Limits of HPLC

Statistical evaluation of HPLC UV MS[19] and CE UV MS[20] methods proves that MS detection of anthraquinone dyes is more sensitive than UV, especially in the case of chromatographic analysis of laccaic acids (almost 20 times) and purpurin (almost 40 times). However, detection limits of HPLC ESI MS determination of alizarin and purpurin (0.03 gg ml ) are about 20 times lower than those of CE ESI MS (0.52 0.58 gg ml x). 

The data presented show a limitation of HPLC-PDA analysis for trace amonnts of CYL in enviromnental samples but also underline the potential of an inexpensive and fast analysis for various purposes. 

One of the limitations of HPLC has been its restriction to the analytical mode of operation. However, increasing attention has been focused upon the development of preparative systems. For example, the use of HPLC in the isolation and identification of the components of complex mixtures of pyrethrum extracts, progesterone, and cholesteryl phenylace-tate was described [265]. Various large diameter columns for preparative work have also been developed such as that available from Du Pont and described by Wolf [266] and by De Stefano [267] in a variety of preparative applications. 

Retinoic acid, an endogenous retinoid, is a potent inducer of cellular differentiation. Because cancer is fundamentally a loss of cellular differentiation, circulating levels of retinoic acid could play an important role in chemoprevention. However, physiological concentrations are typically below the limits of HPLC detection. Sensitive techniques, such as negative chemical ionization (NCI) GC/MS have been employed for quantification, but cause isomerization and also fail to resolve the cis and trans isomers of retinoic acid. Normal phase HPLC can resolve the cis and trans isomers of retinoic acid without isomerization, and mobile phase volatility makes it readily compatible with the mass spectrometer. Based on these considerations, a method combining microbore normal phase HPLC separation with NCI-MS detection was developed to quantify endogenous 13-cis and all-trans retinoic acid in human plasma. The limit of detection was 0.5 ng/ml, injecting only 8 pg of retinoic acid onto the column. The concentration of 13-cis retinoic acid in normal, fasted, human plasma (n=13) was 1.6 +/- 0.40 ng/ml. 

Martin et al. published a paper on the theoretical limits of HPLC which is well worth reading.They used relatively simple mathematics to calculate pressure-optimized columns for which the length L, particle size and flow rate u of the mobile phase were selected such that a minimum pressure Ap is required to solve a separation problem. It has been shown that these optimized colunms are operated at their van Deemter curve minima. Some astonishing facts have emerged from the study, provided that the chromatography is performed on well packed columns (reduced plate height h = 2-3 see Section 8.5). 

A comprehensive presentation of the limits of HPLC was published by G. Guichon as The limits of the separation power of unidimensional column liquid chromatography . ... 

The detection limits of HPLC-NMR are continually being revised downwards as new technical advances are made. Recently, these have... 

Table 1.1 highlights the advantages and limitations of HPLC. HPLC is a premier separation technique capable of multicomponent analysis of real-life samples and complex mixtures. Few techniques can match its versatility and precision of <0.5% relative standard deviation (RSD). HPLC is highly automated, using sophisticated autosamplers and data systems for unattended analysis and report generation. A host of highly sensitive and specific detec-... 

The lower concentration limit of HPLC-FL methods for OA group is generally 10 pg of toxin. In practice, the method is limited by a huge range of interferences and the actual limit of detection for the method apphed to shellfish is 0.1 mg/kg (Quilliam, 1995) howver, in our lab we have tested thousands of samples for OA group toxins by LC-MS to 0.01 mg/kg without any sophisticated cleanup or pre-concentration (McNabb et al., 2005). 

Swartz, M., Murphy, B., and Sievers, D. 2004. UPLC Expanding the limits of HPLC. 

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