NBS

PUBLICATIONS A 11 ID 2 73015b

NBSIR 87-3669

System of Hardware and Software

Developed for Size Exclusion

Chromatography

B. Dickens and F. L. McCrackin

U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards

Institute for Materials Science and Engineering

Polymers Division

Gaithersburg, MD 20899

December 1987

U.S. DEPARTMENT OF COMMERCE

NATIONAL BUREAU OF STANDARDS
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NBSIR 87-3669

SYSTEM OF HARDWARE AND SOFTWARE

DEVELOPED FOR SIZE EXCLUSION

CHROMATOGRAPHY

Research Information Center

National bureau of Standards

Gaithersburg, Maryland 20899

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B. Dickens and F. L. McCrackin

U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards

Institute for Materials Science and Engineering

Polymers Division

Gaithersburg, MD 20899

December 1987

U.S. DEPARTMENT OF COMMERCE, C. William Verity, Secretary

NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Director

System of Hardware and Software Developed

for Size Exclusion Chromatography

B. Dickens and F. L. McCrackin

Polymers Division

National Bureau of Standards

Gaithersburg, MD 20899

March 7, 1987

DISCLAIMER

This system of programs has been written to allow size

exclusion chromatography (data collection and data analysis) to

be performed in the Polymers Division of the National Bureau of

Standards using the MS-DOS system of computers. The programs

are under development. This "manual" accompanies version 1.00

of the programs. The authors reserve the right to make changes

in the programs and in the manual without the necessity to

inform users. The authors do not warrant that the programs are

correct, and indeed they intend to make corrections and

enhancements as the programs evolve.

Users are requested to inform the authors of errors in

calculation and cases where the programs do not do what is

expected. Within our modest capability to supply copies of

disks, the programs will be made available to £ll who are

interested. Copyright belongs to the U.S ; Government. The

programs should not be sold other than by N.T. I.S.
SYNOPSIS OF PROCEDURE

First, run the program SECMENU and select "Enter parameters

for data collection" to go to the SETUP program. Information on

the identity of the specimens is provided to the system of

computer programs in this way and is kept in a small data base on

the hard disk. You may create a new data base, or update the

existing data base. To create a new data base, use the "Provide

new default parameters" route in SETUP. To update the existing

data base, use the "Change titles, run times, etc." option in

SETUP. Save the new information on the hard disk as a new

specimen data base, then exit from the SETUP progr-am.

Get the chromatograph ready to run. When you are ready to

collect data, select the option "Begin data collection" in the

main menu. Data collection will start when the computer receives

a signal, known as a "trigger", from the chromatograph. The data

will be graphed on the screen as they are measured. A better

plot will be provided after data collection has been completed if

you so specified in the "Processing" option in SETUP.

After data have been collected from all the specimens in the

chromatograph, the results may be examined further. Some

processing may already have taken place automatically if you so

specified in SETUP. For this to occur, you must have a sample

file on the disk which has already been treated manually in the

manner in which you want the computer to treat further files of

that type automatically. Select the main menu option "Analyze a

chromatogram interactively" to process chromatograms manually

(interactively) using the program ANALYZE. This option allows

the user to calculate molecular sizes and molecular weights. You

may select "Compare two chromatograms" to examine two

chromatograms visually and calculate a difference chromatogram

using the program COMPARE. In the option "Look at up to 9

chromatograms simultaneously", the chromatograms may be visually

compared, cut, and normalized using the program COMPARES. The

Table of Contents lists all the programs in the system.
TABLE OF CONTENTS

INTRODUCTION ...... . 1

HARDWARE 1

Computer . 1

Interface card 2

Printer 2

SOFTWARE 2

Setting up series of data collections 2

Data collection 4

Plotting of data ..... ......... 4

Data analysis ............ 4

Comparison of chromatograms 5 Calibration of the columns 6

Molecular weight distribution averages 6

DETAILED DESCRIPTION OF PROGRAMS .......... 7

SECMENU - main menu for SEC programs 7

SETUP program to record specimen identification and run conditions ........ 8

SETUP (interactive) 8

SETUP screen 1 - Main menu 11

SETUP screen 2 - Reading in specimen details 12

SETUP screen 3 - Changing titles and run times 13

SETUP screen 4 - Reading changes in titles 14

SETUP screen 5 - Changing injection volumes, etc 15

SETUP screen 6 - Using default values (1) 16

SETUP screen 7 - Using default values (2) 17

SETUP screen 8 - Using default values (3) 18

SETUP screen 9 - Choosing the specimen class (lj 19

SETUP screen 10 - Choosing the specimen class (2) 20

SETUP screen 11 - Choosing the specimen class (3) 21

SETUP screen 12 - Choosing the specimen class (4) 22

SETUP Screen 13 - Choosing the specimen class (5) 23

SETUP screen 14 - Choosing processing (1) ... 1

' 24

SETUP screen 15 - Choosing processing (2) . 25

SETUP screen 16 - Choosing processing (3) ...... 26

SETUP screen 17 - Choosing processing (4) 27

SETUP screen 18 - Choosing column label etc 28

Page 1

SETUP screen 19 - Saving specimen details in data base on disk 29

SETUP screen 20 - Help screen 30

SETUP screen 21 - Leaving SETUP after saving data base on disk 31

SETUP screen 22 - Leaving SETUP after not saving data base 32

Data collection and automatic processing programs 33

DATCOLL (automatic) 33

RUN.NBR (file containing the latest run number) 33

AUTOPLOT (automatic) 34

AUTOPICK (automatic) 34

BACKGRND (automatic) 34

AUTOGP (automatic) 35

l
Utility programs to plot, list titles ..................... 36

PLOTSEC (interactive) ....................... 36

PLOTSEC screen - Menu for plotting all types of SEC files .......... 37

PLOTSEC plot - typical example ....................... 38

TITLES (interactive) ............................ 39

TITLES screen - Menu for selecting range of RAW files ............ 39

Program to monitor steadiness of SEC sensors .................. 40

WATCHGPC (interactive) ........... . ... 40

WATCHGPC screen - Menu to choose time over which to display data ...... 40

WATCHGPC plot - Typical display ....................... 41

Program to analyze a chromatogram interactively ..... ...... 42

ANALYZE (interactive) .................. 42

ANALYZE screen 1 - Start of program ............ 45

ANALYZE screen 2 - Program can not find data file on disk .......... 46

ANALYZE screen 3 - Successful read of data file from disk .......... 47

ANALYZE screen 4 - Function key instructions after reading data file .... 48

ANALYZE screen 5 - After pressing H or h: Menu for help screens ....... 49

ANALYZE screen 6 - Help screen for functions keys FI to F6 ......... 50

ANALYZE screen 7 - Help screen for background correction .......... 51

ANALYZE screen 8 - Help screen for cutting the chromatogram ......... 52

ANALYZE screen 9 - Help screen for further processing of cut chromatograms . 53

ANALYZE screen 10 - Help screen for expanding or contracting the plot .... 54

ANALYZE screen 11 - Help screen for moving the arrow on the plots ...... 55

ANALYZE plot 1 - Chromatogram as input from disk 56

ANALYZE plot 2 - Determining background (with F7) ...... 57

ANALYZE screen 12 - Menu for background correction, after F8 ........ 58

ANALYZE screen 13 - After F8 and 1 (background has been subtracted) ..... 59

ANALYZE plot 3 - Chromatogram with background removed (F8) .... 60

ANALYZE plot 4 - Delineating relevant part of chromatogram (with F9) .... 61

ANALYZE screen 14 - After pressing F10 to cut chromatogram (or M or m) ... 62

ANALYZE plot 5 - "CUT" chromatogram (with F10 then 2) ........... . 63

ANALYZE screen 15 - Normalizing and storing cut chromatogram (M and 3) ... 64

ANALYZE screen 16 - Elution volume to log hydrodynamic volume (M and 4) . . . 65

ANALYZE plot 6 - Distribution of log hydrodynamic volumes (M and 2) 66

ANALYZE screen 17 - Reading "CUT" file ......... 67

ANALYZE screen 18 - Main menu, obtained after reading cut file ....... 68

ANALYZE screen 19 - After F10 or M .............. 69

ANALYZE screen 20 - After M and 4 ...... . 70

ANALYZE screen 21 - After M and 4 part 1 71

ANALYZE screen 22 - After M and 4 part 2 . 72

ANALYZE screen 23 - After M and 5 ............ 73

ANALYZE screen 24 - After M and 5 . . 74

ANALYZE screen 25 - After M and 6 ..................... . 75

ANALYZE plot 7 - Log molecular weight plot (M then 6 then 2) 76

ANALYZE screen 26 - After M and 6 without going through 4 or 5 77

ANALYZE screen 27 - After M and 6 . 78

ANALYZE screen 28 - After M and 6 and "Press any key" 79

Program to compare two chromatograms 80

COMPARE (interactive) . 80

COMPARE screen 1 - Starting menu (1) 81

- ii -
COMPARE screen 2 - Starting menu (2) . 82

COMPARE screen 3 - Reading the data files . 83

COMPARE screen 4 - Plotting menu 84

COMPARE plot 1 - "Clipped" chromatograms as input 85

COMPARE screen 5 - Taking difference chromatogram 86

COMPARE screen 6 - After taking difference chromatogram . . 87

COMPARE plot 2 - Difference chromatogram (after FI) 88

COMPARE plot 3 - Expanding graph frame with Up Cursor key .... 89

COMPARE plot 4 - Replot with FI . . 90

COMPARE plot 5 - Move graph frame down with Page Down key 91

COMPARE plot 6 - Replot with FI 92

COMPARE plot 7 - Scale two chromatograms - as read in 93

COMPARE plot 8 - Multiply second curve by factor of 10 (using F8) ...... 94

COMPARE plot 9 - Result of replotting previous plot with FI 95

COMPARE plot 10 - Multiply by bigger factor ......... 96

COMPARE plot 11 - Result of replotting previous plot with FI ........ 97

COMPARE plot 12 - Normalize curves to unit area with F7 . 98

COMPARE plot 13 - Automatic replot after F7 ..... 99

COMPARE screen 7 - Writing difference chromatogram to disk file . 100

Program to compare up to 9 chromatograms 101

COMPARE9 (interactive) 101

COMPARE9 screen 1 - Reading in the data files 102

C0MPARE9 screen 2 - End of reading the data 103

C0MPARE9 screen 3 - Plotting menu 104

C0MPARE9 plot 1-7 "raw" chromatograms as input, no vertical shift 105

COMPARE9 plot 2 - Move graph frame down with Page Down key 106

C0MPARE9 plot 3 - Replot with FI 107

COMPARE9 plot 4 - Region to cut out selected with F9 108

COMPARE9 plot 5 - Chromatograms cut with F10, replotted 109

COMPARE9 screen 4 - Vertically displacing the curves 110

COMPARE9 plot 6 - Replot shifted curves (arrow on top curve with A) Ill

C0MPARE9 plot 7 - Expand graph frame with Page Up key 112

C0MPARE9 plot 8 - Replot with FI 113

COMPARE9 plot 9 - Normalize to unit areas with F7 114

COMPARE9 screen 5 - Save plot for future hardcopy graph on plotter 115

Programs to calculate and display calibration of columns ... .... 116

Calibration ................................. 116

CALFIT (interactive) ............................ 117

PLOTCAL (interactive) 120

PLOTCAL screen 1 - Reading in the calibration file 120

PLOTCAL screen 2 - Specifying Mark-Houwink parameters for polymer 121

PLOTCAL screen 3 - menu for plotting 122

PLOTCAL plot - Typical calibration plot ..... 123

- iii -

CONTENTS OF DISKS 123
.

..

.
INTRODUCTION

The objective of this work was to adapt the technique of

size exclusion chromatography to the point where it can be easily

and routinely applied to monitor molecular size distributions in

materials

.

Reliable quality control procedures are needed to help

reduce and correct variations which arise in manufacturing

processes. Except for a few research type materials made using

very special techniques, the component molecules of polymeric

materials exist in distributions of sizes. Resin manufacturers

typically monitor the viscosity of the resin as a check on the

degree of polymerization. Although the viscosity of a liquid with

several components is determined by the molecular sizes of the

components of the liquid, the viscosity is not an accurate

measure of the molecular sizes. In fact, the viscosity of a

multi-component liquid can be adjusted by adding more solvent to

decrease the viscosity or more high molecular weight material to

increase the viscosity, as the case requires.

However, since the relative amounts of the different

molecular sizes have a strong bearing on the physical properties

of the material, it is important to monitor any changes in

molecular size distributions in polymeric and pre-polymeric

materials and to include the distribution of molecular size in

the specifications. Such distributions of molecular sizes are

well measured by size exclusion chromatography. Therefore, we

have developed a computerized system which will allow size

exclusion chromatography to be applied routinely and which will

help to tighten specifications on procurements.

HARDWARE

Computer

The computer is a "turbo" IBM-XT type with 640K memory on

the mother board, a 4.77/8MHz switchable clock rate, a 20MB

internal hard disk drive, two 360K floppy disk drives, a calendar

and clock, two serial ports, a keyboard with separate numerical

and cursor control pads, a hard reset button, and the 8088-2

("turbo") processing unit with 8087-2 math chip. The computer

has eight expansion slots and a 135 watt power supply. The

screen operates in both text and IBM color graphics adapter

modes. The operating system is IBM DOS 3.1 The programs will

run under MS-DOS 2.xx and 3.xx. The programs do not require the

math chip. A BASIC interpreter such as IBM BASICA or GWBASIC is

required for the current version of the SETUP program. The other

programs are compiled.

Page 1
Interface card

The interface card in the computer converts the analog

signals from the chromatograph into numbers which are available

to the computer. The card we have installed and developed the

programs for is the Strawberry Tree ACPC"X6-8“T11 , which is of

the integrating type and measures to 18 bits resolution. We do

not mean to imply that this card is the best for the purpose, nor

do we intend to compare the various types of cards available.

The features on the card we have selected fit our needs. It

digitizes at least 2 signals from the chromatograph to a

resolution of 18 bits, detects changes in TTL levels to trigger

the beginning and end of data collection. The card is supplied

with a driver for use with BASIC programs. The card comes with a

terminal box to which connections may conveniently be made, and

the source code for the card driver is made available by the

manufacturer. We are using the card to digitize low level

signals (less than 250 mv)

,

at which level it performs

acceptably. A few statements in the programs DATCOLL and

WATCHGPC must be changed if another card is used.

Printer

The printer is used to provide rapid plots of the results of

data collection, for printing of programs during program

development, and for communicating the results of analyses to the

operator. It is of the IBM graphics printer type.

SOFTWARE

Setting up series of data collections

The process of analyzing specimens using size exclusion

chromatography consists of several main elements. The first is

to prepare solutions of the specimens in a suitable solvent,

typically tetrahydrofuran , and load the vials containing these

solutions into the injector, where, in our configuration, up to

16 vials may be sampled in one run. The injection process is

driven by a microprocessor in the chromatograph. The computer

does not control this microprocessor. It passively accepts the

output of the chromatograph to produce files of information which

are labeled with the identity of the specimen in the solution and

the date and time of data collection. These data files are then

treated further in the computer as the operator specifies.

The computer must therefore be in step with the

chromatograph, in that it must collect data when the

chromatograph is producing data and it must give the appropriate

labels to each data file. The program which conveys the

labelling information to the computer is called the SETUP

Page 2
program. To document the identity of the specimen and the

conditions under which the chromatogram was measured, the data

file collected from the chromatograph for the contents of each

vial contains a title, a time to begin collection of closely

spaced data points instead of merely monitoring the base line of

the chromatogram, a time to end the collection of closely spaced

data points, and a time to end the run, which is usually when the

base line has had time to become steady again. This information

is entered by the operator in a fill in the form type of screen.

The Mark-Houwink coefficients for this specimen to relate log

hydrodynamic volume to molecular weight may also be supplied on

this screen if data treatment to this extent is required. A

similar type of screen allows the operator to enter other

specimen-specific information such as the number of injections

from this vial, the flow rate, the sensitivities (a sort of scale

factor) and the scale factors used in the chromatograph.

Another screen allows the operator to specify the type of

columns and solvent used in the chromatograph, and the name of

the operator.

For quality control, it is necessary to compare a

chromatogram with a standard chromatogram. The correspondence

between the newly collected chromatogram and the standard

chromatogram is made through a variable which specifies the class

of the specimen, e.g., a varnish of a particular manufacturer,

and which is entered into the SETUP program either directly by

the operator or by selection from a table supplied by the

program.

We envisage that a wide variety of specimens will be used.

Some will require only matching of chromatograms, mainly because

they are mixtures of materials which cannot be completely

separated or identified, and for which calculations of molecular

weight distributions are meaningless. Others will be specimens

of well-known molecular weight which will be used to calibrate

the columns. Some will be specimens of a homogeneous material

for which the molecular weight distribution is to be measured.

The type and extent of post data collection processing is

controlled by the "processing" variable in the SETUP program

which is selected from a table displayed by the program.

During the setup of the data collection process, the

operator should print out the complete set of instructions to the

computer to verify that he has entered all the pertinent

information correctly, and then must save the results in the form

of a file on the hard disk which will be read by the data

collection program.

Page 3
Data collection

The process of collecting data representing a chromatogram

begins when the computer receives an electrical pulse from the

chromatograph. Once the run has started, the data collection

program monitors the base line signal from the chromatogram until

the time as specified by the operator in the SETUP program is

reached. Data are then collected more frequently until the

important part of the information has been acquired, at which

point the base line is again monitored at a reduced rate. As the

data are being measured, they are written onto the hard disk. If

data collection terminates normally, the data collection program

updates the file written by the SETUP program to reflect the fact

that data from that specimen has been obtained. If data

collection terminated abnormally, the file from SETUP is not

updated. In this case, the next time the data collection

program is run, it will assume that the data are a repeat of the

run which ended abnormally. The reason for this method of

operation is that the data are processed immediately after data

collection so that the operator does not have to wait for up to

20 hours before seeing the results. After data processing has

finished, the data collection program resumes by measuring the

chromatogram of the next specimen.

Plotting of data

One of the choices in the SETUP program is for the

chromatogram to be plotted out on the printer immediately after

data collection. If this is requested, the plotting program

reads in the file written by the data collection program and

scales the results to fill the plot area.

Data analysis

Analysis of the chromatogram may be carried out

interactively with the operator directing the flow of analysis

from the computer keyboard, or automatically. Analysis is

carried out in the automatic mode immediately after data

collection to an extent which depends on what the operator

specified during setup. Automatic analysis may also be applied

to a series of chromatograms. The general sequence in all cases

is to determine the levels of the base line before and after the

part of the chromatogram which characterizes the specimen, apply

a correction so that the overall base line is effectively zero,

and cut out from the entire chromatogram that part which pertains

to the specimen. The "cut" chromatogram may then be written to a

f ile

.

Page 4
Up to this point, all chromatographic information is in

terms of elution volume. If the operator so specified, and if a

calibration file is available to the program, the chromatogram is

converted to terms of log hydrodynamic volume, which effectively

removes dependence on the particular set of columns used, and, if

the calibration is sufficiently recent, on the age of the

columns. The chromatogram in terms of log hydrodynamic volume

may be written to a file for later use.

If Mark-Houwink coefficients were supplied at setup time,

and if the operator so requested, the chromabogram is converted

into the differential molecular weight distribution of the

specimen, and various averages characterizing this molecular

weight distribution are calculated. The molecular weight

distribution may be written to a file.

These files are not very long. Some 30 uncut chromatograms

will fit on a 360K floppy disk. About 90 cut chromatograms or

log hydrodynamic volume files will fit on a similar disk. If

files of all three kinds are kept together, which is probably not

the most efficient arrangement, the files for about 18 specimens

will fit on one disk, which is about one day’s worth since the

sample carrousel on the size exclusion chromatograph has room for

a maximum of 16 specimens. These considerations are important

only in archiving the data. The available storage on the hard

disk in the computer corresponds to about 45 floppy disks, which

is enough for about 10 weeks of data.

Comparison of chromatograms

When the chromatogram is' to be compared with a standard

chromatogram for that class of specimen, the standard

chromatogram is found from the "class” specified at setup time.

The two chromatograms are matched in a way also specified at

setup time. This does not usually present any difficulty,

because once the procedure has been worked out, the way in which

to match the chromatograms is well-known. Entering this

information at setup time allows the operator to collect data on

various types of specimens in an given run. The comparison may

be carried out in terms of elution volume or log hydrodynamic

volume, as specified.

Unrecognized shifts in the calibration of the columns will

affect the raw data, which are in terms of elution time of the

components of the specimen from the chromatographic columns. Use

of log hydrodynamic volume allows the operator to correct for

such changes in column calibration and to compare specimens taken

at considerably different times, and also to compare specimens

taken with different sets of columns. Log hydrodynamic volume is

therefore is~a more constant quantity to use to specify materials

which will be analyzed at the factory and in the receiving

laboratory

.

Page 5
Methods of characterizing the difference between the two

chromatograms are currently under development. In our initial

approach, we provide an overall mismatch index, and a list of the

areas, heights, and positions of the main peaks in the difference

chromatogram. This difference chromatogram is taken between the

chromatogram of the specimen and the standard chromatogram for

that class of specimens. Such indices will with experience lead

to a quantitative means of deciding whether to accept or reject a

particular material during procurement.

Calibration of the columns

In many of the uses of size exclusion chromatography, the

elution volumes over which the specimen emerges from the columns

and enters the detector must be related to molecular size. The

size of polymer molecules in a solvent depends on the

polymer-solvent interactions as well as the length of the polymer

molecules. The relationship between molecular weight and

molecular size in a solvent is given for a given range of

molecular weight by the Mark-Houwink coefficients, which are

available in table form for many common polymer-solvent pairs.

The procedure of calibration is to find the elution volumes

at which polymer specimens of known narrow molecular weight

range elute, and to use the log hydrodynamic volumes calculated

from the Mark-Houwink coefficients and the molecular weights of

these polymers to relate log hydrodynamic volume and elution

volume, often in terms of a polynomial.

One of the options in the setup program is to automatically

find the elution volume of the highest peak in a chromatogram.

When enough such data have been measured to cover the calibration

range needed for the specimens to be analyzed, a least squares

refinement program is used to compute the appropriate polynomial.

An appropriate least squares procedure has been provided in

the program CALFIT.

Molecular weight distribution averages

The calibration relates elution volume to log hydrodynamic

volume. The Mark-Houwink relationship relates log hydrodynamic

volume to log molecular weight. Thus, with the parameters in

these two relationships available to it, the program is able to

calculate the distribution of molecular weights in a homogeneous

specimen. The distribution is then summarized in terms of

various averages which reflect the position of the distribution

and its width in terms of molecular weight. A graph of the

distribution is also provided.

Page 6
DETAILED DESCRIPTION OF PROGRAMS

SECMENU - main menu for SEC programs

NBS SEC MENU - version 1.01

Move highlight to your choice with cursor keys

then hit <carriage return)

Watch detectors responding on chromatograph

Enter parameters for data collection

Begin data collection

Analyze a chromatogram interactively

Compare two chromatograms, find difference chromatogram

Look at up to 9 chromatograms simultaneously

Automatically process several chromatograms

Browse through chromatograms on disk

View latest chromatogram on disk

View calibration of columns

Redo calibration of columns

Leave MENU program, return to DOS

This representation of the computer screen shows the main menu in

the SECMENU program, which is typically run from the AUTOEXEC.BAT

file. To invoke an option in the program, move the cursor to a line

in the rectangle of options and hit the {Return} key. On the screen,

the line selected by the cursor is in inverse video (backlit)

.

Page 7
SETUP program to record specimen identification and run conditions

SETUP (interactive)

The SETUP program is written in interpreted BASIC because it

consists of f ill-in-the-f orm types of data entry in which cursor

movement is important. The program is started by selecting the

option "Enter parameters for data collection" in the main menu.

The first screen gives the main options (see SETUP screen

1) . The cursor is moved to the appropriate line and the (Return)

is pressed. The usual first step is to read in the data base

file from the disk, which is the first line in the option list

( SETUP screen 2) .

If only small changes to the existing data base need to be

made, the operator should go to the screen to change titles, run

times, etc. ( SETUP screen 3 ). The old information is shown in

yellow on a black background, and the new information is shown

in blue. It is important to note that the new information is

only on the screen and is not available to the program unless the

operator wishes to transfer it to the program. The operator

may transfer the information currently in the form on the screen

into the program by moving the cursor down to the next to the

bottom line and pressing (Return) . The screen is then read

( SETUP screen 4 ) and re-displayed in yellow on black. Moving the

cursor to the bottom line and pressing (Return) returns to the

main menu. Any information still in blue will be lost at this

point

.

Similar screen forms allow the operator to change all the

remaining quantities associated with collecting data for a chromatogram. Titles, number of injections from each vial,

sensitivities, scale factors, flow rates, and injection volumes

are changed as shown in SETUP screen 5 .

If a new series of runs are to be set up, it is probably

more worthwhile to use the default value route to enter the

appropriate information into the program. Default values which

will later be given to all specimens may be entered in the

appropriate form ( SETUP screens 6 and 7 ) . New information (in

blue) may be transferred to the program by pressing (Return) when

the cursor is on the next to the bottom line. After this

operation, the screen will be written entirely in yellow on

black. Note that partial titles can be set up, with the operator

later entering the rest of the title in the appropriate form,

such as "change titles, run times, etc.". The default values may

be transferred to each specimen by pressing (Return) on the

appropriate line in the main menu ( SETUP screen 8 )

.

So far, we have covered the method of providing data on the

chromatographic conditions, such as run times, and other

pertinent information, such as run titles and Mark-Houwink

parameters. The operator should also choose the class into which

Page 8
the specimen falls so that later data processing can

automatically find 1) the desired places at which the background

in the chromatogram should be estimated, and 2) the limits of the

relevant part of the chromatogram in terms of elution volume.

For this automatic processing to be possible, a chromatogram

representing this class must be processed interactively using the

program ANALYZE. Also, the class name, the name of the

representative chromatogram, and the name of the calibration file

to use must be available in a file called STANDARD. CHR

The class of a specimen may be selected, in SETUP from the

table of possibilities presented after selecting "Choose class"

in the main menu. This table is read in by the program from file

SETUP. CLA when the program is first started. Up to twenty

classes are allowed, and several versions of the SETUP. CLA files

may be kept to give a wide choice. The limitation of twenty in

any one run of the SETUP program arises because there is not room

on the screen for more classes. We have not found it necessary

to provide multiple screens of choices. SETUP screens 9 through

13 show the sequence of choosing the specimen class.

The operator should also choose the processing required

after the chromatogram has been measured. A simple example is

"Plot data". The options should be chosen from the table of

possibilities provided after selecting "Choose processing to

occur after data collection" in the main menu. As in the case of

the selection of the class of the specimen, up to twenty types of

treatment are allowed. Each line on the screen in this section

encompasses all previous choices. The list of treatments is read

by the program SETUP from file SETUP. PRO when the program is

first started. SETUP screens 14 through 17 show the sequence for

choosing the processing to occur after data collection.

The documentation of the data file includes the type of

solvent (mobile phase) used, the columns used, and the operator.

This information is entered into the program as in SETUP screen

18

.

After the necessary information has been provided to the

program, the operator should print out the data base and verify

that all data have been given correctly. This is done by

pressing return on the third line of the main menu.

The specimen details must be saved on the disk so that the

data collection program can refer to them and include them with

the appropriate data file. This is done by hitting {return! with

the cursor on line 2 of the main menu. The screen then displays

a message that the information is being saved ( SETUP screen 19 )

.

The help screen is reached from the main menu, and is shown

in SETUP screen 20.

Page 9
When the End program option is chosen in the main menu, the

program displays SETUP screen 21 if no further changes have been

made (or if no possibility of making changes has occurred) since

the data base was last saved. Otherwise, a reminder is provided

( SETUP screen 22 ). To give the user a second chance, both y (or

Y) and {Return} must be given before the program stops and the

information in the computer's memory is lost. The information

must be on the disk in order to survive so it can be used later.

Page 10
SETUP screen 1 - Main menu

Main menu for setting up SEC run data base

Read specimen details in from data base on disk

Write specimen details out to data base on disk

Print out specimen details in data base

Change titles, run times and Mark-Houwink parameters

Change titles, # injectns, sensitivities, scales, inj vols, flows

Choose class for automatic matching of chromatograms

Choose processing to occur after data collection

Change mobile phase descriptor, column description and operator name

Provide new default values

Assign default values to all vials

End program (Save the specimen details by writing them out to the data base!)

HELP!

Move cursor to a line and hit RETURN

This representation of the computer screen shows the main menu in

the SETUP program. To invoke an option in the program, move the

cursor to a line and hit the {Return! key.

Page 11
SETUP screen 2 - Reading in specimen details

Main menu for setting up SEC run data base

Read specimen details in from data base on disk

Write specimen details out to data base on disk

Print out specimen details in data base

Change titles, run times and Mark-Houwink parameters

Change titles, # injectns, sensitivities, scales, inj vols, flows

Choose class for automatic matching of chromatograms

Choose processing to occur after data collection

Change mobile phase descriptor, column description and operator name

Provide new default values

Assign default values to all vials

End program (Save the specimen details by writing them out to the data base!)

HELP!

Move cursor to a line and hit RETURN

Reading in specimen details

This SETUP screen shows that the specimen written

the previous use of the program is now being read into

to the disk in

the program.

Page 12
SETUP screen 3 - Changing titles and run times

Change titles, plot scale. Markvial title

Houwink parameters, and

ymax ymin k

run times

a begin end stop

1 ink specimen 1 .03 -.02 OE-6 0 10 45 50

2 ink specimen 2 .03 -.02 OE-6 0 10 45 50

3 ink specimen 3 .03 -.02 0E-6 0 10 45 50

4 ink specimen 4 .03 -.02 OE-6 0 10 45 50

5 A1 Sicpa S--4 45.8mg .03 -.02 0E-6 0 10 45 50

6 A2 Fountain 44.3mg .03 -.02 0E-6 0 10 45 50

7 A3 Prewipe 43.7mg .03 -.02 0E-6 0 10 45 50

8 A4 Reconstituted 36.7mg .03 -.02 0E-6 0 10 45 50

9 B1 Sicpa S-4 42.8mg dil .03 -.02 0E-6 0 10 45 50

10 B2 Fountain 42.6mg dil .03 -.02 0E-6 0 10 45 50

11 B3 Prewipe 35.0mg dil .03 -.02 0E-6 0 10 45 50

12 B4 Reconstituted 42.8mg dil .03 -.02 0E-6 0 10 45 50

13 B4-A Reconstituted 40.3mg dil .03 -.02 0E-6 0 10 45 50

14 8/27/2A Bk-3795-21-XRD 45.3mgdil .03 -.02 OE-6 0 10 45 50

15 8/27/2 Bk-3795-21-IRD 39.3mg dil .03 -.02 OE-6 0 10 45 50

16 8/27/3 Bk-3795-21-IRD 44.1mg dil .03 -.02 0E-6 0 10 45 50

Move cursor with arrows, type in new values, type over don't

Hit RETURN on this line to read the values on the screen

Hit RETURN on this line to return to main menu

i i i No collection of data if total run time (in stop column)

backspace

= 0 ! ! !

The screen in which the titles, plot maxima and plot minima,

Mark-Houwink parameters, and run times are changed. Each quantity is

displayed in a colored box on the screen to show how long the entry on

the screen may be. The plot maxima and minima are necessary because

the data collection program does not know what the range of the data

will be but must set up the plot frame before any data are collected.

Page 13
SETUP screen 4 - Reading changes in titles

Change titles, plot scale, Mark-Houwink parameters, and run times

vial title ymax ymin k a begin end stop

1 ink specimen 1 .03 -.02 0E-6 0 10 45 50

2 ink specimen 2 *03 “.02 QE-6 0 10 45 50

3 ink specimen 3 .03 -.02 0E-6 0 10 45 50

4 ink specimen 4 .03 -.02 QE-6 0 10 45 50

5 A1 Sicpa S~4 45.8mg .03 -.02 OE-6 0 10 45 50

6 A2 Fountain 44.3mg .03 -.02 OE-6 0 10 45 50

7 A3 Prewipe 43.7mg .03 -.02 OE-6 0 10 45 50

8 A4 Reconstituted 36.7mg .03 -.02 0E-6 0 10 45 50

9 Hi Sicpa S-4 42.8mg dil .03 -.02 0E-6 0 10 45 50

10 B2 Fountain 42.6mg dil .03 “.02 0E-6 0 10 45 50

11 B3 Prewipe 35.0mg dil .03 -.02 0E-6 0 10 45 50

12 B4 Reconstituted 42.8mg dil .03 -.02 OE-6 0 10 45 50

13 B4-A Reconstituted 40„3mg dil .03 -.02 OE-6 0 10 45 50

14 8/27/2A Bk-3795-21-IRD 45.3mgdil .03 -.02 OE-6 0 10 45 50

15 8/27/2 Bk-3795-21-TRD 39.3mg dil .03 -.02 OE-6 0 10 45 50

16 8/27/3 Bk“3795”21“IRD 44.1mg dil .03 -.02 0E-6 0 10 45 50

Move cursor with arrows, type in new values, type over don’t backspace

Hit RETURN on this line to read the values on the screen

Hit RETURN on this line to return to main menu Reading the screen

! i ! No collection of data if total run time (in stop column) = 0 ill

«

The "Reading the screen" line in the lower right hand corner

shows that the operator has hit {return} on the "Read the screen"

line. The screen is yellow on black when first printed* Changes are

shown in blue. When the screen has been read, all the text is in

yellow. Changes may be made and read as long as the form is on the

screen. Hitting {Return} on the next to the bottom line returns to

the main menu.

Page 14
SETUP screen 5 - Changing injection volumes, etc.

Change titles, # injectns, sensitivity,

vial title

1 ink specimen 1

2 ink specimen 2

3 ink specimen 3

4 ink specimen 4

5 A1 Sicpa S-4 45.8mg

6 A2 Fountain 44.3mg

7 A3 Prewipe 43.7mg

8 A4 Reconstituted 36.7mg

9 B1 Sicpa S-4 42.8mg dil

10 B2 Fountain 42.6mg dil

11 B3 Prewipe 35.0mg dil

12 B4 Reconstituted 42.8mg dil

13 B4-A Reconstituted 40.3mg dil

14 8/27/2A Bk-3795-21-IRD 45.3mgdil

15 8/27/2 Bk-3795-21-IRD 39.3mg dil

16 8/27/3 Bk-3795-21-IRD 44.1mg dil

scale, inj vol, flow

# injects flow sensit scale injvol

1 1 -256 99 400

1 1 -256 99 400

1 1 -256 99 400

1 1 -256 99 400

1 1 -256 99 400

1 1

' -256 99 400

1 1 -256 99 400

1 1 -256 99 400

1 1 -256 99 400

1 1 -256 99 400

1 1 -256 99 400

1 1 -256 99 400

1 1 -256 99 400

1 1 -256 99 400

1 1 -256 99 400

1 1 -256 99 400

Move cursor with arrows, type in new values, type over don't backspace

Hit RETURN on this line to read the values on the screen

Hit RETURN on this line to return to main menu

ill Always keep the same injection volume for the same class !!!

The SETUP screen in which the number of injections, flow rate,

sensitivity, scale, injection volume may be changed. Typical value

for these variables are shown. Changes are made and read as in the

previous screen.

Page 15
SETUP screen 6 - Using default values (1)

Current default values

Mark-Houwink K QE-6

Mark-Houwink a 0

Time after which to begin collecting data (mins) 0

Time at which to stop collecting data (mins) 0

Total time of data collection (mins) 0

Sensitivity 0

Scale Factor 0

Injection Volume 0

' Number of Injections 0

Flow Rate 0

Ymax and Ymin in plot on screen 0 0

title is:

class is: Not assigned

processing is: Not assigned

Move cursor with arrows, type in new values, type over don’t backspace

Hit RETURN on this line to read the values on the screen

Hit RETURN on this line to return to main menu

The SETUP screen in which default values may be given. This is

the form as first written by the program if the data base was not read

from the disk. If the data base was read from the disk, the old

default values will be available and are written into the form by the

program.

Page 16
SETUP screen 7 - Using default values (2)

Current default values

Mark-Houwink K . 861e-4

Mark-Houwink a .74

Time after which to begin collecting data (mins) 10

Time at which to stop collecting data (mins) 45

Total time of data collection (mins) 50

Sensitivity -256

Scale Factor 99

Injection Volume 400

Number of Injections 1

Flow Rate 1

Ymax and Ymin in plot on screen .05 -0.02

title is: Black ink test run #

class is: Black ink

processing is:

Move cursor with arrows, type in new values, type over don't backspace

Hit RETURN on this line to read the values on the

Hit RETURN on this line to return to main menu

screen

Typical default values in the SETUP default value screen. Note

the partial title, which will be augmented in the titles screen by

typing in the specimen numbers.

Page 17
SETUP screen 8 - Using default values (3)

Main menu for setting up SEC run data base

Read specimen details in from data base on disk

Write specimen details out to data base on disk

Print out specimen details in data base

Change titles , run times and Mark-Houwink parameters

Change titles, # injectns, sensitivities, scales, inj vols, flows

Choose class for automatic matching of chromatograms

Choose processing to occur after data collection

Change mobile phase descriptor, column description and operator name

Provide new default values

Assign default values to all vials

End program (Save the specimen details by writing them out to the data base!)

HELP!

Move cursor to a line and hit RETURN

Use the default titles? Y or N?

Remember to check the descriptors for mobile phase, columns and operator

starting from the main menu

On return to the main menu from the default screen, the operator

has the choice of using the title in the default screen or keeping the

titles already in the titles screen. The reminder to check the mobile

phase, columns and operator identification is provided because the

default route does not automatically cover specifying these details.

Page 18
SETUP screen 9 - Choosing the specimen class (1)

Choose class for specimens for automatic matching of chromatograms

Titles Class

ink specimen 1 Not assigned

ink specimen 2 Not assigned

ink specimen 3 Not assigned

ink specimen 4 Not assigned

A1 Sicpa S-4 45.8mg Not assigned

A2 Fountain 44.3mg Not assigned

A3 Prewipe 43.7mg Not assigned

A4 Reconstituted 36.7mg Not assigned

B1 Sicpa S-4 42.8mg dil Not assigned

B2 Fountain 42.6mg dil Not assigned

B3 Prewipe 35.0mg dil Not assigned

B4 Reconstituted 42.8mg dil Not assigned

B4-A Reconstituted 40.3mg dil Not assigned

8/27/2A Bk-3795-21-XRD 45 . 3mgdil Not assigned

8/27/2 Bk-3795-21-IRD 39. 3mg dil Not assigned

8/27/3 Bk-3795-21-IRD 44. Img dil Not assigned

change classes? y or n?

n(=no) returns to main menu

The SETUP screen showing the class specified for each specimen.

Answering Y or y to the classes generates the next screen. Any other

answer returns to the main menu.

Page 19
SETUP screen 10 - Choosing the specimen class (2)

Select from table of classes

Type in class names

Hove cursor to appropriate line and hit return

Usually, the operator will choose to select from a table of

classes. If not, the program will generate a form like the one in the

previous screen in which the classes may be typed and then read by

moving the cursor to the appropriate line and pressing {Return}

.

Page 20
SETUP screen 11 - Choosing the specimen class (3)

1 Standard

2 Bodied tung oil

3 Bodied tung oil - Degen

4 Bodied tung oil - Superior

5 Bodied tung oil - Standard

6 Solvar varnish

7 Solvar varnish - Lawter

8 Solvar varnish - Sun-Ray

9 Solvar varnish - Superior

10 Gloss HS

11 Gloss HS - Sun-Ray

12 Gloss HS - Superior Enter number of class at ? mark

13 Gloss HS - Standard Return moves to next vial

14 Aquawipe - Sicpa

15 THF

16 E-beam resin component

17 Green ink - BEP

18 Green ink - Sicpa

19 Black ink - BEP BK62/3A

20 Black ink - Sicpa

21 Not assigned

via] title class number of class in above list

1 ink specimen 1 Not assigned

This SETUP screen in which the specimen class is chosen is

generated by answering y to the question about selecting the class

from a table. The appropriate number from the table in the screen is

typed in the lower right hand corner of the screen at the question

mark, and {return} is pressed to move on to the next specimen, as o shown in the next screen.

Page 21
SETUP screen 12 - Choosing the specimen class (4)

1 Standard

2 Bodied tung oil

3 Bodied tung oil - Degen

4 Bodied tung oil - Superior

5 Bodied tung oil - Standard

6 Solvar varnish • 7 Solvar varnish - Lawter

8 Solvar varnish - Sun-Ray

9 Solvar varnish - Superior

10 Gloss HS

11 Gloss HS - Sun-Ray

12 Gloss HS - Superior Enter number of class at ? mark

13 Gloss HS - Standard Return moves to next vial

14 Aquawipe - Sicpa

15 THF

16 E-beam resin component

17 Green ink - BEP

18 Green ink - Sicpa

19 Black ink - BEP BK62/3A

20 Black ink - Sicpa

21 Not assigned

vial title class number of class in above list

2 ink specimen 2 Not assigned ? 17

Selection of class 17, Green ink - BEP, for the specimen in vial

2 .

©

Page 22
SETUP screen 13 - Choosing the specimen class (5)

Choose class for specimens for automatic matching of chromatograms

Titles Class

ink specimen 1 Green ink - BEP

ink specimen 2 Green ink - BEP

ink specimen 3 Black ink - BEP BK62/3A

ink specimen 4 Black ink - BEP BK62/3A

A1 Sicpa S-4 45.8mg Green ink - Sicpa

A2 Fountain 44.3mg Black ink - Sicpa

A3 Prewipe 43.7mg Aquawipe - Sicpa

A4 Reconstituted 36.7mg Green ink - Sicpa

B1 Sicpa S-4 42.8mg dil Green ink - Sicpa

B2 Fountain 42.6mg dil Black ink - Sicpa

B3 Prewipe 35„Omg dil Aquawipe - Sicpa

B4 Reconstituted 42.8mg dil Green ink - BEP

B4-A Reconstituted 40„3mg dil Green ink - BEP

8/27/2A Bk-3795-21-IRD 45 .3mgdil Black ink - BEP BK62/3A

8/27/2 Bk“3795-21-IRD 39. 3mg dil Black ink - BEP BK62/3A

8/27/3 Bk-3795-21-IRD 44. lmg dil Black ink - BEP BK62/3A

change classes? y or n?

n(=no) returns to main menu

After classes have been chosen for all specimens, the program

generates this screen, which shows the choices which have been made.

The operator may choose again by answering y to the question.

Page 23
SETUP screen 14 - Choosing processing (1)

Choose processing for specimens after data collection

Titles Data processing

ink specimen 1 Not assigned

ink specimen 2 Not assigned

ink specimen 3 Not assigned

ink specimen 4 Not assigned

A1 Sicpa S” 4 45.8mg Not assigned

A2 Fountain 44»3mg Not assigned

A3 Prewipe 43.7mg Not assigned

A4 Reconstituted 36.7mg Not assigned

B1 Sicpa S-4 42.8mg dil Not assigned

B2 Fountain 42,6mg dil Not assigned

B3 Prewipe 35.0mg dil Not assigned

B4 Reconstituted 42*8mg dil Not assigned

B4=A Reconstituted 40.3mg dil Not assigned

8/27/2A Bk-3795-2i-IRD 45.3mgdil Not assigned

8/27/2 Bk-3795-21”IRD 39.3mg dil Not assigned

8/27/3 Bk-3795-21-IRD 44.1mg dil Not assigned

change processing? y or n?

n(-no) returns to main menu

A series of screens similar to the screens in which the classes

are chosen is used to choose the processing to occur after data

collection* This screen shows the initial choices, which are the

choices already in the program*

Page 24
SETUP screen 15 - Choosing processing (2)

Not assigned

Collect data

Plot data

Find elution time of main peak

Subtract background

Compare with class standard

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

vial title

1 ink specimen 1

Enter number of processing at ? mark

Return moves to next vial

treatment # of treatment in above list

Not assigned ?

Answering y or Y to the question in the previous screen results

in this screen, which allows the operator to specify the processing

from the table. Each choice includes all the previous choices. For

example, "Find Elution time of main peak" includes collect data(!)

and plot data.

Page 25
SETUP screen 16 - Choosing processing (3)

Choose processing for specimens after data collection

Titles Data processing

ink specimen 1 Plot data

ink specimen 2 Plot data

ink specimen 3 Plot data

ink specimen 4 Plot data

A1 Siepa S-4 45.8mg Plot data

A2 Fountain 44.3mg Plot data

A3 Prewipe 43„7mg Plot data

A4 Reconstituted 36.7mg Plot data

61 Siepa S-4 42.8mg dil Plot data

B2 Fountain 42.6mg dil Plot data

63 Prewipe 35«0mg dil Plot data

64 Reconstituted 42.8mg dil Plot data

B4-A Reconstituted 40„3mg dil Plot data

8/27/2A Bk"3795-21”IRD 45.3mgdil Plot data

8/27/2 Bk“3795”21-IRD 39.3mg dil Plot data

8/27/3 Bk~3795”21"IRD 44.1mg dil Plot data

change processing? y or n?

n(-no) returns to main menu

This SETUP screen shows the choices typically made when mixture

of materials are being studied, i.e., the data are merely to be

plotted without further processing.

e

Page 26
SETUP screen 17 - Choosing processing (4)

Choose processing for specimens after data collection

Titles Data processing

ink specimen 1 Compare with class standard

ink specimen 2 Compare with class standard

ink specimen 3 Compare with class standard

ink specimen 4 Compare with class standard

A1 Sicpa S-4 45.8mg Compare with class standard

A2 Fountain 44.3mg Compare with class standard

A3 Prewipe 43.7mg Compare with class standard

A4 Reconstituted 36.7mg Compare with class standard

B1 Sicpa S-4 42.8mg dil Compare with class standard

B2 Fountain 42.6mg dil Compare with class standard

B3 Prewipe 35.0mg dil Compare with class standard

B4 Reconstituted 42.8mg dil Compare with class standard

B4-A Reconstituted 40.3mg dil Compare with class standard

8/27/2A Bk-3795-21-IRD 45.3mgdil Compare with class standard

8/27/2 Bk-3795-21-IRD 39.3mg dil Compare with class standard

8/27/3 Bk-3795-21-IRD 44.1mg dil Compare with class standard

change processing? y or n?

n (=no) returns to main menu

This screen shows the choice which could reasonably be made if a

series of single component specimens were being examined and the

operator wanted the program to calculate the molecular weight averages

and log molecular weight distribution for each specimen.

Page 27
SETUP screen 18 - Choosing column label etc.

Change descriptors for mobile phase, columns and operator

THF

Columns go here

B. Dickens & L.Low

Move cursor with arrows, type in new values, type over don't backspace

Hit RETURN on this line to read the values on the screen

Hit RETURN on this line to return to main menu

mobile phase is:

columns are:

operator is:

In a sequence which is typical for SETUP, the operator may

identify the mobile phase, the columns, and the operator.

Page 28
SETUP screen 19 - Saving specimen details in data base on disk

Main menu for setting up SEC run data base

Read specimen details in from data base on disk

Write specimen details out to data base on disk

Print out specimen details in data base

Change titles, run times and Mark-Houwink parameters

Change titles, # injectns, sensitivities, scales, inj vols, flows

Choose class for automatic matching of chromatograms

Choose processing to occur after data collection

Change mobile phase descriptor, column description and operator name

Provide new default values

Assign default values to all vials

End program (Save the specimen details by writing them out to the data base!)

HELP!

Move cursor to a line and hit RETURN

Writing specimen details to data base

The specimen details must be written to the disk before the SETUP

program is ended if they are to be available to the data collection

program. However, there may be cases when the SETUP program is being

demonstrated rather than being used to set up a series of runs.

Therefore, the data file is not automatically written to disk. To

save the data, the operator hits {Return} on the second line of the

menu. The screen then shows that the data are being written to disk.

The DO. BAT file containing a series of DOS instructions for data

collection and further processing is generated and written to disk at

the same time. Data collection is started by typing DO at the DOS

prompt or by selecting the "Collect data" option in the main menu of

program SECMENU.

Page 29
SETUP screen 20 - Help screen

HELP INFORMATION

This program allow you to put into the computer the information to label

and hence identify the chromatograms as they are taken and to give the data

collection program the information it needs to collect data. Each chromatogram

is processed immediately after it has been measured. You may process it again

later if you wish.

First, read the previous data file from the disk, using the first option in the

list. You can alter it as you see fit, using most of the other options.

Generally, you will update the titles, and maybe the run times and other

parameters. The data collection program will only collect data for those

specimens which have a non-zero run time. Hence, to include a vial or remove

a vial from the list, you change its run time. The data collection program

takes a reading about once a minute up to the beginning of data collection

proper, when the specimen is expected to emerge from the columns. After the

specimen has eluted, leave a few minutes for the data collection to continue,

and a few more for the data collection to collect a point a minute to allow it

and you to establish the background again. When you are done, print out your

data, and check them. Then save the data on the disk, using the second option,

so the later programs can read them. After running this program, you start data

collection by typing DO at the DOS prompt.

Press RETURN when ready

This SETUP help screen is generated by hitting {Return

bottom line of the main menu.

on the

Page 30
SETUP screen 21 - Leaving SETUP after saving data base on disk

Read specimen details in from data base on disk

Write specimen details out to data base on disk

Print out specimen details in data base

Change titles, run times and Mark-Houwink parameters

Change titles, # injectns, sensitivities, scales, inj vols, flows

Choose class for automatic matching of chromatograms

Choose processing to occur after data collection

Change mobile phase descriptor, column description and operator name

Provide new default values

Assign default values to all vials

End program (Save the specimen details by writing them out to the data base!)

HELP!

Move cursor to a line and hit RETURN

If you are ready to end the program, press y and then return

otherwise press return

If the specimen details have been written out to disk and no

further chance for editing (such as going to a section of the program

which allows the titles to be altered) has occurred, the program will

give this message to allow the operator to terminate the program.

Page 31
SETUP screen 22 - Leaving SETUP after not saving data base

Read specimen details in from data base on disk

Write specimen details out to data base on disk

Print out specimen details in data base

Change titles, run times and Mark-Houwink parameters

Change titles, # injectns, sensitivities, scales, inj vols, flows

Choose class for automatic matching of chromatograms

Choose processing to occur after data collection

Change mobile phase descriptor, column description and operator name

Provide new default values

Assign default values to all vials

End program (Save the specimen details by writing them out to the data base!)

HELP!

Move cursor to a line and hit RETURN

You have not saved the most recent version of the data on the disk

If you are ready to end the program, press y and then return

otherwise press return

In this case, the operator invoked at least one of the editing

screens after the data were saved to disk and then wanted to end the

program without saving the data to disk. The program warns the

operator that this has occurred.

Page 32
Data collection and automatic processing programs

DATCOLL (automatic)

Data collection is begun by selecting "Begin data

collection" in the main menu or by typing DO at the DOS prompt.

The program SETUP must have been run first to generate a file

DO. BAT which contains the list of programs to be run to

accomplish what the operator specified. Execution of the group

of data collection and automatic processing programs is

terminated by pressing {Control I and {Break} simultaneously (hold

down {Control} and then press {Break}) to set a error signal in

the operating system. Then, if data collection is underway,

press {Escape}. Most of the "automatic" programs can be

terminated by pressing the {Escape} key. Otherwise, wait until

the program which is running has finished. Only the data

collection program and the plotting program run for more than a

few seconds.

Before data collection begins, the specimen details are read

from file SETUP.DAT, which was prepared by the SETUP program.

Much of this information is written at the beginning of each data

file so that it is available to later programs. As the data

collection is successfully completed (i.e., without abnormal

interruptions), the file SETUP.DAT is re-written to the disk to

reflect the fact that data have apparently been collected

successfully for this specimen. If the data collection program

is stopped and re-started, it will not recollect data for runs

which have been marked as being successfully completed.

RUN.NBR (file containing the latest run number)

The data files are named beginning with SEC, for size

exclusion chromatography, and then a number, known as the run

number, and end with an extension of RAW for unprocessed data, or

other extensions such as CUT, HYD , MWD , and DIF. As data are

collected, the run number is increased by one for successive

specimens. The automatic programs look in the file RUN.NBR to

find out the name of the latest data file so they can read it in

and process the data.

These automatic programs may also be used to process many

data files at once. The file names of the data files to be

treated should be put into the file RUN.NBR, using a text editor,

or using the DOS piping command (e.g., DIR SEC97.RAW > RUN.NBR to

put all raw data files with run numbers between 90 and 99

inclusive in the file RUN.NBR) and then editing the resultant

RUN.NBR with a text editor to give the needed file names.

Page 33
The name of the automatic program should be typed at the DOS

prompt >. Then the program will process each of the files named

in RUN.NBR. When data collection is begun again, a program

checks to see that RUN.NBR exists, and makes sure that it

contains the latest run number.

AUTOPLOT (automatic)

The program AUTOPLOT reads in the contents of the file

RUN.NBR and generates from it a file name beginning with SEC and

ending with RAW. It then attempts to read in the contents of

that file, which is usually the latest chromatogram, and plots

it, first on the screen, and then, using a screen dump routine,

on the printer.

AUTOPICK (automatic)

The program AUTOPICK reads the latest run number from the

file RUN.NBR and finds the elution volume of the largest peak in

the chromatogram between the limits given in the file

AUTOPICK. LMT If the program can not find the file AUTOPICK . LMT

,

it finds the largest peak in the entire chromatogram. This is

often not desirable, because the solvent injection peak is

sometimes larger than the peak from the specimen.

BACKGRND (automatic)

This program determines the background level of the signal

in the chromatogram and subtracts it from the data. Any number of

chromatograms may be processed. The program uses two files in

addition to the chromatogram files. The calibration of the

columns must be in file CALIBR.DAT and the file names or run

numbers of the chromatograms must be in file RUN.NBR with one

file per line. If the extensions of the files are RAW, only the

run numbers need be given in file RUN.NBR.

The program uses four positions in the chromatogram to

determine points on the background line. These four positions

are 1) the beginning and 2) the end of the chromatogram, and 3)

the beginning and 4) the end of the calibration range (in

elution volume) of the columns. Positions 3 and 4 are obtained

from the calibration file. Ten points at each position are

averaged to give an estimate of the background level and the

standard deviation is calculated. If the standard deviation is

less than 0.0004, the average is provisionally taken as a point

on the background line, otherwise it is discarded.

Page 34
The surviving average background levels are then fitted to a straight line and the deviation of every level from the line

determined. If the largest deviation is less than 0.0004, the

line so determined is taken as the background line. Otherwise,

the level with the largest deviation is eliminated and the

process repeated. If the number of levels on the background line

becomes less than two, a comment is printed and the background is

not subtracted from the chromatogram. Otherwise , the background

line is then subtracted from the chromatogram and the program

continues with the next file in RUN.NBR or ends if it has read

all the information in file RUN.NBR.

The results of the calculations are written to the screen

and to the printer.

If this program fails to find a background line in a

chromatogram, the chromatogram should be examined using the

program ANALYZE to determine the background interactively. It

may be necessary to reject the chromatogram.

AUTOGP (automatic)

This program reads a data file from the file RUN.NBR and the

classes, standard chromatogram files, and corresponding

calibration files (for the columns) from the file STANDARD. CHR

It then finds the standard chromatogram file corresponding to the

class of the data file, and performs the same analysis on the

data file as was performed on the standard chromatogram file.

The files needed are RUN.NBR, STANDARD . CHR , the calibration

file, the file containing the standard chromatogram, and the data

file. A report is provided on the printer and on the file

AUTOGP. OUT The program is a combination of BACKGRND and ANALYZE

and runs automatically using run number information read from

file RUN.NBR. It is placed in the file DO. BAT by the SETUP

program as needed to fulfill the requirements for data collection

and processing the operator made when filling out the forms in

the SETUP program.

Examples of these files are given below:

RUN . NBR

:

SEC925 . RAW

SEC204 .RAW

SEC930 . RAW

Note that run numbers (such as 925) can also be used if the file

name begins with SEC and ends with RAW.

Page 35
STANDARD . CHR

:

Bodied tung oil - Superior , sec207 . hyd , clonecal .

1

Bodied tung oil - Degen , sec207 . hyd , clonecal .

1

Not assigned , cutchr . std , clonecal .

3

Black ink - Sicpa , sec423 . cut , clonecal .

1

Branched epoxy , cutchr .std, clonecal .

3

PSD/PVME irradiated , pvme . hyd , clonecal .

3

The lines contain the class name, the file containing the

standard chromatogram, and the calibration file-, separated by

commas

.

CALIBRATION FILE:

B DICKENS Calibration of Shodex cols 804 & 802 NOV 1986

20.370 35.355

1

27.022 3.244

“0 . 121440 20E+00 -0 . 40413329E+00 -0 . 85624680E-02

0 . 35396945E-02 0 . 4864007 4E-03 -0 . 86872809E-04

The calibration file contains a title, the range of elution

volumes over which the calibration is valid, the type of

calibration ( ^polynomial ) , the points in elution volume and log

hydrodynamic volume about which the polynomial is fitted, and the

coefficients for a polynomial of up to fifth degree.

Utility programs to plot, list titles

PLOTSEC (interactive)

The program PLOTSEC is a interactive version of the AUTOPLOT

program, except that the operator must specify the file name

which the program will try to find and plot the contents of. Any

type of chromatography data file can ‘be plotted. By responding

appropriately to a question from the program, the operator can

plot the file on the line printer.

Page 36
PLOTSEC screen - Menu for plotting all types of SEC files

Plotting program for individual plots

Give name of file containing data to be plotted.

Remember to include directory as \dir\f ile.ext if required

return without file name means quit sec962.raw

Finding maxima and minima in plot

for file sec962.raw

The PLOTSEC program will plot all types of SEC data files on the

computer screen and will optionally also plot them on the line

printer

.

Page 37
PLOTSEC plot - typical example

The plot generated with PLOTSEC is in high resolution (for

the color graphics adapter) . Answering y to the question at the

bottom of the screen transfers the plot to the line printer. The

x axis is labelled correctly with elution volume, log

hydrodynamic volume or log molecular weight before the plot is

made

.

Page 38
TITLES (interactive)

The program TITLES gives a list on the printer of the

titles, classes and sizes of all the RAW data files available on

the current directory, a specified directory, or a floppy disk.

TITLES screen - Menu for selecting range of RAW files

Print a list of titles of RAW data files on disk

Where are the files ? A: for left hand side floppy disk drive:

B: for right hand side floppy disk drive:

Directory, e.g. \gpcdata\, (Return = current directory) \gpcdata\

From run number (return = from smallest) 950

to run number (return = to largest) 1012

PRINTING LIST OF TITLES

This screen shows that the TITLES program has read files

\GPCDATA\SEC950 . raw to \GPCDATA\SEC1012 . RAW from the disk and is

generating a list of the file names, titles, class, and size in Kbyte

for each file.

Page 39
Program to monitor steadiness of SEC sensors

WATCHGPC (interactive)

To obtain good chromatograms, it is important to

allow the chromatograph to stabilize before collecting data.

The program WATCHGPC allows the operator to observe the

stability of the detector by making a real-time plot of the

detector response on the screen. Sudden changes often occur

after about an hour if the chromatograph has just been

started after a period of inactivity.

WATCHGPC screen - Menu to choose time over which to display data

The sensors in the chromatograph may be monitored continuously

using the WATCHGPC program. This screen shows the choice of the time

window to be displayed. The window is moved to cover the most recent

75% of the display when the trace reaches the right hand side of the

screen

.

Page 40
Plot

0.03

0.03

0.01

0.00

- . 01

- . 02

~

' 80 90 .100 110 120 130 140 150

of GPC sensors 1.2:30:41 01-07-1986

xx x stop plot with <ESC> x-x-x

- 1 —"T~—"~t r i r__ —— Ref pact — Pressure

' U i scos i t

y

1_— X— 1 — i— 1

WATCHGPC plot - Typical display

This screen shows the behavior of three sensors in the size

exclusion chromatograph over the period of an hour. The sensors

have been followed for about 130 minutes. On the screen, the

three lines are in different colors. The bottom line in this

plot is from the pressure sensor, the middle line is from a

prototype viscosity detector which has not yet been implemented

in the software, and the top line is from the refractive index

detector. The program runs until the {Escape} key is pressed.

Page 41
Program to analyze a chromatogram interactively

ANALYZE (interactive)

This program allows the operator to read and analyze a

chromatogram interactively. It allows the background to be

subtracted from the chromatogram, a section to be cut from the

chromatogram, elution volumes in the chromatogram to be converted

to log hydrodynamic volumes, molecular weight averages and the

molecular weight distribution to be computed, and the analyzed

chromatograms to be stored in files.

The program first asks for the name of the file to be

analyzed. If the file is a raw file, i.e. has an extension RAW,

only the number of the file need be entered, otherwise the full

name must be entered. After the file has been read into the

program, it may be plotted by pressing one of the function keys

FI to F6. FI displays the full chromatogram, and F2 to F6 each

display a different section of the chromatogram with the elution

volume scale expanded. F2 and F3 display the left and right

halves, respectively, of the chromatogram and F4, F5 and F6

display the left, middle and right thirds, respectively, of the

chromatogram. The chromatogram may also be expanded to show

greater detail in the vertical direction by pressing the Up

Cursor key one or more times then pressing FI to re-display the

chromatogram. The lower part of the chromatogram will then be

expanded but the upper part of the chromatogram will not be

displayed. Pressing the Page Up key one or more times followed by

FI will display the upper parts of the chromatogram. The Down

Cursor and the Page Down key reverse the effect of the Up Cursor

and Page Up keys, respectively.

An arrow is displayed on the chromatogram. This arrow is

used as a pointer to specify where to define the base line used

to subtract the background from the chromatogram and where to

specify the limits used to cut a section from the chromatogram.

The Right and Left Cursor keys move the arrow one point to the

right or left. The Shift Cursor keys move the arrow 10 points,

the Control Cursor keys move it 100 points. The Home key moves

the arrow to the left of the chromatogram and the End key moves

the arrow to the right or end of the chromatogram.

To modify the chromatogram so that there is zero signal

where none of the components of the specimen is eluting, the

background level in the chromatogram must be determined and

subtracted from the chromatogram. The procedure is as follows.

First, move the arrow to a point near the beginning of the

chromatogram where the signal in the chromatogram is due only to

the background, and press F7 . That point and 7 points to the left

of the point are averaged to determine a point on the background

line. Then move the arrow to a point near the end of the

chromatogram and press F7 again. That point and 7 points to its

right are averaged to determine a second point on the background

line and the line is drawn. Examine the line and then press F8

Page 42
for a menu. Press 1 to correct for background by subtracting the

baseline from the chromatogram or press 2 to redetermine the

background. Then press FI to F6 to re-display the chromatogram.

If the background does not depend on the elution volume

(i.e., it is level but non-zero), the background may be

subtracted by moving the arrow to a point of the chromatogram

before the elution of the sample where the height of the

chromatogram is only due to the background and pressing F7 , then

pressing F8. A horizontal base line will then be subtracted from

the chromatogram.

/ The measured chromatogram generally contains a wider range

of elution volumes than those corresponding to the sample.

Depending on the columns used, some 20 or 30 minutes elapse

before even the largest molecules in the specimen elute from the

columns. We routinely measure the chromatogram until all the

material has eluted from the columns and measure for about

another 10 minutes beyond that point to be able to monitor the

background level at the end of the chromatogram. Before the

chromatogram is analyzed further, the section of the chromatogram

corresponding to elution volumes at which the sample is eluted

must be isolated from the rest of the chromatogram. This is

accomplished by moving the arrow to the beginning of the section

to be cut from the chromatogram and pressing F9 . Then the arrow

is moved to the end of the section and F9 is pressed again. Then

F10 is pressed and a menu appears that allows the section cut

from the chromatogram to be displayed or stored. Other options

are also provided. After F10 has been pressed once to cut the

chromatogram, this menu must be obtained by pressing M or m.

The elution volumes should generally be changed to log

hydrodynamic volumes. Two chromatograms in terms of elution

volume may be compared only if they were obtained before the

calibration of column changed. However, chromatograms versus log

hydrodynamic volumes may be compared even if the calibration of

the columns has changed, or if they were obtained using different

columns. In order to change elution volume to log hydrodynamic

volume, press M and then 4. A file containing the calibration of

the columns must be available. The current version of the

calibration function is kept in a file called CALIBR.DAT but

another calibration file may also be used. See the section

CALIBRATION for a description of a calibration file.

The molecular weight averages and molecular weight

distribution may also be calculated. This requires Mark-Houwink

parameters, which are provided in the RAW file via the SETUP

program.

The section cut from the chromatogram may be stored as a

file for further processing by pressing M and then 3. The file

will have the same name of the raw file but the extension will be

HYD if the elution volume has been changed to hydrodynamic volume

Page 43
and CUT if the chromatogram is still in terms of elution volume.

If the log differential molecular weight distribution has been

calculated, it will be stored in a file with extension MWD

.

To end the analysis of this particular chromatogram, press

Me Pressing 7 will allow the operator to analyze another

chromatogram and pressing 8 will exit the program.

Page 44
ANALYZE screen 1 - Start of program

- The ANALYZE program allows the user to analyze a raw chromatogram.

- The program reads in a RAW chromatogram.

- The user decides where to determine the background.

- The program then determines the background at these points and

subtracts the background.

- The user then decides where to cut the chromatogram in order to

analyze the central part. The program cuts the chromatogram

and analyzes it.

- The background is subtracted, and the chromatogram is analyzed.

Enter file name or run number for RAW chromatogram (quit = stop)

:

This representation of the computer screen shows the initial

display for the ANALYZE program, which is used to correct for

background, cut out the relevant part of the chromatogram, and

calculate averages and distributions based on log hydrodynamic volume

and log molecular weight. The operator must type the name of the data

file to be read into the program at the colon prompt.

Page 45
ANALYZE screen 2 - Program can not find data file on disk

- The ANALYZE program allows the user to analyze a raw chromatogram.

“ The program reads in a RAW chromatogram.

- The user decides where to determine the background.

- The program then determines the background at these points and

subtracts the background.

- The user then decides where to cut the chromatogram in order to

analyze the central part. The program cuts the chromatogram

and analyzes it.

- The background is subtracted, and the chromatogram is analyzed.

Enter file name or run number for RAW chromatogram (quit - stop): sec962.raw

Can not find file sec961.raw

If the operator types the name of a file which can not be found

in the current directory, the program notes this fact and allows the

operator to retry, as shown here. No listing of the files in the

current directory can be obtained from inside ANALYZE. To find where

a file is, ©r what the real name is, the operator would have to type

quit at the prompt to exit the program and then use the DOS DIR

command or a directory listing program.

Page 46
ANALYZE screen 3 - Successful read of data file from disk

- The ANALYZE program allows the user to analyze a raw chromatogram.

- The program reads in a RAW chromatogram.

- The user decides where to determine the background.

- The program then determines the background at these points and

subtracts the background.

- The user then decides where to cut the chromatiDgram in order to

analyze the central part. The program cuts the chromatogram

and analyzes it.

- The background is subtracted, and the chromatogram is analyzed.

Enter file name or run number for RAW chromatogram (quit = stop): sec962.raw

Reading file sec962.raw

The title of the input file is:

Cyclic polystyrenes 2583L Run # 962 vial 1 inj 1 13:10:54 01-30-1986

** READING **

The program has now found a file,

into the computer.

and is reading the content

Page 47
ANALYZE screen 4 - Function key instructions after reading data file

Press a function key

- FI displays the full chromatogram.

- F2 displays the left half of the chromatogram.

- F3 displays the right half of the chromatogram.

- F4 displays the left third of the chromatogram.,

- F5 displays the middle third of the chromatogram.

- F6 displays the right third of the chromatogram.

- F7 marks a place to pass the baseline through.

“ F8 subtracts the baseline from the chromatogram - mark

1 or 2 places with F7 before pressing F8.

- F9 marks where to cut - usually the middle part between

2 cuts is kept.

- F10 cuts out the middle part and keeps it - the rest is

discarded - mark 2 places with F9 before pressing F10.

- Escape prints this menu.

- M goes to the menu for further processing after

the chromatogram has been cut by pressing F10.

Hit 'H' for more help.

After a file has been read successfully, control is transferred

to this menu. The typical sequence is to press FI to plot the

chromatogram, then use the arrow and F7 to select places near the

beginning and end of the chromatogram to use to estimate the level of

the background. Background correction is done by pressing F8. The

part of the chromatogram containing information on the specimen is

then cut out using the sequence: arrow movement, F9 , arrow movement,

F9 , F10. All chromatograms can be saved as a "CUT" chromatogram, and

all can be transformed to log hydrodynamic volume if the columns in

the chromatograph have been calibrated. Chromatograms of specimens

for which Mark-Houwink parameters are available may be transformed to

log molecular weight.

Page 48
ANALYZE screen 5 - After pressing H or h: Menu for help screens

For information on the following :

1) Display the chromatogram

2) Determine and subtract the baseline

3) Cut out a part of the chromatogram

4) Calculate log hydrodynamic volume and molecular weight

5) Expand or contract the region of curve displayed

6) Move arrow on chromatogram

7) Return to main menu

Press 1, 2, 3, 4, 5, 6 or 7

Pressing H or h at the main menu leads to several help screens

which explain the possibilities of the ANALYZE program. The following

screens show the help screens for options 1 through 7 in the menu

displayed here.

Page 49
ANALYZE screen 6 - Help screen for functions keys FI to F6

FI displays

F2 displays

F3 displays

F4 displays

F5 displays

F6 displays

the full chromatogram*

the left half of the chromatogram*

the right half of the chromatogram,

the left third of the chromatogram,

the middle third of the chromatogram,

the right third of the chromatogram.

You should display the chromatogram with various function keys

to get the best view of where you want to mark it to establish a point

to draw the baseline through or a point at which to cut the chromatogram.

The program remembers points you have already specified if you change

views by pressing another function key.

Give Return when ready

This screen is generated following the choice of option 1 in the

H help screen menu.

Page 50
ANALYZE screen 7 - Help screen for background correction

- Move the arrow on the chromatogram, using the cursor keys, to a place to pass the background line through, then press F7.

- To choose a second place to pass the background through,

move the arrow to the second place on the chromatogram

and press F7 again. The background line will then be

drawn so you can see whether or not it is suitable.

- Press F8 to lead to a menu where you have a choice of using

this background or editing it.

Give Return when ready

.in, i aaac j i gsaEB—gaa—assbb—b ebb nag —t a i- m ma u aaasama sssa—

This screen is generated following the choice of option 2 in the

H help screen menu.

Page 51
ANALYZE screen 8 - Help screen for cutting the chromatogram

- The 'meaningful part* may be cut out of the entire chromatogram

by moving the arrow to the beginning of the part to be cut out,

and pressing F9.

~ Then move the arrow to the end of the part to be cut out

and press F9 again.

- Pressing F10 cuts the chromatogram and displays a menu for further

processing.

- Shift with the cursor keys moves the arrow 10 points.

- Ctrl with the cursor keys moves the arrow 100 points.

- Up cursor expands the graph vertically and Down cursor contracts

the graph.

Give Return when ready

This screen is generated following the choice of option 3 in the

H help screen menu.

Page 52
ANALYZE screen 9 - Help screen for further processing of cut chromatograms

- After the chromatogram has been cut, the hydrodynamic

volume distribution may be calculated.

- If Mark-Houwink parameters were provided at setup time,

the molecular weight distribution and molecular weight

averages may also be calculated.

- The resulting distributions may be written to files

on the disk.

Give Return when ready

This screen is generated following the choice of option 4 in the

H help screen menu.

Page 53
ANALYZE screen 10 - Help screen for expanding or contracting the plot

A section of the chromatogram may be shown expanded

by moving the arrow to that part of the chromatogram and

pressing the up cursor key. Then keys FI to F6 will display

the section of the chromatogram magnified by a factor -of 2

in the Y direction. The process may be repeated to further

magnify the chromatogram.

The process may be reversed by pressing the down cursor key.

The Page Up and* Page Down keys move thew viewing area

up and down the chromatogram.

Give Return when ready

This screen is generated following the choice of option 5 in the

H help screen menu.

Page 54
ANALYZE screen 11 - Help screen for moving the arrow on the plots

The arrow on the chromatogram may be moved one point to the

by the right and left cursor keys. It may be moved 10 points to

the right or left by shift cursor keys.

It may also be moved to the left of the chromatogram

by the Home key and to the right of the chromatogram

by the End key.

Give Return when ready

This screen is generated following the choice of option 6 in the

H help screen menu.

Page 55
sec%2 .raw

ANALYZE plot 1 - Chromatogram as input from disk

The display shown here was generated by pressing FI at the main

menu. The operator may proceed to another menu by pressing {Escl to

go to the main menu, H or h to go to the help screens, or M or m to go

to the analysis menu. The analysis menu controls output of the cut

chromatogram, and transformation from elution volume into log

hydrodynamic volume and log molecular weight. It also is the route to

restarting the analysis on this chromatogram or reading in another

chromatogram, and to ending the program.

The arrow shows where the chromatogram will be marked for

background estimation and for cutting. The coordinates of the arrow

are displayed. Note that the background of the chromatogram is not at

zero on the vertical axis. Background correction involves estimating

the heights in the chromatogram near the beginning and end of the

chromatogram, and drawing a line which will be subtracted from the

vertical coordinates of the chromatogram so that regions of the

chromatogram which contain no contribution from the specimen will be

zero

.

Page 56
sec962 .raw

ANALYZE plot 2 - Determining background (with F7)

Here, two places at which the background is to be estimated

have been chosen by moving the arrow and pressing F7 . After the

second press of F7 , the background line is drawn. Before

pressing F8, the operator must verify that this line is

reasonable in that it seems to apply to the whole chromatogram.

Page 57
ANALYZE screen 12 - Menu for background correction , after F8

Background points are at elution volumes of 17.74 and 31.75

Bo you want to:

1) Correct for this background.

2) Redetermine background.

3) Store part of chromatogram without correcting for background.

4) Exit program.

Press 1, 2, 3, or 4.

This screen is provided by the program after F8 has been

pressed. Option 1 leads to the next menu, which explains that the

next step is to cut the chromatogram.

Page 58
ANALYZE screen 13 - After F8 and 1 (background has been subtracted)

- You may now choose the region to be stored in the file

of the 'cut' chromatogram.

- Move the arrow to each of the limits of the region in turn

and press F9 at each limit.

- When you have defined two points using F9, press F10. F10 leads

to a menu which allows several further choices,

including writing the results in files and calculating molecular weights

Press a function key FI to F6 to display chromatogram

This screen follows option 1 in the previous menu, which followed

pressing F8 to subtract the background in the chromatogram. The next

step is to use the F9, F9, F10 sequence to cut out the part of the

chromatogram which pertains to the specimen.

Page 59
sec962 .raw

ANALYZE plot 3 - Chromatogram with background removed (F8)

The non-zero background has now been removed from the

chromatogram so that the level at the beginning and end is zero.

The next step is to cut out the part relevant to the specimen,

which is the peak at about 25 milliliters in elution volume.

Page 60
sec962 .raw

0.0 10.0 2:0.0 30 -0~ 40.0 50.O Relative awount vs el ut i on volume

ANALYZE plot 4 - Delineating relevant part of chromatogram (with F9)

After the effect of non-zer© background has been removed

from the chromatogram using the F7 , F7 , F8 sequence, the part of

the chromatogram pertaining to the specimen may be cut out. This

screen shows limits chosen by moving the arrow and pressing F9,

once for each limit. The next step is to press F10.

Page 61
ANALYZE screen 14 - After pressing F10 to cut chromatogram (or M or m)

Limits at elution or hydrodynamic volumes of 19.88 and 26.06

1) Redetermine limits to cut chromatogram.

2) Look at graph.

3) Store clipped chromatogram versus elution or hydrodynamic volume in a file

4) Change elution volume to log hydrodynamic volume.

5) Compute and print molecular weight averages.

6) Compute differential log molecular weight distribution.

7) Examine another chromatogram.

8) Exit from program.

Give a number between 1 and 8 inclusive

This is the analysis menu. The operator may carry out operations

such as in options 4, 5 and 6, and view the results by pressing option

2. Option 3 allows the data to be stored in a file. The extension of

the file depends on whether the chromatogram is in terms of elution

volume (extension is CUT as in SEC999 . CUT) or log hydrodynamic volume

(extension is HYD as in SEC999.HYD). Chromatograms which are in terms

of log molecular weight are saved to a file on the disk at the end of

option 6.

Page 62
ANALYZE plot 5

- "CUT" chromatogram (with F10 then 2)

The results of clipping the chromatogram with the F9, F9

,

sequence and plotting the result with choice 2 from the analysis

menu are shown here. The analysis menu is obtained by pressing M

(for "m"enu)

.

F1C

or s

Page 63
ANALYZE screen 15 - Normalizing and storing cut chromatogram (M and 3)

Give a number between 1 and 8 inclusive

You may normalize chromatogram before it is stored

1) Normalize chromatogram to unit area

2) Normalize chromatogram so the highest peak is L

3) Multiply heights of chromatogram by a given scale factor

4) Do not normalize chromatogram

Give a number between 1 and 4 inclusive: 1

Chromatogram is being normalized to unit area and stored

The clipped chromatogram has been saved in file sec924.CUT

1) Redetermine limits to cut chromatogram.

2) Look at graph.

3) Store clipped chromatogram versus elution or hydrodynamic volume in a file

4) Change elution volume to log hydrodynamic volume.

5) Compute and print molecular weight averages.

6) Compute differential log molecular weight distribution.

7) Examine another chromatogram.

8) Exit from program.

Give a number between 1 and 8 inclusive

This screen shows the activity following pressing 3 in the

analysis menu. The chromatogram was in terms of elution volume and

hence the program has saved it as a CUT file. The operator chose to

normalize the chromatogram.

Page 64
ANALYZE screen 16 - Elution volume to log hydrodynamic volume (M and 4)

Limits at elution or hydrodynamic volumes of 19.88 and 26.06

1) Redetermine limits to cut chromatogram.

2) Look at graph.

3) Store clipped chromatogram versus elution or hydrodynamic volume in a file

4) Change elution volume to log hydrodynamic volume.

5) Compute and print molecular weight averages.

6) Compute differential log molecular weight distribution.

7) Examine another chromatogram.

8) Exit from program.

Give a number between 1 and 8 inclusive

Do you want to use calibration in file CALIBR.DAT ? (Enter Y or N)

Title of calibration is : B DICKENS Calibration of

Elution volume 19.88 is

Elution volume 19.96 is

Elution volume 20.05 is

Elution volume 20.13 is

Elution volume 20.21 is

Elution volume 20.29 is

Shodex cols

outside the

outside the

outside the

outside the

outside the

outside the

804 & 802 NOV 1986

calibration range

calibration range

calibration range

calibration range

calibration range

calibration range

This screen shows the program flow following pressing 4 in the

analysis menu. To convert from elution volume to log hydrodynamic

volume, the program needs to have the calibration of the

chromatographic columns. The latest calibration is kept in the file

CALIBR.DAT, which is read in automatically on answering Y or y to the

question. The program indicates when elution volumes are being

transformed which are beyond the calibrated range of elution volumes.

This is a dangerous sign, since the calibration is not reliable

outside the limits where it was constrained to follow the data points

which were measured to find the calibration.

Page 65
sec%2 -raw

ANALYZE plot 6 - Distribution of log hydrodynamic volumes (M and 2)

The results of clipping the chromatogram with the F9 r F9, F10

sequence and plotting the result with choice 2 from the analysis

menu are shown here. The analysis menu is obtained by pressing M or m

(for "m"enu)

.

Page 66
ANALYZE screen 17 - Reading "CUT" file

- The ANALYZE program allows the user to analyze a raw chromatogram.

- The program reads in a RAW chromatogram.

- The user decides where to determine the background.

- The program then determines the background at these points and

subtracts the background.

- The user then decides where to cut the chromatogram in order to

analyze the central part. The program cuts the chromatogram

and analyzes it.

- The background is subtracted, and the chromatogram is analyzed.

Enter file name or run number for RAW chromatogram (quit = stop): sec962.cut

Reading file sec962.cut

The title of the input file is:

Cyclic polystyrenes 2583L Run # 962 vial 1 inj 1 13:10:54 01-30-1986

** READING **

The previous analysis sequence demonstrated how to go from a RAW

chromatogram to a chromatogram in terms of elution volume. The

chromatogram was stored in the form of a CUT chromatogram (i.e., in

terms of elution volume, although it could equally well have been

stored in terms of log hydrodynamic volume) . This screen shows that

the stored CUT file can later be read into the ANALYZE program and the

analysis can be continued. In this case,’ there is no need to correct

for the background in the chromatogram. The chromatogram can be

re-cut if desired.

Page 67
ANALYZE screen 18 - Main menu, obtained after reading cut file

Press a function key

- FI displays the full chromatogram.

- F2 displays the left half of the chromatogram.

- F3 displays the right half of the chromatogram.

- F4 displays the left third of the chromatogram.

* F5 displays the middle third of the chromatogram.

- F6 displays the right third of the chromatogram.

- F7 marks a place to pass the baseline through.

- F8 subtracts the baseline from the chromatogram - mark

1 or 2 places with F7 before pressing F8.

- F9 marks where to cut - usually the middle part between

2 cuts is kept.

- F10 cuts out the middle part and keeps it - the rest is

discarded - mark 2 places with F9 before pressing F10.

- Escape prints this menu.

- M goes to the menu for further processing after

the chromatogram has been cut by pressing F10.

Hit 'H

9 for more help.

This is

out plotting

the main menu, which gives information on how

and further analysis.

to carry

Page 68
ANALYZE screen 19 - After F10 or M

Limits at elution volumes of 19.41 and 29.59

1) Redetermine limits to cut chromatogram.

2) Look at graph.

3) Store clipped chromatogram versus elution or hydrodynamic volume in a file

4) Change elution volume to log hydrodynamic volume.

5) Compute and print molecular weight averages.

6) Compute differential log molecular weight distribution.

7) Examine another chromatogram.

8) Exit from program.

Give a number between 1 and 8 inclusive:

The analysis menu is obtained after pressing M or m in the main

menu. This would be the normal route to take. A wise step would be

to plot the input chromatogram with option 2, then press M to return

to this analysis menu for further processing.

Page 69
ANALYZE screen 20 - After M and 4

7) Examine another chromatogram.

8) Exit from program.

Give a number between 1 and 8 inclusive? 4

Do you want to use calibration in file CALIBR.DAT ? (Enter Y or N)

What is the name of the calibration file? clonecal.3

Title of calibration is :

B DICKENS Calibration of Shodex cols 804 & 802 NOV 1986

Elution

Elution

Elution

Elution

Elution

Elution

Elution

Elution

Elution

Elution

volume

volume

volume

volume

volume

volume

volume

volume

volume

volume

19.41

19.51

19.61

19.71

19.81

19.90

20.00

20.10

20.20

20.30

is outside

is outside

is outside

is outside

is outside

is outside

is outside

is outside

is outside

is outside

the calibration

the calibration

the calibration

the calibration

the calibration

the calibration

the calibration

the calibration

the calibration

the calibration

range

range

range

range

range

range

range

range

range

This screen shows the sequence generated by pressing option 4,

the transformation of elution volume to log hydrodynamic volume.

Page 70
ANALYZE screen 21 - After M and 4 part 1

Give a number between 1 and 8 inclusive: 4

Do you want to use calibration in file CALIBR.DAT ? (Enter Y or N) n

What is the name of the calibration file? clonecal.3

Title of calibration is :

B DICKENS Calibration of Shodex cols 804 & 802 NOV 1986

Elution volume 19.41 is outside

Elution volume 19.51 is outside

Elution volume 19.61 is outside

Elution volume 19.71 is outside

Elution volume 19.81 is outside

Elution volume 19.90 is outside

Elution volume 20.00 is outside

Elution volume 20.10 is outside

Elution volume 20.20 is outside

Elution volume 20.30 is outside

Elution volume has been

the calibration range

the calibration range

the calibration range

the calibration range

the calibration range

the calibration range

the calibration range

the calibration range

the calibration range

the calibration range

changed to log hydrodynamic volume

This screen shows that the transformation of elution to log

hydrodynamic volume has been completed. It also shows where the

elution volumes were outside the calibrated range of the columns.

This is a warning to the operator that the results in those regions

may not be trustworthy.

Page 71
ANALYZE screen 22 - After M and 4 part 2

Elution volume 19.61 is outside the calibration range

Elution volume 19.71 is outside the calibration range

Elution volume 19.81 is outside the calibration range

Elution volume 19.90 is outside the calibration range

Elution volume 20.00 is outside the calibration range

Elution volume 20.10 is outside the calibration range

Elution volume 20.20 is outside the calibration range

Elution volume 20.30 is outside the calibration range

Elution volume has been changed to log hydrodynamic volume

Limits at log hydrodynamic volumes of 2.099675 and 7.994616

1) Redetermine limits to cut chromatogram.

2) Look at graph.

3) Store clipped chromatogram versus elution or hydrodynamic volume in a file

4) Change elution volume to log hydrodynamic volume.

5) Compute and print molecular weight averages.

6) Compute differential log molecular weight distribution.

7) Examine another chromatogram.

8) Exit from program.

Give a number between 1 and 8 inclusive:

At the conclusion of option 4, averages based on log hydrodynamic

volume are written to the printer, and the limits of the chromatogram

are presented in terms of log hydrodynamic volume rather than elution

volume

.

Page 72
ANALYZE screen 23 - After M and 5

5) Compute and print molecular weight averages.

6) Compute differential log molecular weight distribution.

7) Examine another chromatogram.

8) Exit from program.

Give a number between 1 and 8 inclusive: 5

Computing molecular weight averages

Limits of log molecular weight values are 3.542915 and 6.930812

Area of chromatogram = .9983551

Mn = 41909.16

Mw = 54068.64

Mz = 87022.96

Polydispersity = 1.290139

Calculated viscosity = .2629267

Press any key

Option 5 in the analysis menu transforms log hydrodynamic volume

(or elution volume) into log molecular weight, providing that

Mark-Houwink parameters were given at SETUP time. These parameters

are available to the program through the data file which contains the

chromatogram. The option provides estimates of the molecular weight

averages, the polydispersity, and the intrinsic viscosity.

Page 73
ANALYZE screen 24 - After M and 5

Mn = 41909 .16

Mw = 54068.64

Mz = 87022.96

Polydispersity ~ 1.290139

Calculated viscosity ~ .2629267

Press any key

Limits at log molecular weight values of 3.542915 and 6.930812

1) Redetermine limits to cut chromatogram.

2) Look at graph.

3) Store clipped chromatogram versus elution or hydrodynamic volume in a file

4) Change elution volume to log hydrodynamic volume.

5) Compute and print molecular weight averages.

6) Compute differential log molecular weight distribution.

7) Examine another chromatogram.

8) Exit from program.

Give a number between 1 and 8 inclusive:

After a key is pressed at the end of option 5, the analysis menu

reappears. The limits of the analyzed range of the chromatogram are

now given in terms of log molecular weight.

Page 74
ANALYZE screen 25 - After M and 6

2) Look at graph.

3) Store clipped chromatogram versus elution or hydrodynamic volume in a file

4) Change elution volume to log hydrodynamic volume.

5) Compute and print molecular weight averages.

6) Compute differential log molecular weight distribution.

7) Examine another chromatogram.

8) Exit from program.

Give a number between 1 and 8 inclusive: 6

Calculating differential molecular weight distribution

Normalizing molecular weight distribution

Differential log molecular weight distribution has been calculated.

Do you want to store the distribution in a file (Enter Y or N) : y

Storing log differential molecular weight distribution

Differential log molecular weight distribution is stored in sec962.MWD

Option 6 in the analysis menu calculates the log molecular weight

distribution and normalizes it. This means that the program makes the

total area under the curve of the log molecular weight equal to one so

the results are independent of the amount of material injected and of

the refractive index difference between the material and the solvent.

Hence, different specimens can be compared meaningfully.

Page 75
sec9S2 -raw

Relative amount vs log mo 1 wt

ANALYZE plot 7 - Log molecular weight plot (M then 6 then 2)

Pressing 2 after option 6 has been carried out generates a plot

of the log molecular weight distribution, an example of which is shown

here

.

Page 76
ANALYZE screen 26 - After M and 6 without going through 4 or 5

Elution volume has been changed to log hydrodynamic volume

Title of data file is : Cyclic polystyrenes 2583L Run # 962 vial 1 inj 1 13:10:54 01-30-1986

Title of calibration file is : B DICKENS Calibration of Shodex cols 804 & 802 NOV 1986

Limits of log molecular weight values are 3.542915 and 6.930812

Area of chromatogram - .9983551

Mn = 41909.16

Mw = 54068.64

Mz = 87022.96

Polydispersity - 1.290139

Calculated viscosity ~ .2629267

Press any key

The next three screens show the sequence generated when option 6

is chosen even though the chromatogram is in terms of elution volume.

The program automatically generates options 4 and 5.

©

Page 77
ANALYZE screen 27 - After M and 6

:

( Mn = 41909 . 16

Mw * 54068.64

Mz = 87022.96

Polydispersity ~ 1.290139

Calculated viscosity = .2629267

Press any key

Normalizing molecular weight distribution

Differential log molecular weight distribution has been calculated.

Do you want to store the distribution in a file (Enter Y or N) : y

Storing log differential molecular weight distribution

Differential log molecular weight distribution is stored in sec962.M¥D

Press any key

Part two of the sequence generated when 6 is pressed immediately

after going to the analysis menu for the first time, when the

chromatogram is in terms of elution volume.

Page 78
ANALYZE screen 28 - After M and 6 and "Press any key"

Limits at log molecular weight values of 3.542915 and 6.930812

1) Redetermine limits to cut chromatogram.

2) Look at graph.

3) Store clipped chromatogram versus elution or hydrodynamic volume in a file

4) Change elution volume to log hydrodynamic volume.

5) Compute and print molecular weight averages.

6) Compute differential log molecular weight distribution.

7) Examine another chromatogram.

8) Exit from program.

Give a number between 1 and 8 inclusive:

Control has now been returned to the

can go to another chromatogram or end the

analysis menu,

program.

The operator

Page 79
Program to compare two chromatograms

COMPARE (interactive)

This program interactively makes a detailed comparison of

two chromatograms. The heights in the chromatograms may be

multiplied by a factor or made to match at a given elution

volume. The chromatograms may be subtracted from one another,

when the difference chromatogram is displayed. It may be stored

in a file for later processing.

The program uses a file called STANDARD. CHR / A chromatogram

may be designated to be a member of a particular class, such as

"polystyrene". The class of each chromatogram is stored in the

file of the chromatogram. A standard chromatogram for each class

may be chosen and all chromatograms of the class may be compared

with the standard. The file STANDARD. CHR contains on each line

the name of the class, the name of the file containing the

corresponding standard chromatogram, and the name of the

appropriate calibration file.

The program first reads the file STANDARD. CHR and then

requests the file name of a chromatogram to read. If the

extension of the file is HYD, only the run number need be given,

otherwise the full name of the file must be typed. The

chromatogram of the file is read and the name of the second file

is requested. A file or run number or {Return} may be given. If

{Return} is pressed, the standard chromatogram corresponding to

the class of the first chromatogram is read, otherwise the

requested file is read.

The chromatograms are displayed by pressing one of function

keys FI to F6. The chromatograms may be normalized to unit area

by pressing key F7 . A curve may be scaled with respect to the

other curve by pressing F8 and giving the curve number and factor

to scale the curve by. Curve 1 may also to scaled to match curve

2 at a specified point without having to input the factor. The

arrow on the curve is moved to the point at which the curves are

to be matched and F8 and then 0,0 {Return} are pressed.

Key F9 subtracts the chromatograms and calculates the

standard deviation of the difference chromatogram. The difference

chromatogram may be displaced by key FI.

Key F10 gives the menu.

The Escape key is used to exit the program or to restart the

program after choosing whether or not to store the difference

chromatogram

.

Page 80
COMPARE screen 1 - Starting menu (1)

COMPARE program

Compare two chromatograms and, if requested,

subtract the second from the first

Which do you prefer?

1) Colored lines in graphs

2) Monochrome lines of different widths

The starting menu of the COMPARE program. If you have a colored

graphics adapter screen, select option 1. The following screen

representations are necessarily in black and white, so option 2 was

chosen

.

Page 81
COMPARE screen 2 - Starting menu (2)

COMPARE program

Compare two chromatograms and, if

subtract the second from the first

The classes and associated files in file STANDARD .CHR are:

Class Standard chromatogram Calibration

4

5

Bodied tung oil - Superior

Bodied tung oil - Degen

Not assigned

Black ink - Sicpa

Branched epoxy

6 PSD/PVME irradiated

sec207.hyd

see207 »hyd

cutchr oStd

sec423 .cut

cutchr .std

pvme . hyd

1

3

1

clonecal.3

clonecal.3

The second screen provided by the program shows a listing of the

standard chromatograms and associated calibration files which the

program reads from the file STANDARD . CHR . The first chromatogram to

be input can then easily be compared with the standard chromatogram if

required

.

Page 82
COMPARE screen 3 - Reading the data files

- The COMPARE program allows the user to compare two chromatograms.

- The first chromatogram may be normalized, expanded, or contracted

to match the second chromatogram which can be a standard chromatogram.

- The second chromatogram may be subtracted from the first

chromatogram and the difference chromatogram may be stored in a file.

Run number is converted to HYD type of file

Enter file name or run number for first chromatogram (quit=stop) : 924

Reading file SEC924.HYD

0 Mrad whole polymer Run # 924 vial 1 inj 1 18:31:49 01-12-1986

Enter file name or run number for second chromatogram:

or RETURN for the corresponding standard chromatogram (quit=stop) : ? 925

Reading file SEC925.HYD

0 Mrad methanol insoluble Run # 925 vial 2 inj 1 19:27:50 01-12-1986

In this example, two chromatograms were read into the program.

If the chromatograms are in terms of log hydrodynamic volume, i.e.,

they are HYD files, the operator need only specify the run number.

Page 83
COMPARE screen 4 - Plotting menu

— — Display Selection Menu ————————

- FI displays the full chromatograms.

- F2 displays the left half of the chromatograms.

- F3 displays the right half of the chromatograms.

- F4 displays the left third of the chromatograms.

* F5 displays the middle third of the chromatograms.

- F6 displays the right third of the chromatograms. - - F7 normalizes the chromatograms to unit area.

- F8 matches chromatogram 1 to chromatogram 2 at the arrow.

Move the arrow with the cursor keys or use shift/ctrl with

the cursor keys. ***** WARNING Press F8 only when graph is displayed *****

“ F9 subtracts chromatogram 2 from chromatogram 1 and

calculates the standard deviation of the difference.

- F10 displays this menu

Esc gives the option of storing the difference chromatogram,

reading in more chromatograms., or quitting the program

Up Cursor expands graph vertically and Down Cursor contracts graph.

Press one of function keys FI to F6 to display the chromatograms.

This screen explains the plotting options and how to match the

two chromatograms. They may be matched at a point specified by the

arrow on the plot (the plot must be on the screen at the time F8 is

pressed) or the chromatograms can be scaled by giving the curve number

(1 or 2 for first curve read in or second curve read in) and the

multiplication factor after pressing F8 . The factor always operates

on the curve as read in and is not cumulative. F7 normalizes the two

chromatograms. This means that the area under each curve is made

equal to one, which is a good general way to compare two

chromatograms. If the comparison is between two materials one of

which has less of one component than the other, it would be more

appropriate to move the arrow to a peak in some region of the

chromatogram which corresponds to a component which is present in

identical amounts in the two cases, and press F8 then 0,0 {Return} to

make the two chromatograms of equal height at that point. The 0,0

means apply no factor to the curves.

Page 84
COMPARE plot 1 - ’'Clipped" chromatograms as input

The two chromatograms read into the program in the example

are displayed here. The label at the bottom of the plot shows

that they are in terms of log hydrodynamic volume. The label at

the top of the plot shows that SEC924.HYD is the lighter line,

and SEC925.HYD is the heavier line.

Page 85
COMPARE screen 5 - Taking difference chromatogram

PLEASE WAIT

Taking difference chromatogram

Pressing F9 takes the difference between the two original

chromatograms and generates this message on the screen.

Page 86
COMPARE screen 6 - After taking difference chromatogram

Chromatograms have been subtracted

Standard deviation of difference of chromatograms = 0.148946

Press one of keys FI to F6 to redraw

Press {escape} to store difference of

Press F10 for the main menu

chromatograms

chromatograms

In subtracting the two chromatograms, the program calculates the

difference in terms of a standard deviation, which is displayed on the

screen as in this example.

Page 87
Press ke

SEC924.

1 . 50—

1 . 00

y F10 f or Menu

ft¥I> points, SEC925 . HYD dashes

i

i

i

i

!I

I

I .

{ /

S 0

1 8 0.5

0,00

arrow -0.50- l 1.0 2.0

A/

AA

A*. 8

I v'llu

I*

06%

location 1

L

246 0 . 005

1JL_

Re lative

3 .

0

anount v

4.0 5.0 6.0 7.0

Lost hydrodynawic vol

COMPARE plot 2 - Difference chromatogram (after FI)

Re-plotting the chromatograms with FI generates a plot which

also contains the difference chromatogram. The plot frame may

be displaced down with the Page Down key, and up with the Page Up

key. The curves may be expanded using the Up Cursor key or

contracted by using the Down Cursor key. The cursor keys

actually change the size of the graph frame. Since the size of

the graph frame determines the scale on the screen, the net

result is to expand the curves when the frame is shrunk and the

plot is automatically rescaled, and to contract the curves when

the frame is expanded and the plot is rescaled.

Page 88
Press ke SEC924

; 1 . 50

F10 for Menu

YI> points, SEC925.HYD dashes

T

fcraph f rahe expanded

Curves contracted by

0 I Range is now -0.524

PI . 00 Graph frane expanded

Curves contracted Ly

0 I Rancre is now -0.621

P0. 50 Graph frane expanded Curves contracted by 0 I Range is now -0.727

0 . &0*PM

-0.50

1 .

0

T

ne t

to

ne t

to

net

to

r

„ f ac top

1 . 402 '

T“

Of

I

\

0.9 0

factor of 0.90

1 . 498

.£s i*

,** f ac top

1 .604

Vi

\

of

1

' 0.90

\Vi

S' —

arrow location 1.246, 0.005

i JL

2 .

0

3 .

0

4.0 5.0 6 .

0

7 .

0

Relative anount vs Log hydrodynawi c vol

COMPARE plot 3 - Expanding graph frame with Up Cursor key

Pressing the Up Cursor key expands the graph frame and shows

the new limits on the screen, as shown here. The frame size

changes about 10% with each press. In this case, the operator

would compare the limits generated after each press with the

limits in the plot as shown and estimate when the frame has been

expanded enough to make the lowest curve clear the legend. At

that point, the plot should be re-drawn with FI.

Page 89
Jab, c <sLs © ©

L&bJ: J* « 'w • VP ® m«P i ® Relative awount vs Lost lny clr^ocly nawi c vol

COMPARE plot 4 - Replot with FI

This is the result of pressing FI to re-draw the previous

screen.

Page 90
Press ke

SEC924.

•2 . 00 T

Frawe shifted

Range

Q I Frawe

Range

1 . 00

m F10 for^ Menu

HYD points, SEC925 H YI> dashes

i s now

shi f ted

is now

0.00l

T— 1—

downward hy -0.843 to

downward J>y -0.960 to

0.117

1 . 487

T

0.117.

1 . 371 f \

i \ ir\

jO( \t,

*•> V/

arrow location 1.246, 0.005 - 1 . 00

~ -1—

1.0 2.0 3.0 4.0 5.0 6.0 7.0 Relative anount vs Log hydrodynawic vol

*

COMPARE plot 5 - Move graph frame down with Page Down key

The graph frame may moved up and down with the Page Up and

Page down keys. This screen shows that the Page Down key has

been pressed twice. Shifts are about 10% of the y axis with each

press.

Page 91
Press key F10 f or menu

SEC924 . HY1> points, SEC925.HVD dashes

COMPARE plot 6 - Replot with FI

This screen shows the plot re-drawn with FI after the frame

was shifted as in the previous screen.

Page 92
Press ke

SEC207

.

1 . 50

1 . 00

0.5

0. 00

y F10 for Menu

R¥I> points, SFC924.HyD dashes “——T“ — ——I "I

arrow location “1.868, 0.013

“0.5 0—— L L —~L„ _

“2.5 0.0 2.5 5.0

Relative amount vs Log hydroclynawic

7 .

5

vo 1

COMPARE plot 7 - Scale two chromatograms - as read in

These two chromatograms are obviously on different scales - because the refractive index of the material in the right hand

chromatogram is nearer that of the solvent than the refractive

index of the material in the left hand chromatogram is.

Page 93
Qiue nuwlber of curve Cl or 2> ^ factor*

or* 8,0 to watch curves at arrow ? 2 , 1 js]

COMPARE plot 8 - Multiply second curve by factor of 10 (using F8)

Pressing F8 with the graph displayed on the screen allows

the operator to specify a curve number (here 2) and a scale

factor (here 10)

.

Page 94
Press F10 for the nenu or FI to F6 tor redraw the chrowatograwst arrow ? 2,10

COMPARE plot 9 - Result of replotting previous plot with FI

As soon as the {Return} key is pressed, the program applies

the scale factor and re-draws the curve.

Page 95
Px*ess F10 for the Menu or FI to F6 tor redraw the chronatograws t arrow ? 2,20

«£=s © V# q V# u o x# « V/ 9 © w Relative anount os Lost Rydrodynawic vol

COMPARE plot 10 - Multiply by bigger factor

This is the result of specifying a scale factor of 20 for

the second curve. The curves are numbered in the order they were

read into the program.

Page 96
- 0.5 8—

——j ~~~~i~— J - 2.5 0.0 2.5 5.0 7.5 Relative amount vs Lost hydrodynawic vol

COMPARE plot 11 - Result of replotting previous plot with FI

e This screen shows the result of reTMplotting the graph by

pressing FI.

Page 97
Press key F10 for Menu

SEC207 . RVD points, SEC924.HVD dashes

COMPARE plot 12 - Normalize curves to unit area with F7

The two curves may be normalized (total area under the curve

made equal to 1) by pressing F7

.

Page 98
COMPARE plot 13 - Automatic replot after F7

The F7 option for normalizing the curves finishes by

re-plotting the curves, as shown here.

Page 99
COMPARE screen 7 - Writing difference chromatogram to disk file

Redo plot with frame in original place ? (Y or N ?) n

Store difference chromatogram (Y or N) ? : y

Give the file name to store the difference chromatogram in- : delta45„dif

Chromatogram is being written to disk

Redisplay chromatograms, enter New chromatograms or Exit program?

Press R, N or E :

Pressing {esc! allows the operator to store the difference

chromatogram, and to continue the analysis, read in more

chromatograms, or end the program.

Page 100
Program to compare up to 9 chromatograms

C0MPARE9 (interactive)

This program displays up to 9 chromatograms simultaneously.

In the display on the screen, the chromatograms may be displaced

so they do not overlap. A section may be cut from the

chromatograms. The displayed cut of the displaced chromatograms

may be stored in a file to be plotted by a plotting program such

as the NBS Polymer Division program GRAPH to produce

publication-quality graphs on a plotter.

/ The desired chromatograms are first read from files. If the

files have the extension RAW, only the run numbers need be

entered, otherwise the full names of the files are entered. A

I Return I with no file name or run number is given after the

desired chromatograms have been read into the program. The full

range of the chromatograms may then be displayed by function key

FI or sections of the full range may be displayed by one of

function keys F2 to F6 . The chromatograms may be expanded in the

vertical direction by keys Up and Down Cursor and Page Up and

Page Down as explained in the section on the program ANALYZE. Key

F7 normalizes the chromatograms to unit area and key F8 displays

the menu

.

Key S allows a displacement of the curves to be given in

units of the vertical axis so that the curves will not overlap.

An arrow is drawn above the first curve. Key A allows the

arrow to be moved to a different curve, which the operator gives

the number of at this point. The arrow may be removed by giving

a curve number of 0 (zero). The arrow is used to indicate where

to cut a section from the chromatograms. The Right and Left

Cursor keys move the arrow one point to the right or left,

respectively. The Shift Right and Shift Left Cursor keys move the

arrow 10 points and the Ctrl Right and Ctrl Left Cursor keys move

the arrow 100 e point to the right or left. The Home key moves the

arrow to the beginning of the curve and the End key moves the

arrow to the end of the curve. To cut a section from the

chromatograms, move the arrow to one end of the section and press

F9 to make the arrow position define the end of the range. Then

move the arrow to the other end of the section and press F9 again

to define the other end of the range. Then press F10 to cut out

and retain the part of the chromatogram between the limits.

The Escape key allows the operator to exit the program, and

provides the option to store the chromatograms to be stored in

files on the disk. If the chromatograms are stored in a file, the

file will be of the form to be plotted by program GRAPH to give

a publication quality-graph.

Page 101
C0MPARE9 screen 1 - Reading in the data files

- The COMPARE program allows the user to compare up to 9 chromatograms.

- The chromatograms may be normalized or the ends cut from the chromatograms.

- The chromatograms as displayed may be stored in a file in a form

suitable to be plotted by program GRAPH after Esc is pressed.

Default type of file is RAW

Press Return after last chromatogram

Enter file name or run number for chromatogram : ? 996

Now reading file SEC996.RAW

HEM- 10 FXm Trans = 8d %I= 1 Run * 996 vial 1 inj 1 00:33:04 02-13-1986

Press Return after last chromatogram

Enter file name or run number for chromatogram : ? 997

Now reading file SEC997.RAW

HEM-10 Plm Trans = 9d %I= 1 Run # 997 vial 2 inj 1 01:29:06 02-13-1986

The initial menu of the COMPARES program allows the operator

to specify the run numbers or file names of the chromatograms to

be read into the program, as shown here.

Page 102
C0MPARE9 screen 2 - End of reading the data

Press Return after last chromatogram

Enter file name or run number for chromatogram : ? 1000

Now reading file SEC1000.RAW

HEM-10 Pirn Trans = lOd %I= 1.5 Run # 1000 vial 5 inj 1 04:17:11 02-13-1986

Press Return after last chromatogram

Enter file name or run number for chromatogram-: ? 1001

Now reading file SEC1001.RAW

HEM-10 Pirn Trans = lOd %I= 2.0 Run # 1001 vial 6 inj 1 05:13:13 02-13-1986

Press Return after last chromatogram

Enter file name or run number for chromatogram : ? 1002

Now reading file SEC1Q02.RAW

HEM-10 Pirn Trans = lOd %I= 3.0 Run # 1002 vial 7 inj 1 06:09:15 02-13-1986

Press Return after last chromatogram

Enter file name or run number for chromatogram : ?

Finding plot limits

After the last chromatogram has been specified, the

operator presses {Return} with no other entry, as shown here.

The program then searches for the limits to use in drawing the

frame round the plot and generates the menu shown in the

following screen.

Page 103
C0MPARE9 screen 3 - Plotting menu

-—

Display Selection Menu — —

- FI displays the full chromatograms.,

- F2 displays the left half of the chromatograms.

- F3 displays the right half of the chromatograms.

- F4 displays the left third of the chromatograms.

- F5 displays the middle third of the chromatograms. . - F6 displays the right third of the chromatograms.

- F7 normalizes the chromatograms to unit area.

- F8 displays this menu

-F9 gives limits to cut chromatograms. To cut chromatograms,

move arrow to lower limit and press F9, then move arrow to upper

limit and press F9 again, then press F10.

- F10 gives the option to cut chromatograms, discarding points at

points at low and high elution volumes.

Esc gives the option of storing the chromatogram,

reading in more chromatograms, or quitting the program

Up Cursor expands graph vertically and Down Cursor contracts graph.

Page Up and Page Down raises or lowers region of graph shown.

S shifts the chromatograms vertically.

A moves the arrow to a chromatogram.

Press one of function keys FI to F6 to display the chromatograms.

Pressing FI from this menu is the first step,

the chromatograms.

This displays

Page 104
Press key F8 for Menu

C0MPARE9 plot 1-7 "raw" chromatograms as input, no vertical shift

The chromatograms are displayed plotted on top of one

another. They may be cut all together with the arrow movement,

F9 , arrow movement, F9 , F10 sequence.

Page 105
P^ess

Frawe

Ranse

F>awe

Range

Fi'awe

Range Frawe

Range

Farawe

Ranye F^awe

Range

I 0,01

0.00

key

sin

i s

slii • » i s

shi

i s

shi

i s

shi

i s

shi

is

F8 £

£ ted

now

£ ted

now

f ted

now

£ ted

now

f ted

now

f ted

now

or Menu downward by -0.000 to downward by -0.00J to downward )>y -0.002 to downward by -0.003 to downward by -0.004 to downward by -0.005 to

0.001 0.019”r

0.001 0.018

0.001 0.017 0.001

0. 016

0.001

0.015

0 . 001 0.014

Eirro w location

0 * 00

0.0 10.0 20.0 30.0 40.0 Elution vohme

re©2

50.0 60.0

*

COMPARES plot 2 - Move graph frame down with Page Down key

This plot shows that the operator decided to shift the

frame down a little by pressing the Page Down key.

Page 106
Press key F8 for Menu

0.02

0.01

0.01

0.00

T

v-^

Vi*

arrow location 0 . 06 0 , 0 . 002 -0 . 01 — 1 — L— 1 —JL——1-

0.0 10.0 20.0 30.0 4O.0 50.0 60.0 Elution voliihe

C0MPARE9 plot 3 - Replot with FI

© The plot has been re-drawn by pressing F10. Now enough

detail can be seen to cut the chromatograms easily.

Page 107
Press key F8 f ex' wen

0.02

0.01

0.01

0.00

” 0.01

aiTOW location 39 . 130

.

jl i i JLLl

0. 004

- _L_

0.0 10.0 20.0 30.0 40.0 50.0 60.0 Elution vokiMe

COMPARES plot 4 ~ Region to cut out selected with F9

The limits at which to cut the chromatograms £ have been

defined using the arrow, F9 key, arrow, F9 key sequence.

Page 108
Pr*ess Ice y F8 for nenu

Elution vol ime

C0MPARE9 plot 5 - Chromatograms cut with F10, replotted

The chromatograms have been cut by pressing F10 f which then

re-plots the cut chromatograms.

Page 109
COMPARES screen 4 - Vertically displacing the curves

Enter vertical shift for plotting curves: .003

Press FI to F6

By pressing the

curves vertically by

S key, the operator is able to shift the

a specified amount.

Page 110
Press key F8 for* Menu

0.04

0.03

0. 02

0.01

T

• vv. *

Afc. ,l s

•V_„_ v*y—'

* .V

9

I I >A~x

'v,w"

V_ vw**S-’J ,'

».*w- J v-V _J*

*V‘\ Vw

arrow location 23 . 320 , 0.002

© . ©0— —J— L- —1—— .. j

20.

0

25.0 30.0 35.0 Elution voluMe 40.0

C0MPARE9 plot 6 - Replot shifted curves (arrow on top curve with A)

The shifted curves may then be re-plotted , and if desired,

re-shif ted

.

Page 111
0. 90

0.041

Press key F8 for Menu Graph frawe expanded Curves contracted hy net factor of

0 I t . Ran ye is now - 0.000 to Graph frawe expanded. Curves contracted by net factor of 0.90

00 * ©3 :f "V

“

-1

Ran ye is now 0.002 to Graph frawe expanded Curves contracted by net factor of

Ran ye is now -0.004 to

0.90

..w -S* 0 . 045 ... *aA. V

0. 01

•S.-V’W-

..A-

•v'n .-A*- V

-V

V

arrow location 23.320,

0 . 00 L

v\

0" 002 _t__

20.0 25.0 30.0 35.0 Elution volime 40.

0

COMPARES plot 7 - Expand graph frame with Page Up key

The operator has decided to contract the curves as plotted

by expanding the graph frame down using the Up Cursor key , which

has been pressed three times.

Page 112
Press key F8 for Menu

0 . 05 —T~ ~J

0.00

arrow location 23.320, 0.002

_ 0 0

... -J 4 — .1 20 1 0 25.0 30.0 35.0 40.0 Elution volune

C0MPARE9 plot 8 - Replot with FI

The curves have now been re-plotted using FI.

Page 113
Pif'ess key F8 for fienu

Elution volume

COMPARES plot 9 ~ Normalize to unit areas with F7

This is the result of normalizing the curves by pressing F7

Page 114
C0MPARE9 screen 5 - Save plot for future hardcopy graph on plotter

Replot with the original scales and a shift of 0 ? n

Do you want to plot the chromatograms later on a plotter (Y or N) ? y

Give file name to store chromatograms for later plotting

by program GRAPH : comp9.grp

Writing GRAPH file

Give title of graph comparison of 7 mixtures

Examine New chromatograms or Exit program?

Press N or E

Several options for ending the program are available when

i Escape} is pressed. One is a recovery if the scaling and frame

movement has got out of hand. A second is the option of saving

the plot for later plotting on a plotter by a graphics plotter

program. Finally, the operator can choose to read in more

chromatograms or exit from the program.

Page 115
Programs to calculate and display calibration of columns

Calibration

The calibration of a set of chromatographic columns used in

size exclusion chromatography consists of a relationship of the

elution volume of the chromatograph to log hydrodynamic volumes

of polymer solutes in a particular solvent. Points on the

calibration are obtained from chromatograms of standard samples

with narrow molecular weight distributions and known molecular

weights in the size exclusion chromatograph. The, elution volume

is determined at the position of the peak in each chromatogram

and the log hydrodynamic volume is computed from the molecular

weight and Mark-Houwink parameters of each sample so that a curve

relating elution volume and log hydrodynamic volume is obtained.

The program AUTOPICK may be used to determine the elution

volumes at the peaks of the chromatograms.

The calibration may be expressed in two ways. For type 1

calibration, the calibrating points are fitted to a polynomial by

program CALFIT. If not used with care, the polynomial approach

gives wrong values, in extreme cases even between the

calibrating points. Also, the polynomial often gives wrong

values outside the range of calibrating points, where the

polynomial function is not constrained. This severity of this

effect may sometimes be reduced by eliminating the squared term

from the calibration. We usually use the polynomial method of

representing the calibration of the columns.

For type 2 calibration, the calibrating points are fitted by

a series of connected straight line segments. This avoids the

spurious maxima and minima of the type 1 (polynomial)

calibration. However, molecular weight distributions computed

using type 2 calibration have spurious peaks due to the

discontinuous slope at the connection of the straight lines.

The programs described in this manual require a file

containing the calibration of the columns used. The first line of

the file is a title for the calibration. The next line contain

the lower and upper values of elution volume for which the

calibration is valid. The next line contains the type of

calibration (1 or 2) . For type 1 calibration, the next line

contains the central point around which the polynomial was

filled, the next line contains the constant, linear, and

quadratic coefficients, and the next line contains the next three

coefficients. A polynomial of up to fifth degree may be used.

For type 2 calibration , these lines are replaced with a line

giving the number of points the connected lines are drawn

through. The calibration points of elution volume, log

hydrodynamic volume are then given one point to a line.

An example of a type 1 calibration:

Page 116
J. PHILLIPS & B DICKENS CALIBRATION OF WATERS COLS April 9 1986

32.422 50.282

1

37.463 3.243

-0.26428 -0.3503 0.9349E-02

0 . 7332E-03 0.16782E-03 -0.1752E-04

An example of a type 2 calibration:

H. Wagner Calibration of Shodex columns Bimodal 8/86

10.2 18.6

2

4

10 10.45

10.55 9.776

17.43 4.394

18.94 0

CALFIT (interactive)

The CALFIT program provides a polynomial of up to fifth

degree to represent the calibration of the columns. The program

reads a file called CALDAT.DAT containing the Mark-Houwink

parameters of the pure narrow molecular weight standard polymers.

This file also contains the elution volumes for the peak in the

chromatograms for each of the narrow molecular weight standards,

and the weighted average molecular weight, (Mn Mw) 0 - 3

, for each

standard. An example is given below. The program writes an

output file CALFIT.DAT which is the calibration file needed by

the other programs.

Example of CALDAT.DAT, the input file to the CALFIT program:

The first line is the title. The next two lines are the symbol

(S) and Mark-Houwink parameters for polystyrene. Two similar

lines for poly (methyl methacrylate) follow. The "E" means that

the input of Mark-Houwink parameters has ended. Similar lines

give the polymer symbol, the molecular weight, and the elution

volume of the peak for each of the narrow molecular weight

standards. This section also ends with "E" (for "end").

B DICKENS Calibration of Shodex cols 804 & 802 NOV 1986

S

0.861E-4 0.74

M

1.04E-4 .697

E

S

510 34.405

S

519 34.402

Page 117
s1811 31.249

S

5330 28.791

M

7670 28.176

S

600 34.0134

S

6200 28.504

S

726 33.637

S

1.574E3 31.594

S

2E3 30.665

S

8.3E5 20.534

S

1.27E6 20 . 3697

S

' 6200 28.494

S

7607 28.215

S

9995 27.474

S

44300 24.419

S

52409 24.365

M

63900 24.137

S

73300 23.476

S

97200 23.126

S

109980 22.819

M

120000 22.900

M

284160 21.695

M

480300 21.060

E
Example of the output file CALFIT.DAT:

One of the two last quantities on the second line must be chosen

by the operator, as explained in the text

B DICKENS Calibration of Shodex cols 804

20.370 34.405

1

27.022 3.244

-0 . 121440 20E+00

0 . 3539694 5E-02

O.OOOOOOOOE+OO

0.00000000E+00

-0 . 40413329E+00 -0

0. 4864007 4E-03 -0

0.00000000E+00 0

0.00000000E+00

& 802 NOV 1986

. 85624680E-02

. 86872809E-04

. 0000D000E+00

Page 119
PLOTCAL (interactive)

The program PLOTCAL allows the operator to examine the form

of the calibration function on the screen of the computer, and to

see how the molecular weights of polystyrene and poly (methyl

methacrylate) vary across the calibration range . Parameters for

a third calibration standard may be given, and parameters for a

polymer being studied provide useful information on how well the

various molecular weight ranges are being resolved by the

chromatographic columns. The behavior of the calibration

function beyond the calibrated range may be seen by giving

appropriate limits to the PLOTCAL program. ,

PLOTCAL screen 1 - Reading in the calibration file

The PLOTCAL program allows the user to plot the calibration

function of the GPC. The program reads in a calibration function

from a file which you specify. You can follow the change in MW

of standards used to generate the calibration data. You can add

your own standard or specimen and follow changes in its molecular

weight with elution volume by giving its Mark-Houwink parameters

and moving the arrow on the graph.

Do you want to use the calibration in file CALIBR.DAT ? (Press Y or N)

Give the name of the calibration file clonecal.3

Title of calibration file is : B DICKENS Calibration of Shodex cols 804 & 802 NOV 1986

Limits are 20.37 to 35.355

Give limits to calculate calibration over 18,38

This is the main menu of the program which plots the

calibration of the columns. The name of the calibration file is

provided to the program, and the limits over which to display the

calculated calibration curve are given.

Page 120
PLOTCAL screen 2 - Specifying Mark-Houwink parameters for polymer

Elution volume 19.80 is outside the calibration range

Elution volume 20.00 is outside the calibration range

Elution volume 20.20 is outside the calibration range

Elution volume 35.40 is outside the calibration range

Elution volume 35.60 is outside the calibration range

Elution volume 35.80 is outside the calibration range

Elution volume 36.00 is outside the calibration range

Elution volume 36.20 is outside the calibration range

Elution volume 36.40 is outside the calibration range

Elution volume 36.60 is outside the calibration range

Elution volume 36.80 is outside the calibration range

Elution volume 37.00 is outside the calibration range

Elution volume 37.20 is outside the calibration range

Elution volume 37.40 is outside the calibration range

Elution volume 37.60 is outside the calibration range

Elution volume 37.80 is outside the calibration range

Elution volume 38.00 is outside the calibration range

Do you want to follow changes in MW in your own polymer? y or n?

Give the Mark-Houwink k and a for the polymer ? .0000861, .74

Your Mark-Houwink parameters are k= 0.0000861, a=0.7400

Is this ok? y or n ?

The program warns which parts of the specified range are

beyond the limits covered by the calibration function read in

from the calibration file. The operator may give Mark-Houwink

parameters for a material of interest and see which part of the

molecular weight range for that material is covered by the

calibration

„

Pace 121
PLOTCAL screen 3 - menu for plotting

Press a function key

- FI displays the full calibration.

- F2 displays the left half of the calibration*

- F3 displays the right half of the calibration*

- F4 displays the left third of the calibration. - - F5 displays the middle third of the calibration.

- F6 displays the right third of the calibration.

- You may move an arrow along the calibration line on the plot.

- The corresponding molecular weights of PS and PMMA standards

are given in the top right hand corner of the plot.

- The coordinates of the arrow in elution volume and log

hydrodynamic volume are also displayed.

- If you gave the Mark-Houwink parameters for your

own polymer, its molecular weight is also displayed

in the upper right hand corner

The plot is generated from this menu. Usually, FI is

pressed

.

Page 122
t <Esc>

DICKENS

5.0

to end. FILE = clonecal Calibration of Shodex cols

nr__

MW380

T

PS 2222009

PhMa MW= 2878901 elution voluwe - 20.00

hMdrodynawic vol~ 6.98

Sour MW= 2222009 “1

20.0 25.0 30.0 35.0 40.0

Log hydrodynawic vol vs elution vol

PLOTCAL plot - Typical calibration plot

The PLOTCAL program shows the relationship between log

hydrodynamic, volume and elution volume as specified in the

calibration file for the chromatographic columns. The molecular

weights of polystyrene and polymethyl methacrylate eluting at the

position of the arrow are shown in the screen plot, as are the

corresponding elution volume and log hydrodynamic volume. If the

user gave Mark-Houwink parameters in an earlier screen menu, the

molecular weight of that polymer is also shown.

Page 123
CONTENTS OF DISKS

DISK 1

Volume in drive A is NBS SEC V 1

ANALYZE EXE 119726

AUTOGP EXE 72890

AUTOPICK EXE 52864

AUTOPLOT EXE 61262

BACKGRND EXE 53530

5 File(s)

12-27-87 7 s 08p

12-27-87 7 : 08p

12-27-87 7:08p

12-27-87 7 » 14p

12-27-87 7 : 09p

0 bytes free

DISK 2

Volume in drive A is NBS SEC V 2

CALFIT EXE 125244

CHKRUNBR EXE 45212

COMPARE EXE 101312

COMPARE9 EXE 80752

4 File(s)

12-27-87 10 : 34p

12-27-87 7 : 09p

12-27-87 7 : lOp

12-27-87 7 : lOp

8192 bytes free

DISK 3

Volume in drive A is NBS SEC V 3

PLOTCAL EXE 69824 12-27-87 7: lip

PLOTSEC EXE 60974 12-27-87 7 : 15p

PRT EXE 39828 12-27-87 7: lip

SECMENU EXE 48990 12-27-87 7 : 42p

TITLES EXE 47004 12-27-87 7: lip

5 File(s) 94208 bytes free

DISK 4

Volume in drive A is NBS SEC V 4

TITLESEC EXE 110654 12-27-87 7 : 37p

DATCOLL EXE • 68414 12-27-87 7 : lOp Recompile these two

WATCHGPC EXE 59064 12-27-87 7 : 12p programs using the

DATCOLL CMP 18169 5-07-87 3 : 58p commands for your A/D

WATCHGPC CMP 13051 5-20-87 12 : 23p data collection card

.

ANALYZE HP1 629 5-04-87 7 : 22p

ANALYZE HP2 535 5-04-87 7 : 22p

ANALYZE HP3 522 5-04-87 7 : 22p

ANALYZE HP4 323 5-04-87 7 : 23p

Page 124
ANALYZE HP5 511 5-04-87 7 : 23p

ANALYZE HP6 304 5-04-87 7 : 23p

SETUP BAS 23117 3-09-87 12 : 30a BASICA is needed to run

SETUP CLA 722 7-08-86 9:47a the SETUP progam

.

SETUP DAT 2780 1-06-87 4 : 21p

SETUP HLP 1390 8-24-86 10 : 55p

SETUP PRO 695 8-25-86 10:30a

STANDARD CHR 338 2-20-87 8:39a

AUTOPICK LMT H/ 1-31-87 3 : 29p

CALDAT DAT 756 11-10-86 3 : 24p

CALIBR DAT 335 2-02-87 12 : lOp

CLONECAL 1 332 1-24-87 12 : 55p

CLONECAL 3 331 11-17-86 4 : 47p

DO BAT 667 2-23-87 7 : 44p

PVME HYD 4242 1-22-87 9 : 40p

RUN NBR 9 2-24-87 7:25a

SEC207 HYD 3518 8-08-86 9:33a

SEC924 HYD 3364 1-24-87 12: 36p

SEC924 RAW 12905 1-14-86 3 : 02p

SEC925 RAW 12905 1-14-86 3 : 03p

CALFIT DAT 226 12-27-87 10 : 35p

30 File(s) 5120 bytes free

DISK 5

Volume in drive A is NBS SEC V 5

SEC1203 RAW 14743 6-01-87 3 : 46p

SEC1216 RAW 14744 6-02-87 8 :14a

SEC1217 RAW 14743 6-02-87 7 : 44p

SEC1387 RAW 12357 10-28-87 12 : 05p

4 File ( s

)

303104 bytes free

Page 125
NBS-114A (REV. 2-80

U.S. DEPT. OF COMM.

BIBLIOGRAPHIC DATA

SHEET (See in struction s)

4. TITLE AND SUBTITLE

1. PUBLICATION OR 2. Performing Organ, Report No. 3. Publication Date

REPORT NO.

System of Hardware and Software Developed for Size Exclusion Chromatography

^

A

^!clLe^n^ and F. L. MeCrackin

6. PERFORMING ORGANIZATION (If joint or other than NBS, see instructions) 7. Con tract/Grant No.

national bureau of standards

DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

8

.

Type of Report & Period Covered

9.

SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS ( Street, City. State. ZIP)

10.

SUPPLEMENTARY NOTES

; Document describes a computer program; SF-185, FIPS Software Summary, is attached.

11.

ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bi bl iography or literature survey, mention it here)

Abstract

A series of computer programs to carry out data collection and

processing for size exclusion chromatography has been written in BASIC

for an IBM XT type computer. This manual documents version 1.0, which

uses a single detector. The detector is assumed to measure the

concentration of the eluting species. Some provision is provided for

quality control by comparing a measured chromatograms with the

standard chromatogram for its class immediately after the chromatogram

has been measured. There is no limit on the number of classes

allowed. The measured chromatogram is automatically processed in the

same way as the standard chromatogram was processed.

12. KEY WORDS (Six to twelve entries; alphabetical order; capitalize only proper names; and separate key words by semicolons)

Computer programs; gel permeation chromatography; molecular size; molecular

weight determination; quality control; size exclusion chromatography

13. AVAILABILITY

Unlimited

1

i For Official Distribution. Do Not Release to NTIS

H Order From Superintendent of Documents, U.S. Government Printing Office, Washington, D.C.

20402.

1 Order From National Technical Information Service (NTIS), Springfield, VA. 22161

14. NO. OF

PRINTED PAGES

15. Price

USCOMM-DC 6043-P80