Reversed phase examples

Successful separations can be carried out only by planning and careful experimentation, the details of which are discussed extensively in Chapter 5. In one sense there are too many ways to achieve a separation in LC. But while this makes the first choice of where to start difficult, the good news is that there are many ways to achieve success. By looking at Figure 4-1 it is obvious that the use of the reverse-phase mode in LC has broad applicability and is, in fact, the most used mode of LC. Reverse phase is used for 80-85% of the separation problems encountered by users of HPLC. For this reason, the majority of Chapter 5, on developing methods, is devoted to reverse-phase examples. Additionally, Chapter 11 is a useful experiment to experience the method development aspect of this mode. Chapter 9 is a useful experiment to experience method development in the normal phase mode. 

Bohman and colleagues described a reverse-phase HPLC method for the quantitative analysis of vitamin A in food using the method of standard additions. In a typical example, a 10.067-g sample of cereal is placed in a 250-mL Erlenmeyer flask along with 1 g of sodium ascorbate,... 

Another example is the purification of a P-lactam antibiotic, where process-scale reversed-phase separations began to be used around 1983 when suitable, high pressure process-scale equipment became available. A reversed-phase microparticulate (55—105 p.m particle size) C g siUca column, with a mobile phase of aqueous methanol having 0.1 Af ammonium phosphate at pH 5.3, was able to fractionate out impurities not readily removed by hquid—hquid extraction (37). Optimization of the separation resulted in recovery of product at 93% purity and 95% yield. This type of separation differs markedly from protein purification in feed concentration ( i 50 200 g/L for cefonicid vs 1 to 10 g/L for protein), molecular weight of impurities (<5000 compared to 10,000—100,000 for proteins), and throughputs ( i l-2 mg/(g stationary phasemin) compared to 0.01—0.1 mg/(gmin) for proteins). 

Many forms of chromatography have been used to separate mixtures of quinoline and isoquinoline homologues. For example, alumina saturated with cobalt chloride, reversed-phase Hquid chromatography, and capillary gas chromatography (gc) with deactivated glass columns have all been employed (38,39). 

Chiral separations have become of significant importance because the optical isomer of an active component can be considered an impurity. Optical isomers can have potentially different therapeutic or toxicological activities. The pharmaceutical Hterature is trying to address the issues pertaining to these compounds (155). Frequendy separations can be accompHshed by glc, hplc, or ce. For example, separation of R(+) and 5 (—) pindolol was accompHshed on a reversed-phase ceUulose-based chiral column with duorescence emission (156). The limits of detection were 1.2 ng/mL of R(+) and 4.3 ng/mL of 3 (—) pindolol in semm, and 21 and 76 ng/mL in urine, respectively. 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

Gels made in this way have virtually no usable porosity and are called Jordi solid bead packings. They can be used in the production of low surface area reverse phase packings for fast protein analysis and in the manufacture of hydrodynamic volume columns as well as solid supports for solid-phase syntheses reactions. An example of a hydrodynamic volume column separation is shown in Fig. 13.2 and its calibration plot is shown in Fig. 13.3. The major advantage of this type of column is its ability to resolve very high molecular weight polymer samples successfully. 

In spite of Such limitations, some examples can be found in literature. For example, a reversed-phase Cjg column has also been coupled to a weak ion-exchange column to determine gluphosinate, glyphosate and aminomethylphosphonic acid (AMPA) in environmental water (28). This method will be described further below. 

One of the first examples of the application of reverse-phase liquid chromatography-gas chromatography for this type of analysis was applied to atrazine (98). This method used a loop-type interface. The mobile phase was the most important parameter because retention in the LC column must be sufficient (there must be a high percentage of water), although a low percentage of water is only possible when the loop-type interface is used to transfer the LC fraction. The authors solved this problem by using methanol/water (60 40) with 5% 1-propanol and a precolumn. The experimental conditions employed are shown in Table 13.2. 

When analytes lack the selectivity in the new polar organic mode or reversed-phase mode, typical normal phase (hexane with ethanol or isopropanol) can also be tested. Normally, 20 % ethanol will give a reasonable retention time for most analytes on vancomycin and teicoplanin, while 40 % ethanol is more appropriate for ristocetin A CSP. The hexane/alcohol composition is favored on many occasions (preparative scale, for example) and offers better selectivity for some less polar compounds. Those compounds with a carbonyl group in the a or (3 position to the chiral center have an excellent chance to be resolved in this mode. The simplified method development protocols are illustrated in Fig. 2-6. The optimization will be discussed in detail later in this chapter. 

Purification of the activation products (PMs). The methylamine activation product dissolved in methanol is purified by chromatography, first on a column of silica gel using a mixed solvent of chloroform/ethanol, followed by reversed-phase HPLC on a column of divinylbenzene resin (such as Jordi Reversed-Phase and Hamilton PRP-1) using various solvent systems suitable for the target substance (for example, acetonitrile/water containing 0.15% acetic acid). 

To retain solutes selectively by dispersive interactions, the stationary phase must contain no polar or ionic substances, but only hydrocarbon-type materials such as the reverse-bonded phases, now so popular in LC. Reiterating the previous argument, to ensure that dispersive selectivity dominates in the stationary phase, and dispersive interactions in the mobile phase are minimized, the mobile phase must now be strongly polar. Hence the use of methanol-water and acetonitrile-water mixtures as mobile phases in reverse-phase chromatography systems. An example of the separation of some antimicrobial agents on Partisil ODS 3, particle diameter 5p is shown in figure 5. 

Many racemic mixtures can be separated by ordinary reverse phase columns by adding a suitable chiral reagent to the mobile phase. If the material is adsorbed strongly on the stationary phase then selectivity will reside in the stationary phase, if the reagent is predominantly in the mobile phase then the chiral selectivity will remain in the mobile phase. Examples of some suitable additives are camphor sulphonic acid (10) and quinine (11). Chiral selectivity can also be achieved by bonding chirally selective compounds to silica in much the same way as a reverse phase. A example of this type of chiral stationary phase is afforded by the cyclodextrins. 

The more dispersive solvent from an aqueous solvent mixture is adsorbed onto the surface of a reverse phase according to Langmuir equation and an example of the adsorption isotherms of the lower series of aliphatic alcohols onto the surface of a reverse phase (9) is shown in figure 9. It is seen that the alcohol with the longest chain, and thus the most dispersive in character, is avidly adsorbed onto the highly dispersive stationary phase, much like the polar ethyl acetate is adsorbed onto the highly polar surface of silica gel. It is also seen that... 

An example of the efficacy of the resin phases used as an alternative to a conventional silica based reverse phase is shown in figure 12 where the separation of the three tocopherols are shown separated on the Cl 8 Polymer Column and The ODA-A 120A silica gel based columns. The columns were 15 cm long, 4.6 mm i.d., operated at a flow rate of 0.5 ml/min at 30°C with a mobile phase of 98% methanol/2% water. 

The basic polymer appears to be a hydroxylated polyether to which octadecyl chains have been bonded and so it behaves as a reverse phase exhibiting dispersive interactions with the solutes. An example of the separation of a series of peptides is shown in figure 15. The column was 3.5 cm long, 4.6 mm i.d. The solutes shown were (1) oc-endorphin, (2) bombesin, (3) y-endorphin, (4) angiotensin, (5) somatostatin and (6) calcitonon. The separation was carried out with a 10 min linear program from water containing 0.2% trifluoroacetic acid to 80% acetonitrile. 

Margarine is an example of a solid sample where the materials of interest are soluble in one solvent (in this case methanol) whereas the matrix materials, largely triglycerides, are not. As a consequence, the sample preparation procedure is relatively simple. The chromatographic separation is achieved by using the dispersive interactions between the hydrocarbon chains of the fatty acids and the hydrocarbon chains of a reversed phase. 

This sample preparation involved, firstly, an extraction and the elimination of the solid matrix by filtration and, secondly, a concentration procedure employing a solid phase extraction cartridge. The compounds of interest were separated solely by dispersive interactions with the reversed phase. In the example given, the corn meal was spiked with the aflatoxins. 

Liquid samples might appear to be easier to prepare for LC analysis than solids, particularly if the compounds of interest are present in high concentration. In some cases this may be true and the first example given below requires virtually no sample preparation whatever. The second example, however, requires more involved treatment and when analyzing protein mixtures, the procedure can become very complex indeed involving extraction, centrifugation and fractional precipitation on reversed phases. In general, however, liquid samples become more difficult to prepare when the substances are present at very low concentrations. 

There are two basic procedures that have been successfully used for the separation of isomers. The first is to add a chiral agent to the mobile phase such that it is adsorbed, for example, on the surface of a reverse phase, producing a chirally active surface. This approach has been discussed on page (38) in chapter 2. The alternative is to employ a stationary phase that has been produced with chiral groups bonded to the surface. 

An example of the separation of a mixture of explosives on a C8 column is shown in figure 7. The column was 3.3 cm long, 4.6 mm in diameter and packed with 3 pm C8 silica based reverse phase. This short column has a potential efficiency of 5,500 theoretical plates. 

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