Normal Phase Columns

Normal phase HPLC consists of methods that utilize a nonpolar mobile phase in combination with a polar stationary phase. Adsorption HPLC actually fits this description, too, since the adsorbing solid stationary phase particles are very polar. (See discussion of adsorption columns in Section 13.5.3.) Normal 

Ion chromatography is used at the City of Lincoln, Nebraska, Water Treatment Plant Laboratory to analyze water samples taken from sampling sites in the distribution system around the city. The common anions determined by IC are not only nitrate, nitrite, fluoride, and sulfate, but also bromate. Bromate is found in the water because the Lincoln plant treats the water with ozone. Adding ozone to the water oxidizes any bromide to bromate. Bromate is regulated at 10 parts per billion (ppb) its concentration must be determined. 

Because the expected concentration level is so low, the standard procedure for bromate using IC calls for a 250-/./L sample loop on the injector, an unusually large volume for a sample loop. The procedure for the common anions listed above utilizes a 50-/./L loop. It is a therefore a common task in this laboratory to change the sample loop regularly as these different anions are determined. 

Eric Lee of the City of Lincoln, Nebraska, Water Treatment Plant prepares to change the sample loop on the IC injector. Notice the 250 /tL sample loop (on the right) ready to be installed. 

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For samples that meet the solubility requirements of the SEC approach, analyses were also reported for additives in polymers such as PVC and PS [28,29]. Direct SEC analysis of PVC additives such as plasticisers and thermal stabilisers in dissolution mode has been described [28,30,31 ]. In the analysis of a dissolved PS sample using a SEC column of narrow pore size, the group of additives was separated on a normal-phase column after elution of the polymer peak [21]. Column-loading capacity of HPSEC for the analysis of additives, their degradation products and any other low-MW compounds present in plastics has been evaluated for PS/HMBT, PVC/TNPP and PVC/TETO (glyceryl tri[l-14C] epoxyoleate) [31]. It was shown that HPSEC can be used to separate low-MW compounds from relatively large amounts of polymers without serious loss of resolution of the additives the technique has also been used for the group analysis of chlorohydrin transformation products of the TETO model compound [32]. 

A chiral GC column is able to separate enantiomers of epoxy pheromones in the Type II class, but the applications are very limited as follows a custom-made column packed with a p-cyclodextrin derivative as a liquid phase for the stereochemical identification of natural 3,4- and 6,7-epoxydienes [73, 74] and a commercialized column of an a-cyclodextrin type (Chiraldex A-PH) for the 3,4-epoxydiene [71] (See Table 3). The resolution abilities of chiral HPLC columns have been examined in detail, as shown in Table 7 and Fig. 14 [75,76, 179]. The Chiralpak AD column operated under a normal-phase condition separates well two enantiomers of 9,10-epoxydienes, 6,7-epoxymonoenes and 9,10-epoxymonoenes. Another normal-phase column, the Chiralpak AS column, is suitable for the resolution of the 3,4-epoxydienes. The Chiralcel OJ-R column operated under a reversed-phase condition sufficiently accomplishes enantiomeric separation of the 6,7-epoxydienes and 6,7-epoxymonoenes. 

Fig. 14A-C Chromatography of the racemic monoepoxy derivatives (I—III) of Z3,Z6,Z9-18 on chiral HPLC columns A Chiralpak AD B Chiralpak AS C Chiralcel OJ-R. The solvent system for the former two normal-phase columns is 0.1% 2-propanol in n-hexane (0.45 ml/min), and that of the third column is 15% water in MeOH (0.45 ml/min). Homo-conjugated dienes, epo3,Z6,Z9-18 H (I) and Z3,Z6,epo9-18 H (III), were detected by UV (215 nm), and Z3,epo6,Z9-18 H (II) was detected by RID. The earlier eluting isomers have a 3S,4R, 6S,7R, or 9R,10S configuration...

A novel development for HPLC is something called bonded reversed-phase columns, where the stationary phase is a nonpolar hydrocarbon, chemically bonded to a solid support. You can use these with aqueous eluents, usually alcohol-water mixtures. So you have a polar eluent and a nonpolar stationary phase, something that does not usually occur for ordinary wet-column chromatography. One advantage is that you don t need to use anhydrous eluents (very small amounts of water can change the character of normal phase columns) with reversed-phase columns. 

Normal phase columns have also been shown to give non-linear van t Hoff plots in many cases. Bidlingmeyer and Henderson [35] found an improved separation of lipophilic amines at high temperature. They were unable to determine whether the lack of linearity was due to absorptive and electrostatic effects or changes in solvation of the silica as a function of temperature. They also noted a degradation of the silica support at elevated temperatures and found it necessary to use a pre-column to pre-saturate the mobile phase with silica. 

Capillary gas chromatography (GC) using modified cyclodextrins as chiral stationary phases is the preferred method for the separation of volatile enantiomers. Fused-silica capillary columns coated with several alkyl or aryl a-cyclo-dextrin, -cyclodextrin and y-cyclodextrin derivatives are suitable to separate most of the volatile chiral compounds. Multidimensional GC (MDGC)-mass spectrometry (MS) allows the separation of essential oil components on an achiral normal phase column and through heart-cutting techniques, the separated components are led to a chiral column for enantiomeric separation. The mass detector ensures the correct identification of the separated components [73]. Preparative chiral GC is suitable for the isolation of enantiomers [5, 73]. 

Cleanup by solid-phase extraction has also been widely employed since it is a simple, fairly inexpensive, and easy-to-perform procedure for purification of the crude extract. The use of disposable solid-phase extraction columns is currently part of most, if not all, modern analytical methods for the determination of anthelminthics in biological matrices at residue levels. Both normal-phase columns based on silica (333-335, 340, 367, 372), alumina (346, 373-375), or aminopropyl (339, 365, 370) materials, and reversed-phase columns based on Ci8 (319, 323, 324, 328, 344, 346, 347, 349-351, 357-359, 364, 367) and cyclohexyl (329, 332, 360) sorbents have been described in analytical applications. 

F. Rabel and K. Palmer, Am. Lab., August 1992, p. 65.] Between uses, reversed-phase columns can be stored in methanol or in water-organic solvent mixtures that do not contain salts. Normal-phase columns should be stored in 2-propanol or hexane. See also R. E. Majors, The Cleaning and Regeneration of Reversed-Phase HPLC Columns, LCGC 2003,21, 19. 

There are numerous papers that refer to the quantification of tocopherols and tocotrienols using HPLC methods. Normal-phase HPLC methods with a silica column as well as reversed-phase HPLC methods with a C18 column are commonly used. A silica normal-phase column is able to separate all eight tocopherols and tocotrienols in a typical chromatographic procedure. Because plant tissues possess most forms of tocopherol and tocotrienol, it is recommended that the normal-phase HPLC method be applied to food samples from plants. In the reversed-phase HPLC method, [3- and y-tocopherol and (3-and y-tocotrienol are not usually completely separated. This method can be used in animal tissues, which either lack or have reduced levels of [3- and y-tocopherol and (3- and y-tocotrienol. The resolution of the normal-phase HPLC method is higher than that of reversed-phase HPLC method however, the reversed-phase HPLC column is more long-lasting than the normal-phase HPLC column (see Critical Parameters and Troubleshooting). 

Column. Although the advantage of the normal-phase column is the high resolution in the separation of all tocopherols and tocotrienols, the silica packing material in a normal-phase column is very reactive to strong polar chemicals. Any high-polarity compounds in the sample extract and mobile phase will diminish the column performance and shorten the column lifetime. It is very important to ensure that the sample extract is free of water and metal ions, and it is also advisable to regenerate the column routinely. 

Mobile phase. Hexane is the major component of mobile phase in the normal-phase HPLC method. The percentage of hexane is up to 99% for silica normal-phase columns. Ethyl acetate, acetic acid, methanol, and isopropanol are used as modifier components (Shin and Godber, 1994). The flow rate of mobile phase is usually controlled at 1 to 1.5 ml/min to completely separate all eight tocopherols and... 

Most HPLC is based on the use of so-called normal-phase columns (useful for class separations), reverse-phase columns (useful for homolog separations), and polar columns (used in either the normal- or reverse-phase mode). Since reverse-phase HPLC columns are generally easier to work with, almost all authors use high-performance reverse-phase liquid chromatography with octade-cyl chemically bonded silica as the stationary phase and nonaqueous solvents as mobile phases (so-called NARP, or nonaqueous reverse-phase chromatography). 

T-S Shin, JS Godber. Improved high-performance liquid chromatography of vitamin E vitamers on normal-phase columns. J Amer Oil Chem Soc 70 1289-1291, 1993. 

Naidong, W., Shou, W. Z., Addison, T., Maleki, S., and Jiang, X. (2002b). Liquid chromatography /tandem mass spectrometric bioanalysis using normal-phase columns with aqueous/ organic mobile phases A novel approach of eliminating evaporation and reconstitution steps in 96-well SPE. Rapid Common. Mass Spectrom. 16 1965-1975. 

Combinations of both reverse-phase and normal-phase columns were used under various sol vent conditions to achieve isolation of the major oligosaccharides. 

Retention changes work exactly the same with reverse-phase column as with normal-phase columns. Increasing the polarity difference between column and mobile phase increases the /c s of the components. However, since the column is nonpolar, we now must add more of the polar solvent to make compounds stick tighter. On our reversed-phase column, our dye mixture would also elute in opposite order, the more polar red dye would have less affinity for the nonpolar column and would elute before the nonpolar blue dye. By controlling the column nature, you control the elution order. Figure 4.6 illustrates the effect of solvent polarity changes on a separation. 

Normal-phase silica packing requires only drying at a uniform temperature to be ready for packing. At 250°C, the fully hydrated silica is produced, while at 300°C water is lost between adjacent silica molecules forming the anhydride form normally packed in normal-phase columns. 

The best way to follow column changes is by way of column standard plate counts. For discussion purposes, we will use the four-standard mixture of acetophenone, nitrobenzene, benzene, and toluene described in the discussion on efficiency factor (Chapter 4). Our column will be a Ci8 reverse-phase column run in 70% acetonitrile/water at 254 nm. In an initial run, we obtain four peaks whose interpeak a s double between each pair. After we discuss reverse phase, we will see how these killers affect normal phase columns. 

Most of the mentioned troubleshooting tools will work with other silica-based columns. With normal-phase columns, you obviously need not worry about bonded-phase removal, but silica still dissolved at high pH and high salt concentrations. Polar materials like some proteins adhere very tightly and require... 

How do you make the decision when to choose a reverse-phase instead of a normal-phase column or an intermediate-phase column such as a cyano column Reversed-phase columns are chosen about 70% of the time, so most compounds can be separated by this partition mode. What in the make-up of the compound being separated selects one column over the other ... 

We have already mentioned solvent solubility. If the compounds are not soluble in nonpolar solvents, there is little chance we will be able to separate them on a normal-phase column. The operating solvent ranges are fairly wide on both columns, as we have seen, and a solvent can usually be found that dissolves our compounds and allows them to be run on the column. 

In the second case, we have the same nonpolar side-chain, but differing polar functions, say p-methylphenol and p-toluidine. We want the phenolic and anilinic functions toward the column, and, therefore, you would select a normal phase column. The nonpolar solvent attracts the aromatic methyl substituents, correctly orienting the molecules for separation. 

One other empirical rule. For some reason, positional isomers seem to be best resolved on anhydrous silica columns. I can t offer you a good reason why this is true. Separation of cis-/trans- and axial/equatorial isomers seems to proceed best on these normal-phase columns. 

Normal-phase columns, either Water s jx Porasil or DuPont s Zorbax-sil , were used for the data of Sheridan s... 

U. R. Cieri, Determination of reserpine, hydralazine HC1, and hydrochlorothiazide in tablets by liquid chromatography on a short, normal phase column, JAOAClnt., 77 1104 (1994). 

Stewart et al. have also reported the efforts at Molecular Nature Ltd. to generate a pure natural product library [41], Compounds for this library were isolated utilizing parallel, normal phase column chromatography followed by C-18 and/or ion exchange chromatography. To be accepted into the library, the compounds must be > 90% pure with structural verification via a combination of HPLC, NMR, MS and GC/MS. 

The monohydroxylated metabolites had large retention volumes (> 15 ml) on the normal phase column when 20% chloroform in heptane was the solvent and could be completely separated from tetrahydrocannabinol on this system. They were resolved from each other with a more polar solvent, 80% chloroform in heptane. 

A MicroPak MCH-10 reverse phase column was chosen for separation of the hexane and ether extracts. The monomolecular bonded phase provides efficient separation of both polar and non-polar substances and rapid equilibration to initial activity after gradient elution programs. The reverse phase column provides symmetrical, narrow peaks for the cannabinoic acids, which tend to tail on polar, normal phase columns (e.g. silica). 

Endogenous substances in the extracts are more polar than the cannabinoids and elute before them on the reverse phase column. On polar, normal phase columns, strong adsorption of endogenous species requires periodic column clean-up. This problem was not encountered with the reverse phase gradient system. 

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