retention order was para, meta, and ortho, as observed with the other systems. However, the meta and ortho isomers were usually very close so that they were eluted as one peak.
CONCLUSIONS The permanently bound peptide can make an interesting stationary phase. It has a pronounced effect on the elution characteristics of the several classes of compounds which we discussed above. The fact that water was used as the mobile phase in all the studies should be emphasized. The effect of other solvents is being investigated. The poly-Gly peptide can be used in separation of amino acids and para isomers from ortho and meta of some classes of solutes. The fact that the retention behavior was the same on all three support-peptide phase systems seems to demonstrate that the peptide had the major role in the separation. In this connection, it would be of major importance to investigate the effect of the peptide chain length and, perhaps more important, the relative amount of peptide per unit weight of the support on the retention behavior and mechanism. In addition, it seems that the
whole peptide chain takes a direct part in the partitioning mechanism and not the terminal group only. This indicates that other polypeptides having different amino acid subunits can be tailored to different separations. These aspects of the peptide phases are now under study. Finally, by using amino acids having asymmetric centers as bound phase, and using, if needed, the recycling techniques recently described by Little (181, separation of D and L amino acids and peptides can be attempted. This is also currently under investigation.
ACKNOWLEDGMENT The authors wish to thank C. G. Scott and K. K. Chan for many helpful discussions and suggestions. We also thank J. A. Faucher of Union Carbide Corporation for the sample of Y-5918. Received for review January 19, 1973. Accepted March 19, 1973. (18) J. N . Little, 164th National Meeting of the American Chemical Society, New York, N. Y . , Aug. 28-Sept. 1, 1972, paper No. Anal. 9.
Rapid, Selective Method for Lead by Forced-Flow Liquid Chromatography M. D. Seymour1 and J. S. Fritz Ames Laboratory-USAEC and Department of Chemistry, lowa State University, Arnes, lowa 50010
Lead(l1) is retained on a small anion exchange column from 0.5M hydrochloric acid and separated from many other metal ions. Then it is eluted with 8M hydrochloric acid and the elution curve is recorded at 270 nm. The amount of lead is obtained from a plot of elution peak height YS. pg of lead. The entire separation sequence requires only 8 min. Several standard samples have been successfully analyzed for lead.
The literature abounds with methods for the determination of lead. This reflects the long-standing interest in the development of more rapid and selective methods for this element in a great variety of matrices. In general, these methods involve a separation step in which lead is removed from interferences and concentrated. Solvent extraction using sodium diethyldithiocarbamate or dithizone followed by photometric determination of the resulting lead complex in the organic phase has proved sensitive and selective (1-3). However, extraction methods often require addition of a number of reagents which require painstaking purification. The high p H values necessary for selectivity often preclude analysis of samples high in metal content and can lead to reagent instability. Ion ex1 Present address, T h e P r o c t e r a n d G a m b l e C o m p a n y , M i a m i
Valley Laboratories, Box 39176, C i n c i n n a t i , Ohio 45239. (1) t i . H. Lockwood. Anal. Chem. Acta. 10, 97 (1954). (2) H . Bode, Fresenius' Z.Ana/. Chem.. 144, 165 (1955) (3) L J. Snyder. Anal. Chem.. 1 9 , 684 (1947).
1632
change methods have enabled separation of lead from bismuth, cadmium, and thallium, which often interfere with extraction techniques, as well as a large number of other metals ( 4 ) . Using strong-base anion exchange resin, separation from a t least thirty-nine ions can be effected in hydrochloric acid media ( 5 ) . This is the basis for several methods utilizing subsequent polarographic, spectrophotometric, and spectroscopic determinations (6-9). Again, much sample manipulation is involved, consuming time and sacrificing accuracy. Recently a forced-flow ion exchange separation of iron from metal ions was described ( I O ) . Iron(II1) was eluted with hydrochloric acid and measured using a UV detector; the amount of iron eluted was determined from a linear calibration plot of peak height us. amount of iron. In the present work, lead is separated from other metal ions by forced-flow chromatography and measured in a manner similar to that used for iron (10). Lead(I1) is sorbed on an anion exchange column from dilute hydrochloric acid and separated from most matrix elements. It is then eluted with more concentrated acid and estimated spectrophoto(4) 0. Samuelson. "Ion Exchange Separations In Analytical Chemistry," John Wiley and Sons, New York, N . Y . , 1963, p 406. (5) K . A . Kraus and F. Nelson, "Metal Separations by Anion Exchange," in ASTM Spec. Tech. Pub/. No. 195. American Society for Testing a n d Materials, Phiiadelphia. Pa., 1958. J R. Nash and G. W . Anslow, Analyst (London). 88, 963 (1963) E. i Johnson and R. D. Pohill. Analyst (London), 82, 238 (1957). E. A . Wynne, R . D.Burdick. and L. H. Fine, Ana/. Chem., 33, 807 (1961) . N . G. Sellers, Anal. Chem., 44, 410 (1972). M D. Seymour, J . P. Sickafoose. and J . S. Fritz, Anal. Chem.. 43, 1T34 (1971).
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 9, A U G U S T 1973
1 1 It
'Hh
For determination of the height equivalent to a theoretical plate (HETP) values, the Coleman Model 101 Hitachi spectrophotometer and Kaylab Microvoltmeter were replaced by a Chromatronix Model 200 UV Detector. Reagents. Dowex 1-X8, 200-400 mesh, Bio Rad Laboratories, capacity 3.2 mequiv/gram of dry resin was used for the analytical separations. The resin was washed with methanol, concentrated hydrochloric acid, dilute hydrochloric acid, and finally acetone prior to air drying. The beads were then immediately sieved isolating the 250-325 mesh fraction. Extreme fines were removed by methanol flotation. Amberlite IRA-900 and IRA-400 and Amberlyst A-26 were obtained from Rohm and Haas for determination of HETP values. Dowex 1-X8, 20-50 mesh, from J. T. Baker Chemical Company was used. After washing in methanol, the resins were subsequently washed in water, 0.5M hydrochloric acid, and again in water. The excess moisture was removed using paper towels prior to grinding to the desired water moist mesh size. The resins were
DACRON 0.25" O.D. TUBING (HELIUM INLET1
n m ? " 1.0 TEFLON TUBING T OUTLET)+
TEFLON
HEAVY POL
CAP
3-E
IJu
Figure 1. Cross section of bottle cap
0.25" DIA.
a
DRILL TAP 28 THREADS
0 . 8 9 4 " 4
C-i-,i--I
PER INCH
0.25" a394" 0.25''
0.125" ,D~A.
END VIEW
CROSS SECTION MATERIALS:
-
CENTER SECTION KEL-F END PLATES TEFLON
-
0.0" Figure 2. Flow-through cell metrically from the height of its elution peak. The method is rapid and nearly specific; accuracy compares favorably with other methods for small amounts of lead.
EXPERIMENTAL Apparatus. The chromatograph design was as previously described with the exception of minor modifications (IO). The caps on the polyethylene eluent bottles were replaced with those shown in Figure 1. The brass valves on the gas pressure manifold were replaced with more corrosion resistant Powell Stainless Steel Glove valves FIG. 1861, Metals 18-83 MO. The pneumatic actuation manifold was pressurized using compressed air a t 90 psi. This enables regulation of the pressure applied to the pressure manifold using a Harris Model No. 92-50 Helium regulator with minimum helium expenditure. The combination of the improved eluent bottle caps and pressure regulator expands the pressure range to 55 psi with a tested safety factor of 3.3. Improved sensitivity is obtained by replacing the quartz tube with a 1.0-cm path length Z-configuration flow-through cell shown in Figure 2. This cell is mounted in the spectrophotometer immediately adjacent to the phototube compartment. Amplification of the spectrophotometer output is accomplished by inserting a Kaylab Model 202B Microvoltmeter between spectrophotometer and recorder.
0.5" SCALE
1.0"
then sieved retaining the respective 150-200 mesh fractions. Again extreme fines were removed by methanol flotation. The resin used in packing the analytical column was dried prior to weighing. The beads, prepared as described above, were again washed with acetone and air dried. The resin was then stored in a desiccator under vacuum over anhydrous calcium sulfate for a t least twenty-four hours before weighing. Samples. The alloys analyzed were standard reference materials obtained from the National Bureau of Standards. The samples were thoroughly mixed before weighing. Each weighed sample was then transferred to an Erlenmeyer flask and covered with 30% hydrogen peroxide solution. A small amount of concentrated hydrochloric acid was then added (1-3 ml) and a watch glass immediately placed on the flask. The samples rapidly dissolved without heating. Excess hydrogen peroxide was driven off by heating to near dryness. A measured amount of concentrated hydrochloric acid was then added such that, upon rinsing and subsequent dilution with water into a volumetric flask, the solution was made to 0.5M HCI. Chemicals employed were reagent grade. It was necessary to analyze Standard Sample 54D (Tin-Base Bearing Metal) soon after dilution t o prevent hydrolysis. Reagent Grade lead, assay 100.0%, was obtained from J. T. Baker Chemical Company and was used to obtain the analytical calibration plot. The lead was dissolved using the same procedure used for the reference materials. All solutions and eluents were prepared from reagent grade hydrochloric acid and distilled, deionized water.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 9, AUGUST 1973
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After 1 minute, the eluent was switched from 0.5M HC1 to 8.OM HCl. During this first minute, most of the nonsorbing matrix was removed. The elution with 8.OM HC1 was continued until 6 minutes after injection, during which time the remainder of the matrix was removed followed by elution of lead. The eluent was then changed to 0.5M HC1 to prepare the column for the next sample. The recorded peak height (in absorbance) was directly proportional to the lead content in the sample. For optimum accuracy, a calibration curve was constructed running standard solutions alternately with unknown samples using the same procedure. To analyze for extractable lead in glazed pottery, the unit was first washed with detergent and rinsed thoroughly with distilled water. The unit was then dried and filled to 75% capacity with 4% acetic acid. The unit was covered with a watch glass and allowed to stand 24 hours at room temperature. The sample was then thoroughly mixed and a 25-ml aliquot taken. The aliquot was taken to near dryness in a 250-ml Erlenmeyer flask. Two drops of concentrated hydrochloric acid were added and the sample was taken to dryness but not baked. The sample was diluted to 25 ml with 0.5M HC1 and analyzed using the same elution sequence as was used for the alloys
0.0
0.7
0.6
0.5
0.4
z
a m a
0.3
m
a
0.2
RESULTS AND DISCUSSION Choice of Method. This method takes advantage of two noteworthy properties of lead in solutions of hydrochloric
0.I
a.o WAVELENGTH (nm)
Figure 3. Spectra of 10 fig Pb/rnl in hydrochloric acid 2
I
t
2
I-
a K
z 2 I-
gLT o I-
?!
0
w
B
3 J
0
>
w -1
0,
HYDROCHLORIC ACID CONCENTRATION (moles/ liter)Figure 4. Distribution of metals vs. Dowex 1-X8
0, Lead(ll); 0 , molybdenurn(V1): D , silver(1); 0 , iron(ll1); Q ,
urani-
um(Vl)
Columns. In all cases, a Chromatronix Model LC-6M-13 column (6.35-mm i.d.) was used. To obtain the bed height desired for the lead analyses, two outlet plungers were employed. The analytical column contained 1.00 gram of resin with a bed length of 7.25 cm. For HETP studies, 10-cm bed lengths were employed. Procedures. For the analysis of lead in alloys, the standard reference materials, after dissolution as described, were diluted with 0.5M HC1 to a concentration of from 0.4 to 40 pg Pb/ml (for optimum precision 10 to 30 pg Pb/ml). The pressure was adjusted to give a flow rate in sorbing eluent (0.5M HC1) of 3.0 ml/min with a 7.25-cm column of Dowex 1-X8 in place. The 0.969-m1 sample loop was filled and the detector was set at 270 nm. After allowing 2 minutes for column pre-equilibration, the sample was injected. 1634
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, N O .
9,
acid to achieve exceptional selectivity and good sensitivity. Lead displays strong ultraviolet absorption at 272 nm having a molar absorptivity of 15,500 in 8M HCl. This peak is sharp with its maximum unshifted with small changes in acid concentration, as is shown in Figure 3. Although 37 of 70 common metal ions surveyed display sufficient ultraviolet absorption in hydrochloric acid to be detected, few have a n appreciable molar absorptivity a t this wavelength. The second important property of lead is its ability to be sorbed to anion exchange resins from low concentrations of hydrochloric acid and eluted at higher concentrations. Only silver(1) and rhodium(1II) share this property. The dual selectivity of sorption a t 0.5M HC1, desorption a t 8.OM HC1 and monitoring a t or around 272 nm makes this a nearly specific method for lead. Choice of Resin. Four resins were compared for efficiency of separation by determining the H E T P for a 4 2 - ~ 1 sample containing 0.1 Fmole of lead at various flow rates of 1.OM HCl. These data are shown in Figure 4. The Chromatronix fixed wavelength detector (254 nm), although not suited for detection of lead(I1) in more concentrated acid, was used for greater sensitivity in 1.OM HC1. This concentration of acid was chosen to ensure a n accurately measurable retention volume on the four resins employed and to provide sufficient absorbance for detection. The resins were all ground and sieved water moist in the chloride form to ensure uniformity in particle size in making this comparison. As is indicated in Figure 4. Dowex 1-X8 displayed the lowest HETP values a t any given flow rate and was, hence, the resin of choice. The volume distribution ratios for lead in 1.OM HCl on Dowex 1-X8, IRA-400, IRA-900, and A-26 were 13.2, 14.8, 12.4, and 10.2 respectively. Choice of Conditions. Although maximum retention of lead on Dowex 1-X8 is obtained a t 1.3M HCl, 0.5M HCl was chosen as the sorbing eluent to obtain greater resolution from ions partitioning a t low acid concentrations. Figure 5 shows the distribution ratios of four metals likely t o interfere with this method. These values were obtained from column retention volumes, assuming 70% packing efficiency. Although antimony(V) and rhodium(III1 are also likely to cause interferences, suitable retention values could not be obtained a t low acid concentrations. Inspection of these data shows that a separation factor of about 10 can be achieved for molybdenum(V1) a t 0.5M HCl, and a n even greater separation factor for all other ions [with exception of rhodium(III)]. Lead, however. is still retained
A U G U S T 1973
6o
Table I. Analysis of NBS Samples for Lead NBS Our analysis, analysis
50
t 40 .-.
Sample type
-
Lead-base bearing metal Ounce metal Sheet brass Tin-base bearing metal Manganese bronze
/A
E
-E 30 20
lo 0
%
NBSNo.
Mean Re1 std error, % dev, pph
37D
84.4 5.20 0.94
85.0 5.18 0.95
+0.7 -0.4 +1.1
1.34 0.95 1.05
54D
0.62
0.61
-1.6
1.64
62B
0.28
0.28
0
538 124d
0.0
I -
0
2 3 4 FLOW RATE (rnl/min)
I
5
6
Figure 5. HETP data
0 , Amberlite IRA-400; A , Amberlite IRA-900; 0 , Amberlyst A-26; 0 , Dowex l-X8
I
8
quence for this column varies from 17 min a t a flow rate in 0.5M HCl of 1 ml/min to 5 min a t 5 ml/min. This corresponds to a 44% decrease in detector response at the elution maximum. As both peak area and peak height are a function of flow rate, it must be carefully controlled for best results (IO). The choice of sample loop size was again a compromise. Larger loop Sizes can be used for greater sensitivity; however, unless the column length is greatly increased, sepa-
0.7
0.6
z a
0
0.5
v)
m
a 0.4
z W
2 0.3 0
n v)
E
0.2
U
w
0.1
MSELINE
I-
9min
u
Y
z
"'
CHANGE 05_M HCl-8hjHCl
4min
TIME
-
5m1n
w
t; a
m
,-I W 5
E3
7min
8min
I
1
SOLVENT CHANGE 1 8g HCI -05 HC I
Figure 6. Typical chromatogram of lead analysis Conditions: Sample NBS 37d 163 mg/100 mi; sample volume, 0.969 ml: flow rate of 0.5M HCI, 3.0 ml/rnm; detection at 279 nm
a t this concentration. The choice of eluent for removal of lead from the column was dictated by the need for resolving the peak maximum from the solvent change signal which is shown with a typical elution curve in Figure 6. Although higher hydrochloric acid concentrations would remove lead more rapidly, 8M HCl was chosen to allow a base line of zero under the peak maximum. Column length was a compromise between time, resolution, and sensitivity. The 7.25-cm column employed allows rapid separation from moderate amounts of molybdenum and other elements a t flow rates u p to 3.0 ml/min. For greater resolution from slightly partitioning elements, slower flow rates or increased column length can be employed. The flow rate of 3.0 ml/min corresponds to an elution sequence of 8 min from injection to injection. The se-
ration from interfering ions is not possible. For example, if the sample volume is increased to 10 ml, resolution of iron and lead can barely be achieved on a 17-cm column a t 2.0 ml/min, although the separation factor for leadliron in 0.5M HC1 is nearly 80. Since the sample loop volume is only approximately proportional to the length of tubing installed, unit volumes are not easily obtained. The dimension of the 0.969-m1 loop reported has no special significance. Accuracy and Precision. Data on accuracy and precision in the analysis of standard reference materials are given in Table I. These data were obtained using a single injection of each of three weighings. The calibration curve was from a single injection of each of three standard solutions run alternately with the samples. A linear least
ANALYTICAL CHEMISTRY, VOL. 45, NO. 9, AUGUST 1973
1635
squares data fit was used to obtain the lead content of the unknowns using a programmable calculator. Graphical methods yielded the same results. As the method of detection is spectrophotometric using no attenuation, precision depends on sample concentration. Concentrations from 0.4 to 40 pg Pb/ml can be detected corresponding to absorbance values of 0.01 and 1.0 or 1%and 100% of the recorder scale. Optimum results were obtained from 10 to 30 pg Pb/ml or in an absorbance range of from 0.222 to 0.647. The relative standard deviation on a single weighing of NBS 124d a t the center of this range was 0.23 pph. A variety of pottery pieces were analyzed for extractable lead. Lead was detected in the leaching solutions from two of the twelve units at levels of 0.4 and 1.4 pg Pb/ml. Standards were treated using the procedure described and a relative standard deviation of 1.6 pph was obtained at 7 ccg/ml. There are two kinds of interference common to this type of analysis. Column overloading can cause alteration of peak shape and retention time and, hence, a dependency of peak height on matrix composition. However, even in the analysis of tin-base bearing metal, where the tin matrix is tightly sorbed to the column while present in a 143 to 1 ratio to lead, no interference was noted. This was due to the small fraction of the column capacity occupied by the tin even after repeated analyses.
The other type of interference is due to ions that are retained in 0.5M HC1 stripped in 8.OM HCl and absorb a t 270 nm. Of the cations tested, only three were found to interfere. Rhodium(In), antimony(V), and molybdenum(VI) were found to cause a 1% error in the analysis with metal/lead ratios of 0.009, 0.181, and 5.30, respectively. Greater resolution from molybdenum can be achieved as previously mentioned. Antimony can be removed during sample dissolution by volatilization. Rhodium(II1) can be oxidized to the tetravalent state which will not be eluted with lead. Nitrate ion can also cause error and should be driven off by taking the sample to dryness in concentrated hydrochloric acid prior to analysis.
ACKNOWLEDGMENT The authors gratefully acknowledge the help of Louise Goodkin with modification of the chromatograph, Dean D. Woods with design and machining of the caps on the eluent bottles, James F. Jensen with hydrostatically testing the eluent bottles, and Raymond H. Bowers with the least squares program. Received for review August 28, 1972. Accepted February 9, 1973.
Use of Chloromethylsilyl Ether Derivatives for the Determination of Hydroxylated Steroids by Gas Chromatography-Mass Spectrometry John R . Chapman’ A.E. 1. Scientific Apparatus Limited. Barton Dock Road. Urmston, Manchester, England
Eric Bailey Medical Research Council Unit for Metabolic Studies in Psychiatry, University Department of Psychiatry, Middle wood Hospital, Sheffield, S6 ITP, England
Halomethyldimethylsilyl ethers, particularly chloromethyldimethylsilyl (CDMS) ethers, are valuable new derivatives for the determination of hydroxylated steroids by combined gas chromatography-mass spectrometry (GCMS) techniques. The mass spectra of the CDMS ethers are particularly useful in distinguishing steroids that differ in the stereochemistry of the 3-hydroxyl group or of the A/B ring junction. Further distinction of compounds, such as the di-CDMS ethers of pregnanediols, that differ only in the stereochemistry of the 20-hydroxyl group is possible on the basis of retention data. The mass spectra of most CDMS ethers also show relatively intense peaks at high mass, particularly the M T - CH2CI peak. Because of this, these derivatives are ideally suited to the quantitative determination of hydroxylated steroids at low levels by single and multiple peak monitoring techniques. Examples of the determination of dehydroepiandrosterone and testosterone in plasma extracts by single peak monitoring are presented. 1 Author
t o whom correspondence s h o u l d be addressed
1636
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, N O .
Following the introduction of halomethyldimethylsilyl ethers as derivatives of hydroxylated steroids ( l - 3 ) , there have been several reports on the use of these derivatives in the gas chromatographic estimation of steroids in urine and plasma. The CDMS ethers have been found especially useful for the determination of 11-desoxy-17-ketosteroids in urine (4, 5 ) and plasma (6) by gas-liquid chromatography (GLC) with flame ionization detection. The bromomethyldimethylsilyl (BDMS) and particularly the iodomethyldimethylsilyl (IDMS) steroid ethers have good electron capturing properties, and these derivatives have been found suitable (1) B. S. Thomas, C. Eaborn, and D. R . M . Walton, Chern. Comrnun.. 2, 408 (1966). (2) C. Eaborn, D. R. M. Walton. and B. S. Thomas, Chern. Ind. (London), 827 (1967). (3) C . Eaborn, C. A . Holder, D. R. M. Walton. and 8. S. Thomas, J . Chern. Soc. C, 2502 (1969) (4) B. S. Thomasand D. R. M. Walton, J . Endocrinoi, 41, 203 (1968). ( 5 ) C. Matthiissen and J. W. Goldzieher, Acta EndocrinoL (Copenhagen), 68, 311 (1971). (6) D. Y . Wang, R. D. Bulbrook, B. S. Thomas, and M. Friedman, J. Endocrinoi, 42, 567 (1968).
9, A U G U S T 1973
I
