Simple device for cleaning the teflon reaction coil in automatic amino acid analyzers

ANALYTICAL

Simple

BIOCHEMISTRY

Device

43, 613-616

(1971)

for

Cleaning

in Automatic

Amino

KENNETH Department

of Chemistry, Bozeman,

the

Teflon

Acid

Reaction

Coil

Analyzers’

D. HAPNER Montana

Montana

Received April

State

University,

69715

21, 1971

Formation of insoluble precipitates within the Teflon reaction coil of the automatic amino acid analyzer has frequently resulted in plugging, high back pressure, flow stoppage, and consequent down time. In addition, insoluble materials which periodically flake off the walls of t.he reaction coil and pass through the flow cells contribute to recorder baseline noise. Analyses requiring the recorder expanded range card are particularly susceptible to this source of background noise. Problems related to coil plugging are usually corrected through tedious flushing procedures with various solvents and/or the purchase of a new reaction coil, procedures both time consuming and costly. This paper describes the use of dichromate solution in a simple method for periodic cleaning of the Teflon reaction coil, which should result in increased coil life, decreased baseline noise, and less instrument down time. MATERIALS

AND

METHODS

The instrument utilized in these experiments was a Beckman automatic amino acid analyzer model 12OC, serial 1193. A “cleaning cell” of approximately 5 ml volume (Fig. 1) was constructed from Teflon rod and stainless steel. The cell was formulated with a detachable top piece containing a threaded seat to accept the stainless-steel swivel tube fitting commonly used with the Beckman analyzer. A similarly sized fitting port was drilled near the bottom of the cell. To install the cell, the reaction coil inlet line (including the fitting) was detached from the analyzer five-port mixing manifold and connected to the bottom port of the cleaning cell. An 18” long Teflon tube (l/is” o.d., I&” i.d.) with stainless swivel tube fittings (Beckman part No. 313336) at each end was then connected to the mixing manifold (from which the coil inlet ‘Supported by a research grant from the Research Corporation. and by the Montana Agricultural Experiment Station. Published as Journal Series No. 266, Montana Agricultural Experiment Station. 613

614

KENNETH

D.

HAPNER

D

==%===

0 C‘J a

0

F

a

G

FIG. 1. Diagram showing various parts of dichromate cleaning cell: (A) five-port mixing manifold in amino acid analyzer; (B) 18” length of Teflon tubing equipped at each end with stainless-steel swivel fittings; (C) stainless-steel top portion of cleaning cell with two O-rings near bottom, diameter at top l”, bottom “a”; (D) central portion of cleaning cell made from bored-out Teflon rod, 1” o.d., “8” i.d., height 21’4’f ; (E) inlet line to reaction coil which is attached to port at bottom of cell; (F) circular Plexiglas base plate, diameter 3”, thickness I’&“, (G) screws which

fasten base plate to bottom line was previously

of cell. For operational

detached)

details see text.

and the top piece

of the cleaning

cell. The

cleaning cell was filled with fresh sodium dichromate2J cleaning solution by means of a disposable syringe and needle prior to attachment of the buffer inlet from the mixing manifold. Eluant buffer was then pumped into the top of the cleaning cell by directing the effluant from one of the analyzer columns to the coil. A three-port Teflon-Kel F valve (No. 315-3, M.E.R. Chromatographic, Mountain View, California 94040) was installed between the reaction coil exit and the inlet to the calorimeter flow cells. During the cleaning ‘Sodium dichromate cleaning solution was prepared by sodium dichromate and 1 liter concentrated sulfuric acid, ’ A nonpolluting cleaning solution was recently described

mixing (1).

35 ml

saturated

DEVICE

FOR

CLEANIh-(;

REACTION

COIL

61 5

procedure the coil exit flow was directed to a waste beaker and away from the calorimeter. The three-port valve was installed permanently and in normal use was positioned to direct the coil exit flow into the flow cells of the analyzer. The valve, as manufactured, accepted the swivel tube fittings and eliminated any need for modification of tubing or fittings. After the cleaning solution was flushed through the coil the buffer flow was stopped and the cleaning cell was dismantled and thoroughly washed to remove any traces of dichromate. The coil inlet line was then reat.tached to the mixing manifold. RESULTS

AND

DISCUSSION

Reaction coil plugging in the automatic amino acid analyzer has resulted in expensive down time and frequently necessitated the purchase and installation of a new coil. We found that periodic purging of the hot coil with dichromate cleaning solution diminished recorder noise and eliminated coil plugging due to the build up and subsequent release of insoluble materials from the coil walls. The oxidizing solution comes in contact only with the cleaning cell, the Teflon coil, the three-port exit valve, and associated stainless-steel fittings, thus excluding the possibility of instrument damage. The entire purging process required less than 30 min and no cleaning solution remained in the instrument. When performing standard amino acid analyses of protein hydrolyzates, we found it, convenient to clean the coil line each time a new batch of ninhytlrin was prepared. When the analyzer was used for the separation of peptides, more frequent flushing of the coil was required due to the rapid formation of precipitated materials in the coil. Peptide separations utilizing sodium citrate buffers formed precipitates less readily than did separations involving pyridine acetate buffers. In the latter case the coil was cleaned after each peptide analysis. Extensive build up of precipitates and eventual coil plugging, as observed by Hill and Delaney (2), did not occur when the coil was flushed frequently. Observation of the color of the cleaning solution as it emerged from the reaction coil indicated the completeness of the cleaning procedure. The initial portion of the solution as it emerged from the coil was green, indicating oxidation of material within the coil. Transition from the green to brown color of the dichromate solution indicated that cleaning was complete. An air bubble was introduced into the coil in front of the cleaning solution (by directing the three-port valve to waste before filling the cell) to avoid initial color change due to reaction with buffer salts. The very last portion of cleaning solution was usually blue, presumably the result of reaction with the buffer solution forcing it through

616

KENNETH

D. HAPNER

the coil. One pass of cleaning solution through the coil was usually sufficient for complete removal of all oxidizable precipitates. SUMMARY

Recorder baseline noise and reaction coil plugging in automatic amino acid analyzers may be minimized through periodic purging of the hot coil with sodium dichromate cleaning solution. A “cleaning cell” containing the dichromate was positioned between the mixing manifold and the coil inlet tube. A three-port valve was installed between the coil exit and flow cell inlet restricting dichromate contact to the coil. Eluant buffer from an analyzer column then forced the dichromate solution through the coil. One 5 ml pass of dichromate solution through the coil completely removed oxidizable materials and resulted in improved performance and longer coil life. ACKNOWLEDGMENT The cleaning cell was constructed University, Bozeman, Montana.

by MSU Instrument

Service, Montana

REFERENCES 1. GODA, G., Amer. Lab. 2, 63 (1970). 2. HILL, R. L., AND DELANEY, R., Methods

Enzym.

11, 339 (1967).

State