ODDC and MULTIGRAPH: programs for spectrophotometer data collection and manipulation using a microcomputer

Computer Methods and Programs in Biomedicine 22 (1986) 171-178 171 Elsevier

CPB 00761

ODDC and MULTIGRAPH: programs for spectrophotometer data collection and manipulation using a microcomputer

Steven M. Ross, Al f red T. Black and C. Patr ick Reyno lds *

Immunohematology Branch, Transplantation Research Program Center, Naval Medical Research Institute, Bethesda, MD 20814-5055, U.S.A.

A software package is described that collects, graphs, performs peak sensing, and prints data from a spectrophotometer interfaced to an Apple II microcomputer with an ADALAB analog-to-digital converter card. Data collection and storage is performed by an interpreted BASIC program with machine language subroutines. Recalling, graphing, and printout of the data is accomplished by a separate compiled BASIC program. Both of these programs, as well as assisting utility programs, are under control of a menu program. The programs take advantage of all available memory in the 64-K Apple II computer to allow for storage of up to 12600 data points (7 hours of data collection at a sampling rate of 1 sample every 2 s). As the data collection program provides peak sensing, and the capability of annotating various time points, the programs are ideally suited for use in column chromatography.

Spectrophotometer Microcomputer Apple II Column chromatography Protein purificaton

1. I n t r o d u c t i o n

Protein separation using column chromatography is a commonly used technique. Dedicated chart recorders and UV monitors, which detect and quantitate protein by measuring the optical density (OD) (usually at 280 nm) can be used to monitor the elution of protein peaks from such columns, avoiding the necessity of monitoring the column by manual sampling [1]. An alternative is to use a flow-through cuvette on a spectrophotometer, thus avoiding purchase of a separate UV monitor; however, such chart recorders can cost as much as a microcomputer and printer. For a laboratory with a spectrophotometer, interfacing a microcomputer to the spectrophotometer provides a cost-effective alternative to the purchase of a dedicated chart recorder. Furthermore, such a sys-

* To whom correspondence and reprint requests should be addressed.

tem has a number of advantages: (1) The microcomputer can also be used for anal-

ysis or storage of a variety of data types in the lab.

(2) The microcomputer can perform peak detec- tion and can be used to sound audible alarms, control fraction collectors, or switch solenoid valves to change buffer solutions in response to the levels of protein eluted from the column.

(3) The microcomputer stores the data in machine-readable form, allowing recall and fur- ther analysis, or output to plotters which can generate presentation-quality graphics. In addition, calculations employing large quantities of data can be performed rapidly. By using the computer to collect data, no keyboard data entry is necessary before calculation can begin. Processed data can be output in the form of automatically scaled graphs or lists.

Several different methods of interfacing micro-

0169-2607/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

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processors [2] or complete microcomputers [3-6] to spectrophotometers have been described; however, none of the previously developed systems are designed for monitoring column chromatography. This paper describes a set of programs for an Apple lie microcomputer that monitor data from a spectrophotometer in real time, stores it on disk, and then allows one to print line graphs of the data. Although these programs have been designed primarily for column chromatography, they can be modified for other uses.

2. Hardware

The programs were developed on an Apple lie with an ADALAB analog-to-digital converter card (Interactive Microware, State College, PA), a disk controller card with two standard 5.25-inch disk drives, a Timemaster II clock-calendar card (Ap- plied Engineering, Carrollton, TX), and an Epson MX-80 dot matrix printer (with Graphtrax), interfaced with a PKASO graphics printer interface (Interactive Structures, Bala Cynwyad, PA). The programs also run on a 64-K Apple II plus with the above peripherals. Similar peripherals may be substituted for the ones listed above, although software modification may be necessary.

The ADALAB circuit board contains an analog-to-digital ( A / D ) converter. The input of the converter is connected to the analog output of a Gilford Model 260 Spectrophotometer so that the ADALAB board receives a variable voltage. The ADALAB board is calibrated so that throughout the necessary optical density (OD) range, a number is output to the computer that is directly proportional to the OD.

The OD data is stored on 5.25-inch floppy disks with a capacity of 128 Kbytes each. The programs can be run with one disk drive, but using two drives provides increased storage capacity for data. The program disk is run in drive 1 and the data disk in drive 2. Screen graphics and data lists are printed using the Epson printer. The use of a real-time clock-calendar card (Timemas- ter II) allows the computer to automatically store the date and time with the data. In addition, a modified disk operating system (David-Dos II,

David Data, Los Angeles, CA) speeds up disk access time, and allows the date to be automatically attached to files when they are stored on disk.

3. Software

Two separate programs were written to optimize storage capacity and program execution speed: ODDC (Optical Density Data Collector) performs data collection, real time graphing, and storage of data to disk; and M U L T I G R A P H allows recall and graphing of stored data, and output of the graph to a printer. The graph is automatically scaled so that maximum detail can be derived from the data. Collection and graphing are performed in separate programs to provide increased RAM for temporary data storage.

To increase execution speed, MU LTIG RA P H was written in BASIC, and then compiled to machine language using the TASC Applesoft BASIC compiler (Microsoft, Bellevue, WA). ODDC is also written in BASIC, but calls a group of machine-language subroutines. For this reason, it is incompatible with the BASIC compiler. Both programs require the David-Dos II operating system, which can be relocated into high memory to provide an additional 10 Kbytes of usable RAM.

A menu program (HELLO) written in BASIC provides for turnkey entry to the system after booting the disk containing the program files. It also allows access to the catalog of files on either disk, and to a utility program for easy reconfigura- tion of the data collection software.

4. Results

4.1. Optical Density Data Collector (ODDC)

ODDC, written by the authors, interacts with a group of machine-language subroutines called QUICK I / O by Interactive Microware. These subroutines are used specifically to facilitate input and output from the ADALAB card. A flow chart of the ODDC program is shown in Fig. 1.

After selecting ODDC from the Main Menu,

i Display ODOC Menu ]

Input menu selection ] (I. 2. 3 or 4} /

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m

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parameters

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Display information reminding I the user of keys that are active

during data collection

Pause until "return" is pressed

/

Get time from real-time I clock card J

? '°O°'anU *,li~. ~o~o.j

t Save data on Disk

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/ j ~ , - ~ o°t . . . . . . " ' - . - . I ~ , l . . . . . 1 . . . . . 7

-

/

s al RA NO stoPag

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sampling r a t e r ~

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{: lear A/D converter on adalab card

Cleor end prepare high reso lu t i on graphics and tex t

s c r e e n s

INput • huBS)or from the I A/D conver ter

Store t h i s n u ~ e r in memory /

Fig. 1. F low chart of the O D D C program.

I Get time from real-timel clock carO

l c~lcul~t~ tot~l tim~ I

Ask user i f data should be saved

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the program displays the time and date, and the O D D C Menu. The following options are available: (1) Save data (2) View/modify title, comments, and labels (3) Collect data (4) Multigraph To collect data, option 3 is selected, and the user is prompted to choose the maximum time (1.75, 3.50 or 7.0 hours) required for data collection.

.This allows a suitable sampling rate to be calculated by the computer based on total storage space divided by maximum time. After the sampling rate has been determined, the program reminds the user of the keys that are active during data collection, and waits for the operator to press 'RE- T U R N ' to start.

The OD is sampled continuously (with fixed time intervals between samples) as the column eluant is channeled through a flow-through cuvette. T h e BASIC program, ODDC, obtains a number that is proportional to the OD from the Q U I C K I / O machine language subroutines, which interact with the ADALAB interface card. The ADALAB card timer (0.1-s increments) insures that the sampling time is accurate.

Each time the OD is sampled, it is POKEd directly into memory. This allows the program to take advantage of every available RAM address, so that 12 600 data points can be recorded. After five points have been stored in memory, their average is plotted on the screen, simulating a chart recorder. Data is graphed with optical density on the Y axis and time on the X axis; the X coordi- nate is incremented each time a point is plotted. Organization of program and data in memory are shown in Fig. 2.

The program also performs peak sensing by setting upper and lower thresholds. When the OD rises above the upper threshold and then falls below the lower threshold, the program sounds a tone through the Apple's speaker. The tone continues to sound until the user presses the space bar, which resets the peak sensor. Data collection continues during this process. Thresholds can be changed by modifying one line in the program.

Another function that is available during data collection is marking and labeling of a particular

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Quick I/0 machine language Program data storage area2

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ODDC Applesoft BASIC program

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Fig. 2. Organization of the ODDC program and data in memory.

part of the graph. Labels should be used to mark the key points on the graph, since these labels can be used for reference points in the MULTI- G R A P H program. To label a graph, one presses the 'T ' key, which marks the current X location on the graph with a short vertical line. The program then prompts the operator to type a label up to 40 characters long. This label is then linked to the X location of the graph when the data is stored.

There are two ways of terminating data collection in ODDC: firstly, if there is no more RAM space remaining (12600 data points have been stored), the program stops collecting data and sounds a tone several times. The tone alerts the operator, who must save the data in memory to disk and restart data collection. Secondly, if the 'ESCAPE' key is pressed during data collection, the program pauses data collection and asks the operator to confirm that data collection should be stopped. This function can be used to pause data collection if, for instance, an adjustment is to be made on the spectrophotometer. After data collection is finished, the data in memory may be saved to disk, and other utility functions on the O D D C Menu may be accessed.

4.2. M U L T I G R A P H

M U L T I G R A P H is a compiled BASIC program that allows the operator to generate graphs of data collected by the O D D C program. Graphs of data can be scaled to emphasize subsets of the collected data. After the graphs are created on the high-res-

InDuE menu sel lct ion (1. 2, 3, or aI ~ m

" E x i t Program - - ~ - - ~ 1 _ l ~

I ................ ? has Deem lombed

I ................. 1 in formatlon

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points

Calculate number of aata oolntsneLl51 to increment each time a oixel

is PlotteO on the s c r e e n |

nemina user which 7 keys are act ve i

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text screens J

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Fig . 3. F l o w chart of the M U L T I G R A P H program.

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content5 of the screen Oe printed

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I T ~

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I to the main menu Return

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olution screen, they can be output to a graphics printer. A flow chart of the M U L T I G R A P H program is given in Fig. 3, and a memory map of MULTI GR AP H is shown in Fig. 4.

After activating MULTIGRAPH, the default printer slot number and the M U L T I G R A P H Menu is displayed. The following options are available: (1) Load data (2) Graph /pr in t (3) Change printer slot number configuration (4) Run ODDC - collect data

Selecting option 1 loads the specified data file from disk into RAM, after which the MULTI- GRAPH Menu is again displayed.

Option 2 graphs the data that has been loaded into RAM on the high-resolution screen, using the markers established by ODDC as reference points, and using automatic scaling. If option 2 is selected, the program checks the computer's RAM to insure that data has been loaded before continuing. The user chooses to either graph all of the data points in memory or to graph a subset of the points. To graph only a portion of the data points, marker numbers must be entered to indicate the beginning and end of the data points selected for graphing. Once the beginning and end points have been defined, the X scale of the graph is calculated. The scale is computed so that the graph fills most of the screen without excluding any of the defined points.

The defined points are displayed on the Apple's high-resolution screen. In most cases, all of the

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EXAMPLE NUMBER ONE

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-1 Label number o n e *2 Label number two *3 Label number t h r e e *4 Label number f o u r *5 Label number f i v e

Fig. 5. Example of graphic output from M U L T I G R A P H show- ing five labels (label markers are vertical bars on X axis). The threshold for peak sensing is indicated by the dotted line just above the X axis. This graph represents data points 0 to 2532 out of a total of 2533 data points in the file: EXAMPLE GRAPH.ODH. The comments are linked to the raw data when they are saved.

data points in RAM cannot be plotted, because of limited graphic resolution. Instead, multiple data points are averaged (1-50, depending on the scale), and the average value is plotted on the screen as one point. Reference markers defined while using ODDC are drawn onto the graph as the points are plotted.

After the graph has been completed, the labels corresponding to any of the markers that are on the screen can be viewed by pressing number keys. If the graph is printed, information not displayed on the screen because of limited space is printed out in addition to the display shown on the monitor. After printing, the user is returned to the Main Menu. An example of output from MULTI- G RA P H is shown in Fig. 5.

5. Speed of program performance

The ODDC program is capable of sampling data at the rate of 20 samples/s, but this is faster than needed for column chromatography. A sampling rate of 2 samples/s can be selected from the menu (suitable for applications such as enzyme kinetics)

which allows the computer to store data in memory for up to 105 min. After data collection is complete, the maximum amount of time necessary for saving data is 20 s (using David-Dos II with the verify mode turned on). Normally, the maximum number of data points (12 600) is not stored, so that loading data from disk to memory usually requires 8 s. A maximum of 4 rain is required to create a graph on the screen, and the graph can be printed in 1.3 min.

6. Discussion

With the programs presented in this paper it is possible to collect, record, manipulate, graph, and print data from a spectrophotometer using a microcomputer. Although developed primarily for column chromatography, with minor modifica- tions the programs described here could also be used for enzyme analysis in a manner similar to a previously described system [6]. The system we have developed has the advantage of interfacing to the spectrophotometer via the ADALAB card, which also can be used to control solenoid valves, fraction collectors, and other peripheral equipment used in column chromatography. For enzyme kinetic studies, the ADALAB card is capable of a faster sampling rate then a previously described system (20/s vs. 5 - 7 / s ) , which uses parallel inter- faces to the Apple computer [6].

One of the main factors that affects the utility of a computer program is the degree of user friendliness incorporated into the program. Both of the programs, O D D C and M U L T I G R A P H , are menu-driven to make the selection of parameters simple and fast. Furthermore, both O D D C and M U L T I G R A P H use the same keys to perform similar functions, so they are consistently struc- tured. These features increase the speed with which new users learn to use the programs. Data stored by O D D C is accessible for use in other analysis or graphic programs. For example, a program could be written to calculate the area under the peaks in the graph, and thereby the total protein in the peak may be derived. Data collected on disk can be transferred to one of several graphics programs that generate high-quality graphs with a pen

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plotter, thus providing graphs for presentation or publication.

As large amounts of software are being developed for the most common microcomputers, interfacing them to laboratory instruments allows use of the microcomputer for more than the single function available with dedicated instrument monitor /recorders . The Apple II computer is useful in word processing [7], statistical analysis [8], database management [9,10] and several types of laboratory instruments have been successfully interfaced to Apple II computers [11-13]. Thus, the software presented in this paper provides a truly flexible and cost-effective system for chromatography, based on hardware useful for many other purposes.

Acknowledgments

This investigation was supported by Naval Medi- cal Research and Development Command Work Unit MF58.527.004.0004. The authors thank Ms. Carole Portis for preparation of the manuscript. The opinions and assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large.

References

[1] L.R. Snyder and J.J. Kirkland, Detectors, in: Introduction to Modern Liquid Chromatography, pp. 125-167 (John Wiley and Sons, New York, 1979).

[2] J.L. Holtzman, Microprocessor-based computer for the acquisition and manipulation of data in rapid kinetic studies, Anal. Chem. 52 (1980) 989-991.

[3] T.R. Sharp, K.R. Gopinath, P.W. Brandt and T.L. Rosenberry, Microcomputer interface for computer-assisted enzyme kinetic studies with UV-VIS spectrophotometers, Anal. Biochem. 116 (1981) 545-552.

[4] C.S. Nichols, J.N. Demas and T.H. Cromarti, Microcom- puter interfaced spectrophotometer for kinetic studies, Anal. Chem. 52 (1980) 205-207.

[5] J. Morton Interfacing a microcomputer to a spectrophotometer for EMIT assays, Ann. Clin. Biochem. 20 (1983) 221-223.

[6] L.A. Schriefer and W.E. Benisek, An inexpensive com- puterized enzyme kinetics system based on a gilford spec- tophotometer and an Apple lie microcomputer, Anal. Biochem. 141 (1984) 437-445.

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[7] P. McDonald, Writing a thesis on a wordprocessor. Br. Med. J. 289 (1984) 242-243.

[8] A.J. Lee, P.J. Mclnerney and P.R. Mullins, STATCALC: An integrated statistics system for the APPLE II microcomputer, Comput. Prog. Biomed. 18 (1984) 265-272.

[9] R.M. Franklin, Microcomputer inventory systems for stored cell lines, J. Immunol. Methods 54 (1982) 141-157.

[10] K. Blochowiak, Apple II file-management systems, Byte 6 (1981) 274-300.

[11] T.S. Ford-Holevinski, B.W. Agranoff and N.S. Radin, An inexpensive, microcomputer-based, video densitometer for

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Documents

ODDC and MULTIGRAPH: programs for spectrophotometer data collection and manipulation using a microcomputer