Sample Concentration

As the HPTLC plate develops, spot diffusion occurs with the result that what was 0.5-1 mm spot at the origin may well be 5 or 6 mm in diameter after complete development. Thus the analyte may well be distributed over 10 times more adsorbent than when at the origin, with the result that when the analytes are introduced into the ion source along with the adsorbent and other extraneous compounds from the plate there is considerably more adsorbent than analyte presented for ionization. Because there is a finite amount of material that can be introduced into the ion source, occasions may arise where there is insufficient analyte available from which good quality mass spectral data may be obtained. Several methods of sample concentration have been devised to overcome this natural spot diffusion phenomenon. For example, an analyte spot can be encircled with a ring of solvent. As the solvent diffuses through the adsorbent the analyte will slowly migrate to, and concentrate at, the centre of the spot. The concentrated spot can then be removed and analysed.

An alternative is where, after chromatographic development on aluminium- and plastic-backed plates, the edge of the plate may be trimmed off using dressmaker's pinking shears. This leaves the plate with a regular saw-toothed edge. The plate is rotated by 180° followed by the application of solvent. The analytes are thus concentrated into the tips of the triangular-shaped segments of the plate. Once concentrated, the zone may be removed from the plate and introduced into the ion source as previously described.

A general method not confined to aluminium- and plastic-backed plates involves scribing a trapezoidal line around the sample spot (Figure 2). A strip, 1-2 mm wide, of the adsorbent on the outside of the

Sample spot Figure 2 Zone concentration.
Figure 3 Zones (A) before and (B) after concentration.

line is removed. Then, from a 100 |L syringe, a quantity of an eluting solvent is slowly deposited on the base of the spot. After several tens of seconds the analyte condenses at the top of the trapezoid (Figure 3). The concentrated analyte and adsorbent are removed from the plate and are introduced into the ion source as described previously.

This method of spot concentration completely overcomes the need to overload the plate, with associated loss of chromatographic resolution, to enable sufficient sample to be introduced into the mass spectrometer for ionization. It is highly satisfactory prior to both vapour-phase and liquid-phase ionization.

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