Microcolumn cleanup

J.B.F. Lloyd and D.A. Parry, Detection and determination of common benzodiazepines and their metabolites in blood samples of forensic science interest. Microcolumn cleanup and high-performance liquid chromatography... 

Law and Goerlitz in 1970 reported the effective removal of co-extractives from water using microcolumns of these three adsorbents for the analysis of chlorinated pesticides. The development of polystyrene resins such as XAD increased the ability to concentrate pesticide residues from water. Large volumes of sample water could be passed through an XAD resin and the pesticide would adsorb on the resin. Elution of the pesticide by an organic solvent such as methanol and subsequent cleanup by the adsorbent materials became the industry standard. 

For the analysis of nonvolatile compounds, on-line coupled microcolumn SEC-PyGC has been described [979]. Alternatively, on-line p,SEC coupled to a conventional-size LC system can be used for separation and quantitative determination of compounds, in which volatility may not allow analysis via capillary GC [976]. An automated SEC-gradient HPLC flow system for polymer analysis has been developed [980]. The high sample loading capacity available in SEC makes it an attractive technique for intermediate sample cleanup [981] prior to a more sensitive RPLC technique. Hence, this intermediate step is especially interesting for experimental purposes whenever polymer matrix interference cannot be separated from the peak of interest. Coupling of SEC to RPLC is expected to benefit from the miniaturised approach in the first dimension (no broadening). Development of the first separation step in SEC-HPLC is usually quite short, unless problems are encountered with sample/column compatibility. 

SC Law, DF Goerlitz. Microcolumn chromatographic cleanup for the analysis of pesticides in water. J Assoc Off Anal Chem 33 1276-1287, 1970... 

This approach utilizes microcolumns of adsorbents to cleanup samples of lOg or less. Various length glass columns of 0.5 to 1.0 cm ID can be used. They should have luer tips and can be obtained with caps to allow low pressure to be applied or solvent to be pumped through. Figure 1. 

Koistinen et al. [58] have applied a Florisil cleanup which uses a partially deactivated (1.25%) Florisil microcolumn (1 g) to separate PCDEs from PCDDs and PCDFs in abiota and biota extracts [33,57,113,114,123-125]. After sulfuric acid cleanup, PCDEs are eluted through a Florisil column together with PCBs using hexane (15 ml). PCDDs and PCDFs are retained in the column and can be eluted with dichloromethane (12 ml). 

It is difficult to conclude what the best analytical method is for PCDEs but, based on the literature, microcolumns such as Florisil [57] seem to be effective in separation of PCDEs from PCDDs and PCDFs. Furthermore they are fast and inexpensive. Before PCDEs can be analyzed by MS, however, an additional cleanup step is needed. This has been performed using column chromatography on carbon, since silica gel and neutral alumina microcolumns have not worked well with fish extracts for this purpose [57]. Activated silica and alumina microcolumns, however, could possibly be alternatives for a carbon column. An activated silica column has been used an additional cleanup step after Florisil and carbon column chromatography in the case of mussel extracts [123]. It is not necessary to separate PCBs from PCDEs, since PCBs have been reported not to interfere in the MS analysis of PCDEs [57,130],... 

Sample preparation. Sulfabromomethazine added to tissue as an internal standard, extraction with water, centrifugation, and cleanup and concentration by a series of solid phase extractions using C-18 bonded silica, acidic alumina, and AG MP-1 anion exchange microcolumns. 

A microcolumn packed with 3 g of activated silica gel is used for sample cleanup and fractionation. An aliquot of the extract (containing about 20 mg oil or TSEM) is then transferred to the silica gel cleanup column to remove polar components and other interferences. The column is eluted first with hexane, which recovers samrated hydrocarbons as Fraction 1 (FI). The mixture of hexane/DCM or hexane/benzene (1 1, v v) is used to elute the aromatic compounds as Fraction 2 (F2). Half of FI and half of F2 are combined into the Fraction 3 (F3). These three fractions are concentrated to appropriate volume (0.5 to 1.0 ml) by nitrogen purge. The quantitation internal standards are 5-a-androstane, di4-terphyl, and C30 17(3(H), 21(3(H)-hopane. The Fraction 3 is analyzed for quantitation of the TPH and the UCM by GC-FID. The Fraction 1 is analyzed for determination of n-alkanes by GC-FID and biomarker terpanes and steranes by GC-MS. The Fraction 2 is analyzed for the determination of alkylated PAH homologues and other EPA priority unsubstantiated PAHs by GC-MS. 

To cite a few examples, HPLC was coupled with SIA for the simultaneous determination of several heavy metals by means of nitro-PAPS (polyfiuoroalkyl phosphate esters) complexes [43]. An SIA—HPLC—atomic fluorescence spectrometry (AFS) system was proposed for As speciation in seafood extracts, implementing standard addition method for simultaneous quantification of four As species [44]. An SIA-HPLC with electrochemical detection was proposed using a homemade microcolumn SPE coupled to SIA in order to automate the sample cleanup, extraction and detection of sulfonamides [45]. 

---