ANALYTICAL CHEMISTRY, VOL. 51, NO. 4 , APRIL 1979 Research Council. Nucl. Sci. Ser., NAS-NS 3107, 25 (1963). (14) M. Epherre and C. Seide, Phys. Rev. C , 3, 2167 (1971). (15) J. A. Cooper, Battelle Northwest Laboratories, Report BNWL-SA-4690.
RECEIVED for review Xovember 20, 1978. Accepted January
575
23, 1979. This work is supported by the National Science and Foundation and the Division Of mental Research of t h e Department of Energy. I t was presented (by S.S.M.) a t the 176th Meeting of the American Chemical Society, Miami Beach, Fla., September 10-15,1978.
Continuum Source Atomic Fluorescence Detector for Liquid Chromatography Darryl D. Siemer,
’ Prabhakaran Koteel, Daniel T. Haworth,
William J. Taraszewski, and Stephen R. Lawson
Department of Chemistry, Marquette University, Milwaukee, Wisconsin 53233
A preliminary study of interfacing of a high pressure liquid chromatography instrument with a continuous source atomic fluorescence detector is reported. The chemical system Investigated is the acetylation reaction of ferrocene by acetic anhydride. The retention data from the conventional UV molecular absorption detector are in excellent agreement with those obtained using the atomic fluorescence detector monitoring the iron fluorescence signal. The metal-containing compounds are more easily quantitated with the fluorescence detector, both because it does not respond to purely organic interferants co-eluting with the compounds of interest and because the integrated response is insenskive to the chemical form of the compound.
High pressure liquid chromatography (HPLC) detectors have been reviewed by several authors (1-3,Detectors based on refractive index, UV or visible light absorption, heat of absorption, electrical conductivity, flame ionization and molecular fluorescence are commercially available. Koen et al. (8) have described a polarographic detector. Other detectors described in the literature include light scattering (9), IR absorption (IO),vapor pressure ( I I ) , and scintillation (12). Metal specific detection methods such as atomic absorption simplify t h e complex chromatograms obtained when other compounds are run with mixtures of organometallic compounds. Gonzales a n d Rose (13)and Longbottom ( 1 4 ) used atomic absorption for the detection of mercury after separation from alkylated mercuric compounds by gas chromatography. Manahan and Jones (15)used atomic absorption as a detector for H P L C , a n d Fernandez (16) reviewed atomic absorption chromatography detectors for use in metal speciation applications. VanLoon (17) was the first to report a line source atomic fluorescence detector for HPLC. Continuum source atomic fluorescence spectroscopy (CSAFS) shows promise for use as a metal specific H P L C detector. Flame atomization CSAFS possesses sub-part per million sensitivity for many elements and is readily amenable for multielement analysis by using either a rapid scanning monochromator or a dedicated polychromator/multiple photomultiplier system. Interfacing of a H P L C instrument with a CSAFS detector system can be accomplished very Present address, Allied Chemical Corporation, CPP-602, 550 2nd Street, Idaho Falls, Idaho 83401. 0003-2700/79/035 1-0575$01.30/0
simply by connecting the outlet of the chromatograph to the nebulizer pick-up tube of t h e burner. Eimac xenon lamp excited CSAFS does not require t h e operator to obtain different source lamps for each element as is t h e case with t h e atomic absorption or lines source AFS detectors previously described. This fact makes the CFAFS detector inherently more flexible than line source based instruments. This paper describes the use of a first generation CSAFS detector with an HPLC instrument t o follow t h e progress of a typical organometallic reaction. This particular reaction (the acetylation of ferrocene giving both acetylferrocene and diacetylferrocene) has been studied by several workers (18-21) using other analytical techniques to follow the relative concentrations of reactants and products throughout the reaction. In Haworth and Liu’s study (21)the reaction was monitored by HPLC using a conventional fixed wavelength (254 nm) UV absorption detector, and both t h e chromatographic system and t h e sample preparation steps used in t h a t work were largely duplicated in this project.
EXPERIMENTAL Equipment. A Tracor 995 isochratic pump with a Tracor 960 UV absorption detector (254 nm) equipped with an injection port was used for the separation of ferrocene (Fc), acetylferrocene(Fc,) and diacetylferrocene (Fez). The outlet of the UV detector from the HPLC instrument was connected to the aspirator of a homemade acetylene-air capillary tube burner which was mounted on a Perkin-Elmer nebulizer scavenged from a PE model 290 atomic absorption unit. The burner was constructed of 20-gage stainless steel disposable syringe needles embedded in high temperature ceramic furnace cement (Sauereisen) in a 1.2-cm i.d. thin wall (0.7 mm), aluminum tube. This tube was centered in a 2.5-cm diameter copper tube, and the gap between the concentric tubes was filled with 18-gage needles embedded in more cement. A flow of argon could be directed through the outer row of tubes to sheath the flame from the atmosphere. A Varian 300 W Eimac xenon continuum lamp powered by a Varian PS 300-1 power supply served as the spectral excitation source. A 2.5-cm diameter, 8-cm focal length quartz lens focused an image of the illuminated portion of the flame onto the entrance slit of the monochromator. Conventional 90’ optical geometry was used. A light baffle made from a 2-inch copper “cross T” plumbing fitting was used to keep stray-light to a practical minimum (Figure 1). Holes (2-cmdiameter) were drilled at right angles to the plane of the fitting a t the juncture of the “T’s” to permit viewing of the signal. No attempt was made to obtain multiple passes of the excitation source beam through the flame through the use of a reflector on the side of the flame opposite of the lamp because of the potential of overheating the lamp. The monochromator used was an f 3.5,lOO-mm efl Oriel ‘C 1979 American Chemical Society
576
ANALYTICAL CHEMISTRY, VOL. 51, NO. 4, APRIL 1979
Table I.
Resolution Data
detection method UVQ CSAF~
Fc/Fc,
Fc,/Fc,
FciFc,
0.4 1.0
1.18 1.41
2.36
1.77
a Flow rate ( 1 . 5 mL/min at 300 psi ( 2 mPa)), UV detector at 254 nm. Fe line a t 248.3 nm. Pulsed source, lock-in at 100-mV sensitivity, C,H, :air burner; standard solution 12, 25, and 25 mg of Fc, Fc, and Fc,, respective-
lv. Flgure 1. Block diagram of instrument. (A) Xenon lamp power supply; (B) Xe lamp housing; (C) quartz lens; (D) copper light shield: (E) top
of burner; (F) monochromator; (G) photomultiplier; (H) PMT power supply; (I) lock-in amplifier; (J) recorder; (K) 50-Hz square wave signal generator; (L) HPLC instrument; (M) exit tube from UV detector Model 7249 equipped with a 2400 lines/mm grating. A variable width entrance slit and a fixed width exit slit giving a bandpass of approximately 1 nm was used for most of the work. The light detector was an IP 28 photomultiplier tube powered with a Fluke regulated high voltage supply. In an attempt to correct for the very considerable molecular fluorescence error signals encountered from PO bands in the vicinity of the iron resonance lines, some experiments were performed using a wavelength modulation system instead of the more conventional source lamp modulation AFS system. This was implemented by vibrating (25 Hz) a 9-cm focal length, 2.5-cm diameter, quartz lens in front of the monochromator exit slit within the monochromator with a miniature (6 V, 50 Q ) relay and using the lock-in amplifier in “ 2 f ” mode. The lens was glued to a 2-cm long strip of thin phosphor bronze which was itself spot welded to the armature of the relay. In these experiments, the xenon lamp was run at a dc current of 22 A. The PM tube was run a t -1000 V with equal voltage decrements between the cathode, down the dynode string to ground. The photocurrent was converted to a voltage and amplified with a homemade 3-stage, tuned amplifier, and that signal was demodulated with a Model 9501 E ORTEC Brookdeal lock-in amplifier. For the conventional AFS experiments, the xenon lamp was pulsed a t about 50 Hz and synchronized with the lock-in amplifier with a 5-V square wave signal generated by one half of a TTL dual flip-flop integrated circuit chip (74107) which was itself fed by a 100-Hz signal from an astable multivibrator integrated circuit chip (Signetics 555), used with appropriate R and C values. The lamp current was pulsed between 30 and 10 A. The UV and CSAFS detectors were each provided with separate strip chart recorders in order to read responses simultaneously. A more complete description of the optical, electronic, and mechanical components used in both the conventional pulsed source and the wave length modulated systems may be found in Lawson’s thesis (22). Reagents. Pure ferrocene, acetylferrocene, diacetylferrocene, and dibenzoylferrocene were dissolved in either chloroform or methanol. Potassium hexacyanoferrate(II1) was dissolved in 80% methanol-water. The organometallic chemicals were obtained from the Aldrich Chemical Company. Analytical Procedure. The acetylation of ferrocene was done with acetic anhydride in phosphoric acid at 70 “C. Details of the workup of the reaction mixture and of the extraction of the ferrocene and its derivatives with chloroform are given elsewhere (19-21). Five samples were obtained from the reaction flask at various time intervals after initiation of the reaction. Two- to eight-microliter aliquots of these samples were injected. The liquid chromatograph was operated under the following conditions: Chromasep S (50 cm X 2 mm) column packed with pellicular 10 fim silica gel; flow rate was varied from 0.5 to 2.0 mL/min; 40:l diethyl ether:methanol solvent mixture; 300 psi; 22 “C; UV absorption detector with wavelength fixed a t 254 nm. The polypropylene pick-up tube of the AFS burner was connected to the small bore stainless steel tube exiting from the UV detector with vinyl electrical heat shrink tubing. The solvent flow rates and total tubing volumes between the UV detector and the AFS burner were such that there was a delay of about 5 s, depending on solvent flow rate, between the appearance of analytical peaks
SECONDS Figure 2. UV detector chromatogram of a standard solution. Flow rate of 1.50 mL/min at 300 psi, UV detector. (1) Fc, (2)Fc,, (3)Fc,. Standard solutions of 12 mg of Fc, 25 mg each of Fc, and Fc, in 25-mL
CHCI,; 5 WL injected. Attentuation 16. Retention times 60, 70, and 105 s for Fc, Fc,, and Fc,, respectively on the two detectors. Aliquots of the crude reaction mixture as well as samples cleaned up by an extraction procedure (22)were chromatographed. The flame was “run” slightly “rich” (about 150 mL/min acetylene, 3 L/min air, 1.50 mL/min solvent (diethyl ether)) for most of this work. This stoichiometry gives a slight green “feather” over the inner flame cones.
RESULTS AND DISCUSSION At low flow rates (0.5 mL/min), the separation of ferrocene, 1-acetylferrocene and 1,l’-diacetylferrocene are accomplished with good resolution using the UV detector. At higher flow rates (greater than 1.0 mL/min) the ferrocene peak is apparently not cleanly resolved and appears only as a shoulder on the acetylferrocene peak. At a flow rate of 0.5 mL/min, although ferrocene is resolved from acetylferrocene, (as indicated by the UV absorption detector), the peaks seen with the AFS detector are broad with a poor signal-to-noise ratio. Increasing the solvent flow rate increases the flux of iron to the burner and gives improved sensitivity but poorer resolution. Table I gives a comparison of resolution between UV and CSAFS chromatograms at solvent flow rates of 1.50 mL/min, and the corresponding UC and CSAFS chromatograms are shown in Figures 2 and 3, respectively. T h e tabulated figures are defined as 2(V2 VI)/ ( W , W,) where V, = retention volume of the compounds compared and W , = width of the base of each peak in volume units. T h e apparently better resolution of the AFS detector is due only to the fact that the much greater ultraviolet molar extinction coefficient of acetylferrocene as compared to ferrocene exaggerates the difference in the size of the big peak upon which the smaller one is situated in the UV detector chromatograms. Most of the noise in the CSAF spectrum is d u e t o the variation in molecular fluorescence background and “flicker” noise originating in the flame. A concentric argon sheath (about 12 L/min of gas passed through the outer ring of tubes surrounding the inner burner tubes) did reduce this “noise” somewhat but proved to be too expensive to implement in view of the rather limited improvement it afforded. T h e iron line
+
ANALYTICAL CHEMISTRY, VOL. 51, NO. 4, APRIL 1979
577
El -
ul
3:
-
10
Z
0 a
10 ? O r
u
a:
1
oc
200
IOC
SECONDS
Figure 3. CSAF detector chromatogram of standard solution. Same
HPLC conditions as in Figure 2. Lock-in at 100 mV, Fe line at 248.3 nm, pulsed Xe arc source, C,H,:air burner: retention times 65, 75, and 110 s for Fc, Fc,, and Fc,, respectively
Table 11. Detector Data UV detector data for extracted Fe samplesa
sample 110.
1 2
3 4 5
weight percentage
Fc 27.9 16.0
-
Fc, 70.5 77.7 91.7 88.9 83.8
Fcl
Fc,lFcl ratio
-
-
6.3 8.3
12.3 11.4 8.0 5.2
11.1
16.2
CSAF detector data for extracted Fe samplesb 69.3 1 28.7 2 11.2 81.0 7.0 11.6 3 91.4 8.6 10.6 4 87.5 12.5 7.0 5 85.1 14.9 5.7 a,b
See footnotes in Table I.
normally used (248.3 n m ) is situated on the shoulder of a molecular (PO) fluorescence background band originating from t h e phosphine present in the acetylene. T h e quantitative data for the ferrocene acetylation reaction as seen with the CSAFS detector are contained in Table 11. T h e agreement between UV detector d a t a and CSAF d a t a obtained simultaneously generally seems to be good. However, quantitation of the acetylferrocene peak with the UV detector is difficult because acetic anhydride co-eluted with it. Acetic anhydride has a similar retention time (70 s a t a solvent flow rate of 1.50 m L / m i n ) to t h a t of acetylferrocene and has a non-zero molar extinction coefficient. T h e peak areas obtained with t h e UV detector must be related t o eluent concentrations with empirically determined calibration factors which correct for t h e different molar extinction coefficients of each compound a t t h e specific wavelength used. Haworth and Liu (21)described how these response factors for t h e various compounds investigated in this study were obtained. From t h e corrected UV detector peak areas, weight percentages were calculated, and the ratio of acetylferrocene to diacetylferrocene in the reaction mixture a t a given time is gathered in Table 11. W i t h t h e CSAFS detector, the peak areas obtained correspond t o t h e iron content only. T h e integrated peak area is a measure of t h e number of moles of each iron-containing
;L\
CSAF detector responses for various iron complexes. Wavelength scan without interfacing with HPtC. (1) Dibenzoylferrocene. (2) Fc (3) K,Fe(CN),. (4) Fc,. Four ppm standard solutions of (1-4) in methanol, run independently. Fe lines shown: (a)252.2 nm, (b) 248.3 nm Lock-in at 100 mV Figure 4.
compound eluted with an uncertainty only of how many iron atoms are associated with each molecule of the compound-a small whole number. Proportionality of peak heights to the concentration of each compound was established in this project by running standard solutions of each compound both separately and in mixtures. A typical AFS detector response for a standard solution is shown in Figure 3. T h e molecular fluorescence interference of phosphorus can be considerably reduced by passing t h e acetylene through a filter before burning it. This was accomplished by passing the acetylene through a series of absorption tubes which contained mixtures of mercury(I1) chloride. iron(II1) chloride, manganese oxide, and diatomaceous earth using a recipe found in Mavrodineanu and Boiteaux's classic text (23). T h e phosphine in t h e acetylene is converted to phosphoric acid which is in turn absorbed by the diatomaceous earth. T h e wavelength modulation system briefly discussed in t h e Experimental portion of this paper did not give as good detection limits for iron as did the source modulated CSAFS system (when used with purified acetylene) a n d was dropped from further consideration for the application. Because ether (a "fuel") was used as one of the mobile phase solvents, the proportion of total fuel t o oxidant in the flame would have varied if solvent programming had been used. This perturbs the flame chemistry and changes the response of the detector somewhat to a given flux of iron. This loss of an i m p o r h i t experimental kariable may prove t o be a serious weakness of flame atomization atomic spectrometric HPLC detectors in general. In order to verify the expected insensitivity of the detector to changes in the chemical environment of the iron, solutions of ferrocene, acetylferrocene, dibenzoylferrocene, and potassium hexacyanoferrate(II1) in methanol were aspirated into the burner and the CSAF response was measured. Each solution contained 4 ppm of iron. T h e monochromator was scanned over a wavelength interval containing two iron lines. T h e response as shown in Figure 4. corresponds to t h e iron content only. These iron lines are 248.3- and 252.2-nm resonance lines. T h e extraction procedure used in some cases t o separate the ferrocenes from the bulk of the reactant mixture (21) is fairly tedious and not easily made quantitative. Excessively
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 4, APRIL 1979
Table 111. CSAF Data for Crude Mixture weight percentage
sample
no.a
Fc
Fc I
Fc,
21.3
78.7 83.0 84.1 75.5 71.4 61.2 57.8 57.2
-
9.8 __
Fc, f F c , ratio
38.8
11.5 5.3 3.1 2.5 1.6
42.3 42.8
1.4 1.3
7.2 15.9 24.5
28.6
t
I.O -
: 0-
Samples 1 to 7 taken a t 15-min intervals. Sample 8 taken after 24 11, Conditions same as given in Table I for CSAF.
large amounts of sodium bicarbonate have to be used to neutralize the reaction mixture. The final resulting aqueous solution is almost saturated with sodium salts. Any organic compound (for example, acetic anhydride) extracted by t h e ether which is less volatile than ether can lead to a complicated chromatogram with t h e UV absorption detector. If the retention times of these compounds are similar to those of the products being monitored, the question arises whether a particular peak is due to a derivative of ferrocene or due t o a n interferent. Identification and quantitation can become difficult in many cases without t h e use of a metal specific detector. T o investigate t h e specificity of the CSAFS detector, the acetylation reaction was run with excess acetic anhydride in phosphoric acid, and samples were drawn a t 15-min intervals and injected into the instrument without any prior separation or chemical treatment. T h e results of this experiment are shown in Table 111. Great difficulty was encountered in the removal of the excess reactants (phosphoric acid and to a lesser extent acetic anhydride), and routine injection of t h e raw reaction mixtures into an HPLC system not incorporating a clean-up pre-column is not recommended for routine work. Both the LJV detector responses and those of the AFS detector seen for this experiment are shown in Figure 5. Acetic anhydride enhances t h e acetylferrocene peak seen with the conventional UV detector giving rise to a false apparent composition. As the reaction progresses, more and more acetic acid is produced giving rise to another peak (peak D in the UV detector response plots) appearing as a shoulder on the acetylferrocene peak. Other smaller peaks are seen a t lower flow rates with the UV detector. T h e corresponding CSAFS response shows only peaks from t h e ferrocenes and, as expected, the acetic anhydride, the acetic acid, and other purely organic compounds in the reaction mixture did not give any response. T h e progress of the reaction can be more conveniently followed and quantitated with that detector. In our work, we did not observe the presence of any iron-containing products other t h a n acetylferrocene and diacetylferrocene. T h e reactant, ferrocene, disappears in the reaction mixture after 30 min a t 70 “C. First there is a gradual increase of acetylferrocene compared to ferrocene. As the acetylation reaction proceeds the diacetylferrocene/acetylferrocene ratio increases until an equilibrium is reached. After 24 h, no appreciable change in t h e ratio was observed (Sample 8 in Table 111). Advantages of using t h e continuum source AFS detector may include any or all of t h e following depending upon the application: considerable reduction of t h e analysis time by reducing t h e amount of sample “clean-up” required (a precolumn is recommended, especially, for “raw” samples); positive identification of t h e compounds of interest in the presence of purely organic interferents; good sensitivity; and easily implemented applicability to the determination of other
30
C
~- -~~
SECONDS
Figure 5. UV-CSAF detector responses for crude mixture. 1-6: Sample numbers. (A) Fc. (B) Fc,. (C) Fc,. (D) Acetic acid. HPLC conditions as in Figure 2. 2-8 pL injected. Interfaced CSAF: Fe line (248.3 nm), pulsed Xe arc source, lock-in at 1 mV for sample 1 and 2 and 100 mV for 3-6
metal atom-containing compounds by simply changing t h e monochromator wavelength setting. In comparison to line source (for example, pulsed EDL or laser) excited AFS, t h e continuum source based detector suffers in t h a t it is not as specific (that is to say, with line source itself, not t h e monochromator, largely determines the resolution and consequently t h e specificity), a n d in t h a t molecular fluorescence and scattering error signals are much larger relative t o the useful atomic fluorescence signal. However, bright line sources suitable for AFS are often quite expensive a n d / o r difficult t o obtain and t o operate reliably, and, generally, as is the case with conventional AAS, a separate source is required for every element studied.
CONCLUSIONS T h e use of HPLC separation with CSAFS detection offers a powerful tool for solving problems in inorganic a n d organometallic chemistry. Possible applications include the monitoring of inorganic chemical reactions involving both transition and nontransition metal; the monitoring of typical organic reactions with organometallic catalysts or reagents to get a better insight of t h e reaction mechanisms; speciation of environmental metal-bearing pollutants; a n d metal speciation of complex products of coal (for example, artificial crude oil). Another interesting application of the detector is t h a t it is possible t o obtain molecular weights of metalcontaining molecules. T h e only requirement is that a known mass of t h e compound be injected and t h a t the detector’s integrated peak response as a function of the amount of t h a t same metal be known. Of course, the “molecular weight” SO obtained is really a measure of the mass of compound per mole of metal in the compound and is subject t o an uncertainty of how many metal atoms are in each molecule. Some of t h e more flexible of the molecular fluorescence detection systems commercially available have optical and electronic capabilities superior to those of the equipment used in this work and could
ANALYTICAL CHEMISTRY, VOL. 51, NO. 4, APRIL 1979
be readily modified (by substituting a nebulizer--burner assembly for the cuvette) t o permit element specific detection.
579
H. N. T e n n e y and F. E . Sturgis, Ana/. Chem., 26, 946 (1954). J . C. Sternberg and W . Kennard, J . Chromatogr.. 2, 53 (1959). E. T. McGuinness and M. C . Cullen, J . Chem. Educ., 47, A9 (1970). J. G. Gonzales and R. T. Ross, Anal. Lett., 5 , 683 (1972). J. E. Longbottom, Anal. Chem., 44, 111 (1972). S. E. Manahan and D. R . Jones. Anal. Lett., 6 , 745 (1973). F. Fernandez. A t . Absorp. News/., 18(2), 33 (1977). J. C. VanLoon, J. Lichwz, and B. Radzuk, J . Chromatogr., 136, 301 (1977). P. T. Graham, R. V. Lindsey, G. W . Parshall, M. L. Peterson, and G. M. Whitman. J . Am. Chem. SOC.,7 9 , 3416 (1957). R . E. Bozak, J . Chem. Educ., 43, 73 (1966). J. E . Herz, J . Chem. Educ., 43, 599 (1966). D. T. Haworth and T . Liu, J . Chem. Educ., 53, 730 (1976). S.R. Lawson, M.S. Thesis, Marquette University, Milwauk , Wis., 1978. R. Mavrodineanu, "Fbme Spectroscopy", R. Mawodineanu dnd H. Boiteux, Ed., 1965, Part I, p 62.
ACKNOWLEDGMENT T h e authors express their thanks to John Naleway of Marquette University, for his continued support of our research efforts.
LITERATURE CITED (1) L. R . Snyder, "Chromatography", 2nd e d . , E. Heftmann, Ed., Reinhold Publishing Corp., New York, 1967, pp 93-96. (2) J. F. K. Huber, J . Chromatog. Sci., 7 , 172 (1969). (3) R. D. Conlon. Anal. Chem., 41, 107A (1969). (4) M . N. Munk, J . Chromatog. Sci., 8 , 491 (1970). (5) H. Veening, J . Chem. Educ., 47, A549, A675, A749 (1970). (6) S. H. Byrne, Jr., Modern Practices of Liquid Chromatography", J. J. Kirkland, Ed., Wiley-Interscience.New York, 1971, Chapter 3. (7) M. N. Munk, in "Basic Liquid Chromatography",N. Haden et al., Ed., Varian Aerograph, Palo Alto, Calif., 1971, Chapter 6. (8) J. G. Koen, J. F. K. Huber, H. Poppe, and G. den Boef, J . Chromatogr. Sci., 8 , 192 (1970). (9) D. L. Ford and W. Kennard. J . OilColour Chem. Assoc., 49, 299 (1966).
RECEIVED for review April 5,1978. Accepted January 25,1979. Acknowledgment is made t o the donors of the Petroleum Research Fund, administered by the American Chemical Society, and t o the Committee of Research, Marquette University, for support of this research.
AIDS FOR ANALYTICAL CHEMISTS Microprocessor-Controlled Digital Integrator for Nuclear Magnetic Resonance Measurements F. Morley, I.
K. O'Neill,'
M. A. Pringuer,* and P. B. Stockwell"
Laboratory of the Government Chemist, Cornwall House, Stamford St., London SE 9NQ, United Kingdom
aspects of the integration procedure and performs all the necessary data processing functions. The system which will be described in detail elsewhere (9) is a plug-in retrofit module which does not degrade spectrometer performance. Integration. For integration purposes, the NMR spectrum is divided into discrete zones; a maximum of six zones can be used. Each zone is positioned so that the beginning and end of each zone can be positioned anywhere within the spectrum but must not overlap another zone. The sixth zone is used t o specify a reference peak for quantitative measurements and can be positioned anywhere within the spectrum and not necessarily sequentially with the other zones. As a spectrum is scanned, the position of the zones is detected by a wiper mounted on the pen carriage and a linear transducer mounted along the top edge of the recorder chart. The NMR signal is amplified, fed to a voltage-to-frequency converter, and the output pulses are gated within the integration zone to a five-decade counter. This displays a digital value representing the area of the peak or multiplet of peaks within a zone and is reset to zero between each zone. Spectrometer Control. The spectrometer parameters are optimized manually. A spectrum of the sample is recorded and the various zone positions and the number of zones to be accumulated are entered using the thumb wheel switches, on the integrator front panel. Automatic control is initiated through a teletype command. The various data input is also entered a t this time; namely, the values of the proton equivalent weight of the analyte under test and the weight of the standard used for reference. The microprocessor sets in motion the pen carriage of the recorder (which is linked to the spectrometer field sweep) and at the end of each traverse returns it for a further scan, until the total number of scans reaches a preset level. In addition, the microprocessor causes a dot to be marked on the chart at the start and end of each integration zone. Data Processing. As each zone is integrated, the integral value is passed to the computer memory store where a running average is maintained of each zone integration value. Then the proton equivalent weights (p.e.w.) for each zone and the ave-aged in-
Continuous wave nuclear magnetic resonance (NMR) spectroscopic observation of 'H nuclei ( I ) is an inherently quantitative phenomenon that has been used for the analysis of pharmaceuticals ( Z ) , polymers ( 3 ) ,and many other materials. Commercial NMR spectrometers, however, are designed primarily for qualitative measurements. Many improvements in sensitivity and resolution have been incorporated into instruments in recent years but there have been no significant advances in integration techniques. Experience of quantitative 'H N M R analyses ( 4 , 5 ) a t the Laboratory of the Government Chemist indicated t h a t significant effort could be saved through automation of the spectrometer integration functions. Commercially available gas chromatographic integrators were unsuitable because data output from the scanning of sequential N M R peaks was too rapid. Subsequently, a digital integrator was constructed (6, 7), primarily to handle N M R data. This paper describes an evaluation of a n automatic system for quantitative measurements, using a microprocessor to effect the various control and data processing steps. A similar system has been constructed (8) for the determination of fluorine by interfacing a commercial computer t o a N M R spectrometer. This is restricted because it will accept only Gaussian peaks free of ringing.
EXPERIMENTAL A JEOL CGO-HL spectrometer was coupled t o the microprocessor controlled integration system shown in Figure 1 . A National Semiconductor IMP 16-C microprocessor controls all 'Presently on secondment to RTZ Services Ltd, York House, Bond Street, Bristol BS1 3PE, United Kingdom. 2Present address, Life Science Research, Stock, Essex CH4 9PE, United Kingdom. 0003-2700/79/0351-0579$01 .OO/O
I
1979 American Chemical Society
