R.W. Frei

On-line trace enrichment in high-performance liquid chromatography using a pre-column

Abstract The potential of on-line trace enrichment on a pre-column in high-performance liquid chromatography is demonstrated using phthalate esters as model compounds. The esters are concentrated onto a very shortr, i.e.,ca. 2 mm long, plug of an apolar chemical bonded stationary phase (5-μm LiChrosorb RP-18). Analysis is performed on LiChrosorb RP-18 with a methanol step gradient as mobile phase; detection is done by UV absorption measurement at 233 nm. Phthalate ester recoveries are 95-100% even after enrichment from sample volumes of 500–1000 ml, with pumping speeds varying between 5 and 25 ml min-1. The single-column and the column-to-column reproductivity of the technique are usually 4–6% (relative standard deviation); band broadening due to the insertion of the pre-column is negligible. As an application, the determination of two esters in tap, distilled, mineral and river water, and in soft drinks, is reported. The trace enrichment of mixtures of polychlorinated biphenyls, and of chloroanilines, is also described.

Use of PTFE coils in post-column photochemical reactors for liquid chromatograph — application to pharmaceuticals

The advantages and the performance of PTFE reaction coils in photochemical reaction detectors for high-performance liquid chromatography (HPLC) are discussed. The excellent performance of these materials at irradiation wavelengths below 300 nm is based on a diffuse radiation transfer and an internal reflectance (light-tube) effect. Optimal coil designs can be obtained from signal vs. flow-rate curves for a particular application. Lamp performance has to be tested periodically and the reactor should be equipped with an efficient cooling system. Application of the principle to the fluorimetric detection of clobazam and phenothiazines after HPLC separation is discussed. The sensitive detection of these pharmaceuticals and their metabolites in serum with a minimum of sample handling demonstrates the potential of photochemical reaction detectors.

Immunoaffinity pre-column for selective on-line sample pre-treatment in high-performance liquid chromatography determination of 19-nortestosterone

A liquid chromatographic column-switching system for automated sample pretreatment and determination of the anabolic hormone beta-19-nortestosterone (beta-19-NT) and its metabolite alpha-19-nortestosterone (19-norepitestosterone) in calf urine is described. The system consists of an immunoaffinity pre-column (immuno pre-column) packed with Sepharose-immobilized polyclonal antibodies against beta-19-NT, a second pre-column packed with C18 bonded silica and an analytical C18 column. Urine (25 ml) is directly loaded on the immuno pre-column, where the analytes of interest are trapped by the immobilized antibodies. Next the analytes are desorbed selectively with a solution containing an excess of the cross-reacting steroid hormone norgestrel and transferred, via the second pre-column, to the analytical column. The recovery of beta-19-NT in spiked urine samples was over 95%. The detection limit was 50 ng/l for a 25-ml urine injection. The system showed no loss of analytical performance over a 6-month period, during which about 100 samples were analysed with the same immuno pre-column. The general applicability of this sample pretreatment method is discussed.

Flow injection determination of hydrogen peroxide by means of a solid-state-peroxyoxalate chemiluminescence reactor

Abstract A solid-state reactor for detection of hydrogen peroxide in aqueous samples by peroxyoxalate chemiluminescence is described. Bis(2,4,6-trichlorophenyl)oxalate in solid form is packed into a bed reactor, which eliminates mixing problems and facilitates the instrumental development. Perylene is added as a sensitizer to a water/acetonitrile (20:80) carrier stream into which the samples (200–600 μl) are injected. Detection limits of 6 × 10 −9 M H 2 O 2 (0.2 μg l −1 ) are obtained with both a commercial and a home-made luminescence detector. Calibration graphs are linear up to 10 −5 M. The r.s.d. for 2 × 10 −7 M (6.7 μg −1 ) hydrogen peroxide ( n = 10) is 2.8%. Sample throughput is ca. 120 h −1 .

Amperometric detection of amino acids in high-performance liquid chromatography with a copper electrode

A copper electrode has been used as an amperometric detector for amino acids in high-performance liquid chromatography. The amino acids are separated in a reversed-phase system, using silica-based and polymer-type column materials. Neutral or alkaline buffer solutions of phosphate and carbonate can be used as mobile phases. Borate buffers are less suitable. The detection method is characterized by a high linear dynamic range, good reproducibility, the absence of electrode poisoning and a sensitivity comparable to that of UV absorption methods after derivatization of the amino acids. Detection limits with conventional-scale columns are in the range 10-100 pmoles. A reduction in the flow-rate in the flow-through cell improves the sensitivity for amino acids that give relatively low signals, such as proline. Therefore, the use of microbore columns is especially advantageous for these compounds. The absolute detection limits decrease by about one order of magnitude on changing to a miniaturized system.

Use of conventinal instrumentation with microbore column in high-performance liquid chromatography

A method to determine extra-column band broadening in microbore high-performance liquid chromatography (HPL) (column diameter, ca. 1 mm) is developed. The extra-column band broadening of several commercially available UV detectors is compared to that of a home-made electrochemical detector. A trace-enrichment method for microbore HPLC is presented and applied to the determination of a series of aromatic nitro compounds. The effect of miniaturization of HPLC on detection limits is discussed.

Supercritical-fluid extraction of aqueous samples and on-line coupling to supercritical-fluid chromatography

The potential of segmented-flow systems for on-line liquid/supercritical-fluid extraction was explored. The use of a phase separator (PS), its design and performance were investigated, utilizing phenol and 4-chlorophenol as the test compounds, water as the liquid phase and supercritical carbon dioxide as the extractant. On-line coupling of such a liquid/fluid extraction (SFE) system to supercritical fluid chromatography (SFC) was demonstrated, as was the feasibility of extracting phenol from an urine sample. The extraction efficiency for the test compounds was over 85%. The repeatability was about 8% relative standard deviation (R.S.D.) (n = 8) for the total SFE-PS-SFC system and 4% R.S.D. for both the SFE-PS and the SFC operation. The potential of coupling SFE to other chromatographic and detection principles is discussed.

Fully automated sample handling system for liquid chromatography based on pre-column technology and automated cartridge exchange.

The design of an automated cartridge exchange module for on-line sample handling in liquid chromatography is described. When combined with a low-cost purge pump, a solvent selection valve and an auto-sampler, a fully automated sample handling system is obtained. Samples are sorbed on a disposable cartridge packed with 40 micron octyl-bonded silica, purged for clean-up and eluted on-line to the analytical column. Unattended operation of the system is demonstrated for various examples, i.e., the determination of anti-epileptic drugs in serum, an anti-cancer drug in plasma, barbiturates in urine, phenylurea herbicides in river water and caffeine in a soft drink.

Design and evaluation of a sandwich phase separator for on-line liquid/liquid extraction

Abstract Sandwich phase separators with various groove dimensions were constructed and as part of a post-column extraction detector for liquid chromatography and for a flow-injection system. The construction materials are stainless steel and PTFE; no membranes are used. The groove volumes vary between 8 and 43 μl; the dimensions of the groove are not critical. Several aqueous (acetonitrile/water and methanol/water mixtures) and organic (1,2-dichloroethane and n-heptane) phases were successfully separated by gravity as well as by wetting. Measurement of statistical second moments showed the total disperson of the extraction system to be 2.5–4 s at the optimum separation efficiency (organic flow through detector/total organic flow) of 0.3–0.4.

Pre-column technology in high-performance liquid chromatography for the determination of phenylurea herbicides in water in the presence of their anilines☆

Abstract Several metal-loaded phases were investigated for their use in the analysis of residues of phenylurea herbicides and their corresponding anilines. A special platinum phase packed in a short pre-column can act as an aniline filter for a group separation of the herbicides from the anilines. Coupling of such a Pt pre-column with a C 18 pre-column permits subsequent pre-concentration of the herbicides and on-line transfer to and separation on a reversed-phase analytical column. The feasibility of this method for the on-line automatable analysis of intact phenylurea herbicides in the presence of their corresponding anilines was demonstrated on polluted river water samples.