Chromatographic conditions flow rate

FIG U RE 15.1 SFC-ELSD of phospholipid mixture with modified COj on the four stationary phases. The modifier consisted of methanol with 5 mM ammonium acetate. Chromatographic conditions flow rate of 2 mL/min, methanol/additive concentration raised from 15 to 55% at 4 min and held for 5 min at 55%. (From Yip, H.S.H. et al. Chromatographia 2007, 65, 655-665. With permission of Vieweg Verlag.)... 

Bulk property detectors function by measuring some bulk physical property of the mobile phase, e.g., thermal conductivity or refractive index. As a bulk property is being measured, the detector responses are very susceptible to changes in the mobile phase composition or temperature these devices cannot be used for gradient elution in LC. They are also very sensitive to the operating conditions of the chromatograph (pressure, flow-rate) [31]. Detectors such as TCD, while approaching universality in detection, suffer from limited sensitivity and inability to characterise eluate species. 

Although not directly pertinent to detectors, the computer that handles the output from the detector should also provide other information in the analytical report. Today, most chromatographic systems, gas and liquid, have a dedicated computer associated with them which, as well as processing the information provided by the detector, will also control and record the operating conditions of the chromatograph. Temperatures, flow rates programs, etc., will be entered via the keyboard of the computer and the information stored for reporting purposes when required. 

A gas-chromatographic column was operated under the following conditions flow rate (50°C) 20cm /min, column temperature 50°C, inlet pressure 820 mm, outlet pressure 760 mm, and volume of stationary liquid phase (50°C) 3.12 cm. The following data were obtained for time of peak maximum from time of injection air, 0.50 min w-hexane, 3.50 min hexene-1, 3.86 min n-heptane, 4.10 min. 

The lower detection limit of a detector is defined as the minimum amount of compound detectable at a given signal-to-noise ratio. Ideally the detection limit should be determined independently of the chromatographic separation system using standard dilution devices. Detection limits are often expressed in g/s (mass flow-sensitive detectors) or g/mL (concentration-sensitive detectors) to obtain a value which is independent of the measuring conditions (flow rate, etc.). 

The choice of operating conditions (flow-rates and switching time) of an SMB chromatographic process is not a simple task. Some constraints have to be met to recover the more-adsorbed species (A) in the extract and the less-adsorbed species (B) in the raffinate. These constraints are expressed in terms of net fluxes of components in each section considering an equivalent TMB. In section 1 species A must move upward to the extract port, in sections 2 and 3 species A must move downward to the extract port and species B must move to the raffinate port and in section 4 the net flux of species B has to be downwards (Fig. 3.4-8). 

Figure 1. Chromatogram showing the separation of the cis and trans isomers of 3-hexen-l-ol (cis/trans 3-H) and a and p-naphthoflavone (NF) on a 25-cm p-CD column using 50% aqueous methanol as the mobile phase. [Conditions flow rate = 1.0 ml/min., 24 C, and chromatographic system II. The wavelength for detection of the 3-hexen-l-ols was 220 nm while that for the naphthof lavone s was 273 nm.]...

SEC measurements were made using a Waters Alliance 2690 separation module with a 410 differential refractometer. Typical chromatographic conditions were 30°C, a 0.5-ml/min flow rate, and a detector sensitivity at 4 with a sample injection volume of 80 fil, respectively, for a sample concentration of 0.075%. All or a combination of PEO standards at 0.05% concentration each were used to generate a linear first-order polynomial fit for each run throughout this work. Polymer Laboratories Caliber GPC/SEC software version 6.0 was used for all SEC collection, analysis, and molecular weight distribution overlays. 

A six-port valve was first used to interface the SEC microcolumn to the CZE capillary in a valve-loop design. UV-VIS detection was employed in this experiment. The overall run time was 2 h, with the CZE runs requiring 9 min. As in the reverse phase HPLC-CZE technique, runs were overlapped in the second dimension to reduce the apparent run time. The main disadvantage of this yu-SEC-CZE method was the valve that was used for interfacing. The six-port valve contributed a substantial extracolumn volume, and required a fixed volume of 900 nL of effluent from the chromatographic column for each CZE run. The large fixed volume imposed restrictions on the operating conditions of both of the separation methods. Specifically, to fill the 900 nL volume, the SEC flow rate had to be far above the optimum level and therefore the SEC efficiency was decreased (22). 

Operation of fhe interface should be compatible wifh all chromatographic conditions which are likely to be encountered, including flow rates from around 20 nlmin to around 2 mlmin solvenf systems from 100% organic phase to 100% aqueous phase, gradienf elution, which is of particular imporfance in... 

The time taken for an analyte to elute from a chromatographic column with a particular mobile phase is termed its retention time, fan- Since this will vary with column length and mobile phase flow rate, it is more useful to use the capacity factor, k. This relates the retention time of an analyte to the time taken by an unretained compound, i.e. one which passes through the column without interacting with the stationary phase, to elute from the column under identical conditions (to). This is represented mathematically by the following equation ... 

The schematic diagram of the experimental setup is shown in Fig. 2 and the experimental conditions are shown in Table 2. Each gas was controlled its flow rate by a mass flow controller and supplied to the module at a pressure sli tly higher than the atmospheric pressure. Absorbent solution was suppUed to the module by a circulation pump. A small amount of absorbent solution, which did not permeate the membrane, overflowed and then it was introduced to the upper part of the permeate side. Permeation and returning liquid fell down to the reservoir and it was recycled to the feed side. The dry gas through condenser was discharged from the vacuum pump, and its flow rate was measured by a digital soap-film flow meter. The gas composition was determined by a gas chromatograph (Yanaco, GC-2800, column Porapak Q for CO2 and (N2+O2) analysis, and molecular sieve 5A for N2 and O2 analysis). The performance of the module was calculated by the same procedure reported in our previous paper [1]. 

In the pneumatic pumping system, the pressure (and not the flow rate) is maintained constant as variations in chromatographic conditions occur. Thus, a change in mobile phase viscosity (e.g. gradient elution) or column back pressure will result in a change in flow rate for these types of pumps. The gas displacement pump in which a solvent is delivered to the column by gas pressure is an example of such a pneumatic pump. The gas displacement system is among the least expensive pumps available and is found in several low cost instruments. While the pump is nonpulsating and hence, produces low noise levels with the detectors in current use, its flow stability and reproducibility are only adequate. In addition, its upper pressure limit is only 2000 psi which may be too low in certain applications. 

Figure 2. Chromatographic profile of DNA hydrolysate from rat liver 12 h after application of 133 mg/18.5 mCi/kg l l-[2f3- H]-nitrosodipropylamine. Key O, A , CPAf. Conditions included column, 30-cm i rBondapak-C18 eluant, 3% methanol/0.05M ammonium formate (pH 3.5) flow rate, 1 mL/min and sample, 10 mg hydrolyzed DNA containing unlabeled 7-propylguarune (41 fig/mg DNA) and 74sopropylguanine (34 fig/mg DNA), which was injected five times in five...

In 2001, Valko et al. reduced the column length to only 50 mm and increased the flow rate to 2mLmin [42]. The gradient time was diminished to 2.5 min with a gradient cycle time of 5 min. Measurement of CHI and evaluation of log P were excellent with a 3-fold improved productivity. In these conditions, the system dwell volume (Vd) becomes essential and only dedicated chromatographic devices with Vjy lower than 0.8 mL can be used [42]. Special attention should be paid to the injected volume, which must remain lower than 3 pL to avoid any overloading or extra-column volume contributions. 

Figure 1 Chromatogram of a neutral compound (toluene) with watenacetonitrile mobile phase. Chromatographic conditions — column 30 cm x 3.9 mm p-Bondapak C18 (10-pm particle size) mobile phase watenacetonitrile (50 50) flow rate 1.5 ml/min column temperature ambient detector wavelength 254 nm.

Figure 14 Separation of 1,2/1,4 ketal with and without protection from oxidative degradation. Chromatographic conditions were column 25 cm x 4.6 mm Zorbax C8 (5-pm) column mobile phase 100 mM KH2P04 (pH 6.5) acetonitrile (50 50) flow rate 1.0 ml/min column temperature 35°C detector wavelength 220 nm. (A) Acetonitrile degassed. (B) Acetonitrile not degassed.

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