considerable doubt about the exact concentration of Al in Orchard Leaves-so much so that the NBS has neither certified nor given a tentative value for it. Using cathode ray polarography, Maienthal (29) obtained a value of 347 8 ppm A1 in Orchard Leaves, while a value of 420 ppm was obtained by activation analysis. Again values from 99 to 401 ppm were reported for this material by 11 non-NBS optical emission spectroscopy laboratories in a roundrobin study (29). Thus, inhomogeneity of A1 in Orchard Leaves standard may be partly responsible for our high value obtained for kale. Morrison and Potter (12) obtained a value of 440 20 ppm for A1 in Orchard Leaves. We determined this element in Orchard Leaves using A1 metal foil as standard and obtained values from 378 to 458 ppm A1 for 18 determinations with an average value of 409 ppm. We have used this value as the standard value for determination of A1 in other materials. Our value for Mg in Bovine Liver is higher than the tentative value given by the NBS; however, our value for the same element in kale is in excellent agreement. Chlorine values are in reasonably good agreement considering the possibility of contamination by this element during handling of the sample. Though our value for Na in Bovine Liver is in excellent agreement with the NBS certified value, our value in kale (0.17%) is lower than the best value of Bowen (0.25%). In this case again, there is a range of values given by Bowen varying from 0.12 to 0.33%. Considering that 24Na dominates the y spectrum shortly after neutron activation of kale, this range and the discrepancy is surprising. Contamination during handling is a distinct possibility. A range of values from 0.5 to 3.5 ppm for Cr was found in Bovine Liver in repetitive samples. No value for this
*
(29) E. J. Maienthal. J. Ass. Off. A n a / . Chem., 55, 1109 (1972)
element has been assigned by the NBS. Values for the remainder of the elements are in good agreement with the certified or literature values wherever available. Values for Al, Ba, Br, La, Cr, Yb, Sb, Ni, Sc, Cs, and Eu in standard Bovine Liver, Yb and Lu in standard kale, and Mg, Rb, Ba, Sr, Mo, Yb, Lu, and Ni in standard tobacco are given here for the first time. The accuracy of most of these determinations is of the order of &5-15%, except for a few elements present in ultratrace amounts. With regard to the capability of determining the essential elements in biological materials, the INAA method has successfully measured Co, Cr, Cu, Fe, Mn, Mo, Se, Zn, Ni, V, Na, K, Mg, and Ca. Because of adverse nuclear properties F, B, P, and S cannot be measured using thermal neutron activation analysis and y spectrometry. Iodine and Sn could not be measured in these samples because they are present below the detection limit of the method. Of the toxicological elements, Ba, Ni, Ag, Hg, As, Sb, and Br can be measured by this method. Li, Be, Pb, and Bi cannot be measured because of adverse nuclear properties, and Cd in these samples is below our detection limit. Finally, the INAA method can also determine Al, Rb, C1, Sr, La, Lu, Yb, Sc, Cs, and Eu in biological samples.
ACKNOWLEDGMENT The cooperation of the personnel of the Nuclear Reactor Facility of the Georgia Institute of Technology, Atlanta, Ga., in performing the irradiations is greatly appreciated. Received for review February 1, 1973. Accepted March 30, 1973. Research was supported in part by the National Science Foundation through the Cornel1 Materials Science Center and the National Institutes of Health under Grant lRQlGM19905.
Determination of Polycyclic Aromatic Hydrocarbons Separation of Benzpyrene Isomers by High-pressure Liquid Chromatography on Cellulose Acetate Columns H.-J. Klimisch Research Institute of the Cigarette Industry, Gazellenkamp 38, Hamburg, Germany
The determination of benzo[a]pyrene (BaP) as a pilot substance for carcinogenic polycyclic aromatic hydrocarbons (PAH) is of special interest in the field of quantitative estimation of PAH. The efficiency of the method lies in the separation of BaP from the isomeric benzo[e]pyrene (BeP) and the other PAH of the so-called benzpyrene fraction, as shown in Table I. Of the methods recently summarized in a review article ( I ) , only the gas chromatographic (GC) and a combined spectroscopic procedure with paper (PC) or thin-layer chromatography (TLC) on acetylated cellulose are partly suitable for the quantitative determination of BaP. With regard to accuracy and reproducibility, there are limits in GC due to operation with capillary columns a t high temperatures. PC and TLC require further steps of elution and bear the risk of sorption and chemical alterations of BaP. All procedures take a considerable amount of time. (1) R. E. Schaad. Chrornatogr, Rev., 13, 61 (1970).
1960
High pressure liquid chromatography (HPLC) is used to gain some advantages. Schmit et al. ( 2 ) described a separation of isomeric benzpyrenes by an extensive procedure of reversed phased liquid-liquid partition chromatography with a linear solvent gradient but did not investigate the separation of other PAH of the benzpyrene fraction. Ledfort et al. ( 3 ) combined a separation on a Durapak OPN column with a separation technique using very special supporting material on glass beads treated with octadecyltrichlorosilane. The separation is followed by a fluorimetric determination. In comparing terms of the maximum number of effective plates per second achieved in HPLC columns with silica gel as supporting material ( 4 ) ,it seems more promis(2) J. A . Schrnit, R. A. Henry, R. C. Williams, and J. F. Diekman, J. Chrornatogr. Sci., 9 , 645 (1971). (3) C . J. Ledford, G. P. Morie, and C. A . Glover, Tobacco Sci., X I V , 158 (1970). (4) J. J. Kirkland, J. Chrornafogr, Sci., 10, 593 (1972).
A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
Table I. Rf Values after TLC Separation of PAH on Cellulose Acetate Plastic Sheets Cellulose acetate sheets 21%
30%
Table II; HPLC Separation of PAH on 45% Cellulose Acetate Column B b Column A" 10%
Ve
Ve
max,
Substancce
I"
Ilb
IIIC
I"
I"
0.79 ... ... ... ... 0.78 ... ... ... ... 0.74 ... ... ... ... ... 0.71 ... ... ... 0.54 0.69 0.62 0.68 0.79 0.61 ... 0.66 0.70 0.83 0.37 0.65 0.53 0.59 0.77 0.73 ... 0.66 0.74 0.80 0.62 0.79 0.66 0.71 0.79 ... 0.72 ... ... ... 0.75 .., ... ... ... a I = solvent system ethanol/dichloromethane 2: 1 . I I = solvent system ethanol/toluene 2 : l . I l l = solvent system ethanol/l.2-dlchloroethane 2 : 1 .
Anthracene Fluorene Pyrene Benzo[a]anthracene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[a]pyrene Benzo[e]pyrene Perylene Benzo[g,h,i]perylene Coronene
ing to separate the isomeric benzpyrenes using a system with high selectivity rather than seeking columns with high numbers of effective plates per second. The preferred system could be cellulose acetate. In analogy to TLC separations on cellulose with different acetate contents (5), the optimal conditions for separating BaP from other PAH of the benzpyrene fraction were investigated. Results obtained by a TLC screening test were subsequently transferred to HPLC.
EXPERIMENTAL Material. Cellulose acetate plastic sheets with 30 and 10% acetate content (Macherey, Nagel & Co., D-516 Duren, Germany) or 21% acetate content (Schleicher & Schuell, D-3354 Dassel, Germany) were used for TLC. As column supports, the following were used: 45% acetylated Avicel (Schleicher & Schuell, 1440145 ac) d, 50-90 pm or 60-120 pm; 21% acetylated Avicel (Schleicher & Schuell, 1440/21 ac) d, 65-110 pm; 30% acetylated cellulose ( M a cherey, Nagel & Co., M N 300 Ac 10) d, 40-120 p m . After distillation, the solvents were purified on alumina columns (W 200 Woelm neutral, acidic, and basic). Instrumentation. Investigations were carried out with the Universal-Liquid-Chromatograph UFC 1000 with a temperature-controlled column oven (Hupe & Busch, D-7501 Grotzingen, Germany) in steel columns of 2 x 500 mm. A spectral photometer PMQ I1 (Carl Zeiss, D-7082 Oberkochen, Germany) with a 8-pl microflow cell was used as a detector. Preparation of Columns. The cellulose acetate was dry sieved, and the fines from each fraction were removed by sedimentation in 99% ethanol. Particle size was measured by a microscopic method. Because of the fibrous nature of the material, it was not possible to prepare sieve fractions of narrow particle size distribution. It was, therefore, decided to signifiy the length of the particles a s the particle diameter. The cross-section diameter varied between 30-40 pm. The dried supporting material was then filled into the columns by a tap-fill method (6) and the columns were then washed for 5-8 hours with solvents. Two columns were coupled. TLC Procedure. PAH (see Table I ) were applied, dissolved in tetrahydrofuran, as spots on the cellulose acetate sheets and developed with the solvents mentioned in saturated N-chambers. The PAH spots were marked under UV irradiation a t 254 nm. HPLC Procedure. Up to 5 pl of the PAH dissolved in tetrahydrofuran or other suitable solvents, were injected into the liquid chromatograph using a 5-pl SGE syringe (Scientific Glass Engineering Pty, Ltd., North-Melbourne, Australia). Retention volumes Ve or retention times t~ were measured. Separation of PAH mixtures were examined with various solvents and a t different pressures (Table 11). (5) R . E. Schaad. Microchern. J., 15, 208 (1970). (6) J. J. Kirk1and.J. Chrornatogr. Sci.. 10, 129 (1972)
Substance
Benzo[e]pyrene Perylene Benzo[klfluoranthene Benzo [b]f Iuoranthene Benzo[a]pyrene
ml
Ve
max, tR,
min
ml
Column Cc
tR,
min
max, mi
tR,
min
2.8 4.0
10.5 15.0
2.5 3.5
3.5 4.8
2.9
13.6
...
, . .
4.7
17.4
4.5
6.2
. . . . . .
5.3
19.6
. . . . . .
4.6
16.8
11.4 42.6 9.7 13.5 6.6 24.3 "Column A: solvent system I ; d , 50-90 p m ; Ap = 15 atm; b = 0.14 cm/sec. Column B: solvent system I ; d , 60-110 p m ; Ap = 15 atm; b = 0.39 cm/sec. CColumn C: solvent system I l l ; d , 60-110 p m : Ap = 102 atm; 'v = 0.14 cm/sec.
RESULTS AND DISCUSSION Solvents for HPLC were selected in the light of two facts. First, the separating efficiency of the various systems was examined in a screening test by thin-layer chromatography on cellulose acetate with different acetate content and the best results were then transferred to column technique. Second, mixtures of a maximum of two solvents were selected which did not influence the detection of PAH. Three solvent systems were selected and examined with regard to their separating efficiency of PAH a t different acetate contents of the sheets (Table I). Special attention was paid to separation of the PAH of the benzpyrene fraction. The solvent system I showed the best separating effect on cellulose acetate sheets. In mixtures I1 and 111, the PAH had higher R f values, but the separation was in fact not so good. Every spot remained smaller. With increasing acetate content, the separation of PAH proved to be more selective and better. System I was, therefore, transferred to the HPLC method, and a t the same time, the acetate content of the packing material was increased to 45%. Columns were filled dry and afterwards washed with solvent. Based on the swelling of the supporting material, the columns showed a somewhat high pressure drop a t high elution velocity. Therefore, permeability K = .L/P was low (column B: K = 0.17 cm2/atm sec., column D: K = 0.88 cm2/atm sec.). As demonstrated in Table 11, there is an excellent separation of BaP in columns A and B. The separation was over-effective and analysis time too long. Two possibilities were available to speed up the analysis time. First the analysis time can be shortened by decreasing term ii in the following equation (7):
But as there were no shorter columns available for the liquid chromatograph, it was decided to vary term iii (the second possibility) by employing another solvent system (column C). As the main interest was in the determination of BaP, a poorer separation was accepted of the other PAH of the so-called benzpyrene fraction. As shown in Table 11, BaP and the other PAHs had smaller elution volumes in column C than in column A. By analogy with results in TLC, the peak width of the PAH was narrower. The best measure of the performance (7) L. R. S n y d e r , J.
Chrornatogr, Sci., 10, 369 (1972).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1 1 , SEPTEMBER 1973
1961
%T
*l*T
%T
C
Rf-
0
3
6
9
COLUMN
R
m
o
1518
F
3
6
o
11
COLUMN
f
n
18
ZIZL
R
G
4. . ( . . , . . , . . I 0
3
6
9
COLUMN
. _ ( . .
12
15
..,. -fR
18
Zl
2L
21 30 33
H
Figure 1. Elution curves of PAH in the system of 30% cetlulose acetate, solvent ethanol/dichloromethane 2: 1 Column F: p = 37 atm; = 0.35 cmjsec; RsBaP = 1.O. Column G: p = 29 atm; = 0.26 cm/sec; RsBap = 1.25. Column H: p = 21 atm; = 0.18 cm/sec; R s ~ a p= better than 1.25. a = solvent. b = benzo[b]fluoranthene. c = BaP.
Table 111. HPLC Separation of PAH on 30% Cellulose Acetate Column Da Substance
Benzo[b]fluoranthene Benzo[a]pyrene
Ve rnax, ml
t R , min
Column E b Ve max, ml
4.4
9.1
4.4
7.3
15.2
6.0
t R , min
23.3 31.9
"Column D: solvent system I; d , 40-120 F; Ap = 29 atm; u = 0.25 cm/ sec. Column E: solvent system 111; d , 40-120 Frn: Ap = 25 atm, Y = 0.1 cm/sec.
of a separation system in HPLC is found in the resolution function R, (Equation 1). The resolution of BaP in column C is Rs = 1.25, which is reasonably satisfactory for a quantitative determination of the separated substance. The peak width is with Ve B~~ = 2.8 ml for 58% narrower than that of column A with Ve Bap = 6.7 ml. In this way, sensitivity of detection as well as accuracy were improved in this system. Term iii can be modified not only by selection of solvents but also by variation of the acetate contents of the supporting material. In this way, it was intended to optimize this separation technique with regard to shorter analysis time and lower pressure drop. Table I11 indicates data of investigations with 30% cellulose acetate. Resolution of BaP from the other PAH in column D is better than Rs = 1.25, the time of analysis is considerably shorter than in column C, and the peak width at V e B a p = 3.6 ml is a little broader than in column C. It will depend on the sensitivity of detection required, as to which column is to be preferred. Column E shows also at Ve = 1.9 ml significantly narrower elution peaks; however, resolution with 1.0 is no longer optimal. At a pressure of 25 atm, times of analysis are also too long. It was not possible to speed analysis by increasing velocity and pressure. Resolution should be increased in accordance with Equation 1. This effect is pointed out in Figure 1. The lowest velocity gave the best resolution of BaP. The lower pressure drop of the columns, when using 1962
30% cellulose acetate, may be caused by the different nature of the supporting material. Microscopic examinations showed that 30% cellulose acetate was more compact. The 21 and 45% cellulose acetate produced from Avicel, a partly hydrolyzed cellulose, seemed to be more irregular, porous, and softer. Upon further reduction of the acetate content, only solvent system I could be finally recommended. The BaP peak had a width of 1.8 ml as the resolution was Rs = 1.25. Also this system is well-suited to quantitative determination. About 200 ng BaP were still detected at a measuring wavelength of 297 nm. CONCLUSION The high selectivity of the cellulose acetate columns enables a good separation of BaP from other substances of the benzpyrene fraction. The selection of a suitable separation system should depend on following general observations. Better separation is achieved when the acetate content of the supporting material is high. Despite this fact, a mean acetate content should be chosen. Since these supports are more stable, the preparation of reproducible columns is facilitated and times of analysis are shorter. When selecting a suitable solvent, the system which caused the lowest retention times of the PAH should be best because peaks become narrower. The measuring accuracy and sensitivity are increased by this effect. The disadvantage due to the low boiling point of CH2C12 is that this solvent in system I can produce peaks by formation of gas. In any case, a screening test as TLC procedure for optimizing systems was approved. In principle, it should be possible that all PAH are similarly separated, as indicated by TLC separation shown in Table I. ACKNOWLEDGMENT I would like to express my sincere appreciation to Miss D. Reese for the autonomous assistance in these studies.
Received for review November 13, 1972. Accepted February 20,1973.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
