Chromatogram isocratic

Figure 2. HPLC chromatograms (isocratic mode, 60% methanol, 40% water) of sediment extracts from 15 study sites in west Florida coastal waters. Migration profile are compared among sediment extracts and crude extract of Nannochloris sp. cell-free culture [See Moon and co-workers (.26) for specific sites].

Isocratic linear development is the most popular mode of chromatogram development in analytical and preparative planar chromatography. It can be easily performed in horizontal chambers of all types. The mobile phase in the reservoir is brought into contact with the adsorbent layer, and then the movement of the eluent front takes place. Chromatogram development is stopped when the mobile phase front reaches the desired position. Usually 20 X 20 cm and 10 X 20 cm plates are applied for preparative separations, and this makes the migration distance equal to about 18 cm. Due to the fact that the migration distance varies with time according to the equation Z, = (Z, c, and t are the distance of the solvent front traveled, constant,... 

Figure 4.10 Typical routine column test chromatogram for a 30 cm X 4.6 mm I. D. column pacXed with an octadecylsiloxane bonded silica packing of lO micrometers particle diameter. The test mixture consisted of resorcinol (0.55 mg/ml), acetophenone (0.025 mg/ml), naphthalene (0.20 mg/ml) and anthracene (0.01 mg/ml) in acetonitrile, 10 microliters injected. The separation was performed isocratically at 23 C with acetonitrile-water (55 45) as the mobile phase at a flow rate of 1.5 ml/min. Detection was by UV at 254 nm (0.1 AUFS).

In a sample containing many different solutes, with isocratic elution it is sometimes impossible to choose a suitable mobile phase that will result in all k values being within the optimum range. If this is the case, the chromatogram may appear as in Fig. 4.3a. 

If this was the case then the chromatogram would resemble the isocratic chromatogram shown in Fig. 4.3a, with poorly resolved peaks at the start and highly dispersed peaks at the end. 

With isocratic elution and a sample having solutes with a wide range of polarity it is sometimes not possible to achieve the desired resolution in an acceptably short time. It may be possible to improve the chromatogram using gradient elution. A practical example of the development of a gradient is discussed. 

For isocratic LC, the solute does not need to fully elute from the second-dimension column prior to the next sampling period. This is illustrated in Fig. 6.4, where it is shown that more than one sample can be resident in the column at one time. Using the chromatograms shown in Fig. 6.5, which show the effect of various injection volumes that will be discussed later, it is not necessary to wait for the full 2 min of sampling time. This significantly helps to speed up the sampling process and is most useful for SEC, where short elution time ranges are typically found and short times between the injection and nonretained peaks are typical of column operation. 

A typical HPLC separation using a 15-cm column of 15,000 theoretical plates produces peak capacity (Giddings, 1991) of about 80-100 under isocratic conditions and up to 150 under gradient conditions in 1 h(Eq. 7.3, n peak capacity, A number of theoretical plates of a column, and fR and t retention time of the last and the first peak of the chromatogram, respectively). An increase in the number of separated peaks per unit time can be achieved by increased separation speed made possible by monolithic silica columns (Deng et al., 2002 Volmer et al., 2002). This has also been shown for peptides and proteins (Minakuchi et al., 1998 Leinweber et al., 2003). 

FIGURE 14.4 Chromatograms of high speed isocratic capillary LC elution of three components. Column 15 cm x 320 /im inner diameter, 5 /im C18 particles. Column head pressure 6800 psi at 48 /(L/min flow rate. System XTS two-dimensional splitless ultrahigh pressure nano UHPLC, Micro-Tech Scientific, Vista, California. 

Fig. 2.3.6. Overlay of reversed-phase chromatograms of four commercial mixtures of alkylphenols and alkyl phenolethoxylates f-OP, OP[EO]8/9) 4-NP, and NP[EOJio. Column 125 X 4 mm2 Lichrospher RP-18 (5 pm), isocratic elution 1.0 mL min-1, mobile-phase 8 2... 

Fig. 2.5.11. (a) APCI-LC-MS(+), (b) ESI-LC-MS(-t-), (c) ESI-LC-MS(+), (d) ESI-LC-MS(+), (e) APCI-LC-MS(—) and (f) ESI-LC-MS(—) reconstructed ion chromatograms (RIC) of methanolic solution of the household detergent mixture as in Fig. 2.5.2. Chromatographic conditions (a), (b), (e), and (f) RP-Cig, methanol/water gradient elution (c) ion-pairing RP-Cla using trifluoro acetic acid (TFA) (5 mmol), methanol/water gradient elution (d) isocratic elution performed on PLRP-column, eluent methanol/water methane...

Fig. 4. Chromatogram of (left) 100 pg of a mixture of (Z)-L-Ala-L-Ala-OMe and (Z)-D-Ala-D-Ala-OMe on a (Z)-L-Ala-L-Ala-OMe-imprinted MIP-phase, gradient elution (right) 1 mg of a mixture of (Z)-L-Ala-L-Ala-OMe and (Z)-D-Ala-D-Ala-OMe on a (Z)-L-Ala-L-Ala-OMe-im-printed MIP-phase, isocratic elution. Reprinted with permission from Kempe M,Mosbach K (1995) Tetrahedr Lett 36 3563. Copyright 1995 Elsevier Science... 

Fig. 2.19. Chromatogram of carotenoid solution in THF. 1 lutein, 2 canthaxanthin, 3 /Tcryptoxan-thin, 4 lycopene, 5 /1-carotene. ODS column, 5/an, 150mm X 4.6mm. Mobile phase eluent A (methanol-acetonitrile 6 11), eluent B (THF) gradient profile 0-5min, isocratic conditions 95 per cent A 5-20min, gradient to 20 per cent A. Flow rate lml/min. Detection 450 nm. Reprinted with permission from C. Tricard el al. [45].

A simple and rapid RP-HPLC method was developed for the determination of retinoid in galenicals. Commercial preparations were diluted, filered and used for separation. Measurements were carried out in an ODS column (150 X 4.6 mm i.d. particle size 3 /xm). Solvents A and B were methanol-10 mM ammonium acetate (75 25, v/v) and methanol-THF (84 16, v/v), respectively. The flow rate was 0.8ml/min. Gradient conditions were 0-25 min, 0 per cent B 35 min, 100 per cent B, isocratic for 10 min. Typical chromatograms are shown in Fig. 2.37. The repeatability of peak area ranged between 0.48 -3.2 per cent for UV-DAD and 0.57 - 3.1 per cent for fluorescence detection. The reproducibility varied between 0.26 - 4.6 per cent. It was found that the method is precise, selective, sensitive and linear, therefore, it can be employed for the routine quality control of this class of drags [85],... 

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