Recording, plate-measuring system for the ultracentrifuge

ANALYTICAL

BIOCHEMISTRY

Recording,

11,

42-47 (1965)

Plate-Measuring

System

for

the

Ultracentrifuge JOHN J. BARTULOVICH From

the Western Development

AND WILFRED

Regional Research Laboratory, Western Division, Agricultural Research Service, of Agriculture, Albany, California

H. WARD Utilization Research and V. S. Department

Received July 27, 1964 INTRODUCTION

Ultracentrifuge experiments recorded by schlieren or interference optical systems yield experimental results as photographs which are interpreted as relationships of solute concentration to position in a column of solution. From such records, fundamental information about molecular size, shape, heterogeneity (in specific terms), and molecular interactions is deduced. Recent advances in technique and theory continue to extend the power and usefulness of the ultracentrifuge for determining and analyzing these molecular properties. For example, weight-average molecular weights can be determined directly by the Archibald approach-to-equilibrium method (1). Procedures for getting distributions of molecular weights from sedimentation equilibrium experiments are described by Fujita (2). Material distribution with respect to sedimentation coefficient can be found in some cases (3) ; if this is not feasible becausethe number of components is small the distribution can be characterized by its standard deviation (4). However, getting out the information which is inherent in the records and which is needed to apply detailed theoretical treatments such as those mentioned require more complete, painstaking measurement--many points on the boundary pattern must be located in two coordinates-and far more complicated computation than is ordinarily feasible. As a very simple example, when a skewed boundary pattern appears, as it often does in sedimentation velocity measurement,s,the easily measured maximum gradient does not satisfactorily represent the boundary position. Instead, a more appropriate measure is the square root of the second moment of ‘Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable. 42

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PLATE-MEASURING

SYSTEM

43

the boundary pattern (5). Consequently, even though computation “by hand” can sometimes be simplified by the use of Rayleigh interference optics (6) or Trautman’s radius-cubed scale (7)) routine complete evaluation of ultracentrifugal results can hardly be done without an electronic digital computer and a quick, reliable way of measuring plates. Here we describe in essential detail a plate-measuring system, assembled especially to overcome problems in characterizing wool proteins but, we believe, of general utility. Somewhat analogous but more complex systems have been developed for determining, for example, the paths of ionizing radiation in cloud chamber photographs (8). DESCRIPTION

AND

OPERATIOK

A block diagram of the syst.em is shown as Fig. 1. The main components are commercially available. The measuring instrument is a Gaertner

Optical

comparator

FIG. 1. Block

diagram

of the data

recording

system.

model 2001M toolmaker’s microscope with fixed (nontilting) axis, projection screen, and 2 X 10 in. plate carrier. Two encoders, Coleman Electronic AP4DT four-place decimal Digitizers, convert the lead screw positions into decimal numbers exactly corresponding to the microscope dial readings. The encoders are connected to the lead screws through Precision Instruments Co. 1: 1 helical gear boxes; side shafts allow both manual and motorized driving of the lead screws. A synchronous stepping motor, Superior Electric Co. Slo-Syn, SS-150, is attached to the s-axis gear-box side shaft for motorized, repetitive incrementing. The encoders, gear boxes, and motor are attached to t.he microscope substage by aluminum alloy brackets. Details are shown in Fig. 2.

44

BARTULOVICH

FIG. 2. Photograph

showing

attachment

AND

WARD

of encoders

and

motor

to the microscope

stage.

Each revolution of the lead screw shaft corresponds to 100 units of the encoder, so that the smallest unit represents a movement of 10 microns. Each encoder has a capacity of 9999, precise to one count. Our present lead screws use only half of this capacity. Two lampbank indicators display the encoder readings and thus the lead screw positions, eliminating reading the scales and vernier dials of the microscope except to check the precision of the assembly. The position defined by each encoder is registered and stored in the corresponding data processor for parallel or serial output as appropriate. A Flexowriter control unit governs output of the stored information through a combination typewriter, tape punch, and tape reader (Friden Flexowriter model Fl-16201. The control unit determines the format and sequence in which information is recorded. It includes a programing patchboard with plug-in wiring allowing the format to be modified. We prefer to record readings in centimeters to thousandths, showing the decimal point. The z reading is recorded first, on signal from a foot switch, then two y readings, on separate signals from a second foot switch. The second y reading is ordinarily a baseline measurement obtained from the

1:LTRACENTRIFUGE

PL.tTE-MEASURING

SYSTEM

45

solvent channel of a twin-channel centrifuge cell. The typewriter carriage then shift.s to a new line for the next set of readings. The control unit will not permit recording another z reading until two y readings have been recorded. The arrangement is arbitrary and controlled through the patchboard. Coleman Electronic Systems provided the encoders, the lampbank indicators (SD 3004), the data processors (SD 2004 series), the Flexowriter control unit (SD 3002), and the comparator (SD 2004 series) used for incremental advance. It is often desirable, especially when evaluating schlieren records to determine molecular weight by the “Archibald” method or distribution of mat,erial as a function of sedimentation coefficient, to measure ordinates at uniform intervals of Z. This procedure is made convenient by motorized incremental advance as follows. A predetermined increment with any value from 0.091 to 9.999 cm in steps of 0.001 cm is set in four dials on the coincidence detector (Fig. 1). When an advance command is given by pushing a switch on the coincidence detector, the x reading registered in the data processor is added to the coincidence detector dial setting and stored. The stored sum is then compared wit.h the value in the x data processor. As Iong as the stored sum is greater, puked signals are transmit,ted to the stepping motor through the matching translator (Superior Electric Co. model ST 250) t,o drive the x lead screw in the positive direction; at coincidence, the motor stops. The y adjustments are then made, the set of readings recorded, and the x position again incremented. The cycle is repeated as often as necessary. One pulse causes the motor to turn l/200 of a revolution, corresponding to a lead screw movement of 5 microns, that is, one-half of the least digit read in t,he encoder. Therefore, each new incremented posit.ion is accurate to 0.0005 cm. The method of incrementing avoids cumulative error over a series of increments because the distance traveled depends only on the stored sum in the comparator and not on the number of pulecs required to reach the position represented by this sum. The plate measurements are automatically recorded through the Flexowriter either as a typed record in specified format, or in eight-channel punched paper tape, or both at the same time. In addition to these data recorded by interrogating the data processors, information needed to identify the record or to be used in computation can be entered in either record through the typewriter keyboard. In fact, selector switches governing the various Flexowriter functions-typing, punching, and reading (that is, typing or punching under control of a punched tape)--give the operator versatile program control. For example, information common to several records can be recorded as a prqrograrn tape. This information is read and punched item by item into the final record tape with only

46

BARTTJLOVICH

AND

WARD

information specific to the given record entered through the keyboard and from the encoders. By the same means, successive sets of data from the encoders can be numbered serially or specially identified, for example as measurements of the meniscus position or of the maximum gradient. The punched tape record is then available for electronic digital computation. DISCUSSION

For the present, in our system, lead screw adjustments are completed manually in at least one coordinate, correct placement being judged visually by inspection of the projected image. Dependence on personal judgment for this setting is our most obvious limitation of accuracy and is one reason for using four-place encoders instead of five-place encoders recording to one micron. In the helical gears maximum backlash amounts to 20’ arc; since the least digit of the encoder corresponds to 3.6” rotation of the lead screw, backlash is not a source of error. Similarly, within existing limitations, lead screw linearity and possible play in the flexible couplings between the lead screws and Digitizers are insignificant. In any case, exact correspondence between the readings of the Digitizers and the corresponding dials of the measuring microscope is readily verified. The convenience of recording facilitates testing the precision of the entire system. Our measuring microscope permits direct measurement only of areas 5 cm square without resetting the plate, except that an additional 5-cm range in the 2 direction is obtainable with a gage block. This arrangement is fully adequate for ultracentrifuge plates but inconvenient for longer records. Most of this description has applied to measurement of schlieren records made with a twin-channel cell to record a baseline. When only one y reading is wanted for each x reading or if only x readings are wanted, as when interference records are being read, the patchboard connections can be changed correspondingly. Alternatively, readings can be recorded in the same format and the computer instructed to ignore the unnecessary data. The measuring system described realizes the following important advantages: It makes plate reading quicker and easier, without sacrificing accuracy. Measurements are recorded mechanically without transcription. The record is suitable for electronic digital computation. It can be input immediately into a computer such as the IBM 1620, equipped with a tape reader, or, after automatic tape to card conversion, into an IBM 7090. Additional features of interest are as follows: A typed record can be provided for reference and checking. Supplementary information can

ULTRACENTRIFUGE

PLATE-MEASURING

47

SYSTEM

easily be incorporated. The record can be duplicated automatically. A preprogram tape can be used to systematize the form in which supplementary information is recorded. The record is stored in a form that can be re-evaluated conveniently to take advantage of new t,heoretical developments. Finally, the system can readily be modified for higher precision. SUMMARY We describe a system appropriate for measuring photographs from ultracentrifuge, free diffusion, or electrophoresis experiments recorded with schlieren or interference optics. The main parts are commercially available. Cartesian coordinates of significant features of the photographs are converted to digital form by means of decimal digital encoders attached to the two lead screws of a two-coordinate measuring microscope. The lead screw positions are continuously displayed on lampbank indicators. On command, the readings are automatically typed, punched into paper tape, or both. Identifying information and supplementary data for computation can be inserted as needed in the typed record or also in the tape. When coordinate readings are wanted at predetermined equal intervals, the abscissa (z) lead screw is advanced automatically to the successive positions at which measurements of the ordinate (y) are wanted. At present, for schlieren records, adjustment of the y lead screw and initial adjustment of the x lead screw are made manually with visual inspection of the projected image. This system increases the rate at which plates can be measured, completely avoids human error in writing down and transcribing measurements, and records measurements in forms convenient for evaluation either by electronic digital computation or by more usual methods. ACKNOWLEDGMENT We are implementing

heavily indebted essential details

to Mr. Tom of this system.

Sanders

of

Sanders

and

Sanders,

for

REFERENCES 1. ARCHIBALD, W. J., J. Phys. Colloid. 2. FUJITA, H., “Mathematical Theory New York, 1962. 3. BALDWIN, R. L., J. Phys. Chem. 63, 4. BALDWIN, R. L., Biochem. J. 65,490 5. GOLDBERQ, R. J., J. Phys. Chem. 57, 6. RICHARDS, E. G., AND SCHACHMAN, 7. TRAUTMAN, R., J. Phys. Chem. 60, 8. BOLZE, E. M., AND AYER, II, F., Rev.

Chem. 51, 1204 (1947). of Sedimentation Analysis.”

Academic

1570 (1959). (1957). 194 (1953). H. K., J. Phys. Chem. 63, 1578 (1959). 1211 (1956). Sci. Instr. 33, 1190 (1962).

Press,