Absorbance detector. HPLC

An HPLC detector is often a modified spectrophotometer equipped with a small flow cell, which monitors the concentration (or mass) of eluting sample components. A number of detectors used in HPLC are discussed below. Most applications utilize absorbance detectors such as UV/Vis or... 

FIGURE 13.9 The HPLC diode array UV absorbance detector. When a mixture component elutes from the column, not only the chromatography peak but the entire UV absorption spectrum for that component can be recorded. 

In Chapter 12, we discussed the need to calculate response factors, specifically when a TCD detector is used (Section 12.8.2). Would response factors need to be calculated in HPLC when a UV absorbance detector is used Explain. 

A PDA detector provides UV spectra of eluting peaks in addition to monitoring the absorbance of the HPLC eluent like the UVA is absorbance detector. It is the preferred detector for testing impurities and for method development. PDA facilitates peak identification during methods development and peak purity evaluation during method validation. Detector sensitivity was an issue in earlier models but has improved significantly (more than ten-fold) in recent years. ... 

CZE = capillary zone electrophoresis EC = electrochemical detector GC = gas chromatography HCD = Hall conductivity detector HPLC = high performance liquid chromatography IDMS = isotope dilution mass spectrometry MS = mass spectrometry RSD = relative standard deviation SEE = supercritical fluid extraction SPE = solid phase extraction UV = ultraviolet absorbance detection... 

The ahquots collected hy the robot were assayed by HPLC. The HPLC system consisted of an extended range LC pump, autosampler and an absorbance detector. 

Although this section provides a brief description of most commonly nsed detectors for HPLC, most of the focus is on a few detection modes. Optical absorbance detectors remain the most widely nsed for HPLC, and are discnssed in some detail. We also focns on flnorescence, condnctivity, and electrochemical detection, as these methods were not widely nsed for HPLC in the past, bnt are especially well suited to micro- and nano-flow instrnments becanse of their high sensitivity in small sample volumes. Mass spectrometry has also come into wide and rontine nse in the last decade, but as it is the subject of another chapter, it will not be fnrther discnssed here. Miniaturization has been particularly important for capillary and chip-based electrophoresis, which often employs sub-nanoliter detection volnmes [36,37]. 

The most commonly used detector for HPLC is still the UV-Vis absorbance detector. The amount of light transmitted through a solution of concentration c in a flow cell with path length ( is given by the Beer-Lambert law. 

HPLC and Isolation of Mutagenic Fractions. Analytical and semipreparative reverse-phase HPLC separations were performed by using a water-to-acetonitrile linear gradient (J2). Separations were carried out on a Hewlett Packard Model 10084 B equipped with an automatic sampling device, a solvent programmer, a variable absorbance detector, and an automatically steered fraction collector. The instrument was fitted with a 3.9-mm X 30-cm prepacked analytical column of 10-/zm silica particles bonded with octadecylsilane (Bondapack-Cis) for analytical scale. For semipreparative scale separations, the HPLC was fitted with a 7.8-mm X 30-cm prepacked column packed with 10-/xm silica particles bonded with octadecylsilane. Samples for HPLC were injected at volumes of 20 /xL (flow rate 1 mL/min) and 80 /zL (flow rate 4 mL/min), and the absorption was measured at 254 nm. Fractions... 

UV/VIS absorbance detector (single wavelength or diode array) placed in-line between HPLC column and mass spectrometer for additional characterization of eluting chlorophylls (optional)... 

Optional Direct the eluate from the HPLC column through a UV/VIS absorbance detector prior to the mass spectrometer for additional sample characterization. 

When determining naturally occurring vitamin D in animal products for nutritional evaluation purposes, 25-hydroxyvitamin D3 should be included, because this metabolite contributes significantly to the total biological activity, particularly in milk. 25-Hydroxy vitamin D3 is present in dairy products, eggs, and meat tissues in sufficient concentration to permit its determination by HPLC using an absorbance detector. In bovine milk the concentration of this metabolite is less than 1 ng/ml (63) hence it is usually determined by a competitive protein-binding assay after fractionation of the extracted sample by HPLC (64). 

Thompson and Hatina (135) showed that the sensitivity of a fluorescence detector toward unesterified vitamin E compounds under normal-phase conditions was at least 10 times greater than that of a variable-wavelength absorbance detector. The relative fluorescence responses of the tocopherols at 290 nm (excitation) and 330 nm (emission), as measured by HPLC peak area, were a-T, 100 /3-T, 129 y-T, 110 and 5-T, 122. The fluorescence responses of the corresponding to-cotrienols were very similar to those of the tocopherols, and therefore tocotrienol standards were not needed for calibration purposes. The fluorescence detector also allows the simultaneous monitoring of ubiquinone derivatives for example ubiquinone-10 has been detected in tomato (136). 

Since cyclamate has poor UV absorbing characteristics, HPLC methods for the analysis of this sweetener require specific detection systems, such as indirect photometry or conductivity. Herrmann et al. (24) used indirect photometry for the detection of cyclamate at 267 nm against a UV-absorbing mobile-phase component, p-toluenesulphonate. Biemer (17) and Wu et al. (47) used a conductivity detector for the determination of cyclamate. According to Biemer (17) the use of this detector offers distinct advantages, since compounds coeluting with cyclamate may not exhibit an electrochemical response and, hence, not appear in the chromatogram. 

Owing to the varied structures of various food dyes, they can often be differentiated from one another by their characteristic ultraviolet/visible absorbance spectra. Using HPLC coupled with a diode array detector (HPLC-DAD) it is possible to collect a compound s absorbance spectrum as it elutes from the HPLC column, which greatly assists in identification. At Reading Scientific Services Ltd (RSSL) this type of detector is routinely used in a range of analyses of such substances as patulin, a mycotoxin found in apple juice, and in the analysis of colours and vitamins, which allows a more certain assignment of a particular peak to a specific compound to be made. 

Pure fluids. Carbon dioxide is often the mobile phase of choice for SFC, since it has relatively mild critical parameters, is nontoxic and inexpensive, chemically inert, and is compatible with a wide variety of detectors including the flame ionization detector (FID) used widely in GC and the UV absorbance detector employed frequently in HPLC (7). The usefulness of carbon dioxide as a mobile phase in many instances is somewhat limited, however, because of its nonpolarity (8), and many polar compounds appear to be insoluble in it. For a sample containing polar compounds, pure carbon dioxide may not be the proper mobile phase. The elution of polar compounds is often difficult and the peak shapes for these polar compounds are sometimes poor. This latter difficulty is commonly observed with nonpolar supercritical fluids and may be due to active sites on the stationary phase rather than any inherent deficiency in the fluid itself. 

A potential alternative for determining UV response factors is to use two HPLC detectors a standard UV absorbance detector and a second detector that has a response uniformly proportional to weight or concentration. For example, if a detector could provide accurate information on the relative amounts of the impurities and parent compound, then this information, combined with the UV peak areas, would supply the desired RRF information without the need for a purified impurity sample. One could reasonably question the need for a UV detector and RRF values at all if such an alternative detector was available, as it would directly provide information on relative amounts of impurities/parent. However, UV detectors are inexpensive, rugged, and readily available therefore, RRF values, once determined, are widely applicable to situations in which no other detector is available. 

Analysis of Tween 80 was performed using a Hewlett Packard 1100 series HPLC equipped with a Sedex 55 Evaporative Light Scattering Detector (ELSD). The mobile phase consisted of 80% acetonitrile and 20% water. Duplicate injections (5 pL) of each sample were evaluated by HPLC. Potassium iodide, used for the 1-D column and 2-D box tracer studies, was analyzed with a continuous flow Isco V4 variable UV wavelength absorbance detector equipped with an EZChrom Chromatography data acquisition system. 

Schematic of a fluorescence monitor in series with an absorbance detector in HPLC... 

Liquid Chromatograph. The liquid chromatograph was comprised of a Waters 660 Solvent Programmer, two Waters 6000A pumps, a Waters U6-K Injector and a Waters 440 absorbance detector (254 nm). Whatman micro-capillary tubing (0.007" ID) was used to transfer the HPLC column effluent from the 254 nm absorption detector to the fluorescence detector. 

Classical LC detectors (refractive index, fixed wavelength UV absorbance at 254 or 280 nm) have lacked the sensitivity to allow direct analysis of cannabi-noids in biological fluids. However, recent development of variable wavelength absorbance detectors extending into the 195-220nm UV region and of fluorescence detectors for HPLC led the authors to initiate... 

---