Normal phase column cleaning

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. 

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. 

Normal-phase sorbents such as silica and Florisil are used to isolate low to moderate polarity species from nonaqueous solutions. Examples of applications include lipid classification, plant pigment separations, and separations of fat-soluble vitamins from lipid extracts, as well as the clean-up of organic solvent concentrates obtained from a previous SPE method or liquid-liquid extraction. Alumina is used to remove polar species from nonaqueous solutions. Examples include vitamins in feeds and food and antibiotics and other additives from feed. Normal-phase chromatography has been used for a number of years, and most applications for normal-phase column chromatography may be easily transferred over to normal-phase SPE. 

Liquid chromatographic clean up [441,443,450] has been used either in normal phase flow using alumina, silica, or florisil [22,189,403,481,484] or with reverse-phase (RP) columns [409,452,480]. In most cases these techniques are well established and are used in an off-line mode, primarily to remove the bulk of co-extracted materials prior to a more refined clean-up prior to the final determination. These columns may be prepared in the laboratory [22,403 -405] or commercial solid phase extraction (SPE) cartridges can be used [409,452, 463,470,485,486]. In both cases, the normal phase cartridges and column materials are disposable since many of the polar co-extractants bind firmly to the substrate surface and are difficult to remove. This has been overcome to some... 

The major difficulty in analyzing OPPs in fatty samples has to do with the wide polarity range for both pesticides and lipids present in the matrix. Normal-phase HPLC is an adequate technique for cleaning up this type of sample using silica gel and modifiers with different polarity. In fact, an automated sample-cleanup system based on normal-phase HPLC using a silica gel column has been reported efficiently to clean up and fractionate chlorpyriphos, chlorpyriphos methyl, and their metabolites in molluscs. The system presents several advantages The procedure is fully automated, from the injection of the extract to the collection of fractions, which are injected directly into the GC system, and a diode array detector (DAD) allows online monitoring of the elution of lipids (68). 

Since the samples run in prep LC are often very crude, it is to be expected that the column packing will become contaminated with chemicals that did not elute. Cleaning can be accomplished with polar solvents like propanol or ethyl acetate (for normal phase systems), or the packing can simply be discarded. The latter alternative may be cheaper, considering the cost of solvents and waste disposal. 

The concept behind the invention is shown in Figure 9.36. It operates with two mechanisms—size exclusion and reverse phase bonded sorption. The purpose is to facilitate the injection of plasma samples without prior clean up to remove proteins that normally clog a reverse phase column. The pore size of the stationary phase is small enough that the large protein molecules cannot enter and the outer surfaces to which they are exposed do not retain them at all, so they are eluted off the column... 

An example is endrin in soil (Fig. 7.7). This highly chlorinated insecticide is extracted by Soxhlet extraction with hexane from a soil that has been mixed one to one with sodium sulfate to remove water. The hexane extract is further dried over sodium sulfate and the hexane is added to a hexane-loaded silica or CN SPE sorbent that has been prepared with hexane. The endrin is sorbed to the silica or CN by normal-phase mechanisms and may be eluted with a solvent that will overcome the interaction, such as methylene chloride or ethyl acetate. The more polar interferences are sorbed to the silica or CN sorbent and are not eluted with endrin. Thus, the SPE column performs both trace enrichment and SPE clean-up. See Chapter 5 for more examples. 

Robot-compatible syringes, standard syringe, maxi-clean cartridge, Novo-Clean membranes (ion exchange and reversed phase), many types of syringe filters, reservoirs, inlet and outlet caps, adapters, frits, and filter columns, vacuum manifolds, reversed-phase, normal-phase, and ion-exchange sorbents. 

If a cyanopropyl column needs to be exposed to solvents of intermediate polarity, for example during cleaning procures, the flow should not be interrupted. The pressure actually holds the particles in place, and a collapse of the bed is less likely to occur. The preferred storage solvent for normal-phase anopropyl columns is a hydrocarbon like hexane. 

In our laboratory we make our own columns for normal-phase chromatography (diol-modified silica). We have noticed that by packing the columns under a high pressure, that is, 950-1000 bar, instead of at the recommended 500-600 bar, we obtained far better separation of the phospholipid classes. Accordingly, this way of packing columns may be a way to achieve better separation of intact polar lipids, if choice of column packing, solvent mixture gradient and sample clean-up have already been optimized. 

Operate the column at half of the maximum recommended flow rate, taking special care to monitor the pressure, because the cleaning solution may be of different viscosity than the normal mobile phase. 

If the column is contaminated with basic compounds, clean it with a concentrated salt in the normal mobile phase, e.g., 0.5-1.0 M K2SO4. Avoid the use of halides, as they will corrode stainless steel over time. Buffered solutions at low pH (2-3) or high pH (11-12) can also be used. 

FIGURE 18.6 Normalization step in the LCIA phase of LCA for SPE11 (D, device S, solvent P, pump A, nitrogen C, columns T, transport W, washing and DC, drain cleaning for the remaining abbreviations, see Table 18.7). 

Another development in HPLC analysis is the interfacing of on-line sample clean-up technologies at the front end of the HPLC analytical column. Using a switching valve, complex samples can be injected directly onto a sample preparation column, the stationary phase of which is designed to bind the analyte(s) of interest to the exclusion of the rest of the matrix, which runs to waste. Then the valve is switched and the mobile phase flows through the sample preparation column where it picks up the analyte(s) and carries them onto the analytical column and through to the detector in the normal way. This automation of sample preparation saves time and effort and reduces errors. There are many applications of HPLC assays in conjunction with on-line sample clean-up methods in the literature ... 

When on-column injection is used the end of the transfer capillary is inserted into the column inlet or retention gap where decompression of the supercritical fluid occurs. Carbon dioxide gas exits through the column and the seal made between the restrictor and septum (unless a closed injector is used). The analytes are focused by cold trapping in the stationary phase. The transfer line must be physically removed from the injector at the completion of the extraction to establish the normal carrier gas flow for the separation. Analyte transfer to the column is virtually quantitative but blockage of the restrictor is more conunon and involatile material accumulates in the injection zone eventually degrading chromatographic performance. The on-column interface is probably a better choice for trace analysis of relatively clean extracts with modest fluid flow rates than the split interface. When optimized both the on-column and split interfaces provide essentially identical peak shapes to those obtained using conventional solution injection. 

The SPE cartridge must always be conditioned with the particular solvent that will be used. This is necessary both to activate the sorbent and to clean the column. Conditioning procedures vary depending on whether normal or reversed-phase sorbents are employed. Ion-exchange conditioning is a function of the polarity of the sample solvent. 

Precise characterization of surfactants in used formulations, such as the recycled solutions used to remove oil after metal-forming operations, normally requires some preliminary separation. For example, HPLC analysis of alkoxylated surfactants is possible if the sample is cleaned up by solid phase extraction on silica. Water is first removed by azeotropic distillation, then organic material is taken up in methylene chloride and passed through the SPE column. After elution of oil and grease with methylene chloride, the nonionic surfactants are removed with methanol (17). 

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