.< •' *« i \

slfe NBS TECHNICAL NOTE 857

"*fAU Of

U.S. DEPARTMENT OF COMMERCE/ National Bureau of Standards

Real-Time Acquisition and

Processing of Fluorimetry Data
NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards 1 was established by an act of Congress March 3, 1901. The Bureau's overall goal is to strengthen and advance the Nation's science and technology and facilitate their effective application for public benefit. To this end, the Bureau conducts

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1 Headquarters and Laboratories at Gaithersburg, Maryland, unless otherwise noted; mailing address Washington, D.C. 20234.

3 Part of the Center for Radiation Research.

> Located at Boulder, Colorado 80302.

* Part of the Center for Building Technology.
IjNAL BUREAU OF STANDARDS OCT 3 1975

> Real-Time Acquisition and

jQCIOD

Peter S. Shoenteld

Processing of Fluorimetry Data £

Analytical Chemistry Division

Institute for Materials Research

National Bureau of Standards Washington, D.C. 20234

*"ffEAU of

U.S. DEPARTMENT OF COMMERCE, Rogers C.B. Morton. Secretary

{j $, , NATIONAL BUREAU OF STANDARDS, Richard W. Roberts, Director

Issued June 1975
Library of Congress Catalog Card Number: 74-600202

National Bureau of Standards Technical Note 857

Nat. Bur. Stand. (U.S.), Tech. Note 857, 45 pages (June 1975) CODEN: NBTNAE

U.S. GOVERNMENT PRINTING OFEICE WASHINGTON: 1975 For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402

(Order by SD Catalog No. C13.46:857). Price $1.05 (Add 25 percent additional for other than U.S. mailing).
TABLE OF CONTENTS

Page

1. INTRODUCTION 1

2. THE ANALYTICAL CHEMISTRY DIVISION COMPUTER SYSTEM . 2

3. THE FLUORIMETRY EXPERIMENT 4

4. THE FLUORIMETRY DATA ACQUISITION PROGRAM 6 A. Experiment and File Structure 6 B. Program Features. . 8

1. Checkpoints and Restarts 8 2. Teletype Output 9 C. Program Description 10

1. Major Variables 10

2. Flowcharts 10

3. Error Codes 11

D. Operating Instructions 11

5. THE FLUORIMETRY DATA PROCESSING PROGRAM 21

A. Program Description 21

1. General Description 22

2. Flowcharts 24

3. Derivation of Formulas 24

B. Operating Instructions 26

6. LITERATURE 30

APPENDIX A: GENERAL RECORD DESCRIPTIONS 31

APPENDIX B: RECORD FORMAT DETAILS FOR FLUORIMETRY

EXPERIMENT 35

APPENDIX C: FLUORIMETRY FILE DESCRIPTION 37

APPENDIX D: SAMPLE DATA PROCESSING OUTPUT 38

LIST OF FIGURES

Figure Page

1. Diagram of Laboratory Instrument Console 5 iii

REAL-TIME ACQUISITION

AND

PROCESSING OF FLUORIMETRY DATA

Peter S. Shoenfeld

The National Bureau of Standards Analytical

Chemistry Division uses a centralized computer

system to automate a number of experiments.

Computer software for a fluorimetry application

is described in this report. The file and program structure used is quite general and can be

applied to other experiments as well.

Key Words: Analytical chemistry; computers;

data acquisition; data processing;

fluorimetry; laboratory automation;

real time.

1. INTRODUCTION

The National Bureau of Standards Analytical Chemistry

Division operates a generalized laboratory automation facility

which has been used to computerize a number of experiments.

The fluorimetry experiment described herein is typical of

a general class of these where minimal control is desired, but

acquisition and special processing of data are extremely

important

.

Considerable effort was expended in devising a file

structure which would accommodate a. variety of experiments.

The data acquisition program can be used for many experiments

with little or no modification.

The major participants in this project were Peter

Shoenfeld, Lawrence Kaetzel, and Fillmer Ruegg. Mr. Shoenfeld designed and programmed the software and wrote this

report. Mr. Kaetzel tested the software, and Mr. Ruegg

designed and implemented the hardware interfacing. The

entire project was under the administrative supervision of

James R. DeVoe.

1
2. THE ANALYTICAL CHEMISTRY DIVISION COMPUTER SYSTEM

The Analytical Chemistry Division Computer System is

built around a Univac Series 60 (formerly known as EMR 6135)

general purpose computer. This computer has 32,000 words of

16-bit main memory, two million words of high speed disk

storage, a magnetic tape drive, and other peripheral devices.

A generalized interface has been developed which allows the

computer to communicate simultaneously with numerous laboratory interfaces via a bidirectional data bus. Information

sent from the laboratory to the computer includes data from

the experiment and parameters dialed in thumbwheels on the

laboratory interface control panel. Information sent from

the computer to the laboratory includes control signals

for the experiment and output to optional Teletypes, CRT

displays, and drum plotters. Communication of experimental

parameters is generally done through the control panel thumbwheels . Data transmissions from the laboratory to the computer

are in the form of standard records of several different

types. All records begin with an address word indicating

the type and origin of the records. Up to fourteen additional words follow, depending on the record type. The

individual record types are described briefly below and in

more detail in Appendix A.

Setup records are sent at the beginning of each experiment to notify the computer of the experiment's onset. A

setup record is triggered by pushing the "Setup" and "Send"

buttons on the laboratory panel when the panel is in its

inactive state. This record consists essentially of the

information dialed in on the panel thumbwheels.

-In no case does the identification of trade names imply recommendation or endorsement by the National Bureau of

Standards, nor does it imply that the equipment identified

is necessarily the best available for the purpose.
Re-setup records are identical in content to setup

records, but are triggered when the "Setup" and "Send"

buttons are pushed while the experiment is running. These

records are generally used to transmit new parametric information and to denote major breaking points in the experiment.

Data records contain up to 32 bits of experimental data.

The triggering logic is generally hardwired for the individual experiment.

Precursor data records (PCD's) contain the time of

year, in milliseconds, and the contents of the input parameter

thumbwheels on the panel. They may also contain several

words of additional information hardwired for the individual

experiment. PCD's are most frequently used to denote the

beginning of a series of data records, but they may be used

for other purposes as well.

Terminate records consist essentially of a single word

and are used to signal the end of an experiment. They are triggered by pushing the "Terminate" and "Send" buttons on

the panel.

Experiments which have their own data buffer, such as multichannel analyzers, may also send data to the computer

in a high-speed, buffered mode. This is done by first

sending the computer a PCD with a special bit set to indicate the presence of buffered data. It is then the computer's responsibility to "read-out" the buffer in the laboratory.

Four major software elements are involved in automating

an experiment; the real-time operating system (ASSET), the

data acquisition system (DACQ) , the file management system

(FMGR) , and the experiment control program. ASSET provides

the multiprogramming, scheduling, and input-output facilities

which make all else possible. The experiment control program is the ultimate recipient of all records transmitted

from the laboratory to the computer and is the source of

control signals and output messages sent back to the labora-
tory. It is written in FORTRAN and is tailored to the

particular experiment. DACQ provides the software interface between each experiment and its associated experiment

control program. FMGR facilitates the dynamic creation of

sequential disk files for the storage of experimental data.

Generally, such a file is created each time an experiment

is run. The experiment control program transfers data to

disk via calls to FMGR.

3. THE FLUORIMETRY EXPERIMENT

An NADH (reduced nicotinamide adenine dinucleotide

)

reference standard is being developed for use in clinical

laboratories. This experiment is used in determining the

theoretical maximum reaction velocity of the NADH sample and

the "Michaelis Constant associated with the NADH-enzyme com- bination.

The fluorimeter is used to measure the fluorescent

intensity of a fluorescing sample, since dimunition in

fluorescent intensity as a function of time serves as a measure of reaction rate of NADH with the alcohol dehyrogenase

and the acetaldehyde substrate. A photomultiplier output

signal is monitored continuously by a strip chart recorder

and a digital voltmeter (DVM) . The DVM is sampled at discrete time intervals, with its output going to the computer.

The sampling rate is set manually and may vary from 0.1 to

20 readings per second.

The experiment, for automation purposes, can be con- sidered to consist of a number of "runs" each containing

three "phases." In a given phase a record of fluorescent

intensity vs. time is obtained, with the light source intensity remaining constant. Typically, a run might be five

minutes in duration with the entire procedure taking several

hours

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In phase one of each run, the fluorescent intensity

of a standard quinine sulfate solution is measured. This

is used to correct for instrumental fluctuations between

runs. In phase two, a mixture containing NADH, a buffer

solution, and an enzyme is placed in the machine and its

fluorescent intensity is measured. In phase three, acetaldehyde is added to the solution, causing a reaction. The

resulting fluorescent decay curve is measured. Values of

initial reaction rate (V n

) and initial NADH concentration

(N ) are calculated from the data obtained for each run. Between runs, the NADH concentration is varied. A plot of

1/Vn vs. 1/N

n

is obtained with each run contributing a single

point.

4. FLUORIMETRY DATA ACQUISITION PROGRAM

FLACQ, the fluorimetry data acquisition program, is the

experiment control program associated with the fluorimetry

experiment. Its generalized structure makes it applicable

to other experiments as well.

A. Experiment and File Structure

An "experiment" consists of that quantity of data collected between the time the control panel is "Set Up" and

the time it is "Terminated." Experiments are identified by

the log number which is dialed into the control panel thumbwheels and is transmitted in the setup record. An experiment

includes a variable number of "runs," each of which includes

a variable number of "phases." A "file" is associated with

each experiment. This file is created when the setup record

is received and is filled with data during the course of

the experiment. After the experiment, the file may be

processed to obtain numeric output, or it may be transferred

to an archive tape for later processing or processing on another computer

.
The control panel "Data Handling" and "Repeat Cycle"

thumbwheels are used to communicate control information

when the experiment is set up. The data handling thumbwheels should be zero unless we are restarting an earlier

experiment (see 4.B.). The right two repeat cycle thumbwheels should contain the maximum number "of phases expected

per run and the left two should contain the maximum number

of DVM values (in hundreds) expected per phase. An absolute

maximum of 500 values per phase is currently assumed.

The information transmitted to the computer for each

phase consists of a PCD followed by a number of data records,

one for each time sampled DVM reading. In addition to the

customary information, the PCD is hardwired to contain the

sampling rate and a scale factor corresponding to instrumental sensitivity (see Appendices A and B) . The last data

record of a phase is marked by a special stop bit. Buttons

marked "Start" and "Stop" are mounted on the DVM panel.

Pushing the Start button causes a PCD to be sent and sampling

to start. Pushing the Stop button causes sampling to cease with the last reading being marked by a stop bit. If the

maximum expected number of points per phase is reached

before the Stop button is pushed, the computer sends back

a signal which has the same effect as pushing the Stop

button

.

Phases are numbered sequentially within each run by

the computer. Runs are numbered sequentially within each

experiment. A new run starts whenever either the maximum

expected number of phases per run is reached or a re-setup

record is sent to the computer.

Each file consists of a label record, also called Record

0, followed by a number of subfiles, also called logical

files, one for each phase. The label record contains certain

standard identifying information, a copy of the setup record,

and the run number and phase number of the last phase currently

in the file. Each subfile contains a header entry followed

7
by the DVM readings for the associated phase in floating point

format. The header entry contains a copy of the PCD record

which initiated the phase as well as the run number and phase

number of that phase. See Appendix C for further details.

B . Program Features

1 . Checkpoints and Restarts

At the end of each phase, certain measures are taken

by FLACQ to facilitate restarts. The fields in the label

record denoting last run number and phase number are updated

with the run number and phase number from the phase just

completed. This information is needed to resume the numbering

sequence in the event of a restart. The file is also checkpointed. If a computer failure occurs, the only information

lost from the file is that which was written since the last

checkpoint

.

Restarts are of two types. One type is used after a deliberate interruption and the other after a computer

failure

.

The experiment may be terminated at any time. If a phase is in progress when the terminate button is pushed,

it is simply truncated at the terminate point. The experiment may be later restarted by sending a "Set Up" with the same log number dialed in the control panel thumbwheels and

the data handling thumbwheels set to 0001. Phases recorded

subsequently will be added to the old file and the numbering

sequence will be resumed at the breaking point.

The second type of restart is used after the system

has failed and has been brought back into service. The run number and phase number of the last completed phase are dialed into the input parameter thumbwheels and the data

handling thumbwheels are set to 0002. Sending a setup then

restarts the experiment, using the dialed in information to resume the numbering sequence.
2 . Teletype Output

Although disk is the primary medium for recording data,

messages are sent to a laboratory Teletype monitoring the

progress of the experiment. These are explained individually

b e 1 ow

.

Event Message

Start of experiment

(Setup)

Start of experiment

PREFATORIAL DATA

Message followed by identifying

information from setup record.

DUPLICATE LOG NO.

Message appears only if log no. dialed in duplicates that of a file already on disk.

Restart

Restart

Start of phase

End of Phase

End of phase

End of Run

End of experiment

(Terminate)

RESTART RUN NO. XX PHASE NO. XX

These are the run and phase nos

.

of the last phase acquired before

restart

.

RESTART- -FILE MISSING

Message appears only if restart

is attempted with a log no. for

which there is no corresponding

file on disk.

RUN XX PHASE XX

VALUE = XXXXXXXXX.

DVM reading in last data record

of phase. Message may not appear

until start of next phase.

END OF PHASE

Message may not appear until

start of next phase.

END OF RUN

Message may not appear until

start of next run. EXPERIMENT TERMINATED
C . Program Description

1 . Major Variables

Major program variables are identified below. Minor

variables, not involved in controlling program flow, are omitted.

NPHASE -- Maximum number of phases per run; obtained from

setup record.

NPTS -- Maximum number of points per phase; obtained from

setup record.

IPHASE -- Current phase number.

IRUN -- Current run number.

IPTS -- Number of points collected so far in current phase.

ACT IV -- Logical switch indicating phase in progress.

FLUSH -- Logical switch indicating that a "Stop" has been

sent to the DVM and that further data should be

ignored

.

VALUE -- Floating point value extracted from data record.

2 . Flowcharts

The basic functions of the fluorimetry acquisition program are shown in the flowcharts which follow. Input-Output

status checks and other minor details are omitted for clarity.

The program is organized around the different possible input

record types and the flowcharts reflect this.

The "End-of-Phase Subroutines" is a separately compiled

external FORTRAN subroutine, called SUBROUTINE EPHASE. The

"Terminate Routine" is a bit different from the other rou- tines in that DACQ enters it directly on receipt of a terminate record instead of passing the record^ to the experiment control program as is done with the other record types.

10
3 . Error Codes

The acquisition program uses Subroutine ERROR to report

input-output failures and other difficulties on the console

Teletype in the computer laboratory. The message typed is

always of the form ERROR 6XX or ERROR 7XX . When ERROR 7XX

is typed, the experiment is automatically terminated. Error

codes typed and their meanings are listed below:

ERROR 600 -- Data record received at time when this is

illegal

.

ERROR 610 -- Unrecognizable record received or PCD record

received at time when it's illegal.

ERROR 720 -- I/O failure.

ERROR 723 -- File manager error, or duplicate log no. on Setup, or file missing on Restart.

D. Operating Instructions

Computer related operating procedures are described

individually below:

To begin experiment -- Insert User I.D. Card. Set

control panel dials as follows:

Experiment Program -- 67 (Identifies FLACQ)

.

Experiment Log No. -- new value (Identifies file).

Data Handling -- 0000

Repeat Cycle -- Maximum number of phases per run in

right two dials, maximum number of points (hundreds)

per phase in left two dials.

The DVM rate dial should be set. Control Panel should

be in ON RDY state. Turn on Teletype. Push "Setup," then

"Send." Control panel should switch to "Operate" state and

the Teletype should type "Prefatorial Data." If "Duplicate

Log No." is typed, try again with a new log number.

11
MAIN ROUTINE

Connect with

DACQ via

L0ADN

G> 200 ^

Get next record

§ select proper

routine via

READT

Control enters here

on receipt of all

records except

Terminates

L0ADN is a system subroutine called at the

"front-end" of experiment control programs

to establish communicationREADT is a system subroutine which reads

records from an input buffer, determines

their type and sends control to the routine

associated with that type.
SETUP ROUTINE

1000

Initialize Teletype,

Type Prefatorial

Data via TPD

Extract NPHASE,

NPTS from setup

Record via

KBCDBN, IEXTR

Subroutine TPD types

Prefatorial Data. ALU, 0PEN, CL0SE, § NTTY are system routines

used in initializing

Teletype

.

KBCDBN is a BCD to binary converter.

IEXTR is a bit

extraction routine,

Generate name for

file via NAMGN

subroutine

Create new file

on FMGR

No

1060

Open File

on FMGR.

IRUN + 1

I PHASE <- ACTIV +- FALSE

TTYPE is a Teletype

subroutine

1040

Type Message

in Lab. via

TTYPE

Subroutine Error

Terminates experiment

when Error code of form 7XX

13
RE-START ROUTINE

1500

Generate Name

§ resume old

file on FMGR

Subroutine Error used,

Type Message

in Lab via

TTYPE

RI0S is ASSET

1/0 routine

1550

Read Record

using RI0S and

extract IRUN,

I PHASE

Type 2 (0002

(System Crash

Extract IRUN,

IPHASE from

Dials

ACT IV * FALSE

Type Message, IRUN,

IPHASE in Lab via

TTYPE

LWRIT is FMGR

logical file handler

Yes

Write logical EOF

on File via

LWRIT

(EOF may be

missing after crash)

14
RE-SETUP ROUTINE

2000

irue

=

IRUN + IRUN + 1 IPHASE *I

Type "End of Run"

Message in Lab

via TTYPE

2050

Extract NPHASE,

NPTS from

Record

Console notified

via ERR0R routine

15
PCD ROUTINE

3000

Console notified

via Error Routine

Compose Header

Record from

PCD Record,

IRUN, IPHASE

I

Write Header Record

via LWRIT

Type Message,

IRUN, IPHASE

in Lab via TTYPE

LWRIT is

FMGR Logical

File Handler
DATA ROUTINE

4000

Subroutine SEND

used to send signals

through interface

Send signal to stop DVM via

SEND

Type Message

in lab via TTYPE

Extract VALUE

§ write on FMGR via

LWRIT

4050. Type Message

VALUE in lab via TTYPE

'Perform

End of Phase

^Subroutine

FLUSH

+ TRUE

17
END OF PHASE SUBROUTINE

Update Record

with current

values for

I RUN, I PHASE

Write Logical EOF

on FMGR via

LWRIT

Type "End of Phase'

Message in Lab

via TTYPE

IRUN + IRUN +1

IPHASE <- I

Type "End of Run"

Message in Lab

via TTYPE

7^->[ Return

j

18
TERMINATE ROUTINE

9000

DJCN is routine by

which experiment control

program relinquishes control

or terminates experiment.

Yes W DJCN

J

9010

Send Stop Signal

to DVM via

SEND

9030

True

False

Perform

End of Phase

Subroutine

Close File

on FMGR

Type Message

in Lab via TTYPE

19
BUFFER EMPTY AND

ILLEGAL RECORD ROUTINES

5000 'Buffer^

Empty

Routint

6000

Console notified

via Error Routine

©Experiment Control Program

relinquishes control

.a DJCN

20
To begin phase -- Push start button on DVM. Teletype

should type out run and phase numbers.

To begin new run before reaching maximum phase number - - Set control dials as at beginning of experiment. Push

"Re-Setup," then "Send." If "Error" lights on panel, try

again. Teletype should respond with "End of Run."

To Terminate Experiment -- Push "Terminate," then

"Send." Teletype should respond with "End of Experiment."

To Restart after normal Terminate -- Follow same procedure as at beginning of experiment, but with 0001 in the

Data Handling dials. Teletype should respond with "Prefatorial Data" followed by the run and phase numbers at which

the interruption occurred. If no file can be found on disk

which matches the Log Number dialed, the Teletype will say so. To Restart after computer failure -- The Data Handling

dials should contain 0002. The Input Parameter dials are used to resume the run and phase number sequence with the run number in the left two dials and the phase number in the right

two. The run and phase number of the last completed phase

should be used unless we are at the beginning of a run, in

which case we use the run number of that run and a phase

number of 0. Procedure is like that immediately above

otherwise

.

Spooling of Teletype messages sometimes results in a delay before their receipt in the laboratory. The user should wait for a message about twenty seconds before con- cluding that the system has failed.

5. FLUORIMETRY DATA PROCESSING PROGRAM

A. Program Description

This program performs computations on the file produced

oy the acquisition program (see Appendix C) . It is run as

a non-resident foreground program on the Univac Series 60

21
(EMR 6135) computer. It reads the file from disk and produces processed output on the line printer.

1 . General Description

The program begins execution by reading the I.D. No.,

Code, and Log*No. needed to identify the file from the

console Teletype. A heading reflecting this information is

printed. Successive phases are then read and processed.

After the last phase has been processed, a summary table is

printed giving the reciprocals of initial reaction rate and

NADH concentration for each run completely processed. The

program expects phases numbered 1, 2, and 3, in that order,

in each run and does not care about the sequence of run numbers. Any sequence errors are detected and handled.

Phase 1 Processing

The average and standard deviation of the intensity

values on the file are computed and printed out using subroutine STAT 2 . The average intensity is saved for phase 3,

where it is used as the normalizing quinine sulfate value.

Phase 2 Processing

The average and standard deviation of the intensity

values are computed and printed out as in phase 1.

Phase 3 Processing

A least squares line is fitted to the values received,

and the slope and y-intercept are printed out. The yintercept is in millivolts and the slope is computed using

a time unit equal to the constant time separation between

points. Residuals and their standard deviation are computed

and printed out in units of volts. A sensitivity correction

is computed using the sensitivity from the header record.

The time unit is converted to minutes using the rate from

the header. The period between readings, the number of

readings, the sensitivity, and the quinine sulfate intensity

22
saved from phase 1 are all printed out. Initial intensity,

reaction rate, and NADH concentration are computed and printed

out using the formulas discussed in (2) . The run number and

the reciprocals of the initial reaction rate and NADH intensity are saved for printout in the summary at the end of

processing

.

The usefulness of the processing program is presently

severely limited by the presence of certain excursions, or "blips", in the phase 3 data. These occur approximately 401

of the time. This problem should be corrected by either

modifying the instrument or by the computational filtering

off of the "blips" in the phase 3 processing routine before

further applying the program to the present apparatus. The

program presently assumes that it is operating on a nearly

linear initial portion of the reaction rate curve. Some

modification of this assumption might also be desirable.

Tables and Constants

A table in the program (CTAB) is used to obtain initial

NADH concentration as a function of initial intensity via

table lookup and linear interpolation. Short tables are also used to interpret the sensitivity and data rate bits

in the header record. The program presently assumes a constant background intensity of .0006.

Error Conditions

Error indications appear on the line printer in the

computer laboratory as listed below:

FILE MISSING -- No file on disk matching identifiers

entered on console Teletype, no recovery.

FMGR ERROR -- I/O error, no recovery.

FILE ERROR -- Error in logical file structure, no recovery

.

23
PHASE NO. BAD -- Phase encountered with phase number

other than 1, 2, or 3. Phase is identified on printout

but not processed.

SEQUENCE ERROR -- Phase encountered out of the expected

1, 2, 3 phase number sequence within each run. Phase

is identified on printout but not processed.

If a run contains less than three phases on the file,

and they are sequenced correctly, the phases present will

be processed, but no entry for that run will appear in the

summary table at the end of the printout.

If a phase contains more than 300 points, the excess points are ignored.

2 . Flowcharts

The general flow of the processing program is shown.

The "Phase 1, 2, and 3 routines** are internal branches.

STAT2 is an external subroutine used to compute the average

and standard deviation of a linear array.

3. Derivation of Formulas

The formulas derived here are used during phase 3 processing for computing initial intensity (I )

, initial

NADH concentration (N

Q

) , and initial reaction rate (V„)

.

Notation

Q = Average fluorescent intensity (volts) obtained with

quinine sulfate in Phase 1; used to normalize

between runs

S = Fluorimeter sensitivity in Phase 3

C = Normalization factor to be applied to Phase 3 intensity data

24
F = y intercept of observed fluorescence curve (volts)

V = -Slope of observed fluorescence curve (volts/minute)

N(t) = NADH concentration as function of time

N

Q = Initial (t = 0) NADH concentration = N

f(n

I(t) = Fluorescent intensity as function of time

(0)

I Q = Initial (t = 0) fluorescent intensity = I

I fiG = Background fluorescence intensity

I = Portion of fluorescent intensity resulting from

reaction = I

- I R p

V = Initial reaction rate = (dN/dt) „ From the observed Phase 3 fluorescence curve we wish to

calculate V

n and NQ

. We use an experimentally obtained table

of NADH concentration vs. fluorescent intensity to obtain N

Q

after calculating I Q

. By first principles we have

(i) dN/dt = -AN for some constant A. We assume fluorescent intensity to be proportional to

NADH concentration. Thus, N(t)/I (t) = constant. Differentiating, (dN/dt)/N = (dl

r /dt)/l

r

. Thus, dl

r /dt = -A I r by (i)

.

Integrating and substituting I-I Rr for I we get

- At

I(t) - I BG

= (I

Q -I BG ) e • Linearizing,

I(t) - I BG

= (I -I BG ) (1-At) or

(ii) I(t) = I Q

-A (I -I BG ) t. Now F and -V are the y- intercept and slope of the

observed fluorescence curve. We apply a factor c = S/(Q x 3.0)

to account for sensitivity and to normalize between runs,

obtaining corrected values F' = cF and V = cV. The factor

25
3.0 arises from an assumed sensitivity during the Phase 1 quinine sulfate observations. We then have I„ = F' by (ii)

and we get N

Q

from I„ by table lookup and linear interpolation.

We have V = A(I

Q -I BG ) from (ii) . Solving for A, we get

(iii) X = V ( 1

)

. BG

Evaluating (i) at t=0 we get

(iv) V

Q = -(dN/dt) t=Q

= A N

Q

Combining (iii) and (iv) we get

(v) Vn - V (r= -—). This is the equation used in i 0" i BG

calculating Vn

.

B . Operating Instructions

1. Enter SNR,45,12 on the console Teletype to begin

execution (schedules non-resident program number 45

at priority 12)

.

2. Console Teletype will request a word on input three

times. Request and proper operator response are given

b e 1 ow

.

ID

I user's I.D. No., in this case 575

CODE

I identifying code word in file name, in this case 00000

LOG NO.

I five digit log number dialed into control panel when

file was created

3. Program runs to completion.

26
Read Log . No .

,

code, 6 ID from TTY

OPEN File Status <0 Error Message

Print Heading

| - NRUN, K

| ± 210 NRUN - LRUN

NPHASE * LPHASE

240 Get Values of

NRUN, NPHASE,

TDELT, SENS from Header

NPTS

_ V 210 _ . „ , r— , T 1 Bad Status or.. r_,.„_

I Read a Point I —-fc Error

FMGR EOF Message

Yes

>,300

NPTS+1 - NPTS Point + Y(NPTS) Perform

PHASE I routine

850Error Message

27
PHASE 1 ROUTINE

Compute Ave § 5td. Dev. from

Y, NPTS

PHASE 2 ROUTINE

Compute Ave

5 Std. Dev. from Y, NPTS

Print Ave

§ Std. Dev.

No

Print

"Sequence Error"

28
PHASE 3 ROUTINE

"NRUN = LRUN\ No

§ LPHASE = 2, Print

"Sequence Error'

C0RR * SENS

QSX3

Find V , F for least squares line

O O

Compute residuals

and Std. Dev.

F X CORR

o

Look up N

in NADH table

as function of F

Vo *V X CORR X N

o o F_ - bG

Print QS, SENS

F„. V , Not residuals, Std. dev. P K + 1

Y oKth row of

Summary Array +Q

29
6. LITERATURE

DeVoe, J. R. , Shideler, R. W. , Ruegg, F. C, Aronson, J. P.,

Shoenfeld, P. S., Anal. Chem. 46, 509 (1974).

ASSET IV Real-Time Monitor User's Manual , EMR Computer,

Bloomington, Minn., 1970.

FORTRAN IV User's Manual , EMR Computer, Bloomington, Minn.,

1970.

30
APPENDIX A. GENERAL RECORD DESCRIPTIONS

The records transmitted from the teleprocessor (lowspeed) to the computer all consist of an address word plus

up to fourteen additional words. Bits 1-8 of the address

word contain the laboratory address of the device sending

the record. Bits 9-10 are unused. Bits 11-16 are control

bits identifying the record type.

1 . Setup Records

A Setup record is sent when the experimenter pushes

his "SETUP" and "SEND" buttons. It contains fourteen words

as well as the address word with contents as follows

Word Contents

* Identifier Octal 100000

address Bits 16 and 15 on (140xxx, x=address) , other control

bits off

1 Bits 1-11, User No. (from I.D. Card); Bits 12-16

No. words in PCD in addition to address word (hardwired), see below

2-4 Time of year to nearest millisecond, see below

5 Experiment log No. (from thumbwheels)

6 Input Parameter (from thumbwheels)

7 No. of high-speed words (Mantissa bits 1-13,

Exponent -1 bits 14-16)

8 Nonresident program number of applications program

(from thumbwheels)

* The Identifier is placed in front of the address word of

the Setup Record by DACQ when it is passed to an application

program It is not transmitted from the teleprocessor.

31
9 Repeat cycle; No. of PCD Records to be processed

before forced termination (from thumbwheels) 4 BCD

digits

10 Data Handling (from thumbwheels)

11-14 Special purpose; not generally used

Time of Year Format

Word Bits Contents

2 15-16 Day; 1st BCD digit

11-14 Day; '2d BCD digit

7-10 Day; 3d BCD digit

5-6 Hour; 1st BCD digit

1-4 Hour; 2d BCD digit

3 12-14 Minute; 1st BCD digit

8-11 Minute; 2d BCD digit

5-7 Second; 1st BCD digit

1-4 Second; 2d BCD digit

4 9-12 Millisecond; 1st BCD digit

5-8 Millisecond; 2d BCD digit

1-4 Millisecond; 3d BCD digit

No. of PCD Words Format (Bits 12-16, Word 1)

Contents No. of Words

10000 14

01000 12

00100 10

00010 8

-

00001 6 32
2 . Re-Setup Records

A Re-Setup Record is sent when the experimenter pushes

his "SETUP" and "SEND" buttons. It contains fourteen words

as well as the address word with contents as follows:

Word Contents

* Identifier Octal 100000

address Bits 16 and 14 on (octal^ 120xxx, x=address),

other control bits off

1-14 Same as Setup Record

* The Identifier is treated the same way as in Setup Records.

3. Data Records

A Data Record is sent when the experimenter pushes his

"DATA" and "SEND" buttons when in manual mode and automatically when in automatic mode. It contains two words as well

as the address word with contents as follows:

Word Contents

* Identifier Octal 2000

address All control bits off

1-2 Data

* PCD and Data Record Identifiers are generated by DACQ and

replace the address word when the record is passed to an application program.

33
4 . PCD (Precursor Data) Records

PCD Records are generally used to transmit control information. They are usually sent periodically according to

a hard-wired scheme which is application dependent. The

number of worcls sent in addition to the address word may be

six, eight, ten, twelve, or fourteen. This number is appli

cation dependent, but fixed for a particular application.

Contents are as follows:

Word Contents

* Identifier Octal 40000

address Bits 16 and 13 on (OlOxxx, x=address) , bit 11

may also be on to signal a high-speed transmission; other control bits off

1-6 Same as Setup Record

7-14 Application dependent when present

* PCD and Data Record Identifiers are generated by DACQ and

replace the address word when the record is passed to an application program.

5. Terminate Records

A Terminate Record is sent when the experimenter pushes

his "TERMINATE" and "SEND" buttons. It contains fourteen

words as well as the address word with contents as follows:

Word Contents

address Bits 16 and 12 on (004xx, x=address) , other

control bits off

1-14 Same as setup record (currently ignored)

.

34
APPENDIX B. RECORD FORMAT DETAILS FOR

FLUORIMETRY EXPERIMENT

Setup Record

Repeat Cycle Dial -- Max. No. Phases/Run in bits 1-8,

Max. No. Hundred Points/Phase in bits 9-16

Log No. Dial -- used for naming file as is customary

Data Handling Dial -- 0000 means normal setup

0001 means restart after normal

terminate

0002 means restart after computer

failure

Input Parameter -- meaningful only when restarting after

computer failure

Bits 1-8 give phase no. to resume with

Bits 9-16 give run no. to resume with

PCD Record

PCD's are nine words long. Words 1-7 are standard and

word 9 is not used. Bits 4-8 of word 8 indicate sensitivity

and bits 9-16 indicate data rate as shown below.

Bits 4-8 Full Scale DVM Value in Volts

00001 1.0

00010 0.3

00011 0.1

01000 0.03

10000 0.01

35
Bits 9-16 Time Interval between Readings in Seconds

00000001 0.05

00000010 0.1

00000011 0.2

00001000 0.5

00010000 1.0

00011000 2.0

01000000 5.0

10000000 10.0

Data Record

Word 1 -- Address word

Word 2 -- Bit 5 -- Stop Bit, 1 means stop

Bit 4 -- Over-range Bit, 1 means over-range

Bit 3 -- Sign Bit, 1 means minus

Bits 1-2 -• Most significant BCD digit (3 is max.)

Word 3 -- Least significant four BCD digits

36
Appendix C. Fluorimetry File Description

1st Phase of 1st Run Last Phase of Last Run

Logical

Rec. 0| | Headerj | Data| E.O.F.

Logical

E.O.F.

FMGR

Header] Data E.O.F.

Record

20 34 35 36

FMGR

Standard Information

Setup Record Info,

inserted by NAMGN I RUN I PHASE

49

IRUN § IPHASE are the Run No. § Phase No. of the last phase on the file

Header

1 9 10 11 12 PCD as received

Run

No. PhaseNo.

Data

Each data block contains an indeterminate number of floating point

(2 word) entries.

Logical E.O.F. 's

These are generated by LWRIT and detected by LREAD.

More than one logical E.O.F. may appear between phases; since an extra logical E.O.F. is sometimes inserted by the Re-start procedure,

37
APPENDIX D. SAMPLE DATA PROCESSING OUTPUT

ID" 575 COOEi

PLUORIHETRY PROCESSING

LOG NO.« 3

RUN NO

INTENSITY

1 PHASE NO.

.3759

1 STO. DEV. , 0020

RUN NO

INTENSITY

1 PHASE NO.

.3069

2

STD. DEV. , 1069

RUN NO

SLOPE

1 PHASE NO.

.3004

3

VINT 315.7137

-.0014 .0019 .0032

RESIDUALS

.0025 .0018 -.0009 -.0016 -.0013

.0000 .0007 -.0.014 -.0021 -.0018 -.0005 -.0022 .0001

.0006 • .0013 .0020 .0003 -.0004 .0009 .0032 .0005

STD. DEV. .0017 PERIOD .01667 NO. DBS. 24

QUININE SULFATE .3759 SENSITIVITY 1.0000

INTENSITY .2799 INIT.RATE 12.0098 CONCEN. 209. 8913

88
RUN NO

INTENSITY

2 PHASE NO.

.3086

1 8TD. DEV. 0017

RUN NO

INTENSITY

2 PHASE NO.

.3020

2 STD. DEV. 0005

RUN NO

SLOPE

2 PHASE NO.

-.9669

3

YINT 308.9615

-.0100 i >.0090 -.0101

RESipUALS

-.0101 -.0061 -.0091 -.0082 -.0092

-.0002 i-.0073 -.0093 -.0093 -.0064 •.0104 -•0074 -.0065

•.0065 •0059 -.0036 -.0076 -.0016 -.0046 -.0017 .0003

.0003 .0002 .0012 .0002 .0021 .0031 • 0071 .0040

.0040 .0060 .0060 .0099 .0079 .0069 • 0108 .0108

.0078 .0097 .0097 .0127 .0136 .0116 .0136 .0135

.0148 .0165 .0155 .0174 .0164 .0184 .0183 .0163

.0223 .9222 .0192 .0222 .0221 .0211 .0241 .0270

.0230 .0240 .0260 .0249 .0279 .0269 -.2352 -.2352

STD, DEV. .0421 PERIOD .01667 NO. 088. 72

QUININE SULFATE .3086 SENSITIVITY 1.0000

INTENSITY ,3337 INIT.RATE 59 e 8335 CONCEN, 317. 4258

39
RUN NO

INTENSITY

RUN NO

INTENSITY

3 PHASE NO.

.6724

3 PHASE NO.

.3054

1 8TD. OEV. .0037

STO. DEV. ,0017

RUN NO 3 PHASE NO,

SLOPE -.3723 YINT 290.7699

-.0014 .0010 -.0007

RESipUALS

-.0003 .0011 -.0005 -.0002 -.0026

-.0014 -.0010 -.0017 .0017 .0001 .0004 .0008 .0012

-.0004 -.0001 -.0007 -.0013 .0030 -.0016 -.0002 .0032

.0005 -.0021 .0003 .0007 .0010 -.0006 .,0002 -.0009

.0005 .0029 -.0017 .0016 .0010 -.0016 .0008 -.0009

-.0025 .0009 .0012 .0026 .0020 -.0006 ,0017 -.0009

.0025 -.0002 .0012 .0006 -.0000 .0023 -.0003 .0001

.0005 -.0012 .0012 -.0024 -.0001 -.0017 -.0003 -.0009

-.0016 -.0002 -.0018 -.0005 -.0001 -.0027 -.0013 -.0010

-.0006 -.0002 .0002 ..0005 .0009 .0023 -.0024 .0000

.0004 .0018 .0011

TO, DEV .0014 PERIOD .01667 NO, OBS 83

QUININE SULFATE .6724 SENSITIVITY 1.0000

INTENSITY .1441 INIT.RATE 4.2765 CONCEN. 55.4318

(RUNS 4-22 OMITTED FROM APPENDIX)

40
SUMMARY

RUN 1/INIT.RATE 1/CONCENTRATX

1 .0833 2.3378

2 .0167 1.4668

3 .2338 1.3698

4 .2581 1.6461

5 .2611 1,5998

6 .2389 2.0528

7 .3069 2.5986

8 .2927 • 0567

9 -580.1123 .0454

10 3.6618 ,0546

11 3.4137 .0516

12 1,2163 ,0538

13 -50.3861 .0545

14 12.5003 ,0544

15 .9596 ,0518

16 2.3376 408.0000

17 1.4668 407.0000

18 1.3698 412.0000

19 1.6481 415.0000

20 1.5998 416.0000

21 2.0528 416.0000

22 2.5986 .0360

END OF JOB

41
|4iBS-114A (REV. 7-731

U.S. DEPT. OF COMM. BIBLIOGRAPHIC DATA

SHEET

1. PUBLICATION OR REPORT NO. NBS TN-857

2. Gov't Accession

No.

3. Recipient's Accession No.

4. TITLE AND SUBTITLE Real-time Acquisition and Processing of

Fluorimetry Data

5. Publication Date June 1975

6. Perfotming Otganization Code

7. AUTHOR(S)

Peter S. Shoenfeld

8. Performing Organ. Report No.

9. PERFORMING ORGANIZATION NAME AND ADDRESS NATIONAL BUREAU OF STANDARDS DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

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Same as number 9

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15. SUPPLEMENTARY NOTES

Library of Congress Catalog Card Number: 74-600202

16. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant bibliography or literature survey, mention it here.)

The National Bureau of Standards Analytical Chemistry Division

uses a centralized computer system to automate a number of experiments. Computer software for a fluorimetry application is described

in this report. The file and program structure used is quite general

and can be applied to other experiments as well.

17. KEY WORDS (six to twelve entries; alphabetical order; capitalize only the first letter of the first key word unless a proper name; separated by semicolons) Analytical chemistry; computers; data acquisition; data processing;

fluorimetry; laboratory automation; real-time.

18. AVAILABILITY KZ Unlimited

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