An automated procedure for circular dichroism and optical rotary dispersion spectroscopy

Ai’i4LTTIC?LL

60, 59-77 (1974)

BIOCHEMISTRT

An Automated and Optical

Procedure for Circular Dichroism Rotary Dispersion Spectroscopy

LEON VISSER, STEVE MINNAAR, Departments

AND

GRAHAM

L. WEBB

of Biochemistry

Pietermaritzburg, University

and Biometry, University of Natal, South Africa, and Computer Centre, of Natal, Durban, South Africa

Received October 22, 1973; accepted December

19. 1973

A method is described whereby CD and ORD spectra are computed and plotted from digital data collected simultaneously with the recording of the initial spectrum on a Jasco Model J-20 automatic recording spectroppolarimeter/circular dichrometer. The procedure eliminates all the customary manual operations in the processing of data, thereby increasing the reliability of the results and saving a substantial amount of time. The determination of circular dichroism (CD) and optical rotatory dispersion (ORD) spectra is fairly common in studies of the configuration and conformation of optically active compounds. Despite the simplicity of the technique and the availability of automatic recording circular dichrometers/spectropolarimeters, the calculation and plotting of corrected spectra from the recorded traces require much extra time. This is brought about by the need to convert observed ellipticities or rotations to a common (e.g., molar) basis for comparative purposes, as well as the design of older-type instruments which does not provide for the direct processing of data by computer. Under these conditions, time-consuming manual operations for the transformation of recorded CD and ORD spectra into suitable format, are unavoidable. However, the latest models of automatic recording circular dichrometers/spectropolarimeters, such as the Jasco Model 520, can be adapted for the computerized analysis of data. We report here on a procedure developed for the fully automated collection of data and the calculation and plotting of CD and. ORD spectra, in the belief that others may find it useful, too. MATERIALS

The following materials were purchased from the respective suppliers: +) -IO-camphorsulphonic acid monohydrate (Merck pro a&y&), polyL-glutamic acid (M, + 10,000; Miles-Yeda) , equine myoglobin (3 x recrystallized), and bovine b-chymotrypsin (six times recrystallized; MilesD(

59 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

60

vIssE~,

MINNAAR

AND

WEBB

Seravac) . The aldobiouronic acid /3-n-glucuronic acid-1,6+galactose was kindly supplied by Professor A. M. Stephen (Department of Chemistry, Cape Town University) and the steroid a-l,Z-epoxy-17-O-acetyl-androstA4-ene-3-one was obtained from Dr. P. R. Enslin (National Chemical Research Laboratory, C.S.I.R., Pretoria). CD and ORD spectra were run on a Jasco Model 520 instrument (Japan Spectroscopic Company, Tokyo, Japan) in Hellma “Suprasil” fused quartz cells. A Beckman Instruments Autopro 311 intercoupler (with collection time intervals of 1, 2, 5, 10, 20, and 50 set) was employed for the analog-to-digital conversion of the instrument’s recorder signal, which was relayed to the intercoupler from the “DS” output on the right-front module. The intercoupler was fitted with an expansion circuit accessory (built by C. Palmer and R. van Rensburg of Beckman Instruments, Durban) whereby the digitized value corresponding to full-scale deflection of the Jasco recorder pen could be preset at convenience. Binary coded decimal data (ASCI) from the intercoupler were collected on paper tape by a punch-teletypewriter combination (International Telegraph and Telephone Co.). The tapes were processed by an IBM 1130 digital computer (16K core) and the final CD/ORD spectra were drawn on an incremental graph plotter (Calcomp Model 563). METHOD

The calculation of comparative CD/ORD spectra requires the determination of values for molar or mean residue ellipticity/rotation at each selected wavelength interval according to the following equation (or its equivalent) :

where [8]~, = molar or mean residue ellipticity in degrees cm2/decimole at wavelength Xi; [m]x, = molar or mean residue rotation in degrees cm2/decimole at wavelength Xi; hi varies from the highest (starting) wavelength to the lowest (final) wavelength of the spectral region under investigation; &brr = observed (recorded) ellipticity in degrees; 0&b,, = observed (recorded) rotation in degrees; M, = molecular weight (or mean molecular weight per residue in the caSe of oligomers and polymers) of the optically active compound; c = concentration of optically active compound in mg/ml of solution; I = cell pathlength in cm.

AUTOMATED

CD

AND

ORD

SPECTROSCOPY

61

All parameters of equation A, excepting cobs (only 6obs will be considered further, but the same arguments apply to cyobs),can be considered as constants for any one CD spectrum. Since circular dichrometers are single-beam instruments, oobs has to be derived from the individually recorded traces for a sample solution and its solvent, and is equal to the product of the difference in signal height (pen tracing) between the sample solution (h,) and reference solvent (h,), and the scale sensitivity (8) employed, i.e., 8oba.Ai= (h - h,)xi X 8, degrees,

W

where 6obs .Xi = observed ellipticity at wavelength Xi; (h, - h,) = difference in signal height, in centimeters, solution and solvent ; S = scale sensitivity in degrees/cm.

between sample

The task of calculating the values of the molar (or mean residue) ellipticity at wavelength intervals covering the whole spectrum in essence, therefore, becomes one of establishing (h, - h,)xi for all hi. Where this difference has to be read off manually from the recorded tracings of sample and solvent, as was the case until recently, it becomes a time-consuming procedure. Such tedium is obviated by the method outlined below, which depends upon the simultaneous conversion of the recorder pen signal to digital data which are stored on paper tape for subsequent offline computer processing. The voltage signal fed to the recorder pen of the Jasco 520 could vary over the range 4 1 V, with the zero position (no signal) corresponding to 0 V, a full-scale (positive) pen deflection to + I V and a full-scale (negative) deflection to - 1 V. The digitized output from the intercoupler was adjusted with the aid of the expansion circuit accessory so that. a zero signal corresponded to a digital value of 500, a full-scale positive recorder pen deflection (equivalent to 20 cm on the chart paper) to 1000 and a full-scale negative pen deflection to 0. Thus, each l-cm deflection of the recorder pen on the chart paper corresponded to a change of 25 (or 500/20) in the digital value punched on the paper tape, e.g., a positive signal of 3-cm height was equivalent to 575 (i.e., 500 -t 3 x 25) and a negative signal of 17-cm height (depth) to 75 (i.e., 506 - 17 x 25j. Although this calibration was found convenient for most routine analyses, it can easily be changed, should this be called for by special situations. In such an event, the equivalence factor (1 cm = 25) in the computer program has to be changed accordingly,

62

VISSER,

MINNAAR

AND

WEBB

The actual determination of a CD or ORD spectrum is then carried out as follows: The spectrum of the sample solution is always recorded first. The frequency at which the recorder pen signal has to be converted to a digital number is preselected by setting the time interval control knob on the intercoupler t.o a value most compatible with the speed at which the spectral region is to be scanned. For scanning speeds of 5 nm/min and 10 nm/min a 2-set time interval is suitable, whereas for slower scans at 1 nm/min digital data need only be collected every 20 sec. The recording is begun at a wavelength 3-5 nm longer than the upper-wavelength limit of the spectral region of interest. When the latter is reached, the collection of data in digital form is initiated by depressing a hand-held contact switch fitted to the leads between the instrument and the intercoupler. When the lower wavelength limit of the spectral region is reached, the collection of digital data is suspended by depressing the contact switch again. A negative number, e.g., -200, is punched on the tape (with the teletypewriter now in “local” mode) to indicate the end of the data set for the sample solution. Subsequently, the solvent is scanned in analogous fashion over the same spectral region and exactly the same number of data are collected, the set of data also being terminated with a negative number. From these two sets of digital data the net signal height, i.e., (ha - h,) of equation B, is readily determined according to the computer program. After data collection, the additional information required for the calculation of molar or mean residue ellipticities/rotations is punched on the paper tape with the teletypewriter again in “local” mode. This includes sample identification, solvent, date, type of spectrum (CD or ORD), molecular or mean residue weight of sample, scale sensitivity (deg/cm), cell length (cm) ! concentration of sample (mg/ml) , chart speed (cm/ min), wavelength expansion (rim/cm), frequency of data collection (set), the wavelength interval (e.g., 1 nm) to be used in the calculation of the CD/ORD spectrum and the starting (upper-limit) wavelength. An example’ of the format of input thus obtained is given in Fig, 1. Upon processing by the computer (see program) the values of [6’] or [m] are calculated at each of the chosen wavelength intervals, extending from the starting to the final wavelength of the spectral region investigated. These values are obtained as print-out. If a peripheral plotter is available the calculated [e] or [m] values are also plotted as a function of wavelength and the points connected by a mathematically determined smooth line to provide a graph of the CDJORD spectrum (see plot Subroutine in the program). A useful feature of the latter is that no information other than that used for the calculation of [0] or [m] is re-

(a) 01 0490 0490 0489 0488 0488 0487 0489 0487 0486 0485 0485 0487 (b) 02 03 04 05 06 07 08 09 10 11 12

0489 0485 0484 0480 0479 0487 0509 0549 0581 0574 0550

0489 0484 0483 0478 0480 0488 0510 0553 0581 0572 0548

0490 0483 0483 0478 0482 0491 0514 0556 0581 0571 0544

0490 0484 0483 0478 0481 0492 0519 0561 0582 0572 0541

0490 0484 0484 0480 0481 0493 0523 0564 0582 0571 0538

0489 0484 0483 0480 0483 0494 0526 0567 0583 0570 0533

0488 0483 0483 0480 0483 0496 0529 0570 0583 0570 0532

0488 0484 0484 0480 0483 0496 0533 0574 0581 0569 0533

0488 0484 0483 0480 0484 0498 0535 0575 0578 0568 0535

0488 0484 0482 0481 0484 0500 0538 0576 0577 0564 0533

0486 0487 0483 0484 0480 0480 0480 0479 0484 0484 0501 0506 0542 0545 0576 0579 0576 0575 0560&55 -200

01 02 03 04 05 06 07 08 09 10 11 12

0497 0492 0491 0485 0491 0501 0506 0511 0520 0523 0520 0522

0479 0491 0490 0484 0492 0501 0507 0513 0520 0524 0520 0521

0497 0491 0489 0485 0493 0504 0507 0514 0520 0524 0520 0523

0498 0492 0486 0487 0494 0507 0508 0514 0522 0522 0519 0522

0497 0492 0485 0488 0494 0507 0508 0514 0520 0521 0520 0522

0479 0491 0485 0488 0493 0507 0509 0514 0521 0521 0520 0521

0497 0492 0486 0487 0492 0507 0509 0515 0520 0520 0520 0521

0495 0491 0486 0486 0493 0507 0509 0515 0520 0521 0520 0522

0495 0490 0487 0486 0498 0508 0509 0516 0521 0522 0520 0522

0499 0491 0489 0488 0498 0507 0510 0516 0521 0521 0520 0522

0492 0491 0488 0488 0498 0506 0510 0518 0522 0521 0521 -200

0493 0491 0487 0489 0499 0505 0511 0520 0522 0521 (9TZ1

*LPHA-CHYMoTRYPsIt?(e! ... (sample HCL PH 3.0 ............. 25/Q/73 ................ CD ..................... 103.0 .................. 0.001 .................. 1.0 .................... 0.6 .................... 5.0 .................... 2.0 .................... 1.0 . . . . . . . . . . . . . . . . . . . . 1.0 .................... 315.0 ..................

name) (solvent) (date) (mode) (molecular or mean residue weight) (scale sensitivity) (cell pathlength in cm) (concentration in mg/ml) (wavelength expansion in rim/cm) (frequency of data collection in set) (calculation frequency desired, in nm) (chart speed in cm/min) (starting wavelength in nm)

(a):

The first line number (01) is preceded by the sequence: carriage return (CR), line feed (LF) and rub-out (RO), which is punched upon pressing the "Reset", "Line number" controls (in this order) on the Intercoupler with the teletypewriter in "On line" mode. The Intercoupler automatically signals the end of each line of (b) : 12 data by the sequence CR, LF, RO. Punch the negative number in "Local" mode, return teletypewriter Cc) : to "On line", press the "Reset" and "Line number" controls on the Intercoupler and proceed to collect solvent (reference) data. Punch the negative number (-200) whilejn "Local" mode, and tkn (d): CR, LF and RO. Stay in "Local" mode. Terminate this line as well as the following ones with CR, LF. (e) : Fro. 1. Format of digital input on paper tape. Explanatory case letters in parentheses. 63

notes appear in lower-

64

VISSER,

MINNAAR

AND

WEBB

quired, since the size of the abscissa and ordinate intervals (i.e., the grid intervals on the graph) is computed from the maximum and minimum values of the wavelength and [0] or [ml, respectively (cf. Interval Subroutine). RESULTS

AND DISCUSSION

Examples of CD and ORD spectra obtained by the above method are given in Figs. 2-8 for a number of optically active substances, which include a steroid, an aldobiouronic acid, two proteins, a polypeptide, and the calibration standard, camphorsulphonic acid. The figures are direct reproductions of the graphs produced on the Calcomp plotter and were selected to illustrate the application of the method under a variety of operating conditions (see figure legends). The CD spectrum of camphorsulphonic acid in the near-uv region (340-250 nm) in Fig. 2 is representative of compounds exhibiting only one fairly strong positive circular dichroism extremum in the spectral region investigated and with little instrument ‘koise” evident in the original recorded tracing. The complementary ORD spectrum of camphorsulphonic acid is shown in Fig. 3 with a positive Cotton effect centered

~:::::::::::::::::::+ 240

260

a39 W0Wkngth

300

320

340

(NM1

FIG. 2. CD spectrum of n( +)-lo-Camphorsulphonic acid monohydrate. Concentration: 0.6 mg/ml of water; pathlength : 1.0 cm; scale sensitivity: O.O2”/cm; soanning speed: 5 nm/min; M, : 250.3; digital data collection every 2 set; calculation of Gel : every 1 nm; servo gain = 4; pen period : 1 sec.

AUTOMATED

CD

AND

ORD

280 wwelenqth

240

65

SPECTROSCOPY

320

360

(NM1

FIG. 3. ORD spectrum of D( +)-IO-Camphorsulphonic acid monohydrate. Conditions were the same as for Fig. 2, except that a scale sensitivity of O.Ol”/cm was used.

280 Wovelength

300 [NM)

320

Fru. 4. Near-ultraviolet CD spectrum of Schymotrypsin. Concentration: 1.0 mg/ml of 10d N-HCl; pathlength : 1.0 cm; scale sensitivity: O.OW/cm ; scanning speed: M, - 103.0; digital data collection: every 2 set; calculation of tel: 5 nm/min; every 1 nm ; servo gain: 6; pen period: 1 sec.

66

VISSER,

MINNAAR

AND

WEBB

near 291 nm, in agreement with the wavelength of maximum ellipticity in the CD spectrum. By contrast, the near-uv CD spectrum of Schymotrypsin in Fig. 4 exhibits three very weak and closely spaced Cotton effects (note the difference in ordinate scale relative to Fig. 2) discernible only at high protein concentration and high scale sensitivity. The well-known far-uv CD spectrum of the synthetic polypeptide poly-n-glutamic acid in o-helical conformation (1) is given in Fig. 5. The application of the method for the determination of the optical behavior of glucuronic acid and steroid derivatives is illustrated by the CD spectra of n-glucuronic acid-,6-1,6-n-galactose (Fig. 6) and m-1,2epoxy-17-P-O-acetyl-androst-A4-ene-3-one (Fig. 7) with their multiple positive and negative Cotton effects. The result of the investigation of an extended spectral region is given by the visible (60&250 nm) CD spectrum of myoglobin in Fig. 8, which shows the presence of extrinsic Cotton effects associated with the prosthetic heme group of that protein. This figure also illustrates the importance of selecting a short enough period or calculation frequency (broken line) if drastic suppression of the smoothed plot (solid line) is to be avoided in regions of high curvature. The above examples clearly demonstrate the general utility of the

FIG. 5. CD spectrum of poly+glutamic acid. Concentration: 0.058 mg/ml of water, pH 4.4; pathlength : 0.10 cm; scale sensitivity: O.O02”/cm; scanning speed: 5 nm/min; M, = 129.0; digital data collection: every 2 see; calculation of Co1 : every 1 nm ; servo gain : 6; pen period : 1 sec.

FIG. 6. CD spectrum of n-glucuronic acid-/3-1,6-n-galactose. Concentration: 5.28 mg of Ba-salt/ml of water, pH 5.5; pathlength : 0.10 cm; scale sensitivity: 0.001”/cm; scanning speed : 5 nm/min; M, (Ba-salt) : 423.7; digital data collection: every 2 set ; calculation of [al : every 1 nm; servo gain: 7; pen period: 1 sec. 2520 ,

-

1680

I

n

FIO. 7. CD spectrum of cul,a2-epoxy-17-P-O-acetyl-androst-A4-ene-3-one. Concentration: 2.0 mg/& of acetonitrile; pathlength: 0.10 cm; scale sensitivity: O.O02”/cm; scanning speed: 10 nm/min; M, : 315.0; digital data collection: every 2 set ; calculation of [el : every 2 nm; servo gain: 6; pen period: 1 sec. 67

68

VISSER,

MINNAAR

AND

WEBB

A::::::::::::::::::+ 280

360 Wavelength

440 LNMI

520

600

FIG. 8. Visible and near-w spectrum of myoglobin. Concentration: 0.9 mg/ml of 0.08~ phosphate buffer, pH 7.5; pathlength: 1.0 cm; scale sensitivity: O.OO!P/cm; scanning speed: 20 nm/min; M,: 114.0; digital data collection: every 5 set; calculation of [el: every 5 nm (solid line) or every 1.25 nm (broken line); servo gain: 6; pen period: 1 sec.

method of computerized calculation and plotting of CD and ORD spectra. Good quality graphs are produced in a fraction of the time demanded by manual procedures and the sheer drudgery of the latter is eliminated. Even if a peripheral plotter is not’ available, the automated calculation of [e] or [m] values greatly facilitates the subsequent manual plotting of the spectra. There is no reason apparent to us why the method cannot be adapted to different makes of instrument, a/d converters, computers and plotters. It will also be evident from an examination of the computer program that it should be relatively easy to modify the procedure so that results from repetitive scans of the same spectrum could be averaged, thus facilitating the interpretation of poorly resolved spectra. ACKNOWLEDGMENTS The financial support of the Department of Agricultural Technical Services and the Natal University Development Fund, and the assistance of Nils Otte of the Natal University Computer Centre during the early part of the investigation are greatly appreciated.

1.

HOLZWARTH,

G.,

AND

DOTY,

P.

REFERENCES (1965) J. Amer. Chem. &x.

87, 218.

AUTOMATED

69

CD AND ORD SPECTROSCOPY APPENDIX

Program for Cahdation

and Plotting of CD/ORD

Spectra

Comments on INPUT: Table of Hs values Table of Hr values The last number of Hr and Hs values must be a negative number, the rest of the field being filled with zeros or blanks as the data are read in in groups of 12. The maximum number of Hr and Hs values is 1800 each. The programme will be terminated if the no of values exceeds 1800. If the first Hr or Hs value on a record is zero, the whole record will be ignored on the assumption that the ecord was empty (i.e. no characters preceded the GR and LF). identification of the sample must be punched in the Thereafter, following way: 30 characters for the sample name. 25 for the solvent. 8 for the date. 3 for the mode.'ORD' or 'CD' The parameters follow in FlO. 3 format and are: MRW=mean residue weight of sample. S=sensitivity setting on instrument in deg/cm. LEN=length of cell in cm. CON=concentration of sample in mg/ml. WE=wavelength expansion in rim/cm. FDC=frequency of data collection in sec. CF=frequency at which calculations have to be performed (nm). CS=chart speed in cm/min. SW=starting wavelength. INTEGER IMODE,MODE(3),SAMPL(30),REFER(25),DATE(8) INTEGER HS(1800),HR(lBOO) INTEGER PT REAL MRW,S,LEN,CON,WE,FDC,CF,CS,SW,SS,WLSPF,FPV of points Comment: YYl - Y-coordinates YY2 - Y-coordinates of points YMIN,YMAX,KMIN,KMAK must be in between calls to 'plot' The core capacity (16K) limits 600.

plotted. plotted (corrected to smooth). common to preserve their values the number of points

pldtted

to

DIMENSION YYl( 6OO),YY2( 600) COMMON SW,MODE,SAMPL,REFER,DATE,WL,YY,YMIN,YMAK,XMIN,XMAX,IPEN COMMON FW DATA IMODE / 'C' / DATA PT / 4 t DATA IBLNK / ' ' / Comment: Treat exclamation mark as rubout character, 12 data from the Beckman Intercoupler is regular rubout character.

since

terminated

each

group

by the

of

70

VISSER, MINNAAB

AND WEBB

1 CALL RBOUT(33) Comment: Read in a11 the information Hs=signal height of sample solvent.

2

4

tape. height

of reference

J=l K=12 READ(PT,l003)(HS(L),L=J,K) Comment:

3

off paper Hr=signal

If

the first

value

IF ( HS(J) ) DO 4 I=J,K IF(HS(1)) 6, CONTINUE J=Ktl K=K+12

6,

2,

6,

4

Comment: Test

to ensure

IF(K-1800)

2,

Comment: If

it

2,

is

zero,

assume an empty record.

3

that

there

are

no

more

than 1800 input

5

does write

out an error

message.

5 WRITE(3,1012) STOP Comment: ISTOR=number

of points

stored.

6 ISTOR-I-l J=l K--12 7 READ(PT,~OO~)(HR(L),L=J,K) Comment: If 8 9

the first

value

is

z'ero,

assume an empty record.

IF(HR(J)) lo, 7, 8 IF(K-ISTOR) 9, 10, l0 J=K+l K=K+12 GO TO 7 Comment: Read in the identification

10 READ (PT,lOOO)

SAMPL

Comment: If heading

is all

blank,

DO 11 J=1,30 IF ( SAMPL(J) - IBLHK) 12, 11, 12 11 CONTINUE GO TO 10 12 READ (PT,lOOl) REFER, DATE, MODE Comment: Write

of the sample.

out a heading.

assume an empty record.

values.

AUTOMATED

71

CD AND ORD SPECTROSCOPY

WRITE(3,1006)SAMPL,REFER,DATE WRITE(3,1002) LINES=K/li DO 13 I=l,LINES

KZI”12

J=K-11 .3 WRITE(3,1004)

(HS(L),L=J,K),

Comment: Read in all

(BR(L),L=J,K)

the parameters

used.

READ(PT,l005)MRW,S,LEN,CON,WE,FDC,CF,CS,SW WRITE(3,1007)MODE,MRW,S,LEN,CON,CS,WE,FDC,SW,CF Comment: Test whether DO 14 1=1,3 IFCIMODE-MODE(I)) 14 CONTINUE Comment: Write

the mode is CD or ORD.

14, 15, 14 a heading

WRITE(3,1008) GO TO 16 15 WRITE(3,lOOR) Comment: Perform the required calculations and plot Calculate the scanning speed (nm/min).

the graph.

16 SS=CSscWE Comment: Calculate the wavelength frequencies (WL,SPF).

span corresponding

to the punch

WLSPF=SS*FDC/60. Comment: Calculate the frequency with which the punched values are to be used in calculations(FPV). Because of the limited range of time intervals that can be selected on the A/D converter for data collection, an excess of punched values appears on the paper tape input. FPV=CF/WLSPF M=IFIX(FPV+O.5) Comment: RAN - range

of X axis.

RAH(I~ToR-~)/M+~)*CF Comment: FW=Final

wavelength

(lower

limit)

of spectrum.

FW=SW-RAN Comment: Determine ellipticity).

max and min values

of Yy (mean residue

rotation/

72

VISSER, MINNAAR AND WEBB

YMAx=o. YMIN=O. JJ-0 DO 20 I=l,,ISTOR,M Comment: Calculate the difference to digital value divided by the

difference between Hs and Hr and convert this cm. In this case the equivalence factor is the corresponding to full-scale deflection of pen chart width = 500120 = 25.

DIFF=FLOAT(HS(I)-HR(I))/25. Y=(DIFF%lRW*S"lOO.)/(LEN"CON) Comment: Store

17 18 19 20

value

of Y in array

for subsequent

plotting.

JJ=JJ+l YYl(JJ)=Y IF(Y-YMIN) 17, 18, 18 YMIN=Y IF(Y-MAX) 20, 20, 19 YHAx=Y CONTINUE Comment: NPP - number of points Plot points calculated

plotted. (actual).

NPP=JJ IVAR=l wL=sw DO 21 JJ=l,NPP YY=YYl(JJ) YY2(JJ)=WL CALL PLOT(IVAR) WL=WL-CF IVAR=2 21 CONTINUE Comment: Tabulate

22 23 24 25

all

points

on printer.

LINES=NPP/S IF(LINES*5-NPP) 22, 23, 22 LINES=LINES+l LENTH=5';LINES DO 25 I=l,LINES IF(I+4~~LINES-NPP) 25, 25, 24 LENTH=4;';LINES WRITE(3,IOll)( YY2(J),YYl(J),J=I,LENTH,LINES) WRITE(3,lOIO) Comment:

Smooth points using 5-point quadratic times to improve the appearance.

DO 28 KSM=l,S NT=NPP-2 DO 26 J3=3,NT YY2(JJ)=(3.4"YYl(JJ)

+ 2.4*(YYl(JJ-l)+YYl(JJ-tl))

fit,

repeat

a number of

AUTO-MATED

AND

ORD

SPECTROSCOPY

- 0.6"(YYl(JJ-2)+yYl(JJ+2)))/7.

1 26

CD

CONTINUE Comment: Points

1 and 2

A~(2.~"(~l(5)+yyl(1))-(~1(4)+YY1(2))-2.QYYl(3))/I4. ~=(2.0~(~~1(5)-~Yl(l))+(YY1(4)-YYl(2)))/10.

YY2(1)=4.O"A-2."B+YY2(3) YY2(2)= A-B+YY2(3) Comment: Points

27 28

NPP and NPP-1.

A=(2.O"(Wl(NPP)+YYl(NPP-4))-(YYl(NPP-l)+YYl(NPP-3)) 1 -2.O"Wl(NPP-2))/14. B=(2.O"(YY1(NPP)-W1(NPP-4))+(W1(NPP-1)-YY1(NPP-3)))/10. YY2(NPP)=4.*A+2.~:BYYZ(NPP-2) YYZ(NPP-l)= A+B+YY2(NPP-2) DO 27 JJ=l,NPP YYl(JJ)=W2(JJ) CONTINUE CONTINUE wL=sw Comment: Plot

smooth line

IVAR=4 DO 29 JJ=l,NPP YY=YYl(JJ) CALL PLOT(IVAR) WL=WL-CF 29 CONTINUE CALL PLOT (3) Comment: Type operator

message

WRITE (1,1013) PAUSE Comment: Test

30

1000 1001 1002 1003 1004 1005 1006

for another

set of data

CALL DATSW(O,J) GO TO ( 30, l),J STOP

FORMAT(30Al) FORMAT(25A1/8A1/3Al) FORMAT (lH0,40X,lgHORD/CD CALCULATIONS/lZHOINPUT DATA-) FORMAT (3X,12(1X,14)) FORMAT(lX,I4,1115,2H ",14,1115) FOPxMAT(F10.3) FORMAT( 16HlIDENTIFICATLON-/1X,16X,6HS~PLE,26X,7HSOLVENT,25X,4HD 1ATE/17X,30A1,2X,25Al,7X,8Al) 1007 FORMAT(1X,5HMODE-/1X,16X,3Al/lX,11HPARAMETERS-/1X,16X,4HMRW=,F6.2, llX,2HS=,T5.3,1X,4HLEN=,F5.3,1X,4HCON=,F5.3,1X,3HCS=,F5.2,lX,3HWE=, 2Fj.Z,lX,4HFDC=,F5.2,lX,3HSW=,F6.2,lX,3HCF=,F5.2//)

73

74

VISSER, MINNAAR

AND WEBB

1008 FORMAT(lH1,9X52HMEAN RESIDUE ROTATION lR./ 238X25HWAVELENGTH (NM) - W.L.// 31X120(1H=)/

(DEG.SQ.CM./DMOLE)

-

M.R,

238X25HWAVELENGTH (NM) - W.L.// 31X120(1H=)/ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1010 FORMAT(lXlZO(lH=)) 1011 FORMAT(lX5(1H"F8.1,Fl3.2,2X)) 1012 FORMAT(lX,35HNUMEER OF INPUT VALUES EXCEEDS 1800/) 1013 FORMAT(45HSET CONSOLE SWITCH '0' ON TO EXIT --START) END

Subroutine to Plot SUBROUTINE PLOT(IVAR) Comment: IVAR = 1 - plots outline, = 2 - plots a point. = 3 - terminates the =4 - plots the best YMIN,YMAx,XMIN,XMAX, must

1 2 3 4

axis

etc.

followed

by 1st point.

plotter. line through the points. be in common to preserve

their

INTEGERMODE(~),SAMPL(~O),REFER(~~),DATE(~) COMMONSW,MODE,SAMPL,REFER,DATE,X,Y,YMIN,YMAX,XMIN,XMAX,IPEN COMMON FW DATA IMODE / 'C' / IF(YMIN) 2, 4, 1 YMIN=O.O GOT0 4 IF (YMAX) 3, 4, 4 YMAx=o.o YMIN=FW xMAx=sw Comment: Direct GO TO (

prograrune

control

to specified

entry

point.

5, 21, 22, 23),IVAR

Comment: Intialise Initialise Calculate Calculate

the the the user

size of the rectangle enclosing the graph. origin. scale along the X and Y axis. unit to cm-conversion factor on each axis.

5 CALL INTS(YMAX,YMIN,DISTY,YO,LNTHY) LNTHY=LNTHY+l YO=YO-DISTY IFCYMIN) 6, 6, 7 6 Yo=o.O LNTHY=LNTHY-1 7 YS=17./(DISTY'cFLOAT(LNTHY)) SCAL=DIST'Y CALL INTS@MAX,XMIN,DISTX,XO,LNTHX) X0=X0-DISTX

values.

AUTOMATED

CD

AND

ORD

SPECTROSCOPY

LNTHX=LNTHX+l XS=13./(DIST%:FLOAT(LNTHX)) XSCAL=DISTX CALL FSIZE(16,ZO) XSS=XO-2.3IXS YSS=YO-1.3/x?

CALL FSCAL(XS,YS,XSS,YSS) CALL FGRID(O,XO,YO,DISTX,LNTHX) Comment: Set up X-axis

and insert

the X values.

XSTRT=XO+FLOAT(LNTHX)"DISTX XST=XSTRT-0.4"DISTX YSTRT=YO-.4/YS XWDTH=O.15 YHGHT=0.2 ANGLE=O.O DO 8 I=1,10 CALL FCHAR(XST,YSTRT,XWDTH,YHGHT,ANGLE) IXT=XSTRT+O.Ol WRITE(7,lOOO)IXT XSTRT=XSTRT-4."DISTX XST=XSTRT-0.4"DISTX IFCXSTRT-X0) 9, 8, 8 8 CONTINUE Comment: Set up the Y-axis

and insert

the Y values.

9 XSTRT=XO-1.2/XS IF(Y0) 11, 10, 10 10 YSTRT'YO GO TO 12 11 L=YO/(DISTY"3.0) YSTRT=-DISTY"3.O~~FLOAT(L) 12 ANGLE=0 DO 13 I=l,lO CALL FCHAR(XSTRT,YSTRT,XWDTH,YHGHT,ANGLE) WRITE(7,lOOl)YSTRT YSTRT=YSTRT+3.Of:DISTY IFCYSTRT-YMAX-DISTY) 13, 13, 14 13 CONTINUE 14 YSTRT=YO+DISTY"FLOAT(LNTHY) CALL FGRID(3,XO,YSTRT,DISTY,LNTHY) Comment: Print

the graph

labels.

YSTRT=YO1. Oh’s XSTRT=XO+5.75/XS

XWDTH=O.17 YHGHT=O.24 ANGLE=O. CALL FCHAR(XSTRT,YSTRT,XWDTH,YHGHT,ANGLE) WRITE(7.1002) YSTRT=Y&.O/YS XSTRT=XO-1.8/XS ANGLE=1.5708

76

VISSER,MINNAAB

AND WEBB

CALL FCHAR(XSTRT,YSTRT,XWDTH,YHGHT,ANGLE) Comment: Test whether on Y-axis.

15 16 17 18

the mode is CD or ORD and print

appropriate

label

DO 15 I=1,3 IF(IMODE-MODE(I)) 15, 16, 15 CONTINUE GO TO 17 WRITE(7,1003) GO TO 18 WRITE(7,1004) CONTINUE XSTRT=XO-1.51XS YSTRT=18.O"DISTY"FLOAT(LNTHY)/17.OtYO ANGLE=O.O CALL FCHAR(XSTRT,YSTRT,XWDTH,YHGHT,ANGLE) WRITE(7,1005)SAMPL,REFER,DATE Comment: If 0.0 is included

on Y-axis

plot

a line

across

graph

at Y=O.O,

IF(Y0) 19, 20, 19 19 CALL FPLOT(l,XO,O.O) XP=XO+FLOAT(LNTHX)*DISTX CALL FPLOT(2,XP,O.O) 20 IPEN=l Comment: Plot

a point.

21 CALL FPLOT(IPEN,X,Y) CALL POINT(2) IPEN=l RETURN Comment: Terminate

- raise

pen and return

to origin.

22 CALL FPLOT(l,XO,YO) Comment: Draws an X at bottom

corner

of page to check plotter

CALL POINT(Z) CALL FPLOT(l,XO,YO) RETURN Comment: Plot

the smooth line.

23 CALL FPLOT(IPEN,X,Y) IPEN=Z RETURN 1000 1001 1002 1003 1004 1005

FORMAT(I4) FORMAT(F7.0) FORMAT(16HWAVELENGTH (NM)) FORMAT(~~HMEAN RESIDUE ELLIPTICITY (DEG.SQ.CM/DM~LE)) FORMAT~~OHMEAN RESIDUE ROTATION (DEG.SQ.CM/DMOLE)) FORMAT(7HSAMPLE-,30A1,2X,8HSOLVENT-,25Al,ZX,5HDATE-,8Al) END

accuracy.

AUTOMATED

Subroutine

for Interval

CD

AND

ORD

77

SPECTROSCOPY

Sire IINTS)

SUBROUTINE INTS(YMAX,YMIN,YINT,YST,N) R=(YMAX-YMIN)120.0 Comment: Find

that multiple

of 5 which

just

exceeds

l/20

of range.

DO 1 I=l,lOOOO X=FLOAT(I)"5.0 IF(R-X)2,2,1 1 CONTINUE Comment: YINT is interval

to be used.

2 YINT=X IF(YMIN)3,3,6 Comment: Find

lowest

value

to be used for non-positive

lowest

value

to be used for positive

minimum.

3 no 4 I=1,10000 X=FLOAT(-I)"YINT IF(X-YMIN)5,4,4 4 CONTINUE 5 YST=X CO TO 9 Comment: Find 6 DO 7 I=l,lOOOO X=FLOAT(I)"YINT IF(X-YMIN)7,8,8 7 CONTINUE 8 YST=X-YINT Comment: Determine 9 DO 10 1=1,40 X=YST+FLOAT(I)"YINT IF(X-YMAX)lO,lO,ll 10 CONTINUE 11 N=I RETURN END

number of intervals

to be used.

minimum.