A MENISCUS SENSOR FOR CONTROLLING THE ONLINE INJECTION OF PTH-AMINO ACID DERIVATIVES FROM A SEQUENCER ONTO AN HPLC SYSTEM.

James Strickler, Dean McNulty, and K. Karl Sonesont. Dept. Macromolecular Sciences, SmithKline Beecham Pharma., P.O. Box 1539, King of Prussia, Pa 19406, and Contract Software Specialist, 531 Westfield Drive, Exton, Pa 19431t.

Control of the injection of PTH-amino acid derivatives from a protein/peptide sequencer onto an on-line HPLC analytical system has historically employed a passive, i.e. time-based, signal from the sequencer. In this article, we describe an inexpensive, active controller that has several advantages over passive methods. The device consists of an electro-optical sensor used to detect an air/water interface at the leading edge of the sample being delivered from the sequencer to a pneumatically actuated injection valve. The sensor is placed on the outlet of the injection valve. When the air/water interface passes through the sensor window, it interrupts a beam of light striking a phototransistor thus inducing a drop in the voltage applied across the transistor. This negative pulse serves to trigger an integrated circuit to actuate a relay for a definable time. The relay acts as a simple contact closure start signal for the HPLC system.

A schematic diagram of the device is shown in Fig. I (16k)along with the specifications of the components(16k) used in our prototype. For convenience, the circuit can be divided into two parts: the output stage and the input stage. The output stage consists of a standard 555 integrated circuit (TLC555) arranged as a monostable timer. The TLC555 functions as a voltage comparator. As long as the trigger voltage at pin 2 is above ~ 1/3 of the voltage at pin 8, the output (pin 3) is low. When the voltage at pin 2 drops below the threshold, however, the output goes high thus activating the relay. Resistor R1 and capacitor C1 control the duration of the output. For our prototype, R1 is 100 kOhm and C1 is 10

Farads, giving a 1 sec duration for the output. The input stage centers on a standard photo-coupled interrupter module (NTE3100). R2 (100 kOhm, 15 turn variable resistor) is used to adjust the voltage across the phototransistor detector of the module to ~ 1/2 of the input voltage. This voltage is the trigger. R3 (2.88 kOhm) and R4 (1 kOhm, 15 turn variable resistor) are used to adjust the current to the light emitting diode (LED) and thus light output and sensitivity of the device. To set R4, the resistance should be decreased to the point at which the meniscus is no longer detected and then increased by 1 /2 to 1 turn. Irradiation of the phototransistor by the LED maintains the trigger voltage above the threshold. When the light beam is interrupted, the trigger voltage falls below the threshold and the output stage is activated.

In addition to the electronics, the design of the flow cell which fits into the sensor is critical to its operation. Most commercially available sensors irradiate too large an area to sense a meniscus moving through HPLC-tubing; therefore, in constructing a flow cell, we tried to minimize the amount of light traversing the fluid path. The flow cell currently being used with this device (Fig. 2) (16k)was made from commercially available HPLC connectors. Briefly, the knurled end of the black Upchurch 1/16" Plastictight male nut was shaved down until it fit tightly into the photo-coupled interrupter module. A .030" hole was bored through this end perpendicular to the tubing path. The cell was completed by running a glass capillary tube (~0.030"i.d.) through the Plastictight nut. The tube was held in place by screwing the nut into a Plastictight adapter (plastic to flange- _ type fitting) which was also used to connect the flow cell to a short length (7 cm) of 0.005"i.d. stainless steel tubing from the injector. Once the interrupter module was correctly positioned on the flow cell, the cell was tacked in place with Duco Plastic cement. (Duco cement will not bond to the Fingertight fitting hence the unit can be disassembled for repair if necessary. The cement will cloud the window of the transducer so care must be taken to avoid contact between the cement and the windows.) The 0.005" tubing was chosen because the sensitivity of the device is somewhat dependent on the rate at which the meniscus traverses the light path. The narrow bore tubing functions as a flow restrictor and insures that the sample does not go through the sensor too fast. Finally, the flow from the 0.005" tubing through the sensor should be downward. We have found that if the flow is arranged upward, small droplets of the sample collect at the 0.005"/0.030" junction and can intermittently produce false start signals. Although this flow cell has functioned well for us, better designs could be readily envisaged which would reduce the dead volume inherent in this design.

The advantages of this device are three-fold. First, since the device is a positive control mechanism, the collect time for delivery of the sample to the injector can be set to a relatively long time (e.g. 60 s); however, the injection will take place when the sample begins to exit the loop. Thus, slight variation in the Ar delivery pressure, partial clogging of the delivery line, or changes in the delivery line (e.g. i.d. or length) will not affect the accurate injection of the sample. With the standard time-based system, any of these problems would result in a partial or blank injection. Second, the injection normally employs loop-fill methodology. In order to insure that the loop is filled, most investigators dissolve the PTH-amino acid derivatives in 1.5 to 2 times the volume of the sample loop. Thus only 50 to 66% of the sample produced is analyzed. By controlling the injection with a positive sensing device mounted on the outlet side of the loop, one can easily minimize the volume used for redissolving the sample and achieve analysis of 80 to 90% of the sample. Finally, with a positive control device, a simple feedback mechanism could be designed to stop the sequencer should there be a problem with injection. For instance, if the delivery line from the sequencer becomes completely clogged, no injection will take place and the HPLC will not be started. A sequencer could be designed to check the status of the HPLC at an appropriate point in the cycle. If an injection had taken place, the sequencer would continue, whereas, if an injection had not taken place, the sequencer would hold at that point. The operator could then fix the problem and continue the program. With the time based delivery system, an injection always occurs, the sequencer continues running, and, if there is a problem, all of the subsequent data are lost.

Our prototype has now been controlling sample injection for >400 cycles. In that time, it has misinjected ~ 10 times. Most of these misinjections were clustered in a single run. We adjusted R4 (decreased the ohms and the sensitivity) after this run and since then, we have not had a misinjection ( > 100 cycles). We would welcome any suggestions on ways to improve the performance of this controller .

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