Methanol as an organic modifier

There is a growing interest on selection of the mobile phase components such as organic solvent, acid, and pH. The results showed that the use of methanol in a mobile phase, induced very slow elution of flavonoids and isoflavones from the chromatography column, and the peaks were not symmetrical. Furthermore, the use of methanol as an organic modifier significantly decreased the efficiency of the chromatographic column, as compared to acetonitrile, which was observed on the basis of the low number of theoretical plates. 

Mobile phase The separation was carried out by the application of two mobile phases with the following components, containing methanol as an organic modifier (p) ... 

S.6 Choice of Organic Modifier. Selection of the organic modifier type could be viewed as relatively simple The usual choice is between acetonitrile and methanol (rarely THF). In Chapters 2 and 4 the principal difference in the behavior of methanol and acetonitrile in the column is discussed. In short, methanol shows more predictable influence on the analyte elution, and the logarithm of the retention factor shows linear variation with the concentration of methanol in the mobile phase. Often for the effective separation of complex mixtures of related compounds, this ideal behavior is not a benefit and greater effect of the type and organic concentration on the separation efficiency is required. Acetonitrile as an organic modifier may offer these variations due to the introduction of a dual retention mechanism. The dual retention mechanism was discussed in Chapter 2. 

However, extrapolated capacity factors may be affected by the nature of the organic modifier. In a study concerning the measurement of lipophilicity indices for monosubstituted benzenes by HPLC, the log values of the more polar derivatives appeared to be lower when methanol was used as an organic modifier, compared to the log values obtained with acetonitrile. In contrast, for nonpolar derivatives, acetonitrile led to lower log fcw values. An analogous decrease in the log fcw values for a series of lipophilic 9//-xanthene and 9//-thioxan-thene derivatives was observed when tetrahydrofuran was... 

Bases Metal ion-free, modem stationary phases suitable for separating bases these include Symmetry, Kromasil, Inertsil, Nucleosil AB, Purospher, Zorbax SB, SymmetryShield, HyPurity Elite, Prodigy, XTerra, Bonus (think of Cg tool). First choice acidic pH (e.g. pH 2.5-3.5) with acetonitrile if retention too little, move to pH 7.5 with methanol as the organic modifier. You may also add triethylamine or octyl-amine (ca. 50-500 ppm) or for strong bases add an ion pair reagent (PIC B), e.g. 50-100 mM octasulfonic acid. 

RPIP chromatography uses a hydrocarbonaceous stationary phase and either an aqueous or aqueous-organic mobile phase which also contains the counter-ion. The stationary phase is usually an octadecyl bonded phase and the mobile phase is usually an aqueous buffer with either methanol or acetonitrile as an organic modifier. The choice of counter-ions depends on the solutes to be separated, but generally for the separation of acids a hydrophobic organic base is added to the mobile phase, while for the separation of bases a hydrophobic organic acid is added. Separations of other compounds are similarly obtained by the addition of an appropriate counter-ion. 

The correlation coefficient was improved to 0.952 when the values for theobromine were excluded. The difference in the p Ta value, 0.770, may be due to solvent effects. The pH of the eluents was measured before mixing with methanol. However, the difference was similar to that reported previously. The pH values of the buffer solutions were influenced by the addition of methanol or acetonitrile as an organic modifier. How to obtain an absolute pH value in eluent containing organic modifier is still unclear for practical purposes. 

Fortunately, the laboratory separation chemist had some experience with the analytical separation of these isomers. It was known that good separation could be achieved on a Cjg stationary phase with methanol as the organic modifier, whereas separation was inadequate using acetonitrile. Optimization of the separation using methanol/water was pursued on an analytical scale column (Cjg) containing an 8 pm particle packing material for preparative separation. 

The most common mobile phase for supercritical fluid chromatography is CO2. Its low critical temperature, 31 °C, and critical pressure, 72.9 atm, are relatively easy to achieve and maintain. Although supercritical CO2 is a good solvent for nonpolar organics, it is less useful for polar solutes. The addition of an organic modifier, such as methanol, improves the mobile phase s elution strength. Other common mobile phases and their critical temperatures and pressures are listed in Table 12.7. 

At least four chromatographic standards prepared at concentrations equivalent to 50-70% of the limit of quantitation (LOQ) up to the maximum levels of analytes expected in the samples should be prepared and analyzed concurrently with the samples. In LC/MS/MS analysis, the first injection should be that of a standard or reagent blank and should be discarded. Then, the lowest standard should be injected, followed by two to four blanks, control samples, fortifications or investigation samples, followed by another chromatographic standard. This sequence is then repeated until all the samples have been injected. The last injection should be that of a standard. In order to permit unattended analysis of a normal analysis set, we recommend that samples and standards be made up in aqueous solutions of ammonium acetate (ca 5 mM) with up to 25% of an organic modifier such as acetonitrile or methanol if needed. In addition, use of a chilled autosampler maintained at 4 °C provides additional prevention of degradation during analysis. 

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