Many of the extraction techniques developed over the past generation tout selectivity among their advantages. In reality, solvent selection and the use of stationary (sorbent) phases are the main mechanisms for providing selectivity. Therefore, selectivity is often limited to isolation of classes of compounds rather than individual structures. In this column instalment, the selective removal of a fat substitute in food products is discussed to demonstrate options for obtaining selectivity during extraction.

Over the past generation or so, myriad extraction techniques were developed that have generally improved yields, lessened the amount of organic solvent used, and minimized time. Additionally, many of these techniques claim advantages concerning selectivity. 

Selectivity is the ability to determine the analytes of interest in preference to other sample components (potential interferents). A recent instalment of this column (1) advocated that selectivity can stem from any point in the analytical process, but as a general rule selectivity arises from separations, selective detection schemes, and selective chemical reactions. These approaches can balance each other. For example, if an analytical separation is not completely sufficient, the use of a selective detection method like mass spectrometry (MS) or fluorescence spectroscopy can offer the balance of the required selectivity provided that the unseparated components do not suppress the detector signal. 

Majors described "just enough" sample preparation (2) in which method selectivity is matched to the qualitative or quantitative analytical requirements. For example, the QuECHERS (quick, easy, cheap, effective, rugged, and safe) method for extracting pesticides from fruits and vegetables combines salting out partitioning with dispersive solid-phase extraction (SPE) to remove matrix components, allowing effective chromatography and MS detection. As Majors points out and illustrates in Figure 1 from his original column, increasing complexity in an analytical procedure typically leads to greater selectivity. 

 Figure 1: Just-enough sample preparation represents a continuum of methodologies.			

Turning our attention back to modern extraction methods, the fundamental driving force of the technique leads to the element of selectivity. A number of sorbent-based methods, such as SPE, solid-phase microextraction, and stir-bar sorbent extraction, use chromatographic stationary phases to isolate solutes of interest from gaseous or liquid samples. Analytes are retained by their attraction to a stationary phase of similar polarity and are selectively eluted via choice of an appropriate solvent. The techniques aimed at solid samples, including supercritical fluid extraction (SFE), pressurized fluid extraction, microwave extraction, and ultrasound extraction, rely on the application of energy (often heat) to drive the analyte into an appropriate solvent. In all of these techniques, both sorbent- and solvent-based, the key to selectivity is the match between analyte polarity and polarity of the extracting phase. In other words, "like dissolves like". Thus, extractions are usually considered crude separation techniques, providing compound class selectivity and less utility for the selective isolation of specific, individual compounds. Of course, volatility is the major contributor to selectivity for gas-phase techniques. 

If the primary selectivity mechanism in extractions is solute polarity (that is, matching solute polarity with the solvent or sorbent following the "like dissolves like" principle), is selectivity possible during chemical extraction? Is selectivity beyond compound class selectivity possible? Do extractions need to be selective or is selectivity solely a function of subsequent chromatography and detection? 

To look at an example of extraction selectivity within the "like dissolves like" polarity context, let's consider the example of fat analysis in food products and, more specifically, the example of sucrose ester fat substitutes. 

The United States Nutrition Labelling and Education Act (NLEA) of 1990 requires the labelling of selected nutrients on prepackaged food products. One issue with this requirement deals with the concept of "total fat". What is a "fat"? Are lipoproteins considered lipid or protein? The next concern is their analysis. If "fats" are based on the fatty acid moiety, how can they be measured? Fatty acids are not volatile enough for gas chromatography (GC) analysis. They do not contain any chromophores necessary for ultraviolet detection in liquid chromatography (LC). (Remember, at the time, 

LC–MS was not as widely accepted as it is currently). The polarity of the acidic group can irreversibly adsorb to active sites on chromatographic stationary phases via hydrogen bonding, depending on the type of chromatography. Consequently, the total fat listed on nutritional labels is based on the acid hydrolysis and formation of methyl esters of an organic extract, using a nonpolar solvent, of the food product. The "total fat" listed on the nutritional label is that of a triglyceride based on the resulting fatty acid methyl ester (FAME) composition (3,4). An overview of the formation of methyl esters from triglycerides is presented in Figure 2. 

Figure 2: Triglycerides are hydrolyzed and esterified with methanol to form FAME. The R groups of the triglyceride are typically fatty acids with a carbon chain length of 14â24 and up to two double bonds.			

In the FAME method, samples are dissolved in a nonpolar solvent and a catalyst like BF

 dissolved in methanol is added. Sometimes methanolic acid or base is used. After mild heating, back-extraction with water removes the polar components. The FAME sample is dried and characterized by GC with flame ionization detection (FID). The esterification is facilitated with an alkylation derivatizing agent to condense the carboxyl group of the fatty acid with the methanol hydroxyl. The catalyst aids the reaction by protonating the acid group to promote the formation of the ester and water. The stability of the methyl ester, or FAME, allows GC separation by boiling point or unsaturation. 

The Procter and Gamble Company began marketing sucrose esters called olestra, or the tradename Olean, as fat substitutes in the mid-1990s. The sucrose ester structure is shown in Figure 3. In this figure, the R group is either hydrogen or any fatty acid. By varying the number of fatty acids connected to the sucrose molecule by ester linkages or by changing the carbon chain length of the fatty acids, the properties of the olestra molecule can be altered. Under appropriate conditions, the olestra molecule can have boiling points, viscosity, mouth feel, and other properties similar to common vegetable oils. The sucrose esters are not subject to enzymatic digestive action, because they are not naturally occurring lipids (even though they are made from naturally occurring compounds). Hence, they can be substituted for vegetable oils in selected applications, such as the frying of potato chips and similar salted snacks. 

Figure 3: Just-enough sample preparation represents a continuum of methodologies.			

If we review the acid hydrolysis and esterification reactions for the FAME analysis, olestra in food products would be hydrolyzed along with triglycerides and other fats. The resulting FAMEs would be indistinguishable regarding their source, olestra or triglyceride. Thus, a selective analysis to determine NLEA "total fats" in the presence of olestra is needed. Here we will present three possibilities to garner the necessary selectivity during the sample preparation process. 

Conceptually, perhaps the easiest method to address the isolation of FAME from total fats from those originating from olestra would be at the level of the extraction, meaning we would selectively extract olestra from the total fats. (That is, we're assuming that we must perform FAME analysis of total fats to comply with the requirements of the NLEA). This brings us back to the issue of solvent polarity or "like dissolves like". Because olestra is designed to have properties substantially similar to vegetable oils, which are composed primarily of triglycerides, the solvents used in the dissolution and extraction of either olestra or triglycerides would likely be very similar. This brings us to the solvent extraction method where we have the most variation in solubility conditions with a single solvent, SFE. SFE almost always uses carbon dioxide, perhaps mixed with small amounts of organic cosolvents, near or above its critical point of 31.1 °C and 72.9 atm. Lipids and lipophilic materials are highly soluble in supercritical carbon dioxide, and the use of this solvent for the extraction and fractionation of lipids is well-reviewed (5–7). By making subtle changes in the operating temperature or pressure, somewhat dramatic changes in solvating ability can occur. These changes may allow the fractionation of members of a single compound class on the basis of either polarity or molecular weight. Because the molecular weights of olestra molecules are at least double that of triglycerides, it is conceivable that SFE could be used to either selectively extract olestra and triglycerides from each other, or to selectively precipitate one from the other. This has not been reported in the peer-reviewed literature, so will remain theoretical for now. No other solvent extraction methods will be able to achieve this level of selectivity as easily. 

The next step up in complexity towards gaining the requisite selectivity during the sample preparation of olestra-containing food products would be to use a sorbent-based method to separate olestra from total fats postextraction. SPE is the most basic of these techniques and perhaps the most directly applicable to our hypothetical scenario. SPE can, in many ways, be regarded as an elementary form of LC. A stationary phase is placed onto a support material and put into a cartridge, disk, or other vehicle. Liquid samples are placed onto the SPE sorbent where total retention is achieved. Then analytes and interferents are isolated from each other by the judicious elution with selective solvents. 

Tallmadge and Lin (8) used reversed-phase LC to determine the percent olestra in lipid samples. They found an octadecylsilane column (Zorbax, Agilent Technologies) appropriate to separate olestra from other lipophilic sample components in samples of soybean-oil olestra and heated or unheated cottonseed-oil olestra in soybean oil. The percentage of olestra in these samples varied from 5% to 90% and relative recoveries of 99.2% to 106.0% were reported. Thus, it seems possible that with minimal additional method development a protocol could be developed that involves an extraction of total fats and olestra from the sample food product, SPE separation of olestra from the total fats, and FAME analysis of the total fats. 

Simultaneously, perhaps the most obvious and the most direct means of addressing the proposed situation is to explore the fundamental chemistry behind the problem. Again, olestra is created by esterification of sucrose with fatty acids, but because it lacks the glycerol backbone, it is not subject to enzymatic digestion as are triglycerides. Can this resistance to digestion be exploited in the conversion of total fats to FAMEs to the exclusion of olestra? This is the approach taken in a method validated under the Association of Official Analytical Chemists (AOAC) Peer-Verified Methods Programme (9). A modified version of AOAC Method 983.2.3 was used, in which a chloroform–methanol extraction of olestra-containing snacks was performed. This extract contained both the total fat and olestra. The hydrolysis portion of the FAME analysis used a lipase to hydrolyze the total fats, leaving the unaltered olestra. The fatty acids resulting from the lipase hydrolysis were precipitated as calcium salts and the olestra was extracted with hexane. The fatty acid salts were redissolved and esterified before GC analysis. Recoveries of 101% (6% relative standard deviation [RSD]) for total fat and 104% (6% RSD) for saturated fat were reported. Repeatability and reproducibility were also studied and the method was standardized for fatty acid carbon chains of 6–24. This represents the official method for the determination of total fats in packaged food products containing olestra fat substitute. 

Analytical selectivity can occur during any step of an analytical method, but typically it occurs during the separation or detection steps rather than during sample preparation. Selectivity during chemical (analytical) extractions is almost exclusively limited to solute polarity. Consequently, selective extractions beyond compound class separations will be difficult. Although selective sample preparation is not the typical case, increasing complexity of the procedures can lead to selective analysis. This column instalment presented a scenario in which selectivity during the characterization procedure was not possible, but selectivity during solvent extraction (SFE) and sorbent-based extraction (SPE) or via selective reactions was shown. 

is an Associate Research Professor at South Dakota State University. His research interests include green chemistry, alternative solvents, sample preparation, high resolution chromatography, and bioprocessing in supercritical fluids. He earned his PhD in 1990 at Brigham Young University under the direction of Milton L. Lee. 

"Sample Preparation Perspectives" Editor 

 is an analytical consultant and is a member of

editorial advisory board. Direct correspondence about this column should be addressed to "Sample Preparation Perspectives", 

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

				(3) AOCS Method Ce 1-62, "Fatty Acid Composition by Gas Chromatography," American Oil Chemists Society Official Methods (2005).			

(4) AOAC Method 996.06, "Fat (Total, Saturated, and Unsaturated) in Foods," 18th edition Association of Official Analytical Chemists Methods. 

(6) F. Sahena, I.S.M. Zaidul, S. Jinap, A.A. Karim, K.A. Abbas, N.A.N. Norulaini, and A.K.M. Omar,

(9) D. Schul, D. Tallmadge, D. Burress, D. Ewald, B. Berger, and D. Henry, 

Articles

Sample Preparation Perspectives

In this column, the selective removal of a fat-substitute in food products is discussed to demonstrate options for obtaining selectivity during extraction.

The Role of Selectivity in Extractions: A Case Study

A recent stimulus to the review process article by the United States Pharmacopoeia (USP) expert committee proposes a major change to the way regulated laboratories develop, validate, and control analytical procedures. Is this Quality by Design (QbD) for the chromatography laboratory? 

In this column I want to focus on the heart of chromatographic analysis: The analytical method or procedure. Analytical procedure development, validation, and transfer are key parts of the traditional process that starts, if you're lucky, with defining what you want the method to do and ends up with the operational use of the procedure. In the middle is the stuff that should be done properly but usually is not, as evidenced by how poorly a method operates in routine use. This is typically because the method development and validation is performed by one group and the operation of the procedure is carried out by another. In many instances the two groups rarely talk to each other except when things go wrong — and blame each other for the problems. Of course, this never happens in your organization, does it? 

Validation of analytical procedures in the pharmaceutical industry was harmonized with the agreement of the International Conference on Harmonization (ICH) quality publications Q2A and B covering the text and methodology of validation of analytical procedures (1,2) in 1994 and 1996, respectively. These two publications were merged into a single revised document ICH Q2 (R1) in 2005 without any text being amended (3).  

Robustness of an analytical procedure is defined in ICH Q2(R1) as: 

A measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

In the methodology section there is more information on the topic of robustness:  

The evaluation of robustness should be considered during the development phase and depends on the type of procedure under study. It should show the reliability of an analysis with respect to deliberate variations in method parameters. If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure. One consequence of the evaluation of robustness should be that a series of system suitability parameters (for example, resolution test) is established to ensure that the validity of the analytical procedure is maintained whenever used (3). 

This is the interesting part — for a document focused on the validation of analytical procedures we have the statement that robustness is actually a job to be performed during method development. How many people actually conduct robustness studies when under time pressures? However, it does say that robustness depends on the method but then goes on to mention some variables that could be considered when looking at high performance liquid chromatography (HPLC) procedures: 

In the case of liquid chromatography, examples of typical variations are: 

Influence of variations of pH in a mobile phase;

Influence of variations in mobile phase composition; 

Different columns (different lots and/or suppliers);

Therefore, in my view, robustness should be undertaken for chromatographic methods, especially those involved in generating data for making critical decisions. 

The aim of ICH Q2(R1) was to provide guidance in the validation of any analytical procedure but this document has tended to take on the role of regulation. Don't think, just do it. ICH Q2(R1) has been incorporated into the United States Pharmacopoeia (USP) <1225> Validation of analytical procedures (4), for example. Note the chapter numbering: It is an informational (strong guidance) but not a mandatory general chapter (5). 

In the current version of the USP there are three general chapters involved with analytical procedures:  					

USP <1224> Transfer of analytical procedures (6);

USP <1225> Validation of analytical procedures – this is ICH Q2(R1) (4);

USP <1226> Verification of compendial procedures (7).

Interestingly, there is no mandatory chapter on the validation of analytical procedures, only guidance. The aim of all three chapters is to allow an analytical procedure to become operational in a regulated laboratory, as shown in Figure 1. However, there is no consideration of the importance of method development except where discussed in ICH Q2(R1) (as mentioned earlier). As you can see, method development remained outside of the control of pharmacopeias — despite its importance to the operational method. 

 Figure 1: Current USP general chapters on analytical procedures.			

Since the publication of ICH Q2(R1), the International Conference on Harmonization has issued three key documents that impact the way that the pharmaceutical industry operates. It changes the industry from a reactive to a proactive industry — or should do if these documents are implemented correctly. The three documents are: 					

ICH Q10 on Pharmaceutical Quality Systems (8): This document describes a quality management system based on ISO 9001 but adapted for the pharmaceutical industry. A key change is the introduction of continual (not continuous as pharma is a regulated industry) improvement. In response to the publication of ICH Q10, EU GMP Chapters 1 (9) and 7 (10) have been issued to incorporate this new approach.

ICH Q9: Quality Risk Management (11): This covers an overall approach to the assessment and management of risk in the pharmaceutical industry.

ICH Q8(R2): Pharmaceutical Development (12): This is the most important document for the remainder of our discussion as it provides the framework for the approaches that the USP have taken for analytical procedure development, validation, and application.

To understand the approach taken in ICH Q8(R2) we have to step back in time and outside the pharmaceutical industry. 

A catchy phrase is it not? However, this is key to understanding the overall approach proposed by the USP paper. We need to consider what is Quality by Design (QbD)? It is a concept that was outlined first by Joseph Juran, a well-known expert and consultant in quality, who stated that "

product features and failure rates are largely determined in planning for quality

" (13). Put simply, quality must be designed into a product or a process but quality cannot be tested into it, that is, the analytical procedure. Therefore without adequate quality designed into the procedure during the development phase, there is not much point putting it into operation because it would not be very effective. This ineffectiveness would be typically evidenced in the operational use of the method with more out of specification (OOS) results when compared with a similar procedure where time and effort has been taken to understand how the chromatographic method works.  

Some of the key terms and definitions from ICH Q8(R2) are presented in Table 1. Although ICH Q8 is focused on the development of a pharmaceutical product, there are several topics that are pertinent to the development, validation, and operation of an analytical method such as the life cycle, design of experiments, or the robustness that form the basis of the USP proposed approach, which is why it is important to understand the role and terminology of ICH Q8. We will meet the modified versions of these terms as we look at the new USP approach.  

Table 1: ICH Q8 key terms and definitions. 			

 (14), a USP expert committee published a stimulus to the revision process paper entitled:

 Lifecycle Management of Analytical Procedures: Method Development, Procedure Performance Qualification, and Procedure Performance Verification

. This paper proposes a QbD approach to method development, validation, and operational use using a life cycle concept. The importance of this stimulus paper is that for the first time there would be a formal link between method development and method validation within a Pharmacopoeia. Just from the title of the paper we can see the adaption of two ICH Q8 terms: Life cycle management (Q8: Life cycle) and procedure performance verification (Q8: Continuous process verification). However, there are other terms that are a little strange such as procedure performance qualification, and just where is method validation in the title?  

In the stimulus to the revision process paper it is proposed that the three existing USP general chapters dealing with analytical procedure validation, verification, and transfer will be replaced by two general chapters. There will be a mandatory general chapter, <220>, dealing with the minimum requirements for method validation and an informational general chapter, <1220>, covering the whole life cycle of analytical procedure development, validation, use, and modification best practices.  

Overview of the Analytical Procedure Life Cycle:

 Following on from the principle of ICH Q8, analytical procedure development, validation, use, and modification is a life cycle process with defined activities and deliverables. The outline of the three stage process is shown in Figure 2. 

Figure 2: Proposed stages in the analytical procedure life cycle (14).			

The first stage of the process is method development and understanding. The last word is important! This goes back to Juran: Quality is designed into an analytical procedure during the method development stage and not tested during the validation part of the life cycle. You need to therefore understand how the method works by knowing the key variables and how these influence the analysis. The KISS principle applies here. KISS? Keep it simple, stupid. Don't make an analysis more complex than it needs to be.  

The second stage is procedure performance qualification — what? This is method validation under another name and has been taken from the US Food and Drug Administration's (FDA) updated guidance for industry on process validation (15). In essence, this stage demonstrates that the procedure or updated procedure is fit for the intended purpose of the assay. 

The third stage is the procedure performance verification which checks how the method operates in actual use and remains under control. This can be achieved through routine monitoring and by charting performance using either Shewart or Cusum plots and reviewing variations in the performance of the procedure. If data indicate that the method is not functioning as expected, it should be investigated to see the cause of the variation. Outcomes of an investigation may be a change to the method which we will discuss later. 

As the aim of this column is to discuss quality by design, I want to look in more detail at the proposals for the method development stage. Within this stage there are a number of activities, as outlined in the USP stimulus paper (14).  

Define the Analytical Target Profile (ATP) for the Procedure: 

The ATP is the interpretation of the Quality Target Product Profile under ICH Q8 for quality control and analytical development laboratories. An example of an ATP may be measuring analyte X in a specified matrix over the concentration range between A% and B% with a pre-defined uncertainty for the reportable result. The ATP may also define the matrix of the sample or any limits of detection if measuring impurities and in the presence of any potentially interfering compounds. In a move away from the typical ICH Q2 definitions, the USP stimulus paper talks about uncertainties, not precision and accuracy, therefore bringing the pharmaceutical industry into the ISO world. Defining the ATP is an important first step because it is the specification for the method against which the procedure will be validated. The first stage of quality by design is therefore to put a marker in the sand with the specification for the analytical procedure. 

Typically, an ATP would not define the analytical technique used but as this column is published in a chromatographic magazine it will be safe to assume that we're looking at a separation method of analysis, typically gas chromatography (GC) or HPLC. However, the responsible analyst should select an appropriate technique that is able to meet the ATP, and in doing so the ATP will be broken down into more detailed critical performance characteristics. Keep in mind that although I'm putting the emphasis on the chromatographic technique, there is also a sample preparation element of any analysis as the samples may be liquid, semi-solid, or solid. 

 Once the ATP has been defined and the analytical technique selected, the potential method can be designed in outline. To ensure that the method is reliable and meets the requirements in the ATP, a risk assessment should be undertaken — including of the overall procedure. The stimulus paper recommends the use of process mapping the stages of the procedure (as the method is equated to a process) or Ishikawa (fishbone) diagrams to identify potential variables that could impact on determining a reliable reportable result. One way of ranking these variables is a technique known as CNX (Control, Noise, Experimental). Each variable is ranked as a factor to be controlled (C), those that are noise (N), and the ones that need to examined experimentally (X). Naturally, for a regulated industry all these classifications should be justified and documented. Further risk analysis tools can be used to screen variables and reduce the number of experiments. 

Questions of Quality

A recent stimulus to the review process article by the United States Pharmacopoeia (USP) Expert Committee is proposing a major change in the way regulated laboratories develop, validate and control analytical procedures. Is this Quality by Design (QbD) for the chromatography laboratory?

Life Cycle and Quality by Design for Chromatographic Methods

A simple and fast analytical method using ion-pair chromatography with suppressed conductivity detection was developed to simultaneously detect and quantify sodium decylsulphate (C

) in environmental water. The effect of the mobile phase parameters (NH

 concentrations) on the retention factors has been studied in detail, and the separation of the seven analytes was achieved using gradient elution. 

Surfactants are widely used in personal and home care products including household detergents, shampoos, and cleaning products. They are also used in oil, pesticides, and metal processing (1–3). The annual worldwide consumption of surfactants is steadily increasing. Surfactants are classified into four groups according to their chemical characteristics: Cationic, anionic, non-ionic, and amphoteric (4). Anionic surfactants account for more than 40% of the total surfactants. Once in effluent sewage water, these anionic surfactants produce foams, emulsify, and suspend. They prevent the oxygen exchange between air and environmental water, resulting in the deterioration of water quality and potential harm to aquatic ecosystems (5–6). Recently, surfactants have been receiving more attention because of their growing occurrence in environmental water (7–8).  

Several methods have been reported for the determination of anionic surfactants by spectrophotometry (9), liquid chromatography (LC) (10–12), and by electrochemical means (13). The spectrophotometry method is used for detections at low µg/L levels and suffers from chemical interferences. The LC method can detect the substances that contain a UV chromophore; and this method has been reviewed by Sohrabi (9). The surfactants which have to be derived can be detected by LC–UV. The electrochemical method can detect the compounds with oxidation and reduction. It is unsuitable for anion surfactants without oxidation and reduction potential.  

This work opted for the suppressed conductivity detection based on the intrinsic properties of anion surfactants. Some papers have been published on ion chromatography investigations. Levine studied sodium dodecylsulphate in wastewater (14). Porter detected quantitatively poly disperse polyethylene oxide (PEO) and sodium dodecylsulphate (SDS) in water (15). In J. Weiss's publication, only five sodium alkyl sulphates have been detected (16). The focus of these studies were directed towards the traditional method and a limited number of analytes (17–18). 

As a result, the aim of this work was to develop an ion chromatography method for the separation and precise quantification based on ion-pair chromatography with suppressed conductivity detection. This method offered three advantages: Firstly, it should be noted that the separation of seven species was achieved in less than 20 min. The effect of mobile phase parameters (NH

 concentration) and the optimization of a gradient elution on separation were studied. To the best of our knowledge, this is the first report on the separation of these seven analytes; secondly, the environmental water samples were injected into the ion chromatography instrument without any pretreatment; finally, suppressed conductivity is known to detect simple cationic, anionic compounds such as F or Cl

. Few ion-pair methods with suppressed conductivity detection for the separation of sodium alkyl sulphates are available. As a result of these advantages, this method can become a technique capable of the simultaneous determination of seven sodium alkyl sulphates in environmental water and can potentially be applied to environmental monitoring.  

The ion chromatography (IC) system consisted of a 850 professional IC separation centre, a 872 gradient pump, a 771 IC compact interface, a 858 professional sample injector equipped with a 20 µL sample loop, and a suppressed conductivity detector (Metrohm). Conductivity suppression was performed by an anion chemical suppressor with a carbon dioxide suppressor (Metrohm). The chemical suppressor was operated in the external chemical mode, with 3 mM sulphuric acid as a regenerant. All instrument control and data collection was performed by MagIC Net 2.2 for Windows chromatography software (Metrohm).  

The analytical column was a neutral polymer column, 4 mm × 250 mm, IonPacNS1 (Dionex). The resin in the columns was composed of ethyl vinyl benzene cross-linked polystyrene-divinylbenzene substrate (EVB-DVB) (Dionex). 			

) were obtained from Alorich (purity>99%).  

HPLC-grade acetonitrile and analytical-grade ammonia were used (Merck). Deionized 18.2 MΩ water (Watsons) was used for eluent and standard solution preparation. 

Stock solutions and standard solutions for calibrations were prepared with deionized water. A mixed working solution at a concentration of 2500 mg/L was prepared by dissolving an accurately weighed quantity of 0.25 g of seven sodium alkyl sulphates into deionized water and diluting to 100 mL. The stock solution was serially diluted with deionized water to the concentrations of 200 mg/L, 150 mg/L, 100 mg/L, 50 mg/L, 30 mg/L, and 10 mg/L as the working solutions. They were kept in glass vials at 4 °C. 

Samples of river water and sewage were collected from various locations in Lanzhou (China). The river samples were collected at surface level from discrete sections of the Yellow River in Lanzhou. Sewage samples were collected from living areas, industrial parks, and university areas. A total of 11 samples were obtained. 

 The separation column was a 4 mm × 250 mm, NS1 column (Dionex). A gradient elution was adopted by eluent A and eluent B. Eluent A was composed of a 5 mmol/L ammonium hydroxide +22% acetonitrile +0.3 mmol/L sodium carbonate; eluent B was 5 mmol/L ammonium hydroxide +80% acetonitrile. The following gradient programme was used throughout the experiment: 100% eluent A was increased to 100% B by a concave 2 mode at 20 min. The column oven was maintained at 30 °C. The sample injection volume was  20 µL. The flow rate was 1 mL/min. At 23 °C, the pH of eluent A and eluent B was 10.63 and 8.82, respectively. The chromatogram of an optimized separation of seven anions spiked in a 200 mg/L standard solution is shown in Figure 1. 

 Figure 1: Optimized separation chromatogram of seven anions spiked in a 200 mg/L standard solution.			

Ion-pair chromatography is based on the use of an apolar stationary phase, such as EVB-DVB polymers, which is dynamically modified by an ion-interaction reagent added to the mobile phase. Anions can be retained because of the formation of an electroneutral complex between the anionic sample and the ion-pairing reagent of the opposite charge (15,19). However, the mechanism of ion-exchange separation depends on the exchange of ions between a stationary phase and a mobile phase (20). According to the difference of retention in the stationary phase, the analytes can be separated from each other. Through ion-pair mechanism, the effects of following four parameters were investigated and are presented in this paper. These are organic solvents, ion-pairing reagents, inorganic additives, and gradient type selection.  

Separation of the seven sodium alkyl sulphates is very difficult because of their similar physicochemical properties. In this study, the effects of organic solvents were to modify retention time and improve separation were investigated.  

Common organic solvents are acetonitrile, methanol, and isopropanol. They make it difficult for surfactants to produce foams, and therefore achieve good reproducibility. The polarity of acetonitrile is lower than that of methanol. As a result, acetonitrile was added to reduce the mobile phase polarity. Isopropanol has a higher viscosity, leading to higher column pressure and probably lower column efficiency (21). Acetonitrile can obtain a better separation and has a shorter retention time in comparison with methanol and isopropanol. Acetonitrile was therefore adopted as an organic solvent. 

Figure 2 shows the effect of acetonitrile in a range of concentrations from 15%, 18%, 20%, 22%, and 30% (v/v) on the retention time for the analytes. The increase of acetonitrile from 15% to 22% in the eluent can reduce retention times of the seven sodium alkyl sulphates. For C

 sodium alkyl sulphates, acetonitrile at a concentration of 22% to 30% (v/v) had less effect on their retention times. Longer alkyl chains of sulphates had stronger hydrophobic interactions. It was observed that the retention time was at an optimum when adding 22% (v/v) acetonitrile to the mobile phase for the separation. 

Figure 2: Effect of acetonitrile concentration on retention time for the analytes studied. Column: IonPac NS1 (4 Ã 250 mm). Eluent B: 80% acetonitrile. Eluent A: acetonitrile as shown.			

 The most commonly used ion-pairing reagents presented a large hydrophobic moiety and a small ionized group whose charge is opposite to that of the samples. The effect on the retention of ionic samples is interpreted by the adsorption of the ion-interaction reagent on the hydrophobic surface, leading consecutively to the formation of charged sites. Ionic samples are then retained by electrostatic forces in the layer adjacent to the surface of the particles in the stationary phase (15). Owing to the presence of ion-pair reagents, sample ions can be exchanged and retained on the consecutive ion-exchange surface of the stationary phase. 

Hydrophilic ion-pairing reagents are suitable for hydrophobic analytes separation. The reagents of smaller molecular mass have more effect on separation than larger molecular mass. Ammonium hydroxide is the strongest hydrophilic and has the smallest molecular mass in comparison with other ion-pairing reagents such as tetrabutyl ammonium hydroxide (TBAOH). Ammonium hydroxide is the preferred ion pairing reagent for analysis of high molecular masses and long alkyl chains such as sodium alkyl sulphates. 

This study contrasted three concentrations with one another. The results showed that the effect on retention time was less significant with a change in the NH

OH concentration range 4–10 mmol/L. Seven analytes were not completely separated and had longer retention times at 4 mmol/L. Both 5 mmol/L and 10 mmol/L NH

OH obtained complete separation. However, the peak height of each component reached the maximum value when 5 mmol/L NH

OH was used for the determination of sodium alkyl sulphates. It obtained an efficient separation, with sharp and symmetrical peaks (Figure 3). 

Figure 3: Effect of NH4OH concentration on retention time for the analytes studied. Column: IonPac NS1 (4 Ã 250  mm). Eluent A: 22% acetonitrile; eluent B: 80% acetonitrile. The concentration of chromatogram (a): 4 mmol/L; (b): 5 mmol/L; (c): 10 mmol/L NH4OH.			

 The effect of organic solvents was evaluated at 22% (v/v) acetonitrile and 5 mmol/L ammonium hydroxide. Alkyl sodium sulphates are two valences, long alkyl chains, and strong hydrophobic anions. Because of the selectivity exhibited by the analytical column, a Na

 eluent was initially chosen to improve separation and selectivity. 

The team studied the effect of concentration of Na

 at a concentration of 0.1 mmol/L, 0.3 mmol/L, and 0.5 mmol/L. The results showed the influence of a small amount of Na

 sodium alkyl sulphates. It was observed that retention time increased as the inorganic additives concentration of the analytes was increased. It was 0.3 mmol/L Na

 that achieved optimum retention times and a complete separation.  

 The number of the analytes and their similar structure made the optimization of the separation a difﬁcult task. If a low concentration of leachate is chosen, the seven sodium alkyl sulphates cannot be separated thoroughly in 20 min. Strongly reserved substances will have a long retention time. From the experiments, it was found to be necessary to optimize the eluent composition gradients. There are nine kinds of gradient types: linear; concave 1; concave 2; concave 3; concave 4; convexity 1; convexity 2; convexity 3; and convexity 4.  

The acetonitrile content ranged from 22% to 80% in 20 min. Linear gradient was not able to separate the C

 sodium alkyl sulphates well and caused broad peaks, with the result that the elution power was significantly decreased (concave 1) in a shorter period of time. In subsequent experiments, concave 2, concave 3, and concave 4 gradients were tested. The gradient of concave 3 had broad peaks. C

 sodium alkyl sulphates had longer retention times when concave 4 was used. However, the concave 2 gradient ensured that all the compounds separated thoroughly and achieved sharp and symmetrical peaks. As a result, a gradient type of concave 2 was chosen (Figure 4). 

Figure 4: Effect of different gradient types on retention time for the analytes studied. Column: IonPac NS1(4 Ã 250 mm). (a): Linear; (b): Concave 1; (c): Concave 2; (d): Concave 3; (e): Concave 4.			

 The linear range was calculated with the slope variation. Different concentrations of working standard solutions from 10 mg/L to 200 mg/L were injected. The results showed that the seven sodium sulphates had good linear regression and a wide linear range. Detection limits were evaluated at three times the background signal and were determined injecting 20 µL of working standard solutions. The detection limits were 10 mg/L for the seven sodium alkyl sulphates. The results are shown in Table 1. 

The repeatability of the peak areas and the retention times for the seven sodium alkyl sulphates were studied by repeatedly injecting nine replicate analyses of mixed 10 mg/L standard solutions. It was observed that both retention times and peak areas were reproducible and all the relative standard deviation (RSD) (n = 9) values obtained were satisfactory (<4.0%). The results are shown in Table 1. 			

To calculate the reproducibility conveniently, the study selected the Yellow River in Lanzhou (China) without sodium sulphates as the blank sample. The RSD (n = 9) spiked with three concentrations of 30 mg/L, 50 mg/L, and 100 mg/L into the blank sample were in the range 0.62–4.24%. The analytical procedure showed good precision and accuracy for the determination of the standard solution in different concentrations. The results are shown in the Table 2. 

Table 2: Relative standard deviations and recovery results when adding different concentrations.			

 After optimization, the ion-pair chromatography method was applied to the simultaneous determination of the seven sodium alkyl sulphates in the river samples from the Yellow River in Lanzhou (China) and the sewage samples from living areas, industrial parks, and university areas. 

Table 3: Analytical results of seven sodium alkyl sulphates in water samples (mg/L). 

The work also selected the Yellow River without alkyl sodium sulphates as the blank sample. These samples have been divided into three groups which have been spiked with 30 mg/L, 50 mg/L, and 100 mg/L of a mixed standard solution of seven sodium sulphates, respectively. These were measured in accordance with the optimization of the experimental method and compared to a blank experiment. Recoveries of 92.8–100.5% (n = 3) were obtained. The results of analytical samples are shown in Table 3. The chromatogram of the real sample from the Xigu (Lanzhou, China) industrial estate is displayed in Figure 5. 

Figure 5: Chromatogram of a real sample in Xigu (Lanzhou, China) industrial estate.			

The chromatograms achieved a good application to real sample analysis without time-consuming pretreatment. The results demonstrated that industrial wastewater had the higher concentration of sodium tridecylsulphate (C

). Most sewage samples contained octadecylsulphate (C

Separation was achieved after a study of the effect of mobile phase parameters (NH

concentration) and an optimization of gradient elution. The application of ion-pair chromatography in analyzing polar organic compounds was able to make up the deficiencies of LC. It should be noted that suppressed conductivity was coupled to an ion-pair mechanism, and the technique was developed for the determination of seven sodium alkyl sulphates. The average recoveries for samples were in the range of 92.8% to 100.5% (n = 3) and the RSD (n = 9) was less than 4.24%. The method can therefore be applied for the analysis of numerous surfactant materials such as sodium decylsulphate (C

is currently studying for a PhD at the School of Mechanical Engineering and Automation, Northeastern University, Shenyang, China. 

 is the director of the Central Laboratory of Technical Center of Gansu Entry-Exit Inspection and Quarantine Bureau, Lanzhou, China. He sits on the CIQ Technical Committee of Food Inspection Professional, and the council in Chinese Society of Fourth Chromatography. He is also a professor at the Northwest Normal University and Gansu Agricultural University. 

 is a professor at the School of Mechanical Engineering and Automation, Northeastern University, Shenyang, China. He is the director of the Process Equipment and Environmental Engineering Research Institute. He is also a member of the International Water Association; the China Environmental Science Society; and the Japan Society of Water Environment. 

 works in the Central Laboratory of Technical Center of Gansu Entry-Exit Inspection and Quarantine Bureau in Lanzhou, China. 

(2) S. Tai, Z. Gao, X. Liu, and Q. Zhang, 

Feature

A simple and fast ion-pair chromatography method to detect sodium alkyl sulphates in environmental samples using conductivity detection is described.

Determination of Seven Sodium Alkyl Sulphates in Environmental Water by Ion-pair Chromatography with Suppressed Conductivity Detection

Several high performance liquid chromatography (HPLC) approaches can be used in the analysis of polar small molecules that are classified as pharmaceuticals, metabolites, or biomarkers. Many of these polar compounds can be challenging for traditional reversed-phase separation methods. One approach for analyzing these types of samples is the use of columns having polar retention capabilities such as ion-exchange, hydrophilic interaction liquid chromatography (HILIC), polar-embedded, or polar-endcapped columns. Another approach is aqueous normal phase (ANP) chromatography. This mode has the advantage of having both reversed-phase and normal-phase retention. In a recent web seminar, Joseph Pesek, a professor of Chemistry at San José State University (San José, California, USA), explained the mechanisms, advantages, and disadvantages of these approaches to the analysis of polar compounds. Below, he answers questions raised during the web seminar. 

If hydrophilic interaction liquid chromatography (HILIC) works now, why did it not work 30 years ago? 

 HILIC was not well understood 30 years ago. However, some of the shortcomings of HILIC were noted at that time and for that reason it was not pursued vigorously.  

How is HILIC different from working with plain silica columns? 

 Silica is just one column material used in HILIC. There are many others, each having somewhat different properties. Therefore, as in reversed-phase chromatography, in HILIC it is important to select the right type of column to match the properties of the analyte.  

What is the difference between polar-endcapped and HILIC columns?

 Polar-endcapped columns are similar to traditional reversed-phase columns but instead of using a nonpolar endcapping reagent like a trimethyl group, a more polar group is used to cover some of the remaining silanols. HILIC columns generally are significantly more polar since they are based on bare silica or have bonded polar groups that cover a significant fraction of all of the available bonding sites. 

Is silica really a HILIC stationary phase? Several studies suggest that it is very different from bonded phases.

 It is if you believe that the mechanism for HILIC retention is the presence of an adsorbed water layer on the surface that serves as a medium of analyte partition. It may be different from bonded phases but for most of these, the presence of a layer of water at or near the surface is used to describe retention.  

What is your experience with the robustness of most HILIC columns?

 Most HILIC columns are less robust than traditional reversed-phase columns. This results in part from the fact that the surface can be easily contaminated and is hard to clean. In other cases, the bonded group is not very robust and is susceptible to being cleaved from the surface in mobile phases used for HILIC retention.    

Are HILIC or similar columns compatible with ultrahigh-pressure liquid chromatography (UHPLC)?

 All of the columns discussed in the seminar, including aqueous normal phase (ANP) and HILIC columns, are compatible with UHPLC. 

Can I use silica hydride columns for mixed analysis of polar and nonpolar compounds or amphiphilic compounds? If so, is there a possibility that the retention times of polar and nonpolar compounds might be the same (that is, their respective affinities are equal), thus yielding the same retention times? 

 This is relatively unlikely because that would require that both mechanisms be operating at equal efficiency for those particular compounds. In most cases one mechanism would be more predominant than the other and retention would be different. However, even in the unlikely event that both were equal, that would only occur at one mobile-phase composition. Therefore, you could switch to a different isocratic composition or a different gradient and that would shift the relative contributions of the two mechanisms, allowing separation.   

Why are there different bonded phases using silica hydride if it is possible to retain both polar and nonpolar analytes on any silica hydride column?

 The degree of retention in reversed-phase and aqueous normal phase modes on silica hydride columns depends on the degree and type of modification. For columns with no or minimal modification the ANP mode is stronger than the reversed-phase mode. As the degree of modification becomes greater — that is, in columns modified with larger groups or that have a greater degree of surface coverage — the reversed-phase properties increase.   

What type of columns can be used to separate both polar and nonpolar compounds? 

 The most versatile columns for this type of separation are silica hydride columns because the amount of retention in both modes can be adjusted by the type of modification and the mobile-phase composition. Polar-endcapped and polar-embedded columns have this same capability because they are also classified as mixed-mode stationary phases. The degree of polar retention depends on the endcapping group or the embedded group. 

What type of gradient do you suggest for ANP analysis of hydrophilic and hydrophobic molecules in the same run? I have a large hydrophobic active and a prodrug, as well as five small polar metabolites. 

 The usefulness of ANP separations is that you have two options in selecting the gradient. It would be best to try both a reversed-phase gradient (from 80% to 20% aqueous) and then an ANP gradient (from 20% to 80% aqueous) as an initial screening process. From the preliminary results you can determine which mode is most likely to produce the best separation. Once the direction has been selected, then the gradient can be modified to reach the desired retention and separation of the components in your mixture. 

How much efficiency do ANP columns have? Does efficiency have detrimental effects for biological matrices for liquid chromatography–mass spectrometry (LC–MS) analysis? 

 Normal-phase retention generally has lower efficiency than reversed-phased methods. It is highly dependent on the compound being retained. Efficiency doesn't have detrimental effects for biological matrices for LC–MS analysis, but sometimes lower ionization of the compounds is observed in MS detector when compounds are being analyzed in biological matrices. The use of internal standards (usually deuterated compounds) will help to assess how much lower the peaks are. 

Are there other bonded phases for ANP chromatography?

 Yes. As stated in the seminar, polar-embedded, polar-endcapped, and fluorinated phases have some ANP capabilities. Also, graphitized carbon phases have demonstrated some ANP behaviour. 

Carbon columns, or porous graphitized carbon columns, also possess dual retention capabilities. Their structure is porous particles composed of flat sheets of hexagonal carbon. Selectivity in the reversed-phase mode is somewhat different than with traditional C18 columns. The absorptive nature of the carbon surface results in the polar retention observed.  

What manufacturers make low-carbon silica hybride columns? 

 The current suppliers of low-carbon silica hydride carbons are Microsolv Technology and VWR in the United States and Hichrom Ltd. in Europe and the UK.  

How stable or durable are silica hydride columns?

 Silica hydride columns are just as durable, and in some cases more durable, than traditional chemically modified stationary phases based on organosilane chemistry. This is due to the direct silicon-carbon at the surface. For columns that are used primarily for polar retention in the ANP mode, these materials are more durable than typical HILIC columns because the hydride surface is not as easily contaminated as ordinary silica and the surface is less susceptible to attack by aggressive mobile phases because of its more hydrophobic nature. 

Can you compare silica hydride and ionic liquids? Are there any important advantages of one over the other for retaining polar analytes?

 It is assumed you are referring to ionic liquid stationary phases. All of these materials contain a charged group that is part of the bonded material. The charged site is usually within the bonded chain, but not always. It is most similar to the polar-embedded phases discussed in the presentation. They have been shown to retain both polar and nonpolar compounds. To date, the range of compounds analyzed with ionic liquid stationary phases is not as broad as with silica hydride phases so the same versatility has not been demonstrated. A good review of ionic liquids in chromatography can be found in the book by Mun and Sim (1). 

Why are polar compounds retained more strongly when they are ionized and not when they are neutral, as in reversed-phase chromatography? 

 This occurs because the highest polarity is generally found in ionized compounds, as a result of the fixed charge on the compound. Therefore, in both aqueous normal phase chromatography and HILIC, maximum retention is attained for compounds with the highest polarity, in other words, those that are ionized.  

Have you had any issues with ion suppression when using ion-pairing agents? If so, how did you remedy the issue?

 The issue of ion suppression for the analysis of polar compounds when using ion-pairing reagents in reversed-phase chromatography is a prime reason to not use this approach. In general, ANP chromatography with silica hydride stationary phases offers the most versatile approach for the analysis of polar compounds. It has been demonstrated that ANP chromatography can analyze positively or negatively charged compounds having molecular weights below 100 to larger molecules, such as peptides. Polar uncharged molecules can also be analyzed by this approach. In every case the mobile phases do not contain more than 10 mM of an additive, such as acetic or formic acids, or ammonium acetate or formate, all of which are MS-compatible.  

Peak shape is usually the main problem I encounter with HILIC, mixed-mode, or ion chromatography, even when I'm matching the strength of the sample solvent with the mobile phase. Do you have any tips on improving peak shape?

 It is generally better to have the sample solvent strength stronger that the mobile-phase solvent, especially at the beginning of a gradient. In addition, silica hydride phases are less susceptible to peak distortions than HILIC. With the proper gradient, most compounds will give good peak shape. Certain compounds like polyprotic acids and phosphate-containing species have special considerations because they can be affected by metal ions in the high performance liquid chromatography (HPLC) system. Sometimes the addition of 0.5% formic acid or ammonia to the sample (depending on the compound) can improve peak shape.  

Given that ANP media do not appear to form a water layer, does the ionic strength of the mobile phase have a significant impact on the retention characteristics of ANP chromatography, as it does in HILIC?

 The ionic strength of the mobile phase has a smaller effect in ANP chromatography than in HILIC because it appears that the mechanism is some type of competitive adsorption on the surface. Ionic strength would be expected to have some impact on this process.  

Up to what percent of acid can you use to acidify silica hydride columns? 

 Generally speaking there is not much need to go above 0.5% acetic or formic acids for most ANP applications. If it is trifluoroacetic acid for biological applications, the usual limit is about 0.1% when using UV detection. Trifluoroacetic acid is not recommended when using MS detection because of ion suppression.  

Can you inject aqueous samples using ANP columns?

 Yes, it is possible to inject aqueous samples for many applications. Each specific analysis would have to be tested to determine if it is possible in that case.  

Are all of these techniques (HILIC, ANP, and ion-exchange chromatography, and reversed-phase chromatography with polar-embedded or polar-endcapped columns) compatible with mass spectrometry?

 Yes, to some degree. It all depends on the additive and the concentration of the additive in the mobile phase. In ANP chromatography, most applications reported to date use mobile phases that are MS-compatible.  

Joe Pesek answers questions about IEX, HILIC, polar-embedded, and polar-endcapped columns, and aqueous normal phase chromatography.

HPLC Analysis of Very Polar Compounds in Bioanalysis

What kind of adjustments need to be made when scaling an isocratic method?

Today we are often confronted with many different types of liquid chromatography (LC) methods. These may use conventional 250 or 150 mm × 4.6 mm, 150 mm × 2.1 mm, 50 mm × 2.1 mm, and many other column configurations packed with particles generally ranging from <2 µm to 5 µm, and sometimes even 10 µm in diameter. One of the challenges this variety presents is transferring a method from one column configuration to another and still obtaining the same resulting separation. For example, you may use an ultrahigh-pressure LC (UHPLC) system to develop methods quickly in your research and development (R&D) laboratory, but want to transfer it to a conventional LC system for routine use. Or your conventional method with ultraviolet detection (LC–UV) may need to be transferred to an LC system with mass spectrometry detection (LC–MS). Alternatively, you may want to adjust a pharmacopeial method to use a different column configuration. In many of these cases, the method must be moved from one column size to another, yet maintain the same separation. The conversion is not difficult, but you do have to be careful to make the appropriate adjustments. Isocratic separations, in which the mobile-phase concentration is constant, are simpler to convert than gradient methods, where special care has to be taken to avoid inadvertent chromatographic changes. This month's "LC Troubleshooting" discussion focuses on isocratic separations, and next month we'll look at gradients. 

Equivalent separations require that the resolution stays the same when conditions are changed. In the method development classes we teach, we use what is often referred to as the "fundamental resolution equation" as a guide for the method development process. We can use this same equation to guide us in method conversion: 

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

 is the retention factor. The first caveat is that the chemistry of the system cannot change when a change in the column or other conditions changes. This means that the mobile phase must remain the same, as well as the column temperature and column chemistry. With today's columns, it is usually valid to assume that the same brand name description of a column (for example, ACE C18 [Advanced Chromatography Technologies Ltd.]) will have the same column chemistry, no matter what the particle size is (2, 3, 5 µm, and so forth). You'll recall that the retention factor is defined as: 

 is the column dead time (retention time of an unretained peak). If we keep the chemistry of the system constant, the retention time relative to the dead time should stay constant, so 

 will remain unchanged. Any change in retention because of a change in column length, diameter, or flow rate will have a proportional change for 

 will stay constant in this case as well. For example, doubling the flow rate will halve

The separation factor α is simply the ratio of 

 constant, as discussed above, α will be unchanged. If 

LCGC Europe-02-01-2014

The Role of Selectivity in Extractions: A Case Study

Life Cycle and Quality by Design for Chromatographic Methods

Determination of Seven Sodium Alkyl Sulphates in Environmental Water by Ion-pair Chromatography with Suppressed Conductivity Detection

HPLC Analysis of Very Polar Compounds in Bioanalysis

LC Method Scaling, Part I: Isocratic Separations

Troubleshooting Real GC Problems

Georges Guiochon: Separation Science Innovator

Vol 27 No 2 LCGC Europe February 2014 Regular Issue PDF