Gradient elution precision

Use of 10 pm LiChrosorb RP18 column and binary eluent of methanol and aqueous 0.1 M phosphate buffer (pH 4.0) according to suitable gradient elution program in less than 20-min run time with satisfactory precision sensitivity of spectrophotometric detection optimized, achieving for all additives considered detection limits ranging from 0.1 to 3.0 mg/1, below maximum permitted levels Simultaneous separation (20 min) of 14 synthetic colors using uncoated fused silica capillary column operated at 25 kV and elution with 18% acetonitrile and 82% 0.05 M sodium deoxycholate in borate-phosphate buffer (pH 7.8), recovery of all colors better than 82%... 

Implementation of SFC has initially been hampered by instrumental problems, such as back-pressure regulation, need for syringe pumps, consistent flow-rates, pressure and density gradient control, modifier gradient elution, small volume injection (nL), poor reproducibility of injection, and miniaturised detection. These difficulties, which limited sensitivity, precision or reproducibility in industrial applications, were eventually overcome. Because instrumentation for SFC is quite complex and expensive, the technique is still not widely accepted. At the present time few SFC instrument manufacturers are active. Berger and Wilson [239] have described packed SFC instrumentation equipped with FID, UV/VIS and NPD, which can also be employed for open-tubular SFC in a pressure-control mode. Column technology has been largely borrowed from GC (for the open-tubular format) or from HPLC (for the packed format). Open-tubular coated capillaries (50-100 irn i.d.), packed capillaries (100-500 p,m i.d.), and packed columns (1 -4.6 mm i.d.) have been used for SFC (Table 4.27). 

The traditional HPLC instrument is composed of two different parts the first part separates the components of the sample and the other part accomplishes the detection of the components separated. The part of the HPLC carrying out the separation contains a column, an injection device and the eluent delivery system (pump with filters, degasser and transfer tubing, eventually a mixer for gradient elution). One or more detectors, a signal output device coupled with appropriate software, are responsible for detection and primary data evaluation. Pumps deliver the eluent or the different components of the eluent into the column with a precise, constant and reproducible flow rate. 

Most commercially available HPLC analyzers have been designed to provide precise and accurate analytical information on the composition of samples containing small molecules by using conventional columns. The time scale of the separation by isocratic or gradient elution under these conditions corresponds to standards established about a decade ago. 

Various improvements in HPLC systems have been reported and include an inexpensive injection and pressure relief device [283], automatic gradient elution devices [284, 285], and a high precision sampling device [286]. Some idea of the potential sophistication possible in HPLC can be judged from the description of the use of an on-line computer coupled to a liquid-chromatograph [287]. 

For measurement of 5-HIAA in plasma, HPLC methods with electrochemical or fluorescence detection have been described. The latter uses diethyl ether for preliminary extraction of heparinized plasma. The internal standard and 5-HIAA are then separated by gradient elution on a C18 column and detected with a spectrofluorometer (excitation wavelength, 280nm emission wavelength, 345nm). This method is linear to I90ng/L, and the interassay precision, expressed as a coefficient of variation, is less than 6% at a 5-HIAA concentration of 30ng/L. 

Because of the many requirements which the solvent must meet in a given separation, it frequently happens that no pure solvent can be found which is really suitable. The use of solvent mixtures immensely expands the number of possible solvent systems to choose from and greatly favors the selection of just the right solvent. Solvent mixtures, besides permitting increased control over all solvent properties, permit a precise adjustment of solvent strength to fit individual separations. Solvent mixtures are also a necessary adjunct to such techniques as gradient elution. 

The internal standardisation technique actually increases the analytical error due to the measurement of two peak areas and should be reserved for samples undergoing pretreatment of pre- or post-column derivatisation to account for variable sample recovery or conversion. Quantitative analysis when applied to gradient elution systems affords reduced accuracy and precision due to the practical disadvantages of constancy of flow, reproducibility of gradient formation and solvent mixing-demixing. 

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