Volatile eluents

Huber, C.G., Premstaller, A. (1999). Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography-electrospray ionization mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry of proteins. I. Liquid chromatography. J. Chromatogr. A 849, 161-173. 

Another approach is to separate the solvent from the sample before a spectrum is obtained. Because HPLC is often used with a combination of volatile eluents and organic compounds that are not volatile, the solvent can be removed and the isolated component analyzed by FTIR [13]. FTIR is not particularly useful for the identification of inorganic components, particularly ions. 

For preparative ion-exchange separations of nucleotides a volatile eluent is desirable and the use of ammonium carbonate and ammonium acetate have been described (Linz, 1983). 

MS coupled with LC is the most selective detection for alkaloids. As all-volatile eluents are needed for LC-MS, the acidic eluents often contain formic acid, acetic acid, or trifluoroacetic acid. 

Nonionic detergents are probably the most suitable additives when further purification by lEC is required. Winkler et al. [26] used SDS in the elution buffer for SEC and were able to purify the eluted proteins further by RPC without prior removal of the SDS. We were less successful using a similar procedure for the purification of Sendai virus proteins. All Sendai virus membrane proteins containing SDS were eluted at the same position in RPC with low yields. In some cases [27,28], a volatile eluent has been used for chromatography, which facilitates further purification by one of the other modes of HPLC. 

Figure 3.295 Preparative loading of a 250 mm x4mm i.d. DNAPac PA100 using a volatile eluent. Eluent (A) water/MeCN (90 10 v/v), (B) 1.5 mol/L ammonium acetate/MeCN (90 10 v/v) gradient loading at 10% B, step to 60% B at...

The extent and type of sialylation on IgGs is variable and has been characterized by HPAE-PAD [81]. Weitzhandler et al. [80] used a volatile eluent for the monitoring of immobilized papain digestion of mAb and the subsequent Protein A purification of F b and Fc subunits on ProPac WCX-10 (Figure 4.94). Such volatile eluent system enables collected fractions to be directly dried to allow further... 

Any type of fraction collector with a drop counter will do. When the solvent is volatile, attention has to be paid so that the solvent does not dry from the concentrated eluent at the tubing vent. 

More commonly, a fraction, based on chemical type, molecular weight or volatility, is heart-cut from the eluent of the primary column and introduced into a secondary column for more detailed analysis. If the same mobile phase is used in both dimensions, fractions may be diverted by means of pressure changes-an approach first used in 1968 in GC-GC by Deans (35), and applied by Davies et al. in SFC-SFC (36). If the mobile phases are different, valves are employed, and special... 

The retention gap method (1, 2) represents the best approach in the case of qualitative and quantitative analysis of samples containing highly volatile compounds. The key feature of this technique is the introduction of the sample into the GC unit at a temperature below the boiling point of the LC eluent (corrected for the current inlet pressure), (see Eigure 2.2). This causes the sample vapour pressure to be below the carrier gas inlet pressure, and has two consequences, as follows ... 

A layer of condensed eluent is built up ahead of the evaporation site which acts as a thick layer of retaining stationary phase, thus blocking the further movement of all but the most volatile compounds into the column. Solvent evaporation, therefore proceeds from the rear towards the front of the sample layer (see Eigure 2.2). 

The retention gap techniques, essential for the analysis of very volatile components, are often replaced by concurrent eluent evaporation techniques, due to their simplicity and the possibility of transfering very large amount of solvent. In this case, the solvents are introduced into an uncoated inlet at temperatures at or above the solvent boiling point. 

Traditionally, LC and GC are used as separate steps in the sample analysis sequence, with collection in between, and then followed by transfer. A major limitation of off-line LC-GC is that only a small aliquot of the LC fraction is injected into the GC p. (e.g. 1 - 2 p.1 from 1 ml). Therefore, increasing attention is now given to the on-line combination of LC and GC. This involves the transfer of large volumes of eluent into capillary GC. In order to achieve this, the so-called on-column interface (retention gap) or a programmed temperature vaporizor (PTV) in front of the GC column are used. Nearly all on-line LC-GC applications involve normal-phase (NP) LC, because the introduction of relatively large volumes of apolar, relatively volatile mobile phases into the GC unit is easier than for aqueous solvents. On-line LC-GC does not only increase the sensitivity but also saves time and improves precision. 

Preliminary purification of a starting band contaminated with plant oil should be performed by predevelopment with a nonpolar solvent such as benzene or n-heptane, delivered from the eluent container. Weakly retained ballast substances (e.g., lipids) move with the solvent to the edge of the adsorbent layer, covering the glass plate where the volatile solvent evaporates. The contaminants can then be removed (scraped out with the adsorbent) from the layer or adsorbed on the strip of blotting paper placed on the upper edge of the layer. 

Eluent components should be volatile. Solvents such as ethyl acetate, isopropyl ether, diethylketone, chloroform, dichloromethane, and toluene as modifiers and n-hexane as diluent are recommended for normal phase chromatography. For reversed-phase systems, methanol or acetonitrile are used as modifiers. Such components as acetic acid or buffers, as well as ion association reagents, should be avoided. 

The above methods work well for LC fractions in volatile and relatively nonpolar organic solvents. It is much more difficult to apply the same techn to reversed-phase eluents containing an... 

Other kinds of bloassays have been used to detect the presence of specific allelochemical effects (8), effects on N2 fIxatlon (9), the presence of volatile compounds (10) and of Inhibitory substances produced by marine microalgae (11). Putnam and Duke (12) have summarized the extraction techniques and bioassay methods used In allelopathy research. Recent developments In high performance liquid chromatography (HPLC) separation of allelochemlcals from plant extracts dictates the need for bloassays with sensitivity to low concentrations of compounds contained In small volumes of eluent. Einhellig at al. (13) described a bloassay using Lemna minor L. growing In tissue culture cluster dish wells that maximizes sensitivity and minimizes sample requirements. 

---