The Analysis Time

The equations for the analysis time are given in chapter 13. Reiterating equations (15), 

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Now t[i is a minimum when k = 2, that is, when = 3t . There is little increase in analysis time when k lies between 1 and 10. A twofold increase in the mobile-phase velocity roughly halves the analysis time (actually it is the ratio Wu which influences the analysis time). The ratio Wu can be obtained from the experimental plate height/velocity graph. 

Procedure. Select a volume of sample requiring less than 15 mL of titrant to keep the analysis time under 5 min and, if necessary, dilute the sample to 50 mL with distilled water. Adjust the pH by adding 1-2 mL of a pH 10 buffer containing a small amount of Mg +-EDTA. Add 1-2 drops of indicator, and titrate with a standard solution of EDTA until the red-to-blue end point is reached. 

Minimizing Electrolysis Time The current-time curve for controlled-potential coulometry in Figure 11.20 shows that the current decreases continuously throughout electrolysis. An exhaustive electrolysis, therefore, may require a long time. Since time is an important consideration in choosing and designing analytical methods, the factors that determine the analysis time need to be considered. 

The relationship between capacity factor and analysis time can be advantageous when a separation produces an acceptable resolution with a large b. In this case it may be possible to decrease b with little loss in resolution while significantly shortening the analysis time. 

If the capacity factor and a are known, then equation 12.21 can be used to calculate the number of theoretical plates needed to achieve a desired resolution (Table 12.1). For example, given a = 1.05 and kg = 2.0, a resolution of 1.25 requires approximately 24,800 theoretical plates. If the column only provides 12,400 plates, half of what is needed, then the separation is not possible. How can the number of theoretical plates be doubled The easiest way is to double the length of the column however, this also requires a doubling of the analysis time. A more desirable approach is to cut the height of a theoretical plate in half, providing the desired resolution without changing the analysis time. Even better, if H can be decreased by more than... 

Time, Cost, and Equipment Analysis time can vary from several minutes for samples containing only a few constituents to more than an hour for more complex samples. Preliminary sample preparation may substantially increase the analysis time. Instrumentation for gas chromatography ranges in price from inexpensive (a few thousand dollars) to expensive (more than 50,000). The more expensive models are equipped for capillary columns and include a variety of injection options and more sophisticated detectors, such as a mass spectrometer. Packed columns typically cost 50- 200, and the cost of a capillary column is typically 200- 1000. 

Examining equation 12.41 shows that we can decrease a solute s migration time (and thus the total analysis time) by applying a higher voltage or by using a shorter capillary tube. Increasing the electroosmotic flow also shortens the analysis time, but, as we will see shortly, at the expense of resolution. 

Thus, for significant values of (k") (unity or greater) the optimum mobile phase velocity is controlled primarily by the ratio of the solute diffusivity to the column radius and, secondly, by the thermodynamic properties of the distribution system. However, the minimum value of (H) (and, thus, the maximum column efficiency) is determined primarily by the column radius, secondly by the thermodynamic properties of the distribution system and is independent of solute diffusivity. It follows that for all types of columns, increasing the temperature increases the diffusivity of the solute in both phases and, thus, increases the optimum flow rate and reduces the analysis time. Temperature, however, will only affect (Hmin) insomuch as it affects the magnitude of (k"). 

Finally, the speed of response of the detector sensor and the associated electronics once played an important part in optimum column design. The speed of response, or the overall time constant of the detector and associated electronics, would be particularly important in the analysis of simple mixtures where the analysis time can be extremely short and the elution of each peak extremely rapid. Fortunately, modern LC detector sensors have a very fast response and the associated electronic circuits very small time constants and, thus, the overall time constant of the detector system does not significantly influence column design in contemporary instruments. The instrument constraints are summarized in Table 2... 

The minimum solvent consumption will be obtained from the product of the optimum flow rate and the analysis time. 

The solvent consumption is simply obtained by multiplying the analysis time by the optimum flow rate as shown below. 

Employing equation (22) the analysis time was calculated for a series of different separation ratios and the results obtained are shown in Figure 15. 

Figure 15. Graph of the Log of the Analysis Time against the Separation Ratio of the Critical Pair...

It is seen from Figure 15 that the analysis time ranges from about 10,000 seconds (a little less than 3 hr) to about 30 milliseconds. The latter, high speed separation, is achieved on a column about 2 mm long, 12 microns in diameter, operated at a gas velocity of about 800 cm/second. Such speed of elution for a multicomponent mixture is of the same order as that of a scanning mass spectrometer. 

The date of occurrence is an important element since it verifies whether the equipment failure occurred during the analysis time frame. The equipment type is necessary for... 

The Shodex GPC KF-600 series is packed with 3- im styrene-divinylbenzene copolymer gels in a column having a volume of about one-third compared to standard-types of columns, which are best suited for reducing the organie solvents eonsumption, shortening the analysis time, and lowering the detection limit (Table 6.5). 

The linear column (PSS SDV 5 /mm linear) has a wider molar mass fractionation range while keeping the analysis time roughly the same. Therefore the slope of the calibration curve is much steeper and the resolution will be poorer in this case. The second column with a single pore size (PSS SDV 5 /mm 1000 A) separates only below 50,000 Da, but does this very efficiently in the same time. 

The efficiency of many CSPs increases dramatically when liquid eluents are replaced with sub- or supercritical fluids. During a comparison of LC and SFC performed with a Chiralcel OD CSP, Lynam and Nicolas reported that the number of theoretical plates obtained was three to five times higher in SFC than in LC [26]. The separation of metoprolol enantiomers by LC and SFC on a Chiralcel OD CSP is illustrated in Fig. 12-2. Although impressive selectivity is achieved by both techniques, resolution is higher in SFC (R = 12.7) than in LC (R = 4.8), and the higher flowrate in SFC reduces the analysis time. The increased efficiency of SFC also improves peak symmetry. 

The curve exhibits a minimum, which means that there is an optimum mobile phase velocity at which the column will give the minimum HETP and consequently a maximum efficiency. In practice this usually means that reducing the flow rate of a column will increase the efficiency and thus the resolution. In doing so, however, the analysis time will also be increased. As seen in figure 5, however, there is a limit to this procedure, as reducing the column flow rate so that the mobile phase velocity falls below the optimum will result in an increase in the HETP and thus a decrease in column efficiency. 

The above considerations apply to samples where all the components are of interest and all need to be separated and quantitatively assessed. In practice, for many samples, only specific components of the mixture are important and only those need to be separated from the matrix and be analyzed. The components of the matrix need not be resolved and they are of no interest. It follows, that under these circumstances the critical pair will be comprised of the component of interest that has the closest neighbor and the neighbor itself. Such a situation usually greatly simplifies the separation problem but it should be noted that the last peak must still be eluted before the next analysis can be carried out and so the analysis time may not be significantly reduced. 

MALDI-ToF is a technique that allows the molecular weights of proteins and peptides to be determined. It is less susceptible to suppression effects than electrospray ionization and thus is able to be used for the direct analysis of mixtures. In the case of a crude tryptic digest, MALDI-ToF will provide a molecular weight profile of the polypeptides present without the analysis time being extended by the need to use some form of chromatographic separation. 

Titration. This method obviously requires physical removal of a sample from the plant and results in the sample being thrown away. In modem equipment samples can be taken at specified intervals from a flowing sample stream using Flow Injection Analysis, enabling the analysis time to be shortened. 

OS 94] [R 13] [P 74/The analysis time for one sample, less than 1 min, is considerably faster than that of a conventional system, which needs about 2 h [28],... 

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