Focus the Department of Laboratory Medi cine at the Albert Einstein College of Medicine, described the low sample throughput one encounters in liquid chromatography (HPLC) and gas chromatography (GC) because of the serial nature of sample introduction with those two techniques. "We tried to automate LC and GC sample intro duction and experimented with run ning samples overnight, but this still is not the best way to do things," said Karmen. "We thought if we could use a system like HPTLC, in which we could analyze a number of samples si multaneously and just automate the readout, we would probably be ahead." Karmen ultimately found that HPTLC adapted well to the demands of the clinical environment: "The sen sitivity is beautiful. The analytical re covery is more than acceptable. The precision (coefficients of variation) are well within anybody's reasonable de mand. And so far we have had no dif ficulty correlating HPTLC results with our EMIT system or with HPLC."
closed column, or bed, is used.) In HPLC the separation is per formed by elution chromatography, in which each sample component has to traverse the entire column bed to be detected. HPTLC, on the other hand, is a development process, in which the total separation time depends only on how long it takes for the solvent to reach a predetermined point on the plate. Since there is no waiting for strongly retained components, a con siderable savings in analysis time can be realized with development chroma tography compared to elution chroma tography. "Poisoning" of the HPTLC plate is not a problem, if only because the plate is designed to be disposed of after each analysis. In a closed bed system such as HPLC, the resolution may degrade when contaminants be come irreversibly adsorbed on the col umn. A further savings in time is af forded by the fact that sample analy sis is simultaneous in HPTLC, where as samples must be loaded serially into a closed bed chromatographic system.
Both the advantages and the disad vantages of HPTLC show up when the technique is systematically compared with HPLC (see Table II). HPLC is admittedly still more efficient than HPTLC. Poole pointed out at the meeting that conventionally packed HPLC columns can have twice the theoretical plates currently possible in HPTLC and that special HPLC col umns, such as capillary and microbore columns, are capable of even higher efficiencies. But HPTLC still has a number of advantages, most of them based upon the open bed nature of the HPTLC separation. (In HPLC, a
Separation of Amino Acid Deriv atives. One application of HPTLC discussed at the symposium involved the separation of phenylthiohydantoin-amino acids (PTH-AAs). P T H AAs are generated in the Edman deg radation reaction, used in automated protein/peptide sequenators. In this process, each succeeding N-terminal amino acid is cleaved from the peptide chain as a PTH derivative. A reliable analytical method is then needed to identify which amino acid has been cleaved at each stage of the process. Poole pointed out that his success in separating out 18 of the 20 common
Table II.
Comparison of HPTLC and HPLC 1
Parameter
HPTLC
Type of chromatography
Open bed
Development technique Mobile phase velocity control System equilibration
Development chromatography Fixed by capillary forces 2
time Simultaneous sample analysis Sample capacity (throughput) Plate height Number of effective theoretical plates Separation number Mode of detection Detection limits 1 2 3
HPLC
Closed system Elution chromatography Easily adjusted
Small
Small to large 3
Yes
No, serial
High
Low
12 μνη
