Chromatograms bonded-phase

These separations can be carried out using a silica-based bonded phase however, the important advantage of organic polymer stationary phase materials is their chemical stability. The columns can be washed by using an alkaline solution after a certain number of injections. According to the chromatograms, the proteins in serum are completely eluted and nothing remains inside the column. However, the pressure drop in this type of analysis... 

The pKa of the imidazole ring is near 6 (16) so histamine would only exist as an ion in the acidic (pH = 2-3) mobile phase. One would predict no retention on a bonded phase column under this condition however, it does occur. Figure 3 is the simplest way to account for this retention. Here, the mineral acid acts as the counter-ion, as well as the buffer. All of the histamine in the mobile phase is in the ionic form and is in equilibrium with the ion-pair which is only soluble in the stationary phase chemically bonded to silica. Histamine only elutes in the ionic form and is then derivatized for detection. A sharp peak in the chromatogram with good shape and no change in retention time with variation in sample concentration indicates a working system. However, if the paired ion has some solubility in the mobile phase, peak tailing occurs. 

Analytical Methods. All of the sorbent extracts were analyzed by using GC-MS. The solutions were chromatographed on a 30-m X 0.32-mm i.d. Supelcowax 10 capillary column with a film thickness of 1 /um (Supelco). The Supelcowax 10 is a Carbowax PEG 20 M bonded-phase capillary column. The instrument conditions were as follows injector, 250 °C separator oven, 250 °C column over initial, 40 °C programmed to 250 °C at 6 °C/min linear velocity of helium carrier gas, 35 cm/s at 40 °C column head pressure, 6 lb/in.2 mass range, 33-333 amu scanned in 1-s intervals. Two-microliter aliquots were injected in the split mode at a 10-to-l split ratio. A typical chromatogram appears in Figure 1. 

Other workers have successfully applied these principles to the optimization of their separations, and their papers attest to the value of this method Antle67 has separated steroids by normal phase on an amine bonded phase, and Lehrer68 has separated phenols and cresols by reverse phase on a C8 bonded phase. Both provide interesting chromatograms. 

Only the HPLC using a bonded phase in the reverse-phase mode accomplished the separation of the hemoglobins. The chromatogram is shown in Figure 2-5. 

In the following example the assumption was made that two compounds were to be loaded onto a bonded-phase cartridge and, in the eluent that was chosen, peaks A and B had a k of 10 and 30, respectively (Figure 6-32). These conditions imply that if a continuous stream of liquid containing components A and B is pumped across a cartridge, a frontal chromatogram results, as is illustrated in Figure 6-32. 

A recent modification of the atmospheric pressure ionization technique involving a special low dead volume interface was described by Thomson etal. [27]. They employed packed microbore columns (170 p, 320 p, and 500 p I. D. with lengths ranging from 5 to 15 cm) in conjunction with a low-volume, wall-coated capillary column as an interface. The total ion current chromatogram of the tryptic digest sample of about 1 picomole of human growth hormone is shown in figure 29. The column was packed with an octadecyl bonded phase... 

Figure 1.5. A reversed-phase HPLC chromatogram of three organic components eluting in the order of polar first and nonpolar last. The basic pyridine peak is tailing due to a secondary interaction of the nitrogen lone-pair with residual silanol groups of the silica based bonded phase. Figure reprinted with permission from reference 8, Chapter 2.

Figure 3.8. Comparative chromatograms illustrating the effect of selectivity of C8, cyano-, and phenyl-bonded phases. Diagram courtesy of MAC-MOD Analytical.

Figure 3.17. Comparative chromatograms of a low-coverage C18-bonded phase designed for the separation of polar water-soluble analytes versus that of a conventional high-coverage CIS-bonded phase. Chromatograms courtesy of Waters Corporation.

Guidelines on mobile phase selection are discussed in Chapter 2, Section 2.3 and are illustrated in the case study below. After evaluation of the first sample chromatograms, further method development to fine-tune the separation will be performed until all method goals are achieved. Other bonded phases or column configurations can be selected to enhance method performance. These are discussed in the Section 8.6. 

Figure 8.20. Three comparative chromatograms of three different bonded-phase columns using the optimized mobile phase conditions shown in Figure 8.15. The surprise finding of peak shape problems with two specific impurities might be attributed to the higher silanophilic activity of the column. Data presented in parts at Eastern Analytical Symposium, Somerset, New Jersey, November 2003.

In order to reduce the time of analysis for routine reversed-phase analysis, GC conditions were optimized for each bonded phase chain length. Figures 7a and 7b show chromatograms of test mixtures for mono-, dl- and trlfluoro derivatives of octyl and octadecyl phases. Including the internal standards. Analysis times were reduced to 11 minutes and 18 minutes, respectively, for each phase type. The response factors for both the octyl and octadecyl phases were found to be nearly unity for an FID detector. 

Figure 7. Optimized separation conditions for both octyl (top) and octadecyl (bottom) bonded phases on an SE-30 capillary (12 m) column. The octyl separation conditions are 65 C to 135°C at 6 C/mln. The octadecyl separation conditions are 180°C to 210 C at 2 C/mln. Identification of compounds In chromatograms are ...

Fignc 11.1 Sqnration of sugars on an aminopropyl bonded phase. Peak identification (1) fiructose (2) glucose (3) sucrose (4) maltose (S) lactose. (Chromatographic conditions Column Waters High-Performance Carbohydrate Column. Mobile phase aoetonitrile/water 7S-2S v/v.) Chromatogram courtesy of D. J. Phillips, Waters Corp.)... 

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