Reversed-phase columns loading

It is seen that the polymer resin does not have the same retentive capacity as the conventional reverse phase column and thus, will not exhibit the same resolution or the equivalent loading capacity. Nevertheless, the polymer column will function over a wide range of pH whereas the silica based columns will be restricted to operating within a pH of 4.0 to 8.0 at the most. 

Liquid chromatography cleanup has been further described for purification of lincomycin residues from milk and tissue extracts (146). This procedure involves loading the sample extract onto a reversed-phase column (Supelcosil LC-... 

Figure 4.8. MuDPIT platform. The MuDPIT or multidimensional gel protein identification system is an multidimensional liquid chromatography (LC)-based system of separation prior to tandem mass spectrometry (MS/MS). It is not necessary to purify proteins prior MuDPIT, although a reduction in protein complexity by some prior purification is helpful in obtaining interpretable spectra. Protein lysates are digested into peptides that are loaded onto a strong cation exchange (SCX) support. Peptides are sequentially eluted onto a reverse-phase column for a second separation. Eluted peptides from the reverse-phase column are electrosprayed into a tandem mass spectrometer for amino acid sequencing and identification of proteins in the sample.

In our laboratory we have equipped the Famos autosampler (LC Packings, Amsterdam) so that samples of up to 85 pL can be analysed. This means that the samples do not need to be dried down to decrease the volume of sample before loading the peptides onto the reverse phase column. The drying-down step results in significant losses of peptides and should be avoided if possible. 

Each stored sample is then loaded on a reversed-phase protein trap column attached to a second six-port valve located on the capillary LC system. Proteins and peptides in the stored sample are captured on the trap column and washed with a solution of water-acetonitrile-fomic acid (94.9 5.0 0.1) at a flow-rate of 20pLmin for 3 min. Most of the ampholyte, urea and DTT are removed during this washing step. The protein fraction is then eluted from the trap column with mobile phase and further separated on a C4 reversed-phase column (5 cm X 300 pm i.d.). Mobile phase A [water-acetonitrile-fomic acid (94.9 5.0 0.1)] and mobile phase B [acetonitrile-water-acetic acid (94.9 5.0 0.1)] are delivered at a flow-rate of lOpLmin using a two-step gradient of 40min (phase B from 20 to 60%) and 2 min (phase B from 60 to 90%). The sample eluted from the RPLC column is sent directly into the ESI-QTOF mass spectrometer. 

The method of preparation of the crude extract pnor to loading on the solid phase extraction column will depend on the compatibility of the column and the solvent. For example, if an aqueous methanol extract is loaded onto a normal phase column (e.g., silica gel, diol, or alumina), the sample must first be evaporated to dryness to remove water, which is incompatible with normal phase adsorbents, and then the residue must be dissolved in a nonpolar solvent such as ethyl acetate, toluene, or hexane that has limited solubility for the product. Conversely, if a toluene extract is loaded onto a reverse phase column (e.g., C8, Cl8), the sample should be evaporated to dryness and the residue dissolved in a minimal volume of solvent compatible with the mobile phase. 

Toda procedure for obtaining enantiomeri-cally pure compounds will find broad application very soon. This development could make preparative HPLC with chiral columns obsolete and be applied to distillable amino acid derivatives as well. After all, analytical resolution of amino acids was quite successful by host/guest complexation chromatography with reversed-phase packings loaded with Cram s chiral 1,1 -binaphthyl crown ethers (similar to 1). [20]... 

Fig. 14. The calculated concentration profile for bovine trypsin undergoing dynamic interconversion whilst chromatographed on a reversed phase column. It is assumed that the interconversion processes occur by a two-state reversible transition with overall forward conversion and reverse conversion rates r /t = r /t =1.25X10 sec and that at / = 0 and equilibrium mixture of two inteiconverting components of equivalent mole fractions is initially loaded onto the column. It is further assumed that the migrating zone for each component generates a Gaussian distribution profile with a = 0.1 and that the effective diffusion coefficients of both forms are the same. From (23].

Combine supernatants and load on a reverse phase column. 

Short (3-10 cm) HPLC columns can also be used for rapid sample concentration. Microbore reversed-phase columns have been used for preconcentration of proteins prior to microsequence analysis, after collection and dilution of milliliter fractions from conventional HPLC columns [36]. Gradient elution at low flow rates (0.1-0.2 mL/min for 2-mm-ID or 0.02-0.04 mL/min for 1-mm-ID columns) permits recovery of proteins in volumes as small as 25 pL, with concentration factors up to 80-foId. The high capacities of porous microparticulate packings enable 50-100 pg to be loaded onto microbore columns as long as the sample is introduced in a weak solvent. 

---