Off-line fraction collection

A mechanized off-line fraction collection from a microbore LC column on an indented sample holder, which is subsequently analysed by pulsed laser desorption MS, was described in 1983 by Huber et al. [68],... 

To date, the vast majority of experiments in which liquid separation techniques have been coupled with MALDI have utilized off-line fraction collection. The availability of MALDI sample preparation robots has facilitated the preparation of hundreds of samples as discrete spots on the target plate in a short time frame. If the fraction collection is triggered by detection of a UV signal (or other analytical detector), then MS analysis can be focused only on the chromatographic regions which contain the most abundant analytes. In addition, fraction collection allows... 

Conroy and Griffiths (45) developed a solvent extraction device that could be employed with a LC/FTIR combination. The device involved an extraction procedure that took the column eluent and continuously extracted the desolved solute into dichlormethane. The dichlormethane was then concentrated and finally dispersed onto a plug of potassium chloride powder. TTiis device appeared to be a little clumsy and in fact, was really an off-line fraction collecting procedure. 

The use of extraction cartridges in the separation of azines, discussed in the last Section, is an example of on-column concentration using off-line column switching. A chromatogram can be cut off-line by collecting the zones of interest at the detector outlet followed by reinjection of the collected fraction onto a secondary column. The mobile phases used with the two columns should be compatible, eg they should be miscible and the mobile phase used with the first column should not have too high an eluting power in the second column. If the mobile phases are incompatible it may be possible to evaporate the primary mobile phase and redissolve the sample in a suitable solvent. 

FIGURE 16.12 Enzymatically synthesized amylose"-type nb/lcb glucans ( ) with a significant amount of the substrate glucose-1-PO4 separated on Sephacryl S-SOO/S-IOOO (60 + 9S x 1.6 cm) 3-ml fractions were collected for further analysis normalized (area = 1.0) eluogram profiles (ev) constructed from an off-line determined mass of carbohydrates for each of the pooled fractions flow rate 0.42 ml/ min V,xd = 126 ml, V , = 273 ml eluent O.OOS M NaOH. 

In coupled LC-GC, specific components or classes of components of complex mixtures are pre-fractionated by LC and are then transferred on-line to a GC system for analytical separation. Because of the ease of collecting and handling liquids, off-line LC-GC techniques are very popular, but they do present several disadvantages, e.g. the numerous steps involved, long analysis times, possibility of contamination, etc. The on-line coupled LC-GC techniques avoid all of these disadvantages, thus allowing us to solve difficult analytical problems in a fully automated way. 

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. 

Online LC-ESI-TOF-MS experiments are carried out in a very similar fashion to the off-line NPS-HPLC separations described above, with a few notable exceptions. Firstly, 0.3% (v/v) formic acid is added to each mobile phase to counteract the ionization suppression induced by TFA. Because of the formic acid UV detection must be carried out at 280 nm (as opposed to 214 nm). To aid in normalization between runs 1 jag of Bovine insulin (MW = 5734 Da) is added to each chromatofocusing fraction prior to injection onto the column. Finally, the flow is split postcolumn directing 200 JlL/min into the ion source and the remaining 300 JlL/min through the UV detector and fraction collection. 

There exist essentially three categories of SCX/RP/MS/MS approaches. In one approach, SCX is run off-line followed by on-line RP/MS/MS (Fig. 11.1). In the offline SCX approach, fractions do not directly elute onto RP material but rather are collected. In one of the two in-line approaches, SCX is run in line with RP/MS/MS using different columns for SCX and RP (Fig. 11.2). In the multidimensional protein identification technology approach (MudPIT), SCX and RP are run in line in the same column, and this column serves as the ion source for a tandem mass spectrometer (Fig. 11.3). Both the in-line approaches are true SCX/RP/MS/MS approaches the first approach could be abbreviated as SCX—RP/MS/MS where... 

In an off-line configuration, a complex peptide mixture from a proteomic sample is loaded onto a SCX column and fractions collected (Fig. 11.1). After the collection of fractions, they are then loaded into an autosampler and analyzed via the traditional RP/ MS/MS approach. Using this system, a variety of buffers and elution conditions may be used (Table 11.1). For example, one may use a volatile salt such as ammonium formate (Adkins et al., 2002 Blonder et al., 2004 Fujii et al., 2004 Yu et al., 2004 Qian et al., 2005a and b) or ammonium acetate (Cutillas et al., 2003 Coldham and Woodward, 2004), collect SCX fractions, lyophilize, resuspend in low acetonitrile and acid, and then directly analyze via RP/MS/MS. In most of the cases, when ammonium acetate or ammonium formate are used, a 20-minute wash period is used to remove the ammonium acetate or ammonium formate prior to the reversed-phase gradient (Table 11.1). However, because fractions are collected and can be buffer exchanged,... 

RPLC-CZE, fractions are collected off-line from the first dimension and subsequently subjected to CZE analysis (Issaq et al., 1999 and 2001 Shen et al., 2001 He et al., 2002 Sanz-Nebot et al., 2002 Rodriguez et al., 2003 Yang et al., 2003). Details of selected applications will be discussed subsequently. 

He et al. (2002) used an off-line HPLC/CE method to map cancer cell extracts. Frozen ovarian cancer cells (containing 107 cells) were reconstituted in 300 pL of deionized water and placed in an ultrasonic bath to lyse the cells. Then the suspension was centrifuged and the solubilized proteins were collected for HPLC fractionation. The HPLC separation was carried out on an instrument equipped with a RP C-4 column, 250 mm x 4.6 mm, packed with 5-pm spherical silica particles. Extracted proteins were dissolved in 300 pL of DI water, and lOOpL was injected onto the column at a flow rate of 1 mL/min. Buffer A was 0.1% TEA in water and buffer B was 0.1% TFA in acetonitrile. A two-step gradient, 15-30% B in 15 min followed by 30-70% B in 105 min, was used. The column effluent was sampled every minute into a 96-well microtiter plate with the aid of an automatic fraction collector. After collection, the fractions were dried at room temperature under vacuum. The sample in each well was reconstituted before the CE analysis with 10 pL deionized water. The... 

---