ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
ACKNOWLEDGMENT The authors thank the Japan Spectroscopic Co. for technical assistance. LITERATURE CITED (1) Bylina, Andrzej; Sybilska, Danuta; Grabowski, Zbigniew R.; Koszewski, JBzef. J . Chromatogr. 1973, 83, 357-62. (2) Denton. Mark S.;DeAngelis. Thomas P.; Yacynych, Alexander M.; Heineman, William R,; Gilbert, T, W, ~ ~Chem, ~ 1976, l 48,, 20-24, (3) Milano, M. J.; Lam, S.;Grushka, Eli. J . Chromatcgr. 1976, 125, 315-26.
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(4) McDowell, Alan E.; Pardue. Harry L. Anal. Chem. 1976, 48, 1815-17. (5) McDowell, Alan E.;Pardue. Harry L. Anal. Chem. 1977, 49, 1171-76. (6) Saitoh, Koichi; Suzuki, Nobuo. J . Chromatogr. 1975, 109, 333-39. (7) Suzuki, Nobuo; Witoh, Koichi; Shibukawa, Masami. J . Chromatogr. 1977,
138, 79-87. (8) Saitoh, Koichi: Suzuki, Nobuo. Bull. Chem. Soc. Jpn. 1978, 51, 116-20.
RECEIVED for review January 3,1979. Accepted May 23,1979. The authors thank the Ministry of Education for a Scientific Research Grant.
Electro-Optical Ion Detector for Capillary Column Gas Chromatography/Negative Ion Mass Spectrometry Bjorn Hedfjall and Ragnar Ryhage" Laboratory for Mass Spectrometry, Karolinska Institute,
S-104 0 1 Stockholm,
An LKB 2091 GWMS instrument has been equipped with a microchannel plate electron multiplier array and a P-20 phosphor coated glass fiber optical window. The image obtained on the window is detected by a photodiode array controlled by a computer via an interface. The detector system has a true parallel spectrum measurement for each scan; and the time that the sample is exposed, as compared to the time it is actually measured, Is approximately the same. The ratio of the highest to the lowest simultaneously detectable masses is 1.08. The advantage of using an electro-opticalion detector In capillary column work along wlth the sensltlvity of the system and other data are discussed. The instrument was run In a negative chemical ionization mode with ammonia as reagent gas.
Electro-optical ion detectors (EOID) for mass spectrometers are still in the experimental stage. Only a few papers presenting mass spectral data from this type of detector have been published (1-6). The advantage of using a mass spectrometer with EOID as an integrating detector is that all ions in the collected spectra are detected simultaneously. An EOID system includes a channel electron multiplier array (CEMA). The CEMA has been carefully tested on mass spectrometers where organic ions in the mass range m / z 31-614 were studied. The minimum detectable ion current and the maximum obtainable resolution were determined (5, 7). The CEMA used in mass spectrometry applications is equipped with a phosphor screen and an optical fiber glass window. Phosphor is coated directly onto the window which is used to guide the light image out of the vacuum system. In papers published on EOID systems, an optical lens and a vidicon camera have been used to detect the light image on the window ( 2 ) . Other systems also include an optical glass fiber to format the image directly on the vidicon camera ( 4 ) or via an optical lens system to the camera ( 1 , 3, 5 ) . Some authors are planning for future development to use a photodiode array instead of a vidicon camera ( 4 ) . None of the above references describe the computer and the interface used for data collection. Therefore no conclusion can be drawn as to whether or not the spectra are collected with a long or short time interval between each spectrum. A long time interval 0003-2700/79/0351-1687$01 .OO/O
Sweden
will result in a loss of information from the sample. Nor is there any mention if, or how, the integration time, the CEMA, or the phosphor screen voltage are to be changed to record weak and strong peaks in the same spectrum. The present paper describes an EOID system used in capillary column GC/MS work, where the EOID is equipped with a photodiode array.
INSTRUMENTATION GC/MS Instrument. The single focusing mass spectrometer used in this experiment was an LKB 2091 instrument equipped with a capillary column inlet, a one-stage jet separator, a heated inlet system and an electronic mass marker. The instrument operates in both electron impact and chemical ionization modes and in both cases either negative or positive ions can be detected. A glass column 25 m X 0.3 mm i.d. coated with SP-lo00 was used at a temperature of 190 "C. The carrier gas flow rate was 5 mL He/min and a make-up gas at 15 mL He/min. The accelerating voltage was kept at -3.5 kV, the electron voltage a t 250 eV, and the total electron emission current at 0.5 mA. EOID System. Figure 1 shows the principles involved in transferring data to the computer from the CEMA connected to the mass spectrometer. The CEMA is angled 45" to obtain an approximate focal plane over the detected mass range. It consists of many individual channels placed in parallel, to comprise an active area of 5 cm2 with a diameter of 25 mm and a thickness of about 1mm. In our work, two CEMA plates are used and they are placed in close proximity to each other with channel bias angles of 8"/8" front/back. A voltage of maximum 2 kV can be maintained across the channel plates. The optical window consists of glass fibers having a diameter of 6 pm. The inside of the window is coated with aluminum and P-20 phosphor for a viewing of the CEMA output. The distance between the second CEMA and the phosphor screen is determined to give an optimum resolution at 5 kV. The total spatial resolution of the CEMAs is 16 lpmm-'. The CEMAs were delivered by Galileo Electro-optics Corp., Sturbridge, Mass. The coupling between the vacuum window and the photodiode array is made of rigid optical glass fibers (5 mm in height X 25.5 mm width X 10 mm length) with a fiber diameter of 7-8 pm. The fibers are black coated and have a hexagonal transverse section. The optical glass fiber was delivered by Volpi AG, Urdorf, Switzerland. 0 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
Phosphor Fiber optic screen /vacuum window
ChaAnel electron multiplier arrays
\
Photodiode array
Amplifier
Ion source
Multiplexer
.
Analog subtracter
DAC
ADC
! ! I
!
'
1
1
Overflow tester
Spectra adder
f
I
-
4 Digital adder
Background compensator
Controller
I
Interface bus
I
J
Multiplexer
! 7 [
Buffer
I
DMA interface DRSll -C
I
Buffer UNIBUS PDP 11/34
Flgure 1. Schematic diagram of mass spectrometer, electro-optical ion detector, and computer interface system
The photodiode array is a Reticon 1024 C/17, which has 1024 photodiode elements with a rectangular section width of 12.5 pm and a height of 0.4 mm. The center to center space is 25.4 pm. The photodiode array was delivered with a protecting glass, which was dismounted. One end of the rigid optical glass fibers is placed in close contact with the photodiodes and the other end in direct contact with the fiber optic window. The ratio of the highest to the lowest simultaneously detected masses was 1.08 for an accelerating voltage of -3.5 kV. T o minimize the dark current from the photodiode array, the array can be refrigerated to -35 OC. C o m p u t e r a n d P e r i p h e r a l Equipment. The computer used in this application is a PDP 11/34 processor with a mos memory capacity of 48K, 16-bit words and with memory management. T o obtain a faster arithmetic operation, a floating-point processor is also included. The disk memory consists of two RK06 disk storage devices with a capacity of 7M words each. A nine-track magnetic tape unit is used for the permanent storage of data and for the exchange of data with other computers within or outside the laboratory. For the presentation of the spectra, the chromatograms, and for the operator communication with the computer, a Tektronix 4012 alpha-numeric-graphic display terminal is used. The hard copy form of the spectra and the chromatograms are presented on an electrostatic line printer/plotter. A second communication with the computer is provided via a DECwriter, type 11. The link between the UNIBUS of the computer and the constructed GC/MS-interface is a general direct memory access interface, type DRS11-C. The operating
system in use is the real-time operating system, RT-11 VOX M S X o m p u t e r Interface. For each scan 4 1 s are required to process one element in the photodiode array and this is the only time during which no measuring is made on the element. The relation measuring time/interval time in percent is 100 X T / ( T + 0.004) where the value of T is between 5 and 320 ms. Thus the measuring time will be close to 100%. When the accelerating voltage or the CEMA voltage is changed between scans, a delay of 3 ms is necessary for voltage stabilization. A dummy reading of the photodiode detector must be done to reset the detector. This requires 4 ms, which is also the shortest usable integration time. Alternating between two memory buffers eliminates the loss of measuring information during the transfer of spectra to the computer. T o enable the photodiode detector to operate a t selectable integration times of 5, 10, 20, 40, 80, 160, and 320 ms, the interface sums up the corresponding photodiode signal intensities from a selected number of scans to form a sum spectrum which is transferred to the computer. The scan summing sequence must be in decreasing order with regard to integration times. A maximum of 15 scans can be summed together. The intensity can be transferred, either as an integer value with a 16-bit dynamic range, or as a 4-bit shift factor representing the number of shifts which have been made to obtain the 12-bit intensity value. Working with a shift factor gives a dynamic range greater than 16-bits, but with a 12-bit accuracy. T o build up a spectrum, each individual scan requires parameters, such as: function control, integration time, accelerating and CEMA voltages. These parameters are
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
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transferred from the computer to the control memory in the interface. The interface was designed a t our laboratory.
SOFTWARE Data Processing. The intensity values can be smoothed with a selectable odd number of points between 5 and 21. A peak is determined by searching for a maximum intensity, which is temporarily stored together with the element number. The peak end is found when the intensity has dropped to 50% of the maximum value. Only the highest peak within an integer mass interval is saved. The element numbers, which limit an integer mass interval, are calculated from a calibration value and the mass a t the central point of the detector. The calibration value is a percentage of the central mass, which has been found to be detectable with the photodiode array. The central mass is read from the mass marker on each occasion. The data reduction is done during the data collection, because in GC/MS work it is necessary to be able to store as many final spectra as possible. During the spectra data collection, the total ion current or the spectra can be presented on the display terminal. S p e c t r u m Manipulation. From the spectra files on the disk memory, a total ion current chromatogram, a mass chromatogram, a spectrum or a selected sum of spectra with or without a selected background subtracted, can be plotted. Spectra in table form with a normalized or absolute intensity can also be presented. All forms of presentation can be done on the display terminal or on the electrostatic line printer/ plotter. In a maintenance mode the calculation functions, the digital functions, the analog functions, the memories, the ADC and the DAC in the interface can be tested with the program. A complete plot of the analog signal from the photodiode array can also be made.
~
-r
where t l = recording time of a peak (exponential scan), t2 = scan return time, and M,,, = resolution of the mass spec= 600, tl = 5 ms and trometer. A mass range of 5C-500, M,,, t 2 = 200 ms gives F = 1580. However, this advantage is overshadowed if any of the parameters are changed during data collection. The most serious drawbacks to the EOID system are the noise level from the photodiode array and its small dynamic range of about 1-100 for a specific integration time. T o obtain a greater dynamic range for the spectrum, several different integration times, or different CEMA, or phosphor screen voltages must be used. If a weak and an intense peak are measured a t the maximum integration time and the intense peak causes overload of the photodiodes, a shorter integration time must be set or the CEMA voltage must be decreased to measure the stronger peak without overloading the photodiodes. When this becomes necessary, the smaller ion current is not detected with maximum sensitivity during the whole data collection and the signal-to-noise ratio will be decreased. The data collection time for an intense peak, which causes overload, will decrease in accordance with the total selected data collection time. For the integration, during which the intensity cannot be measured, an extrapolated value will be calculated from the intensity measured a t the shorter integration time. The extrapolation is done to correct the peak relation between weak and strong peaks measured with different integration times and then summed together to give one spectrum.
z x
522
-
-
-
4
M Z
Figure 2. Simuttaneousiy recorded negative ion mass spectrum of PFK in the high mass region. Integration time was 160 ms
30C 350
RESULTS AND DISCUSSION A mass spectrometer with an EOID system will collect the ions in a given mass range for a much longer time than a scanning spectrometer with a single ion detector! or by a factor F
_-
------
L
30C
t- SFEITRA hJIJBE2
350 i
290 WZ
Figure 3. Negative ion cwent detection of 100 fg of hexachkrobenzene in the mass range 275-293. Column temperature was 190 OC. Integration time was 320 ms and the CEMA voltage was -1.2 kV. (a) Total ion current. (b) Ion chromatogram of m l r 282, 284, and 286. (c) Normalized mass spectrum
2
Frgure 4. Negative ion current detection of 10 fg of hexachlorobenzene in the mass range 275-293 obtained at different integration times. Column temperature was 190 OC and the CEMA voltage was -1.2 kV. 320 ms (two spectra added). (b) 320 ms. (c) 160 rns (a) 320
+
Figure 2 shows the mass region of PFK from m / z 490-525. The resolving power of the mass spectrometer and EOID system is shown t o be greater than 600 a t 10% valley. The integration time used was 160 ms. The sample was introduced into the heated inlet system. Since the CEMA is angled 45' a t the focal plane of the ions, the mass range is reduced by a factor of 2lI2, as compared to an unangled CEMA ( 5 ) .
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benzene dissolved in 1 pL of methanol. Three different integration times were used: 320 320 ms, 320 ms, and 160 ms. The signal-to-noise ratio decreased significantly with shorter integration times and the chromatographic resolution increased as expected. The high noise level is due mainly to the photodiodes. Figures 5a-5c show the partial mass spectra from the total ion current peaks 1-3 in Figure 4a. Peak 1 and 2 represent unknown impurities with base peaks at m / z 281 and 283, respectively, and peak 3 shows the characteristic pattern of hexachlorobenzene. Three different purified solvents of methanol were tested, and all of these showed impurities especially for masses below m / z 250. From our experience with the GC/MS and EOID system, we have found that the solvent used must be carefully purified when substances in the sub-picogram or femtogram range are to be measured. It is also of great importance to use low bleeding columns and to keep a low background in the spectrometer to prevent overloading of the photodiodes. Impurities will limit the full use of the sensitivity in the EOID system when weak peaks from a substance are to be measured. It is also difficult to measure very small amounts of sample using the direct probe inlet since the sample must be dissolved in a solvent before it is introduced into the probe. In LC/MS analysis the difficulties will be increased since a great amount of solvent is introduced into the ion source and ionized. A mass spectrometer with a larger mass range, equipped with an EOID system, including a new type of photodiode arrays, will soon be completed.
+
275
230
275 290
M/Z
V/Z
M/Z
Figure 5. Normalized mass spectrum of the mass range 275-293 of peaks 1-3 in Figure 4a. (a) Peak 1. (b) Peak 2. (c) Peak 3
However, the resolution of the angled CEMA is increased by approximately the same factor. The small mass range covered by the described detector, shows only a part of a mass spectrum and limits the structure information from the cracking pattern of the molecules. However, it can advantageously be used for detection of known compounds. Figure 3a shows the intensity of the total ion current of negative ions in the mass range 275-293 as a result of introducing 100 femtograms (fg) of hexachlorobenzene into the capillary column, kept a t a temperature of 190 "C. The CEMA voltage used was -1.2 kV. An integration time of 320 ms was used to obtain the maximum sensitivity for a single scan of the photodiode in the system used. This is also the approximate maximum integration time that can be used in high resolution capillary column work. Figure 3b shows the three most prominent ions in the detected peak and Figure 3c shows the normalized mass spectrum. Figures 4a-4c show the total ion current in the mass range 275-293 for the introduction of 10 fg of hexachloro-
LITERATURE CITED (1) C. E. Giffin, H. G. Boettger, and D. D. Norris, Int. J . Mass Spectrom. Ion Phys., 15, 437 (1974). (2) . . J. H. Bevnon, D. 0. Jones. and R . G. Cooks. Anal. Chem.. 47. 1734
(1975). (3) D. D. Norris and C. E. Giffin. Proc. SPIEISPSE Techn. Symp. East., 77, 103 (March 1976). (4) H. G. Boettger, G.E. Giffin, D. D. Nonis, W. J. Dreyer, and A. K u p p e m n n , A&. Mass Spectrom. Biochem. Med., 2, 513 (1976). (5) H. H.Tuithof, A. J. H. Boerboom, and H. L. C. Meuzelaar, Int. J . Mass Spectrom. Ion Phys., 17,299 (1975). (6) J. Yinon and H.G. Boettgec, 25th Annual Conference on Mass Spectromeby and Allied Topics, Washington, D.C., May 29-June 3, 1977,paper 711. (7) H. H.Tuithof and A. J. H. Boerboom, Int. J . Mass Spectrom. Ion Phys., 15, 105 (1974).
RECEIVED for review March 28, 1979. Accepted May 8,1979. Work supported by grants from the Swedish Board for Technical Development and from Knut and Alice Wallenbergs Stiftelse.
Quantitative Thin-Layer Chromatography and Mass Spect romet ry of I satinyImethy I Esters of Car boxy Iic Acids G. Gubitz" and W. Wendelin Institut fur Pharmazeutische Chemie der Universitat Graz, A-80 10 Graz, Austria 1-Chlormethylisatin (CMI), a new derivatization reagent for the determination of carboxylic acids by chromatography has been studied. With crown ethers as catalyst, CMI reacts with carboxylic acids under mild reactlon conditions in less than 10 min. The derivatives were separated by TLC and determined in situ by reflectance measurement.
The determination of carboxylic acids by chromatographic methods often requires the use of derivatization reagents. For 0003-2700/79/0351-1690$01.00~0
GC separation, for example, the preparation of the methyl esters (1, 2 ) , benzyl esters ( 3 ) ,pentafluorobenzyl esters (41, and p-bromophenacyl esters ( 5 ) has been reported. Only a few derivatization reagents for enhancement of the sensitivity of detection in TLC or HPLC have been described. Politzer and Griffin (6) used 1-benzyl-3-p-tolyltriazeneto prepare the benzyl esters of carboxylic acids for liquid chromatographic analysis. With 1-p-nitrobenzyl-3-p-tolyltriazene (7), esters with higher UV absorption have been obtained. p-Nitrobenzyl esters have also been prepared using 0 1979 American Chemical Society
