Detection with HPLC, detector linearity

In contrast with UV methods where the linear range is approximately 1-2 orders of magnitude at best, the HPLC method with UV detection typically has a linear range of 3-4 orders of magnitude due to the narrow path length of the detector flow cell. For example, a simple... 

A multiresidue preparation technique—MSPD—has also been applied to the analysis of CAP residues in meat samples. Two fractions were collected by elution with methylene chloride and ethyl acetate. No additional purification was necessary. Diode assay detection and fluorescence detectors were recommended for the multiresidue analysis of sulfonamides, benzimidazoles, nicarbazin, furazolidone, and CAP. The percentage recoveries and linearity of the method were evaluated. The method was linear from 50 to 250 /tg/kg of CAP. Not only do the authors recommend the MSPD multiresidue procedure for HPLC analysis, but it could be associated with several detection modes, such as immuno- or receptor assays. The MSPD technique represents a new approach in the field of biological-matrix extraction and provides a great possibility for the analysis of a wide range of compounds (20). 

A variety of detectors have been used for the HPLC determination of NOC in foods. These include UV, fluorescence, electrochemical, TEA, and various postcolumn denitrosation detectors. To be applicable for the low-ppb detection of these compounds in foods, an HPLC detector should meet the following criteria high sensitivity and specificity, responsive to all classes of NOC, linearity over a fair range of concentration, compatibility with both normal- and reversed-phase mobile phases, and minimal interference from changes in solvent composition, thereby making it amenable to solvent programming. As will be seen from the following discussion, none of the detectors currently available meet all these criteria. 

Cassidy and Frei [23] designed a microflow cell for the Turner Assoc. Model III fluorimeter for use with HPLC. Nanogram quantities of fluorescent materials could be detected. The volume of the flow cell was only 7.5 jul. The detector was unaffected by the flow-rate or composition of the solvent. This gives this detector a decided advantage over refractive-index or UV detectors. The peak shapes were symmetrical and the linear range of response was 2-3 orders of magnitude. 

Cetin et al. [29] used a HPLC method for the determination of lomox-icam. The measurements were made by using Perkin Elmer 200 Series with UV detector. Cig column was used as an analytical column and analysis were detected at 364 mn. The mobile phase was methanol acetonitrile and aqueous solution of diammonium hydrogen phosphate. Retention time of lornoxicam was 1.87 min. The drug was separated on Ci8 column (ODS Hypersil 5 pm 25 cm x 4.6 mm) with a flow rate of 1.6 ml/min. The linear calibration range was found to be 1-30 pg/ml. 

High performance liquid chromatography (HPLC) has been widely used for many years in industrial laboratories but its use in environmental laboratories has usually been restricted to analyses such as the determination of polyaromatic hydrocarbons and linear alkylbenzene sulphonates. Traditionally gas chromatography (GC) has been the first choice technique and HPLC only used when GC has proved unsuitable, due to thermal lability or other reasons. This reliance on GC is despite the fact it has been reported that 80-90% of the total organic carbon content in waters is non-volatile and not amenable to GC. Probably the reason for the lack of use of HPLC lies in the poor sensitivity of its most common detector (UV spectrophotometric) compared with GC detectors and the often demanding limits of detection required for environmental analysis, where sub-pg 1 limits of detection are the norm. 

Once the chromatographic separation on the column has been conducted, the composition of the eluent at the column end must be determined using a detector. In all HPLC detectors, the eluent flows through a measuring cell where the change of a physical or chemical property with elution time is detected. The most important parameter of the detector is sensitivity, which is influenced by the noise and baseline drift, the absolute detection limit of the detector, the linearity, the detector volume (band broadening), and the effects of pressure, temperature and flow (pulsation, gas bubbles). 

The availability of both GC and HPLC detectors in SFC means that derivatization to enhance selectivity and sensitivity should be an important analytical tool. Thus, David and Novotny showed [25] how nitrogen thermionic detection of quinoxalinol derivatives of a-keto acids was sensitive at the pg level with a response linear over 3 4 orders of magnitude. The derivatives were formed by reaction with o-phenylenediamine ... 

Some of the comparisons are described below, but the results must be interpreted with due caution particularly in view of continuing improvements in detector design. One report compared two coulometric detectors for catecholamine analysis and concluded that they were equivalent to current amperometric detectors. Another study compared wall-jet and thin-layer cell configurations. Other more comprehensive studies have been controversial. Forzy et al compared 11 detectors for the analysis of 5-hydroxyindoleactic acid (5-HIAA). They used the same HPLC system with each detector to determine linearity, repeatability, absolute sensitivity, limit of detection and stabilisation time. Driebergen and Benders evaluated 10 detectors with respect to their suitability for routine use in a pharmaceutical company using tetramethylbenzidine as the test compound. Both of these reports found similar relative results with respect to sensitivity and ease of use but stressed the importance of matching instrument to application. 

Electrochemical detection in HPLC has become established for some, more specialized, applications such as catecholamine analysis though it can be exploited for a far wider range of compounds. The work in this paper has attempted to investigate some of the basic properties of an electrochemical detection system and some more difficult applications. The detector has a linear dynamic range and precision that are comparable with those of other detectors for HPLC. It is, however, more dependent on temperature than, for example, the UV absorption detector and must be operated in a temperature controlled environment to obtain the lowest detection limits. For many electroactive compounds with moderate oxidation potentials, the electrochemical detector can yield sub-nanogram detection limits. 

The on-line measurement of reducing capacity can be performed with either a single or a series of electrochemical detectors, and linear correlations have been demonstrated between total antioxidative activities determined by the electrochemical detection and those determined by DPPH- reduction or by the ORAC assay (Guo et al, 1997 Peyrat-Maillard et al, 2000). The reducing capacity must also be quantified by post-column reactions, either with DPPH- or by the reduction of phosphomolybdenum complexes followed by UV-VIS-detection (Bandoniene and Murkovic, 2002 Cardenosa et al, 2002). A combination of HPLC and semi-automatic ORAC analysis has also been described (Caldwell, 2001). 

A phosphorus-specific thermionic detector was also adapted from GLC (See Section III.3.b) for use with small-bore HPLC columns208,307,330,334. Based on an electrically heated rubidium salt bead, it permits detection limits of 0.2-0.5 ng of phosphorus and its response is linear with the amount of phosphorus over several orders of magnitude. This detector yields good results with phosphates which cannot be detected by UV spectrophotometry or by fluorescence measurements. 

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