Quaternary gradient

Where binary, ternary or quaternary gradient elution (p. 91) is required, a microprocessor controlled low-pressure gradient former is the most suitable (Figure 4.31(c)). The solvents from separate reservoirs are fed to a mixing chamber via a multiport valve, the operation of which is preprogrammed via the microprocessor, and the mixed solvent is then pumped to the column. For the best reproducibility of solvent gradients small volume pumps (< 100 gl) are essential. 

Most chromatographers limit themselves to binary or tertiary gradient systems however, it should be noted that pumps capable of quaternary gradient are available (see reference 3, Chapter 3) and should be considered in the equipment selection process to allow greater versatility in method development. 

Pump The pump should be capable of a flow rate between 0.50 and 5.00mL/min. The pump should be a quaternary gradient pump and have a compositional accuracy of +1.5% of the theoretical values for the four channels. The pump should have relative standard deviation (RSD) of <2.0% for six successive readings from a calibrated flowmeter. 

Figure 6.14 shows the resulting quaternary gradient and the resulting chromatogram for the 14-component mixture to which the 7 gradients described in figure 6.13 and table 6.4 have been applied. 

Interpretive methods will generally arrive at the global optimum after a limited number of experiments. However, (by definition) the recognition of the individual solutes is required in each experimental chromatogram. Also, the computational requirements are relatively high, especially if the simultaneous optimization of several parameters is considered. For example, (linear) ternary gradients (one parameter) will be much easier to optimize than quaternary gradients (two parameters). 

The system shown is capable of performing a quaternary gradient and shows all of the elements to be considered for industrial scale separations. For instance, a solvent reservoir size of 1-2 m3 would allow continuous operation for several hours. Separations carried out at this scale will be extremely well-defined with respect to fraction size and point of collection so very few fraction collection vessels would be required. 

AQC (6-aminoquinolyl-A-hydroxysucdnimidyl carbamate) Fluorescent (ex. 245 nm, em. 395 nm) UV 245 nm 160 fmol 35-50 min precolumn Quaternary gradient elution required for complex, nonhydrolysate samples Tolerates salts and detergents, rapid reaction, stable product, good reagent separation, high sensitivity and accuracy... 

Lesellier, E. Tchapla, A. Sequential optimization of the separation of a complex mixture of plastic additives by HPLC with a quaternary gradient and a dual detection system. Chromatographia 1993, 36, 135-143. 

Functional specifications Pump Detector Autosampler Column oven Computer Quaternary gradient pump with online degassing and a flow range of 0.01-5 mL/min UV/Vis diode array, 190-600nm with 1-nm resolution >100 sample vials, 0.5-2,000 i.L injection volume 15-60°C, Peltier System control and data acquisition by a computer workstation or a network with remote access... 

HPLC quaternary gradient pump, with flow splitter. 

Figure h. Twenty minute quaternary gradient profile as entered into the gradient generator. Reproduced from Ref. 22. Copyright 1985, American Chemical Society. 

Finally, a multiple linear quaternary gradient was run consisting of a first segment from 6.0% THF, 8.6% and 20.6% methanol to 10.0% THF, 15.6% acetonitrile and 15.6% methanol In 10.0 minutes followed by 10.0 minute segment to 11.0% THF, 17.4% acetonitrile and 14.4% methanol. Like the previous quaternary gradients the THF and acetonitrile concentrations rise from the Initial to the final concentrations while the methanol concentration falls. The results for the multiple linear quaternary gradient are presented In Table VI b. 

The overall accuracy of the quaternary gradient experiments was slightly worse than for the simple binary gradients, but In no case was the error between predicted and observed more than 6%. It was felt, given the complexity of the system, that these errors were sufficiently small for prediction purposes. 

V ioop + 5pl LGP Low pressure gradient pump (quaternary gradient pump)... 

HPLC analysis was performed on a Surveyor (Thermo Finnigan) HPLC system consisting of a quaternary gradient pump with an integrated degasser, a PDA detector, and an autosampler. Compounds were separated on a Knauer Eurospher 100-C8 (5 pm) reversed-phase colunm (250 x 3 mm i.d.) integrated with a precolunm (5x3 mm i.d.). The colunm temperature was 25 C and the injection volume 5 pL. The mobile phase was 30 70 (v/v) acetonitrile-0.1 % trifluoroacetic acid. The flow rate was 0.8 mLmin. The lobeline peak was identified by the addition of authentic standard (lobeUne base) and by diode-array detection [40]. 

S.3.2.2 Chromatographic Separation The liqttid chromatograph was equipped with a Sirrveyor autosampler model Plus and a Sirrveyor quaternary gradient HPLC-pump (Thermo Fisher Scientific, Breda, The Netherlands). The separation of the compoimds was performed either on a 3-p,m Omnisphere C18 (Varian-Chrompack, Middelbirrg, The Netherlands) colirrrm with dimensions 100 x 2.0 mm i.d. (2006-2007 series) or a 150 x 2.1 mm i.d. Xbridge C18-colunm (Bester,... 

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