Gas Chromatograph-Minicomputer System Design and Application to Biomedical Problems S. P. Levine and J. L. Naylor Veterans Administration Hospital. D e p a r t m e n t of Medicine-Gastroenterology Division, Denver, Colo.
J. 6. Pearcel Ball Brothers Research Corporation and D e p a r t m e n t of Astrogeophysics, University of Colorado, Boulder,
Digital computers have been used in many laboratories for several years for automatic collection and reduction of gas chromatographic (GC) data. Most present day systems fall into four main categories: (1) hybrid systems that include an electronic digital integrator connected to a minicomputer; (2) on-line time-shared computer systems for multiple GC installations; (3) multichannel dedicated systems; and (4) dedicated GC-computer systems, the socalled one-to-one systems. The system described here is of the latter type. The costs of the above systems have, until recently, ranged from $10,000 to $400,000 (1-6). They have, therefore, been out of the range of financing available to the small laboratory. The increasing availability of inexpensive, small computer systems (in the $3000 range) has provided our laboratory with the opportunity to utilize a one-to-one dedicated system for a variety of analyses including the quantification of each component of GC metabolic profiles in urinary acids and the GC quantitation of bile salts present in various biological fluids. Our GC system consists of a Hewlett-Packard 7620 gas chromatograph equipped with a Model 7670A automatic sample injector and a Heathkit IR-18M recorder. The basic components of our computer system are a Digital Equipment Corporation PDP-8 computer (with 4096 words of 12 bit memory) equipped with a 12 bit analog-to-digital converter (Model 189) and a teletypewriter with paper tape. This computer system can be purchased complete for about $3000. Peak detection, separation, and integration are accomplished by an assembly language program (VAPKS-1) which makes extensive use of the floating point arithmetic interpreter, supplied with the computer as a subroutine. The real time portion of the program samples data from the GC detector once per second. These data are smoothed and differentiated using a weighted, odd integer smoothing routine ( 7 ) . When the derivative exceeds an operator selected value, a “GC peak” is provisionally established. If the peak satisfies the selected minimum width criterion, the location and area of this event are either printed out immediately on the teletypewriter or stored in memory for further processing, if there is evidence that the peak is incompletely resolved. When the GC data returns to within a selectable vicinity of the base line, or a maximum number of peaks (five, at 1To w h o m dressed.
inquiries concerning system software s h o u l d be ad-
S. P. Perone, Ana/. Chem., 43. 1288 (1971). S . P. Perone, J . Chromatogr. Sci.. 7, 714 (1969) J . M . Gill, J. Chromatogr, Sci. 7 . 731 (1969). J. M . Gill. J. Chromatogr, Sci.. 10, 1 (1972) I . E. Bush. Amer. J. Clin. Pathol.. 53, 755 (1970). R . A Landowne. R W.Morosani. R. A . Hermann, R . M . King, Jr.. and H . G. Schmus. Anal. Chem., 44. 1961 (19721 (7) A . Savitsky and M . J. E. Golay A n a / . Chem., 36. 1627 (1964)
(1) (2) (3) (4) (5) (6)
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present) is exceeded, the perpendicular drop method is used to resolve them (8, 9 ) , and the results are printed out. If desired, a paper tape recording of the results can also be generated. In most laboratories, as in ours, automatic data reduction is as important as peak area integration. Reduction of the acquired data is accomplished using the same equipment in an off-line mode. This second segment of the program accepts the paper tapes generated by the first segment, as well as additional mode information from the keyboard. This program also uses the floating point interpreter. The interpreter thus remains resident in core and only the driving segments of the program need be exchanged.
SYSTEM OPERATION The voltage us. time output of the GC are collected by the computer and reduced to peak area us. time by the first, on-line, segment of the program. Certain parameters needed for proper operation are entered by the operator. Mode. A digit from 0 to 3 is entered by the operator to control the various combinations of operations possible. The options include repetitive run and the slope printouts. In repetitive run mode, the system is geared to analyze multiple GC runs without operator intervention. Once the initial information is entered, the system repeats its sequence endlessly until interrupted manually. This is useful for comparative analyses, using an automatic sample injector on the GC. If repetitive run is not specified, the program restarts itself a t the completion of a run and waits for the operator to reenter the run parameters. If the slope printout mode is selected, the slope of a line drawn from the start of a peak to the end of a peak (or a multiple peak envelope) is printed out for each peak identified along with the area and time of occurrence of the peak. Run Length. The operator specifies the time in seconds from the sample injection until the end of the run. At the end of this time, the program either reinitializes or automatically starts another identical run, depending on the repetitive mode switch. If the repetitive run mode is chosen, the first off-scale peak (the solvent) following the end of the previous chromatogram triggers the new run. Timing starts a t that point with a presettable time added to correct the difference between the actual start of the chromatogram and the start of the solvent peak. Minimum Derivative. The operator enters the minimum value of the slope of the data (measured in volts/ second) necessary for a given feature to be recognized as a GC peak. Features with slopes below this value are rejected as noise. ( 8 ) J. Novak. K . Petrovic, and S. Wicar, J , Chromatogr.. 55. 221 (1971). (9) K Kishimoto. H . Miyauchi, and S . Musha J. Chromatogr Sci.. 10. 220 (1972).
RF'S O R PEAKS?l NEW DATA?l READY LIST?O DELETE (N) 0 ADD AT NO (N,T,A)O LIST?O T SCALE (N.T,N.T) 5 13.19 17 17.75 MODE 0 OR l ? l CORR (N,AN) 5 ?172.5X400X1.38X ,625 / 1.42 = 419.1 SCALE ( X , Y ) ?500X1395/419.1X 50 = 33.29 = X ?33.29X5/627.75= ,2651 = Y IND PK (N.Z) 0 N 1 2 3 4 5 6 7
a 9 10 11 12 13 14 15 16 17 18 19 20 21 22
T 790 900 1020 1120 1180 1480 1560 1760 1800 1910 2050 2130 2240 2350 241 0 2530 2616 2680 2900 2940 2980 3136
CT 11.95 12.30 12.68 12.99 13.19 14.14 14.39 15.03 15.15 15.50 15.95 16.20 16.55 16.90 17.09 17.47 17.75 17.95 18.65 18.77 18.90 19.40
Type 0 for RF calculations, 1 for peaks Questions are answered O=no. 1 =yes Data entered by keyboard or paper tape For operator to check data entry For data editing
MODE 0 OR 1?0 GIVE INJ DATA ?32 X lOX.5/1.96=81.6326
GIVE ALL RFS 633603510574566 PRINT: 4 MG=.548 PRINT: 0
=
RF=574
Time scale
Normalization to give area of pk N at 100% recovery Scaling factors applied to normalized area
DELETE (N) 4 DONE DELETE (N) 0 GiVE MMF
?2X.576/.548=2.1021
Factor(s) applicable to individual peak(s) 0 indicates no corrections A 25.60 118.40 25.60 51.20 473.60 92.00 72.00 20.00 65.00 10.00 27.00 34.00 13.00 57.00 114.00 43.00 89.00 67.00 14.00 86.50 176.00 90.00
AN 22.65 104.77 22.65 45.30 419.10 81.41 63.71 17.69 57.52 8.84 23.89
30.08 11.50 50.44 100.88 38.05 78.75 59.28 12.38 76.54 155.74 79.64
AX 754.15 3487.96 754.15 1508.30 13951.80 2710.24 21 21.05 589.18 1914.84 294.59 795.39 1001.61 382.96 1679.17 3358.34 1266.74 2621.86 1973.76 412.42 2548.21 5184.81 2651.32
AY 6.00 27.77 6.00 12.01 111.10 21.58 16.89 4.69 15.24 2.34 6.33 7.97 3.04 13.37 26.74 10.08 20.87 15.71 3.28 20.29 41.28 21.11
Figure 1. Typical report of data entry mode and mode 1 for a urinary acid metabolic profile N = peak No., T = retention time (sec), CT = corrected time, A = peak area, AN = normalized area, AX = p g component excreted/hour, AY = p g excreted 'mg creatinine
End Derivative. This quantity establishes the criterion for determining the end of a peak. Minimum Width. The operator sets the half width (in seconds) to be used as a noise rejection criterion. Blend Slope. After a peak has ended, the slope of a line joining the beginning and end of the peak is calculated. If the calculated value exceeds the entered BLEND SLOPE, this peak is considered to be part of a family of unresolved features. The parameters describing this peak are then stored in memory for later processing. If the slope of the base line is less than the entered value, its location and area are printed out immediately. Incompletely resolved peaks are indicated by an asterisk in the printout. Solvent Delay. The operator sets the program to ignore all data until this time has expired. In this way, the solvent peak area and location do not appear in the output. After choosing the appropriate mode and entering the desired parameters, the operator readies the syringe for injection. A t the moment of injection, the operator presses the return key on the teletypewriter keyboard, which starts the integration program.
N 1 2 3 4
T 1074 1589 1959 2730
CT 1071.99 1576.68 1946.46 2716.00
A ,0900 1.4200 1.6599 2.3800
Injection data & GC conditions Response factors are entered By giving peak operator can determine how much of a certain component (internal standard, for example) is present Operator can delete internal standard from final tabulated results Correction to 100% recovery of internal standard and to unit volume (mg/ml)
RF 633.0000 603.0000 509.9999 565.9999
MG ,0116 ,1922 ,2657 ,3432
MM ,0243 ,4041 ,5585 ,7215
%T ,0142 ,2375 ,3268 ,4223
Figure 2. Typical report in mode 0 for bile salt analysis of gall bladder bile RF = response factor, MG = milligrams, M M = milligrams/milliliter of bioiogical fluid, %T = weight per cent
CALCULATION AND REPORTING OF RESULTS With the peak times and areas calculated, the VAPKS2 program is now called to compute the final results. VAPKS-2 operates in three modes (Figures 1 and 2 ) . Data Entry Mode. In this mode, the time and area data are entered from the teletypewriter or using the paper tape record produced by the VAPKS-1 program. Data editing can be performed; peaks can be deleted, added, or changed from the keyboard. If desired, an arbitrary time scale can be set up by choosing two peaks and assigning them arbitrary time values. The program then linearly interpolates and extrapolates time values using these two points and establishes the new time scale. Mode 0. This mode is designed to process samples for which the GC detector response factor (RF) is known for each component measured (IO). In Part A, the response factors are calculated and multiple analyses are averaged. Part B performs the internal standard normalization, weight, and weight per cent calculations. The internal standard(s) value is(are) deleted by the operator before the weight per cent calculation. Mode 1. In mode 1, all component areas are first corrected to 100% recovery of the internal standard. All normalized data are then scaled by two separate factors. A third factor can be separately applied to each individual component of the normalized data to correct differing detector responses and recoveries of the components (if, in fact, they are known). The normalization and scaling factors used in each mode are calculated from pseudoarithmetic statements entered from the keyboard.
(10) H M McNair and E J Bonelli Basic Gas Chromatography ian Aerograph Walnut Creek Calif 1967
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technique to the quantitation of urinary acids has been undertaken in order to study the effects of alcohol metabolism on other oxidative processes and to compare these effects in young normal adults and in chronic alcoholics with and without cirrhosis (12). The arbitrary time scale utilized in these experiments is the methylene unit scale which is useful for inter- and intralaboratory retention time comparisons (13, 14). The quantification by VAPKS-1 of each urinary acid represented in a typical GC metabolic profile is compared in Figure 3 to that obtained by integration with a planimeter. The mean of the difference of the results and the standard error of the mean show that there is no systematic bias in the computer results. The peak time and area data are then reduced by VAPKS-2; Mode 1 prints the results as an eight-column matrix (Figure 1). These two programs, when used together, make possible the quantification and comparison of the metabolic profiles of large numbers of subjects. The combination of VAPKS-1 and VAPKS-2, Mode 0, is used in the analysis of bile salts extracted from serum, ascitic fluid, and bile (15).This is illustrated in Figure 2. PEAK
TIME
A B C D E F
5aa 620 690
G H I
J K L M N
0 P
0 R S T U V
aoo a99 1017 1102 1151 1452 1516 1760 2007 2131 2197 2295 2347 2469 2558 2625 2864 2927 3052
COMPUTER AREA*
PLANIMETER AREA
4.35 20.1 13.8 2.84 15.0 2.89 17.0 100 5.02 4.53 8.88 1.06 0.62 1.07 10.4 20.0 6.44 8.08 10.4 23.4 10.8 20.0
3.35 17.7 13.7 3.36 14.4 2.64 15.6 100 5.28
4.80 10.6 0.96 0.72 2.03 9.83 19.2 8.87 8.15 10.8 22.5 9.60 18.5
Mean of Difference (C-P) ~ 0 . 2 1 3 Standard , Error of Mean=0.261, Standard deviation=l.ZO
Figure 3. Metabolic profile of urinary acids and comparison of
computer and manual integration results *Accuracy improving as VAPKS-1 evolves
BIOMEDICAL APPLICATIONS The temperature-programmed gas chromatographic analysis of each component of a mixture containing a class of compounds extracted from a biological fluid is called a metabolic profile (11). The application of this (11) E. C . Horning and M. G. Horning, J. Chromatogr. Sci., 9, 129 (1971 ) , and references contained therein.
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CONCLUSIONS The advantages of this system are fourfold: (1) low cost -this is the first true GC-computer system capable of peak integration and data handling available a t a price within reach of most small laboratories; (2) accuracy-the accuracy of this system has been demonstrated on very complex metabolic profiles (hand repetition of VAPKS-2type calculations is very susceptible to human error); (3) speed-it is a near real time, on-line system; and (4)flexibility and easy expandability-programs are available from Digital Equipment Users Society (DECUS) to adapt this computer to many other types of calculations. VAPKS programs are written in easily understood and modifiable configurations. We are, a t present, modifying VAPKS-1 to use a second derivative peak sensing routine. ACKNOWLEDGMENTS The contributions of P. J. R. Boyle in software systems development are gratefully acknowledged. We also wish to thank A. Savitsky for helpful discussions early in this project. Received for review November 20, 1972. Accepted February 12, 1973. This project was supported by the Division of Gastroenterology, Department of Medicine, Veterans Administration Hospital, Denver, Colo., under the direction of T. A. Witten and J. E. Struthers, Jr. A portion of the support for this project came from the B. F. Stolinsky Laboratories of the University of Colorado Medical Center, Denver, Colo. (12) T . A . Witten. S. P. Levine. M . Killian, and S. P. Markey, Gastroenterology. 62, 777 (1972) (13) T. A . Witten, S. P. Levine, S. P. Markey, and M Killian, submitted to Clin. Chem. (14) H. Vanden Do01 and P. D. Katz, J. Chromatogr.. 11.463 (1963) (15) J. E. Struthers and J. L. Naylor. unpublished results.
