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Anal. Chem. 1985, 57,2409-2412
Table 111. GC-ECD Characteristics of the Methyl, Hexafluoroisopropyl (HFIP), and Pentafluorobenzyl (PFB) Esters of Captopril N-Ethylsuccinimide (11)and S-Benzoylcaptopril (V)
a
compound
no. of peaks
re1 responsea
methyl ester of I1 HFIP ester of I1 PFB ester of I1 methyl ester of V HFIP ester of V PFB ester of V
2 2 2 1 2 1
2 8 14 1 3 5
When the number of peaks is equal to two, the sum of the two used. The area count for 50 pg of injected methyl ester of V
was was
10000.
response. The methyl esters of the S-benzoylanalogues V and VI also gave excellent ECD response, with sensitivity equal to about half of that of I1 or IV. The 4-thiophenyl analogue of captopril (VIII) behaved like captopril in that the methyl ester of the NES derivative (IX) or S-benzoyl derivative (X) exhibited excellent ECD response whereas the methyl ester of the compound (VIII) had little ECD response. Table I11 summarizes the GC-ECD characteristics of the methyl esters of I1 and V vs. those of the hexafluoroisopropyl (HFIP) and pentafluorobenzyl (PFB) esters. There was no dramatic increase in the ECD response of I1 or V as a result of introducing the fluorinated esters. In addition, the chromatograms of the methyl esters were cleaner than those of the PFB or HFIP esters. A similar behavior was noticed with the methyl, HFIP and PFB esters of the 4-thiophenyl analogues, compounds IX and X. While disulfides and trisulfides are known to be electrophoric (19,201, reports of electrophoric sulfides are rare (21). As classified by Vessman (22), the methyl ester of I1 would fall into the category of conjugated electrophores, due to the NES group. We have verified that the thiobenzoyl derivative of captopril possesses good electrophoric response while the thiol (19,20) or thioacetyl (22,23) derivative has no response. While thioarylacyl derivatives could yield sufficient sensitivity to determine captopril in blood, this reaction is not exclusive to thiols and, hence, contributes to the background. Because the NES derivative is stable, it can be formed in body fluids and then isolated intact. The use of a selective reagent such as NEM, coupled with a specific extraction technique and high-resolution capillary column GC, greatly improves the chances of successfully using the electron capture detector for
the measurement of low levels of sulfhydryl compounds such as captopril in complex matrices such as blood, plasma, or urine. Registry NO.I, 62571-86-2; 111,72180-18-8;V, 75107-57-2; VII, 64838-55-7;VIII, 75176-37-3;X, 81872-10-8;NEM, 128-53-0.
LITERATURE CITED Peliizzarl, E. D. J. Chromatogr. 1974, 96, 323-361. Poole, C. F. HRC CC, J. High Resolut. Chromatogr. Chramatogr. Commun. 1982, 5 , 454-471. Conkill, J. A,; Joppich, M.; Kuttab, S. H.; Glese, R. W. Anal. Chem. 1982, 5 4 , 481-485. Zlatkls, A,; Poole, C. F. “Electron Capture. Theory and Practice in Chromatography”; Elsevier: Amsteldam, 1981. Blau, K., King, G. S., Eds. Handbook of Derivatives for Chroamtography”; Heyden: London, 1977. Knapp, D. R. “Handbook of Analytical Derivatization Reactions”; Wiiey: New York, 1979. Ziatkis, A.; Poole. C. F. Anal. Chem. 1980, 52, 1002A-1016A. Funke, P. T.; Ivashkiv, E.; Malley, M. F.; Cohen, A. I . Anal. Chem. 1980, 52, 1086-1089. Cohen, A. I.; Deviin, R. G.; Ivashklv, E.; Funke, P. T.; McCormick, T. J. Pharm. Sci. 1982, 71, 1251-1256. Ivashklv, E.; McKlnstry, D. N.; Cohen, A. I.J. Pharm. Scl. 1884, 73, 1113-1 117. Fontana, A.; Tonlolo, C. “The Chemistry of the Thiol Group”; Patai, S., Ed.; Wiley: New Yrok, 1974; pp 294-296. Pontanova. J. P.; Shrift, A. J. Chromatogr. 1977, 139, 391-394. Cohen, A. I.; Kripalani, K. J. US. Patent 4 179568, December 8, 1979. Migdalof, B. H.; Singhvi, S. M.; Kripalani, K. J. J. Liq. Chromatogr. 1980, 3, 857-865. Matsukl, Y.; Fukuhara, K.; Ito, T.; Ono, H.; Ohara, N.; Yui, T.; Nambara, T. J. Chromatogr. 1980, 168, 177-183. Bathala, M. S.; Weinstein, S. H.; Meeker, F. S., Jr.; Singhvi, S. M.; Migdalof, B. H. J. Pharm. Scl. 1984, 73, 340-344. Drummer, 0. H.; Jarrott, B.; Louis, W. J. J. Chramatogr. 1984, 305, 83-93. Tu, J.; Liu, E.; Nickoloff, E. L. Ther. Drug Manit. 1884, 6, 59-65. Oaks, D. M.; Hartmann, H.; Dlmlck, K. P. Anal. Chem. 1964, 36, 1560- 1565. Satouchl, M.; KoJlma, T. Anal. Left. 1972, 5, 931-942. Clozel, J. P.; Caille, G.; Taeymans, Y.; Theroux, P.; Biron, P.; Besner, J. G. J. Pharm. Scl. 1984, 73, 207-209. Vessman, J. J. Chromatogr. 1980, 184, 313-324. Gyilenhaal, 0.; Hawig, P. J. Chromatogr. 1980, 769, 351-357.
Mohammed Jemal* Allen I. Cohen Squibb Institute for Medical Research P.O. Box 191 New Brunswick, New Jersey 08903
RECEIVED for review April 19,1985. Accepted June 17,1985. Part of this work was presented at American Pharmaceutical Association Academy of Pharmaceutical Sciences National Meeting, Miami Beach, FL, Nov 13-17,1983, in a symposium entitled “Recent Advances in Capillary Gas Chromatography”.
AIDS FOR ANALYTICAL CHEMISTS Differential Density Method for Determination of Carbon Load on Chromatographic Packings Wei Cheng Beckman Instruments, Inc., 1716 Fourth Street, Berkeley, California 94710 Reverse-phasematerials are now the most popular packing materials in high-performance liquid chromatography (HPLC) and are widely used in routine analysis. Commercial silicabased packings are available with methyl-, n-propyl-, n-octyl-, and octadecyl-bonded functional groups.
Carbon load is generally considered as the most important characteristic of packings and its determination mainly relies on the conventional elemental analysis. Recently Hartwick et al. reported an alternative method based on the hydrolysis of packings and gas chromatography (1). Elemental analysis
0003-2700/85/0357-2409$01.50/00 1985 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
However, it can be calculated from a known carbon load. After predetermination of the do value, a carbon load can be easily obtained by measuring the density of an original silica and the corresponding bonded silica.
s i l i c a matrix
~
s i l y l group
I
Figure 1. Two portions in bonded silica.
requires expensive equipment and skill so as to be mostly performed in professional analytical laboratories. Hartwick's method possesses considerable variation for carbon load although it offers some particular advantages for checking mixed phases. A simple, rapid, and accurate method for determination of carbon load is presented in this paper. Since silanizing reagents have a lower density than the true density of silicas, a bonded silica packing would possess a lower density in comparison with the original silica. Strictly speaking a bonded silica consists of two portions, i.e., the bulk silica and the surface bonded silane (shown in Figure 1). During silanization the hydrogen atoms in silanol groups form a new product with the function groups (usually chlorine atoms) in silanizing reagents to liberate, usually, HC1. As a result, the density of silica and silyl moiety should be theoretically different from those before the silanization. However, the weight of the liberated hydrogens is negligible in comparison with the bulk silica matrix. For instance, the typical specific surface area for small mesopore silica is about 200 m2/g, the typical silanol surface concentration is about 8 pmol/m2, and maximum coverage is about 50%, so that the maximum weight percentage of the librated hydrogen atoms would be 0.08%. Since hydrogen is very small, the librated hydrogen atoms would result in little decrease in volume. Hence it can be reasonably assumed that the density of silica moiety in bonded silica equals that for the original silica. In contrast, the weight percentage of the liberated functional group in the silanizing reagent would be considerably higher so that the density of a silyl moiety would be quite different from the original reagent but should be constant for a particular silanizing reagent. The density of a bonded silica, db, can be expressed by the following formula:
where d, and do are densities for silica and silyl moieties, respectively, and V, and Vo are corresponding volumes, respectively. Equation 1 can be rearranged in the following form:
VO ---da - db _
(2) db - do The carbon load percentage, C%, can be expressed as vs
(3)
EXPERIMENTAL SECTION Determination o f Carbon Load. The packing materials used in this study were Ultrapack ODMS (octadecyldimethylsilyl), OTMS (octyldimethylsilyl),and Me3Si (trimethylsilyl)(Beckman Instruments, Inc., Berkeley, CA). The silica used was spherical, from a single lot, with a nominal particle diameter of 10 pm and a pore diameter of 8.5 nm at the maximum value of pore volume distribution as well as the specific surface area of 160 m2/g (determined by BET method, Quantachrome Corp.). The same gravimetric method as described in the literature (2) was applied to determine the carbon load for primary bonding and end capping of these bonded silicas. The carbon load for these materials after end capping was also measured by elemental analysis (Galbraith Laboratories, Inc., Knoxville, TN). Determination of Densities. The silica and the corresponding bonded silica were dried under vacuum a t 180 "C and 70 "C, respectively. Then the dried materials were immediately stored in sealed glass vials. The volumes of three 10-mLvolumetric flasks were calibrated carefully by the conventional weighing method. About 3.5 g of silica and corresponding bonded silica were accurately weighed in two tared dry flasks, respectively. Then methanol (analytical grade) was filled into those flasks as well as the empty flask which was dried and tared also. All these flasks with stoppers were placed in an ultrasonic cleaner (Bransonic Model 12,50W) with water level to the neck of the flasks for 40 min. Then the wet portions of the flasks were rinsed by methanol several times and uncapped flasks were placed in a vacuum chamber for 30 min. The vacuum should be applied to the chamber gradually to avoid any splashing or entrainment of the solid particles. The flasks were then taken out of the vacuum chamber and allowed to stand for temperature equilibration for 1.5 h. Methanol was again added to adjust volume to exactly 10 mL for all flasks. Then the flasks were weighed accurately again. The density of an original silica and the correspondingbonded silica can be calculated by the following formulas:
w,
d, = v1
- Wlv3/
v2
-
(5) w3
w b
db =
w 2 v 3 / w3
where W, and w b are weights for bare silica and the bonded silica, respectively, Vl, V,, and V3are the calibrated volumes of flasks for the bare silica, the bonded silica, and the empty flask, respectively, and W1, W,, and W3 are corresponding methanol weights in these flasks.
RESULTS AND DISCUSSION The gravimetric method for determination of carbon load was more sensitive and accurate than the elemental analysis so that the density of a silyl moiety should be calculated based upon the carbon load from the gravimetric method. The carbon loads and densities for Ultrapack materials are shown in Table I. Obviously there was excellent reproducibility in determination of the densities and the standard deviation was estimated to be less than 0.2%. Equation 4 was differentiated to obtain the maximum possible error for the carbon load
where n is the number of carbons in the silyl moiety and Mo is the molecular weight of the silyl moiety. By some mathematical manipulations, eq 3 can be transformed into the following form (see Appendix): (4)
In eq 4,d, and db are measurable, and do is constant for a specific silylating reagent but is not directly measurable.
It was reasonable to assume the variation of density Adb = Ad, = 0.005 g/cm3 and amorphous silica density d, = 2.2 g/cm3. The maximum variations of the carbon load under various conditions were then estimated and the results are summarized in Table 11.
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
Table 111. Calculated Parameters for Some End-Capped Reverse Packings
Table I. Carbon Loads and Densities for Ultrapack Materials
C%"
exptl density, g/cm3 run average
silyl density, g/cm
original silica
0.00
2.162 2.170 2.172
2.168
ODMS-silica
10.72
1.745 1.740 1.737
1.741
0.8607
OTMS-silica
6.43
1.865 1.869 1.867
1.867
0.8625
Me&-silica
2.20
2.028 2.035 2.030
2.031
0.8638
phase
ndo/Mob
Vll VoC
k
B
ODMS-silica OTMS-silica PMS-silican
0.05524 0.05035 0.04325
0.05522 0.03762 0.01647
0.641 0.703 0.813
0.981 0.989 0.997
PMS-silica is propyldimethylsilyl group. nldl/Ml = 0.0354. cFrom the reaorted results in ref 2. perature. Nevertheless these problems do not affect the predetermination of the densities of silyl groups on silica. The close densities of the silyl moieties with various alkyl chain length offers particular advantage to the differential density method for determining the carbon load of mixed phase. Most commercial reverse-phase packings are endcapped by trimethylchlorosilane and are virtually mixed phases. Equation 1 should be modified for an end-capped phase as follows:
From the gravimetric method.
+ dOV, + d,Vl v, + v, + v,
d,V, db =
Table 11. Calculated Maximum Variation under Various Conditions ODMS-silica
2411
OTMS-silica
Me3Si-silica
C%
AC%
AC% / C%,%
AC%
AC% / C%,%
AC%
AC%/ c%,%
15 10 5 3 1
0.0042 0.0038 0.0035 0.0034 0.0032
2.8 3.8 7.0 11.3 32.0
0.0039 0.0036 0.0032 0.0031 0.0030
2.6 3.6 6.5 10.3 30.0
0.0031 0.0027 0.0024 0.0022 0.0021
2.1 2.7 4.8 7.5 21.2
The explicit trend in Table I1 was that the higher carbon load and the silyl groups with longer alkyl chains led to higher absolute variation. In contrast, the higher carbon load and the silyl groups with a shorter alkyl chain led to the lower relative variation, which was generally more important. Clearly, the carbon load was the critical factor governing the relative variation whereas the alkyl chain length merely had a minor effect. It is well-known that the silyl group with a longer alkyl chain would cause a higher carbon load and vice versa. As a result, Table I1 should be divided into two regions by a diagonal line decreasingfrom left to right with the upper region being applicable practically. It could be seen that even for a carbon load as low as 3%, the maximum relative variation was still less than 8%,which indicated the present method was accurate and reliable. The calculated densities of different silyl moieties were very close and showed a slight decrease with an increase in the alkyl chain length. This phenomenon was quite understandable. The silicon atom is relatively heavier than carbon and so has a higher density, and the more carbon atoms attached to the silicon, the lower bulk density a silyl group will possess. On the basis of the experimental data in Table I, the quantitative relation between the density of a silyl group and the number of carbons in the alkyl chain (excludingdimethyl group) could be expressed approximately by the following formula: dnt = 0.86394 - 0.00018n'
(8)
where n' is the number of carbons in the alkyl chain. Equation 8 may be utilized to estimate the density of a silyl moiety other than C1, Cg,and C18without experimental calibration. No effort was made to determine experimentally the densities of silanizing reagents (not silyl groups) due to some difficulties such as low purity, high hygroscopicity, and the solid state of octadecyldimethylchlorosilaneat ambient tem-
(9)
where dl and Vl are density and volume portion for the trimethyl moiety, respectively. The values of do and cl, are very close and the ratio Vl/ Vo is generally less than 0.06 (see Table 111). Hence, eq 9 can be reduced to the simple form db =
+ doV4 v, + v,,
d,V,
where V,l = V, + Vl. Equation 3 for the end-capped phase must be accordingly modified as follows:
C% =
+ db(Vs + vo + vi)
12ndoVo/Mo 12n1d1V1/M1
(11)
where nl and Ml are the number of carbons and molecular weight for trimethylsilyl moiety, respectively. Equation 11 can be then rearranged in the form
where k = dlnlMo/donMl. The k values for various silanizing reagents are calculated and shown in Table 111. Furthermore, B is defined as (Vo + KVl)/(Vo Vl). The B values for several typical silanizing reagents are computed and shown in Table I11 using the reported primary and end-cappingalkyl surface concentrations (2). Apparently B is very close to unity and (Vo + Vl) can be substituted for ( Vo KVJ in eq 12 with little deviation. Noting V,l = Vo + V,, eq 12 can be reduced to the simple form
+
+
It can be noticed that eq 10 and eq 1 3 are identical with eq 1and eq 3, respectively. As a result, eq 4 can be applied to calculated carbon load for end-capped reverse-phase packings. Although the values of Vl/V,, may vary depending on the silica pore structure and silanization conditions, the deviation would be generally small from the typical values listed in Table 111. Hence eq 4 is applicable for all end-capped reverse-phase packings. For a typical mixed phase with two different types of silyl groups, eq 4 would still be appropriate if B is close to 1. In
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ANALYTICAL
CHEMISTRY,
VOL. 57, NO. 12, OCTOBER iga
Table IV. Comparison of Carbon Loads from Different Methods
Table V. Calculated Thickness of Silyl Group on Silica Surfacea
dh
phase ODMS-silica end capped OTMS-silica end capped
C%" 11.071
6.602
C%b
run
average
C%C
11.3
1.783 1.778 1.779
1.780
11.053
6.8
1.899 1.895 1.897
1.897
6.612
Gravimetric method. Elemental analysis. Differential densitv method. the case that B deviates from 1,the B value may be considered as a correction coefficient in eq 4 if the B value is or near a constant. The carbon load for the end-capped Ultrapack packings analyzed by the gravimetric, elemental, and differential density methods are shown in Table IV. Evidently the carbon load from differential density method was very close to that from the gravimetric method and the relative variation was less than 0.17%. This confirmed that the present method was suitable for both single phase and end-capped phase. It should be also mentioned that removing completely the physically adsorbed water on silica surface is difficult even at 200 "C under vacuum (3). Hence a trace amount of the residual water would most likely remain on silica surface all the time and consequently affect the densities for both bare silica and bonded silica to a very small degree. However the surface water could be considered as a permanent portion of the silica and virtually would have no effect on the determination of carbon load unless the amount of water on a bare silica is quite different from that on the corresponding bonded silica. On the other hand the elemental analysis analysis consistently showed slightly higher values of the carbon load. As reported before ( I ) , it was probably attributed to embedded organics in the silica matrix and the residual organic solvent adsorbed on pore surface. It is difficult to completely avoid these factors so that a carbon load from elemental analysis often does not represent the true surface ligand concentration. However, similar to the residual surface water, these factors would contribute equally to the densities of both original silica and the corresponding bonded silica so that their effect on the densities can cancel out each other. Thus the carbon load determined from the present method would reflect the true surface silyl concentration. Besides the carbon load, the present method would provide an estimate of thickness of silyl groups on silica surface. The minimum average thickness, t , can be estimated by the following formula (see Appendix):
t, A
ODMS-silica
OTMS-silica
Me3Si-silica
24.00
8.64
3.38
ONote: calculated from the data without end carmine.
of a bonded phase can be also employed to obtain the corresponding bare silica ( 1 , 5 ) . The present method merely requires two density measurements and a complete determination takes about 3 h. Several determinations can be carried out at the same time to reduce greatly the analysis time for each measurement. Furthermore, it may be possible to apply a pycnometer with gas permeation to measure densities. Conceivably it would greatly speed up the analysis because slow liquid permeation and temperature equilibration could be eliminated. The surface of a chromatographicsilica is hydrophilic while the surface of a reverse phase is generally hydrophobic. Hence methanol, which can wet both silica and the bonded silica easily, is chosen as the solvent. Other organic solvents such as 2-propanol and acetonitrile can be also selected as solvent. However, owing to slightly different wettability the density of a silyl group should be calibrated again. The present method is not limited to silica-based reverse packings and it can be applied to packings with different base materials provided that the density of a bonded phase is considerably different from that of the base material.
ACKNOWLEDGMENT I thank Arthur Furst for some useful discussion. Ali Raji's help in some experimental work is gratefully acknowledged.
APPENDIX Both numerators can be added to the corresponding denominators in eq 2 and one obtains
--VO
V, + Vo
CONCLUSION In a broad sense, the differential density method is the extension of the gravimetric method, which must include a small scale silanization process so as to be often inapplicable. In the case of the original silica being unavailable,the bonded silica can be converted to the bare silica by simply heating at 500 OC for 3 h, rehydrating with water, and then redrying by using the foregoing method. The acid and base hydrolysis
- db
d, -do Inserting eq A1 into eq 3, one then obtains
The thickness of a silyl layer on silica surface can be calculated by the formula t = vo/s (A3) Inserting eq 2 into eq A3, one obtains
Substituting l / d s for V , in eq A4, one then obtains
t= where S is specific surface area of the original silica. The calculated results listed in Table V were in fairly good agreement with the previously reported data ( 2 , 4 ) .
--ds
da - db Sd,(db - do)
Registry No. C, 7440-44-0.
LITERATURE CITED (1) Crowther, J. 8.;Fazio, S. D.; Schiksnls, R.; Marcus, S.;Hartwlck, R. A. J . Chromatogr. 1984, 289, 387-375. (2) Cheng, W.; McCown, M. J. Chromatogr. 1985, 318, 173-185. (3) Unger, K. K. Porous Slllca"; Elsevler: Amsterdam and New York,
1979. (4) Roumeliotis, P.; Unger, K. K. J. Chromatogr. 1978, 149, 211-224. (5) Verzele, M.; Mussche, P.; Sanda. P. J. Chromatogr. 1980, 190,
313-31 5.
RECEIVED for review February 25, 1985. Accepted May 28, 1985.
