Agilent instruments and improvements in MS quantitation, peak integration, and reporting. Figure 3 shows a screen shot of the Agilent OpenLAB CDS with four tiled panels for data processing, chromatograms, peak analysis, and processing methods.

ChromPerfect SL-ChromPerfect introduces a CDS for small or mobile laboratories that controls up to four chromatographs with web-based and Windows 10 operating systems. It is compatible with tablets with data processing, reporting, and analysis functions.

	DataApex Clarity v.7.2 CDS-The Clarity v.7.2 advanced CDS operates under Windows 10 and can be connected to four instruments and up to 32 data channels. Its optional control modules provide integrated control of selected instruments. Extensions are available for diodeâarray detectors, gel permeation chromatography (GPC) analysis, MS (single quadrupole and time of flight), and system suitability testing.

	Waters Empower Cloud-Waters announced a release of cloudâbased Empower 3 (feature release 4) available through Amazon Web Services in late 2017. This cloudâbased CDS platform is particularly suited for pharmaceutical companies to reduce costs for implementation and maintenance and can globally support all subsidiaries and contract organizations.

	ACD/Labs AutoChrom 2016 version-This automated HPLC method development software uses predictive simulation and can interface with both UV and MS detection. This version includes overlaid logD curves, better peak tracking, and an enhanced column data base with Tanaka parameters and superficially porous particle columns. It also has better connectivity to Chromeleon 7, Empower, and other MS data systems.

	ACD/Labs LUMINATA-LUMINATA multitechnique software offers comprehensive data aggregation for impurity characterization and knowledge management applied to pharmaceutical development applications. Figure 4 shows a screen shot from this software displaying multiple panels including the overall synthetic scheme of the drug substance, a table of potential impurities, and the data associated with each impurity (HPLC chromatogram, UV, and mass spectra).

ACD/Labs MetaSense-MetaSense software uses LC–MS data as part of its workflow in metabolite identification. It facilitates faster, easier, and more accurate detection and identification of predicted and unexpected metabolites. Built on the ACD/Spectrus platform, MetaSense offers functionalities including the facilitation of decision-support and effective knowledge sharing throughout organizations as well as the ability to support LC–MS analytical data from most instrument vendors.

	JASCO method scouting software-JASCO offers a method scouting software for automated solvent and column screening with up to 10 solvents and 10 columns. It also supports multiple detectors and can be configured for use with up to 30 columns and parallel screening.

	S-Matrix Fusion QbD method development software v. 9.8.0-The Fusion QbD software for automated LC method development using principles of quality by design (QbD) and design of experiments (DOE) has been updated to include an enhanced user interface with an integrated chromatogram viewer, new project management capabilities with extended data security, additional support for GC and capillary electrophoresis (CE) method development, and increased instrument support for most HPLC and UHPLC systems, including hybrid systems.

	Thermo Scientific Chromeleon 7.2 SR5 CDS-The Chromeleon CDS is a multitechnique, multivendor scalable platform for GC, LC, IC, and MS applications and includes a unique workflow automation called eWorkflows. The new platform is optimized for large-scale installation for thousands of users and instruments with data integrity features for regulatory compliance. This latest platform is designed particularly for MS analysis including biotherapeutics and is equipped with capabilities for automated target mass analysis for both LC–MS and GC–MS.

	Thermo Scientific AppsLab methods library-The latest AppsLab library is a searchable on-line repository for easy access to chromatography and MS application databases including analytical methods, chromatograms, and related compound information (1886 applications and 380 readyâtoâuse eWorkflows for Chromeleon CDS).


	
	Most analytical chemists come to Pittcon for professional development and networking. My schedule this year included teaching several short courses on HPLC, UHPLC, and method development, and chairing an invited symposium on UHPLC method development in pharmaceutical analysis. My networking events included a Chrom Mixer sponsored by Chrom Forum Delaware Valley in memory of Jack Kirkland (more than 500 attendees), a networking dinner event with the Chinese American Chromatography Association (CACA) attended by more than 120 conferees (featuring a lecture by Dr. Perry Wang of the US FDA on the perfect marriage of HPLC and MS), and the annual Pittcon presidential reception, where I personally thanked many Pittcon staff and programme chairs.

	I spent considerable time on the exposition floor to verify information on new HPLC products for this column instalment. I returned home Thursday night, slept late, and woke up to find 4 inches of snow on the ground in Connecticut. We were lucky as the weather can be unpredictable in early March in Chicago, as well as the Northeast. Rumour says that next year will be the last year for Pittcon in Orlando because the vendors complained about conference attendees visiting Disney World instead of the exposition. I love Orlando because of the excellent restaurants and the warmer weather-and I plan to visit both the exposition and the theme parks.

	This instalment provides a brief and nonexhaustive overview of new HPLC product introductions (systems, modules, MS, CDS, software, and related products) at Pittcon 2017 and during the prior year. Readers should contact manufacturers for additional technical details. Note that new HPLC columns are covered separately in LCGC’s “Column Watch” annual review by David Bell. The opinions expressed in this instalment are the author’s own and bear no reflections on those of LCGC magazine, the Pittsburgh Conference, or any other organizations.

	I wish to give thanks to the marketing and editorial staff of LCGC and all HPLC manufacturers for their timely responses to my numerous requests for information. I am particularly grateful to Davy Guillarme and Szabolcs Fekete of U. Geneva, Harika Vemula of U. Missouri at Kansas City, David Van Meter from EAG Laboratories, Hernan Fuertes from Ardelyx, and Matt Mullaney from Pentec Health for their invaluable editorial and technical input.

		D. Guillarme and M.W. Dong (Eds.), UHPLC: Where We Are Ten Years After Its Commercial Introduction, 

 is a principal of MWD Consulting, which provides training and consulting services in HPLC and UHPLC, pharmaceutical analysis, and drug quality. He was formerly a Senior Scientist at Genentech, Research Fellow at Purdue Pharma, and Senior Staff Scientist at Applied Biosystems/PerkinElmer. He holds a Ph.D. in analytical chemistry from City University of New York. He has more than 100 publications and a best-selling book in chromatography. He is an editorial advisory board member of LCGC North America. Direct correspondence to: 

Perspectives in Modern HPLC

This instalment highlights new high performance liquid chromatography (HPLC) systems, modules, and software that were introduced at Pittcon 2017 and during the year prior, and describes their innovative features and significant benefits.

New HPLC Systems and Related Products: A Brief Review

The 30th International Symposium on Polymer Analysis and Characterization (ISPAC) will take place in Linz, Austria, on 11–14 June 2017.

is a non-profit scientific organization formed to provide an international forum for the presentation of recent advances in the field of polymer analysis and characterization methodologies. This unique symposium brings together analytical chemists and polymer scientists who are involved in the analysis and characterization of polymeric materials. Meetings are held annually, rotating to venues in the USA, Europe, and Asia, with ISPAC 2016 taking place in Singapore.

 sessions comprise a three-day programme and feature invited lectures, poster sessions, and discussion and information exchange sessions, all focused towards polymer analysis and characterization approaches, techniques, and applications. Each session features multiple lectures and a one-hour open discussion period where participants from academic, industrial, and government settings can discuss different aspects of polymer analysis and characterization approaches, techniques, and applications. The symposium offers a perfect networking platform, enabling the free exchange of ideas and tips about different techniques, as well as an opportunity to learn about the latest developments firstâhand.

 will focus on several topics including a broad spectrum of polyolefin-related themes, specific topics on advanced fractionation, natural polymers, solar applications of polymers, and electrical properties of polymers. Esteemed scholars from different institutions will give plenary lectures that will be followed by invited and contributing papers. The first day of the conference will be a workshop on advanced characterization methods and is specifically designed for graduate students and young researchers. The topics and speakers of the workshops are as follows:


		Broadband Dielectric Spectroscopy as a Complementary Analytical Tool to Characterize Polymer Systems

Manfred Wilhelm, Karlsruhe Institute of Technology, Germany 


		Rheology: From the Basics up to Complex Polymers

Stepan Podzimek, University of Pardubice, Czech Republic


		Determination of Molar Mass Distribution and Branching of Synthetic and Natural Polymers Using Separation Techniques with Advanced Detection

Antje Potthast, UniversitÐt fÐ¯r Bodenkultur, Tulln, Austria


		Analyzing Lignocellulosic Biopolymers-Challenges, Requirements, and (some) Solutions

Clemens Schwarzinger, Johannes Kepler University, Linz, Austria 


		Advanced Mass Spectrometry of Polymers

	A number of invited speakers have already confirmed their attendance from institutions across the world as well as a number of sponsors who are supporting the conference and will be providing an accompanying instrument exhibition.

	More details on the ISPAC 2017 symposium including registration and abstract submission can be found at 

	As the organizers of this conference we hope to see you there and look forward to celebrating the 30th anniversary of ISPAC with you.
	
	

Prof. Harald Pasch (University of Stellenbosch, South Africa)

Prof. Clemens Schwarzinger (Johannes Kepler University, Linz, Austria)

Events

ISPAC 2017: 30th International Symposium on Polymer Analysis and Characterization

Issue PDF

Click the title above to open the LCGC Europe April 2017 regular issue, Vol 30, No 4, in an interactive PDF format.

LCGC Europe-04-01-2017