Automated Isocratic Development

Obviously, to make this happen we have to 1) have a way of measuring the completeness of a separation, 2) define what constitutes the best separation, and 3) define a systematic pattern for making changes. 

When we look at a separation to judge whether two peaks are separated, we look at the centers of the peaks, but more importantly, we look at the valley separating the peaks. An ideal separation is one in which all peak pairs are baseline separated the peak valleys all come down and touch the chromatographic baseline. If we were to draw a line connecting the two peak tops (Fig. 14.2, line 1-2), then drop a perpendicular line from the center of this connecting line to the baseline (A-B), the length of the resulting line would represent a standard of baseline separation for these two peaks. 

If the valley did not touch the baseline, it would have a length (A-C) that would be less than the distance to the baseline. If we divide the length of A-C by the length of A-B, we have a mathematical measure of the baseline resolution for the two peaks. Summing these ratios of baseline resolution for every peak pair gives us a resolution sum for the whole chromatogram. 

Once we can put a relative value on the degree of baseline resolution between two peaks, the rest is easy. The next step is to work to make the resolution sum number as large as possible, moving us toward a better separation. The computer changes one system variable, has the autoinjector make a new injection, and calculates a new resolution sum.Through repeated variable changes, new injections, and recalculation, it works to maximize this value. 

Left to itself, the computer will carry this optimization to ridiculous extremes, optimizing for every baseline pair it finds no matter how long it takes. We would like the final chromatogram to run in a reasonable time, so we set limits on the length of the expected run time. We also need to specify the expected number of peaks in this run time. Now the computer will search until it has maximized the baseline resolution for the expected number of peaks in the preset run time. 

---

There are four basic system types. Type I are basic isocratic systems used for simple, routine analysis in a QA/QC environment often for fingerprinting mixtures or final product for impurity/yield checking. Type II systems are flexible research gradient systems used for methods development, complex gradients, and dial-mix isocratics for routine analysis and standards preparation. They fit the most common need for an HPLC system. Type III systems are fully automated, dedicated systems used for cost-per-test, round-the-clock analysis of a variety of gradient and isocratic samples typical of clinical and environmental analysis laboratories. Type TV systems are fully automated gra-... 

Recently, Alexander et al. [44] reported the use of an automated separation system developed for micro-LC and CEC using both isocratic and gradient elutions. The complete system is shown in Fig. 2.15. An enlarged view of the coupling of the column to the injection valve presents Fig. 2.16. The mobile phase was delivered by two micro-LC pumps at a flow rate of 30 pL/min to a post injection splitter that houses the column inlet. In the CEC mode, pressure was not applied (no restriction on... 

LC-MS-MS methods for the determination of 5-HIAA in urine have been developed. one of which uses an automated solid phase extraction, isocratic LC elution, and quantification against a stable isotope-labeled internal standard.With these procedures, sample preparation is automated, chromatographic interferences are eliminated, and analytical time is reduced (2 minutes per sample). 

To address development of chiral separations by SFC, Villeneuve and Anderegg have developed an SFC system using automated modifier and column selection valves. Columns (250 x 4.6 mm i.d., 10 pm) packed with Chiralpak AD, Chiralpak AS amylose derivative, Chiralcel OD cellulose carbamate derivative, and Chiralcel OJ cellulose ester derivative (Chiral Techologies, Exton, PA) were connected to a column-switching valve. Candidate samples were run successively on each column using fixed isocratic, isobaric, and isothermal conditions of 2 ml/min, 205 atm pressure, and 40 °C with the vari-... 

Therefore, if a fast, automated, routine method for the measurement of multi-species/elements is the desired analytical goal, it is often best to attempt an isocratic separation method first, because of the complexity of method development and the low sample throughput of gradient elution methods. In fact, a simultaneous method for the separation of As, Cr, and Se species in drinking water samples was demonstrated by Neubauer and coworkers they developed a method to determine inorganic forms of arsenic (As, As+ ), chromium (Cr , Cr ), and selenium (Se, Se and SeCN ) by reverse-phase ion-pairing chromatography with isocratic elution. Details of the HPLC separation parameters/conditions they used are shown in Table 18.2. 

---