1844
Anal. Chem. 1982, 5 4 , 1844-1849
Nondestructive X-ray Fluorescence Spectrometry for Determination of Trace Elements along a Single Strand of Hair T. Y. Torlbara" and Davld A. Jackson Department of Radiation Biology and Biophysics and The Envlronmental Health Sciences Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Walter R. French Department of Physics and Astronomy, Nebraska Wesleyan University, Lincoln, Nebraska 68504
A. C. Thompson and J. M. Jaklevlc Lawrence Berkeley Laboratory, University of Callfornia, Berkeley, California 94720
Concentrations of trace elements In halr are flnding lncreasihg value In nutrltlonal studles (essential trace elements) and in toxicology (toxic metals). Previously avallable methods have been tedious, tlme-consumlng, and expensive. We describe here a new development In X-ray fluorescence Instrumentatlon that provldes automatic longltudlnal scannlng for some 16 elements In consecutive halr segments. The mass (In pg/ mm) Is determlned simultaneously at each point, and the absolute concentrations are computed In parts per million for the elements. The method Is nondestructlve, and results (lncludlng mass) show much reproducible detail. The mllllmeter by mllllmeter longitudinal analysis allows recapltulatlon for previous toxic metal exposures for months or years, dependlng upon the length of the hair strand. This Instrument, in a cilnlcal settlng, opens up many new research opportunltles.
The fact that many metals appear in the hair in amounts related in some way to the amount ingested (arsenic is an infamous classic example) is well established. In recent years there has been much interest in the use of hair as a biopsy material, and a number of reviews and detailed tabulations of the elements found in hair have been published (1-3). Studies with methylmercury have shown that the level found at a given location in the hair reflects the concentration in the blood at the time the hair was formed. With a knowledge of the growth rate, the time of the maximum concentration in the body could be calculated from the location of the peak in the hair if an analytical method sufficiently sensitive for measuring the element in a small length could be found. In the last part of 1971, a tragic occurrence of mercury poisoning occurred in Iraq when a large number of natives ate bread made from seed grain treated with a methylmercury fungicide. In addition to the immediate direct effects on those consuming the bread, it became apparent later that many fetuses being carried by pregnant women who consumed the bread were also affected. In order to determine at which stage of pregnancy the ingestion of the high dose of mercury affected the fetus, it was necessary to establish the date of that dosage as well a~ the date of birth of the baby. Studies involving these children, now in school, are still being carried out, In 1977 a prototype X-ray fluorescence instrument was built to do longitudinal analyses on a single strand of human hair (4). The goal in the present research was to develop a computer-automated analytical instrument with associated computer software and operating techniques such that elemental 0003-2700/82/0354-1844$01.25/0
concentrations could be easily and routinely scanned along a single strand. We desired an instrument that could operate in a clinical setting with no special facilities and was so automated that it could function 24 h/day. Our design goal was to have sufficient sensitivity to be able to measure Hg levels directly in ppm (parts per million or gg/g) to an uncertainty of *10 ppm in a counting time of 300 live time seconds per determination. We have surpassed that goal in the instrument described here. It is now possible to produce more information in a shorter period of time with at least as good accuracy as in previous methods without destroying the sample.
EXPERIMENTAL SECTION The Instrument. The automatic scanning hair analyzer is an energy dispersive X-ray fluorescence spectrometer system that was specifically developed to do elemental analyses on small longitudinal samples such as human hairs. The system uses a small molybdenum anode X-ray tube which is placed close to the sample and the detector to achieve high sensitivity. A guard ring is used on the detector, but it is the close proximity of the collimator which is primarily responsible for the good signal to noise ratio. The details of the X-ray tube and sample geometry are similar to those of a prototype instrument ( 4 ) and will not be described in detail here. The excitation X-rays are primarily the 17-keV Kru photons from the Mo anode, and a Mo filter is used between the anode and the sample to reduce the primary X-rays below 17 keV. Four different collimators are arranged on a circular wheel so they may be computer selected. In practice, only two of these have been carefully calibrated and are routinely used. The smaller has an aperature of 1.2 mm and the larger, 2.3 mm. Collimator selectability is a convenient feature which permits broader scans with shorter times for most applications but provides the option of more detailed looks with better spatial resolution if desired. The automatic sample changer is in the form of a stack of 16 sample trays which accommodate 2.00 in. wide 0.125 in. thick Lucite sample mounting frames. These are made in three lengths, 3, 6, and 12 in., respectively. A hair to be analyzed is mounted by stretching it between two small holes accurately centered on the frame ends so that the hair will track precisely over the center of the detector collimator. The hair is held in place with Scotch magic mending tape, and any excess length is taped down to the top of the frame. Using this mounting method, it is possible to scan hairs to within 20 mm of the end. With care, hairs may be mounted, unmounted, and remounted with no observable changes in the results. If it is desired to scan closer to an end, the hair can be cemented to another fiber such as a fine thread with a cyanoacrylate adhesive. This permits analysis to within 2 mm of the end. Special 3 in. frames have been devised to hold a hair from one end so that analysis of the bulb end can be achieved. The spectrometer uses a Texas Instruments 9900 microprocessor, a Tektronix 4051 minicomputer, and a Tektronix4631 hard copy unit to provide most of its control, acquisition, output, and data storage functions. A RS-232 port is available to drive a 0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
standard printer if hard copy is desired. The TI 9900 controls the various motors for collimator position, sample tray selection, sample insertion and removal, and the high voltage-current combinations of the X-ray controller. It also has in its (volatile) memory all1 of the calibration constants of the instrument including a background spectrum, and shape spectra for all elements for which the spectrometer has been calibrated. All of these data are also stored on a system backup cassette tape through the Tektronix 4051. The operator selects the various functions of the machine through the Tektronix 40M. The software is written in an interactive style so that the screen displays questions to which the operator responds. Both hardware and software protect the instrument and the user firom mistakes. Various programs are available to the operator via cassette tape. These are labeled running, setup, marking, plotting, test, and alternate running. Each program contains several options related to that function. In routine operation the operator inserts up to 16 samples in the sample trays of the instrument. The marking program is then used to mark a cassette tape with all details of how each sample should be run. These include the high voltage (HV) and collimator selection, the number of data points to be taken, the millimeter advance between each data point, how far to advance before the first data point, and the elements for which data is desired. Analyses are actually done for each of the following elements at each point: Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, IZn, As, Se, Br, Rb, Sr, Hg, Pb, and MS (mass/length of the sample), but only those previously selected are to be stored on magnetic tape or printed out. The alternate running program displays on the Tektronix 4051 screen the various X-ray spectral peaks besfore and after the spectral stripping operation. This can be very useful as a diagnostic tool. Status lights on the front panel provide the operator with the status of various mechanical and electrical parameters to assist in spotting difficulties should a malfunction occur. Also, digital displays on the front panell indicate elapsed time on one display and count rate (or, if selected, stepping motor steps for checking on the advance of a sample) on the other. This reduces the time taken to setup unusual samples; for if the mount or thread is in the instrument’s field of view, the count rate is much increased over that normally observed. The operating program is then used to actually take the data. The 4051 accesses the first data tape file to be used and sends this information to the T I 9900 which carries out these steps sequentially: (1) selects collimator, (2) selects HV setting, (3) lowers desired sample tray into the insert position, (4) inserts the sample into the instrument to a “located” position determined by a microswitch,(5) turns on the X-ray tube current, (6) advances the sample with a stepping motor the desired distance before the first analysis (in 0.2 mm steps), (7) turns on the analog to digital converter (ADC) and takes data for the desired live time, storing the data in buffer 1of the T I 9900 memory. When the preset counting time has been reached, analysis stops, and the sample is advanced to the next point and the ADC again turned on, this time storing the data in buffer 2 of the TI 9900. The 4051 then proceeds to do the analysis on the data in buffer 1. The upper portion of Figure 1shows a plot of the raw data from the buffer and represents the spectrum of one point from a hair of a healthy individual with no occupational exposure to heavy metals. The lower portion of the figure shows the background spectrum which was derived from a real hair with the elemental peaks subtracted. The twlo plots are scaled nuch that the argon peaks are the same height. The Fe peak in the background spectrum results from a contaminant present in the X-ray chamber and does not charige from point to point or from sample to sample. In order to correct for any spectral shifts due to temperature variations, the analysis routine first checks the centroid of the Ar peak (from Ar in the (air) and also the location of the Mo coherent peak. If these are not in the correct locations, these two centroids are used to do a spectrum shift. The shift routine continues until the two centroids are each within 2~0.04channel of the correct position or until eight attempts have been made. If it fails to accomplish the shift routine, the spectrometer is shut down and the fatal error message is printed. Typically, the
1845
35000r NORMAL HAIR
‘““oo“#l~r I5000
5000
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‘50
V -I d
SOOOr
t-
6000
cr0
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7000tI
BACK GR 0 UNO
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Fe %
J
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0 4ob
CHANNEL NUMBER
Flgure 1. Spectra of normal hair (top)and background (bottom)of real
hair wlth elements subtracted. instrument will make one to three spectral shifts on each point. The mass is then determined for that point by integrating the incoherent scatter peak and the mass is calculated by using a linear equation with constants determined in the calibration procedure. During the setup procedures, a background was stored in the TI 9900. A background for a given analysis is subtracted in three steps, each of which covers a definite region of the spectrum. The appropriate section of the stored background is scaled after determining the appropriate factor by comparing the stored values with those observed at a carefully selected spectral interval. The overlapping nature of the K a and KP X-ray energies for various elements, and the even more difficult overlap between the K series for some elements and the L series for heavier elements, makes it necessary to accurately “strip” all X-ray peaks due to a given element from the net elemental spectrum as that element’s concentration is determined. For example, the As Kal peak at 10.543 keV is not resolved by the spectrometer from the Pb La1 at 10.549 keV. Only a careful spectral stripping routine can provide a determination of As in the presence of Pb. The quantitative elemental amounts in pg/mm are then determined in an iterative manner with the highest peak in a series of elements being subtracted first. A standard peak shape, stored in the TI 9900 memory, is used to subtract all peaks due to that element to reduce interelement effects. Elements are sequentially subtracted from the original data in order of decreasing intensity, and the concentrations in pg/mm are computed for each element along with the statistical uncertainties of that measurement. These data are displayed on the Tektronix 4051 screen until the next analysis procedure begins. When all analyses from a given sample are complete, each pg/mm datum is divided by the corresponding mass value (in pglmm) to give concentrations in ppm. These ppm data are stored in a tape file and/or printed out as desired. Various plotting routines are available in the data presentation programs. The most useful program provides automatic plotting and copying on the Tektronix 4631 of elemental concentrations in ppm vs. the distance along the hair in mm. Some of these plots will be presented in subsequent sections. The most used HV-current combination for the X-ray tube is 56 kV at 600 pA or an anode dissipation of approximately 35 W. This heat is dissipated by a small fan blowing on the anode heat sink. This amount of power is sufficient to provide about 3500-4500 counts/s for a single hair with the routinely used 2.3 mm collimator and about loo0 counts/s for the 1.2 mm collimator. Counting times of 300 s per point are normally used unless higher resolution or better statistics are required. Mass Measurement. While relative measurements of elemental concentrations in environmental or biological samples are
1846
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
Table I. Elemental Concentrations of a 1 mm Point as a Function of Counting Time (all concentrations in pg/g except for MS which is kg/mm) 300 s MS
Ca Ti V Cr Mn Fe Ni
cu Zn As Se Br Rb Sr Hg Pb
6.74 209.3 f 15.1 -3.3 f 10.6 2.3 f 8.2 6.5 f 3.2 6.4 f 2.6 22.1 ?: 2.7 1.8 f 2.8 15.1 I 1.5 154.1 i. 2.7 -4.4 i 3.6 -2.4 4.2 -1.5 f 4.3 1.6 f 5.0 3.4 f 4.9 2.2 f 8.5 11.8 f 15.4
600 s
6.74 216.6 1.0
f
1000 s
6.55 10.8
7.5 -1.1 f 5.8 3.1 f 4.2 6.0 f 1.8 17.9 f 1.9 2.1 f 1.0 12.4 f 1.0 148.6 f 1.9 -0.5 f 2.6 -3.8 f 3.0 -2.7 f 3.0 -2.2 f 3.5 1.8 f 3.5 1.0 i 6.0 6.3 f 10.9 k
informative, it is much more useful if measurements can be quantified in parts per million (ppm). An exact conversion of the elemental level in the segment measured requires a simultaneous determination of the mass of that segment. We have utilized the counts under the incoherent peak for this determination. The incoherent scattering peak results from Mo K a photons from the Mo anode of the X-ray tube undergoing Compton scattering in the sample. This results in a broad peak in the spectrum at an energy slightly below the exciting radiation at 17.4 keV. I t has been reported earlier (4)that this incoherent scattering intensity is linearly related to the mass per unit length for a variety of small diameter linear organic fiber samples. Several hairs with different but reasonably uniform linear densities were mounted and scanned at 5-mm intervals over their entire lengths, and the XRF-mass/length was determined by taking the mean of the values. Then the same section of hair scanned was cut from the mounting frame, the length measured, and the mass determined on a Perkin-Elmer AD-2Z autobalance. A linear relationship (slope = 0.97 and r2 = 0.99) was obtained for hairs ranging from 2.5 kg/mm to 10 rg/mm and the absolute calibration is probably good to about *5%. The reproducibility of the XRF measurements,based upon repeated scans of the same hair, is approximately A 1 %. Calibration for Elemental Analyses. Several techniques are used to calibrate this instrument for absolute elemental analysis. I t is first useful to determine the relative sensitivities for the different elements. For this purpose, thin samples can be prepared on Mylar f i which have known stoichiometricratios. The sensitivity is a smooth function of the atomic number but it is different for the K and L lines, and so the system should then be calibrated against some other reliable techniques for at least one element in the K series, and one, but preferably two, heavier elements using the L series X-rays. As we specifically designed the instrument to do Hg analyses with high sensitivity, much attention was given to calibration for that element. Zn was chosen as a convenient element for the K series calibrations. It should be noted that this procedure does not provide absolute calibration for all the elements in samples other than Mylar film. There is attenuation of both the K and L series X-rays due to self-absorption from the matrix (substrate). For our instrument this error for hair samples is no greater than 20% for elements between Ca and Fe and no greater than 10% for those of greater mass than Fe. For quantitative results on any particular element the system should be calibrated against some independent analytical technique. For calibration purposes, strands which were relatively constant in Zn along their length were selected. It was useful to artifically remove Zn from some hairs and to spike others in order to extend the calibration range. The process is similar to that used in the mass calibration in that these hairs were scanned over their entire 270 mm length, with data points taken every 5 mm. A counting time of 300 live time seconds was adequate to give approximate uncertainty due to counting statistics of k2%. This same length
216.4 f 10.8 -1.5 f 5.9 -1.8 ~t4.6 2.9 ~t.3.3 5.9 f 1.4 20.7 f 1.5 3.4 f 0.8 14.2 i: 0.8 155.0 f 1.5 -1.3 f 2.0 -2.2 f 2.3 -2.92 2.4 -1.2 f 2.8 0.6 f 2.8 2.3 f 4.8 5.4 f 8.6
2000 s
5000 s
6.57
6.54
210.3 f 0.4 f 0.4 f 2.2 i 5.5 f 22.3 f 2.2 f 15.2 f 150.7 f -4.7 * -2.0 * -0.6 f 1.3 f 4.5 f 2.5 ?r 14.8 k
6.0 4.2 3.2 2.3 1.0 1.1 0.5
0.6 1.0
1.4 1.6 1.7 2.0 1.0
3.4 3.4
210.0 f 3.8 1.0 f 2.6 -1.0 f 2.0 0.9 i 1.5 5.6 f 0.6 20.0 i: 0.7 1.8 i: 0.3 14.1 ?r 0.4 151.7 f 0.7 -4.9 f 0.9 -1.3 f 1.1 -1.7 1.1 1.2 f 1.2 4.1 i: 0.6 3.8 i: 1.0 15.0 f 2.1
was then cut out, weighed, and dissolved in concentrated nitric acid and the solution was diluted to volume. The zinc content was then determined with a Varian AA-5 atomic absorption spectrophotometer. Comparison of the XRF measurements with the atomic absorption measurements showed a linear relationship with a slope of 0.99, an intercept of 3.4 ppm, and a correlation factor of 0.99. Standardizations for Hg were carried out in a similar manner to those for zinc with the exception that the hairs chosen contained no naturally acquired Hg. Different amounts of Hg were deposited on the hairs by soaking them for different periods of time in different concentrations of methylmercury chloride. After drying at room temperature, the hairs were scanned by XRF and sections were cut to permit analysis by cold-vapor atomic absorption (5). The slope of the calibration line was found to be 0.97, the correlation factor was 0.96, and the Y axis intercept was offset by +8.9 ppm. The two instruments were brought then into even closer agreement by small changes in the background subtraction routine and by adjusting the Hg sensitivity constants. R E S U L T S AND DISCUSSION Reproducibility. Table I shows the recults from a series of measurements on a single hair strand of linear density -6.6 kg/mm for different counting times. The sample was analyzed repeatedly using all of the same parameters except counting time. The sample was inserted under computer control each time using the regular running program, and the uncertainties were computed automatically from the counting statistics. For this reason, occasionally a slight increase in the standard deviation with a longer counting time will be observed. A slight negative offset is apparent for As, and likely in Br and Se. As we have not been analyzing for these elements, we have not been concerned with these slight systematic errors. The data in Table I can be readily converted to pg/mm by multiplying the ppm data by the mass in the pg/mm. In the analysis of single hair strands, only a relatively few elements have trace element abundances in measurable ppm ranges with reasonable counting times. The elements normally observed are Ca, Mn, Fe, Cu, Zn, Br, Sr, Hg, and Pb, the concentrations of the latter four elements varying greatly from individual to individual. Figure 2 shows the reproducibility of the mass measurements. All but the bottom curve were taken by using the same instrument parameters. This bottom scan is with the smaller 1.2 mm collimator instead of the routinely used 2.3 mm one. The time span from the first to the last measurement is more than 9 months. Note that there is no significant improvement in the detail of the mass plot using the 1.2 mm collimator even though one might expect better spatial resolution. It is evident that the observed mass variations are real and not due to the
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
1847
1200c
3
~~
o
eo
40
EO
160
zoo
240
D I S T A N C E FROM CUT E N D ( m m )
Figure 2. Mass profile of the same strand of heir taken at different dates. Curves A, B, and C: were obtalned by use of the 2.3 mm collimator with readings taken every 2 mm. Curve D was obtained by use of the 1.2 mm collimator with readings every 1 mm.
DISTANCE FROM CUT END
(mm)
Flgure 4. Mercury profiles of three different strands of hair from the one Iraqi woman.
l000~
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DISTrPNCE F R O M C U T E N t l ( m m )
Flgure 3. Mercury profile of the same strand of hair from an Iraqi woman taken at different dates (A, 7/19/81; B, 7/17/81; C, 7/17/81; 0, 1/13/81). Successlve pbts are each displaced upward 100 pg/g.
uncertainties in the analyses. Variations in the mass along the hair emphasize the importance of performing mass scans as opposed to using averaged mass measurements. The absolute values for the 7/17/81 plot are somewhat high as data were taken during recalibration following instrument repair. Figure 3 shows four Hg concentration curves acquired on the same strand from an [raqi peasant woman who ate wheat made from seed grain treated with methylmercury as a fungicide. These curves, like those for mass, were taken over a 6-month interval and show both the reproducibility and the nondestructive nature of’the analyses. Successive plots are each displaced upward 100 ppm (pg/g) for clarity. The mean of peak values for the four curves is 554 f 3.6 for a reproducibility of better than f l % To investigate further the value of data taken with the 1.2 mm collimator and 1 mm steps, we took 64 points bracketing the peak on this same hair sample with 600-s counting times. No new details were observed. Hence we conclude that the 2.3 mm collimator with 2 mm steps provides as much detailed information in one-fourth the counting time as compared to the 1.2 mm collimator aind 1 mm steps.
.
40
80
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D I S T A N C E F R O M C U T END ( m m )
Flgure 5. Profiles of the calcium, zinc, and copper contents of the hair of an Iraqi woman from one particular region.
To justify the need for such precision, it must be shown that a single hair strand is representative of the biological status. Figure 4 shows the Hg concentration plots for three hairs from a different Iraqi woman than the one utilized in Figure 3. Note there was alignment error in the taking of the original hair samples, as evidenced by the position of the rapid rises indicating the time of ingestion of the methylmercury. It should also be noted all curves show the same somewhat exponential decay shape, with a biological half-time of approximately 22 mm which corresponds to 70 days at an average growth rate of 10 mm/month. Using the rapid rise of the Hg peaks as a time indicator, it is possible to accurately align different hair strands from the same individual and look at similarities in the variations for mass and the elemental concentrations. Absolute values of mass, for example, will be quite different but ppm levels of elements very similar. Trends are also very similar, even in the mass plots, so we conclude that a single, hair strand is usually a reliable biopsy sample. Multielement Applications. Figure 5 shows an example of multielement profiles for an Iraqi peasant woman from one particular region. There is what seems to be an annual cycle in the Zn levels, a feature not seen in scans of samples from
-
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982 lOOr
Table 11. Comparison of Uncertainties in PIXE Analysis (8)with That of XRF Analysisa PIXE, ppm Fe Ni
cu Zn
24.0 i 20.0 i: 13.5 'r 143 i:
5.5 2.5 2.5 5.5
XRF, PPm 22.1 'r 1.8 t 15.1 f 154 i:
2.1 2.8 1.5 2.7
a The PIXE data were obtained on a 400-pg digested hair strand; the XRF data came from a 15-pg section of a different hair strand, nondestructively.
T , 0
I
40
,
I
80
120
160
200
240
D I S T A N C E F R O M CUT END ( m m )
Figure 6. Profiles of the mercury, zinc, and copper contents of the hair of an individual whose diet was prominent in fish.
220
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ZINC
-
-z \ 0
a
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2 w 5
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Flgure 7. Profiles of the mercury, iron, and zinc contents of the hair of a Canadian Inuit fishing guide who had no clearly diagnosed Hg poisoning symptoms.
other districts in Iraq. The cause of this apparent cycle is not known, but presumably it is related to seasonal changes in diet. Figure 6 shows a multielement profile from an individual from an area where fish is prominent in the diet. The relatively constant but elevated Hg levels are evident. Figure 7 shows a multielement profile for a Canadian Inuit who died a t the age of 72 of pneumonia without having any clearly diagnosed Hg poisoning symptoms. The cyclic nature of the Hg plot is explained by this individual's life style as a fishing guide. The high Hg levels presumably correspond to those times when he was away from home on a fishing trip and eating substantial amounts of fish. COMPARISON W I T H O T H E R TECHNIQUES At present there are four techniques that are used for hair analyses. These are neutron activation analysis (NAA), atomic absorption (AA), proton-induced X-ray emission (PIXE) and
photon-induced X-ray fluorescence (XRF) using an X-ray tube as the exciting source as described in this paper. In both the NAA and AA techniques the method is destructive and usually involves a minimum of several strands of hair if any longitudinal analysis is to be attempted. NAA gives results for many isotopes of elements, but in a complete analysis for each point by this technique the sample must be counted several times with a high-resolution Ge(Li) detector system to get all of the elements because of the wide range of half-lives and activation cross sections involved. A relatively high-flux research reactor must be available to carry out these measurements or the samples must be sent to a location having such a facility for the measurements to be done, and final results can be delayed for weeks. In the AA technique, only one element can be measured at at time, and analysis for each element requires a bundle of hairs to be aligned and cut into segments. Thus there is no way of accurately comparing several elements in a longitudinal study along hair strands, as different samples are used in each elemental analysis. This may provide useful averages, but is also very time-consuming, and multielemental analysis is not practical on a routine basis as it is so labor intensive. This leaves two methods with the required sensitivity to do multielement analyses on single strands of hair: PIXE and autoscanning XRF. Horowitz and Grodzins (6) used PIXE in some preliminary work to show the feasibility for the technique for measuring the presence of mercury and other elements in single strands of hair. Henley et al. (7) used the technique to examine the roots of single hair strands. A recent paper by Campbell et al. (8) describes in some detail the techniques used in the PIXE method. It is appropriate to make some comparisons. Sensitivities. These are difficult to compare accurately as the necessary data are not given for scanning strands with protons. However, in Table I1 a rough comparison is presented of the elemental sensitivity of the PIXE system of Campbell et al. (8) and our XRF instrument. The PIXE sensitivities are based on a digested 400-pg sample with a 1 nA/mm2 proton beam and a counting time of 300 s. The XRF sensitivities are based on nondestructive measurements of a typical hair segment (15 pg) with the 2.3 mm collimator and a counting time of 300 s. As can be seen from the table, the sensitivities are very similar for these elements. Sample Preparation. In calibrating our instrument for mercury and zinc in hair, we noted how readily these two elements could be incorporated into hair immersed in the respective solutions. In the case of zinc, acidic solutions would remove the element from strands of hair. Therefore, for our method we recommend a first analysis with no treatment other than wiping off any obvious dust or dirt. Any particle of dust will be very evident in the concentration plots as a sharp spike. Since our method is nondestructive, the same strand of hair may be analyzed again after any indicated need for cleaning. Most of our samples are usually ready for analysis in the short time required for mounting in our frames. Mass Measurement. The simultaneous precision mass measurement is a very strong feature of the automatic scan-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
ning XRF technique. It permits an immediate ppm calculation, which is very important in medical and biological applications, In the PIXE technique, the mass measurement is ambiguous, and cumberrnome a t best, and destructive of the sample. Efficiency. One of the most important aspects of any analytical method is the number of samples that can reasonably be done to the necessary precision in a given amount of time. The automatic scanning XRF instrument described here can operate unattended for up to 3 days at a time, running 24 h/day. At the routine counting time of 300 live time seconds it is quite reasonable to take 1200 multielement and mass data points in H 1-week period. This allows time for plotting the results and marking cassette tapes for future analyses. By comparison the PIXE method is at a severe disadvantage. First, the capital costs of the required accelerator are very great, and it probably is not available for use exclusively for hair analysis. The costs of energy, maintenance, and the technicians required to keep such a system operating are very high and probably could not be justified on a long-term basis unless there is available time on an existing machine that is kept operating for other purposes. Speed. In medical caiies of suspected ingestion of toxic substances for a period of' sufficient duration for the hair to emerge or where the symptoms have shown up a t some time after a single sizable intake, the doctor would like to obtain verification as soon as posnible. In the XRF system described herein, the ppm results of a 10-point scan can easily be available in less than 2 h after receipt of the sample. Advantages of Nondestructive Analysis. The fact that there is no deterioration of the sample which is measured while exposed to the room atmosphere has practical as well as scientific advantages. On the practical side, the design of the sample compartment is greatly simplified because no special chamber to contain an inert atmosphere is necessary. This feature allows ease of sample insertion and removal which results in a great saving of time. A further saving in time is made because an initial rapid survey may be made at intervals of 5-10 mm with short clounting times to locate regions of interest which can then be scanned in greater detail. A method which results in partial or complete destruction of the sample must be scanned in great detail over the entiire length in order to avoid missing the regions of interest. From the scientific standpoint, the nondestructive nature permits repeated scannings of the same sample a t any desirable time. The use of the same sample gives a rapid check concerning the status of the instrument at any time. Since any number of measurements may be made for any single
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location or over any desired length of the same strand of hair, the repeatability or precision may be determined with great confidence. The ability to make repeated scans on the same strand of hair enables us to investigate different washing techniques. If the solutions used for washing contain the elements of interest, under certain conditions the hair may pick up the metal from solution. Our experience with zinc has also shown that under other conditions the metal may be removed. These observations have convinced us that the kind of treatment the hair samples receive before analysis should be carefully investigated for each element of interest. In general the handling of hair samples with bare fingers should be kept to a minimum. A first scan on the sample as received followed by scans after selective washing procedures could reveal the extent and nature of surface contamination. We are continuing to investigate the numerous washing techniques used by other medical researchers.
ACKNOWLEDGMENT The technical assistance of members of the LBL Department of Instrument Science and Engineering, particularly William Searles, is appreciated.
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RECEIVED for review January 28, 1982. Accepted June 21, 1982. This paper is based on work performed under Contract No. DE-AC02-76EV03490 with the U.S. Department of Energy, the National Institute of Environmental Health Center Grant No. ES01247, and FDA Contract No. 223-77-2242 at the University of Rochester Department of Radiation Biology and Biophysics and has been assigned Report No. UR-34902003. The work a t Lawrence Berkeley Laboratory was supported by the Director's Office of Energy Research, Office of Health and Environmental Research, Pollutant Characterization and Safety Research Division of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098 and under intragency agreement with the Food and Drug Administration NO.FDA 224-78-2450.
