HPLC equipment detectors

Colorplate 12 shows a photo of an HPLC equipped with a diode array detector. 

By applying an extension of the clearance concept 30, 31), in vitro metabolism was used to predict in vivo toxin elimination. Hepatocytes were incubated with 0.5 to 10 pg unlabeled PbTx-3 containing 0.1 pg radiolabeled toxin as tracer. Disappearance of parent compound and the appearance of metabolites were measured by HPLC equipped with a Radiomatic isotope detector. (1.6 nmol/min/g liver)... 

A Waters 2690 Alliance HPLC equipped with a 996 photodiode array and a 896 IJV/Vis detector was used for carotenoid analysis. The column (Phenomenex, Torrance, CA) was a 250x4.6mm Ultracarb 3 pm C-18 stationary phase and elution was carried out isocratically at a flowrate of l.OmL/min with 85 15 (v v) acetonitrileimethanol (HPLC grade) containing 0.1% triethyl amine to prevent on-column carotenoid decomposition. 

HPLC equipment has been designed and produced to assure correct volumetric delivery of the mobile phase, including the injected sample, and has low-noise detectors so that low concentrations of samples can be analyzed conveniently. Discussed below, briefly, are some of the important considerations for the HPLC equipment. More detailed discussion can be found in a recent text (see Chapter 3 of reference 3). 

Measurements were performed on a Waters 470 HPLC fluorescence detector equipped with a JASCO cuvette accessory and connected to a Perkin Elmer 561 strip chart recorder. Excitation and emission band-widths were 18 nm. Emission spectra were measured for the three excitation wavelengths mentioned above and emission starting from 10 nm higher than excitation up to 700 nm. Fluorescence at fixed wavelengths was measured four minutes after cuvette insertion and expressed as per-millage of the 275/303 fluorescence of 3.0 pM tyrosine in 50 mM HEPES, pH 7.4. Corrections were made for buffer- and blank collagenase fluorescence, and for signal attenuation. 

Experimental Techniques. Chromatography was performed on a Varian model 5060 HPLC equipped with a RI-3 refractive index detector. A Vista Plus Gel Permeation Chromatography (GPC) data system was used consisting of a Vista 401 chromatography data system serially connected to an Apple II microcomputer. The Vista 401 performs data acquisition and allows data storage and automations capability while all SEC data processing is performed on the Apple II by means of user-interactive GPC software for automated, on-line calibration and polymer analysis. 

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. 

Part II shows you how to make the best use of the common columns and how to keep them up and running. (Chapter 6 on column healing should pay for the book in itself) It discusses the various pieces of HPLC equipment, how they go together to form systems, and how to systematically troubleshoot system problems. We will take a look at the newest innovations and improvements in column technology and how to put these to work in your research. New detectors are emerging to make possible analysis of compounds and quantities that previously were not detectable. 

A second alternative to the conventional chromatographic column is to use a narrower column bore, typically 1-3 mm ID. For these narrow-bore columns, the primary advantage is an increase in mass sensitivity [44] along with reducing the volume of the eluent. In contrast to the 1-2-mm-ID columns the 3.0-mm-ID columns can be used with conventional HPLC equipment. The most likely role for narrow-bore columns will be in pharmaceutical analysis as an interface to detectors such as a mass spectrometer. ... 

They are then quantitated by High Performance Liquid Chromatography (HPLC) equipped with an amine column and UV detector. This procedure is a modification of the Ciba-Geigy AG-417A method. 

Equipment for recycle operations differs from conventional HPLC equipment. For a recycle system to be useful, the extracolumn band spreading must be small relative to the band spreading of the column. This involves the solvent delivery system, transport tubing, and detector(s). Also, because a recycle system is a closed system with a finite volume, the operator must be aware that fast-moving materials could eventually overtake slower-moving materials and remix. To prevent peak overlap, a means must be provided to allow the operator to remove a portion of the sample components before overlap can occur. 

Figure 4.3 Arrangement of HPLC equipment for termination of reaction by direct injection of sample. A sample is removed from the reaction mixture and transferred directly to the injection port for introduction onto the column. The HPLC column is protected by a guard column, which removes debris. The eluent flows through the detector, from which a signal is displayed on a recorder. The area of each peak is electronically integrated.

Protein digests were analyzed by reversed-phase on a 1090A/M Hewlett Packard HPLC equipped with a diode-array detector and Chemstation. They were analyzed (100-200 for PVDF and 20-30 p.L total volume for in-gel digests) on a SynChrom C4 (2.1 x 50 mm) column (3) at a flow rate of 0.146 mL/minute at ambient temperature, and monitored at 215 and 280 nm. When used, the anion exchange column was placed in series before the C4 column and removed approximately 7 minutes into the isocratic portion of the gradient. Buffer A was aqueous 0.1% TFA. Buffer B was 0.1% aqueous TFA in 90% acetonitrile. The gradient used was 3%B (0.01-10 minutes), 3-18%B (10-26 minutes), 18-55%B (26-86 minutes), 55-80%B (86-100 minutes). Peptides were collected manually for further analysis. 

Thus, iV-ethylpiperidine was added to a suspension of tin(II) trifluoromethanesulfonate in anhydrous CH2CI2 at — 50°C under Ar, After addition of a solution of 3-acetyl-(4/ ,5S)-MPOT (30) in CH2CI2, the mixture was stirred at — 50 to — 40°C for 3 hr. A solution of isobutyraldehyde in CH2CI2 was then added at - 78°C and the mixture was stirred at the same temperature for 20 min. Usual workup of the reaction mixture gave a mixture of diastereoisomers 32 and 33, the ratio of which was readily checked by HPLC equipped with a UV detector. Chromatographic separation of each diastereoisomer gave optically pure 32 and 33 (Scheme 5). 

The radio-HPLC equipment considered of 2 Waters model 6000 A pumps, a Waters 660 solvent programmer, a Waters U6K injector, a Varian Varichrom UV-VIS spectrophotometric detector, a FMI LB 5031 scintillation cocktail pump, a Berthold Radioactivity Monitor LB 504 fitted out with an 800-pl flow-cell and a Spectra-Physics SP 4100 computing integrator. Dried extracts were dissolved in minimal amounts of dimethylsulphoxide for injection. 

This method uses the high-performance liquid chromatography (HPLC) equipment for sample handhng and requires molar mass sensitive detectors (such as light scattering and/or viscometry) to obtain a mean property values from each detector (Mw and/or IV, respectively). The FIA result from a concentration detector yields polymer content in a sample, which can also be determined with other well-established methods. The FIA approach requires expensive and well-maintained equipment, and will not save much time or solvent furthermore, no distribution information is available. 

The detection and identification of phenolic compounds, including phenolic acids, have also been simph-fied using mass spectrometry (MS) techniques on-hne, coupled to the HPLC equipment. The electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) interfaces dominate the analysis of phenohcs in herbs, fmits, vegetables, peels, seeds, and other plants. In some cases, HPLC, with different sensitivity detectors (UV, electrochemical, fluorescence), and HPLC-MS are simultaneously used for the identification and determination of phenolic acids in natural plants and related food products.In some papers, other spectroscopic instmmental techniques (IR, H NMR, and C NMR) have also been apphed for the identification of isolated phenolic compounds. 

A computer associated with an HPLC equipment may control the instrument or part of it, e.g., the photodiode array detector, or acquire data generated by the instrument, such as the peak area of analytes. 

Rapid quantification of products and substrates in a fermentation process is essential for process development and optimization. Most fermentation laboratories have access to HPLC equipment with possibilities to couple them to quite inexpensive diode-array-detectors, and this equipment could be used for quantitative monitoring of the process. Because HPLC can allow multi-component analyses, i.e., several analytes in the same sample can be determined virtually simultaneously, and since it is often necessary to monitor more than one substance at a time, this technique is an important tool for bioprocess monitoring. HPLC coupled to expensive MS does not represent standard equipment at fermentation laboratories. Even if mass spectrometers are available, DAD is often sufficient for quantification because product concentrations are relatively high, so the MS could be used for other issues. In paper II the goal was to develop and validate a method for analytical quantification of both the product and the substrate to enable the proper characterization of the kinetics of the process i.e., the determination of the values of substrate conversion and product formation. 

Detectors used in the initial experiments with capillary electrophoresis were simple absorbance and fluorescence detectors that had been adapted from HPLC equipment. However, it soon became apparent that these instruments yielded poor... 

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