Chromatogram, HPLC experiment

Problems that arise with HPLC experiments are usually associated with abnormally high or low pressures, system leaks, worn injectors parts, air bubbles, or blocked in-line filters. Sometimes these manifest themselves on the chromatogram and sometimes they do not. In the following subsections, we address some of the most common problems encountered, pinpoint possible causes, and suggest methods of solving the problems. You can also refer to the troubleshooting guide in Chapter 12 for possible solutions. 

MICROPELLICULAR AND POROUS STATIONARY PHASES. In order to compare the features of micropellicular and porous stationary phases in rapid protein HPLC, experiments were conducted with two columns of similar size, each one of which was packed with different stationary phase and operated under comparable conditions. In this experiment the results of which are shown in Figure 10, the operational conditions were optimized for the micropellicular stationary phase (conditions A) and used subsequendy for separation of the same mixture with the porous stationary phase under identical conditions. Thereafter, the elution conditions were optimized for the column packed with the porous stationary phase (condition B) and the experiment was repeated with the column packed with micropellicular stationary phase. The chromatograms are depicted in Figures 10 and 11 and the results of the two approaches are summarized in Table III. 

HPLC experiments were carried out using a Model 510 Liquid Chromatograph equipped with a Model 481 UV detector (Waters Associates, Milford, Massachusetts, U.S.A.). The chromatograms were processed by means of a chromatographic workstation (Baseline 810). Separation was performed on a reversed-phase Supel-... 

HPLC experiments were carried out using a Model 510 Liquid Chromatograph equipped with a Model 481 UV detector (Waters Associates, Milford, Massachusetts, U.S.A.). The chromatograms were processed by means of a chromatographic workstation (Baseline 810). Separation was performed on a reversed-phase Supelcosil LC-18 column (250 x 4.6 mm I.D., 5 jim particle size) (Supelco, St. Louis, Missouri, U.S.A.). The samples were injected into a 50 jl1 loop using a Rheodyne 7125 valve (Rheodyne, Cotati, California, U.S.A.). 

Dendritic molecules 33 and 34 were then incubated with PGA in PBS (pH 7.4) at 37 °C. Control solutions were composed of buffer without the enzyme. The sequential fragmentation illustrated in Fig. 5.31 was monitored by observing the disappearance of dendrons 33 or 34 and the release of 4-nitroaniline by RP-HPLC. As expected, dendron 33 could not be activated by PGA and remained intact for 72 h (data not shown). However, dendron 34 showed clear activation upon incubation with PGA and its corresponding peak completely disappeared from the HPLC chromatogram as 4-nitroaniline appeared (Fig. 5.32). No 4-nitroaniline was observed in the control experiment when dendron 34 was incubated in the buffer without PGA. 

A gSe of two Waters ultrastyragel columns, designated 10 A and 10 A and a Waters pump (Model 590) for HPLC were used in this study. The elution solvent was tetrahydrofuran (THE) which was distilled in the presence of a small amount of CaH in order to remove the peroxide. The flow rate was maintained at 1 ml/min. The sample injection volume was -30 pi. The chromatogram detected by the differential refractometer (Waters R401) was recorded on a strip chart recorder. All experiments were performed at room temperatures with concentrations below the over-loading condition. 

If the retention times of the analytes are known, or there is an efficient method for their detection on-line, such as UV, MS or radioactivity, stop-flow HPLC-NMR becomes a viable option. In the stop-flow technique, all the usual techniques available for high-resolution NMR spectroscopy can be used. In particular, these include valuable techniques for structure determination such as 2-dimensional NMR experiments which provide correlation between NMR resonances based on mutual spin-spin coupling such as the well-known COSY or TOCSY techniques. In practice, it is possible to acquire NMR data on a number of peaks in a chromatogram by using a series of stops during elution without on-column diffusion causing an unacceptable loss of chromatographic resolution. 

Fully automated analysis is also an option wherein the samples are placed in an autosampler and predefined HPLC-NMR experiments are performed. This is covered in detail elsewhere in this volume, but in summary, the software allows automatic detection of UV peaks in the chromatogram based on predetermined time-windows or peak intensities. The successful detection of each UV peak triggers the system to stop the flow at an appropriate time to isolate the peak in the NMR flow probe. Then data relating to the peak (intensity, retention time, etc.) are transferred to the NMR host computer and used to define the parameters for the automatically acquired NMR spectrum. This automatic NMR operation includes field homogeneity optimisation, setting and optimisation of all NMR acquisition parameters, and the predefinition of the resultant signal-to-noise ratio required in the spectrum. The measurement of 2-dimensional (2D) NMR spectra can also be performed. 

Figure 5.2.3 depicts the HPLC chromatogram of a tomato peel extract monitored by UV absorbance at 469 nm. The separation was performed on a 150 x 4.6 mm C30 column (ProntoSil, 3 xm, 200 A, Bischoff, Germany) at room temperature and a flow rate of 1 ml/min with a binary mixture of acetone/ water, developed for LC-NMR experiments. The 50-min gradient elution was performed in four steps, i.e. (1) an initial 3 min with 75/25 (v/v) acetone/water, (2) a 24-min gradient to 100% acetone, (3) an isocratic step from 27 45 min with 100% acetone, and (4) a 2-min gradient back to the initial conditions. 

Figure 2. Radioactivity chromatogram of sulfur compounds derivatized with monobromobimane. The reversed-phase HPLC separation is based on the hydrophobic properties of the bimane-sulfur adducts but peak area is based on "S-radioactivity of the compounds. At time 0 sulfite and thiosulfate impurities are present before addition of the hepatopancrease tissue homogenate. This was a 60 min experiment to determine the sulfide detoxifying functions of the hepatopancrease of the hydrothermal vent crab Bythograea thermydron. During this time the proportion of radioactivity in sulfide rapidly decreases and thiosulfate and sulfate accumulate as end products. Two intermediates, pi and p2 accumulate then decrease during the experiment. The two intermediates are believed to be polysulfides based on similar elution times of polysulfide standards. (Figure is the unpublished chromatograms from the data in Vetter et al. (24)-) continued on next page.

The chromatograms presented in Figures 4 and 8-12 were obtained by using the HPLC unit depicted in Figure 2 Results shown in the other figures were from experiments carried out with Hewlett Packard Model 1090 liquid chromatograph. 

Fig. 9 Radio-HPLC chromatogram for the chirality experiment. The single peak at 12.5 min is due to [99mTc(HYNIC-BA)(tricine)(PSA)], where HYNIC-BA is AT-benzyl-6-(2-sulfobenz-aldehydehydrazono)nicotinamide and PSA is pyridine-3-sulfonic acid, and the peaks at 19.8 and 20.4 min are from [99mTc(HYNIC-MBA)(tricine)(PSA)], where HYNIC-MBA is N-((R) (+)-a-methylbenzyl)-6-(2-sulfobenzaldehydehydrazono)nicotinamide...

A reaction mixture was prepared that contained a metal at concentrations in excess of that of ATP. The reaction was started by the addition of the enzyme, and samples were taken and analyzed by the HPLC method. Surprisingly, the chromatograms for the experiments that included metals were different from the ones obtained earlier. In the original experiments, only two peaks were present, those representing ATP and AMP. However, in the experiment that included the metal calcium, the chromatograms showed the elution of an additional peak jsut after the ATP emerged (Fig. A.5B). Further studies showed that the new peak had a retention time identical to that of ADP, and therefore we assumed that this second peak was ADP. From these findings, we speculated that the metal had stimulated the activity of an enzyme that catalyzed the formation of ADP. 

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