Practical Protein Chemistry - A. Darbre 1989

Current state of automated liquid-phase amino acid sequence analysis
Reagents and solvents

In automated sequence analysis, Reagents account for The most significant portion of operating expenses, costing approximately $15,000 annually to supply a single Sequencer. The scientific literature and laboratories are continuously debating the best sources of reagent supply. Opinions vary widely: some argue that end-users must perform the final purification of all reagents [1, 11], while others suggest purchasing the cheapest commercial batches and testing them individually in-house. The majority view lies somewhere between these two extremes. Our laboratory relies on several different supply sources, and our strategy is outlined below.

We purchase all of our reagents from Beckman. Although historically the most expensive among all available sequencer-grade reagents, Beckman products are the only ones guaranteed to be pre-tested on a sequencer. Because each individual reagent undergoes sequencer testing and exhibits minimal batch-to-batch variation, using them minimizes the risk of failure during structural analysis.

However, their high cost has driven many laboratories—including our own—to look for alternative suppliers. Overall, our experience indicates that reagents from Pierce are of good yet variable quality, with PTC being a key reagent. In our view, most of the step-by-step yield variations reported in the literature stem precisely from this inconsistent quality of PTC. It has been proven that commercial preparations of "anhydrous" PTC contain Water, and a Procedure for its further drying has been described [1]. In our opinion, this is generally unnecessary, as we observed no distinct advantages in reducing the water content from 30 to 4–6 ppm^-1.

Substantial savings can be achieved by purchasing Solvents from suppliers other than Beckman. We have never encountered any difficulties using benzene from alternative sources, although Burdick and Jackson remains our regular supplier. Certain issues may arise when using different batches of ethyl acetate, as many batches contain impurities that degrade amino acid ATZ-derivatives.

We recommend a simple test for ethyl acetate. Take a standard amount of amino acid ATZ-derivatives (we typically use our standards intended for GLC or HPLC), mix them with 1.5 mL of the ethyl acetate batch being tested, leave at room Temperature for 30 minutes, and then dry under a stream of nitrogen. The residue is subsequently analyzed quantitatively by GLC or HPLC by measuring peak heights. Dissolution in ethyl acetate either has no effect on the peak heights of the ATZ-Amino Acid Derivatives or completely destroys these derivatives, leaving no detectable peaks.

On rare occasions, we encountered solvent batches that yielded intermediate results. This simple test produced favorable outcomes for both relatively inexpensive ethyl acetate (Burdick and Jackson) and moderately priced ethyl acetate (Pierce).

The addition of various protective agents to Reagents and solvents—such as hydrochloric and ascorbic acids—is practiced less frequently today than in the past. Similarly, the addition of reducing agents to chlorobutane is applied less often, especially when HPLC is used to analyze Ser and Thr derivatives*.

Purifying reagents requires dedicated distillation equipment. Spinning-band distillation units cost approximately $4,000–30,000 (B/R Instrument Corp.).

* The addition of dithiothreitol to all reagents and solvents used in the thiazolinone Cleavage and conversion steps into ATZ is now considered mandatory for high-sensitivity analysis. — Translator's Note.