Column chromatography sample pretreatment

Development of a micro-pre-column method for pretreatment of samples. Journal of Chromatography 152(2), 341-348, 1978. 

A. Farjam, A. E. Brugman, A. Soldaat, P. Timmerman, H. Lingerman, G. J. de Jong, R. W. Frei and U. A. Th Brinkman, Immunoaffinity precolumn for selective sample pretreatment in column liquid chromatography immunoselective desorption , Chromatographia 31 469-477 (1991). 

A. Farjam, R. de Vries, H. Lingeman and U. A. Th Brinkman, Immuno precolumns for selective on-line sample pretreatment of aflatoxins in milk prior to column liquid chromatography , Int. J. Environ. Anal. Chem. 44 175-184 (1991). 

There are two basic approaches off-line and on-line. The off-line method, as discussed in the chapters on sample pretreatment, are most often used because they involve either manually or automatically collecting a fraction from a sample cleanup sorbent. The appropriate fraction is transferred and then assayed by a second chromatographic method. The manual steps are time-consuming and potentially introduce significant error to the j precision and accuracy of the method. The on-line method, when fully automated, would have the chromatography system perform sample pre- j treatment by column switching between two or more columns. j... 

Mobile phases useful for suppressed conductivity detection of anions include sodium hydroxide, potassium hydroxide, and the sodium and potassium salts of weak acids such as boric acid. In nonsuppressed conductivity detection, the ionic components of the mobile phase are chosen so that their conductivities are as different from the conductivity of the analyte as possible. Large ions with poor mobility are often chosen, and borate-gluconate is popular. For cations, dilute solutions of a strong acid are often used for nonsuppressed conductivity detection. For more information on the application of electrochemical detection to inorganic analysis, see Ion Chromatography Principles and Applications by Haddad and Jackson,17 which provides a comprehensive listing of the sample types, analytes, sample pretreatments, columns, and mobile phases that have been used with electrochemical detection. 

Chromatography is a very versatile technique offering a wide range of solid phase materials and detector types which can deal with very complex mixtures. In practice all materials and conditions used in the instrument are carefully chosen to match the type of sample mixture involved. This includes selection of stationary phase (chemical and physical properties) column type and length sample pretreatment, operational temperatures, pressures, and flow rates physical and chemical nature of mobile phase detector type and so forth. Detection to nanogram level is quite common and some systems can detect to picogram level using very small volumes of sample. 

In contrast to GC, liquid chromatography hyphenated with mass spectrometry (LC-MS) does not require a derivatization step before sample analysis. Separation of metabolite from sample matrix is achieved using chromatography columns with various stationary phases of different physicochemical characteristics. LC-MS is more often used than GC-MS because it is more suitable for unstable compounds, compounds difficult to derivatize, and nonvolatile compounds [6, 7]. Therefore, a wider range of metabolites with various physicochemical properties can be determined using LC-MS. Moreover, the sample pretreatment procedure is much simpler, which can have a great impact on minimization of analytical variability. 

This chapter is not meant to be a short course in LC. Some aspects of LC, important in relation to combined liquid chromatography-mass spectrometry (LC-MS), are discussed, e.g., column types and miniaturization, phase systems and separation mechanisms, and detection characteristics. In addition, important sample pretreatment techniques are discussed. Special attention is paid to new developments in LC and sample pretreatment. 

A Farjam, GJ De Jong, RW Frei, UA Brinkman, W Haasnoot, ARM Hamers, R Schilt, FA Huf. Immunoaffinity pre-column for selective on-line sample pretreatment in high-performance liquid chromatography determination of 9-nortes-tosterone. J Chromatography 452 419-433, 1988. 

J. De Jong, A.J.F. Point, U.R. Tjaden, S. Beeksma and J.C. Kraak, Determination of catecholamines in urine (and plasma) by hquid chromatography after on-line sample pretreatment on small alumina or dihydroxyborylsilica columns, J. Chromatogr., 414, 285-300 (1987). 

Because of the detrimental effect of the presence of water on the column substrate, and the risk of irreversible interactions of some matrix constituents with the column material, direct application of an untreated biological sample is not advisable. Consequently, a sample pretreatment step is always required. This step may involve the removal of most of the matrix constituents by ion-exchange chromatography, ultrafiltration and/or dialysis followed by solvent extraction and derivatization of the species under study. The complexity of the method chosen would depend on the stability of the species and the extent to which other matrix constituents interfere with its isolation. 

The results obtained for the vast majority of the laboratories carrying out the ISO 15061 IC method were considered to be fit for the purpose. In addition to this method, five alternative methods suitable for trace bromate determinations were also considered, namely on-line IC-ICP-MS, simple on-line column chromatography ICP-MS, IC with chlorpromazine post-column reaction and colorimetric detection, and fluorescence quenching with Carbostyril (with pre-treatment), which are all capable of achieving a bromate detection limit below 1 xg/L. A field method with methylene blue and fluorescence quenching with Carbostyril without sample pretreatment did not lead to satisfactory results at this level of bromate concentrations. 

The second method uses preconcentration to increase the analyte concentration in the absorption solution to detectable levels prior to analysis. One example is the use of solid-phase extraction with an anion exchange column as a pretreatment technique to remove interference from the sample matrix and to enhance the detection sensitivity prior to determination of the concentrated iodide anion through reversed phase high-performance liquid chromatography (HPLC) using an ultraviolet (UV) detector. The results show more than 90% recovery and good reproducibility for the determination of trace iodine in iodine-enriched eggs. 

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