Metodo Pico Tag

Journal of Automatic Chemistry of Clinical Laboratory Automation, Vol. 8, No. 4 (October-December 1986), pp. 170-177

An evaluation of the Waters Pico-Tag system for the amino-acid analysis of food materials
j. A. White, R. J. Hart and J. C. Fry
Leatherhead Food Research Association, Randalls Road, Leatherhead, Surrey K T22 7R Y, UK

Introduction
The Pico-Tag method, recently described byHeinrikson and Meredith and developed commercially by Waters Associates, is an integrated technique for amino-acid analysis. Phenylisothiocyanate (PITC, or Edman’s reagent) is used for pre-column derivatization, while reversed-phase gradient elution high-performance liquid chromatography (HPLC) separates the phenylthiocarbamyl (PTC) derivatives which are detected by their UV absorbance. Figureillustrates the chemistry of the derivatization reaction. Cohen, Tarvin and Bidlingmeyer [2] from Waters Associates have reviewed the potential of the Pico-Tag method. The technique was developed to improve the speed and sensitivity of amino-acid analysis. This need for faster analysis times and greater sensitivity arises from the recent popularity of HPLC for rapidly separating very small amounts ofcomplex peptides and protein mixtures.

The generation of numerous peptides and proteins for amino-acid analysis has thus placed a great strain on current instrumentation. Hence, there is a requirement for a fast and sensitive technique which does not waste valuable samples.
The traditional method of amino-acid analysis is separation by ion-exchange chromatography (IEC) developed by Moore,Spackman and Stein [3]. Samples are applied to the column at low pH, and eluted with a series of buffers of increasing pH and/or ionic strength. Postcolumn derivatization with ninhydrin then gives a product which is detected colorimetrically. The majority of current amino-acid analysers employ this technique.

The Pico-Tag method appears to have quite substantial advantages over the conventionalion-exchange analyser. The most important of these are speed, greater sensitivity, excellent reproducibility and general reliability of both the results and the hardware.
The separation of the PTC derivatives of the common amino-acids takes 12 min (total analysis time, including column regeneration time, is 20 min) with the Pico-Tag method, compared with about 90 min for a conventional ion-exchangeanalyser using sodium-based buffers. The manufacturer claims very good reproducibility, whic should allow automated data collection and calculation of results. With ion-exchange separation this is often not possible owing to variability of retention times.

R
NCS
+

NH2--CH--CO0Amino acid

Phenytisothiocyonote
(PITC)

One noteworthy feature of the analysis with PITC is thequantification of secondary amino-acids, such as proline and hydroxyproline. Unlike post-column ninhydrin detection or ortho-phthalaldehyde (OPA) derivatization, the proline and hydroxyproline PTC derivatives have the same chromophore and approximately the same molar response as other amino-acids. There is thus no need to suffer reduced sensitivity or to employ a second detector for these secondaryamino-acids.
The Pico-Tag system can, with the appropriate column and solvent(s), also be used as a general-purpose HPLC, offering greater flexibility compared with the dedicated amino-acid analysers.
Conventional ion-exchange analysers using post-column derivatization are complex and hence sometimes temperamental instruments, usually with quite intricate fluidics and expensive electronics in closeassociation. In contrast, the Pico-Tag system is of a simpler, modular design, allowing easy access to the individual units. Hence, the HPLC system should be more reliable mechanically.

S R N H--- C--- N H-----C H

C 00-

Phenytfhiocarbamyt (PTC) amino acid
Figure 1. Production
170

of PTC amino-acids.

j. A. White et al. Waters Pico-Tag

The Pico-Tag system was originally developed...