Water treatment by disinfection processes is considered to have been the major public health achievement of the twentieth century. Many drinking water utilities have changed their primary disinfectant from chlorine to alternative disinfectants, which reduce regulated organic by-products such as trihalomethane levels, but at the same time often increase the level of other potentially toxicologically important compounds. The hazardous inorganic oxyhalide by-products are bromate, chlorite and chlorate. The most important of these is bromate, which is formed when source waters containing bromide are ozonated. Bromate is considered a possible human carcinogen. The article is a short review of separations of chlorite, chlorate and bromate in water using ion chromatography (IC) and their determination using various modes of detection. 

In the 19th century, chlorine was introduced to water treatment as a chemical disinfectant, followed by remarkable reductions in cholera, dysentery and typhoid worldwide. The importance of drinking water quality and its influence on human health was well known, but the specific contaminations would not be identified for centuries because the analytical methods available were not appropriate for specific applications. This situation has changed when modern instrumental analytical techniques and methods were introduced into routine analyses. The number of chemicals determined in drinking water has grown exponentially; however, out of hundreds of them, only very few have been studied or have documented proof of their health effects.  

In the 1970s, it was discovered that chlorination of drinking water produced carcinogens, such as trihalomethanes and haloacetic acids. Since 1974, the presence of more than 500 disinfection by-products have been determined in drinking water.

  Since that time, environmental regulatory agencies and drinking water treatment technologists have performed extensive research for alternative disinfection methods that reduce the generation of organic by-products with significant health risks. 

Many drinking water utilities are replacing the most popular chlorine, with alternative disinfectants, such as ozone, chlorine dioxide and chloramines. Ozonation has emerged as one of the most promising alternatives to chlorination. In the early 1980s, it became obvious that the application of ozonization in drinking water treatment not only resulted in the formation of oxygenated compounds but also in the formation of bromide-containing water; brominated organic compounds and bromate as well.  

The identification of new, possibly hazardous compounds in drinking water has become an important task for water suppliers. In an ideal situation, where standards for different intake routes of exposure are fully adjusted to each other, regular monitoring of these compounds should only be necessary when these compounds are carcinogens, or if the relative contribution of drinking water to total exposure or to the tolerable daily intake is high. If formation, toxicity and methods of analysis of metals and selected organic compounds in drinking water were well known, determination of inorganic disinfection by-products at the required low levels was a new challenge for researchers in the end of the 20th century. 

Recently chlorite, chlorate and bromate are the most important inorganic oxyhalide by-products and their concentrations in drinking water have to be controlled. Chlorite is a disinfection by-product that is formed when chlorine dioxide is used for disinfecting drinking water, while chlorate is formed when chlorine dioxide or chloramine is used.

  Chlorination using hypochlorite acid solutions, which contain some ClO

  as a product of HClO disproportionation, may also contribute to ClO

  contamination in disinfected water. Even low levels of chlorite may lead to hemolytic anemia and can have dangerous effects on the nervous system in infants and young children. There is no limit for chlorate due to limited knowledge about its toxicity; however, the World Health Organization (WHO) recommends minimizing the level of chlorate as much as possible for as long as there is no reliable toxicological data.

The most dangerous inorganic disinfectant by-product — bromate, is formed when water containing bromide is ozonated. Bromate formation can be restricted by careful adaptation of the ozone dosage to disinfectant demand. Other options include lowering of the pH, the use of ozone and hydrogen peroxide, addition of ammonia, removal of bromide before ozonization, and the use of membrane filtration or anaerobic processes.

Bromate has been identified as an animal, and possibly human, carcinogen. The International Agency for Research on Cancer (IARC) has classified bromate in group B-2 (the agent is possibly carcinogenic for humans).

  In 1993, bromate was judged by the WHO as a potential carcinogen, initially at a 25 μg/L level, which was associated with an excessive lifetime cancer risk of 7 × 10

  on account of the limitations in the available analytical and treatment methods. Soon, health effects research indicated that it can be a human carcinogen that poses a potential 10

  risk of cancer after a lifetime exposure in drinking water at a 5.0 μg/L level and a potential 10

The US Environmental Protection Agency (EPA).

  as well as the Commission of the European Communities

  has recently issued new rules that require public water suppliers to control some of the previously unregulated microorganisms and cancer-causing disinfection by-products in drinking water. According to these regulations, the maximum admissible level (MAL) is 10 μg/L for bromate and 1000 μg/L for chlorite. 

Bromate and chlorite are two of the chemicals parameters included in EU Directive with a maximum allowed limit of 10 μg/L and 1000  μg/L, respectively. That Directive specifies under Annex III, 2.1: 

"performance characteristics are that the method of analysis used must, as a minimum, be capable of measuring concentrations equal to the parametric value with a trueness, precision and limit of detection specified"

. Both trueness and precision criteria were set at ± 25% of the parametric value.  

Determination of Chlorite, Chlorate and Bromate by using Ion Chromatography

The idealized method for bromate, chlorite and chlorate determinations should meet the following requirements: 					

detect the target ions in drinking water with a limit of determination at 25% of the maximum acceptable concentration

The most useful analytical technique to determine inorganic anions and cations seems to be IC, which is a significant addition to the ever-expanding field of chromatographic analysis. IC has seen phenomenal growth in most areas of analytical chemistry since it was introduced in 1975. It has become a versatile and powerful technique for the analysis of a vast number of ions present in the environment and in biological tissues and fluids.

  IC is now considered a well-established, mature technique for the analysis of ionic species. Many organizations, such as the International Standardization Organization (ISO) and the US EPA have standards or regulatory methods of analysis of anions and cations using IC.

In the mid-1990s, the ISO worked on Method 15061 for bromate

  for chlorite, chloride and chlorate determination. The ISO 15061 standard specifies a method for the determination of dissolved bromate in drinking water, raw water, surface water, partially ozonated water and swimming pool water. Measurement of bromate is done in the range from 0.5–1000 μg/L, with or without sample preconcentration. If preconcentration is necessary, sample is passed through three cation-exchange cartridges, in the Ba

  forms, to reduce the total ionic strength. This standard consists of two parts — a direct method and a preconcentration method, but suffers from the existence of interferences such as chloride and chlorite, which require time-consuming steps for elimination, clean-up and separation before the instrumental measurements. 

Figure 1: Pretreatment steps for samples, calibration and blank solution according to ISO 15061.10			

The ISO 10304-4 standard specifies a method for the determination of dissolved chlorite, chloride and chlorate anions in water with low contamination (e.g., drinking water, raw water, swimming pool water). An appropriate sample pretreatment and the use of conductivity, UV or amperometric detectors make the working ranges from 0.03–10 mg/L (chlorate) to 0.01–1000 mg/L (chlorite). 

The methods of bromate, chlorite and chlorate determination using IC can be generally divided into:

direct methods (suppressed conductivity detection)

indirect methods (UV/Vis detection after post-column derivatization)

hyphenated techniques (ICP–MS and MS detection). 

The direct methods are based on the selective separation of ClO

  ions in the presence of other anions in the sample and their detection in the conductivity detector after suppression. 

These methods are relatively simple and inexpensive but the main disadvantage is the proper resolution of ClO

  ions, whose concentrations in real samples differ significantly.  

Figure 2: Separation of Fâ, ClO2â, BrO3â, Clâ, NO2â, ClO3â, NO3â, PO43- and SO42- in standard solution.			

Better bromate limits of quantification are obtained with indirect methods (derivaization methods) consisting of converting the determined substance, before or after its separation in the analytical column, into derivatives which can then be detected with UV/vis detector. Reagents such as fuchsine, 

-dianisidine, chlorpromazine, potassium bromide or potassium iodide are applied for the bromate determination with the derivatization methods.

Presently, the technique based on the triiodides formation seems to be the most promising one amongst all the derivatization methods for bromate determination by IC.

  In this connection, in 2009, the Work Group 33 of Technical Committee 147 of ISO completed the development of the new bromate determination method based on this post-column derivaization reaction with the use of triiodides reaction. It was thoroughly described in 2009 in the ISO standard 11206 entitled 

Water quality - Determination of Dssolved Bromate — Method using Liquid Chromatography of Ions and Post Column Reaction (PCR)

When its verification process within the proficiency testing framework is concluded, it should become the most important standard used for the bromate determination in water. Moreover, it may be expected that it will gradually replace the already used direct method described in the ISO Method 15061. A chromatogram of bromate determination using an indirect method according to reference 15 is shown in Figure 3.  

Figure 3: Chromatogram of ozonated drinking water.			

The best results in terms of bromate detection and quantification limits in water can be obtained with the hyphenated techniques. Among them, the most popular ones are IC–ICP-MS (Ion Chromatography with Inductively Coupled Plasma Mass Spectrometry) and IC–MS (Ion Chromatography with Mass Spectrometry).

  The obvious disadvantages of MS-based detection techniques are that they each add considerable complexity and significant cost to the analysis.  

The use of hyphenated techniques requires an in-depth understanding of the analytical methodologies and knowing the apparatus in minute detail. Such systems are expensive and for this reason, they are currently mainly used for scientific research rather than routine analyses. Additionally, up till now no international IC standard-based ion MS or ICP-MS detection has been declared for the regulatory monitoring of bromate or any other disinfection by-products anions.  

Global and national agencies are continually striving to monitor bromate, chlorite and chlorate levels in drinking water to establish appropriate regulatory limits. Depending on the results of further research, a risk model could indicate a more definitive guideline value for oxyhalides in drinking water.  

Compared with non-ion chromatographic methods of inorganic disinfection by-products determination, IC is usually faster, more accurate and reliable, less susceptible to sample matrix effects and gives much more information about sample composition. 

Furthermore, for the moment IC is the only accepted standard method for inorganic disinfectant oxyhalide by-product analysis. 

The problem of inorganic oxyhalides also concerns swimming pool waters

  and bottled water which has become a healthier choice than tap water for many people because they believe that bottled water contain fewer contaminants, or dislike the taste of chlorinated tap water.

  The next serious problem which has to be solved is using a KBrO

 in the baking industry and detecting the presence of bromate residues in food samples.

The selection of a proper bromate determination method using IC depends on many factors and is usually affected by the availability of appropriate equipment, the number of analysed samples,the expected analyte content and the required detection and quantification limits.

  Among three main groups of IC methods recently available (suppressed conductivity, UV/vis and MS detection modes) in the majority of routine laboratories all over the world, methods based on suppressed conductivity detection are used. They are good enough for chlorite and chlorate determination, but are criticized because of costs, and difficulties in bromate determination (< 2.5 μg/L in samples with chloride content over 50 mg/L). 

Hyphenated techniques such as IC–ICP-MS and IC–MS are still very sophisticated and not popular in routine laboratories. In this connection, derivatization methods for bromate analysis based on UV detection seems to be the most attractive, alternative, reliable, robustness and low operational cost option. 

 works in Institute of Environmental Engineering of Polish Academy of Science in Zabrze (Poland). He is engaged in research on the application of ion chromatography for the determination of inorganic disinfection by-products in water samples. He is the author of several books and a few hundreds articles about theory and applications of ion chromatography in environmental research. 

1. S.D. Richardson, Drinking Water Disinfection By-products,

The Encyclopedia of Environmental Analysis and Remediation

, R.A. Meyers, Ed.; Wiley: New York, 1398–1421 (1998). 

4. IARC. Some Naturally Occurring and Synthetic Food Components, Furocoumarins and Ultraviolet Radiation: Potassium Bromate, 

Monographs on the Evaluation of the Carcinogenic Risk to Human

Stage 1: Disinfectants and Disinfection By-Products Rule. A Quick Reference Guide,

Articles

A review of separations of chlorite, chlorate and bromate in water using ion chromatography and their determination.

Analysing Inorganic Disinfection By-products by Ion Chromatography

Ghost peaks can be difficult to eliminate when minor peaks are important in an LC separation. This case study shows how to isolate the source of such peaks.

Ghost peaks can be referred to as artifact (erroneous) peaks, system peaks, pseudo peaks, vacancy peaks, eigenpeaks, induced peaks and spurious peaks.

  These are often observed unexpectedly in a chromatogram and can pose a challenge to the analytical scientist. These peaks may arise from unknown impurities or artifacts within the liquid chromatography (LC) system (such as a dirty injector needle, an air bubble in the pump, or a trapped contaminant in the guard column), from the mobile phase, from an extractable contaminant, from autosampler vials and caps or from a contaminant carried over from a previous injection. 

The occurrence of ghost peaks is seen more frequently in the gradient mode. The presence of a ghost peak in a sample chromatogram, especially at the elution time of a known impurity, may trigger an out-of-trend (OOT) and perhaps an out-of-specification (OOS) investigation. An OOT or OOS investigation will lead to efforts to identify an assignable cause, assess the impact of the result and propose further corrective or preventive action, if required. Because the contamination or artifact causing the ghost peak may come from many sources or may be transient in nature, the investigation can be time-consuming. In certain cases, the cause of the ghost peak may remain unresolved. 

Furthermore, in some company standard operating procedures (SOPs) in a good manufacturing practice (GMP) environment, if the cause of a random ghost peak cannot be identified and the peak is ruled out as a non-sample-related impurity, the data may need to be reported "as is." In some situations a blank subtraction can be justified when reporting the data with ghost peak problems; however, this is usually not preferred in routine quality control (QC) labs. The biggest problem with significant ghost peaks is that it makes automatic integration of chromatograms difficult. Hence, when manual integration or blank subtraction is used, it may potentially cause inconsistent and inaccurate impurity results. The problem eventually intensifies if the inconsistent impurity result has been used for trending purposes in stability studies.  

Many articles in the past have cited ghost peak problems and perspectives.

 In this study, we outline a step-by-step approach we have used for investigating the source of ghost peaks in a gradient LC method and share our empirical perspectives as quick approaches for solving ghost peak problems in LC. As a case study, we'll offer our experience with a reversed-phase (RP) LC method that was used to analyse a non-polar, small-molecule active pharmaceutical ingredient (API) in an early development stage.  

Whenever gradient methods are used, it is a good idea to run a blank gradient, in which no injection is made or only the sample diluent is injected, to check for any ghost peaks that might be present. In this study, acetonitrile was used as the sample diluent. An overlay chromatogram is shown in Figure 1 comparing injections of an acetonitrile blank with multiple ghost peaks and an injection of the API sample containing related substances. The analyses were performed on Waters LC Alliance 2695 and Waters photodiode array (PDA) detector model 2996 (Waters Corporation, Milford, Massachusetts, USA) with Phenomenex Luna C8, 250 mm × 4.6 mm, 5 μm column (Phenomenex, Torrence, California, USA) operated at 25 °C and 1 mL/min, with 264 nm (ultraviolet) UV detection. A linear gradient was run from 95% 50 mM ammonium acetate buffer, pH 4.75 (mobile phase A) to 95% acetonitrile (mobile phase B) in 30 min, with a final 5 min hold at 95% acetonitrile.  

Figure 1: Overlay of chromatograms with ghost peaks (circled) from a blank (lower chromatogram) that interfere with an impurity sample analysis (upper chromatogram).			

The overlay chromatogram of Figure 1 clearly shows that some of the ghost peaks in the blank (lower chromatogram) were eluted at the same time as some of the related substances (upper chromatogram); these will interfere with the determination of the related substances.  

As shown in Figure 2, multiple runs on different days indicated that these ghost peaks showed up inconsistently in various acetonitrile blank injections. This random appearance of ghost peaks made related substance peak identification difficult. Furthermore they led to an elevated baseline (or a bump) which made peak integration more difficult.  

Figure 2: Overlay of chromatograms of acetonitrile blanks injected on different days, showing inconsistent nature of the number and size of ghost peaks.			

To isolate the source of the ghost peaks, we eliminated one potential source at a time until we found the root cause. 

Systematic experiments were performed to rule out mechanical sources within the LC system by cleaning the LC system components that may potentially cause ghost peaks. Generally these experiments are described in the HPLC system manufacturer's troubleshooting guidelines.

  In our investigation, we took the following steps: 

1. Clean the LC system by flushing with 25/25/25/25 water/isopropanol/acetonitrile/methanol at flow-rate of 1 mL/min for 1–2 hours (without the column attached). This procedure helps to flush away any containments over a wide polarity range. 

2. Change to a new in-line filter (and/or guard column), if applicable. This can be done to ensure that the LC system is free from the any potential contaminants deposited on the in-line filter or guard column. 

3. Clean and purge the injector needle (to rule out a dirty injector or air peak). Typically, a needle wash solution mixture of equal volumes of water and organic (acetonitrile, methanol or isopropanol) was used to clean the needle or needle seat manually or automatically, depending on the LC system used. The cleanliness of the injector needle can be checked by running a blank gradient run (with or without doing the actual injection). For Waters' LCs, a "condition column" function performs a no-injection gradient. 

4. Clean and wash the UV detector cell. This can be done by following the detector manual instructions. Be careful not to exceed the UV cell's pressure limit. 

5. Check the intensity of UV lamp (to rule out a failing lamp). 

After ensuring that the LC system modules were clean, blank acetonitrile was injected into the cleaned system using our gradient method. Ghost peaks were still observed with freshly prepared mobile phases, indicating that the causes of the ghost peaks were not due to mechanical sources from the LC system.  

Contamination from Glassware and Filtration of Reagents

To rule out any contamination from glassware, we tested several mobile phase bottles by rinsing them and by testing the mobile phase either with or without sonication. Initially, a blank injection was performed by using the mobile phase contained in a bottle (without sonication). Subsequently, the same bottle containing the mobile phase was placed into a water-bath sonicator. After 30 min of sonication at ambient temperature, a second blank injection using the sonicated mobile phase was performed, and the results from the two runs were compared. 

From the sonication we expected to extract any additional amounts of the source contaminant from dirty glassware (the bottle) into the mobile phases. This study was repeated several times with different preparations of mobile phases. No significant changes were observed in sonicated and unsonicated mobile phases, which indicated that the mobile phase glassware was not the source of ghost peaks. 

A blank injection was also performed using a freshly prepared mobile phase A (ammonium acetate buffer) that was passed through a 0.45 μm nylon filter to evaluate whether filtration introduced any contaminants. As a control, the acetonitrile blank was injected using unfiltered mobile phase A. Filtration of buffered mobile phases actually reduced the areas of ghost peaks slightly, and that suggested that mobile phase reagents could be the source of ghost peaks. 

Figure 3: Comparison of blank chromatograms comparing acetic acid brands when all factors except the source of acetic acid were held constant.			

Contamination from Mobile Phase A Reagents and Solvents

The effect of HPLC-grade water on ghost peaks was evaluated by using Milli-Q water (Millipore Corp., Bellerica, MA, USA) and commercially available HPLC-grade bottled water in preparing mobile phase A (ammonium acetate buffer). We did not see any significant differences between the two blank injections when comparing sources of water in the mobile phase. This led us to suspect that the ghost peaks could be arising from the organic reagents or additives in the mobile phases.  

Figure 4: Blank chromatograms from two sources of ammonium hydroxide; all other factors were held constant.			

First we evaluated the glacial acetic acid that we used in preparing ammonium acetate buffer. Five HPLC-grade acetic acid brands were evaluated while keeping the ammonium hydroxide and acetonitrile (mobile phase B) brands unchanged. Figure 3 demonstrates that some of the ghost peaks (at 23 min, 25 min and 30 min retention times) resulted from impurities in a specific acetic acid brand (#1) used to prepare the mobile phase A. Another ghost peak at 15 min showed up primarily in brand #2 and to a minor extent in brand #3.  

Figure 5: The effect of elimination of ammonium acetate in mobile phase A.			

The next step was to use the glacial acetic acid (brand #5; the cleanest) that gave no ghost peaks, and to compare sources of the next reagent used in preparation of mobile phase A. Acetonitrile blanks were injected while changing the brands of ammonium hydroxide. Interestingly, another ghost peak at about 11 min retention time resulted from one source of ammonium hydroxide, as shown in Figure 4. At this point, we had successfully pin-pointed the source of five ghost peaks (at about 11 min, 15 min, 23 min, 25 min and 30 min) that came from the particular brands of acetic acid and ammonium hydroxide used to prepare the mobile phase A buffer when the problem originally appeared. This can be seen by comparing the chromatograms generated with acetic acid brand #1 (Figure 3) and ammonium hydroxide brand A (Figure 4) with the blank runs of Figures 1 and 2.   

Figure 6: Comparison of blank chromatograms obtained from two different sources of acetonitrile; all other factors were held constant.			

Contaminant from Mobile Phase B (Organic Solvent)

By a process of elimination, the remaining unidentified ghost peak that eluted at 24 min (Figure 1) was suspected to come from the mobile phase B (acetonitrile). To prove that this peak was not coming from mobile phase A, a blank was injected using Milli-Q water instead of ammonium acetate buffer (as mobile phase A) and acetonitrile (as mobile phase B), as shown in Figure 5. Then two HPLC-grade brands (X and Y) of acetonitrile were compared as shown in Figure 6. Based on these overlay chromatograms, we are certain that acetonitrile brand X (our original source) contributed to the ghost peak that eluted at 24 min. Lastly, by choosing the selected ammonium hydroxide, acetic acid and acetonitrile brands with no ghost peak contributions, a blank sample was injected to produce a clean baseline (Figure 7). The tiny peak at 30 min did not interfere with impurities that had been identified in our samples, so this was acceptable.  

Figure 7: Successful elimination of interferences (top chromatogram) after selecting uncontaminated reagents (bottom chromatogram).			

We successfully isolated the source of all major ghost peaks in our LC method by using a systematic elimination approach. In our case, the ghost peaks were coming from both mobile phases. After ensuring that all the mechanical aspects of the LC system are functioning properly, it is appropriate to shift the investigation directly to the mobile phases. We recommend evaluating different brands of HPLC-grade reagents, because some can contain impurities that can potentially cause ghost peaks in specific LC methods.

As preventive action, we suggest specifying the vendors and part numbers of solvents and reagents used in preparing mobile phases in the analytical procedure. This information is very useful during method development and especially in method transfer so that ghost peak problems can be prevented. However, if the method specifies an "equivalent" brand of reagent, it is not always necessary that the replacement reagent be of optimal grade or expensive. In such a situation, one needs to evaluate the most suitable reagents for the assay because each contaminant arising from reagent's impurity may have a different UV absorption and may only be a problem under specific conditions and not others. Sometimes the cause of ghost peaks remains unresolved and the peaks are impossible to eliminate. In such cases, three possibilities exist:  

1. For a ghost peak that is consistently present and elutes at a specific retention time, but does not overlap with any peak of interest, the contribution of the ghost peak's area (obtained from blank injections) can be ignored. 

2. For a ghost peak that is inconsistently present, but elutes at a specific retention time and does not overlap with any peak of interest, the identified contaminant can be used as a retention time marker in system suitability testing and excluded from reporting as an impurity. This approach may only be used after identifying the source of the contaminant. For example, a common contaminant may come from widely used plasticizers that may easily (but inconsistently) leach and extract out from a variety of products into the blanks and sample solutions. 

3. For a ghost peak that is consistently present and co-elutes with a peak of interest, the ghost peak's average area response in blank injections bracketing the sample injections can be subtracted from the sample chromatograms to obtain the net peak area, but only if the overlapping ghost peak has consistent area response throughout the entire LC analysis batch. This approach can be used provided that the ghost peak is present in all blank injections and is not sample-related. It should be noted that such background subtraction is much less desirable than the elimination of the interfering ghost peak by isolation of its source or moving it out of the way in the chromatogram. Nevertheless, and in any case, the outcome of the ghost peak investigation should be well documented and reflected in the test procedure if necessary. 

 is a formulation development scientist at SuperGen in Pleasanton, California working with early LC method development, pre-clinical and clinical pre-formulation and formulation development.  

 is an analytical scientist at SuperGen working with R&D formulation and analytical personnel with regard to R&D and clinical stability studies. 

 is the director of analytical development at SuperGen and is responsible for the release and stability testing of drug substances and drug products.  

 is the vice president of manufacturing and preclinical development at SuperGen, a pharmaceutical company dedicated to the discovery and development of novel cancer therapies.  

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

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

, 4A Bridgegate Pavilion, Chester Business Park, Wrexham Road, Chester, CH4 9QH, UK, or e-mail the editor, Alasdair Matheson, at 

LC Troubleshooting

Ghost peaks can be tough to eliminate when minor peaks are important. This case study shows how to isolate the source of such peaks.

Ghost Peak Investigation in a Reversed-Phase Gradient LC System

The development of chiral column technology since the review in 2008 is discussed by Thomas Beesley, a pioneer in the chromatographic separations of enantiomeric compounds. He discusses the use of generic chiral stationary phase (CSP) screening to find the optimum column, the use of smaller diameter columns for high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC), clones of off-patent phases and new concepts in CSP development.

The chiral business continues to grow, albeit at different rates in different parts of the globe. A handful of successful chiral stationary phases (CSPs) recently developed have prompted a reliance on a generic approach to screening for selectivity. The primary goals of speed and efficiency have been demonstrated through the application of supercritical fluid chromatography (SFC) and columns of smaller dimensions with high solvent throughput. Standard HPLC still dominates the methodologies because of its perceived robustness, transferability and wide instrument availability. A number of new clones of the popular cellulose and amylose derivatives have entered the field with a special emphasis on a 3-chloro-5-methylphenyl carbamate derivative of both cellulose and amylose. Two new concepts in chiral stationary phase development have been introduced: one based on bonded derivatized cylcofructans and the second on a derivatized spiral chiral polymer. Several instruments introduced to support chiral screening and detection have also furthered the goals of achieving speed and efficiency. 

The success of chiral separations is highly dependent on several factors, including the availability and performance of the appropriate CSPs, the knowledge base of the operator and the methods used to obtain results, including the instrumentation. In this review we would like to cover each of these aspects to reflect on what has occurred in the industry since the last chiral review in April 2008.

Continued discoveries of significant differences in effectiveness and toxicity of individual enantiomers in biological systems has maintained the importance of chiral separations. In addition, global governmental rules for dealing with compounds that have stereogenic centres has put a mechanism in place for the rapid advancement of chiral technology. As a result of these factors, the chiral knowledge base has grown exponentially. Developments in broadly applicable CSPs and continued expansion of the applications data base have created a more routine analytical methodology, which has also been aided by appropriate developments in instrumentation. 

The current available classes of CSPs offer more generic approaches, although there are still certain structural types that require new concepts in CSP development. For example, it has been found to be difficult to separate compounds with stereogenic centres that are nonpolar — offering no hydrogen bonding or interactive groups — by HPLC, as well as a significant number of chiral primary amines. In addition, multiple chiral centres offer unique challenges — both analytically and preparatively. A call to expand the menu of chiral selectors was heard from many experts in the field at Pacifichem 2010.

  On the plus side, advances in screening technologies — such as the use of SFC combined with MS, multiple column switching and parallel column screening systems — has offered better and faster outcomes: a major focus and goal of the pharmaceutical industry. The Express LC systems of Eksigent (Dublin, California, USA) offer an eight microcolumn screening system that claims to reduce method development time by factors of 10 with 1/10 the solvent consumption. Sepiatec GmbH (Berlin, Germany) also offers an eight parallel column HPLC screening system trademarked Sepmatix, as well as a similar eight column system for SFC. 

Numerous publications on a range of chiral screening strategies and their evaluation,

  have all helped to accelerate the advances in the chromatographic separation of stereoisomers. The cited papers represent only a small sample of the many publications available. The first book dedicated solely to the mechanisms of various CSPs, edited by Alain Berthod, was published by Springer in May 2010 called 

Chiral Recognition in Separation Methods, Mechanisms and Applications

. While the emphases on single enantiomer production has remained in tack as a strategy, the pharmaceutical industry in the United States has now placed a greater emphasis on proteomics and biologics, while the small molecule effort has seen a major shift to Europe and Asia. Europe has seen a significant increase in chiral analysis. China is making a major effort to take its long history of effective natural products and bring their production into the 21st century, the development of which typically requiring chiral evaluations. Chiral publications in China from 2000–2009 exceeded the US by 177 to 142.

  China and India are only now in the early stages of developing the necessary expertise, with India showing a somewhat more advanced progress in chiral analysis. Even though the apparent numbers of chiral compounds in the US pipeline has not increased significantly, the stress on analytical chemists to come up with chiral methods quickly and efficiently has not diminished. Little time or effort is given to optimizing a separation or searching for the optimum CSP. Speed and efficiency remain the targets. This seems like a flawed strategy when method redevelopment has to be done when the needs of the method changes, such as requiring a change in mobile phase type from normal phase (NP) to reversed phase (RP) in manufacturing processes or polar organic to SFC for preparative chromatography needs. Aspects of drug development require changes to tolerable mobile phase components. 

The State of Chiral Screening Strategies 

The success of chiral methods development is often complimented by the introduction of appropriate instrumentation. This includes everything from column heater-coolers — a very important operating parameter in chiral chromatography — to screening and detection systems, such as optical rotation (LC–OR), circular dichroism (LC–CD) and mass spectrometry (LC–MS). 

In the last coverage of this subject, a number of approaches were being investigated, including sequential screening of four to as many as twelve CSPs under an established protocol, and the introduction of a number of parallel screening systems as well as packed capillary screening systems described in the previous review. Statistics are not available, but from a brief survey of users in the field, these options appear to have now settled down in general to a screen of four or eight chiral stationary phases with backup schemes when the first pass through fails. A number of recent publications have also introduced the concept of using an SFC tandem column screening system.

  This seems to be a growing trend. In the one case,

  a two by four column system was operated in parallel, claiming that for five sequential samples it took 29 min on average for the method development of each sample. Screening of each four channel columns typically took 5 min and each optimization took ~15 min. Of the more than 100 samples processed by this method, the authors claimed remarkable success. The system was comprised of two banks of four columns (150 mm × 2.1 mm) that included ChiralPak AD-H, AS-H, ChiralCel OD-H, OJ-H (All available from Chiral Technologies, West Chester, Pennsylvania, USA) and the Chirobiotics V, T, R and TAG (from Sigma-Aldrich/Supelco, St Louis, Missouri, USA). 

To further expand this generic screening activity, PDR-Chiral (Lake Park, Florida, USA) has developed a 12- or 24-column automated method screening system with a 10- or 20-bottle solvent mixer. The system can be operated as an HPLC or SFC system purchased with, or without, heating and cooling and is equipped with both UV and laser polarimeter detectors to identify chiral components and elution order. In a recent paper, four currently available HPLC chiral detectors; three polarimeters and a circular dichroism detector, where evaluated for their performance in chiral screening systems.

  The CD detector was considered the most useful as its response is both linear and sensitive except for non-UV analytes where the polarimeter offers a distinct advantage.

The battle for fast chiral analysis has taken many paths to date looking for the optimum solution. In the absence of sub-2 μm CSPs availability, a recent paper demonstrated the effectiveness of UHPLC for fast chiral separation of drugs by first derivatizing the analytes; rac amphetamine, rac methamphetamine and several β-blockers with AITC and Marfey's reagent respectively. The separation of the resulting diastereoisomers was run on the sub-2 μm Acquity C18 (Waters Corp. Milford, Massachusetts, USA) resulting in the separation of up to ten enantiomers in less than 3 min.

  While this may be a useful methodology for pure substances it presents its own analytical problems, especially for the clinical analysis of amines. 

Of the more than 110 CSPs that have been introduced in the last 10 years, there are currently a handful of CSPs that have appeared to satisfy the majority of chiral separation needs of the industry. The derivatized cellulose and amylose phases still dominate the chiral market, while the macrocyclic glycopeptides applications have significantly expanded and taken on a complimentary role. Cyclodextrin derivatives, like in the Cyclobond (Sigma-Aldrich/Supelco) line, and π-complex CSPs like the Whelco-01 (Regis Technologies, Morton Grove, Illinois, USA) have largely fulfilled the balance of the needs, the choice depending on compound structure and the particular mobile phase requirements of the application. In some instances, as in the area of clinical applications, the protein phases can be added to the preceding list. From a review of the literature, many of these latter applications outperformed the cellulose and amylose phases based on sample solubility due to mobile phase components, selectivity and/or separation of multiple chiral centres or degradation products within the sample. 

At the time of the last review, Chiral Technologies (Daicel), West Chester, Pennsylvania, USA had just introduced the Chiralpak IC, a bonded 3,5-dichlorophenylcarbamate derivative of cellulose. This phase had never been made available as a coated version due to its high solubility in organic solvents. As a bonded column it has demonstrated good selectivity for those analytes previously not resolved or poorly resolved by the other immobilized cellulose and amylose derivatives. This column is now considered to be complementary to the other two bonded phases. More complex method development protocols and the expanded use of high levels of methyl-

-butyl ether MTBE with this phase, however, limits its applicability in certain applications. Chiral Technologies has also added two new 5 coated derivatives to its line, the methylchlorophenyl carbamate of cellulose (Chiralpak AY-H) and of amylose (Chiracel OZ-H). These phases were originally available coated on 20 μm silica particles for preparative applications only. Figure 1 and 2 demonstrate a comparison to the original more standard dimethylphenyl carbamate derivatives of cellulose (OD) and amylose (AD) demonstrating enhanced selectivity. In addition, they have extended the Chiracel OD and Chiralpak AD to 3 μm material for LC–MS applications. A 50 mm × 4.6 mm column producing 7000 plates offers high-speed analysis with manageable pressures. Stability testing up to 250 bar (3625 psi) showed no deterioration in aqueous mobile phase systems.  

Figure 1: Separation of methyl 1-benzyl-5-oxo-3-pyrrolidinecarboxylate enantiomers. Red trace = Chiralcel OZ-H. Blue trace = Chiralcel OD-H. Column dimensions = 250 mm Ã 4.6 mm; mobile phase = 80:20 hexaneâethanol; flow rate = 1.5 mL/min; temperature = 25 Â°C. (Courtesy of Chiral Technologies).			

An HPLC method development strategy and applications update had been published in 2009

  for the macrocyclic glycopeptides (Chirobiotic phases) from Supelco/Sigma-Aldrich. The article cites their unique breath and versatility in a variety of mobile phase conditions suitable for clinical applications using LC–MS methods, drug discovery screening and preparative methods. The article identifies the potential mechanisms at work and the mobile phase that drives those specific interactions. For these and other phases in their line, Sigma-Aldrich/Supelco has developed a new search engine for obtaining chromatographic information on a particular chiral analyte of interest, compound class or applications on a particular chiral column. This information can be found on the Sigma-Aldrich website under chiral e-Times. They also now offer ideas, tips and tricks to chiral chromatographers on Twitter. For more information on this latest innovation in chiral communication go to 

Figure 2: Separation of cyclandelate diastereomers. Red trace = Chiralpak AY-H. Blue trace = Chiralpak AD-H. Column dimensions = 250 mm Ã 4.6 mm; mobile phase = 90:10 hexaneâethanol; flow-rate = 1.5 mL/min; temperature = 25 Â°C. (Courtesy of Chiral Technologies).			

Cyclodextrin technology has a long history of development dating back to the 1970s. It is ubiquitous in its breadth of applications both for chiral and achiral analytes. It continues to find its mark in current chiral processing. A surprise has been the unique opportunities it has afforded in SFC

 for the π-acidic derivative Cyclobond DNP (Supelco). Another unexpected discovery was the ability of the Cyclobond DM, a fully methylated version with no hydrogen bonding capabilities, demonstrating good selectivity for single ring structures with bulky side arms and fused ring structures with a dominance of steric interactions as the driving force.

With the increasing interest in SFC, both for analytical and preparative applications, the π-complex phases have seen an increase in activity due primarily to their success in normal phase conditions. The Whelk-01 and -02 have been the work horses in this area. A recent study by Regis Technologies demonstrated the effect of varying co-solvents in SFC on a Whelk-O1 CSP. The study showed that and increase in polarity of the modifier correlated with a decrease in selectivity and a decrease in retention (Figure 3).  

Figure 3: Effect of varying co-solvents in SFC method development on a Whelk-O 1 CSP. Group 1 = Troger’s base; Group 2 = chlormezanone; Group 3 = indapamide; Group 4 = devrinol. (Courtesy of Regis Technologies).			

While SFC has taken a very substantial role in the chiral business it has been mostly used so far, though not exclusively, in preparative applications for its green benefits, as well as for its speed. Comments from analysts in the field indicated that the major reason that HPLC still seems to be the method of choice is the robustness, transferability and wide instrument availability of standard HPLC equipment. The rule seems to be that methods based on typical NP solvents or, to a lesser degree from the polar organic mode, can be transferred to SFC, while those developed with a RP system were less successful. In addition to speed and the reduction in solvent consumption it has substantially reduced the solvent waste stream. Development of a fraction collector for the inline concentration of purified enantiomers by Modular SFC, (North Attleboro, Massachusetts, USA) will certainly further its use and cost effectiveness. 

There are still a significant number of applications requiring conventional HPLC and the recent introduction of a versatile bench-top simulated moving bed (SMB) system: the Semba Octave by Semba Biosciences (Madison, Wisconsin, USA) should help bridge the gap in cost effectiveness. Supelco (Bellefonte, Pennsylvania, USA) was a test site for this device and has now introduced a set of eight, 15 μm particle size, 5 mm × 10 mm columns designed to operate with this system or any bench-top HPLC system (Figure 4).  

Figure 4: Columns = Astec Chirobiotic V2, 5 cm Ã 10 mm, 15 Î¼m (set of eight); sample feed = 0.15 mL/min; mobile phase = methanol; sample = 5-methyl 5-phenylhydantoin (10 mg/mL); productivity = 20 mg/h for each enantiomer under above conditions. Increased throughput up to 60 mg/h was achieved by increasing concentration and focusing on either raffinate or extract. (Courtesy of Supelco/Sigma-Aldrich).			

With expiration of the Chiral Technologies (Daicel) patents on their highly successful coated versions of the derivatized cellulose and amylose phases, an increasing number of companies have attempted to produce generic versions with varying degrees of success.  

The successful Kromasil AmyCoat, a 3,5-dimethylphenyl carbamate derivative of amylose, and CelluCoat, the 3,5-dimethylphenylcarbamate derivative of cellulose from Akzo Nobel (Bohus, Sweden), who have announced an exclusive agreement with SigmaAldrich/Supelco for exclusive distribution in the United States, Canada and Puerto Rico. In a recent poster, Akzo Nobel demonstrated the optimization of small-scale chiral HPLC preparative separation of tropic acid on a Knauer bench top SMB system (Berlin, Germany) using eight 4.6 mm × 150 mm (25 μm) AmyCoat columns. Total development time was said to be two working days with the Knauer SMB-Guide software.  

Phenomenex (Torrance, California, USA) continues to expand the Lux chiral line that includes a 3-chloro-4-methylphenyl carbamate derivative of cellulose (Lux Cellulose-2) and 5-chloro-2-methylphenylcarbamate derivative of amylose (Lux Amylose-2). There is also, as part of this line, a replacement claimed to match the Chiracel OD-H from Chiral Technologies referred to as Lux Cellulose-1. At Pittcon 2011 they introduced Lux Cellulose-3, the 

(4-methylbenzoate) derivative and Lux Cellulose-4, the 

4-chloro-3-methylphenylcarbamate). All these phases show complimentary selectivity. 

ES Industries (West Berlin, New Jersey, USA) has also introduced a 3-chloro-5-methylphenyl carbamate derivative of cellulose referred to as Chromega Chiral CC2 coated on a 5 μm spherical 1000 Å silica that they claim has improved efficiency. This line has now been expanded to include a Chromega Chiral CC4, a 4-chloro-3-methylphenylcarbamate derivative.  

Table 1: Examples of racemic separations on Epitomize CSPs.			

Orochem Technologies (Lombard, Illinois, USA) has now introduced four products in this area, trade name Epitomize, that include the typical 3,5-dimethylpheny carbamate derivatives of both cellulose (CSP-1C) and amylose (CSP-1A) as well as an interesting 3-chloro-4-methylphenyl carbamate derivative, again of both cellulose (CSP-1Z) and amylose (CSP-1K). For a comparative enantiomeric response see Table 1. In addition to the usual variety of particle sizes and column lengths Orochem has added a UHPLC version for LC–MS using 1.7 μm particles (Figure 5). This format is available for all four coated versions with a pressure limit of ~3500 psi.  

Figure 5: Separations achieved using Epitomize CSP-1C 1.7 and 5 Î¼m. Left: Column = 250 mm Ã 4.6 mm, 5 Î¼m; mobile phase = 10% isopropanolâheptane; flow-rate = 0.5 mL/min; back pressure = 330 psi; mobile phase used = 4.6 mL. Right: Column = 50 mm Ã 2.1 mm, 1.7 Î¼m; mobile phase = 10% isopropanolâheptane; flow-rate = 0.5 mL/min; back pressure = 3620 psi; mobile phase used = 0.4 mL. (Courtesy of OraChem Technologies).			

A number of the earlier attempts to introduce new concepts in CSPs have all but disappeared, including chiral molecular imprinting. Chiral ionic liquids have been developed for a wide variety of achiral applications including their recent successful application in the chiral area for GC

  One of the consistent failures in the chiral separation business has been the separation of a significant number of primary amines. The crown ethers do a very good job in separating many of these types of compounds, but not all, and they fail to deliver on preparative, clinical and some manufacturing processes because of the lack of mobile phase volatility.  

Dr Daniel Armstrong of the University of Texas at Arlington has now published on a new series of bonded CSPs based on derivatized cyclofructans and appears to have more than solved the problem of difficult chiral amines.

  His new company AZYP (Arlington, Texas, USA) has produced four products in this series that have been launched this year at Pittcon. The first is based on an isopropyl-derivatized C6 cyclofructan, designated Larihc CF6-P, that efficiently separates chiral primary amines. The second is a highly aromatic derivatized C6 cyclofructan that loses its ability to separate these chiral amines but has excellent selectivity towards a broad spectrum of other types of racemates and is called Larihc CF6-RN.

  The interesting fact is that the dominant mode of separation for both these phases is either NP or polar organic mode, both of which allow for direct transfer to efficient SFC methodologies. (Figure 6) With the isopropyl derivative on the CF6 over 20 chiral primary amines were separated to baseline in one day. Stability was addressed, indicating no change in performance in all common organic solvents even after over 1000 injections. These stationary phases also seem to have good sample capacity as demonstrated by the 4200 μg in 100 μL resolved to baseline on an analytical column, in this case limited only by the sample solubility.  

Figure 6: SFC separation on isopropyl-CF6. Column = Isopropyl-CF6; mobile phase = 80:20 carbon dioxideâmethanol with 0.1% diethylamine; flow-rate = 3 mL/min. (Courtesy of Dan Armstrong, University of Texas, Arlington, USA).			

Toyko Kasei (TCI) from Tokyo, Japan has introduced a new concept in chiral stationary phase development with a spiral chiral polymer that offers steric recognition as the primary mechanism. Three different products result when chiral side groups are attached to the backbone adding additional mechanisms. In addition to steric recognition from the side chain, the three offer electrostatic interactions between an analyte containing a carbonyl group and a nitrogen side chain and π–π interaction between an analyte and the side chain. The polarity of the three products ranges from low for the TCI Chiral MB-S, to moderate for the TCI Chiral BP-S, to high for the TCI Chiral CH-S covering a broad range of chiral applications in either normal or reversed phase conditions. The products come in 3 μm and 5 μm formats. 

A new approach for biomedical analysis of chiral compounds was demonstrated with the direct separation and quantification of lactic acid enantiomers in human urine. This was reported using for the first time a highperformance immunoaffinity LC–MS system. The antibody was immobilized onto a Poros-OH 20 μm support material from Perceptive Biosystems (Cambridge, Massachusetts, USA) activated and packed into a 4.6 × 150 mm stainless steel column. Pure alpha hydroxyl acids were injected and eluted with a buffer demonstrating the selective binding of the D-enantiomer but not the L. It was determined that the limit of detection when binding the D-hydroxy acid antibody for lactic acid enantiomers using MS as a detector was 63 μM.

Chiral analysis continues to expand and to gain in speed and efficiency driven by the use of SFC and small column design at high linear velocity. Generic screening for selectivity remains the method of choice with four to eight columns operated either sequentially or in parallel with the availability of new screening instrumentation. New generic versions of the popular coated cellulose and amylose derivatives continue to enter the market. Two new concepts in CSP development have been introduced based on a bonded and derivatized six carbon cyclofructan and a derivatized spiral chiral polymer. 

 is founder and former CEO of Quantum Industries, which designed pre-absorbent and channeled TLC, which was sold to Whatman in 1978. He was founder and CEO of Advanced Separation Technologies which manufactured chiral, HPLC, Cyclobond and Chirobiotic HPLC columns and Chiraldex capillary GC columns. The company was sold to Sigma-Aldrich in 2006. He is currently a lecturer, expert witness and contract problem solver with a speciality in the separation of chiral molecules. 

Column Watch

The development of chiral column technology since the review in 2008 is discussed by Thomas Beesley.

Review of Chiral Stationary Phase Development and Chiral Applications

Comprehensive two-dimensional liquid chromatography (LC

LC) is a relatively new and expanding technology which provides enhanced separation power by combining two LC modes with different separation mechanisms. In this review, practical principles and guidelines for designing LC

LC methods are given. Selected applications from food, plant, polymer and bioanalytical areas from the last five years are presented.

In the quest for better separation efficiency, comprehensive two-dimensional liquid chromatography techniques (LC×LC) have been increasing in popularity recently. In 2D-LC, the sample is subjected to two different separation mechanisms, which are practically achieved by using two columns with different stationary phases. In a comprehensive mode, all fractions pass through the whole analysing system. This is the essential difference compared with heart-cutting liquid chromatography (depicted by LC–LC), in which only selected fractions are analysed by the second column. In LC×LC, two separation modes are used to increase the separation efficiency. The instrumentation and operation of LC×LC has been described in recent literature.

  In short, the heart of an LC×LC system is the interface that connects the two columns.  

The interface is typically a simple multiport high pressure switching valve, which ensures the collection of the first dimension effluent in aliquots of predefined volumes and allows the transfer of these fractions on the secondary column in an automated way. Low flow-rate employed in the first dimension enables the filling of one sample loop and at the same time flushing the second one by fast flow-rate into the second dimension within one valve cycle. Prior to the introduction of a successive fraction onto the secondary column, the analysis of the previous fraction should be finished. Thus, the second dimension analysis time should be equal to (or less than) the duration of a modulation period. This sample cycle, (i.e., the modulation time or sampling frequency) is thus determined by the analysis time of the second column. To maintain the separation achieved in the first dimension, a sufficiently large number of fractions have to be taken from a peak eluting from the first column. Consequently, fast 2D analyses are essential in LC×LC.  

Prior to coupling, the LC methods in both dimensions should be optimized with respect to the sample characteristics and taking into account all parameters that influence peak capacity, such as orthogonality, sampling frequency and compatibility of both dimensions.  

The optimization should start with selection of the separation mode and the two columns, and then by optimization of the second dimension separation. The design of the LC×LC methodology is summarized in Figure 1. The separation mechanisms used in the two dimensions of a two-dimensional separation process must have different selectivities for the sample components. The sample components should be spread over as broad a separation space as possible in both separation dimensions. The best separation is achieved when there is no relationship between the retention data of the components of the sample in the two separations. Various combinations of reversed-phase (RP) and normal phase (NP), ion-exchange (IEC), hydrophilic interaction liquid chromatography (HILIC) or size-exclusion (SEC) or LC modes can be used in LC×LC. In these combinations, the separation is based on the differences in the molecular structure of separated compounds, such as size, shape and polarity or the specific charge of ionic compounds.  

Figure 1: Selection of LCÃLC conditions. 

In practice, the separation selectivities in the two LC modes can be more or less correlated because the interaction mechanisms of the analytes with stationary phase and mobile phase are seldom based on a single factor only, and the secondary interaction mechanisms can be correlated in the two separation modes. This must be taken into account when developing real world LC×LC separations. Since the mobile phases used in RPLC×RPLC, IEC×RPLC, RPLC×SEC, NPLC×SEC or HILIC×RPLC separation systems are usually fully miscible and have similar physicochemical properties, these are in practice the most commonly used combinations. 

For example, the combination of IEC and RPLC can be used for the separation of ionic compounds, acids or bases. The combination of SEC with either NPLC or RPLC is useful for the separation of macromolecules, such as synthetic polymers and biopolymers. An NPLC×RPLC combination is more demanding due to eluent incompatibility, and is useful for the separation of samples with two or more types of structural elements that differ in lipophilic and polar properties. For polar compounds, using HILIC for the second dimension separation is a viable option, particularly when stationary phases that are less polar than silica gel (e.g., aminopropyl silica) are used because then mobile phases containing up to 70% water can be applied. Table 1 shows the compatibility of different LC modes.  

Table 1: The compatibility of different LC modes. 

The selection of column dimensions is crucial in LC×LC. The inner diameter (i.d.) of the second column is typically larger than that of the first column to minimize extra column peak broadening, although it is also possible to use a narrow-bore column with high permeability in the second dimension. The first column is typically long and is operated at lower flow-rates than the second column, which is short and uses a high-speed mobile phase. Here, the speed of the second dimension is a key feature of successful separation and the separation should be as fast as possible, with sufficient resolution.  

Choosing the proper sampling time is essential in designing an LC×LC method.

  The first-dimension separation is maintained more efficiently when the sampling frequency is sufficiently high. To maintain the resolution obtained in the first dimension, a large number of fractions should be transferred along a peak eluting from the first dimension. To achieve sufficient amount of fractions, the first dimension peaks are occasionally broadened by operating the primary column at a lower (than optimal) flow-rate. In this way, a sufficient amount of samplings across the primary peak are obtained. However, the first dimension peak capacity is significantly reduced by operating at a flow-rate below the Van Deemter's optimum. Some guidelines for selection of optimal modulation frequency are available. Three or four samplings across the first dimension peak are often considered sufficient

  and in practice even two sampling periods can usually be tolerated.

  The effect of proper sampling time is demonstrated in Figure 2. Here, selected phenolic compounds were analysed with an RPLC×RPLC combination.

  When three fractions for each peak were sampled [Figure 2(a)], the best overall separation was obtained. With longer modulation time, only 1–2 fractions were samples and the resolution obtained in the first dimension was partly lost. Conversely, when the modulation time was too short, significant wrap-around took place and the peaks were tailing. The wrap-around effect is typical in 2D separations when the modulation time is shorter than the compounds' second dimension retention time and the compounds, consequently, only elute to the detector during the next modulation period and can coelute with the compounds in that fraction. This results in a loss of resolution.  

Figure 2: Effect of modulation time to the overall separation of phenolic compounds in an RPLCÃRPLC system. (a) Optimal sampling time with three fractions for each first dimension peak; (b) modulation time is too long with 1â2 fractions/1st dimension peak, (c) modulation time is too short with 3â4 fractions/1st dimension peak, resulting in wrap-around. Conditions: First dimension:  C18, 150 Ã 2.1 mm i.d., 3 Î¼m, gradient with acetic acid and acetonitrile at 0.1 mL/min, 2nd dimension: + Ultrasphere CN, 75 Ã 4.6 mm i.d., 3 Î¼m, eluent 65/35 0.5% (v/v) acetic acid in water/ acetonitrile at 1.9 mL/min. For details, see reference 11. 

After selection of the sampling frequency, the first dimension separation can be optimized, with the flow-rate optimized, taking into consideration the modulation time.  

The inner diameter of the second column should be equal to — or larger than —that of the first column. Short columns packed with small particles (3 μm or less), non-porous, superficially porous or monolithic columns are especially suitable for this purpose. In addition, the column material should withstand high pressure, because high flow-rates are used in the second dimension. Typically, columns of 2–5 cm of length and with i.d. of 2.1–4.6 mm are used. High speed separation can be achieved by using monolithic columns, an ultra-high performance liquid chromatography (UHPLC) approach

  or columns packed with partially porous particles. Monolithic columns are also suitable for LC×LC. It should be noted that the use of columns packed with sub-2 μm particles at high flow-rates requires sophisticated instrumentation capable of delivering high pressures (600–1000 bar). The interface valves should also tolerate such high pressures. It is also possible to increase the speed by using elevated temperatures in the second dimension. At elevated temperatures, the viscosity of the mobile phase is reduced and the flow-rate of the eluent can be increased. At 40 °C the viscosity of an eluent is already significantly reduced. In some applications, even higher temperatures have been used, but then the stability of the stationary phase and analytes must be carefully considered.  

The selection of the mobile phases in the first and second dimensions is very important because the whole stationary/mobile phase system controls the retention and the separation selectivity. If possible, the mobile phase used in the first dimension should have low elution strength in the second dimension to focus the fraction transferred from the first dimension in a narrow zone on the top of the second-dimension column before the elution step. This is important, particularly if the transferred fractions are large. Of course, the compatibility of the mobile phases in the first and second dimensions should also be considered — mainly mutual miscibility, solubility of analytes, adsorption of mobile phase components and viscous fingering. In addition, the second-dimension eluent must also be compatible with the detection system. 

In the first dimension separation, gradient elution is generally used to obtain high peak capacity. In the second dimension, isocratic separation is often used because the time necessary for the re-equilibration of the column before the transfer of the next fraction decreases the time available for the separation if repeated gradients are run with each transferred fraction. This problem can be avoided in some cases by using parallel gradients spanning over the entire gradient time, both in the first and second dimensions.

In this review, practical principles and guidelines for designing LCxLC methods are given.

LCGC Europe-05-01-2011

Analysing Inorganic Disinfection By-products by Ion Chromatography

Ghost Peak Investigation in a Reversed-Phase Gradient LC System

Review of Chiral Stationary Phase Development and Chiral Applications

Comprehensive Two-dimensional Liquid Chromatography (LC×LC): A Review

New Gas Chromatography Products at Pittcon 2011

Event News