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Microcomputer-AssistedAcquisition and Reduction OfTLC Scanning DensitometricDataRobert G. Foss a , Carl W. Sigel a , Robert J.Harvey a & Richard L. Deangelis aa Departments of Medicinal Biochemistry andMicrobiology , Wellcome Research LaboratoriesResearch , Triangle Park, North Carolina, 27709,USAPublished online: 06 Dec 2006.

To cite this article: Robert G. Foss , Carl W. Sigel , Robert J. Harvey & RichardL. Deangelis (1980) Microcomputer-Assisted Acquisition and Reduction Of TLCScanning Densitometric Data, Journal of Liquid Chromatography, 3:12, 1843-1852,DOI: 10.1080/01483918008064774

To link to this article: http://dx.doi.org/10.1080/01483918008064774

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JOURNAL OF LIQUID CHROMATOGRAPHY, 3(12), i843-i852 (1980)

MICROCOMPUTER-ASSISTED ACQUISITION AND REDUCTION OF TLC

SCANNING DENSITOMETRIC DATA

Robert G. Foss. Carl W. Sigel, Robert J. Harvey and Richard L. DeAngelis Departments of Medicinal Biochemistry and Microbiology

Research Triangle Park, North Carolina 27709 USA Wellcome Research Laboratories

ABSTRACT

The rapid microcomputer processing of quantitative TLC scanning data is described. The microcomputer, using BASIC language, is based. on the Intel 8080A microprocessor IC and has 64K bytes of RAM. Data is acquired via a 16-bit integrating ADC. A TLC plate with up to 18 samples may be analyzed in as little as 5 minutes. Graphics capabilities include automatic analysis and depiction of peak heights and peak areas. Display of normal or log-normal plots of data for reference compounds and samples with least-squares linear regression is available. Graphics are directly accessible to permit independent plotting of data.

INTRODUCTION

Current systems for the acquisition of TLC scanning densitometric

data can be interfaced with computing integrators; however, this practice

is not entirely satisfactory since these integrators have restrictions

as to rigidity of integration procedures and inability to manually

overide integrations. Furthermore, proper laboratory practice and

government regulations mandate the retention of large volumes of

unprocessed data in support of analytical work. Such documentation is

easier to use and more readily understood when available in a concise

form. The automation of TLC data analysis addresses these problem

areas. flicrocomputers, user-programable in a simple language such as

BASIC, provide both analytical flexibility and condensed data storage.

1843

Copyrrpht Q 1980 by Marcel Dekkn. Inc.

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1844 FOSS ET AL.

EXPERIMENTAL

Microprocessor technology provides a means to automate TLC analyses,

both conventional and high performance, in a package that is also fully

compatible with GC and HPLC equipment. These demands for both data

manipulation and instrumentation control have been satisfied by a

microcomputer system developed by Wooduard and Reilly' (Fig. 1).

System components consist of an INTEL 80806 microprocessor IC, a 64-K

byte random access memory, and a 16-bit integrating analog to digital

converter for data acquisition. A television is used as a visual

monitor while a flat-bed X-Y recorder serves as a printer-plotter. A

h

FIGURE 1

Microcomputer-based system described in text for data acquisition and reduction.

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MICROCOHPUTEBIZING TLC SCANNING DATA 1845

conventional cassette recorder is ured for storage of data and programs

on magnetic tape. The microcomputer in interfaced with a Schoeffel

SD 3000 spectrodensitometer operating in the ringle-beam reflectance

mode.

converter.

Output to the microcomputer is obtained from the SD 300 density

DISCUSSION

An essential portion of a successful instrument automation scheme

is the development of a versatile computer program that is adaptable to

the needs of the analyst. With this goal in mind a BASIC program has

been developed in this laboratory to increase the data-handling efficiency

of methods involving quantitative thin-layer chromatography. The

program will analyze and display data in any of several formats selected

by the user. Data may be gathered in either absorbance or fluorescence

modes. Plates may be scanned either parallel or perpendicular to the

direction of development (termed normal-scan and cross-scan, respectively)

Cross-scanning permits the rapid analysis of TLC plates provided that

the solute R 's are consistent. Data obtained by cross-scanning is

normally acquired continuously; however, the accumulation of data may

be controlled manually to permit optimization of the detector. Optimi-

zation of the spectrodensitometer is achieved by manually aligning the

scanning stage so that the light reflected from the analyte on the TLC

plate elicits the maximum detector response. If both samples and

standards are prepared in the same matrix, it is necessary to optimize

the detector response of only the most intense spot in a series to be

cross-scanned. If the standards are not in the same matrix, the response

for both the most intense standard and sample should be optimized.

Normal scanning requires optimization for each channel on the plate.

During the scanning operation acquired data is displayed on the monitor.

An operator-determined "scaling factor" normalizes this display to

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FOSS ET AL. 1846

u t i l i z e b e t t e r t h e e n t i r e viewing a r e a of t h e moni tor .

a n a l y s i s of unprocessed d a t a p rov ides number, r e t e n t i o n t i m e , a r e a and

h e i g h t above b a s e l i n e f o r each peak i n t a b u l a r form on t h e moni tor .

Data can then be shown i n t h e ana log form i n which it was a c q u i r e d ,

w i th de t ec t ed peaks numbered s e q u e n t i a l l y and marked t o show a r e a s of

i n t e g r a t i o n (F ig . 2 ) . The program normal izes t h i s d i s p l a y t o t h e

l a r g e s t peak d e t e c t e d , t h u s , a l lowing a c c u r a t e measurement of compounds

i n t h e low nanogram/spot range.

a l s o be i n t e g r a t e d manually. Th i s d a t a may be reproduced i n t a b u l a r

and ana log forms us ing t h e X-Y r e c o r d e r .

Automated

Any peak d i s p l a y e d on t h e moni tor may

The c a l c u l a t e d d a t a may b e f u r t h e r t ransformed t o y i e l d normal o r

log-normal p l o t s o f peak a r e a s ve r sus peak amounts ( F i g . 3 ) . The u s e r

de te rmines which r e f e r e n c e peaks and sample peaks a r e t o be p l o t t e d .

Appropr ia te d i l u t i o n f a c t o r s and r e f e r e n c e peak amounts a r e next i n p u t t e d ,

cho ices of normal o r log-normal p l o t and v i s u a l d i s p l a y o r copy a r e

made, and t h e normalized r e f e r e n c e d a t a i s p l o t t e d . An o p t i o n is

a v a i l a b l e t o superimpose on t h e d a t a t h e l e a s t - s q u a r e s f i t parameters

and a l i n e r e p r e s e n t i n g t h i s r e g r e s s i o n . A d d i t i o n a l l y , sample d a t a is

f i t t e d t o t h e l i n e a r r e g r e s s i o n equa t ion , and sample amounts and concen-

t r a t i o n s a r e dep ic t ed on t h e moni tor .

The g raph ics p o r t i o n of t h e program may be accessed t o pe rmi t

manual i n p u t t i n g of d a t a , independent ly of t h e scanning d a t a a c q u i s i t i o n

s e c t i o n .

and sample d a t a , such a s drug plasma concen t r a t ion - t ime curves (F ig . 4 ) .

In t h i s manner, p l o t s may be o b t a i n e d of any set of r e f e r e n c e

The automation system desc r ibed h e r e i n accomplishes o b j e c t i v e s

f r e q u e n t l y pursued by most modern a n a l y t i c a l l a b o r a t o r i e s . Compliance

wi th government r e g u l a t i o n s mandate t h e r e t e n t i o n of l a r g e amounts of

d a t a t o document a n a l y t i c a l p rocedures . Th i s t a s k i s s i m p l i f i e d by

s t o r a g e of d a t a on magnetic t a p e ensu r ing a means f o r r a p i d r e c a l l

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1850 FOSS ET AL.

whi le p rov id ing a convenient method f o r permanent a r c h i v a l s t o r a g e o f

d a t a i n a condensed form. The i n c l u s i o n o f processed d a t a i n t h e

a r c h i v a l d a t a set encourages good l a b o r a t o r y p r a c t i c e . Thus, d e p i c t e d

a r e a s of i n t e g r a t i o n and peak h e i g h t s a r e a v a i l a b l e f o r f u t u r e s c r u t i n y .

Furthermore, t h e unprocessed, o r raw d a t a , i s n o t r e a d i l y a l t e r a b l e ,

t he reby p rec lud ing q u e s t i o n s o f d a t a tamper ing . Th i s system a l s o

p rov ides r ep roduc t ions o f d a t a v i a t h e p r i n t e r - p l o t t e r t h a t a r e s u i t a b l e

f o r d i r e c t i n c l u s i o n i n p r e s e n t a t i o n s , such a s p rogres s r e p o r t s and

p u b l i c a t i o n s . To s u i t i n d i v i d u a l requi rements t h e s e formats may be

a l t e r e d through mod i f i ca t ion o f t h e BASIC program.

The combination of t h i s computer -ass i s ted au tomat ion wi th t h e

technique of c ros s - scann ing conven t iona l TLC p l a t e s has served t o

dec rease a n a l y s i s time. The observed i n c r e a s e i n t h e i n s t r u m e n t a l

s e n s i t i v i t y of t h i s method over manual TLC scanning is der ived from t h e

a b i l i t y t o e n l a r g e t h e p o r t i o n of t h e chromatogram be ing ana lyzed ( F i g .

5a-c) . This c a p a b i l i t y has s i g n i f i c a n t l y inc reased t h e p r e c i s i o n and

accuracy of t h e in s t rumen ta t ion v i t h which i t has been used . Although

t h e presence of r e f e r e n c e and sample d a t a s imul t aneous ly i n t h e micro-

computer memory makes TLC p a r t i c u l a r l y a d a p t a b l e t o t h i s d a t a r educ t ion

scheme, o t h e r chromatographic equipment, such a s HPLCs and G L C s , i n t e r f a c e s

e a s i l y . Thus, t h e a d a p t a t i o n of au tomat ion techniques o f f e r s many

advantages t o t h e a n a l y t i c a l l a b o r a t o r y . The e x t e n s i v e a v a i l a b i l i t y of

microprocessor i n t e g r a t e d c i r c u i t r y makes f e a s i b l e t h e development and

widespread use of microcomputer-based au tomat ion sys tems.

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MICROCOMPUTERIZING TLC SCANNING DATA 1851

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1852 FOSS ET AL.

FIGURE 5

Successive enlargements of portions of a typical TLC chromatogram demonstrating potential for enhanced sensitivity of automated system described in text. (a) Display of peaks 1-26; (b) enlargement of section containing peaks 14-26; (c) further enlargement of section containing peaks 16-18.

REFERENCES

W.S. Woodward and C.N. Reilley, Pure and Appl. Chem., so, 785 (1978).

A facsimile of this program is available from our laboratory upon

request.

J. Ripphahn and H. Halpaap, J. Chromatogr., I&, 81-96 (1975).

1.

2.

3 .

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