Beginner's Guide to Convergence Chromatography

Due to the expanded selectivity range of convergence chromatography described previously, the technique is suited for a wide variety of applications (Table 5).

Regardless of the market area or exact nature of the analytes tested, CC helps overcome analytical challenges in three key ways:

• Convergence chromatography simplifies the workflow
• Convergence chromatography separates compounds with structural similarity
• Convergence chromatography is an orthogonal mode of separation to reversed-phase LC

Next we explain the key benefits of CC using some application examples.

One of the most useful discoveries made during the evolution of SFC to CC is how compressed CO₂ mixes with a wide range of organic solvents to conduct chromatographic analyses in a way that was not possible before. In this section we will explain how CC can drastically simplify the work flow in an analytical laboratory.

Simplifying a workflow, from initial sample collection through to the final analysis, generally has the greatest business impact in any analytical laboratory. CC has the ability to drastically simplify the workflow for many applications, resulting in cost and time savings, reduced potential for error, and increased productivity. Some of these simplifications include:

• Combining multiple techniques (LC and GC)
• Combining multiple methods (normal-phase LC and reversed-phase LC)
• Reducing sample preparation time

Combining Multiple Techniques – Lipid Analysis

Analyzing lipids is important for many reasons. In the pharmaceutical industry, lipid profiles are studied to determine the impact of drug efficacy in control and dosed subjects.

In clinical research, lipid levels are studied as biomarkers in different diseases, as well as for efficacy of treatment. In food applications, certain lipid classes such as triglycerides are profiled for nutritional purposes or to determine product authenticity. In chemical materials, fatty acids, and triglycerides are analyzed in petroleum products (e.g., biodiesel). Depending on the desired outcome, analyzing lipids requires different techniques. For free fatty acids that’s typically GC and requires derivatization of the free fatty acid to the fatty acid methyl ester (FAME) in order to improve peak shape and detection limits, especially for longer carbon chains. Derivatization can take several hours, and the resulting GC analysis can take up to thirty minutes. More polar lipids, such as phospholipids and sphingolipids, often require HILIC or normal-phase LC to separate the different lipids classes (based on the nature of the polar head group). Then, reversed-phase LC is used to target more hydrophobic lipids within a class based on the carbon chain length and/or number of double bonds. As we can see, a complete lipid profiling requires multiple techniques. That’s not the case with CC which separates all classes of lipids in a single injection. Figure 40 shows the comprehensive lipid profile of a mouse heart extract using the ACQUITY UPC² System. In this example, a BEH column and a generic gradient separate the different lipid classes, resulting in a separation similar to that given by normal-phase LC or HILIC.

For the neutral lipids (i.e., triacylclycerol (TG), diacylglycerol (DG), cholesterol esters (CE), and free fatty acids), simply changing the column and gradient conditions retains and separates the different lipids within each class based on the fatty acid chain length and number of double bonds (Figure 41).

Not only is CC faster than GC (up to 10 times faster) for this application, no derivatization is required, thus dramatically simplifying the overall workflow for lipid analysis. The absence of derivatization saves time and minimizes potential errors when adding these extra steps into the workflow. Without CC, this type of profiling and targeted analysis can require up to three different techniques, resulting in lower sample throughput, higher solvent usage, longer run times, and overall increased cost of analysis.

Combining Multiple LC Methods – Fat-Soluble Vitamins

Fat-soluble vitamin analysis is important to the pharmaceutical industry, clinical research and diagnostics, and food and fuels industries. Analysis of fat-soluble vitamins and carotenoids is typically done by either reversed-phase or normal-phase LC (Table 6). Due to the difficulty in separating these compounds in a single injection, they are analyzed individually using different columns and mobile phases, with analysis times between 10 and 30 minutes. Contrast that with CC which analyzes all vitamins and related compounds in less than 10 minutes (Figure 42). Unlike traditional methods of analysis listed in Table 6, the streamlined CC method calls for the use of a single column, one mobile phase condition, and one detector. With CC solvents are directly injected often using the same solvents used to extract or dissolve these compounds (e.g., isooctane and hexane), and does not require the solvent exchange that is typically required for reversed-phase analysis.

Reducing Sample Preparation Time

In addition to separating analytes faster by virtue of being able to combine multiple techniques and methods into one, CC can often reduce sample preparation time. The compatibility of CC with organic solvents leads to the following benefits:

• Elimination of hydrolysis and/or derivatization steps
• Elimination of evaporation and reconstitution for solvent exchange
• Reduction in the number of handling steps, thus reducing experimental error and improving data quality 

Consider, for example, the multiple sample preparation steps and analyses required for fat-soluble vitamins in food. A typical sample workflow for analysis of vitamins A, D, and E in food is shown in Figure 43. Notice that vitamins A and E require a different sample preparation procedure as compared to vitamin D. Also, three separate HPLC analyses are required (both normal and reversed-phase) for each vitamin. The sample preparation for vitamin D analysis is particularly complex and can contain many steps, including a semi-preparative HPLC step in some cases.

The sample preparation procedure for analyzing the same vitamins using CC is much simpler (Figure 44) due to the compatibility of the technique with the nonpolar organic solvents used early in the extraction process. In the example, by injecting the sample directly from the hexane extraction, we can quantify the vitamin E. It is then concentrated for the analysis of vitamins A and D3, making the run time twenty-fold faster than traditional methods of analysis. In addition, CC requires only three sample preparation steps, one method, and a single instrument, whereas the traditional workflow shown in Figure 43 requires 12 sample preparation steps and three methods on two different instruments. Table 7 summarizes the advantages of CC for the applications discussed, and highlights others where analytical scientists would benefit from similar simplification.

Fast Separation Of Structurally Similar Compounds

Isomers and structural analogs are sometimes difficult to separate due to structural similarity, especially for optical isomers. We now discuss the use of CC for the following structurally similar compounds:

• Chiral separations (enantiomers and diastereomers)
• Positional isomers (differing in location of functional groups)
• Structural analogs
  • Biomarkers (conjugated/unconjugated)
  • Drugs (metabolites, impurities, degradants)

Often, different enantiomers of a compound can have varying potency and toxicity profiles, and thus need to be monitored throughout the research, development, and production phases. Chiral separations are predominantly performed by normal-phase LC using cellulose or amylose-based stationary phases. In normal-phase LC, gradient separations are not easy to pull off. They require different, isocratic analyses on different columns, with different mobile phase combinations and often with highly toxic solvents. The method development process can be quite time consuming. With CC, the ability to run gradients and cover a wide selectivity space, with nontoxic solvents, gives analytical scientists the ability to develop chiral separations in a single day.

Purification chemists have recognized the value of SFC for this type of separation for many years. Analytical SFC separations, though difficult to get right, are highly desirable for fast chiral screening, chiral method development, enantiomeric excess determination and chiral inversion studies. Unlike normal-phase LC, CC is highly compatible with mass spectrometry detection, providing the ability to identify and characterize enantiomers and their formation during reactions, manufacturing processes, and in biological systems (Figure 45).

Figure 46 compares normal-phase and convergence chromatography for the separation of warfarin enantiomers. CC baseline resolves the enantiomers in a fraction of the time versus normal-phase (up to 30 times faster). Also, eliminating toxic solvents, which are costly both to purchase and dispose of, reduces the cost of chiral separations by as much as 100 fold per analysis. All of these advantages make CC the preferred technique for chiral analyses of all types.

Positional Isomers and Structural Analogs

CC is useful for separating positional isomers and other structural analogs. Positional isomers are compounds with the same molecular weight (isobaric), but differ in the location of their functional groups. They are often found in applications involving the analysis of starting materials, reaction monitoring, and asymmetric catalysis. Often, these compounds are derivatized prior to GC analysis to aid in separation of the isomers. Normal-phase LC methods are inherently less robust and slower. On the other hand, the selectivity of CC separations easily separates positional isomers without derivatization under a generic set of conditions.

The ACQUITY UPC² System (Figure 47) separates isomers so quickly, it can help assess in real time the optimization of reaction starting materials, intermediates, and final products. Structural analogs are difficult to separate due to their similarity to each other, and can include conjugated or unconjugated (e.g. glucuronides, sulfates) biomarkers, as well as metabolites, degradants, and impurities of drug compounds. Steroids are one of the most popular classes of structural analogs (Figure 48). The structural similarity between different steroids makes them difficult to separate and analyze, even when using MS detection, because of small mass differences. Resolving them is easy with CC using a generic screening gradient on multiple columns in fewer than two minutes (Figure 49). This separation is challenging for reversed-phase LC because of the non-polar nature of the compounds, and GC requires derivatization in order to improve peak shape and detection limits. Coupling the ACQUITY UPC² System with MS detection is a sound method for identifying and quantifying steroids.

Conjugated structural analogs are more difficult to separate. Free steroids (Figure 48) are water insoluble, so the body converts them to water-soluble derivatives by converting them to their sulfated form. This process results in a negatively charged hydrophilic side group, making them water-soluble (Figure 50). Isolated from natural sources for therapeutic use, these compounds are used as biomarkers to study disease and determine efficacy of treatment. Analysis of these compounds presents two major challenges.

First, it takes 2.5 hours to prepare a sample for a 30-minute GC analysis requiring enzymatic hydrolysis of the sulfate group followed by derivatization. Second, mass spectrometry cannot distinguish some of these estrogens since they are isobaric (same m/z). Thus chromatography is needed to separate different forms of the isobaric compounds.

With CC, all 10 sulfated estrogens can be separated in 15 minutes (Figure 51) including two closely eluting peaks (peaks 6 and 7) that cannot easily be separated by a 30-minutes GC analysis (Figure 52). With CC, the sample does not have to be hydrolyzed and derivatized because the sulfated compounds can be analyzed in their native form. This significantly reduces the amount of needed steps to analyze therapeutic formulations thus increasing throughput and productivity.

Table 8 summarizes the advantages of using CC for the applications discussed above, and highlights other application areas for separating enantiomers, positional isomers, and structural analogs.

Orthogonality

Orthogonal modes of separation are complementary to one another yet unique in the way they retain peaks differently and, by doing so, generate more information about a sample than a single separation mode alone. The ability to resolve analytes using different techniques is extremely important for the following reasons:

• Confidence that an impurity, degradation peak, or similar compound (e.g., isobaric compounds or co-eluting compounds) can be identified and characterized
• Ensuring full characterization of a sample
• Ability to obtain more information about a sample
• Separating desired compounds from matrix interferences

Examples of orthogonal separation techniques include complementary modes such as normal and reversed-phase chromatography. CC selectivity is similar to that of normal-phase chromatography, but it is much more robust, reliable (see Chapter 2), and reproducible than any normal-phase method. The following section shows examples of how CC is used as an orthogonal mode of separation to achieve the goals listed above.

The separation of an active pharmaceutical ingredient (metoclopramide) from its related substances using both ACQUITY UPLC and ACQUITY UPC² systems demonstrates this orthogonality (Figure 53). Peaks that are not resolved by one technique are resolved by the other, and vice versa. CC is able to retain polar compounds longer than RPLC (for example, peaks 1 and 2). In this example, the ACQUITY UPC² System resolves the critical pairs (peak 5 and metoclopramide), thus facilitating larger scale purification and isolation of unknown compounds for subsequent identification and characterization. All this makes CC an ideal technique to use in parallel with other, routine techniques to help solve a variety of separations challenges.

Orthogonal methods are also important for separating analytes of interest away from matrix interferences, for instance in bioanalysis or food analysis.

Figure 54A shows a typical example of an LC-MS/MS chromatogram of clopidogrel extracted from human plasma using protein precipitation. Due to the hydrophobicity of clopidogrel, it elutes rather late during the analysis. Interfering phospholipids (with a choline-containing head group) also elute in this same region (Figure 54B), potentially causing ion suppression of the clopidogrel peak and variable quantitation. Interestingly, the interfering phospholipids elute in about the same region on the ACQUITY UPC² System (Figure 54C). Due to the orthogonality of CC with respect to reversed-phase LC, the analyte of interest elutes much earlier, and away from these interfering phospholipids (Figure 54D). This minimizes the chance of matrix effects, ensuring more accurate and precise quantitation.

These benefits all illustrate the three key attributes of CC, as mentioned earlier in this chapter:

1. Convergence chromatography simplifies the workflow
2. Convergence chromatography separates compounds with structural similarity
3. Convergence chromatography is orthogonal to reversed-phase LC

• Combines multiple techniques into one
• Reduces sample preparation and analysis times
• Can be used for direct injection of organic solvents/extracts

• Enantiomers (chiral)
• Positional isomers
• Structural analogs and conjugates

• More confidence in identifying impurities/ degradants
• Full sample characterization
• Separation of analytes from matrix interferences