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

 will be held in Amsterdam, The Netherlands from 

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

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

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

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

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

 Another hot area in separation science is mass spectrometry (MS). The application of MS for the analysis of macromolecules has been — and still is — making amazing progress.  

, which can be easily and rapidly reached from central Amsterdam. There will be a large exhibition, which will be integrated with posters sessions, luncheons and coffee breaks. There is also an opportunity for exhibitors to present new developments in the form of vendor seminars. 

promises to be every bit as informative, challenging and enjoyable as the previous meetings in this series.  

Details of the conference programme and further information can be found on the conference website at 

XII Chemometrics in Analytical Chemistry Conference (CAC 2010)

Recent Advances in Sample Preparation Techniques 

Departments

Event News

Ron Majors, editor of "Column Watch" and "Sample Prep Perspectives," has been with 

 for over 26 years. Currently a senior scientist with Agilent Technologies, Wilmington, Delaware, Ron is known industry-wide as one of the premier chromatography experts in the field. He is also a member of 

The technique of QuEChERS (quick, easy, cheap, effective, rugged and safe) is only seven years old, yet it is revolutionizing the manner in which multiresidue, multiclass pesticide analysis (and perhaps beyond) is performed. Columnist Ron Majors interviews inventors Michelangelo Anastassiades and Steve Lehotay about the successes, challenges and potential future directions of this growing sample preparation technique.

Sample preparation and analysis of trace compounds in difficult matrices has always been a challenge. In today's society, the drive to expand food production to feed an ever-increasing population has led to the increased use of a multitude of toxic pesticides in all countries of the world. Analysts have had to keep up with techniques to measure low levels of these pesticides, sometimes used indiscriminately for a wide variety of crops, including those for which they were not intended. Hence, methods for the trace analysis of multiresidues for multiclasses of pesticides have received a great deal of attention, especially in view of recent scares of contaminants showing up in our food supplies. 

Sample preparation traditionally used liquid–solid extraction followed by liquid–liquid extraction and more recently, solid-phase extraction (SPE) to isolate pesticides of interest. In 2003, building on extraction techniques of the past, chemists working at the US Department of Agriculture introduced a technique that was simple, yet effectively isolated trace pesticides from a variety of fruits and vegetables.

  Relying heavily on marked improvements in high sensitivity, high selectivity tandem techniques of gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) (and more recently on their MS–MS counterparts), the technique of QuEChERS (quick, easy, cheap, effective, rugged and safe, and pronounced 'catchers') was developed (Figure 1). An acetonitrile salting-out extraction of a solid sample in an aqueous environment is followed by liquid–solid extraction (called dispersive SPE, abbreviated d-SPE) to remove a majority of the remaining matrix interferences. The sample is then analysed by one or more of the tandem techniques.

 Figure 1: Modern GCâMS and LCâMS instruments have been important in the development and growth of QuEChERS for sample preparation.			

The two chemists who, along with their coworkers, developed and piloted the technique through the international validation and accreditation organizations [AOAC International and European Committee Standardization (CEN), respectively] were Doctors Steven J. Lehotay of the USDA in Philadelphia, Pennsylvania, USA and Michelangelo Anastassiades of Chemisches und Veterinaruntersuchungsamt, Stuttgart, Germany, who worked together in the USDA laboratory from 2000 to 2002. The technique has grown to the extent that it is becoming the standard sample preparation technique for pesticides in fruits and vegetables in laboratories across the globe. In addition, QuEChERS is rapidly expanding beyond its traditional applications area to address a wide variety of contaminants, antibiotics, pharmaceuticals, drugs of abuse and other compounds in a wider variety of matrices such as meat, fish, whole blood, wine and even soil. For this instalment of "Sample Preparation Perspectives", 

 columnist Ron Majors interviewed Anastassiades and Lehotay about the technique, its successes and challenges and where it might be heading in the future. 

The original QuEChERS publication was in 2003. What would you both consider to be the three biggest improvements in these extraction protocols since the original method was developed?

During the conception of QuEChERS, we conducted many experiments to isolate each facet of the extraction and clean-up process for a wide range of representative pesticides and commodities. The original method from the 2003 publication

  is fine and even advantageous over current modifications, depending upon the analytes and matrices. When we independently conducted validation experiments for hundreds of pesticides in different commodities, we both found that there were about 10 pesticides that showed pH dependence in the recoveries, depending on the matrix.

  Buffering at pH = 5 was the best compromise to achieve acceptable recoveries for those few pH-dependent pesticides and we each introduced buffering during extraction in our preferred way.

  This was the first 'improvement' (however, buffering also leads to more coextractives in some matrices, so we prefer to call it an 'adjustment' rather than an 'improvement').  

Secondly, the addition of C18 along with the primary-secondary amine (PSA) in the d-SPE step definitely helps improve clean-up for some samples, particularly those that contain lipids such as olives,

  and it does no harm in any case. Lehotay regrets that he did not uniformly include C18 in the AOAC Official Method 2007.01 protocol, while in the European Standard Method EN 15662, C18 was made an option. Some chemists employ a freeze-out step to reduce lipid coextractives, but C18 in d-SPE is faster and easier, and has been shown to work equally well in removing lipids, although freezing out also precipitates additional matrix components having limited solubility in QuEChERS extracts.

Thirdly, the reduction of chlorophyll coextractive from green commodities and carotenoids via the addition of a small amount of graphitized carbon black (GCB) as in the CEN Standard Method 156627 or ChloroFiltr product (UCT Inc., Bristol, Pennsylvania, USA) constitutes a third prominent adjustment, at the sacrifice of ≈25% reduced recovery of some structurally planar pesticides. Another option of note entails the use of a greater amount of PSA in d-SPE to remove more fatty acid coextractives from cereals and grains (at the expense of ≈20% lower recovery of certain polar pesticides).

What have been the three biggest setbacks or difficulties in advancing the QuEChERS methodology?

We don't think we've really had any setbacks in advancing QuEChERS concepts and the number of vendors selling QuEChERS and d-SPE products has continued to increase (Table 1). QuEChERS generated much excitement from the day it was introduced at the 

 (2002 in Rome) and it has continued to gain popularity since (Figure 2). For example, Anastassiades has compared QuEChERS with the traditional method extensively used throughout Europe for 15–20 years

  and calculated an approximate 95% reduction in solvent consumption (10 versus 535 mL), an approximate 95% reduction in consumable costs, and an approximate 90% reduction in time. Looking at the reports of the official EU-proficiency tests for pesticides in fruits and vegetables with 130–150 participants each year, the number of laboratories using QuEChERS has been continually increasing to reach approximately 40% in 2009. Among private laboratories, where financial aspects weigh even more, the adaptation rate to QuEChERS is reportedly even greater and close to 70%.

Table 1: QuEChERS vendors and web addresses.			

The biggest difficulty with QuEChERS arises from its greatest strength: it is extremely flexible and rugged in the sense that dozens of different small modifications will still give high recoveries for hundreds of pesticides in hundreds of commodities. There is an old expression that "analytical methods are like toothbrushes and everybody prefers to use their own." We can see numerous modifications of the original method or the two official versions of QuEChERS in the literature that are based solely upon personal preferences or limited experimentation. Sometimes modifications are necessary to better adjust the method to a specific application, but in many cases the impression is that the introduction of amendments arises merely from the need to do something different to better justify projects or publications. In various cases, modifications actually make the method less rugged, longer, or more expensive to perform. 

Figure 2: Number of citations to reference 1 by year and publications with topic "QuEChERS" or dispersive solid-phase extraction, according to ISI Web of Knowledge search conducted on June 3 2010. The asterisk indicates partial results for 2010. Total = 249 citations and 158 publications.			

We went through this divide ourselves when Anastassiades preferred to use citrate buffering (at a relatively low buffering capacity)

  while Lehotay preferred acetate buffering (at higher concentration to give greater buffering strength).

  We each have our reasons, but ultimately both methods have been extensively validated for many pesticides in many foods by many laboratories.  

Another difficulty inherent in the flexibility of the method is that it accommodates different sample sizes and different injection volumes and solvents. Therefore, the vendors provide many products to accommodate the many preferences and options that laboratories use throughout the world. Too many details and too many choices also lead to misinformation and confusion among novices in the pesticide analysis field.  

Perhaps the greatest difficulty that all multiclass, multiresidue methods possess is that certain pesticides (for example, captan, captafol, folpet and chlorothalonil) don't give high and consistent recoveries with the method. Acidic conditions help to reduce losses of these pesticides, but these analytes degrade readily in nonacidic samples even before extraction. Unfortunately, these troublemaker compounds are not amenable to LC–MS–MS and their GC analysis is notoriously problematic. The GC–MS conditions have to be well controlled for their high-quality detection, which is not always possible in routine monitoring operations. Therefore, QuEChERS faces criticism by some for not being perfect. The use of analyte protectants (APs), another interesting feature introduced in the original QuEChERS paper, improves GC analysis of these troublemakers.

You both worked on the original methods, yet as you have gone your separate ways, your refinements have led to two additional and different extraction protocols. Currently, there are three popular "standardized" methods: original, AOAC Official Method 2007.01 (attributed to Lehotay and coworkers) and CEN Standard Method EN 15662 (attributed to Anastassiades and coworkers, see Figure 3), along with many homemade modifications. Is there a movement to 'harmonize' these methods to provide the users (and the manufacturers who supply kits) a more generic choice?

According to Lehotay, AOAC International has approved moving forward with another collaborative study to 'harmonize' the QuEChERS method in which the best of both current official methods are combined into a single method for a variety of foods. This entails the use of acetate buffering during extraction and d-SPE (or disposable pipette extraction, DPX, DPX Laboratories, Columbia, South Carolina, USA) clean-up with 50 mg PSA, 50 mg C18 and 7.5 mg GCB (plus 150 mg MgSO

 drying agent) per millilitre of extract. For cereals and grains, a 2.5 or 5 g sample (plus 10 mL water) is extracted and 150 mg of PSA is used rather than 50 mg. The study protocol still needs to be written and reviewed by the AOAC Int. Chemical Contaminants and Residues in Food Community. 

Figure 3: Steps in the original and official versions of QuEChERS sample preparation of pesticide residue analysis of food commodities.			

Although QuEChERS started out as a method for the extraction of multiclass, multiresidue pesticides from fruits and vegetables, it now is being used for a wide variety of analytes (for example, vet drugs, PAHs and antibiotics) in a wide variety of matrices (for example, plasma, meat and soil). Do you think that it is expanding too quickly without the careful interlaboratory testing that you performed during your early work? 

We are glad to see the exploration and expansion of QuEChERS concepts in different applications. In terms of quality of the work, it is a case-by-case basis, which boils down to good science or not, independent of QuEChERS. In all endeavours, people have to critically assess the quality and validity of the information they are given. 

From the beginning, we did not try to patent or control the use of QuEChERS concepts, foremost because we did not think the chemical approach or d-SPE could be patented (after all, Tswett used sorbents in tubes for chromatography prior to the idea to use a column), but also because philosophically, we both feel that QuEChERS would be more widely used if we (or our respective employers) did not become involved in business. Neither of us personally nor our organizations profit monetarily from the QuEChERS name or selling of commercial products.  

What we have done to "protect" QuEChERS is work through the auspices of international official method validation and accreditation organizations (AOAC International and CEN). AOAC Official Method 2007.01 and CEN Standard Method EN 15622 are gold standard methods and modified versions are acceptable if they are properly validated and fit-for-purpose in the particular application.  

However, we caution that many methods achieve high and consistent recoveries for spiked samples during method development and limited validation experiments, yet the method may not work well in real-world samples in which the analyte–matrix interactions are stronger or routine analysis poses instrument maintenance problems. For example, we are sceptical of methods using QuEChERS for soil analysis because stronger extraction conditions are typically needed, such as pressurized liquid extraction (PLE) or Soxhlet extraction, rather than just shaking to overcome the strong binding characteristics of the soil–sediments. Pesticide extraction from many foods does not entail strong matrix interactions, and shaking has been demonstrated to be acceptable for incurred as well as spiked samples.  

Along those lines, the original QuEChERS was devoted to trace pesticides, mainly in pesticide laboratories that were very familiar with the techniques of statistical and representative sampling. As QuEChERS finds its way into nonpesticide laboratories that are doing trace analyses but without the wisdom that pesticide analysts have in that area, do you think that QuEChERS might get a "bad rap" because of nonrepresentative samples being analysed? After all, the result is only as good as the sample supplied.

Again, representative sampling issues are fundamental considerations with any analysis, not just with QuEChERS. In our original paper,

  subsequent papers and all presentations we've given about QuEChERS, we emphasize that the original sample collection and homogenization step is integral to the QuEChERS concept due to the smaller subsample size and use of shaking for extraction. In this regard, the $5000 chopper used for sample comminution is more important than the $300000+ worth of LC–MS and GC–MS instruments used for analyses.  

With pesticide laboratories now encountering large numbers of samples, the question of automation of the entire QuEChERS process naturally arises. In its generally practiced form, QuEChERS is still a manual procedure with lots of shaking and sample manipulations. How do you see the automation of the technique taking hold?

In our views and experiences, QuEChERS is so fast and easy that, rather than making it quicker and simpler, automation adds to the time and effort for sample preparation, and certainly adds to the expense. A chemist can often complete the procedure by hand for a batch of samples faster than a person can load and unload the tubes in automated systems. Fortunately, we have large enough hands to hold 6–10 extraction tubes (50 mL) at a time, and enjoy the 1 minute exercise from the vigorous shaking. However, one of the most frequent complaints from bench chemists is that they don't like the shaking step (despite the suggestion for them to put on some music and do the QuEChERS dance!). Also, some people just don't shake as vigorously as others.  

Therefore, a strong shaker can be a beneficial tool in the laboratory for the extraction step, depending on analyst's capabilities, and commercial shakers are an option for QuEChERS. Anastassiades is currently studying the impact of extraction conditions on the extraction yields of incurred pesticide residues from food commodities. In many cases, 1 minute extractions are just fine, but there are also cases where there is a notable increase in the extracted amounts when extraction time is extended. For example, this effect may concern residues that have diffused into wax layers. Wax particles obviously need some time to soak for those enclosed residues to become accessible to the extractant. Automated shakers thus become very interesting for QuEChERS applications from this point of view as well. 

Otherwise, the d-SPE step does not require vigorous shaking and it only takes 30 s, thus it does not need automation. The semi-automated DPX device can handle 20 samples at a time, which does not slow sample throughput versus manual d-SPE. The use of automated robotics to conduct d-SPE may save time in theory, but the logistics, costs and added instrument downtime seem daunting for little benefit. 			

The most laborious and time-consuming sample processing and sample weighing steps seem beyond the ability to automate and the little details of entering information about samples and keeping track of them are also of critical importance and require careful human inputs.  

Now that QuEChERS is commonly used for sample preparation, the limiting step for sample throughput involves data analysis — reviewing the results for hundreds of pesticides in the analyses. The data processing software is still not trustworthy enough in our opinion to allow it to generate results without human review. Residue chemical analysis does not currently entail the same degree of high-throughput sample analyses that clinical or pharma tests have employed, but if QuEChERS is used in those type of applications, then its automation becomes more important. Gerstel (Germany, UK and US) is working with DPX Labs and others to automate QuEChERS. 

Extractions of all kinds are often matrix dependent. An analyte in one matrix can behave well but in another matrix be difficult to extract. Have you seen cases like this in QuEChERS? If so, are there some recommended modifications in the QuEChERS protocols to handle these situations?

Food samples in most cases do not have pesticide–matrix interactions to affect recovery (certain pesticides in dry and oily samples are some exceptions). However, there are some complex matrices (such as teas, herbs, spices, liver, citrus oil and the like) that give a tremendous number of coextractives no matter what method is used. It is inherent in multiclass, multiresidue methods that the sample preparation process cannot be so selective to remove many matrix chemical compounds if those chemicals have similar properties as the diverse range of analytes being monitored. Sure, modifications can be made by adjusting solvents, pH, salts, volumes, water content and clean-up sorbents.  

In terms of pH adjustment, for example, Anastassiades found out that the quantitative extraction of nicotine from mushrooms requires pH values in the range of 10–11.

  However, the extraction of chlorothalonil from certain commodities, such as onions and leeks, requires pH values close to 2 to minimize losses caused due to interaction with the matrix. Similarly low pH values are needed to achieve good recoveries for pesticides belonging to the nereistoxins class.  

Acidic herbicides, such the phenoxyalcanoic acids, tend to form covalently bound residues. As these are typically included in legal residue definitions, they have to be released before extraction. This is conveniently achieved by introducing an alkaline hydrolysis step (30 min at pH 12) before reneutralization and normal QuEChERS extraction.

Individual pesticide classes can be better isolated through more intensive clean-up, but this inevitably reduces recoveries of other analyte classes and typically leads to fractionations into multiple extracts and multiple injections.  

For such complex commodities, we thus very much rely on high quality chromatography–MS analysis using highly selective and sensitive modern instruments. High sensitivity instruments allow us to reduce the 'sample concentration' in the final extract (for example, to 0.1 g/mL in the case of spices) and thus also the sample equivalents injected, whereas the high chromatographic and MS selectivity allows us to largely 'fade-out' matrix components coeluting with pesticides (for example, terpenes in GC applications). If ruggedness or detection limits are compromised too much, then traditional methods involving gel permeation chromatography (GPC) or fractionation of extracts might be a better option. Such decisions depend on the required data quality objectives that need to be met and the practical matters of time, cost, labour and instrumentation in the laboratory.  

We have never claimed that QuEChERS works well for everything, and we only say that it is fast and easy to try. If it is found to work well in the application, then it saves a tremendous amount of method development time and labour. 

Sample Preperation Perspectives

An interview with the inventors about the successes, challenges and potential future directions of this technique.

The QuEChERS Revolution

For more than half a century, gas chromatography (GC) and especially capillary gas chromatography (capillary GC) has been the technique of choice for the analysis of volatiles and semi-volatiles. Progress made in column technology and injection systems during the past decades extended the application range to molecular weights up to 1500 Da. Significant evolutions in GC instrumentation — including electronic pressure control, fast GC ovens, miniaturized devices for splitting, back-flushing, column switching and comprehensive GC — combined with fast detectors (including sensitive and low/high resolution benchtop mass spectrometers) turned capillary GC into a very mature high-resolution technique in a wide variety of disciplines and industries.

Taking into account the current advantages of capillary GC and its inherently green character compared with LC, LC is often selected for 'typical GC' applications.  

The analysis of polycyclic aromatic hydrocarbons (PAHs) by LC–fluorescence detection is a typical example. Capillary GC–MS using deuterated PAHs as internal standards is far superior for PAH analysis in environmental samples. Other examples where GC should be preferred over LC are the analysis of triglycerides in chocolate products to elucidate the presence of cacao butter equivalents, analysis of sterols in plant material and analysis of triazines in environmental samples, for example. 

 Considerations such as the analyst's expertise or skill or the availability of a particular instrument often prevail over the green character of GC compared with LC. We have to note, however, that the opposite is also true (i.e., GC is used for typical LC applications). For example, for the analysis of amino acids, analytical productivity is much higher with LC compared with GC. Nowadays, amino acid analysis can be performed by LC–MS without derivatization, which contributes to principle 8 of green analytical chemistry. The same holds true for metabolomic studies: GC–MS analysis is currently quite popular but requires derivatization by oximation and silylation whereas LC–MS analysis can be performed without derivatization.  

GC is often not selected for 'thermostability' reasons. There is, however, no clear definition of what a 'volatile' and 'thermostable' solute is. Partly due to this perception, GC is not widely applied in several industries, for example, in the pharmaceutical industry, where the ratio of LC analyses versus GC analyses in quality control (QC) is probably 10/1. Lack of knowledge in GC method development makes transfer of methods from LC to GC difficult and LC is preferred, even if resolution is lower, detection not universal and less sensitive, or identification of impurities is more challenging because electron impact (EI) ionization in LC–MS is not feasible.  

Solutes that are considered thermolabile, and used as examples to illustrate degradation in elevated temperature LC, can often be analysed by capillary GC. This is illustrated with the analysis of a standard mixture composed of methiocarb, thalidomide, nifedipine, ethinylestradiol, torcetrapib and maraviroc (Figure 1).  

Figure 1: Optimized GC chromatogram for some thermolabile pharmaceuticals. Compounds: (1) methiocarb, (2) torcetrapib, (3) thalidomide, (4) nifedipine, (5) ethinylestradiol, (6) maraviroc. 

The mixture was analysed on an apolar column (30 m × 0.25 mm i.d. × HP-5MSUI) (Agilent Technologies, Folsom, USA) using cool-on-column injection and MS detection in scan mode. Helium was used as carrier gas at 1.5 mL/min constant flow and the temperature programme was from 50 °C (1 min) to 150 °C at 50 °C/min, then to 250 °C at 3 °C/min and to 330  °C at 10  °C/min (10  min hold). All solutes are eluting with good peak shapes and without any degradation.The chromatogram was recorded with state-of-the-art optimized GC conditions. It is of the utmost importance to avoid degradation by using cool-on-column injection, proper column selection, constant flow and a relatively slow temperature gradient. The use of splitless injection with a glass wool packed liner and a very fast temperature programme was dramatic in terms of solute stability.  

Helium is the most widely used carrier gas in GC because it is inert, non-flammable and available in high purity. However, for several years there has been concern over helium supplies. Helium is non-renewable and it is estimated that the earth will be virtually helium-free by the end of the 21st century. Nitrogen is sometimes used in GC as a cheap alternative, but this choice should not be made. Due to the slow mass transfer, the use of nitrogen reduces analysis speed. For the same efficiency (expressed by plate number) or resolution, nitrogen is about three times slower than helium. Increasing linear carrier gas velocity results in a fast and significant loss of column efficiency (and consequently of resolution). 

The best alternative is hydrogen. All methods using helium as the carrier gas can be translated into methods using hydrogen without sacrificing resolution, sensitivity and analysis speed. To the contrary, for the same resolution (and plate number), hydrogen is approximately 30% faster than helium (analysis time × 0.7). Moreover, the availability of hydrogen gas generators can overcome concerns about safety of hydrogen gas bottles. In general, the gas generators produce high-quality hydrogen gas using only water as 'fuel'. 

Hydrogen can also be used in combination with MS detection. We have experienced, however, a slightly lower sensitivity (approximately 30%) when using hydrogen in GC–MS compared with helium, but mass spectra are very similar. 

Reduction of analysis time in GC and, consequently, increasing sample throughput is contributing to green analytical chemistry. Analysis time in GC can be reduced while maintaining separation and resolution (obtained on a standard 0.25–0.32  mm i.d. column) by using columns with smaller i.d. Plate number and column efficiency in GC depends on the capillary column i.d. (N = L/d

), in the same way as column efficiency in LC depends on the particle size (N = L/2d

). Narrow bore capillary columns have more plates per metre and the same resolution can therefore be obtained using a shorter column length. Moreover, the optimum linear velocity of the carrier gas is increased, which also contributes to a further reduction of analysis time. 

The gain in analysis speed can be predicted using method translation software (MTS).

  The use of the MTS calculator is illustrated by the analysis of polychlorinated biphenyls (PCBs).

  Using a standard method on a 30  m × 0.25  mm i.d. × 0.25  µm HP-5MS column, a good separation is obtained for a mixture of PCB congeners, as illustrated in Figure  2(a). For this analysis, hydrogen was used as carrier gas at 1.35 mL/min (40  cm/s) and using a temperature programme from 70  °C (2 min) at 25  °C/min to 150  °C, at 3  °C/min to 200  °C and at 8  °C/min to 320  °C (5  min). Analysis time was about 30  min. The method was translated to a 10  m × 0.10  mm i.d. × 0.10  µm HP-5MS column (Agilent Technologies, Folsom, USA). Hydrogen setting was at 0.54  mL/min (60  cm/s) with a temperature programme from 70  °C (0.44  min) at 113  °C/min to 150  °C, at 14  °C/min to 200  °C and at 36  °C/min to 320  °C (1  min). The chromatogram is shown in Figure  2(b). The resolution, which is vital for the identification and quantification of the most important Ballschmitter congeners, is maintained while analysis time is reduced by about a factor of 3 to 10 min. 

Figure 2: Method transfer of PCB analysis from conventional to fast capillary GC. 

As stated before, GC is by definition 'greener' than LC because no solvents are used as the mobile phase. However, in GC, the footprint of the instrument and the energy consumption of a classical GC oven can be substantial. 

In 2001, low thermal mass (LTM) technology was introduced by R. Mustacich. The principles and applications have been reviewed in reference  3. With this technology, the capillary column is resistively heated. Power consumption is drastically reduced and extremely fast temperature programme rates (>  1000 °C/min) can be obtained. This technology can be used for ultra-fast GC, however, one should be aware that beyond a certain programme rate (10 °C per void time)

  resolution decreases. An application that is suitable for LTM technology is mineral oil analysis. In this application, also called hydrocarbon oil index (HOI), environmental samples (water, soil, sediment) are screened for the presence of C10 to C40 hydrocarbons. Only limited solute resolution is needed and a simulated distillation profile is obtained. The analysis of a calibration sample, containing a diesel oil (C10–C25) and a motor oil (C20–C40) is shown in Figure 3. 

Figure 3: Analysis of mineral oil reference sample by fast capillary GC using LTM technology. 

Using classical methods, the analysis time is approximately 30 min. Here, fast oven programming from 40 °C (0.5 min) at 200 °C/min to 240 °C and at 100 °C/min to 340 °C (0.5 min) was used. Analysis time was 3 min. Moreover, LTM technology allows a rapid cool-down from 340 °C to 40°C in about 1.5 in, resulting in total turnaround time of 5 min.  

Over the years, a trend towards on-site measurement has been observed. Several portable or transportable GC and GC–MS systems are now commercially available that allow on-site analysis. All those systems can be considered as contributing to green analytical methods, because the expensive, energy-consuming transport of samples to laboratories is reduced. On-site sampling also has the advantage that remediation or re-sampling can take place immediately after the first analytical results are obtained. 

Recently, a transportable system combining a LTM–GC with a standard MSD system was introduced.

  Using this system, on-site analysis is possible using the same technology and performance as available within a laboratory and sample injection can be performed by liquid split/splitless injection, gas sampling valve, static headspace or solid-phase microextraction (SPME). 

Supercritical fluid chromatography (SFC) has been around for over 25 years, but despite its many claimed 'theoretical' advantages over normal phase (NP) HPLC, is only intensively used for some niche applications, for example, chiral separations in the pharmaceutical industry. In recent years, its popularity has increased. This has been ascribed by system advances but, in our opinion, one of the main reasons is its green character. SFC is environmentally friendly and minimizes the use of toxic organic solvents and additives, with their concomitant risks of laboratory worker exposure and disposal problems. Readers can object that CO

-based techniques can never be considered "green" because the gas causes global warming, but CO

 used in SFC is reclaimed from the atmosphere. Moreover, for preparative processes, the CO

 is recycled. In this framework, it is worthwhile to note that International Conferences on Supercritical Fluid Chromatography are organized by the Green Chemistry Group.

Present interest in supercritical fluid technology mainly originates from the needs in pharmaceutical drug discovery and development. Analytical SFC is not only a fast green separation technique but can easily be up-scaled to semi- and preparative dimensions. Recovering solutes is much easier using CO

-based mobile phases compared with liquid-based semi- and preparative systems. 

Notwithstanding its many features, people still consider SFC more a hype than a happening. There are many reasons that can explain why SFC could not keep the promises advanced (and illustrated!) 25 years ago.  

The main reason is undoubtly the past instability in the SFC instrumentation market. Big manufacturers that originally began producing SFC instrumentation were disappointed by the unit sales (hundreds per year rather that the expected thousands) and were concentrating more on developments in HPLC, resulting in ultra high pressure and elevated temperature LC, and electrodriven separation methods. However, the small niche suppliers did not have the means to produce robust and easy to validate SFC instrumentation, which is a must to penetrate into QA/QC and regulated applications. Fortunately, the big companies re-entered the SFC market (Agilent Technologies — Aurora, Waters — THAR and JASCO) and it is expected that installation qualification (IQ), operational qualification/performance verification (OQ/PV) and system suitability testing in SFC will soon be at the same level as for state-of-the-art GC and HPLC. We recently presented the validation study for the determination of thiourea in a pharmaceutical intermediate at the 0.01% (w/w) level using packed column SFC on a JASCO system.

  Another example of state-of-the-art SFC is illustrated with the determination of the enantiomeric excess of 

-thalidomide. Figure 4 shows the enantiomeric separation of the racemic mixture and of the pure 

-enantiomer on a 25 cm × 4.6 mm i.d. × 5 µm Chiralcel OD-H column (Chiral Technologies, Illkirch, France) installed on an Agilent 1260 Infinity Analytical SFC system (Agilent Technologies, Waldbronn, Germany). Operating conditions were: CO

 flow rate = 2 mL/min, modifier = 17% MeOH containing 0.1% TFA and 0.1% DEA, temperature = 30 °C, outlet pressure  =150 bar. Note the excellent UV-stability at 220 nm. The enantiomeric excess was 99.72% and the 

-enantiomer could easily be measured down to the 0.05% level. 

Figure 4: Determination of the enantiomeric excess in S-thalidomide. 

Without any doubt, chiral separations in drug discovery are the most important niche application of SFC. Systems have been described with parallel columns, sample pooling and mass spectrometric detection to enhance productivity.

  A system presently used in our laboratories consists of a SFC-pump operated at constant CO

-flow (SandraSelerity Technologies, Kortrijk, Belgium), a standard HPLC pump to deliver the modifier in a programmed way, short columns packed with 3 µm particles and a high resolution TOF-MS. Sample pooling is applied to allow analysis of chiral solutes simultaneously. Figure 5 shows the analysis of a sample containing six solutes from drug discovery on two serial coupled 5 cm × 4.6 mm i.d. × 3 µm d

 with LUX-1 Cellulose (Phenomenex, Torrance, USA) and Amy-Coat (Akzo Nobel/Kromasil, Bohus, Sweden), respectively. 

Figure 5: High-throughput chiral screening by SFCâAPCI-TOF-MS (+ and -) on serial coupled columns. 

 flow rate = 2 mL/min, modifier = MeOH containing 0.1% TFA and 0.1% DEA programmed from 0.2 to 0.8 mL/min (12 to 35%) in 3 min, temperature = 40 °C, and a fixed restrictor at 100 bar at initial conditions. The sample was twice injected with operation of the MS in the positive and negative mode. Total analysis time is 2 × 5 min which means 1.6 min per sample.  

A very important feature of SFC is its unique selectivity. In fact in chiral SFC analysis, speed of analysis, by higher diffusivity and lower viscosity, is advanced as the most important characteristic of a supercritical medium compared with HPLC, but in our experience CO

 enhances the selectivity so that the separation can be sped up. CO

 definitely contributes to chiral recognition and in diastereomeric interactions. SFC is by definition a normal-phase mechanism but in practice we experience a multimode mechanism when CO

 is the main mobile phase ingredient. Figure 6 shows the separation of the 16 EPA polycyclic aromatic hydrocarbons (PAHs) on a reversed-phase column (Cosmosil p-NAP, Nacalai, Kyoto, Japan) of 15 cm × 4.6 mm i.d. × 5 µm particles. 

Figure 6: PAH analysis by SFC in hydrophobic interaction mode. Compounds: (1) naphthalene, (2) acenaphthylene, (3) acenaphthene, (4) fluorene, (5) phenanthrene, (6) anthracene, (7) fluoranthene, (8) pyrene, (9) benz(a)anthracene, (10) chrysene, (11) benzo(b)fluoranthene, (12) benzo(k)fluoranthene, (13) benzo(a)pyrene, (14) dibenzo(a,h)anthracene, (15). indeno(1,2,3-c,d)pyrene, (16) benzo(g,h,i)perylene. 

 flow rate = 2 mL/min constant, modifier = EtOH programmed from 0.0 (1 min) to 0.8 mL/min in 7 min, temperature = 40 °C, a fixed restrictor at 100 bar at initial conditions, and UV-detection at 254 nm. Through p–π interactions all solutes are separated in a very green way compared with the classical water–acetonitrile gradient applied in HPLC for PAH analysis. The figure also opens the discussion on "

Is what we are presently doing supercritical, subcritical or enhanced fluidity chromatography?

" Most of the noteworthy SFC applications published (> 90% of the chiral SFC separations) are not under 'supercritical conditions'! In Figure 6, starting from around 2 min, subcritical conditions are used and this seems to not have a big influence on the performance of the system. 

 as a mobile phase ingredient, hardly any differentiation is made between supercritical, subcritical and enhanced fluidity chromatography and regardless of the concentration of modifier, pressure and temperature used, the term SFC is applied. We recently proposed to use SFC for simplified fluid chromatography

  to embrace the different modes. Although the instrumentation used is identical and phase transitions between the different states are hardly detectable, the consequences cannot be neglected. In the fluid state, diffusivity and viscosity are less favourable than in the supercritical state and optimal velocities are closer to HPLC conditions. As an example, the enantioselective analysis of 

-stilbene oxide was performed on a LUX-1 Cellulose column (50 mm × 4.6 mm i.d. × 3 µm) (Phenomonex, Torrance, USA) with CO

 modified with 25% MeOH containing 0.1% TFA + 0.1% DEA. The flow was modified from 1 mL/min to 5 mL/min, the outlet pressure between 100 and 200 bar and the temperature from 30 °C to 60 °C. This embraces the conditions commonly applied in chiral SFC analysis. The best separation in terms of enantio-resolution was obtained at the longest analysis time (30 °C and a flow rate of 1 mL/min) while the outlet pressure had nearly no influence on the resolution while the analysis time was only reduced by 12% from 100–200 bar outlet pressure.  

This partly explains why SFC could not keep its promises in terms of faster analysis compared with HPLC. Moreover, developments such as ultra high pressure LC (UHPLC), elevated temperature LC and sub-2 µm particles in recent years overshadowed the speed argument of SFC.  

As in HPLC, the stationary phase selectivity is of utmost importance and dedicated SFC stationary phases have been developed. Additionally, all HPLC columns can be used in SFC, giving the user a vast selection of columns to chose from possibly too many to make an informed decision. Stationary phases applied in SFC have been characterized using the solvation parameter model by C. West and E. Lesselier.

  Selection of a stationary phase is particular challenging in developing a generic method for drug discovery. We evaluated and synthesized different stationary phases over the last five years and came to the conclusion that state-of-the-art bare silica is presently the best choice. Our results on bare silica SFC were presented at HPLC 2009

  and recently confirmed in the literature.

  Advantages of silica over other SFC phases are that the material is very well characterized, batch-to-batch reproducibility is excellent and, moreover, columns are readily available in all lengths, i.d.s and particle sizes. Fine-tuning of the selectivity can further be done by varying the modifier and additive concentrations, the temperature, the outlet pressure and eventually the flow. The impact of the different parameters on the selectivity is not yet fully understood and quite complicated. Notwithstanding this, generic 'green' conditions for the analysis of polar small molecules on bare silica, at the same time compatible with different detection systems including mass spectrometric detection, could be developed and they are listed in Table 1. 

Figure 7: SFCâAPCIâMS analysis of pharmaceutical compounds. Compounds: (1) caffeine, (2) theophylline, (3) cortisone, (4) sulphamethaxole, (5) prednisone, (6) sulphadimethoxine, (7) hydrocortisone, (8) sulphaquinoxaline, (9) prednisolone, (10) sulphamerazine, (11) adenine, (12) sulphamethizole, (13) hypoxanthine, (14) sulphaguanidine, (15) cytosine. 

Some points of the table need some clarification. Constant CO

-flow delivery simplifies the instrumentation and guarantees a stable baseline in UV-detection. The pump can easily be installed on standard HPLC instrumentation, from which the modifier is mixed in the gradient mode, without any modification. That the total flow is increased during the analysis, has only a minor influence on the chromatographic performance of the system because the van Deemter curves are relatively flat in this velocity domain. The selected modifiers and additives are not only green but are perfectly suited for the different detection systems and properly deactivate the stationary phase for basic and acidic solutes. Note that water in the range of 1 to 5% is added to the modifier. This not only suppresses silanol-activity but also provides very fast re-equilibration. The water concentration can be adapted to a given application as it has an influence on the selectivity. A fixed outlet restriction of 100 or 150 bar, made from stainless steel tubing of 0.12 mm i.d. and placed after the UV-detector is advantageous for mass spectrometry (MS), evaporative light-scattering detector (ELSD) and charged aerosol detector (CAD). No special interfacing after the classically used back pressure regulator (BPR) in packed column SFC, is required and the complete column effluent is introduced in the universal detectors. For MS detection, atmospheric pressure chemical ionization (APCI) is preferred and APCI can tolerate the high flow rates. We have noted that by making the decompression in the MS source, a perfect spray is formed resulting in very clean MS spectra and high sensitivity.  

Some examples of green simplified fluid chromatography using columns packed with 1.8 µm silica particles and the generic conditions are presented in Table 1. 

Figure 7 shows the analysis by SFC–APCI-TOF-MS in the positive mode of some solutes of pharmaceutical interest. The column was 5 cm × 4.6 mm i.d. × 1.8 µm Zorbax Rx-SIL (Agilent Technologies, Waldbronn, Germany) and the operating conditions were: CO

 flow rate = 2 mL/min, modifier = EtOH/water (95/5) containing 5 mM ammonium formate and 0.05% formic acid programmed from 0.25 to 1 mL/min in 10 min, temperature = 40 °C, and a fixed restrictor at 100 bar at initial conditions. 

Table 1: Generic simplified supercritical fluid chromatography (SSFC!) 

The quantity injected on the column was around 2 ng per compound. Much higher detectability was noted in MS compared with UV. This is an interesting feature of the instrumental set-up as MS is the detector of choice in drug discovery. The insert of the figure shows the spectrum recorded for sulphamethizole with a molecular mass of 270.3. 

Regular Article

The second part of this series on green chromatography describes characteristics of GC and SFC

Green Chromatography (Part 2): The Role of GC and SFC

John Dolan has been writing "LC Troubleshooting" for 

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

There is currently considerable interest in speeding up liquid chromatography (LC) separations through the use of smaller particles, changes in column dimensions and higher flow rates. Some of these gains are made possible through the use of sub-3  μm packing particles and the availability of ultrahigh-pressure liquid chromatography (UHPLC) instruments capable of pressures greater than 400  bar (> 6000  psi). For isocratic methods, the separation time can be reduced with nearly any LC instrument simply by increasing the flow rate. However, a change in flow rate without some additional change can produce surprising changes in the chromatogram in gradient methods. This month's "LC Troubleshooting" takes a look at how to speed up gradient separations while avoiding some common errors that result in selectivity changes. 

Based upon our experience with isocratic separation, it might seem that the simplest way to speed up a gradient run is to do the same thing that we do with an isocratic one — just increase the flow rate. Take, for example, the partial chromatogram shown in Figure  1(a). Here, the peaks come out in the 7  minute region of a 10  min gradient run at 1  mL/min. This run has well-spaced peaks and is a good candidate for increased throughput. So we increase the flow rate from 1  mL/min for the run of Figure  1(a) to 3  mL/min and get the results shown in Figure  1(b). This looks pretty good. Yes, we've lost some resolution between the last two peaks, but perhaps we can live with it. However, as we continue to work with the method, we realize that the peak order of the last two peaks has reversed between Figure  1(a) and 1(b). What has happened? We're used to some change in column efficiency with flow changes in isocratic separations, but we don't expect peak spacing to change, especially not peak reversals. Is this just one more mystery of gradient elution that causes you to swear off using the technique?  

Figure 1: Simulated chromatograms of what can go wrong when changing the flow rate of a gradient separation. (a) 10 min gradient at 1 mL/min, (b) 10 min gradient at 3 mL/min, (c) 3.33 min gradient at 3 mL/min. 

The results of Figure  1(b) are not at all surprising if you consider the behaviour of solutes in gradient separations. In isocratic runs, we calculate the selectivity, or relative peak spacing α as  

are the retention factors for the two peaks of interest, calculated as  

is the column dead time (retention time of the solvent front). Note that if we change the flow rate, both 

change in proportion to the flow change, so 

 remains constant with flow rate changes. If 

 is constant, then α will be constant for an isocratic separation — gradient separations don't follow the same rules. 

For gradients, the equivalent of the isocratic retention factor, 

 is a constant (a value of 5 is a good value for making estimates with compounds < 1000  Da). From Equation  3, it is easy to see that a change in the flow rate will result in a proportional change in 

 in isocratic separation), it is common to see changes in relative peak spacing α. So we should not be surprised to see that there is a change in selectivity when only a change in flow rate is made under gradient conditions. This is a problem that can be overcome easily by making some compensating change in the gradient conditions to keep 

* constant. For example, when the flow is increased, we could decrease the gradient time. Or when the gradient range Δ%

 is reduced, we could reduce the gradient time. Or if the column length and diameter are changed, we can make another change to compensate. The key is to make compensating adjustments so that 

If gradient conditions are changed such that 

* is kept constant, the same separation (in terms of relative peak spacing) should be observed. The most common gradient change is to change the flow rate or gradient time, and these changes are easily adjusted for by changing the other factor. Another way to think of this is that the gradient volume (

) must be constant. The effect of this is illustrated in Figure  1(c), where the flow rate was changed from 1.0 [Figure  1(a)] to 3.0  mL/min, and the gradient time was simultaneously reduced by 1.0/3.0 to 3.33  min. The two gradient volumes are the same: (1.0  mL/min × 10  min) = (3.0  mL/min × 3.33  min) = 10  mL. Now the same sample peak order and relative retention are observed in both runs.  

The example of Figure  1 is based upon simulated chromatograms obtained from DryLab software (Molnar Institute, Berlin, Germany). Does this predicted behaviour really happen with real samples? The examples of Figure  2 show that it does indeed. In each chromatogram progressing from Figure  2(a) to 2(f), the flow rate was increased by 1.4-fold, with an appropriate adjustment of the gradient conditions. A quick glance at Figure  2 shows that the retention time of the last peak drops from ≈ 6  min in Figure  2(a) to ≈ 2  min in Figure  2(f) as the flow rate is changed three-fold from 0.35  mL/min to 1.05  mL/min. Let's take a look at the process in a little more detail.  

Figure 2: Chromatograms for flow-adjusted gradient runs summarized in Table 2.  (Data Courtesy of Dionex) 

The original gradient was run on a 100  mm × 2.1  mm Acclaim C18 column packed with 120  Å pore, 2.2  μm particles (Dionex Corp., Sunnyvale, California, USA). The A-solvent was water and the B-solvent was acetonitrile, with an initial flow rate of 0.35  mL/min. The gradient conditions are summarized in Table  1. There is an initial 0.2  min hold, followed by a 4.2  min gradient of 10–95% B, a 2.0  min hold and return to the initial conditions for re-equilibration. Whenever the flow rate is changed to speed a gradient, all the "working" gradient steps need to be scaled accordingly, so that the gradient volume is kept constant. By "working", we mean the portion of the gradient responsible for sample elution (the first two segments in the present example). The re-equilibration steps do not need to be scaled, but it is convenient to do so, and when increasing the flow rate, as in this case, this further reduces the run time.  

To obtain the different gradients of Figure  2, the gradient conditions were adjusted as shown in Table  2. For example, when the flow was changed from 0.35 to 0.49  mL/min, a 1.4-fold change, the isocratic segment at the beginning of the run was reduced 0.200/1.4 = 0.143  min. Each of the steps was adjusted in this way. As a check on this, compare the gradient segment of the run shown in Figure  2(f) with that of Figure  2(a). The gradient volume for Figure 2(f) is (1.467 – 0.067  min) × 1.05  mL/min = 1.47  mL, which is equal to the volume for Figure  2(a) of (4.400 – 0.200  min) × 0.35  mL/min = 1.47 mL. So it looks like the scaling worked correctly. Do the results confirm that we've scaled properly?  

Table 2: Adjustment of gradient conditions* 

Table 3 shows the retention times of each peak in each run, normalized to the retention of the first peak. If the selectivity was exactly the same between runs, the relative retention should be the same. At the bottom of Table  3 is a calculation of the percentage relative standard deviation (%RSD) for the runs shown in Figure  2. You can see that the %RSD is less than 1% in all cases, an excellent match. A close examination of the data, however, shows that the variation does not seem to be completely random. In fact, the highest-flow runs, Figures  2(e) and 2(f), are the only runs that would be considered at all different from the others. We already checked the scaling and the calculations look OK. What else could be happening to influence the retention times of these runs? One possible factor could be the column temperature. As the flow rate is increased, especially with the smaller-particle columns, frictional heating of the mobile phase takes place. An increase in the column temperature would be expected to lower retention times, and this is consistent with the results, although no effort was made to confirm a temperature change experimentally. Even with this taken into account, the relative retention agreement between runs is impressive.  

Table 3: Relative retention times for runs of Figure 2. 

A final observation based upon the runs of Figure  2 and the related data of Table  2 is the system pressure. Because the viscosity of water is greater than that of acetonitrile, the pressure under the starting conditions (10% acetonitrile) will be higher than at the final conditions (95% acetonitrile) — this is reflected in the observed pressure range listed in the right-hand column of Table  2 for each flow rate. The pressure should increase in direct proportion to the flow rate and this is observed for the acetonitrile-rich mobile phase (333/109 bar = 1.05/0.35  mL/min = 3-fold). A similar, but slightly smaller increase is observed for the water-rich mobile phase (689/254  bar = 2.7-fold); the reason for this was not investigated. 

The maximum flow rate of 1.05  mL/min with the 2.1  mm i.d. column gives the same linear velocity as ≈ 5  mL/min with a 4.6  mm i.d. column, which means that the two flow rates are equivalent. The combination of high relative flow rate and sub-3  μm particles generates pressures beyond the capacity of traditional LC equipment, with pressure limits of 400  bar (6000  psi). Only for the runs with flow rates of < 0.5  mL/min (equivalent to ≈ 2.5  mL/min with 4.6  mm i.d. columns) could conventional LC equipment have been used. Therefore, these experiments were run on a UHPLC capable of pressures in excess of 400  bar. With this added pressure capability, it was simple to increase the flow rate while keeping the gradient volume constant so that the method run time could be reduced from 8.6 to 2.9  min while maintaining the same selectivity. A benefit of the 2.2  μm particles used for this separation is that their performance does not change much with flow rate, so the same resolution was obtained at the higher flow rate as was seen under the initial conditions. 

Scaling of gradient runs for changes in flow rate, column size and other gradient conditions can be performed successfully if the conditions are adjusted so that the gradient retention factor 

* is kept constant. The relationship shown in Equation  3 is a useful tool to guide the adjustment of gradient conditions. In the example separation of Figure  2, the gradient volume of each gradient segment was kept constant by making the adjustments summarized in Table  2. When proper adjustments were made, an equivalent separation was possible over a three-fold change in flow rate. 

 is a technical sales representative with Dionex in Olten, Switzerland. 

 is vice president of LC Resources, Walnut Creek, California, USA; and a member of 

 editorial advisory board. Direct correspondence about this column to "LC Troubleshooting", 

, Poplar House, Park West, Sealand Road, Chester CH1 4RN, UK, or e-mail Alasdair Matheson at 

LC Troubleshooting

How to make gradient separations faster without changing the separation

Speeding Up Gradients

 (red clover) is a medicinal herb, traditionally used in the treatment of chronic skin diseases and whooping cough, which contains at least four estrogenic isoflavones: formononetin, biochanin A, daidzen and genistein (Figure 1).

  Supplements containing isoflavones derived from red clover isoflavones are promoted worldwide for the treatment of menopausal symptoms and the maintenance of health and welfare after the menopause.

In recent decades, red clover has been extensively investigated in phytochemistry, and the results indicated that formononetin, biochanin A, daidzen and genistein were the main active components. Pharmacological studies on the components showed that they all had good biological activities. Biochanin A has been reported to have beneficial health effects including antioxidant, anti-inflammatory and anticarcinogenic effects.

  Daidzen and genistein have a structure similar to the hormone estrogen and share some of its physiological properties. They may also act as endocrine disruptors.

To our knowledge, previously reported analytical methods were developed to quantify only one or two types of components in red clover such as studying pharmacokinetics and bioavailability of the isoflavone biochanin A in rats with HPLC,

  determination of daidzen and genistein by GC–MS and HPLC,

  biochanin A and formononetin by HPLC–ELSD or diode array detector (DAD)

  In this study, a HPLC–DAD method was developed to quantify four components simultaneously in red clover. The parameters validated were linearity, quantification and detection limits, accuracy and precision. 

Formononetin, biochanin A, daidzen and genistein were all of HPLC grade and purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile and methanol were of HPLC-grade and purchased from Fisher Scientific International (St Louis, Missouri, USA). Distilled and deionized water were used throughout the study. Red clover was collected in natural growth sites in Hubei, China. The samples were crushed and dried in an oven at 50 °C for 24 h before being subjected to extraction procedures. 

Experiments were performed on a Shimadzu LC-10ATvp system (Shimadzu, Kyoto, Japan) consisting of a LC-10ATvp binary pump, a SPD-M10Avp diode array detector (DAD), a CTO-10Asvp column oven, a SCL-10Avp system controller and a Class-VP workstation. Data were collected and processed by Class-VP 6.10 software (Shimadzu). For chromatographic analysis, a Kromasil C18 column (4.6 mm × 250 mm, 5 µm) was used. The mobile phase was acetonitrile:water containing 0.5% phosphoric acid (40:60). The flow rate was 1.0 mL/min and column temperature was maintained at 30° C. The detection wavelength was set at 254 nm for acquiring chromatograms. 

Based on the solubility of each component in methanol, stock standard solutions were prepared in methanol at concentrations of 0.25 mg/mL, 0.1 mg/mL, 0.75 mg/mL and 0.5 mg/mL for daidzen, genistein, formononetin and biochanin A, respectively. The working solutions for standard curves at specific concentrations were prepared by diluting the stock solutions with methanol:water (50:50) containing 0.5% phosphoric acid. 

About 500 mg of the powdered sample was accurately weighed in a 50 mL volumetric flask followed by the addition of 20 mL of extraction solvent (methanol:water, 80:20 v/v, containing 0.5% phosphoric acid). The flask was shaken and placed at room temperature for 30 min, and then sonicated for 15 min. After cooling to room temperature, the mixture was made up to volume with the extraction solvent. An aliquot (1 mL) of the final mixture was further diluted with 1–10 mL of methanol:water (80:20) containing 0.5% phosphoric acid to appropriate concentrations, and then followed by centrifugation at 10000 g for 10 min. An aliquot (20 µL) of the supernatant was injected into the HPLC for analysis. The experiment was conducted in triplicate. 

The development of a fast HPLC system with a sufficiently high efficiency is currently in demand in the field of chromatography. It has been well recognized that the column efficiency in the high flow rate ranges of the mobile phase solvent primarily rests on the mass transfer resistance in the stationary phase.

  When full-porous and non-porous fine spherical particles are used, there is a limitation to the mobile phase flow rate, suggesting that they are not suitable for fast HPLC. The pellicular spherical particles of a relatively large diameter and the full-porous cylindrical fibre have a separation speed advantage over the fine particles because they can make the size of macro-pore channels between the stationary phase materials wider. The reduction in the thickness of the superficial porous layer of the pellicular type particles and in the radius of the cylindrical fibre is effective for improving the column efficiency. Although the efficiency of the non-porous spherical particles is sufficiently high, they have two drawbacks; the extremely small retention strength and the narrow flow rate range of the mobile phase. These results suggest that the performance of the separation media should be comprehensively studied because the chromatographic behaviour is affected by their intrinsic characteristics concerning the retention strength, the mass transfer kinetics and the permeability against the mobile phase flow. In order to achieve simultaneous separation and determinate the four analytes, we chose a full porous column due to the column efficiency and peak capacity.

A new and simple HPLC method was developed and validated for the quality control of red clover. The developed HPLC procedure is specific to four components in the red clover products. The chemical structures of the four components were shown in Figure 1. Under the current chromatographic conditions, the components were satisfactorily separated. Figure 2 shows representative HPLC profiles of red clover detected at 254 nm for four estrogenic isoflavones. No interference was observed under the assay conditions. The peak of each analyte was identified by comparing the retention time with that of standards and further confirmed by comparison of online UV spectra. Typical retention times for each component are listed in Table 1. 

 Figure 1: Chemical structures of the major active components of red clover. 

Linearity, Limit of Detection (LOD) and Limit of Quantification (LOQ)

A simple rapid HPLC method for simultaneously determiing formononetin, biochanin A, daidzen and genistein in Red Clover

LCGC Europe-09-01-2010