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Click the title above to open the LCGC Europe March 2014 Analytica Guide Supplement, Vol 27 No s3, in an interactive PDF format.

Vol 27 No s3 LCGC Europe March 2014 Analytica Guide Supplement PDF

As Mozart's birthplace, Salzburg, Austria, is well-known for its connections to classical music. However, on 14–18 September it will become host to the 

30th International Symposium on Chromatography (ISC 2014)

 attracting chromatographers from around the world. 

 is a major liquid chromatography/gas chromatography LC/GC) conference that was first held in 1956 in London, and since then has been held biannually in various European cities. 

Chromatography in its various forms has become an eminently powerful analytical technique that is now indispensable for combating challenges in fields such as metabolomics, proteomics, pharmaceutical analysis, food control, environmental analysis, clinical chemistry, and doping control. Hyphenation with sophisticated mass spectrometric detection has led to the possibility of identification of unknown analytes with quantitation limits in a range that was beyond reach only a few years ago. Gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) technologies now represent a gold standard for analysis methods. 

Progress in chromatography will always be an interplay between academic research performed at universities and the production of sophisticated and reliable instrumentation by manufacturers. Some may remember that sub-2 μm particles were investigated decades ago as stationary phases for LC, but dedicated ultrahigh-pressure liquid chromatography (UHPLC) instrumentation only became commercially available in 2005. Monolith-type stationary phases and core–shell particles have been developed, highlighting the continuous advances in column and instrument technology that are occurring. Similarly, supercritical fluid chromatography (SFC) has been the focus of research for many years, but its acceptance by chromatographers was quite slow until recently when a new generation of SFC instruments started a renaissance of this technique. 

 will be a forum for discussing fundamentals, applications, and the latest developments in instrumentation. The internet is an immense source of information for all fields of research, but identifying the most relevant information has become tricky. Personal communication and networking are required more than ever to keep the momentum going. It is important to know the faces behind the names of authors of scientific papers. Personal communication is a prerequisite for progress, as is reflected in the motto of 

ISC 2014: "Communicating Separation Science for the Future".

A broad knowledge of separation science is required to become an expert and to judge the best option for tackling and solving a given analytical problem. As well as modern LC and GC instruments, it is important not to forget capillary electrophoresis (CE and CE–MS), which can sometimes be a more appropriate approach. The selection of the most suitable analysis methodology depends on the analytical challenge at hand including consideration of the crucial step of sample pre-treatment. 

 will cover all aspects of modern separation science, including hyphenation with MS–MS instrumentation, and will, therefore, go beyond the scope of some other conferences that are dedicated to single fields of chromatography. 

 will provide a "one-stop shop" for analytical chemists and attendants to see a complete picture of current progress. It will bring together a wide range of international scientists from both academia and industry so that attendees can learn the most up-to-date information about theory and practice from each other to accelerate progress in the field. 

 will enjoy an exceptional scientific meeting in the unique and beautiful cultural surrounds of Salzburg! 

Chairpersons: Wolfgang Buchberger, Michael Laemmerhofer, and Wolfgang Lindner

  International Symposium on High Performance Liquid Phase Separations and Related Techniques

				Hilton Riverside Hotel, New Orleans, Louisiana, USA 			

ExTech2014: 16th International Symposium on Advances in Extraction Technologies

				Center of Mediterranean Architecture, Chania, Crete, Greece 			

Technical University of Crete, Greece 			

Recent Advances in the Applications of Chromatography–Mass Spectrometry to Environmental Matrix Analysis

				The Auditorium, British American Tobacco Group Research and Development, Southampton, UK 			

Environmental Mass Spectrometry Special Interest Group of the BMSS 			

20th International Symposium on Separation Sciences

				Hotel DAP, Prague, Czech Republic 			

The Chromatography and Electrophoresis Group of the Czech Chemical Society 			

Departments

Event: ISC 2014: The Sound of Chromatography

The demonstration of drug substance (DS) or drug product (DP) stability over the shelf life is a regulatory requirement In the pharmaceutical industry. To fulfill this requirement, a stability-indicating method (SIM) must be developed and validated to separate and quantify both the active pharmaceutical ingredient (API) and its related compounds (process impurities and degradation products). This article discusses the characteristics and relevant considerations for the development and validation of a SIM.

Concerns over the inability of a drug product (DP) to meet quality standards over the course of its shelf life have been present since the early 1970s. In 1975, the United States Pharmacopeia (USP) added a clause about the expiration dating of drug products. The US Food and Drug Administration (US FDA) provided the first stability guideline in 1984. The US FDA took further steps in 1987 by issuing guidelines for the submission of stability information and data for Investigational New Drugs (IND) and New Drug Applications (NDA). The International Conference on Harmonization (ICH) created the Q1A guideline in 1993 to harmonize the requirements for international marketing in Japan, USA, and the European Union. Additional guidelines that followed from ICH included Q7A, GMP guide for active pharmaceutical ingredients (API). More recently, the World Health Organization (WHO) has released guidelines on the stability of pharmaceuticals (1).  

Despite all of the requirements from the regulatory and governing entities, there is still not a clear consensus of what constitutes a stability-indicating method (SIM). The guidance documents also do not provide details about the scope and degradation study practices (1). Unfortunately this leaves many in the industry with a requirement that must be achieved but without clear direction. This article will review existing literature and current best practices for a SIM. The objectives for a SIM, the process for development and validation, and the critical characteristics will be discussed. 

The method objectives should be defined early, so the development process can be clearly established. For example, the analytes that need to be separated should be established early in the process. Stress testing or forced degradation studies can be useful in defining degradation products and the major degradative pathways. In general, for ANDA (Abbreviated New Drug Application) development of generic drug products, only compounds that exceed the ICH threshold for reporting should be investigated unless special toxicology concerns (for example, genotoxic impurities) are known. Once it is known which compounds are of interest, further objectives such as desired resolution, limit of quantitation (LOQ), precision and accuracy, analysis time, and adaptability for automation can be defined (2).  

For biologics, a series of methods based on orthogonal approaches may be required to achieve a SIM (1). Biologics degrade in a much different manner than small molecules. To identify all of the degradation pathways requires a variety of biochemical, biophysical, and biological methods, which can lead to a lengthy process (3). While small molecules generally degrade following first order kinetics, biologics can follow secondary or higher order kinetics that may not fit to linear or exponential curves. Degradation of biologics can be both chemical (oxidation, de-amidation, disulphide bond rearrangement, hydrolysis) and physical (aggregation, adsorption, loss of three-dimensional structure) (4).  

The Process of SI Method Development and Validation

There are three major steps to undergo when developing a SIM: Obtaining a suitable sample, developing the method, including the separation technique and detector, and the final validation of the method (1,5). This process was further broken down into seven steps by Bakshi and Singh (6,1). Snyder, Kirkland, and Dolan (7) and Kazakevich and Lobrutto (8) also provide extensive details on high performance liquid chromatography (HPLC) method development processes. According to the authors, the following steps must occur for a SIM: 

1. Understanding of the drug substance (DS) chemistry; 

2. Preliminary separation method development; 

3. Defining degradation products and reference substances; 

The focus of step 1 is on having a complete understanding of the DS active pharmaceutical ingredient (API) chemistry including physicochemical properties and anticipating its degradation products. For instance, if a new API is a derivative of one previously used and well established, it is likely that the degradation pathways will occur in a similar fashion and therefore several degradation products can be anticipated (6). Understanding of the intrinsic properties of the API is very useful to determine a starting point for step 2. Several aspects of the SIM, such as diluent choice, sample preparation, separation technique (column choice), and detection technique, are dependent on the intrinsic properties of the API. Dissociation constants and partition coefficients can be used to determine an efficient extraction solvent and a proper pH for the mobile phase to provide the best separation.  

The extraction solvent can be selected by matching it with the most polar functional group on the molecule. An understanding of fluorescent properties, chromatographic behaviour, spectrophotometric properties, and oxidation–reduction potentials can be useful to identify the best means for measuring the analytes. A compatibility study known as a "drug-excipient interaction study" may be needed to properly assess the API and excipient interactions (9,2) for method development of a SIM for a drug product. However, it is important to note that this is merely a starting point and an API's true degradation products will not be known until forced degradation studies are conducted. For example, if the API has a nucleophilic centre, it is expected to react with an electrophile to form an addition product during base degradation. However, without performing the actual base degradation study, this cannot be positively determined. 

One overlooked phenomenon, especially for solid dosage forms, was discussed by Arindam et al. (10). A solution compatibility study should be considered in addition to the drug excipient study. This type of study evaluates solution chemistry and potential solution stability issues. 

Preliminary Separation Method Development: 

Step 2 is related to the development of the separation process and can be the most time-consuming step. Within the pharmaceutical industry, reversed-phase LC is the most commonly used separation technique. The sample solvent, mobile phase composition and pH, column type, and temperature can all be varied to achieve the appropriate separation. Adjustment of the mobile phase pH can be a very good tool for the separation of ionizable compounds and is often overlooked during development (11). With advances in technology, ultrahigh-pressure liquid chromatography (UHPLC) systems equipped with small particle size columns can enable shorter run times and a faster development process without sacrificing resolution within the analysis. Software calculators to convert standard HPLC conditions to UHPLC conditions are a very useful tool if an existing HPLC method is suitable but a higher efficiency or shorter run times are desired. Sometimes it is very helpful to develop the method using UHPLC and convert it to HPLC for routine use. The development process can be performed more quickly by virtue of having shorter experiment times using the UHPLC approach. A review of the UHPLC facts and myths was recently published by Michael Dong (12). 

Michael Dong (13) also recently discussed a three-pronged template approach to HPLC method development. This included fast isocratic LC, generic broad gradient, and multi-segment gradient. For SIMs, fast isocratic methods are generally not acceptable because most are not capable of separating all components and degradation products. A generic broad gradient is commonly used for in-process monitoring in organic synthesis laboratories using a gradient such as ~5–95% B. Analytical development and quality control (QC) laboratories generally develop a custom method for each new chemical entity, which can lead to several methods each with a different column and mobile phase. A multi-segment gradient is more complex but is useful when trying to separate isomers or degradation products with similar structures to that of the API. A shallow gradient may be used to maximize resolution around the API region. A steep gradient is then used to elute the strongly retained components (13).  

The detectors used for liquid chromatography (LC) are UV, fluorescence, charged aerosol detector (CAD), evaporative light scattering (ELSD), refractive index (RI), amperometry, and conductivity. While mass spectrometry (MS) detection is becoming more common, particularly for genotoxic impurities, UV detection is still the most common for SIMs. The use of a diode array detector (DAD) is advantageous to obtain spectral information (maximum absorption wavelength and peak purity) of the parent compound and each separated impurity. It should be noted that spectral differences can only be observed from compounds which show different UV spectra in the region selected. If the spectra are similar from multiple components, peak purity by DAD may not offer any additional information. An MS detector is quite useful to determine the identity of unknown compounds, which is step 3 of the process. For this reason, it is ideal to use a mobile phase that is compatible with MS (5). For a complex formulation, which contains many UV active excipients, the challenge is to minimize interferences. Detectors which are very specific, such as fluorescence and MS, can be very useful to suppress response from excipients when used properly. Alternatively, a UV wavelength programme can reduce the interference as well. 

Biologics may require multiple methods for demonstrating the stability-indicating profile. Assays must be able to detect changes in identity, purity, and potency (if biological activity can be defined and measured). Typical purity assay methods include electrophoresis, immune-electrophoresis, chromatography methods, and peptide mapping. These methods must be able to detect changes as a result of de-amidation, oxidation, sulphoxidation, aggregation, and fragmentation (14).  

Defining Degradation Products and Reference Substances: 

Step 3 focuses on finding degradation products to optimize the development process (step 4). Once there is an understanding of the likely degradation products from the API(s), samples or standards are needed to determine the actual retention time of the degradation products.  

The use of forced degradation studies is the primary way to obtain an adequate sample containing potential degradation products. Ideally, the DS is allowed to degrade no more than 10% using any variety of heat, acid, base, light, or oxidation. All avenues of potential degradation should be explored to ensure that all possible degradation products are being identified. Degradation above 20% is generally not acceptable as this can result in secondary degradation products that may not be seen under normal storage conditions (5). Many authors have suggested that 20% may be too much degradation and recommend the API to be degraded between 5% and 10% (2). Based on the knowledge that several assay methods have a specification of 90.0% to 110.0% over stability shelf life, a 10% target for degradation under forced degradation conditions is deemed appropriate. The anticipated assay specification and definition of a "major change of the content" from ICH Q1A should be taken into consideration when determining a proper amount of degradation. Stress testing of pharmaceuticals was thoroughly discussed by Baertschi, Alsante, and Reed (15). 

Degradation products that exceed the ICH thresholds for identification or qualification encountered in stability studies require special attention, including complete identification. A number of techniques can be used for the identification of unknowns (impurities or degradation products). When the unknowns cannot be isolated in pure form, any combination of HPLC coupled with diode-array detection (DAD), LC–MS, LC coupled to nuclear magnetic resonance (NMR), and gas chromatography (GC) coupled to MS can be used for complete structure elucidation. Singh and Rehman (16) explored the uses and limitations of each of these techniques. NMR and the synthesis of the reference compound remain the gold standard in structure confirmation of the identified impurities. 

HPLC–DAD will provide the UV spectrum of an unknown as well as the peak purity of any peak (if significant in size). A similar UV spectrum to that of the API indicates that the degradant has the same chromophores as the API. A degradant with a significantly different spectrum indicates a change in the chromophoric portion of the molecules (16).  

LC–MS has become a powerful tool in the identification process of unknowns in pharmaceuticals. As stated earlier, an LC–MS friendly method is ideal for a SIM. Methods that contain nonvolatile components (phosphate buffers, etc) should be avoided if possible to allow for analysis by LC–MS during the unknown identification process. LC–MS is advantageous for its sensitivity, selectivity, speed, and ability to identify trace components within a complex mixture. Based on the polarity of the compounds being analyzed, different ionization techniques should be used. For nonpolar compounds, atmospheric pressure photo ionization (APPI) is the best technique. Electrospray ionization (ESI) can be used for compounds with low polarity to ionic molecules. Atmospheric pressure chemical ionization (APCI) should be used for compounds with weak to medium polarity. Matrix-assisted laser desorption–ionization (MALDI) is primarily used for large biomolecules or biopolymers such as proteins and peptides. Atmospheric pressure matrix-assisted laser desorption–ionization (AP–MALDI) is similar to MALDI but performed at atmospheric pressure and can be used to ionize large molecules (16). Deuterium-exchanged LC–MS has been shown to be very useful for the identification of isobaric compounds (for example, N-Oxides and hydroxides). Cation enhanced mass spectrometry is very helpful to increase ionization efficiency for some molecules using electrospray ionization, for example, vitamin D derivatives (17, 18, 19). For structure elucidation, use of exact mass spectrometers such as time-of-flight (TOF)-MS and sequential multi-stage fragmentation using ion trap is becoming indispensable. 

Along with LC–MS, LC–NMR has also become a technique of choice for some challenging identification of unknowns. LC–NMR can not match the sensitivity of LC–MS but can definitively provide a molecular structure when LC–MS data is insufficient or isolation of the unknown is not possible. As with LC–MS, the LC method must be compatible with this LC–NMR technique. Protons from the mobile phase components can cause interference. For this reason, deuterated solvents should be used (16). 

Combinations of techniques such as tandem mass spectrometry (MS–MS) or LC–NMR–MS are additional tools that can efficiently provide important information about unknown compounds. 

While the majority of unknown identifications are performed by any of the described LC techniques above, some compounds (such as raw material and critical reagents) may be better suited for GC analysis. This may be a result of the volatile nature of the molecule or its lack of polarity making it unsuitable for LC separation or detection. In these cases, GC–MS may be an appropriate method. Electron ionization (EI) and chemical ionization (CI) are two techniques used. EI is an aggressive ionization technique that may not produce a molecular ion, thereby making identification difficult. For this reason CI, which is a soft ionization technique, is preferred (16). 

During initial method development, it is recommended to make significant changes in the separation variables to create significant changes in the chromatographic profile, so step 4 can be carried out quickly as needed. Smaller adjustments can be made after the significant separation requirements have been achieved (5). The fine-tuning step or method optimization often involves changes to the mobile phase (pH, buffer type and ionic strength, pH), gradient profile (gradient time, number of segments), flow rate, injection volume, and sample concentration to ensure that the method can separate all impurities and degradants and is compliant to ICH guidelines in sensitivity and other requirements.   

The final stage of step 4 should be a method prequalification to ensure that the newly developed method is "validatable". Several of the experiments that are to be performed during validation should be completed at this point to ensure the method is fully capable of meeting the validation requirements. Since method validation is a good manufacturing practice (GMP) process and method development is not, this is an important step to ensure that the validation step proceeds smoothly without unexpected failures or anomalies (13). 

 The final step of the SIM process is validation of the finalized method. The ICH guidelines (Q2A, Q2B) provide detailed validation requirements (20). Validation should include specificity, linearity, accuracy, precision, intermediate precision, LOQ, and robustness. If a thorough and complete development process has already occurred, the validation step is merely a formality of repeating and documenting several experiments that should have already occurred. To establish specificity of a SIM, it is critical to show separation of all known key components, including process impurities and degradation products, and to demonstrate peak purity on the API(s) when possible. However, it is important to note that while peak purity is a useful tool for large peaks, the same cannot be said for peaks at or near the LOQ of the method. At these low levels, the baseline noise can interfere with peak purity calculations and data is unreliable at best. An example of a typical method validation process, except specificity, is given in Figure 1.  

Figure 1: Typical validation characteristics of a method. Note: Please see Figure 2 for specificity.			

This approach is a more general approach. Phase-specific strategies are normally performed by different companies for method development and validation, especially for ANDA development. For generic drug product development, the timeline is condensed and attention is given to the similarity profile of the drug in development with the commercial innovative drug.  

Recently, the USP Validation and Verification Expert Panel published a stimuli article on life cycle management of analytical methods. According to this paper, instead of current discreet steps of development, validation, or verification, life cycle management of analytical methods should be based on ICH Q8, Q9, and Q10. Analytical target profiles, quality by design concepts, control strategies, knowledge management, and risk-based approaches are the keys to this new concept for life cycle management. It also emphasizes an understanding of the variability in the data, probability, and confidence interval. As a result of these recommendations, the Validation and Verification Expert Panel proposed that the concepts addressed in USP 1225, 1226, and 1224 be revised to integrate the processes for demonstrating that an analytical procedure is fit for purpose throughout its life cycle, and that these three chapters be compiled into a single general information chapter 1220 and a new general chapter 220 that specifies the basic requirements (21).  

The primary characteristic of a SIM is its ability to separate degradation products or impurities from the active(s) and each other (when needed). The SI nature can be established by the following approach, as shown in Figure 2.  

Figure 2: Characteristics of the stability-indicating nature of a method.			

 During forced degradation, the most likely degradation products are generated. These degradation products must be separated from each other and from the active ingredient. Generally peak purity is demonstrated by using a DAD detector for the homogeneity of a chromatographic peak when using UV detection. 

When structurally similar compounds with very similar UV spectra are expected, mass spectral peak purity can be obtained for the separations that use MS-compatible mobile phases. 

If a different detection technique is used, an orthogonal separation or a stretch gradient is appropriate to show peak homogeneity. A special case in this category would be a drug product where multiple DSs are present. In those cases a forced degradation study should be done at a minimum with each DS, placebo, and the drug product. A design of experiment study may be undertaken to understand the inter API reaction chemistry. It is important to note that when a new impurity is observed during stability study, retention time for that impurity should be examined for all the methods to demonstrate specificity. Impurity detected by the assay method may be examined but not necessarily be investigated if a corresponding unknown is seen by the impurity method. 

 The purpose of the forced degradation is to obtain the most likely degradation products. For a mature product, if during the life cycle management process a gap is identified regarding the stability-indicating nature of the method, a complete forced degradation study is not necessary in most cases. 

Expired samples from different lots may be used instead of forced degradation. The same strategy of peak purity, orthogonal separation, or stretch gradient approach can be utilized for the confirmation of separation of the degradation products using expired sample when compared to release data. 

Normally separation of known degradation products by itself does not constitute a SIM, unless forced degradation or expired sample analysis is performed. 

Table 1 shows generic forced degradation conditions for a SIM. As stated above, the appropriate stress concentrations and times should be determined for each product or API to achieve approximately, but not more than, 10% degradation. Some APIs may be unresponsive to some of the forced degradation conditions. This is acceptable in these cases so long as due diligence is shown.  

Table 1: Mass balance calculation from typical forced degradation conditions or expired sample analysis.			

Mass balance is another tool that can be used to demonstrate the SI nature of SIMs in the absence of forced degradation data. The idea is that for any loss observed in the API over time, a corresponding increase in the degradation products should also be observed. This approach can be used on either expired samples or forced degradation samples as long as the "initial" (that is, release or time zero) assay and degradation results are available for comparison. The assay loss (final–initial) in weight (%) is added to the degradation increase (final–initial) to obtain a mass balance percentage. A negative value indicates more API loss; a positive value indicates more degradation; and a value of 0.0% would be a perfect correlation. 

Analysis of Recombinant Protein Therapeutics

There are many aspects to consider when evaluating biologics. Absolute purity is very difficult to determine because of heterogeneity. Shelf life of a biologic has to be supported by long-term, real-time, and conditional stability data. Degraded product may be active but may present issues because of immunogenicity or bioavailability (14). 

Cover Story

In the pharmaceutical industry, the demonstration of drug substance (DS) or drug product (DP) stability over the shelf life is an important aspect of the regulatory requirements.

Current Practices and Considerations for a Stability-Indicating Method in Pharmaceutical Analysis

Analytica returns to Munich this year, with a focus on the latest analytical techniques and innovative applications. The conference will take place from 1–3 April at the Internationales Congress Center München, and the trade fair will be held from 1–4 April at the Munich Trade Fair Center in Germany. 

The Analytica conference is held biannually in the beautiful city of Munich bringing together internationally renowned experts around the world to present more than 120 lectures in the constantly evolving field of analytical chemistry. 

The scientific programme has been organized by the German Chemical Society (GDCh), the Society for Biochemistry and Molecular Biology (GBM), and the German United Society for Clinical Chemistry and Laboratory Medicine (DGKL). The conference will cover all areas of analytical chemistry, with a focus on food analysis, water analysis, polymer analysis, environmental analysis, and pharmaceutical analysis.  

The first day features a full-day symposium on 

 Fourteen speakers from Germany, Finland, Great Britain, Norway, Australia, Canada, the USA, and Switzerland will take an in-depth look at the characterization and effects of aerosols. In this session, Per Schwarze from the Norwegian Institute of Public Health in Oslo, Norway, will reveal how to handle the cocktail of contaminants that is produced when diesel fuel is burned. Robert Healy from the University of Toronto (Toronto, Canada) will describe single-particle mass spectrometry, a technique that can detect individual particles and identify pollution sources. 

Testing for traces of pesticides, medications, and contaminants is no longer enough because analysts now need to identify decomposition products that can also be toxic. Confirmed speakers in this exciting area of research include Damia Barceló from the Institute of Environmental Assessment and Water Research in Barcelona, Spain. Barcelo will talk about a method that combines turbulent-flow LC with an Orbitrap MS system to identify transformed pharmaceuticals in wastewater, and Juliane Hollender from the Federal Institute of Aquatic Science and Technology (Eawag) in Dübendorf, Switzerland, will describe an LC–MS–MS-based technique for analyzing water and sediment samples. 

Bioanalysts are also venturing into areas that appeared unreachable just a few years ago. The 

 symposium will feature Kannan Balasubramanian from the Max Planck Institute for Solid State Research in Stuttgart, Germany, presenting a lecture on sensors that can detect attomolar quantities of nucleic acids, and Andrew deMello from ETH in Zurich discussing microfluidic systems for high-throughput experiments with DNA, living cells, and multiple-cell organisms.  

All three days of the conference will include innovative developments in separation science, but two half-day symposia on the third day will focus on 

Hyphenated Techniques in Modern Analytical Chemistry 

Modern Trends in Separation Sciences: Exploring the Limits. 

Confirmed speakers in these sessions include Karl-Siegfried Boos from the University of Munich (Munich, Germany) presenting a lecture on an LC–MS–MS analyzer for the fully automated analysis of blood and other body fluids, and Wolfgang Goetzinger from Amgen (Massachusetts, USA) discussing the challenges facing analysts in pharmaceuticals research.  

The complete programme and abstracts of the conference are available online at 

Alongside the scientific programme, more than 1100 exhibitors are expected to present the latest products and developments in the field. In the Live Labs, presentations are planned three times a day — 11:00, 13:00, and 15:00 — and will cover routine analysis applications, as well as specialist analysis techniques. In the Analytica forums application-based exhibitor lectures will give routine-analysis professionals tips that are relevant to business and actual applications. 

All visitors attending Analytica may participate in the conference free of charge. For the first time ever, the conference is being held at the ICM — Internationales Congress Center München, directly next to the exhibition halls. 

Features

A look at some highlights for chromatographers at Analytica 2014.

Analytica 2014

If scaling isocratic separations is so simple, why is gradient scaling so confusing?

In last month's "LC Troubleshooting" instalment (1), we looked at how to scale isocratic separations when the column size or packing particle size is changed. The process is quite simple. First, find a column with approximately the same plate number, then adjust the flow rate to give an acceptable pressure. The most common problems that result from mistakes in this scaling process give somewhat lower resolution than is expected or higher pressures. With gradients, unexpected consequences may occur from the changes that may be relatively unimportant in isocratic methods. In this month's discussion, we turn our attention to the proper scaling of gradient methods. 

Last month (1) we looked at the fundamental resolution equation for isocratic conditions:  

 is the column plate number, α is the separation factor, and 

 is the retention factor. A similar equation can be stated for gradient separations:  

* is the effective plate number under gradient conditions, α* is the gradient separation factor, and 

* is the gradient retention factor. As with isocratic conditions, we must be careful to keep from changing the chemistry of the system by keeping the same brand and series of column packing, the same mobile phase, and the same column temperature. We'll see below that we have some additional factors to be careful of with gradients. If we keep these things constant, 

* values for two adjacent peaks) should remain constant. If α* is unchanged, we will obtain the same resolution if we keep the same column plate number. 

The column plate number cannot be measured easily under gradient conditions, so we measure it under isocratic conditions. Because the plate number is a characteristic of the column, the use of isocratic conditions is not a problem. We use the same approach we used with isocratic conditions to select a column with an equivalent plate number so that we maintain the same resolution with the scaled method. We saw that we could obtain approximately the same plate number if we kept the column length-to-particle-diameter ratio constant within a range of +50% to –25%. Thus, we can determine the desired column length from:  

 are the particle diameters of the original and new column, respectively. So far, nothing is different between the isocratic and gradient scaling process. 

With isocratic separation, we saw that the flow rate had no influence on the retention factor, 

, and only a minor influence on the plate number, 

. So, although we often start the isocratic scaling process by keeping the linear velocity of the mobile phase the same, from a practical standpoint the flow rate is adjusted to maintain an acceptable pressure. With gradients, however, a change in the flow rate without some other compensating change can generate problems. This is because 

 is the flow rate (millilitres per minute), 

 is the column volume (millilitres), ΔΦ is the gradient range (for example, 5–95% = 0.9), and 

 is a constant related to the molecular weight and is characteristic of each analyte. For the present discussion on scaling separations, we will not be changing the gradient range or the analytes, so ΔΦ and S will be constant and can be dropped for a simpler relationship:  

With isocratic separations, the separation factor, α, is an expression of the peak spacing of two peaks with 

As long as we do not change the chemistry of the system (mobile phase composition or column chemistry) or the column temperature, α will remain constant, as will the peak spacing. Isocratic 

 is changed by nonchemical or temperature means, 

 stays constant. This is why we can change the column length, the column diameter, or the flow rate and not affect 

 is constant, α will also remain constant. On the other hand, if we do something that changes 

, we expect that α will change because it is rare for a variable that changes 

 in exactly the same manner for all the other compounds in the separation. Thus, a change in the mobile phase or column temperature usually results in a change in peak spacing — in fact, we count on this effect as a tool for moving peaks relative to each other during method development. 

A similar requirement of keeping α* constant holds for gradient separations, where α* is expressed as follows:  

As with isocratic separation, chemical or temperature changes are not likely to change 

 in the same way, so these variables must be kept constant with gradients too. But with gradients, the added complication is that the variables on the right-hand side of equation 5 must also be kept constant, or they should be changed in a manner that allows α* to stay constant. Next, let's consider how this requirement influences the scaling of gradient methods.  

We'll start with a simple linear gradient of 5–95% acetonitrile over 20 min at 1 mL/min with a 150 mm × 4.6 mm, 5-μm particle diameter (

) column. Let's say that we want to get the same separation under ultrahigh-pressure liquid chromatography (UHPLC) conditions on a 1.8-μm 

 column with an internal diameter of 2.1 mm. 

The first step is to find a column with approximately the same plate number. We can use equation 3 to help us. The new column length (

) should be (150 mm × 1.8 μm)/5 μm = 54 mm long. Because 54-mm columns aren't available, we can use any length between 41 mm (–25%) and 81 mm (+50%) and be within our –25% to +50% guidelines. I would probably choose a 50 mm × 2.1 mm column in the present case. 

As with isocratic separations, with gradients it usually is a good idea to start with a flow rate that gives us the same linear velocity. We use the same technique we discussed last month:  

 is the column internal diameter for columns 1 and 2. In the present case, the new flow rate should be 1 mL/min × (2.1 mm/4.6 mm)

  = 0.208 mL/min; we'll round this to 0.2 mL/min for convenience. So, our new method will run on a 50 mm × 2.1 mm, 1.8-μm 

Let's check to see if we are done. Because we want k* to remain constant, we can restate equation 5 as follows:  

We can calculate each half of equation 10 for the proposed conditions and see how close we are. For the column volume, we'll use the approximation of (

), because these are the only two variables that change between the columns. For the original conditions, (20 × 1)/(150 × 4.6

) = 0.00630, and for the proposed conditions, (20 × 0.2)/(50 × 2.1

) = 0.0181. The value for the new conditions is approximately three times too large. Because we've already defined the column size and the flow rate, the only variable we can adjust to fix this problem is the gradient time. If we reduce the gradient time to 7 min, the calculated value drops to 0.0635, which is close enough. So the gradient equivalent to the original conditions is a 7-min gradient at 0.2 mL/min on a 50 mm × 2.1 mm, 1.8-μm 

 column. This means that the switch from the high performance liquid chromatography (HPLC) to the UHPLC column reduces the run time by approximately threefold (20 min to 7 min). 

But if we're running under UHPLC conditions, we can operate at higher pressures and because we're using sub-2-μm particles, flow rate will not affect 

, so a higher flow rate may help to further speed the separation. We use the same equation to calculate the system pressure as we did last month for isocratic conditions:  

 are the original and new pressures, respectively; all the other variables were defined above. Let's assume that the original method generated a 2000 psi (140 bar) back pressure. For the proposed conditions, 

  × (0.2/1) ≈ 4935 psi. For UHPLC, we can probably tolerate twice this pressure, and the easiest way to do this is to double the flow rate so that a new pressure of ~9875 psi would be expected. Wait! We've changed the flow rate, so equation 5 tells us that this will change 

*, and we may find a change in peak spacing — not something we want. So we need to make another compensating change using equation 5 as a guideline. If we double 

* will stay constant. Now our method would be a 3.5-min gradient at 0.4 mL/min on a 50 mm × 2.1 mm, 1.8-μm 

 column. This would reduce the original run time by approximately sixfold (20 min to 3.5 min). 

The example above used a simple, one-segment linear gradient. Let's add a bit of complication and see how a two-segment gradient is handled. We'll use a 5–50% gradient in 10 min followed by a 50–95% gradient in 5 min, the other conditions (column and flow rate) are the same as before. Let's scale this to the same 2.1-mm i.d., 1.8-μm 

 column. The same column, flow rate, and pressure scaling results will be obtained: Scale to a 50 mm × 2.1 mm column operated at 0.2 mL/min resulting in a back pressure of ~4935 psi. 

When it comes to scaling the remaining gradient conditions, we proceed in the same manner as with a single-segment gradient, but we need to treat each segment as a separate gradient. Thus, we have two gradients to consider: 5–50%/10 min and 50–95%/5 min. Because we're not changing the gradient range, we can scale the gradient times so that equation 10 is balanced. For the first segment, the original conditions give (10 × 1)/(150 × 4.6

) = 0.00315 and the proposed conditions give (10 × 0.2)/(50 × 2.1

) = 0.00907, approximately three times too large. A change in the gradient time to 3.5 min gives a value of 0.00317. For the second segment, our original gives (5 × 1)/(150 × 4.6

) = 0.00158. We would expect the same gradient time ratio adjustment to hold for the second segment as for the first, so we can use it in our trial calculation. So we'll use (3.5/10) × 5 min = 1.75 min, rounded to 1.7 min for convenience: (1.7 × 0.2)/(50 × 2.1

) = 0.00154, which is close enough. Now our new method will be 5–50% in 3.5 min followed by 50–95% in 1.7 min at 0.2 mL/min on a 50 mm × 2.1 mm, 1.8-μm 

If we want to increase the flow rate to accommodate the higher pressure capability of a UHPLC system, we use the same technique as discussed above with equation 11, but remember to adjust the gradient time of each segment by a factor of two using equation 5 if you change the flow rate by a factor of two. 

Don't Forget to Adjust the Injection Volume

One thing I didn't mention in last month's discussion of isocratic scaling is the potential need to adjust the injection volume when the column size is changed. The same rules hold for both isocratic and gradient conditions. As a general rule, the injection volume should be adjusted in proportion to the column volume. We can use the following relationship as a guideline:  

 are the injection volumes of the original and new conditions, respectively. If our original method used a 25-μL injection, the new method should use an injection of (25 × 50 × 2.1

) = 1.75 μL, which we can round to 2 μL. 

Often the injection volume for the original method may not have been optimized, so although equation 12 gives the proper scaling, I suggest using an empirical test to determine the injection volume of the new method. I would try the calculated volume plus additional injections at twice and half the recommended volume. In the present case, I would try injections of 1 μL, 2 μL, and 4 (or 5) μL and observe the chromatogram. If there is no unacceptable degradation of the chromatogram, especially in the first part of the run, it is likely that the injection volume can be tolerated. I like to have a safety margin in my conditions, so the injection volume should not be so large that it is right on the edge of failure. This can be achieved by allowing a safety margin of 50–100%. For example, if in the above example, 5 μL still looked OK, try 8 μL or 10 μL. If these injection volumes are OK, 5 μL is safe. 

As you can see from the discussion above, when the number of segments in the gradient increases, the work in calculating new conditions increases because each gradient segment must be scaled individually. What happens when there is a curved (convex or concave) gradient? I recommend strongly against using this type of gradient, because they are hard to reproduce from one instrument to another, and very difficult to scale. I don't know of any way to scale such gradients, because they are essentially composed of an infinite number of very small segments with different slopes. So, if you have an existing curved gradient that needs to be scaled, use the single-segment rules and hope that the new gradient works. 

Those of you who are familiar with gradients will notice that I ignored the instrument dwell volume (gradient delay volume) in this discussion. The new dwell volume requirements can be calculated if the dwell time (the time to wash out the dwell volume) is considered as a gradient segment. The complication is that the dwell volume is seldom adjustable. Usually when you are moving a method from a conventional column to a small-volume or UHPLC column, you also will be moving from a conventional LC system to a UHPLC or a newer LC system, either of which is likely to have a reduced dwell volume. Dwell volume differences are most likely to affect the peak spacing for peaks eluted early in the gradient, so watch for such changes when the method is scaled. You may need to adjust the initial conditions to compensate for such changes. Dwell volume, of course, is not important in isocratic methods. 

Finally, as with the isocratic examples last month, gradient calculations are tedious. You can simplify the process by using one of the on-line calculators to do the work for you. Some of these also calculate scaling of the injection volume and take dwell volume into account. Search the internet for "HPLC method transfer calculator" and you'll find several to choose from. I suggest that you try several and settle on the one that seems like the easiest for you.  

 is vice president of LC Resources, Walnut Creek, California, USA. 

LC Troubleshooting

LC Method Scaling, Part II: Gradient Separations

This month's column discusses the safe storage and use of calibration, carrier, and detector gas cylinders for small or large organizations.

Carrier and detector gases, when not generated directly on-site, are transported to laboratory facilities in gas cylinders and stored there until used. A recent "GC Connections" instalment (1) explored the sources, both natural and synthetic, of carrier and detector gases for gas chromatography (GC). When put into service, secured gas cylinders or laboratory generators are connected to pressure- and flow-regulating devices that establish appropriate flow and pressure conditions for delivery to one or more gas chromatographs. The delivery system may be a simple length of tubing or it may be a more complex manifolded arrangement with intermediate regulation and valving. Ideally, the gas stream passes through a final purification stage positioned close to each chromatograph. 

Much of the equipment associated with gas supplies in the laboratory ensures the purity and control of carrier and detector gases; the rest provides necessary safety measures for day-to-day encounters with flammable or asphyxiant gases that come in heavy high-pressure containers. Both the correct equipment and the procedures associated with gas deployment are critical for achieving the safest work environment and the best possible results. 

In the United States, both the federal and state government authorities promulgate regulations and guidelines for the safe handling and use of gases according to their delivery system and chemical nature. In 29 

1910.101, the United States Occupational Health and Safety Administration (OSHA) incorporates standards issued by the Compressed Gas Association (CGA) (2). Several organizations have made their policies and procedures for compressed gas handling available on-line; among these, the State University of New York (SUNY) campus at Stony Brook Compressed Gas Safety Guide (3) is one that is easily accessible and useful for typical chromatography laboratory scenarios. 

Industrial gas consumers — including both companies and their employees — are responsible for the safe storage and use of gas cylinders and their contents from the moment the tanks come off a delivery truck until the gas supplier collects the empties for return. During this period of responsibility, certain procedures and equipment are used to mitigate the hazards and get the best use out of the cylinders' contents. In addition, companies should enact specific training programmes to qualify employees to handle compressed gases safely and effectively. The contents of this article are intended as general professional advice and are not meant to reduce or replace any requirements, procedures, or regulations imposed by companies or their local authorities. 

Gas cylinders are ubiquitous in chromatography laboratories. Their presence in nearly all chromatography work environments, combined with the rarity of accidental injury or property damage, engenders a sometimes too-relaxed attitude towards them on the part of laboratory workers and managers alike. Other hazards, such as burns from hot GC inlets or punctures suffered while cutting fused-silica columns, are much more common and can be mitigated by following easily understood and simple procedures. It is safe to say that the gas cylinders in a chromatography laboratory present an array of hazards that, short of the presence of toxic chemicals, comprise the greatest personal and property safety challenges chromatographers are likely to encounter. 

Gas cylinder users do not always follow safety procedures. I often see cylinders that are not properly restrained or a lack of valve-protection caps that should be installed when the cylinders are not in use. Sometimes I observe incorrect transfer of cylinders from one location to another. For example, years ago at a university a gas delivery worker tilted two cylinders 15° from vertical, one with each gloved hand, and proceeded to roll them on their bottom edges down a hallway and into a laboratory. The cylinders did not fall, but I wonder how many times they had fallen during his tenure in that job and what kind of damage had been caused. 

And how about moving tanks with a regulator attached and the main valve open, or without a protective valve cap? Not a good idea, but I've seen it done more than once. So, why not handle and use compressed gas cylinders without following the prescribed safe procedures? There are lots of reasons not to do so, and they can be grouped into three categories: Physical hazards, energy hazards, and chemical hazards. 

Gas cylinders are massive. A 49-L capacity steel cylinder, such as what is commonly used for helium or nitrogen carrier gas, weighs about 63 kg (138 lb) and is physically unstable in its normal vertical position. Such a mass of steel can do considerable damage to limbs and toes that happen to be in the way as it falls. 

In addition to being bulky, massive, and unstable, compressed gas cylinders constrain a considerable amount of potential energy as pressurized gas. Should that pressure be released suddenly, a cylinder can become a spinning projectile that can penetrate walls and potentially inflict deadly harm. In an earlier "GC Connections" instalment (4), I calculated that a full helium cylinder could attain a velocity of more than 108 km/h (67 mph) in a few seconds if the gas were released along the cylinder's main axis. The result depends on how quickly the gas is released, but by an estimate that is enough energy to do a lot of damage. 

 In the case of hydrogen, a gas cylinder represents considerable combustible chemical energy as well. Hydrogen concentrations can reach the lower explosive limit (LEL) of only 4% in air in any enclosed space into which sufficient quantities are released. 

Cylinders that contain gases other than air also pose an asphyxiation hazard that results from the potential displacement of oxygen should the gas be released more rapidly than ambient air can be replaced by ventilation. 

Some laboratories also use cryogenic liquefied gases such as liquid nitrogen or carbon dioxide. The proper use and storage of cryogenic liquids is a separate topic about which the interested reader can learn more from the references at the end of this article. Specific personnel protective equipment such as face masks, gloves, and boots should always be used when handling cryogenic liquids. 

Gas cylinders for the laboratory include some built-in safety measures. They are protected against over-pressurization caused by heating or inadvertent over-filling by a burst or rupture disk in the cylinder valve stem. Safety burst disks will open at pressures greater than the normal operating pressure for the cylinder, but less than the cylinder's test pressure. For a laboratory carrier-gas tank, the disk will open when the tank pressure exceeds about 225 MPa (3200 psi). This pressure is greater than what would be attained should a completely full cylinder be stored at up to 60 °C; the maximum recommended storage temperature for nonreactive gases is around 50 °C. A ruptured disk will release the contents of the cylinder safely (and loudly). A so-affected cylinder must be returned to the supplier for inspection and refurbishing. 

Now let's examine how to manage and handle gas cylinders safely. A cylinder passes through three stages at a laboratory site: Receipt and storage, in-use, and ready to return. These stages usually represent three separate locations within a facility; the first and last have nearly identical requirements, while the in-use stage is different. 

Regardless of the stage of a cylinder's lifetime, store inert laboratory gases separately from reactive gases, including hydrogen fuel gas or any other gases used for other purposes. Hydrogen has some special venting and storage requirements that are detailed in the standards; keeping different types of gases separate helps avoid mix-ups. 

 Receiving personnel should check the labelled contents of all cylinders upon delivery, as well as the overall appearance and condition of the cylinders. Any exceptions should be brought to the attention of the supplier as soon as possible. Suppliers don't deliver damaged or incorrect cylinders intentionally, but a quick check is in order. Ideally the date of the most recent hydrostatic pressure test should be verified; however, this information can be difficult to ascertain from the markings on a cylinder. Cylinder pressure can be verified if the valve assembly includes an indicating regulator; otherwise it's better to wait until a cylinder is put into service before checking the fill pressure. 

Gas cylinder contents generally are colour-coded on the cylinder, but the colour scheme is not consistent from supplier to supplier. Thus, it is not possible to rely on the colour of a cylinder for identification. An attached label or tag will give the necessary information. If there is no tag or label, then the cylinder must be returned immediately to the supplier — or another supplier if the original supplier is unknown — for proper disposition.  

Gas cylinders must be moved and stored properly from receipt until they are returned to the supplier. Outdoor and indoor storage are both possible. Indoor storage is more convenient if space is available, while outdoor storage should provide protection from weather and dirt. Cylinders must be kept dry in all cases to avoid corrosion.  

Suitable cylinder storage locations include any area with limited access where they can be properly restrained. For small organizations the laboratory itself is often the best spot. Storage in a public hallway is not desirable, of course, while too-limited access might cause a problem should quick access be required in an emergency. Each location where gas cylinders are used is unique and, if at all possible, the question of proper cylinder locations should be considered carefully. 

Often, and especially in situations where a larger number of cylinders are used, there may be more than one location for cylinder storage. For example, some companies store full cylinders in a location near a loading dock while keeping in-use cylinders in the laboratory or in a shared area that feeds multiple locations.  

Cylinders are classified as in-use if a regulator is attached or the protective valve cap is removed, whether or not gas is flowing at any particular time. With the protective cap removed or with a regulator attached, the high-pressure cylinder valve is exposed and represents an increased hazard. A cylinder that falls on its side with the cap or protective collar in place is unlikely to decompress explosively, whereas one with an exposed valve is much more likely to do so. Therefore, the in-use category requires very careful attention to cylinder restraints. 

Used cylinders ready for return should be kept in a dedicated empty-cylinder area that is convenient for their removal from the building, or at least the empties should be marked clearly as such. Once empty, or otherwise no longer needed, cylinders fall back into a category with the same requirements as full cylinders waiting to be put in use — the protective valve cap must be installed; otherwise the valve could be damaged. Even non-obvious damage to the valve can be a problem for the supplier when the cylinder is refilled. 

If possible, gas cylinders should not be emptied completely before they are returned, since doing so may expose the inside of the cylinder to ambient air and moisture. The worst case is for an empty cylinder to be stored outside with an open valve. It will be difficult for the supplier to completely purge and clean the cylinder before the next filling. A residual pressure of 175–700 kPa (about 25–100 psi) is suitable. 

The image of two large cylinders being rolled by hand on their edges down a hallway remains with me more than 30 years after I witnessed it. The right way to move cylinders is of course with a cylinder cart or hand-truck. These are available in single- and dual-cylinder models with a variety of configurations. The better designs include static-dissipating wheels, which are much appreciated when moving hydrogen cylinders. Never move a cylinder with the regulator attached or the valve cover missing. And never use a gas cart as a permanent cylinder stand. Some tilting and rolling is required to manoeuvre a cylinder onto the cart, so caution is appropriate. Wear gloves and protective goggles and footwear.  

If a cylinder starts to fall from upright toward prone, do not attempt to catch it. Jump back quickly out of the way. Return a fallen or otherwise physically stressed cylinder to the supplier immediately. A fall can introduce weaknesses in the cylinder that may make it potentially unsafe for continued use. 

Proper restraint of gas cylinders is extremely important for their safe use. When set into position, cylinders must be prevented from tipping over, especially when in use with regulators attached. Gas cylinder restraints provide protection from accidental tippage that could compromise attached gas lines or, in the extreme case, cause a regulator or the tank valve to break or detach.  

Several types of restraining systems are available, including single- or dual-tank straps that attach to a fixed object or a wall; individual free-standing cylinder floor stands; small multicylinder corrals with a restraining chain; and completely enclosed and lockable cylinder cages. 

Do not place cylinders on their sides: Moving them down to the floor or back to the upright position is a lifting hazard at the least, and the movement greatly increases the chances for dropping the cylinder. 

These are available in several forms. They all feature an attachable strap or brace that encircles one or two cylinders approximately 1 m above the floor. The chain, woven belt, or metal strap is attached with a snap, buckle, or bolt so that the cylinder is prevented from falling over or moving out of position. Tank straps should be affixed permanently to an immovable object such as a wall or a laboratory bench that is bolted to the wall or floor. Some straps feature a clamp that attaches temporarily to a benchtop and allows the strap to be relocated easily. Although this type does not provide strict immobility it may be sufficient in some locations, but only if the bench is secured. 

 Floor stands are single-cylinder stands that clamp or strap onto a cylinder and provide a larger footprint than the cylinder itself. They provide improved stability but are not immune to tipping or movement, so they are recommended only for temporary cylinder placement. 

A cylinder corral is affixed permanently to the floor or wall. The corral admits two or three cylinders across and two or three deep, and the cylinders are restrained by a chain that attaches onto several hooks welded to the corral. This arrangement is suitable for stored or in-use cylinders where single tank straps are not enough — for example, in a gas analysis laboratory where multiple calibration gas cylinders plus carrier gas, hydrogen, and air are required. A cylinder corral is the most flexible arrangement and requires the least amount of linear wall space for multiple cylinders. 

 A cylinder cage or locker is a large box with heavy mesh walls and usually a floor as well. The door is firmly secured with a lockable bolt. Cylinder cabinets are similar, but they have solid walls. A cylinder cage holds up to 16 or more laboratory gas tanks, but only for storage purposes as it is not a good practice to snake gas lines through the cage mesh. It is the most secure method of cylinder storage. Cages or cabinets are also the most expensive storage method, but they are well worth the investment in safety and security for organizations that have to manage a large number of cylinders. 

After verifying gas cylinders and their content upon receipt, safely moving and securing them in place, and organizing them by their current use, the next step is to attach the appropriate gas regulators, tubing, filters, and fittings to bring the high-purity gas supplies up to one or more gas chromatographs. The next "GC Connections" instalment will discuss these topics and more, as we continue to follow the multiple gas paths through a GC system. 

is a senior scientist at Serveron Corporation in Beaverton, Oregon, USA, and is a member of the

editorial advisory board. Direct correspondence about this column should be addressed to "GC Connections", 

, 4A Bridgegate Pavilion, Chester Business Park, Chester, CH4 9QH, UK, or email the editor-in-chief, Alasdair Matheson, at 

This instalment discusses the storage and use of carrier and detector gases, gas filtration, and the means by which pressures are controlled on the way from the gas supplies to a GC instrument.

LCGC Europe-03-01-2014

Vol 27 No s3 LCGC Europe March 2014 Analytica Guide Supplement PDF

Event: ISC 2014: The Sound of Chromatography

Current Practices and Considerations for a Stability-Indicating Method in Pharmaceutical Analysis

Analytica 2014

LC Method Scaling, Part II: Gradient Separations

The Storage and Use of Gases for Gas Chromatography

The Evolution of Chiral Stationary Phases from HPLC to UHPLC

Mixed-Mode HPLC Separations: What, Why, and How

Vol 27 No 3 LCGC Europe March 2014 Regular Issue PDF