I-









.









                                         FIGUW 4
                             GAS CIIROMATO(;fiAPiI OF URINARY ACID PROPILE
                             A. PATIKIT ACIDOTIC (Z-17-73)
                                   I\" !`A'I'T)`yr' vfq."; 1, ( 2,. 1 (l-71 1


l.fIf!
n.52
3.51
f-l.23
Q.4P
-!I.51
').7c,
2.7')
n.74
I , :: 1
3 "I,

Areas

A r-as

Arr-as

Areas

Arwis

hrfvs

Areas

Arcas

At-pas

Art-as

Areas

11349.2/  21382.5

l&LX/ 109h7.3

6272.4/  6054.7


39;. 21  I;!i53.7


R35,1/  67621.3


7cn3.1/-330es.r

116?.7/ 2712.q

3293.4/  31f?4.7


2920.1/  5971.8


22El.E/  5751.0


lcZE3.1;/  19353.8

PATIC! =   0.5308 Locattan Error - 9

?????? ?? o 0.1771 Locatifh rrrar =`-2

F4T

PAT

PAT

PAT

I

1.0277

0.107G

0.1235

-0.2238

0.4270

        FIGURE 5
ANALYSIS OF 12 AMINO ACIDS IN URINE
   USING MASS FRAGMXNTOGRAPHY

Location Erroi = 2



Lccntfnn Error - -1



loc?tinn Frrnr = 2


Locatfon Crror = 2


LocatIon Error = 0



Location Error - 0

Lor?&an 'rror = -2


Locattnn rrror = 0


Loci+tlan Frror - -1


THE SIMULTANEOUS QUANTITATIPN OF TEN AMINO ACIDS IN SOIL EXTRACTS
       BY MA& FRAGMENTOGRAPHY

W. E. Pereira, Y. Hoyano, W. E. Reynolds, R. E. Summons and A. M. Duffield

    Department of Genetics, Stanford University Medical Center

               Stanford, California

Running Title:  Mass Fragmentography of Amino Acids

Address for Proofs: Dr. A. M. Duffield
            Department of Genetics
           Stanford University School of Medicine
            Stanford, California 94305

Received

P- 5-z


  The analysis of amino acids from terrestrial and extraterrestrial
sources is becoming increasingly, important (l-5) ,  The need for a specific,
                                    I

sensitive and rapid method of quantitation is desirable.  The methods
currentiy employed for amino acid analysis involve ion exchange procedures
(6,7) or gas chromatography (8-10). These techniques, although of

immense value, are limited by their non-specificity for the absolute

identification of any substance responsible for a gas chromatographic

peak.

  In the present communication we report an absolute, unambiguous

method for the positive identification rnd quantitation of ten amino acids

present in soil extracts using GLC-mass fragmentography. In mass

fragmentography the mass spectrometer is used only to detect certain

preselected ions known to be characteristic for each compound being
quantitated, and the internal standard.  The technique of mass fragmentography
                                           . .

using sector mass spectrometers is usually restricted to the simultaneous

monitoring of up to three integer mass values (11, 12), although with

one instrument five ions were used

spectrometer up to eight ions have

analog signals monitored (14). We

(13) o  Using a.quadrupole mass

been selected and their respective

now wish to report the modification

of the gas

previously

conqo~ of

chromatography-quadrupole mass spectrometer-computer system

described (15) for the simultaneous monitoring under computer
                          ., . . .                                               ,

          the ion currents from 2.5
  cc\nje br*w-- WIICS C Ld 750 i- con+tst 2 r;sc.l;Eetdcnteger mass _ values. -pjcsc via us cc-
                                ;yL  UVIIlCbIL 4or n`$l &8y--io+j ;I:;*(j SC'
P*r.tr'XIf required this number could be increased by suitable alteration of the

computer control programs.  Specifically we wish to report the application
                                .

of thii system to the quanti'tation of ten of the amino acids present in soil
                           . ___. --. _-
                                   .,
extracts .


Reagents:  A deuterated amino acid mixture was supplied by

Merck Laboratory Chemicals (New Jersey). 1.25N HCl in n-butanol,

25% (v/v) trifluoroacetic anhydride in methylene chloride and Tabsor

column packing were obtained from Regis Chemical Co., Illinois. A

standard amino acid solution was purchased from Pierce Chemical Co.,

Illinois.

Equipment:  A Varian model 1200 gas chromatograph was coupled

by an all glass membrane separator (16) to a Finnigan 1015 Quadrupole

mass spe-trometer which was interfaced to the ACME computer system of

the Stanford University Medical School (15). CLC separations were

conducted using a 6 foot by 4 mm. (I.D.) coiled glass column packed

with Tabsorb (Regis Chemical CO.).  The flow rate of the carrier gas

(helium) was 60 ml/minute.

. .

The uniqueness of the mass spectrometer instrumentation lies in the
                                                                        -_-

modified computer software (program) used.  The hardware is the system
previously described (15) and as:umes an operating cycle of:

(a) transmission of a control number, N, from the computer to an

interface controller which sets the quadrupole mass analyser to

  a particular mas-s point in the m/e continuum.
                                        --
(b) an integration of the ion.signal for a pre-set period, T

  (integration time = 8 milliseconds in our work), and
(c) computer reading of the integration value with a twelve bit A + D

      conversion.
     .                         * '
For the recording of normal mass spectra N is selected such that successive


cycles result in m/e values of 1,2, . . ...750. At the beginning of each
                   --

day the instrument is calibrated using a reference compound. Idiosyncracies

of the IBM 360,/50 to IBM 1800 computer data paths dictate that the mass
                                     s

values be buffered into groups of 250.%

   For normal g.c.-m.s. procedures the operator is allowed to select

a mass range of 1 to n x 250 (n = 1,2, or 3 buffers).  For mass fragmento-
graphy n is set to zero and instead,a "precision collect" buffer of 250
control-data acquisition cycles is employed.  The operator must then enter
the pre-selected m/e values he wishes to scan. When the precision collect
                   --

buffer is constructed, 10 cycles are allocated to each m/e value selected.
                                                               --

The first of the 10 cycles sets N to K -4.
                                           In   The returned integrated ion

measurement is discarded; this cycle serves only to slew the quadrupole
electronics from anywhere in the m/e continuum to the mass region of
                                      --

InterFst. The additional 9.cycles are used with N = N,-4....Nm.....Nm+4.

The returned values represent a set of readings about the m/e value of
                                                                  --

Interest + 0.5 amu.  The center three points are then smoothed with a

five point qudratic function (17).  The highest value of these three

smoothed points is then selected,as the precision collect value.  Thus
                                      ,

small drifts in calibration are corrected and a signal average obtained.
Finally ,i the abbreviated "spectrum" of 25 precision intensities for each

m/e are filed on disc.
a-

  Such a "spectrum" is recorded every 2 seconds and a summation of all

the ion intensities is used to cou$truct the ion chromatogram shown in Fig.

2.  Inditidual ion chromatograms can also be constructed if required

(Fig. 3).  A threshhold is established from the ion currents before and

aftet each gas chromatographic peak and a computer program performs

integration of the ion currents under each peak.


Procedure

  1 g of sieved, air-dried soil (Stanford University garden soil)

was refluxed with 6N HCl (10 ml) for 20 hrs.  The mixture was filtered
and the residue washed kth 1N HCl (5 ml).  The combined filtrate

and washings were extracted with chloroform (4 x 10 ml) and the

aqueous phase evaporated to dryness.  The residue is dissolved in

water (5 ml) and passed through a column of "Ion Retardation Resin"

AG 11-A8 (So-100 mesh, 1 x 21 cm).  The amino acids were eluted with
water (50 ml) and the eluate evaporated in vacua to dryness. The
residue is dissolved in water (5 ml) and placed on a column of

p-57


cation exchange resin (AC SOW-X12, SO-100 mesh, 1 x 21 cm) and washed

with water (50 ml) to remove neutral and anion contaminants.  The amino
acids were eluted with.4N NH4?H (80 ml) and the eluate evaporated to

dryness. The residue was disiolved in water and made up to a volume of

4 ml.  A portion of this solution (1 ml) was used for the amino acid

analysis using an amino acid ,analyser.  To another 2 ml of the processed

solution was added 2 ml of the deuterated amino acid standard solution

(100 mg in 100 ml of O.lN HCl) and the mixture evaporated to dryness.

The residue was refluxed with 1.2 N HCl in n-butanol (1 ml) for 30 min.

and evaporated to dryness in vacua.  To the residue trifluoroacetic

anhydride in methylene chloride (0.7: ml) was added and refluxed for 10

min.  The solution was evaporated to dryness at room temperature and the

residue dissolved in ethyl acetate (100 ~1). An aliquot (1 ~1) was
injected into the injector port of the gas chromatograph and the oven

kept at 100" for 1 min. when it was programmed at 4"/min. to 220'.

  To each of 4 tubes containing 2 ml of the deuterated amino acid
standard solution (100 mg in 100 ml of O.lN HCl) was added 150, 200,
250 and 300 ~1 respectively of a standard amino acid solution (2.5 ,,moles

                                                             -e
of each amino acid per ml).  The solutions were mixed and evaporated 'to

dryness.  Each residue was derivatized by the above method and an
aliquot of each (1 l.J) injected into the gas chromatograph which

was operated under the conditions described above.  This procedure

                                     . . . .

was used to construct a standard curve for the quantitation of each
am&o acid.  A typical standard curve is shown (Figure 1) for glutamic.

acid.

p- 5-z


RESULTS

The N-TFA, 0-n-butyl derivative was chosen for the derivatization
                              s

of amino acids for two reasons.. Firstly, these derivatives have
                 14

excellent glc separation characteristics @$j and secondly the selected
                                    .

characteristic fragment ions of the deuterated and non-deuterated

derivatives do not interfere with each other, nor with other a-amino

acids.  Table I records the individual ions monitored for quantitation

in the mass spectra of each of the deuterated and non-deuterated amino

acids.  Thezomput~tegrates-the-intensity of the deuterated and

Be-ratio-of-their-respective-peak-areas7

Our results of a typical soil analysis are compared with those

from an amino acid analyser in Table II. The higher value obtained

with lysine by the amino acid analyser is due to a ninhydrin positive

substance in soil interfering with the quantitation of lysine. In

this respect mass fragmentography is superior to the amino acid analyser

in that using a mass spectrometer as detector only characteristic

pre-selected ions are detected and quantitated and any impurity present'

under the same gas chromatographic peak is not measured*  A summation of

20 such characteristic ions was plotted as an ion chromatogram of a

derivatized soil sample and is shown in Fig. 2.

Preliminary experiments showed that when the deuterated amino

acid mixture was added directly to the soil sample extensive hydrogen-

deuterik exchange occurred during acid hydrolysis of the soil extract.

The removal of the isotopic label was catalysed by the hot mineral acid

in presence of excess mineral used in the soil hydrolysis step. Fox


and collaborators have reported (4) a similar finding concerning the
                                                                           _ -

decomposition of amino acids in soil upon direct acid hydrolysis. In

the present work the deuterated amino acid mixture was added just before

derivatization (i.e. after hydrolytic extraction of the soil) in order

to avoid this problem.  However, in cases where it is necessary to

quantitate the free amino acid content of complex mixtures, such as

In serum or urine samples, the deuterated amino acid mixture may be added

sample before processing without any deleterious

Although only ten amino acids present in soil were quantitated

the method can be extended to all the normal amino acids found in

protein.  The deuterated analogs of arginine, histidine, serine,

threonine and tyrosine are commercially available.  Appropriate

deuterated analogs of methionine, tryptophane, cysteine and cystine

would have to be chemically synthesized from the appropriate precursors.

In these instances at least two deuterium atoms should be incorporated

in non-exchangeable positions so that for the characteristic ion

chosen the P + 2 peak is separate from the 13 C isotope contribution

of the unlabeled amino acid.  Furthermore,  the deuterium substitution

need not be quantitative (>90%) provided the same characteristic ion

of that deuterated analog is used for the construction of a standard

curve such as Figure 1.
   -Lx


  Instrument analysis time is approximately one hour and with our

system we have been able to achieve accurate quantitation with samples
containing as little as 10 nano'grams of an amino acid,

SUMMARY

  A specific and sensitive method for the identification and

simultaneous quantitation by mass fragmentography of ten of the amino

acids present in soil has been developed.  The technique uses a computer

driven quadrupole mass spectrometer and a commercial preparation of

deuterated amino acids is used as internal standards for purposes of

quantitation.  The results obtained are comparable with those from an

amino acid analyser.  In the quadrupole mass spectrometer-computer

system used up to 25 pre-selected ions may be monitored sequentially.

This allows a maximum of 12 different amino acids (one specific ion
in each of the undeuterated and deuterated amino acid spectra) to be

quantitated.  The method is relatively rapid (analysis time of

approximately one hour) and is capable of the quantitation of nanogram

quantities of amino acids.

. . . . .

ACXNOWLEDGMEXTS

This research was funded by the Planetology Program Office, Office

of Space Science, NASA Headquarters under grant NGR-05-020-004.


REFERENCES

1.

2.


3.

4.

5.

6.

7.

8.

9.

10.

Il.

12.

'13.

14.

POCKLINGTON, R., Anal. Biochem. 45, 403 (1972).

TAYLOR, R. and DAVIES, M. G., Anal. Biochem. 51, 180 (1973).
                                                   -

HALPERN, B., WESTLEY, J. W., LEVINTHAL, E. C. and LEDERBERG, J.,

Life Sciences and Space Research - North Holland, Amsterdam, 239 (1967).

HARE, P. E., HARADA, K. and FOX, S. W., Proc. Apollo 11 Lunar

Science Conf. 2, 1799 (1970).
               =

KVENVOLDEN, K., LAWLESS, J. G., PERING, K., PETERSON, E., FLORES, J.,

PONNAMPERUMA, C., KAPLAN, I. R. and MOORE, C., Nature, 228, 923 (1970).
                                                               Z

SPACKMAN, D. H., STEIN, W. H. and MOORE, S., Anal. Chem. 3&, 1190 (1958).

HAMiLTON, P. B., Anal. Chem. 35, 2055 (1963).

GEHRKE, C. W., KUO, K. and ZUMWALT, R. W., J. Chromatog. 57,
                                                                 =

193 (1971).

GEHRKE, C. W., ZUMWALT, R. W. and WALL, L. L., J. Chromatog. 37,

398 (1968).                         . . .
                                     ._
JijNSSON, J., EYEM, J. and SJGQUIST, J., Anal. Biochem. 51, 204 (1973).
                                                               -

HAMMA!, C. G., HOLMSTEDT, B. and RYHAGE, R., Anal. Biochem. 2, 532

(1968).                                                 _-

HAMMAR, C. G. and HESSLING, R., Anal. Chem. 43, 298 (1971).
                                     --  -

SNEDDEN, W., PARKER, R. B. and WATTS, R. E., Int. Conf. on Mass
                                                   --
Spectrometry Brussels, 31&. i 4 Sept. 1970, Vol. 5, Institute
                          -

of Petroleum, London, and Elsevier, Amsterdam, 1971. p. 742.
                                . .

KNIGHT, J. B., Finnigin Spectra, &, No. 1 (1971).

.lS.. REYNOLDS, W.: E.; BACON;& A;, BRIDGES, J.. C., COBURN;T. C.,.

HALPEPN, B.,
.

LEDERBERG, J., LEVINTHAL, E. C., STEED, E. and TUCKER,

R. B., Anal.

        .`, .
Chem. 42, 1122 (1970).
      =


  16.  HAWES, J. E., MALLABY, R. and WILLIAMS, V. P., 2. Chromatog. Sci.
   2, 690 (1969).
(%H. GEHRKE, C. W. and STALLING, i. L., Separation g. 2, 101 (1967).
1q.6  PEREIRA, W. E., BACON, V. A., HOYANO, Y., SUMMONS, R. and DUFFIELD,
   A. k., Clin. Biochem. (In Press).


Table I.  CHARACTERISTIC FRAGMENT IONS SELECTED FOR MASS FRAGMENTOGRAPHY OF UNDEUTERATED AND-DEUTERATED
  .

N-TFA-0-IJ-BUTYL-AMINOrACIDS.

Amino
Acids i

Fragment Ion

VAL


GLY


ILEU


LEU

PRO

PHE


ASP


GLU


LYS

CH3CH=kICOCF3 (m/e 140)      CD3CD(NH2)COOH
I-C3H7CH=zHCOCF3 (m/e 168)     I-C3D7CD(NH2)COOH
CH2&COCF3 (m/e 126)        NH2CD2COOH
C2H5CH(CH3)CH=zHCOCF3 (m/e 182)   C2D5CD(CD3)CD(NH2)COOH
i-C3H7~2CH=kXOCF3 (m/e 182)    I-C3D7CD2CD(NU2)COOH

kCOCF3 (m/e 166)

C6H5CH=CHCODHl + (m/e 148)
BuOOCCH2CH&XOCF3 (m/.e 240)
HOOCCH2CH2CH=kKOCF3 (m/e 198)
CX2-CHCH2CH2CH-&COCF3 (m/e 180)

Deuterated Amino Acids

D

C6D5CD2CD(NH2)COOH
HOOCCD2CD(NH2)COOH
HOOCCD2CD2CD(NH2)COOH

Fragment Ion

CD3CD&COCF3 (m/e. 144)
                       . .
I-C3D7CD=hCOCF3 (m/e 176)
CD2=iHCOCF3 (m/e li8)
C2D5CD(CD3)CD=zHCOCf+3 x+/e 1%)
i-C3D7CD2CD=hCOCF~~~(m/e 192)

D     D
K2 hi -COG3 (m/e 173)
DD ; D

C6D5CD=CDC001q+ (m/0.155)
                      ,
BuOOCCD2CD=hCOCF3 (m/e 243)
HOOCCD2CD2CD=$HCOCFj, (m/e 203)
CD2-CDCD2CD2CD&O~~3 (m/e 188)


LEGENDS TO FIGURES

Fig. 1. Standard curve for the quantitation of Glutamic acid.

Fig. 2: Typical ion chromatogram of soil amino acids.
Ftg.3. fv\',sr Fc=p e-2          Q+ub& -i--*u-   O- f



                                .


Table II.  ANALYSIS OF AMINO ACIDS IN SOIL (pg/g SOIL)

Mass Fragmentography

Amino Acid

Ala

Val

GUY

Ileu


LeU


Pro


Phe

Amino Acid Analysis

    206.5

    148.3

    215.4

     95.4

    154.2

    143.4


     80.3


    218.3

    227.0

    129.7

Bl


198.7


151.0

ASP

Glu

LYS

S96.8

100.4

152.1

141.4


80.5


217.1

217.2

115.3

#2

202.7


150.5


201.6

100.2

149.7


142.8


80.7


219.8

215.6

113.5

83


198.3

149.9


201.3


92.3


154.2

141.2

80.0


219.9

214.1

114.9

P-Lb


Fig. 1.

GLU
- 4dded
D5^GLU -


                             3
                     J

3           E         i, I


IIC. 3


A Lomputer Uperated Mass Spectrometer System

W. E. Reynolds, V. A. Bacon, J. C. Bridges, T. C. Cohurn, Berthold Halpern,
Joshua Lederbcrg, E. C. Levinthal, Ernest Steed, and R. B. Tucker
Department o$ Genetics, Stan$ord University School of Medicine, Stanford, Calif. 94305

An integer resolution mass spectrometer-computer
system has been developed in which the computer
controls the "scan" of a mass spectrometer. In this
system, the computer queries the user for operating
parameters which are then translated into control
functions which operate the mass analyzer. The
spectral information acquired from the mass spec-
trometer is made available to the chemist within min-
utes in an on-line graphic system.
processing of GLC effluent are given. Examples of the

THE USE OF htAss SPECTROMETRY has been hampered by the
lagging development of a fast and convenient method of re-
ducing the spectral output of the mass spectrometer (MS)
to numerical data.  Usually the operator must convert a MS
chart recording, which is an analog plot of intensity cs. time,
to a digitized plot of intensity cs. mass number.  Because of
instrument instabilities, wide range of signal amplitudes,
large amounts of data, and other operational ditficulties,
(I; 2), it is often dificult and time-consuming to establish all
the correct mass peak identifications. One aid is to use a
reference compound (3) tither prior to the run or as an in-
ternal standard with the unknown sample.  By counting from
known mass peaks, unknown spectral peaks can bc idcntificd.
However, the processing of data by this tczhnique is still a
formidable task and it may take several days to accumulate
all the information from a gas chromatograph-mass spec-
trometer (GLC-hIS) run.

Several workers have demonstrated MS-computer systems
in which the computer monitors and records digital data
from a MS. In most of these applications the mass spcc-
trometer has operated indepcndcntly oi the computer, scannmg
in some time dcpcndcnt mode. mcnsuring ion intensities at
all points within the range of (500 to 5000 samples per second)
and afterward performs the computations required to reduce
the large amounts of digital data to useful information
(4-7). Much instrument time and sampling effort is ex-
pended in the intervals between integer peak positions where
there is little or no information.  One system that improved

(1) K. Biemann, "Mass Spectrometry." McGraw-Hill, New York,
1962, p 10.

(2) J. Lederberg, E. Levinthal, and Staff. "Cytochemical Studies
of Planetary Microorganisms Explorarlons in Exobiology,"
IRL Report No. 105-1, Instrumenration Research Laboratory,
Department of Genetics, Stanford University School of Medi-
tine, April 1966 to October 1966. NASA Accession No. N66-
34195. -
(3) J. H. Beynon. "Mass Spcctrometry and Its Applications to
Organic Chemistry," Elsevicr Publishers. Amsterdam, 1960,
p 44.

(4) R. A. Hitcs an K. Biemann. ANAL. CHEhq., 39,965 (1967).
(5) ILGd., 40, 1217 (196s).
(6) R. A. Hitcs, S. Markey, R. C. Murphy, and K. Biemnnn, 16th
Ann. CO/I./. ,lfrw Spcc/rarwrr.v Aliid To,Cs, ASTM E-I J,
Pittsburgh, Pa., hla>, 196s.
(7) R. B. Tucker, "A hlass Spcctromater Data Acquisition and
Analysis System."  IRL Report No. IOG?, Instrumcntntion
Research Laboratory. Depnrtmcnt of Gcncticb. St:tniorcl Um-
versity School of Medicine, NASA Acc:ss~on No. NOS-25743,
1968.

upon this latter ineficiency used step switches to step the
scan from position to position (8).

We now describe a MS-computer system, suitable for
routine laboratory use, in which the computer controls the
operation of a quadrupole mass spectrometer (9, 10). In
this system the "scan" is calibrated by relating known mass
positions of a reference compound to a computer g~ncxtcd
control voltage (V,).  R, is gencratcd as the result of a number
N, serit from the computer to a Digital-to-Analog (D-to-A)
converter in a MS-computer interface. The parameters of
this V,, or the N for each integer mass position, arc de-
termined by a computer program and stored in memory.
The subsequent use of this information allows the computcr-
directed MS output to be recorded directly as mass,chnrgz
(nz/r) cs. intensity.  On request, this data is then made al,ail-
able to the operator in an on-line system.
The use of this computer-.MS interaction, combined with
the decision-making ability of the opcra+?r, permits n sig-
nificant saving in data processing costs.  Furthermore, a
much larger duty cycle of analyzer "on peak" time is obtain-
able, resulting in the detection of more ions for a given rnxs
position than is possible in conventional time based scanning.
The new MS-computer system has at least thrcs unique
features. There is a hardware control intcrfncc to connt':t
the MS intimately with the computer; there is an improv:d
etlicicncy of information acquisition from spectral psahs that
are limited in ion production rates; and there is a uscr-
oriented control and data presentation system that csnc.xls
the foregoing details from the operator, but prssen:s 1i7c gxr
with prompt and concise data which include normalirsd mass
spectral plots.

The described system has evolved through three mass
spectrometers, three computers, and two basic computer pro-
grams (I I, 12).  The later systems have greater range. scnsi-
tivity, and convenience, but they all have a common concept.
Therefore the description that follows will be concrprunl
rather than specific to any one configuration.

(8) H. L. Friedman, H. W. Goldstein. and G. A. Griffith. "\lnjs
Spectrometric Thermal Annllsis of Polymrr DCCOnlpOhiIiJn
Products," 15th d~vr. CU/I~: .\fcrss S~cctronre!r.v rlllrc*ti 7'0,~jcs.
ASTM E-Id, Denver, Colo., hfay 1967.
(9) W. E. Reynolds, "A Small Computer Approach to Lou
Resolution hlnss Spectrometry." Pacitic Conference on Chc:l:ls:r>
and Spectroscopy, Anaheim. Cnlif.. November 1967.
(IO) W. E. Reynolds. T. B. Coburn. J. Bridges. and R. Tuc`ker.
"A Computer Operated ,Ilass Spectrometer S~stcm." IRL
Report No. 1062. lnstrumcnration Research Laboratorv [)c-
partment of Genetics, Stanford Univcwty School of \lc::c!zc.
NASA Accession No. N68-l ! S69. Nov. 1967.
(11) W. E. Reynolds. R. B. Tucker, R. A. Stillman. and J. C.
Bridges, "hlass Spcctromcrers In a Time Shxcd C:xn:~u!~r
Environment,"  I7 t h rlrur. Co,rjI iIl:,ss Sprcrror,~c*tr~ .~li!;,x
Topics, ASThl E-14. 1969.

(I?) J. Lectcrberg. E. Lcventh:ll. and StatT. "C! tochmxnl Stt~d~c; ot
Planetary hlicroorgcanisms Esplorshons in t\;olxoioc~ ." II;L
Report No. 1076. Instrumcntntion Rcscarch Labornror). I):-
partmcnt of Gcnctizs. Stnnf'urd C'mvcrsity School of X1:, I;,!::.
October 1967 to r\prll IYM.  Appcndiu A IS a ~LI!~~I~IICLI IC;':.IIL
of the abovc "hlass Spcctrometcrs in 2 Tuw Shurcd t. II\ iron-
menl," NASA Accession No. N6S-ZYS-IG.

   Reprinted from ANALYTICAL CHEMISTRY, Vol. 42. Papc 1112, Scptentbcr 1970
Copyright 1970 by the Amcricm Chemical Society and rcprintcd by permission of the copyright owner

P-7

1 ,


The present system is operating with a Finnigan 1015
quadrupole MS and a Varian Aerograph GOOD chromato-
graph.  The same computer programs and a similar interface
were also operated successfully with a Bendix Time-of-Flight
(T-o-F) MS (13) and an EAI qundrupole (14) MS. In all
cases the GLC-MS, the teletypew/riter, and the digital plotter
were situated in a wet chemical laboratory.

A schematic diagram of the GLC-MS combination is
shown in Figure 1. The emuent from the gas chromato-
graph, equipped with a flame ionization detector, first passes
through a variable splitter that diverts between Ii3 and *I?
of the flow through a Biemnnn separator (15) and into the
MS.  A solenoid-actuated valve in this line helps to keep the
large initial solvent peak from entering the MS system. A
reference gas reservoir containing a fluorine compound at a
vapor pressure of approximately 3 X IO--' Torr is also in-
corporated in the system and is connected to the MS by
another solenoid valve. The computer, tin the interfacing
electronics, has direct control of gas valves, and can valve in
or shut off the reference gas whenever it is needed for the
calibration routine.

These valves were constructed in our shops in such a way
that the back side is open to the vacuum system when the
valve is closed.  This avoids the common pressure burst when
conventional valves are opened to a vacuum.

The right side of Figure 1 illustrates the major components
and functions of the interface.  This computer-MS interface
was built in our Instrumentation Research Laboratory (16)
and contains all the electronics not normally supplied with a
standard configuration IMS or computer.  Al1 of thcl operating
parameters of the hlS are, or may be. controlled by a digital
word (binary number) sent from the computer.  The principal
control is ricl fhe "N" register to the D-to-A converter.  The
analog signal, V,, from the D-to-A sets and holds the mass
analyzer to pass ions of a predetermined ))I e.  Alternately
the digital output may be coded to operate auxiliary control
functions, such as actuate valves. set amplifier gains, the low
speed multiplexer, or enable the digital plotter.

STlEV'3 ONll33NN03 UOHS
      1

The characteristic method of controlling the nl:e passband
and taking measurements while the mass analyzer dwells
upon a m/e value converts what is normally measured as a
time dependent parameter, to a stationary signal. This
statistically stationary property of the signal enables the em-
ployment of full integration to enhance signal to noise.  Both
the electrometer and the integrator are standard commercial
FET operational amplifiers of the 550.00 class. The time
allowed for integration and the operation of the integrator
reset are controlled by signals (numbers) from the computer
to the "T" register.  The output of the integrator is sampled,
held, and read ciu the Analog-to-Digital (A-to-D) converter.
Auxiliary signal sensing is provided by the low speed
multiplexer. This is useful to determine the automatic
settings for self calibration,or may be used to record tempcra-
ture, pressure, etc. These sense functions, plus some valve

(13) D. B. Harrington and R. S. Gohlkc. "High Resolution Time of
Flight Mass Spectrometers." 10th .Irw. Coqj: ,Iltrss Sp~wrum-
erry A//id Topics, ASTM E-14. New Orlcnns. La.. 1962.
(14) W. I'& H. I'. Keinhard, and U. van Znhn, Z. Plrys., 152,
143 (1958).

Figure 1. The GLC-AIS instrumentation and the electronics
interface to enable computer systems integration

control and checkout functions, are controlled by the "C'
register.

(IS) J. T. Watson and K. Bicmnnn. ANAL. CIIEM., 37, WI (1965).
(16) W. E. Kqnolds, J. C. Hric!gcs. R. B. Tucker, and T. U. Co-
burn, "Computer Control of Xfass AnalyLcrs," 16th .&III. Co\!/:
Mass Sprclrowe~ry d//M Topics. ASTXl E-14, Pittsburgh. Pa..
1968.

      Thcrr are no manual opcrntor control functions in :lny o'
      the above steps.  The control is nccompli>hcd iIt tl~ I<!:[> 11
     writer keyboard. This keeps the system llcsiblc ant! IILI,L,.
     it indcpendcnt of the idiosyncrasies of indi\,idual con~put~ I
                                   P-1'
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970 . 1123


    COMPUTER DIALOGUE
  (The rcser's respome is underlined)
OPTION = collect
EXPERIMENT; = test
               -

T=g

MODE = single

PLOT, TYPE, or FILE = plot
                   -

BY MASS, AMP. or ENTIRE = entire

FROM MASS = 1
g,;g n

PLOT, TYPE, or FILE = 1
T=I
EXPExIMENT,+' = MS26x15
              --~
CONTINUATION = yes
T=6            -
MODE = continuous

# OF SPECTRA = 130
FILED IN POSITION 930 to 1070
T=/
EXPE`i%IMENT; = /
OPTION = sum -
EXPn = MS26x15
      _~---
FROM SPECTRU,M = 9-U)
TO = 1020          -
FROMXSS = ilo

TO MASS = 500
PLOT = yes -

EXP,+ =, 1
OPTION = plot
EXPERIMEtii+ = XlS26x15
              _- -
FRO&M SPECTRU.M = 957
TO = 957           -

&OM%iASS = 1
TO MASS = 340 -
          -

interrupt lines and/or individual computer characteristics.
This straightforward system definition makes the software
design much like conventional computer programming rather
than encouraging intricate techniques highly dependent upon
the specific hardware.
Thus the system is not oriented specifically to any given
computer.  It has operated on an early model LINC (17) com-
puter with 2K words of 12 bits. memory, and on a time-shared,
locally programmed, IBM 360;50, butTered with an IBhl 1800
(18). In all cases the computer was somewhat remote,
separated by some 500 ft of cable from the rest of the in-
strumentation.  The system is very economical of computer
resources.  Most of todak's small general purpose computers
would be able to operate the described functions if it were
desired to avoid time-shared computer dependency. Some




(17) R. W. Stacy and B. Waxman. "Computers in Biomedical Re-
search." Vol. II. Academic Press. New York. N. Y.. 1965. DD
35-66..                                     . .

(18) W. J. Sanders, G. Breitbard. G. Wledcrhold. er (I/.. "An Ad-
vanced Computer for hlcdicnl Research." Full Joiut Cortrpwr
Colt/. Proc., ACM, Anaheim, Calif., 1967, p 497.

COhlMEm

The user reauests the data collection chase.
A catch-all name, "test". is given; the spectrum
will be used simply for a systems check.

An integration time of 35 milliseconds per
peak is requested.

Only one spectrum will be taken (single mode).
The data is acquired after this answer.
The user can plot, type. or file the data col-
lected; here a plot is requested.
The user can plot selected masses, the
highest intensities, or the entire
spectrum within requested limits.
The user indicates the limits.

A QUICK plot omits annotation, etc.
Note that "y" and "n" mean "yes" and "no".
The user completed the checkout and now
wishes to proceed with the experiment.
The "/" is used to backup through the
conversation.

An existing experiment name is given here.
The user confirms that the spectra are
to be added to the eslsting experiment file.

The user requests that spectra be collected
continuously until 130 are taken.

Data collection is complete.

The user then wishes to sum the elements of
each spectrum to produce a "total ion"
plot, analogous to a GLC trace.

Mass position 40 to 500 are summed for each
of the collected spectra.

The "total ion" curve is now plotted.
(Figures 6 and 7 are illustrative of this
&tiple dialogue.)

Guided by the total ion plot. the user
will plot interesting m3ss spectra.
Only the spectrum filed in position 957
is chosen.

The plot (Figure 17) is drawn and normalized
to the base peak, nlje = 31.

Figure 2.  An example of the user-computer dialogue during operation

sort of magnetic storage for object code programs and data
storage is most desirable. DEC-t)-pe tapes ha;-c be:n uzd
on the LINC system and disc packs on the IB\l s\-stem.



        THE SOFIV.IRE STRUCTURE

The objectives of the software are to operate and conrrol
the MS, acquire data from the MS, process and prcsznr rhis
data in a manner useful to the chemist, and provide ctrtn:n
control and information to aid in maintaining and ser\-l:ing
the instrument.
With the program loaded into the computer, the Csfr
requests any one of several functions (see Table I) by t!Fing
the name of that function. The computer responds \x::h a
series of prompts (see Figure 2) to elicit user m;lcrocomm:!ntis.
The computer then generates the detailed conrrol funz!ons
to perform the assigned task.  At the comp:<tion of the :.:,i.
requests are made for new parameters.  By striking the si.tlh
("/"), the user can "backup" through anj' conversntion to
correct errors or to go to a JilTercnt function.  This con\.::>.!-
tion technique makes the system both tlesiblrt an;l r<ason.l>l)
self-instructing.

1124  .  ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970                        P- 72


      Table I.  A List of Program Options
(1) CALIBRATE: Creates an accurate N Table. The .V "urn-
hers which correspond to the peaks in the reference gas are
used as the end points of a piecewise linear interpolation pro-
cedure for calculating a complete .V Table.
(2) COLLECT: Is the primary data collection step. It is here
that the 750 N Table values are sent to the MS and the 750
m/e intensities rccordcd. This operation can be repeated at
five-second intervals as the data are tiled on disk under an e.y-
periment name.
(3) TYPE: Allows the user to print out spectral data by indicating
what spectra in a given file are to be reviewed.  The user can
request that the amplitudes of particular rtr/e positions be
typed ; that a given number of the highest amplitudes be
typed, or that a consecutive number ot' them over a given range
be typed.
(4) PLOT:  Enables the user to have bar graphs produced by the
computer controlled digitai plotter. The amplitudes to be
plotted can be selected with the same flexibility as described in
  TYPE.
(5) SUM:  Produces a plot of the total ion current over a series
of gathered spectra.  All responses of a spectrum are summed
to produce one datum point on the plot. This plot corre-
spondsclosely with the GLC output when runrung with theGLC.
(6) TRACE: Produces a record of a spectrum similar to the
normal chart recording output. The analyzer is sampled at
all N values (about 10 per amu) over a given range and the
result is plotted as a "broken line." (Used for system check
out)

(7) MONITOR: Provides for inspecting the peak profiles by
sampling the ,$>ctrum around a given or position.  The
gathered dara are then typed out.  (Normally used for system
service or service log)
(8) DISPLAY:  Enables the user to display a given mass position
(or N numbcrj in the center of the console oscilloscope.  (Used
in the adjustment of the mass spectrometer)
(9) GAS:  Allows the user to remot+ turn the reference gas on or
  off.  This is helpful when operating the system from a remote
position.

n 31      69
:! 1'"     1212
         .0600

2060
.7600

&
2507
.260I

.il.
   169     :
   3327 1
D    1.300 #

PRES  Jr lo'7/orr     T FACTOR    40

rlICA '  40,&U.      123351    L/he
           I

IONV   20 r&,;r's      691218    dafe

          Figure 3.  A monitor plot indication of instrument scr\iccability

The example of a user-computer conversation gi*:cn in
Figure 2 represents the day-to-day conlput-r-r~sc:!r~her
dialogue given to direct the system's olxrarion. D:?per
level programming may be done at the terminal to rcdctine
these functions or to add new modes. AddiIionnl b!`;tcm
development may be done by the chemist-user, or his program-
mer in a manner typical of general purpcse ccrnptit<r soft-
ware. In normal daily practice, the user first rcqucsts the
calibrate function and then proceeds to data acquisition,
analysis, and presentation. Usually the calibrntion is done
once every four hours.

It is this calibration subsection of the prcgrnm rhnt nssigns
to each integer mass position a value Nwhich when wnt to the
D-to-A converter in the interface, will set the mass analyzer
to pass that particular species oinz.`e.  During this caiibration
phase a reference compound (perfluorotributylnmine. i-C-13),
is introduced into the ,&IS. The calibrarion proxdure in
addition to determining the N values, makes dltn ?.~~ailnble
that will aid the operator in making qua1itstii.e judgments
about the stability, sensitivity, and resolution oi thz \fS.
-41~0 a service or maintenance reccrd plot is n!x~l~ble (xc
Figure 3), that, at least indirectly, shows these and other im-
portant instrument conditions.  Figure 3 is acttxiiy 12 trxsd
segments of a complete spectra, each segnent co\ erin_r a szan
of about 4 amu and each taken at a diffsrcnt int<gratlon time
(gain).  The m/e value, its position. and a parnmetrrr indicat-
ing the gain is automatically printed be!ow each peak.  The
date and time is printed by the computer, but at prssent thz
operator must insert the sample pressure and icnimion pa-
rameters.  The file of these plots represents an exc?!lent rec-
ord of the instrument's serviceability,  The czlibrntion is
automatic and its use less complicated than ths dsscrlption.
It takes about 5 minutes, after which the refcrcnce gss is
pumped out of the system.  In the IBM 360-1600 system, the
time is used to compile the main program.

L+x
    264
. .
&I    5381  :;
    .se00 2.

I

I

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ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970 . 1125


Total spectral data acquisition time depends upon the
number of m/e positions measured and the integration time
allowed per position.  It may be calculated:
    Spectrum acquisition time = P X (T, + 2")    (1)
where P is the number of m/e positions to be measured (they do
llot have to becontiguous or sequentiai), TI is a transition time
(2 msec in our system), and T is the integration time per peak
(nominally 6 to 17 msec, but we have usefully used 1 to 1000
msec).

Normally data are collected at each integer m/e position I
through 750.  The 750 N values arc sent through the D-to-A
converter to the MS and the 750 responses (a full spectrum)
are recorded by the computer cia the A-to-D converter.  This
process can be repeated approximately every 5 to 10 seconds
for an arbitrary number of times.  The spectra thus gathered
are stored by the computer on magnetic disks or tapes.  Pro-
gram changes may be made lo measure any subset of the 750
nr/e positions and thus achieve faster repetitive spectra.  Con-
versely more measurements may be made at any specific
peak position, a technique which may be used for accurate
isotopic ratio measurements.

Since many spectra are taken and stored during a GLC run
or a solid probe experiment, the user requires fast methods to
evaluate the data. The more useful data abstracting pro-
grams we use are:
`THE MATRIX SEALH. The user specifies which group of
spectra, what range of mass values in each spectra, and how
many large peaks he wants abstracted from each spectrum.
An abstract of these highest peaks is then typed out and in
many cases this abstract contains useful chemical information
or at least indicates the spectrum of interest.
THE TI~IE PRUEXATION (PLOT).  This is a computer drawn
plot of certain peak intensities or a sum of all peak intensities
ofeach spectrum (total ion current) plotted against time.  The
latter gives a good reproduction of the GLC curve and also
indexes the spectra of interest (19).

NOR~~ALIZED SPECTRUXI PLOTS. Conventional bar graphs
of mass US. time, normalized and annotated, are routinely
available.

AU data outputs are in the laboratory and are available
immediately after data acquisition.  All spectra are filed and
may be recalled at a later time or date and reprocessed in any
way desired.

Involved programs of these magnitudes are specifically
dependent upon the language of a given computer. The
logic may be easily transferred. but in general the specific pro-
gram may not.  We have about 4 man-years of programming
invested in this system.

THEORY OF OPERATION

During spectrum data acquisition, the computer directs the
mass analyzer to a program-selected mass position and reads
the output intensity of the MS. The mass analyzer is not
swept in a conventional sense.  As indicated in Figure 1, it is
controlled by a voltage (V,) such that

           m/e = WC)           (2)

where m/e is the mass/charge ratio and f(V,) is a monotonic
function characterized by the MS.  For every Mi, (M = ~!e),
to be passed by the mass analyzer, the computer has (according
to the prior run calibration program) a digital number N,

(19) R. A. Hires and K. Biemann, "Advances in Mnss Spectrometry
IV," Elsevier Publishers, Amsterdam, 1965, p 37.

which is transformed by the D-to-A converter to the voltage
VW

The determination of these values, Nr. is accomplished b)
the calibration program.  The value of IV for 12 keq ptxks of
the reference compound are known to approximately 1 amu
from prior calibrations.  The actual ,V value for the ccntrold
of each of these peaks is then determined by detailed examinn-
tion of the rll/e continuum in each of these areas.  Sutficlcnt
detail is obtained by designing the D-to-A resolution to be
10 or more values per peak width.
After determining these exact 1 2 Iv VaIues. linear interpola-
tion. superimposed uF:on the analyticai function, 111'e = jiVCj,
is used to expand the list of 12 experimentally determined
values to a full tabie of 750 entries. (The analytical func-
tion of tnie to control voltage is linear for the quad-
rupole and parabolic, m/e = k(Vcz), in the case of the T-o-F
MS.)

Thus the procedure to measure the intensity at any .Cli is as
fol1ows:  a. The number NI which corresponds to the se-
lected rll/e ratio (1Mi) is loaded from the computer into the D-
to-A converter. This sets the control voltage, 5/,, to the
mass analyzer. The output of the mass spectrometer is
proportional to the quantity of ions, IV,, passed from the
sample.

6. An analog circuit, reset and released by the computer,
integrates the output of the MS.

c. Several milliseconds after the integrator is released. (the
choice of integration time was initially supplied by the user
upon program request), the computer samples the output of
the integrator by means of an A-to-D converter.  This digital
value is stored as the intensity of Mi.

Steps (a) through (c) are repeated to acquire a complete
spectrum.

The fundamental restraint upon this system is the drift of
the function 1~ = j(V,) following calibration.  Our experience
with the Finnigan 1015 afld a Bendix T-o-F instrument and
our interface, is that this drift causes an error in ;V of less than
1,`) the value from one Nentry to the next in a l-hour period.
This is sufficiently small to allow an unambiguous mass idrnti-
fication.

Table II contains comparisons of signal-to-noise ratios and
the following defined figures of merit.  The comparisons are
made between the described control system. a linear scan m
time, a parabolic scan in time such as the T-o-F, and the ex-
ponential time scan characteristic of magnetic instruments.

Uniform conditions are used to give realistic values for
comparison; it is assumed that in each case the peak jha?es are
uniform if scanned in time, and that they are gaussian. and
that the resolution is commensurate with the 10% va!lcy (5 ",
points on a single peak side) definition (3).  In order to give
typical comparison figures. it is further assumed that a spt'c-
trum will be taken from mass 50 to 500 in 4500 milliseconds.

The first column in Table II is the time the mass snnlqzer
is on or about the mass position.  In the case of the computer
control system, the 4500 milliseconds is divided equally into
450 periods of 10 milliseconds each. Two milliseconds nre
allowed for each transition, and the mass analyzer will dwsll
on the peak position for 8 milliseconds.  In the CCISC` of a con-
ventional linear scan, the analyzer will enter a peak nren and
leave it 10 milliseconds I;lter.  By the 10% vnllc!~ con\`c`niLzn.
this means the time from the beginning 5 x level to the end 5 "g
level of a single peak.  However for the parabolic C:ISC (the
T-o-F) it bill be found th;u the resolution of the mstriimcnt
will have to be set for the uork case, peaks 499 and YlO.  It
will be found that there is 6.6 milliseconds between thcbc pc'ak

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1126  o ANALYTICAL CHEMISTRY. VOL. 42, NO. 11, SEPTEMBER 1970                                     . ,


Type of scan
Control and
integrate
Linear scan
m = kf
Parabolic scan
m = kta
Exponential scan
~ m=m.ekl

Table II. A Comparison of Attributes Affecting Signal-to-Soise Efficiencies
Typical Operation Condition:  Scan from nr/e 50 to 500 in 4500 Xlilliseconds
  Time on,
  or between                  Ions detected:
  5 % points,      Time constant    (Peak intensities     Effective noise
of a peek, msec   of amplifiers, msec   of n ions/m@    bandwidth, Hz

Figure of merit:
1000,`~ X de:ccte;l
ions,`bandv.tdth

8          VA         8.011            40          200

10            2            5.1 n           80           64

6.6           1.3          3.4 n          120           28

3.9          0.8           2.0 n          200            10

centers, and the theoretical 5% heights must be at the mid-
point. Since all peaks are similar, all peaks of the scan will be
just 6.6 milliseconds from 5% point to 5% point. During
34% of the scan time, the analyzer is not in the area of any
peak position at all; but is mostly between peaks in the low
mass range. The case of the exponential scans is similar.
In this case it will be found that the peaks are 3.9 milliseconds
wide and 61% of the time no information can get through the
instrument.

The next column indicates the time constant (7) of the ampli-
fier channel appropriate to the scan pammctcrs.  The control
`system uses a full nitegrator, so the entry is not applicable.  In
the conventional scanning system, the time constant is usually
chosen as large as skewing permits to integrate signal and
discriminate against noise. The relationship between 7 and
the 3-db bandwidth (jhB) of an amplilier is simply r =
I/(2&). If 7 is chosen to be large, peak skewing and
broadening as illustrated in Figure 3 will occur.  If T is chosen
small, the bandwidth with its attendant noise is excessive and
there is little integration of the signal.

This is the dilemma always faced by the user of linear ampli-
fier circuits:  the desire to limit amplifier bandpass to smooth
the signal, as opposed to the need for a wide bandpass to pass

the signal without distortion. Since the purpose here is to
compare our described amplifier and integrator system with
conventional linear amplifiers, a T of 0.2 is assumed for the
conventional case.  This T is still large enough to cause deg-
radation of resolution in the conventional output signal (25
to 35% depending upon the definition used). It is felt that
this choice represents a fairly typical operational psrametsr.
The assumption of a rigorous lower value would rcsalt in an
unnecessary, and perhaps unrealistic, comparison ndiantngz
for the described control and inteprate signal skstcm.

The column "Effective Noise Bandwidth" is the !I:. for the
time dependent scans. However an equivalent 3-cib band-
width is not as well defined for the integrator.  It can be
shown that for an integration interval. T, (8 miliiseconds in
this example) an .& may be determined such that a linear
amplifier of bandwidth I;, would pass the same amount of
"white" noise as the integrator.  The actual bandpxs oi an
integrator is a sin(.y)`x type function.
The white noise power passed by either qstem may be es-
pressed as an integration of the white noise model, r'*`, (29)

(20) W. B. Davenport, Jr., and W. L. Root, "Random Signals and
Noise," McGraw-Hill, New York, 1958, p 88.

OUTPUT OF A UNITY     r-v-l

AMPLIFIER

TIME CONSTANTS

T

T

i  \?t 2=0.15 XT

T milliseconds

V'
In

R&=2

          1.35 T

Figure 4. Peak broadening and skewing effect of narrow bandr$idth amplifiers         p- 7<
                                                                          

                ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970  o  1127