Detection methods in HPLC

In this chapter, developments and applications of the CL detection methods in HPLC are reviewed. 

Fluorescence is one of the most sensitive detection methods in HPLC analyses. However, as ginsenosides do not contain a suitable fluorescence chromophore they have to be derivatized before detection. Shangguan... 

Overall, a broad choice of detection methods in HPLC has provided pharmaceutical researchers with great flexibility in solving the dissolution... 

Ultraviolet (UV) absorbance has been the most commonly used detection method in HPLC determination of carbamate pesticides (18,61-64). Normally, the absorption maxima occurred at... 

Pharmaceutical analysts often have no experience in direct polarographic or voltammetric methods, but almost all will have used high-performance liquid chromatography (HPLC) for the determination of drugs and/or metabolites in biological matrices or drugs and/ or their degradation products in pharmaceutical formulations. Spectroscopy (ultraviolet and fluorescence) is the most common detection method in HPLC, but for molecules that do not possess a suitable chromophore or when increased sensitivity or specificity is required, electrochemical detection offers a suitable alternative. ELCD is applicable to any molecular species capable... 

The most widely used detection methods in HPLC analysis of anilines and phenols are UV (especially diode array) and electrochemical detection (ED). UV detectors provide very good signal stability and in the case of diode-array detectors they can be used for analyte tentative confirmation purposes using UV spectra libraries. Electrochemical detectors are more sensitive than the UV detectors however, their performance is highly dependent on the type of samples analyzed. Components from dirty samples are deposited on the electrochemical cell and the detector sensitivity is rapidly decreased. ° ° The heterocyclic amines can be measured with UV, electrochemical, and fluorescence detectors. 

Shaw et al. [64] described a (D)-penicillamine detection method in blood samples that had been treated with EDTA, deproteinized with trichloroacetic acid, and analyzed within 1 h. Penicillamine was detected at a vitreous-carbon electrode operated at +800 mV after HPLC separation. A linear calibration graph was obtained, and the method had a limit of detection equal to 5-20 ng. The method was useful in clinical and in pharmacokinetic studies. 

One of the most severe shortcomings of PO-CL reactions is their relatively low selectivity this problem can be overcome in two ways by physical discrimination (i.e., by sequentially separating the analytes prior to their determination), or by mathematical discrimination, the usefulness of which is restricted to only a few components. The former approach is the more interesting in fact, PO-CL is widely used as very sensitive detection method for HPLC [46, 47], The latter approach is a novel alternative in CL analysis it is discussed in detail in Sec. 4. 

This preliminary work demonstrated well the use of CL as a highly sensitive and selective detection method in CE by its application to the separation of luminol and ABEI using the same experimental conditions cited previously for the CL reaction of luminol in HPLC. Detection limits (S/N = 3) of 100 amol and 400 amol were obtained for the compounds mentioned, respectively, achieving an improvement in sensitivity of 2-3 orders of magnitude with respect to the ones obtained using UV absorption for detection. 

There is a strong limitation in the concentration range due to the logarithmic relationship between transmission and concentration (optical densities reasonably to measure range from 0.1 to 1.5). Nevertheless, protein quantification by direct UV-measurement or after staining with dyes in the visible range is a very robust method and can be found, e.g., as a common detection mode in HPLC or other chromatographic techniques. 

The most frequently used detection method for HPLC is UV spectrophotometry. Routine detection in HPLC is typically based on measurement of UV absorption, or visible absorption in the case of anthocyanins. No single wavelength is ideal for all classes of flavonoids since they display absorbance maxima at distinctly different wavelengths. The most commonly used wavelength for routine detection has been 280 nm, which represents a suitable compromise. 

Mass spectrometry as a detection method following HPLC separation provides a much better solution to the problem of imeqnivocally identifying microcystins, as microcystins produce characteristic ions in their mass spectra (Namikoshi 1992 Kondo 1992 Rinehart 1994 Lawton 1995 Ynan 1998, 1999). 

The refractive index detection (RID), often used in high-performance liquid chromatography, is an interesting detection method in CE with a laser light source and a limit of detection (LOD) in the micromolar range. Electrochemical detection (ECD) and pulsed amperometric detection (PAD) of sugars are common and effective methods used in HPLC. Some recent communications show that the sensitivity of these detection methods in CE have an approximately 1000-fold better LOD than RID. Unfortunately, these de-... 

Comparative studies were carried out to determine the efficiency of the various detection methods in the analysis of phenolic compounds. On-line SPE of sixteen priority phenol pollutants in water on polystyrene was followed by HPLC separation and detection. The sensitivity of ELD was higher than that of UVD. LOD down to the ppt level was attained by ELD on 100 mL samples for all the chorinated phenols however, nitrophenols could not be equally determined because they require working potentials different from those... 

Only a few methods for urinary catecholamines have been published that do not require preextraction prior to analysis." " These methods minimized sample preparation by making use of different precolumn derivatization procedures. The selection of a suitable method for sample preparation prior to analysis by HPLC depends on a number of factors, such as the biological source, the type of column used and the selectivity of the detection method. In cases where the analyte concentration is very low and the analyte is present in a complex matrix (urine or plasma) with interfering compounds, an exhaustive pretreatment may be unavoidable. Sample pretreatment is also essential to ensure the sensitivity and specificity of the assay and protect the analytical column from contamination. 

Both GC and HPLC have been used to separate MBOCA and its metabolites from urine. Most recently, HPLC has become the method of choice to selectively detect MBOCA and its metabolites in urine. The most sensitive and specific detection methods for HPLC are ED (Ichikawa et al. 1990 NIOSH 1986b Okayama et al. 1988 Trippel-Schulte et al. 1986 Vantulder et al. 1981) and photoconductivity detection (PCD) (Ducos et al. 1985). Of these, ED has been the most frequently used detection method. Ultraviolet detection (UV) has also been paired with HPLC (McKerrell et al. 1987 Angerer and Schaller 1985 Trippel-Schulte et al. 1986) but is less sensitive and less selective than either ED or PCD (Trippel-Schulte et al. 1986). 

Supercritical Fluid Chromatography. The origin of SFC with packed columns goes back over two decades (17-26). The recent interest in SFC has been due in large part to the limitations in both chromatographic efficiency and detection methods with HPLC. The introduction of fused silica capillary columns with nonextractable stationary phases for SFC (O and the potential compatibility with gas phase detection methods (13) has served to further increase the attention given these methods. 

For more complex samples with a number of electroactive species to be determined, separation by HPLC, or other methods such as ion chromatography or capillary electrophoresis, followed by coulometric detection is better suited. Its applications in HPLC are usually to oxidizable organic species that cannot be determined by ultraviolet absorption, the standard detection technique in HPLC. Examples for such species include amines and phenols, catecholamines (such as the neurotransmitters adrenaline and... 

As detection method in separation techniques, especially in thin-layer chromatography, high-performance liquid chromatography (HPLC) and electrophoresis. In HPLC applications for non-fluo-rescent compounds, either precolumn or postcolumn fluorescent derivatization is used. In capillary electrophoresis, the use of a laser to directly excite fluorescent compounds allows a extremely high sensitivity (femtomole to attomole levels of analytes can be detected in nanoliter volmnes). 

Some parameters affect the conversion of nonflu-orescent compounds into fluorescent ones, e.g., UV irradiation time and the nature of the solvent used (see Table 1). For example, pesticides such as fenvalerate, diflubenzuron, and deltamethrin are efficiently converted into fluorescent products (high signal with short irradiation time) in protic solvents, while for fenitrothion and chlorpyriphos polar apro-tic solvents are the choice. Comparing the analytical performances of the photoconversion method with other derivatization approaches, it can be considered a rapid, sensitive, and precise method for quantifying several classes of pesticides in stationary media and as postcolumn detection mode in HPLC and in FIA. 

HPLC coupled with a mass detector has been shown to be suitable for determining minor differences in molecular masses, and thus can satisfactorily replace the UV-Visible detection method. In this destructive detection system, ideal separation (resolution below 1.5) is not required for completing the validation of identification and quantification procedures. However, the HPLC-mass-mass technique is not cost-effective and is more suitable for research centers. However, at present, the HPLC-UV technique is easily affordable in both developed and developing countries. 

In chromatography, CDs have been used in both stationary and mobile phase methods. In HPLC applications, CDs can be incorporated into stationary phases either by adsorption or bonding onto silica. As an example of the adsorption method, Thuaud et alP first grafted a ff-CD derivative onto a polyvinyl-imidazole (15) that was then absorbed onto silica and used to separate racemic mixtures of amino acid derivatives, among other species, using UV detection. The structure of one )8-CD-containing polymer is shown in Figure 18. 

The detection system in HPLC is often based on ultraviolet (UV) light measurement of the effluent. Since the concentration of cobalamins in human samples is very low, our laboratory has developed a sophisticated method to determine low-level concentrations of HPLC-separated cobalamins (Hardlei and Nexo 2009). After immunoprecipitation of haptocorrin or transcobalamin followed by a proteolytic extraction and reversed-phase HPLC in darkness (to avoid conversion of methyl- and adenosyl-cobalamin into hydroxo-cobalamin), the fractions are vacuum-dried, re-dissolved in buffer and the cobalamin in the fractions is measured either by employing a sensitive cobalamin specific assay or an assay recognizing also the cobalamin analogues capable of measuring levels as low as 4pmol/L. 

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