Isocratic separation

Two variations of the technique exists isocratic elution, when the mobile phase composition is kept constant, and gradient elution, when the mobile phase composition is varied during the separation. Isocratic elution is often the method of choice for analysis and in process apphcations when the retention characteristics of the solutes to be separated are similar and not dramaticallv sensitive to vei y small changes in operating conditions. Isocratic elution is also generally practical for systems where the equilibrium isotherm is linear or nearly hnear. In all cases, isocratic elution results in a dilution of the separated produces. 

Fig. 19. Efficiency of RPC in protein separation. Isocratic RPC separation of murine epidermal growth factor (after elution as a single peak from a diethylaminoethyl cellulose column). Column RP 18 (300x7.8 mm dP = 10 pm) 40 °C eluent 0.04 M triethylamine acetate in water — acetonitrile (74 26), pH 5.6 flow rate 0.8 ml/min UV detection at 254 nm, 0.05 AUFS injection 100 pg in 100 pi. The a peak was due to the polypeptide of 53 amino acid residues (M =6040 g/mol), the p component did not contain the asparagine residue in position 1. (From Ref.77) with permission)...

Reversed-phase chromatography also separates, isocratically, vitamin D2 or D3 from their respective previtamins and inactive isomers (207), but, unlike normal-phase chromatography, it can separate vitamin D2 from D3 using nonendcapped stationary phases (198). The 25-hydroxylated metabolites of vitamins D2 and D3 can be separated from one another using a Vydac 201 TP column (37). The separation of vitamin D2 from vitamin D3, and 25-hydroxyvitamin D2 from 25-hydroxy vitamin D3, allows the D2 form of the vitamin or its metabolite to be used as an internal standard for quantifying the corresponding D3 form. 

Normal-phase HPLC is capable of separating isocratically all of the eight unesterified tocopherols (T) and tocotrienols (T-3) that occur in nature, and it has been utilized in determining the distribution of these vitamers in a wide variety of fats, oils, and foodstuffs. The elution sequence... 

An optoelectronic image device has been used as an HPLC detector to obtain UV absorption spectra of shale oil aromatic hydrocarbons separated isocratically (10). Since isocratic separations maintain a constant mobile phase composition, the... 

Figure 5. HPLC absorption chromatogram (254 nm) of a contaminated two component mixture separated isocratically...

The approach of Jandera and Chura ek allows the optimization of the resolution of one given (arbitrary) pair of sample components and the minimization of the retention volume of another (arbitrary) solute. It requires knowledge of the isocratic retention vs. composition relationships of these three solutes. The information needed may be acquired from gradient elution experiments performed as part of the optimization procedure, or from separate isocratic experiments. The selection of the three arbitrary solutes considered during the optimization process appears to have a large effect on the result and the resolution cannot be optimized throughout the chromatogram. 

Figure 4. HPLC separation of phenolic components of an aqueous leachate from the surface of corn leaves infected with Colletotrichum graminicola. a) untreated leachate b) base hydrolyzed leachate c) acid hydrolyzed leachate. Compounds 1 and 2 are isomers of p-coumaric acid. Compounds separated isocratically on a reversed phase C-18 column with a 70 % to 30 % mixture of absolute methanol and 1 % acetic acid. Reproduced with permission from Ref. 69. Copyright 1989 Academic Press, Inc.

A mixture of six compounds with different acid and base properties was separated isocratically on a reversed Cig phase. The influencing parameters that needed to be optimized simultaneously were the methanol content (%B) and the pH of the mobile phase. For systematic optimization it was necessary to perform a limited number of... 

The quality of an HPLC analysis is often equated with the resolution of the peaks achieved, and as previously discussed this may be optimised by adjusting the solvent composition to obtain appropriate capacity factors for the solutes. For difficult separations, isocratic elution may be inadequate and the desired resolution may only be achieved using gradient elution... 

A mixture of six compounds with different acid and base properties was separated isocratically on a reversed C ig phase. The influencing parameters that needed to be optimized simultaneously were the methanol content (% B) and the pH of the mobile phase. For systematic optimization it was necessary to perform a limited number of experiments within the boundaries 45-65% B and pH 3.0-6.0. The results, i.e. the minimum resolution of the critical peak pair as a function of these parameters, were evaluated mathematically and represented as a surface in three-dimensional space (similar to Fig 18.15). It could be... 

Various sulfur-containing species in the scrubber solution of a flue-gas desulfurization plant (see also Section 9.2) are to be analyzed. According to Table 1-1, non-polarizable ions such as sulfite, sulfate, and amidosuMbnic acid with pfC values below 7, are separated isocratically by HPIC using a conventional anion exchanger and are detected via electrical conductivity. A suppressor system may... 

Ricci et al. [38] used gradient elution separation for the determination of five organic and inorganic species of arsenic. Although the method is effective and separates all five species in one run, the column must be restabilized with the weaker eluant for 1 hour after each run. For this reason, the authors suggested using two separate isocratic approaches with two eluant buffer systems. The method was applied for the determination of organoarsenic compounds in air, but could be used for other environmental samples. 

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