Detectors, HPLC drift

It is recommended that OQ test the following on an HPLC system flow accuracy, pump compositional accuracy, pressure pulsations, column oven temperature accuracy/stability, detector noise/drift and wavelength accuracy, autosampler injection precision and carryover. 

Absorbance detection, either UV/visible or photodiode array, has broad but specific applicability, especially for styrenic polymers, epoxies, pheno-lics, polycarbonates, polyurethanes, aromatic polyesters, and many additives. When other HPLC modes are used, additional separating capability is sometimes achieved by changing the solvent composition during the analysis (gradient elution). For this work the UV or PDA detector is essential, since the RI detector would drift excessively as the composition, and therefore the refractive index, changes. See Sec. ILF. 

An ELSD converts the HPLC eluent into a particle stream and measures the scattered radiation. It offers universal detection for nonvolatile or semivolatile compounds and has higher sensitivity than the RI detector (in the low ng range) in addition to being compatible with gradient analysis. ELSD is routinely used in combinatorial screening. Response factors are less variable than that of other detectors. An ELSD consists of a nebulizer equipped with a constant temperature drift tube where a counter-current of heated air or nitrogen reduces the HPLC eluent into a fine stream of analyte particles. A laser or a polychromatic beam intersects the particle stream, and the scattered radiation is amplified by a photomultiplier. Manufacturers include Alltech, Polymer Laboratories, Shimadzu, Waters, Sedere, and ESA. 

The samples were raw ewe s milk, raw cow s milk and raw goat s milk. Cream was separated from milk by centrifugation, and TGs were extracted with n-hexane. For the HPLC analysis, two stainless steel columns, 25 cm and 15 cm X 4.6-mm ID with 3 /zm Spherisorb ODS-2 (Phase Separations, Queensferry, United Kingdom Symta, Madrid, Spain) were connected in series. The mass detector (ACS 750/14 The Arsenal, Macclesfield, United Kingdom) drift tube temperature was 45°C, and the inlet gas pressure was 1.38 bar. The mobile phase consisted of a... 

They studied the effect of the mass detectors drift tube temperature on the low-molecular-mass TGs. Solutions of 10 mg/ml of tributyrin, tricaproin, tricaprylin, tricaprin, and trilaurin were injected twice at each of the following drift tube temperatures 20,25, 30,45, and 60°C. Five replications of the HPLC analysis were performed for one sample of ewe s milk fat to determine the reproducibility of the HPLC method. The TG composition was estimated in accordance with the method based on the calculation of the equivalent carbon numbers (ECNs) of the HPLC chromatographic peaks and in the molar composition in fatty acids, analyzed by GLC, collected at the HPLC chromatograph outlet. The HPLC fractions were collected every 40 s at the outlet of the column after 14 min there were no peaks before that time. 

Detectors. A detector capable of continuously monitoring effluent from the column is essential for efficient HPLC analyses. Considerations in connection with detector performance include absolute and relative sensitivity, drift characteristics, noise, linearity, specificity, and band spreading resulting from detector design. The selection of a proper detector is essential for successful analysis, both from the standpoint of sensitivity and elimination of effects of interfering compounds (specificity). 

Typical Response Peaks. Figure 2 shows some typical response peaks for the HPLC separation of the saturates and aromatics in a typical vacuum gas oil. The curve in the upper portion of the figure is the response for the sample using the RI detector. After the saturates peak appeared, the backflush valve was switched and the attenuation changed as indicated in the figure. The peaks were very sharp and symmetrical and appeared in less than 10-min lapsed time from the point of injection. Base line drift was minimal and the areas of the response peaks were obtained with a ball and disc integrator on the strip chart recorder. 

The performance of all HPLC detectors can be characterised by certain parameters such as sensitivity, noise, drift, limit of detection, linear and dynamic range, and detection volume. Other factors are more specific to individual types of detectors, and are discussed in their respective sections. 

Electrochemical detectors are generally incompatible with gradient elution, except for the multichannel coulometric CoulArray detector, which is controlled by a software compensating for the gradient baseline drift during the elution. This detector allows highly sensitive and selective detection of oxidizable or reducible compounds in gradient HPLC. 

Most commercial ELSDs employ a standard or modified HPLC nebulizer (Venturi flow type). It was believed that this nebulizer was not necessary for SFC because nebulization of the SFC mobile phase is accomphshed by gas expansion in a restrictor which controls pressure and mobile-phase flow rates. To counter the cooling effects of CO2 decompression in the linear fused-silica restrictor and improve heat transfer, Nizery et al., using a Cunow Clichy Model DDL 10 detector, placed the restrictor tip into a heated brass ring and applied heat to a small section of tubing between the restrictor and the drift tube... 

Noise and drift are measured in static (dry detector cell) and in dynamic mode at different wavelengths, e.g., 200, 254, and 390 nm. The change in the absorbance as a function of flow rate at the same wavelengths reflects flow sensitivity. Noise is expressed in AU/cm, drift in AU/hr, and flow sensitivity in ALf min/mL. Some equipment units can automatically perform calibration for accuracy. For example, some HPLC-UV/Visible detectors include holmium oxide filters for measurement and calibration of the wavelength accuracy. 

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). 

For the determination of the dynamic detector noise it is necessary to equilibrate the whole HPLC system for at least 30 min according to the conditions described in the autosampler test. The recording unit is set to 0.5-1.5 X 10 AU and the detector signal is recorded during 10 min. The detector noise is determined graphically (peak-to-peak) and documented. Note that this is only a test of short-time noise. A possible drift (long-time noise) is not included. 

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