High performance liquid chromatography detector calibration

The determination of the molecular weight of nanoparticles is performed by gel permeation chromatography (GPC). The experimental setup consists of a high performance liquid chromatography system with a size exclusion column and a refractive index detector. The nanoparticles are usually freeze-dried and dissolved in tetrahydrofuran for analysis on the system. Poly(styrene) or poly(methylmethacrylate) standards are used to calibrate the column, to enable the determination of number average molecular weight (Mn), as in... 

An important feature of modern high-performance liquid chromatography (HPLC) is its excellent quantitation capability. HPLC can be used to quantify the major components in a purified sample, the components of a reaction mixture, and trace impurities in a complex sample matrix. The quantitation is based on the detector response with respect to the concentration or mass of the analyte. In order to perform the quantitation, a standard is usually needed to calibrate the instrument. The calibration techniques include an external standard method, an internal standard method, and a standard addition method. For cases in which a standard is not available, a method using normalized peak area can be used to estimate the relative amounts of small impurities in a purified sample. 

A solvent module (Varian model No. 5000) with a UV detector coupled to an on-line Nal(Tl) detector was used for high performance liquid chromatography (HPLC) analysis. For radioactive measurements, a dose calibrator (Capintec CRC-7, USA), a solid scintillation counter (ORTEC, USA) with a plane (7.62 cm x 7.62 cm) Nal(Tl) detector, an automatic well type gamma counter (Compac-120, Picker, USA) and a multichannel analyser coupled to a Nal(Tl) detector (7.62 cm x 7.62 cm) were used. 

Recent developments in the sensitivity and resolution of NMR spectrometers permitted us to use one as a real-time detector for high-performance liquid chromatography as demonstrated in recent review articles.332,333 Recently we developed an on-line GPC/NMR system in which a 500-MHz 1H NMR spectrometer was used as a detector.334-339 By using on-line GPC/NMR the molecular weight of a polymer can be determined without a calibration curve if the polymer sample contains a known amount of end-group per polymer molecule. 

As an approximation to a universal response detector, an ultraviolet (UV) detection with a high concentration of in vivo sample or in vitro incubation can be used to calibrate the mass spectrometric response of the metabolites. Josephs et al. (2009) recently reported the use of high-performance liquid chromatography (HPLC)-UV detection to get area responses of a 30-tiM in vitro microsome or hepatocyte incubation. The incubated sample was then diluted in matrix to create a single point calibration for mass spectrometric quantitation of the metabolites. The results from this method were successfully verified using buspirone and proprietary compounds for which the synthetic standards for their metabolites were available. 

Many components of SEC systems are well known and ubiquitous in an analytical lab. Size exclusion chromatography systems require at least an isoaatic pump, a high-pressure injection systan (manual or automated), one or more SEC colunm(s), one or more detector(s), and data evaluation units with software to acquire, calibrate, and analyze data. Other components, such as degassers or column compartments are often optional and depend on the particular application and on the lab environment and conditions. Although SEC and high-performance liquid chromatography (HPLC) seem to be very similar in instrumentation requirements, the theoretical background and separation mechanism of both techniques are entirely different. The major differences in the practice of SEC are outlined in Table 9.2. 

Seppanen, C.M., Rahmani, M., and Csallany, A.S., Simultaneous determination of chlorophylls, pheophytins, beta-carotene, tocopherols, and tocotienols in olive and soybean oils by high-performance liquid chromatography. J. Food Sci., 68, 1644, 2003. Torsi, G., Chiavari, G., and Lippolis, M.T., Quantitation without calibration curves using HPLC with nondestructive detectors, LC-GC Inti., 5, 37, 1992. 

Fig, 4. A typical size exclusion calibration curve and chromatogram of the separation of a protein mixture. Column BIOSEP-SEC-S3000. Mobile phase pH 6.8 phosphate buffer. Detector UV abs. at 280nm. Reproduced from W.J. Lough I.W. Wainer (eds), High Performance Liquid Chromatography, 1996, first published by Blackie Academic Professional,... 

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