Microcomputer Programs for Size Exclusion Chromatography - ACS

Chapter 3 Microcomputer Programs for Size Chromatography

Exclusion

Brian Dickens and Frank L. McCrackin

Downloaded by NORTH CAROLINA STATE UNIV on January 1, 2018 | http://pubs.acs.org Publication Date: August 29, 1989 | doi: 10.1021/bk-1989-0404.ch003

Polymers Division, National Institute of Standards and Technology, Gaithersburg, MD 20899

An implementation of automatic processing in a system of computer programs to perform size exclusion chromatography on IBM-compatible microcomputers is described. The programs allow identifying vials in an auto-injector, data collection from two detectors, and manual and automatic processing to compare chromatograms and calculate molecular weight averages. Chromatograms may be compared in terms of elution volume, log hydrodynamic volume and/or log molecular weight. The programs also perform chromatographic column calibration and various house-keeping activities. The user may assign each specimen to a class and thus determine the type of chromatogram matching and automatic processing for that specimen. A system of computer programs to perform size exclusion chromatography has been written for the IBM-type family of microcomputers. The programs are written i n BASIC and are used i n compiled form. Using the programs, chromatograms are measured and stored on a disk for l a t e r processing. The data c o l l e c t i o n module measures outputs from two detectors. Subsequent processing includes subtraction of a baseline and transformation of the relevant part of the chromatogram from e l u t i o n volume to log hydrodynamic volume and log molecular weight. Many of the data processing functions may be c a r r i e d out i n t e r a c t i v e l y by an operator or i n "automatic" mode, where operator input i s not required once data c o l l e c t i o n has been started. Automatic data processing has been implemented by assigning each specimen to a class. The chromatogram for that specimen can be processed automatically i n the same way that the chromatogram of a standard member of the class was previously processed i n t e r a c t i v e l y by the operator. This chapter not subject to U.S. copyright Published 1989 American Chemical Society

Provder; Computer Applications in Applied Polymer Science II ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

24

COMPUTER APPLICATIONS IN APPLIED POLYMER SCIENCE II


Other modules i n the system of programs are concerned with determining the c a l i b r a t i o n of the chromatographic columns, comparing up to 9 chromatograms v i s u a l l y or two chromatograms q u a n t i t a t i v e l y , and reviewing chromatograms already on the disk. The programs are described at length i n the NBS p u b l i c a t i o n NBS-IR 87-3669, System of Hardware and Software Developed for Size Exclusion Chromatography by B. Dickens and F.L. McCrackin, National Bureau of Standards, 1987, and available from NTIS under order number 88153697. Most of that report i s concerned with i n t e r a c t i v e operation of the programs. Apparatus The programs are used on an IBM-compatible turbo XT-type microcomputer with color graphic adapter graphics card, color monitor, hard disk, one floppy drive, and a l i n e p r i n t e r compatible with the IBM graphics p r i n t e r . An analog to d i g i t a l conversion card i s necessary to be able to read voltages into the computer. The only connections from the computer to the chromatograph are to the outputs of the detectors and to a signal which denotes the s t a r t of each run. The chromatograph i n our implementation i s c o n t r o l l e d by i t s own microprocessor. The data c o l l e c t i o n sequence can accommodate any number of injections from a maximum of 16 sample v i a l s i n an auto-injector. Automatic Processing The unusual feature of the programs i s the automatic processing based on sample c l a s s . The operator provides the relevant parameters to the system of programs using the program SETUP, which contains several f i l l - i n - t h e - f o r m screens. These parameters include the "class" of the specimen and the required automatic processing, as described below, and other pertinent information such as Mark-Houwink parameters, v i a l number, i n j e c t i o n number, and documentation of the experimental conditions under which the chromatogram i s to be measured. Up to 20 classes are provided by the SETUP program so that various types of specimen can be processed i n the same run. The program-supplied options are read from a text f i l e on the disk and hence are e a s i l y changed. The operator may type i n the name of any class not provided as a choice by SETUP. The type and extent of automatic processing to be c a r r i e d out immediately a f t e r a chromatogram has been measured i s c o n t r o l l e d by a "processing" variable selected from a table displayed by the SETUP program. In our implementation, the choices are

1) 2) 3) 4) 5) 6)

ignore t h i s v i a l c o l l e c t data, no p l o t c o l l e c t and p l o t data c o l l e c t and p l o t data, f i n d e l u t i o n time of main peak do 4) and then subtract baseline do 5) and then compare with class standard.



3.

DICKENS & McCRACKIN

Microcomputer Programs for

SEC

25

Thus, chromatograms can be processed to various amounts, and the comparison with the class standard may be c a r r i e d out i n terms of e l u t i o n volume, log hydrodynamic volume or log molecular weight. In option 3, the peak positions of narrow molecular weight standards are found routinely without operator intervention. Many of the options i n t h i s system of programs are performed both by small programs which do not require user input and by larger programs which are c o n t r o l l e d from the keyboard. SETUP passes on the choice of processing by naming the appropriate small programs i n a BATCH f i l e which i s executed by the main menu option "Begin data c o l l e c t i o n " . The parameters which control the programs are obtained from the header of the f i l e containing the chromatogram. Data C o l l e c t i o n The f i l e written by the SETUP program indicates which v i a l s to use i n the auto-injector and the number of injections to be expected from each v i a l . This f i l e i s read by the data c o l l e c t i o n program before data c o l l e c t i o n begins and i s updated after data c o l l e c t i o n terminates. The process of c o l l e c t i n g data begins when the computer receives an e l e c t r i c a l pulse from the chromatograph. The data c o l l e c t i o n program f i r s t c o l l e c t s data at a f i x e d rate of one reading per minute to monitor the baseline signal from the chromatograph before the specimen elutes. Then data are c o l l e c t e d every second to record the e l u t i o n of the specimen. After the specimen has eluted, the baseline i s again monitored at one point per minute u n t i l the run i s complete. The times at which to change the rates of measurement are supplied by the operator to the SETUP program and are read by the data c o l l e c t i o n program from the SETUP file. The frequencies of measurement are fixed. I f measurement of a chromatogram terminates normally, the data c o l l e c t i o n program updates the f i l e i n i t i a l l y written by the SETUP program to r e f l e c t the fact that data have been obtained from that specimen. I f data c o l l e c t i o n terminated abnormally, the f i l e from SETUP i s not updated. In this case, the next time the data c o l l e c t i o n program i s run, i t w i l l assume that the data are a repeat of the run which ended abnormally. A l t e r n a t i v e l y , the SETUP f i l e may be changed using the SETUP program and a new series of injections started. The data c o l l e c t i o n program always writes a f i l e containing any data to the disk whether or not the termination was abnormal. In the case of abnormal termination, i t also provides a signal which stops a l l subsequent processing i n that series of i n j e c t i o n s . A f t e r any automatic processing of a chromatogram has finished, the data c o l l e c t i o n program measures the chromatogram of the next specimen. Data f i l e s are automatically numbered i n sequence by updating a run number saved i n a disk f i l e . The data c o l l e c t i o n program i s configured for a p a r t i c u l a r A/D board. The parts of the program s p e c i f i c to t r i g g e r i n g of data c o l l e c t i o n and to A/D conversion are contained i n separate subroutines to allow easier adaption to a d i f f e r e n t A/D board. These programs are used with a chromatograph which i s c o n t r o l l e d by a microprocessor programmed by the manufacturer of the chromatograph. Therefore, the programs do not control the chromatograph. However, a l l the information necessary to set the options on the chromatograph i s present as documentation of the run


26


conditions i n the f i l e written by the SETUP program (the pertinent d e t a i l s are written i n each chromatogram f i l e by the data c o l l e c t i o n program to provide a permanent record). Control of the chromatograph would not be d i f f i c u l t to implement i n those cases where the chromatograph gracefully accepts external control.


Data Analysis Analysis of the chromatogram i s c a r r i e d out i n t e r a c t i v e l y with the operator d i r e c t i n g the flow of analysis from the computer keyboard, or i n the automatic mode immediately after data c o l l e c t i o n to an extent s p e c i f i e d using SETUP. Automatic analysis may also be applied to a series of chromatograms at any time by putting the run numbers or names of the f i l e s i n the run number f i l e . Before the next set of data c o l l e c t i o n s , the programs check that the number i n the run number f i l e i s the l a t e s t run number f o r a l l the f i l e s on the disk. The f i r s t step i s to determine the l e v e l of the baseline before and a f t e r the part of the chromatogram which characterizes the specimen, apply a s t r a i g h t - l i n e correction so that the o v e r a l l baseline i s e f f e c t i v e l y zero, and cut out from the entire chromatogram that part which pertains to the specimen. The "cut" chromatogram may be written to a f i l e which i s named automatically by using CUT as an extension of the f i l e name. Up to t h i s point, a l l chromatographic information i s i n terms of e l u t i o n volume. I f a c a l i b r a t i o n f i l e i s available to the program, the chromatogram may be converted to log hydrodynamic volume. The chromatogram i n terms of log hydrodynamic volume may be written to a f i l e (with extension HYD) f o r l a t e r use. I f Mark-Houwink c o e f f i c i e n t s were supplied at setup time, the chromatogram may be converted into the d i f f e r e n t i a l molecular weight d i s t r i b u t i o n of the specimen. Various averages characterizing t h i s molecular weight d i s t r i b u t i o n are then calculated. The molecular weight d i s t r i b u t i o n may be written to a f i l e . Comparison of Chromatograms When the chromatogram i s to be compared "automatically" with a standard chromatogram f o r that class of specimen, the standard chromatogram i s found from the "class" s p e c i f i e d at setup time. The f i l e STANDARD.CHR contains a l i s t of classes, corresponding standard chromatograms, and the c a l i b r a t i o n f i l e used to process the standard chromatogram. The newly measured chromatogram i s processed automatically before the comparison i s made. The places i n the chromatogram to use f o r baseline estimation and f o r cutting out the relevant part are available i n the header of the standard chromatogram f i l e f o r that class. The a b i l i t y to specify the processing and class information at setup time allows the operator to process chromatograms from various types of specimens i n one series of i n j e c t i o n s . The comparison may be c a r r i e d out i n terms of e l u t i o n volume, log hydrodynamic volume, or log molecular weight. Unrecognized s h i f t s i n the c a l i b r a t i o n of the columns w i l l a f f e c t the raw data, which are i n terms of e l u t i o n time of the components of the specimen from the chromatographic columns. Use of log hydrodynamic volume allows the operator to correct f o r such


3.

DICKENS & McCRACKIN

Microcomputer Programs for SEC

27


changes i n column c a l i b r a t i o n and to compare specimens taken at considerably d i f f e r e n t times, and also to compare specimens taken with d i f f e r e n t sets of columns. Log hydrodynamic volume i s therefore a more constant quantity to use to specify materials which w i l l be analyzed at d i f f e r e n t times or d i f f e r e n t locations. To characterize the difference between two chromatograms, an o v e r a l l mismatch index and a l i s t of the areas, heights, and positions of the main peaks i n the difference chromatogram are provided. The difference chromatogram i s the r e s u l t of subtracting the standard chromatogram f o r that class of specimen from the chromatogram of the specimen. C a l i b r a t i o n of the Chromatographic Columns One of the options i n the SETUP program i s to automatically f i n d the e l u t i o n volume of the highest peak i n a chromatogram. When enough such data f o r narrow molecular weight standards have been measured to cover the c a l i b r a t i o n range needed f o r the specimens to be analyzed, the operator may use the least squares refinement program i n the system to compute a polynomial r e l a t i n g log hydrodynamic volume to e l u t i o n volume. Other Programs i n the System Although the convenient quantitation of size exclusion data was the primary purpose f o r writing this system of programs, there are cases where p i c t o r i a l representations are invaluable. A program has been included which displays the shape of the c a l i b r a t i o n function r e l a t i n g e l u t i o n time to log hydrodynamic volume. The program also shows the molecular weights of polystyrene and poly methyl methacrylate which correspond to the point on the c a l i b r a t i o n curve designated by an arrow. The arrow i s moved along the c a l i b r a t i o n curve using the cursor keys. This allows the operator to see the behavior of the c a l i b r a t i o n function over the c a l i b r a t i o n range and to select standards could be used to improve the c a l i b r a t i o n function. The operator may include Mark-Houwink parameters f o r a t h i r d polymer and thus follow e l u t i o n of a p a r t i c u l a r material. Another program allows the operator to compare graphically and hence v i s u a l l y up to 9 chromatograms, to correct a l l these chromatograms simultaneously f o r non-zero baseline and to chop o f f the ends. When plotted on the screen, the chromatograms may be superimposed or spaced v e r t i c a l l y , subject to the l i m i t a t i o n that they must be equally spaced. F i l e s can be written to disk to transfer the processed chromatograms to p l o t t i n g routines or to other programs i n the system. A t h i r d program allows p l o t t i n g of selected or a l l data f i l e s on the disk. A fourth program i s used routinely to watch the responses of the detectors before data c o l l e c t i o n i s started. I t plots the l a t e s t responses on the screen f o r a time range selected by the operator.


28


Conclusion We have designed and written a system of computer programs f o r size exclusion chromatography which run on a readily available microcomputer. The programs have been i n use f o r about 2 years. This a r t i c l e describes the concepts we have used to implement automatic data processing i n s u f f i c i e n t d e t a i l to allow the ready i n c l u s i o n of these or similar concepts i n other systems of programs. February14,1989


RECEIVED


Microcomputer Programs for Size Exclusion Chromatography - ACS

Recommend Documents