Separation Stronger solvent

However, consider the separation of solutes that are more polar than the aromatic hydrocarbons, for example mixtures of ethers or aliphatic esters. If it were attempted to separate a series of aliphatic esters on silica gel employing n-heptane as the mobile phase it would be found that the retention of the later eluting solutes was inordinately long. If a slightly stronger solvent, such as chloroform was used as an alternative to n-heptane, it would be found that the less polar esters... 

We have our conditions if all the peaks are accounted for and separated. If not, we can do k development, control pH by buffering, or change the stronger solvent or the type of column to produce an a change. We have a starting point, and that is half of the battle. 

The most common variable used to control a is the stronger solvent in the mobile phase. The stronger solvent is the mobile phase component most like the column in polarity. Changing the chemical nature of this stronger solvent will produce shifts in the relative peak positions. For instance, if we are unable to achieve the desired separation on a C18 column using acetonitrile in water, we can produce an a effect by shifting to methanol in water an opposite effect occurs on switching to tetrahydrofuran in water (Fig. 4.8). 

Injection samples need to be as concentrated as possible and this leads to problems. A column acts as a sample concentrator. If the solution starts out saturated, it will supersaturate on the column, precipitate, and plug the column. I have seen a column with a 3-cm-deep plug that had to be bored out with a drill bit and a spatula. A couple of injector loops full of the stronger solvent in a mixed mobile phase will clear this if there is still some flow, but the separation will have to be repeated. It is better to dissolve the compound, then add a half volume of additional solvent, ensuring that there will be no precipitation on injection. 

In another study [45], the quantitation of risperidone and its 9-hydroxy metabolite in EDTA-anticoagulated plasma was reported. After protein precipitation with acetorritrile, the superrratant was injected onto an on-line SPE-LC-MS system. The sample was loaded in a weak solvent (15% acetorritrile in 10 rmnol/1 aqueous AmOAc) to a 12.5x4.6-mm-ID Cig SPE-colurrm (5 pm) for 1 min at 0.7 rtil/min. After valve-switching, the SPE colurtm was backflushed with a stronger solvent (80% acetonitrile in 10 mmoFl aqueous AmOAc) to the 30x2.1-mm-ID Cig analytical colurrm (3.5 pm) at 0.35 ml/min. Positive-ion ESI-MS was applied in SRM mode, monitoring transitions for risperidone, 9-hydroxy risperidone, and an ANIS. No separation of the 7-hydroxy and 9-hydroxy metabolites was achieved under these conditions. The method was validated. 

Insertion/introduction of the needle into the GC port, depression of the plunger, and thermal desorption of the analytes. Alternatively, the analytes are washed out of the fiber by the HPLC mobile phase via a modified HPLC six-port injection valve and a desorption chamber that replaces the injection loop in the HPLC system. The SPME fiber is introduced into the desorption chamber, under ambient pressure, when the injection valve is in the load position. The SPME-HPLC interface enables mobile phase to contact the SPME fiber, remove the adsorbed analytes, and deliver them to the separation column. Analytes can be removed via a stream of mobile phase (dynamic desorption) or, when the analytes are more strongly adsorbed to the fiber, the fiber can be soaked in mobile phase or another stronger solvent for a specific period of time (e.g., 1 min) before the material is injected onto the column (static desorption) (Fig. 6). 

Complex mixtures, i.e. those that contain 20 or more components, in most cases present separation problems. Under isocratic conditions (Figure 18.1)the initial peaks are likely to be poorly resolved and the final peaks will probably be broad and flat and may be swamped by background noise. If a weaker solvent is used, the initial peaks show improved resolution but the final ones are not eluted at all. A stronger solvent compresses the early peaks together more, so that some components can no longer be distinguished. 

In reversed-phase and ion-exchange chromatography it is a common procedure to run a gradient scouting run if the conditions for a successful separation are unknown. Such a mn is performed from 10 to 100% B solvent (stronger solvent) with a linear profile. (As already explained, a 100% A mobile phase is often not recommended in reversed-phase chromatography because the alkyl chains are collapsed and equilibration with... 

A retention-order reversal would not occur if all three molecules underwent the same interactions with the mobile and stationary phases. Different separation mechanisms must occur in each of the two chromatograms. The dichloromethane interacts differently with each PAH, as shown by different slopes in a plot of the logarithms of relative retention versus dichloromethane concentration (Figure 5). If the molecules had the same retention mechanisms, these plots would be parallel lines. The three plots are nonparallel, and only the one for dibenzo[cd,/ra]perylene is linear. The curvature in the other two plots could indicate different interactions in each at low or high dichloromethane concentrations. This behavior could be due to the stronger solvent having increased interaction with the PAHs so that the two larger PAHs become more nonplanar. 

For the separation of decaborane on silica, Hermanek (42) has noted that tetrahydrofurane and ethyl ether appear anomalously weak as solvents. The apparent eluotropic series is tetrahydrofurane, n-hexane i% cyclohexane, ethyl ether, CCI4 (cf. Table 8-1), followed by several stronger solvents in approximately their normal order. The greater adsorption of decaborane from the two ether solvents is attributed to the formation of a polar decaborane-ether adduct, which is expected to be more strongly adsorbed than the nonpolar decaborane. 

An eluotropic series can be used to find an optimum solvent strength for a particular separation. Using a solvent of constant composition is called isocratic elution. If an isocratic solvent is too strong (if the k values for the solutes are too small), a weaker solvent is substituted. On the other hand, if the initial solvent is too weak (the k values are too large), a stronger solvent is selected. This trial-and-error approach to finding the optimum solvent can be done more rapidly by TLC than by column chromatography. 

Alternatively, retention can be predicted from solute log Po/w values. The most suitable organic solvent to be used as modifier of the mobile phase should be chosen according to the polarity of the eluted compound. For SDS, a low propanol content ( 1%, v/v) is useful to separate compounds with logPo/w< — 1> such as amino acids. A larger amount of propanol ( 5-7%) is needed for compounds in the range -1 < log Po/w < 2, such as diuretics and sulfonamides. Other alcohols (<10% butanol or <6% pentanol) are required for apolar compounds with log Po/w >3, such as steroids. This rule of thumb is, however, not always valid propanol is too weak for cationic solutes, such as phenethylamines (0 < log Po/w < 1-7) or j -blockers (1 electrostatic attraction to the anionic surfactant molecules adsorbed on the stationary phase makes a stronger solvent necessary. 

It will become clear in the following that it is necessary to ensure that the sample extract to be injected on column uses a solvent that is either the HPLC mobile phase itself (the t = 0 composition in the case of gradient elution) or a solvent of lower elution strength (Section 4.4.2a) otherwise, the chromatographic peak is broadened by the interference with the intended separation by partitioning between mobile and stationary phases, since now refers to the equilibrium distribution of A between the stationary phase and a stronger solvent than the mobile phase. 

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