Automated Data Analysis System for a Gel Permeation

Size Exclusion Chromatography Downloaded from pubs.acs.org by CALIFORNIA INST OF TECHNOLOGY on 12/24/17. For personal use only.

4 Automated Data Analysis System for a Gel Permeation Chromatograph with Multiple Detectors M.

E.

KOEHLER,

A.

F.KAH,T.

F. NIEMANN, C.KUO,and T. PROVDER

Glidden Coatings and Resins, Division ofSCMCorporation,

Strongsville,

OH 44136

A Waters Model 150C ALC/GPC was interfaced to a minicomputer system by means of a microcomputer for automated data collection and analysis. Programs were developed for conventional molecular weight distribution analysis of the data and for liquid chromatographic quantitative composition analysis of oligomeric materials. Capability has been provided to utilize non-standard detectors such as a continuous viscometer detector and spectroscopic detectors for compositional analysis. The automation of the instrument has resulted in greater manpower efficiency and improved record keeping.

E f f i c i e n t use o f a modern h i g h performance g e l permeation chromatography (HPGPC) instrument r e q u i r e s computer aided a n a l y s i s i n order t o take f u l l advantage o f both the q u a l i t y and the q u a n t i t y o f i n f o r m a t i o n the instrument i s capable o f p r o v i d i n g . Commercial data a n a l y s i s packages f o r t h i s purpose are, f o r the most p a r t , s i m p l i s t i c and i n f l e x i b l e . This i s p a r t i c u l a r l y t r u e when m u l t i p l e or non-standard d e t e c t o r s are r e q u i r e d . T h i s work d e s c r i b e s an automated data a n a l y s i s system used i n c o n j u n c t i o n w i t h a Waters A s s o c i a t e s Model 150C ALC/GPC to read o p e r a t i o n a l parameters from the instrument, t o c o l l e c t data from m u l t i p l e d e t e c t o r s , and t r a n s m i t the data t o a minicomputer system f o r s t o r a g e , a n a l y s i s , r e p o r t i n g and plotting. Data A c q u i s i t i o n System Automated data a n a l y s i s f o r the chromatograph i s achieved by i n t e r f a c i n g the instrument and d e t e c t o r s t o a microcomputer f o r data a c q u i s i t i o n . The microcomputer i s connected t o the I n t e l i n k 0097-6156/ 84/ 0245-O057S06.00/0 © 1984 American Chemical Society


58

SIZE E X C L U S I O N

CHROMATOGRAPHY

i n t e r f a c e of the instrument so that the o p e r a t i o n a l parameters for each sample a n a l y s i s can be transferee! to the minicomputer. The microcomputer i s r e s p o n s i b l e f o r a l l r e a l - t i m e a c t i v i t i e s i n v o l v e d i n data c o l l e c t i o n . At the completion o f the experiment, data are t r a n s f e r r e d v i a a s e r i a l l i n e t o the minicomputer f o r storage and a n a l y s i s . Report generation and p l o t t i n g may be done at any time a f t e r the completion of the experiment. The minicomputer system uses a D i g i t a l PDP 11/44 processor running the RSX 11 —M o p e r a t i n g system. Programming f o r communications and data a n a l y s i s on the minicomputer i s done i n FORTRAN-77. The microcomputer uses an 8080A processor and i s composed p r i m a r i l y of standard Pro-Log c i r c u i t cards. Programming f o r the microcomputer i s done i n assembly language and cross assembled on the minicomputer. D e t a i l s o f the mini-microcomputer system and i t s o r g a n i z a t i o n have been reported elsewhere (1,2,3). Automated Instrument A n a l y s i s Process There are four stages i n an automated instrument a n a l y s i s . In the f i r s t stage, the instrument operator i n i t i a t e s the experiment by means o f d i a l o g programs on the minicomputer. Examples of the d i a l o g s f o r the HPGPC o p e r a t i o n are shown i n F i g u r e s 1-4. D i a l o g 15, shown i n F i g u r e 1, i s used f o r sample d e f i n i t i o n . This i n c l u d e s i d e n t i f i c a t i o n o f the l o c a t i o n of the sample i n the automatic i n j e c t o r , the column set i n use, the data c o l l e c t i o n r a t e , the d e t e c t o r s t o be used, the o p e r a t o r s i n i t i a l s and the sample i d e n t i f i c a t i o n . This d e f i n i t i o n f i l e may be modified and d i s p l a y e d on the t e r m i n a l , or p r i n t e d . The f i l e i s updated during operation t o show the current s t a t u s of the samples. Before i n i t i a t i n g an a n a l y s i s , the instrument must be programmed for automatic operation and the samples placed i n the appropriate p o s i t i o n s o f the i n j e c t o r . D i a l o g 16, shown i n Figure 2, s t a r t s operation o f the microcomputer. I n t e l i n k communication w i t h the instrument i s e s t a b l i s h e d and the parameters f o r the f i r s t sample are taken from the sample d e f i n i t i o n f i l e on the minicomputer and are t r a n s m i t t e d t o the microcomputer. The microcomputer turns on a ready s t a t u s l i g h t at the instrument to s i g n a l t o the operator t o begin automatic o p e r a t i o n of the instrument. The second stage i s data a c q u i s i t i o n . This stage i s entered when the operator s t a r t s the instrument. The instrument makes the f i r s t i n j e c t i o n and s i g n a l s the microcomputer v i a I n t e l i n k . A f t e r a delay p r o p o r t i o n a l t o the void volume of the column s e t , data are c o l l e c t e d on a time b a s i s (constant flow r a t e assumed) at the predetermined r a t e from each o f the d e t e c t o r s s e l e c t e d , up to a maximum o f three simultaneous d e t e c t o r s . When the sample run i s complete, the instrument again s i g n a l s the microcomputer which places the instrument i n a hold s t a t e w h i l e i t reads the o p e r a t i o n a l parameters from the instrument f o r t h a t sample and

4.

MA

KOEHLER ET AL.

Automated

Data Analysis for

GPC

59

15

Instrument

No.

34

- HPGPC Sample

Definition


Option*: C A D Ε Τ Ρ X

-

C r e a t e new file Add sample d a t a t o o l d f i l e D e l e t e sample f r o * f i l e E d i t sample d a t a Type f i l e on t a r t i n a i P r i n t f i l e on p r i n t e r Exit

Option...T Pos 1 OK >DIA

Job Err No 6797 0

InJ No 1

Tot InJ 1

Col Set 3

Flow • 1/m 1.0

DATA

RI

UV

IR VI

Opr

0

AFK

Sample

ID

Pt/» 1 0

30

0

5872t

PHOSPHATE

15C

Instrument

No.

34

- HP6PC Sample

Definition

I n i t i a l s . . . AFK Column S e t . . * 3 Default

Values:

D e t e c t o r s ( R i t UVt IRt VI)... Flow Rate ( m l / m i n ) . . . 1 Data C o l l e c t i o n R a t e . .. 2

30

Position 1 Job No. 6803 Sample ID... TEST SAHPLE · 1 Customer... KU0 Position 2 Job No. 6804 Sample ID... TEST SAMPLE * 2 Customer... KU0 OK

Figure 1. Sample d e f i n i t i o n d i a l o g f o r automated instrument o p e r a t i o n .

DIA

16

INSTRUMENT

NO*

S T A R T I N G HPGPC

34

-

HPGPC

SAMPLE

ANALYSIS

OK

>

Figure 2. D i a l o g f o r i n i t i a t i o n of automated instrument operation.

HON

SIZE EXCLUSION

CHROMATOGRAPHY

DIA 17 I n s t r u e e n t No* 34 - HPOPC Column S e t D e f i n i t i o n


Option»: A D Ε Ρ X

-

Add net* c o l u a n s e t D e l e t e column s e t f r o * f 1 1 · E d i t coluan s e t data P r i n t f i l e on p r i n t e r Exit

Option*••A DEFINE NEW HPOPC COLUHN SET Column S e t No*

4

V o i d Voluae ( a l > . . * 3 T o t a l Voluae ... 8 D e s c r i p t i o n * *· TEST COLUHN SET OK >DIA 17E Instrument No* 34 - HPOPC Coluan S a t D e f i n i t i o n E d i t OPC Coluan S e t Coluan s e t * * * 4 Moid v o l u a e ( a l ) < 3*0>*** 3*2 T o t a l v o l u a e C a l ) < 8*0>*·· D e s c r i p t i o n OK >DIA 17D I n s t r u a e n t No* 34 - HPOPC Coluan S e t D e f i n i t i o n D e l e t e OPC Coluan S e t F r o a

File

Coluan s e t * * * 4 Coluan S e t 4 TEST COLUHN SET Delete t h i s coluan set? Y OK >

Figure 3 . D i a l o g f o r column s e t d e f i n i t i o n .

Automated Data Analysis for GPC

KOEHLER ET AL.

>DIA 18 Instrument No* 34 - HPGPC C a l i b r a t i o n

Curve

Definition


Options S A D Ε Ρ X

-

Add new c a l i b r a t i o n c u r v e D e l e t e c a l i b r a t i o n c u r v e from E d i t c a l i b r a t i o n curve data P r i n t f i l e on p r i n t e r Exit

file

O p t i o n * «.A DEFINE NEW

HPGPC CALIBRATION CURVE

Calibration

Curve No*

12

Colu»n Set»·· 1 D e t e c t o r (RI» UV, IR, MI)*,* IR Flow r a t e DIA 18D Instrument No. 34 - HPGPC C a l i b r a t i o n D e l e t e HPGPC C a l i b r a t i o n

Curve

Definition

Curve

Curve No*·· 12 Curve

12

Column S e t

1

Detector 3

22-0CT-82

Delete t h i s c a l i b r a t i o n curve? Y OK >

Figure M.

D i a l o g f o r d e f i n i t i o n o f c a l i b r a t i o n curves*


62

SIZE E X C L U S I O N C H R O M A T O G R A P H Y

combines t h i s i n f o r m a t i o n w i t h the raw data and sample d e f i n i t i o n information i n memory. The t h i r d stage i s data t r a n s m i s s i o n d u r i n g which the microcomputer t r a n s m i t s the e n t i r e data set f o r the sample t o the minicomputer. The data i s stored on d i s k u n t i l the operator i n i t i a t e s the f o u r t h stage, data r e d u c t i o n . I f m u l t i p l e samples and i n j e c t i o n s have been programmed, the minicomputer sends to the microcomputer the i n f o r m a t i o n i n the sample d e f i n i t i o n f i l e for the next sample, and o p e r a t i o n continues without f u r t h e r operator i n t e r v e n t i o n . C a l i b r a t i o n i s performed by u s i n g narrow molecular weight d i s t r i b u t i o n p o l y s t y r e n e standards. A polynomial up t o s i x t h order i s f i t t o the l o g ^ ( m o l e c u l a r weight) vs r e t e n t i o n volume data f o r the standards using conventional polynomial r e g r e s s i o n methods, and the c o e f f i c i e n t s of the best f i t polynomial ( u s u a l l y f o u r t h order or l e s s ) are used to d e f i n e the c a l i b r a t i o n curve. Dialogs 17 and 18, shown i n F i g u r e s 3 and 4, are used by the operator to d e f i n e column s e t s and c a l i b r a t i o n curves. This i n f o r m a t i o n i s stored i n f i l e s on the minicomputer u n t i l modified or d e l e t e d by the operator and i s used by the data a n a l y s i s programs. An example of operator i n t e r a c t i o n w i t h the primary a n a l y s i s program, GPC, i s shown i n F i g u r e 5. The job number assigned by the computer during sample d e f i n i t i o n i s entered along with the detector s e l e c t e d f o r a n a l y s i s . The operator then s e l e c t s the b a s e l i n e and the l i m i t s f o r data a n a l y s i s by e n t e r i n g the times of the d e s i r e d p o i n t s . The p l o t s d e s i r e d and the d i s p o s i t i o n of the r e p o r t f i l e are chosen. The most recent c a l i b r a t i o n curve on f i l e f o r the column set i s used by d e f a u l t but others may be s e l e c t e d at the operator's o p t i o n . I n t e g r a t i o n of the data f o r the c a l c u l a t i o n o f molecular weight d i s t r i b u t i o n averages i s performed i n time-volume space using Simpson's Rule (assuming constant flow r a t e ) . Molecular weight averages are c a l c u l a t e d u s i n g the equation

L

/

U

/

M

M

M

L

j

1

M " (V)F(V)dV

M

J

i-2 ^(V)F(V)dV

H

where j = 1, 2, 3, and 4 correspond to the N, W, Ζ and Z+1 averages, r e s p e c t i v e l y ; M(V) represents the molecular weight c a l i b r a t i o n curve as a f u n c t i o n of r e t e n t i o n volume and F(V) i s the normalized chromatogram height as a f u n c t i o n of r e t e n t i o n volume. The weight 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 , f ( l o g M ) , i s c a l c u l a t e d according to the method o f P i c k e t t et w

1 Q

a l . ( 5 ) u s i n g the equation

K O E H L E R ET

4.

AL.

f (log M) w

1 0

Automated Data Analysis for

F(V)

GPC

63

(2)

2.303 (dlog M(V)/dV) 10

where dlog M(V)/dV i s the slope of the molecular weight c a l i b r a t i o n curve. An example of the weight 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 p l o t i s shown i n Figure 8 along w i t h the weight cumulative molecular weight d i s t r i b u t i o n . The p o s i t i o n of the molecular weight averages, Μ , M M M ^ and R p on the l o g ^ M a x i s a l s o are i n d i c a t e d i n Figure 8. Examples o f t h e p l o t s ana r e p o r t generated by the program are shown i n F i g u r e s 6 - 9 . The r e p o r t shown i n Figure 9 i s composed o f four s e c t i o n s : molecular weight d i s t r i b u t i o n s t a t i s t i c s , sample i n f o r m a t i o n , raw chromatogram s t a t i s t i c s and column set i n f o r m a t i o n . The v a r i a n c e , skewness and k u r t o s i s s t a t i s t i c s which i n v o l v e moments about the mean are c a l c u l a t e d from equations r e l a t i n g moments about the mean to moments about the o r i g i n ( 6 ) . Customized p l o t p r e s e n t a t i o n s and c o p l o t t i n g of data from m u l t i p l e samples can be generated when r e q u i r e d . Other a n a l y s i s methods dependent on m u l t i p l e d e t e c t o r s can be implemented using t h i s automated system. Two methods under development are the use of a continuous viscometer d e t e c t o r w i t h a r e f r a c t i v e index d e t e c t o r to y i e l d absolute molecular weight and branching, u t i l i z i n g the u n i v e r s a l c a l i b r a t i o n curve concept ( 4 ) , and the use of a UV or IR d e t e c t o r w i t h the r e f r a c t i v e index d e t e c t o r t o measure compositional d i s t r i b u t i o n as a f u n c t i o n o f molecular weight. Oligomer a n a l y s i s i s performed by a separate program OLIG by a method analogous to conventional l i q u i d chromatograph peak a n a l y s i s . This program u t i l i z e s the D i g i t a l Equipment Corporation s c i e n t i f i c subroutine PEAK. Since the subroutine operates on p r o g r e s s i v e l y broadening peaks, the data i s analyzed i n reverse order, t h a t i s , from long to short r e t e n t i o n times. The operator can s e l e c t a b a s e l i n e , or l e t the program s e l e c t and adjust the b a s e l i n e a u t o m a t i c a l l y . Response f a c t o r s may be c a l c u l a t e d at the operator's d i s c r e t i o n , or c o n c e n t r a t i o n s can be c a l c u l a t e d from known response f a c t o r s on an area b a s i s . The operator i n t e r a c t i o n w i t h OLIG and samples of the r e p o r t and p l o t from t h i s program are shown i n F i g u r e s 10-12.


1Q

t

f

+

+

Conclusions B e n e f i t s have been r e a l i z e d from the automation of the Waters Model 150C ALC/GPC i n s e v e r a l areas. F i r s t , a s i g n i f i c a n t amount of time has been saved by performing automated data c o l l e c t i o n w i t h automatic i n j e c t i o n during n i g h t o p e r a t i o n w h i l e unattended. Secondly, record keeping i s more complete and accurate. This has

SIZE E X C L U S I O N C H R O M A T O G R A P H Y

64


>RUN t G P C JOB NUMBER » RUN NUMBER SAMPLE 9165: 1306-12-B PMMA D e t e c t o r : R i t UVt o r IR CURVE 10F 13-AUG-82 - CHANGE ? PLOTS: RAW* VOLUMEF M O L W T F CHAIN BASELINE IN MINUTES START < 23.93 > STOP < 79*87 > DATA LIMITS IN MINUTES START < 36.00 > STOP < 75.00 > OUTPUT F I L E (PRINTt SAVE) < DELETE > F I L E HPGPC.LST CREATED PLOT F I L E S PRODUCED: RAWDAT RVOL MOLWT F i g u r e 5.

9034 RI Ν RVM 36 75 40 63 Ρ

Operator i n t e r a c t i o n w i t h program GPC. - RAW DfiTft

HP6PC

O JOB 9 0 3 4 . 1 - DETECTOR = REFRRCTOMETER Ο 9 1 6 5 : 1 3 0 6 - 1 Z - B PMMR

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R E T E N T I O N VOLUME (ml) F i g u r e 6. Raw HPGPC data w i t h o p e r a t o r s e l e c t e d

baseline.

96.99


4. KOEHLER ET AL. Automated Data Analysis for GPC 65


66 SIZE E X C L U S I O N C H R O M A T O G R A P H Y


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70 SIZE EXCLUSION CHROMATOGRAPHY

4.

K O E H L E R ET A L .

Automated Data Analysis for G PC

71


s i m p l i f i e d accurate reproduction o f experimental r e s u l t s and has helped d i s c e r n s u b t l e or long term v a r i a b i l i t y i n the o p e r a t i n g c h a r a c t e r i s t i c s o f the instrument. F i n a l l y i t has f a c i l i t a t e d the development o f experimental methodology f o r non-standard detectors.

Literature Cited 1. Niemann, T. F.; Koehler, M. E.; Provder, T. "Microcomputers used as Laboratory Instrument Controllers and Intelligent Interfaces to a Minicomputer Timesharing System" in "Personal Computers in Chemistry"; Lykos, P., Ed.; John Wiley and Sons: New York, 1981; pp. 85-91. 2. Kah, A. F.; Koehler, M. E. ; Niemann, T. F.; Provder, T.; Eley, R. R. "An Automated Ferranti-Shirley Viscometer" in "Computer Applications in Applied Polymer Science"; Provder, T., Ed.; ACS SYMPOSIUM SERIES No. 197, American Chemical Society: Washington, D.C., 1982; pp.223-241. 3. Kah, A. F.; Koehler, M. E.; Grentzer, T. H.; Niemann, T. F.; Provder, T. "An Automated Thermal Analysis System for Reaction Kinetics" in "Computer Applications in Applied Polymer Science"; Provder, T., Ed.; ACS SYMPOSIUM SERIES No. 197, American Chemical Society: Washington, D.C., 1982; pp. 197-311. 4. Malihi, F. B.; Kuo, C.; Koehler, M. E.; Provder, T.; Kah, A. F. "Development and Application of a Continuous GPC Viscosity Detector for the Characterization of Absolute Molecular Weight Distribution of Polymers", this volume. 5. Pickett, H. E.; Cantow, M. J. R.; Johnson, J. F. Appl. Polym. Sci. 1966, 10, 917. 6. Aitken, A. C. "Statistical Mathematics"; Oliver and Boyd: London, 1962; Chap. 2. RECEIVED

October 4, 1983

Automated Data Analysis System for a Gel Permeation

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