ANALYTICAL CHEMISTRY, VOL 51, NO. 1, JANUARY 1979
CONCLUSION
7
Atomic Energy Agency, Vienna, 1974. Vol 1. p 149. (6) R S. Yalow and S. A. Berson, in "In vitro Procedures with Radioisotopes in Medicine". International Atomic Agency, Vienna, 1970, p 455. (7) R. S. Yalow and S. A . Berson. in "Principles of Campetitive Protein-Binding Assays". W . D Odell and W. H. Daughaday, Ed , Lippincott, Philadelphia, P a , 1971, p 1 (8) R . P Ekins, in 'In vitro Procedures with Radioisotopes in Medicine". International Atomic Energy Agency, Vienna. 1970, p 325. (9) R Ekins and B. Newman. Acta Endocrinol., Suppl., 147, 11 (1970). (10) R P. Ekins. G B. Newman. and J.. L H. O'Riordan, in "Radioisotopes in Medicine: in vitro Stixlies", R. L.. Hayes. F. A. Goswitz, and B E. Pearson Murphy. Ed., U S Atomic Energy Commission. Oak Ridge, Tenn., 1968, P 59 (11) R . P. Ekins, G . B. Newman, and J. 1. H. O'Riordan, in "Statistics in Endocrinology''.J W McArthur and T CoRon, Ed., M.1.T Press. Cambrage. Mass.. 1970, p 345. (12) 0. Rodbard and Y. Feidman, Immunochemistry, 15, 71 (1978). (13) H J. Schuurman and C. L de Ligny. Clin Chim. Acfa. 89. 191 (1978). (14) D. Rodbard, in "Principles of Competitive Protein-Binding Assays". W. D. Odell and W. H Daughaday. Ed.. Lippincott. Philadelphia. Pa., 1971, p 204. (15) D Rodbard, Adv. Exp. Med Biol.. 36, 289 (1973). f 16) D Rodbard and D. M. Hutt. in "Radioimmunoassayand Rebted Procedures in Medicine", International Atomic Energy Agency. Vienna, 1974. Vol. 1. p 165 (17) J C. Hendrick and P. Franchimont, Prof.Bid. fluids (Proc. Coll. Brugge), 24, 735 (1976). (18) I. E M Miles. Prof. Biol. Fluids (Proc Coll. Brugge). 24, 695 (1976).
T h e models of Yalow and Berson and of Ekins et al. for the competitive inhibition radioimmunoassay yield divergent values of t h e conditions for maximal sensitivity. For solid phase RIA of IgA, calculations based on t h e model of Yalow a n d Berson a n d t h e case of Ekins' model in which both t h e bound and the free fraction are counted agree reasonably well with experimental results, Hence, these models are useful to predict initial values of t h e experimental conditions for a sensitive assay t h a t can be subsequently refined in a n experimental optimization procedure. According to a simplified physical model of t h e sandwich assay, t h e minimal detection limit can be lower than that of t h e traditional (labeled antigen) competitive inhibition radioimmunoassay.
LITERATURE CITED (1) R. S. Yalow and S A Berson. Nature (London). 184. 1648 (1959). (2) W. M. Hunter, in "Handbook of Experimental Immunology" 2nd ed.. D. M. Weir, Ed., Biackweli, Oxford, 1973, Chap 17 (3) D. S. Skeiley, L P. Brown, and P K. Besch. Clin. Chem ( Winston-Salem. N C ), 19. 146 (1973) (4) L Wde, in Radioimmunoassay Methods K E Kirkham and W M Hunter Ed Churchill Livinastone Edinburah 1971 D 405 (5) L E M Miles, P Bieber L- F Eng and D A Ltpschitz in Radioimmunoassay and Related Procedures in Medicine International
RECEIVEDfor review April 17. 1978. Accepted October 12, 1978
Determination of Hydroxyl Groups in Poly(ethy1ene glycols) D6nes F. Fritz, Araksi Sahil, Hans-Peter Keller, and Ervin
sz. Kov6ts"
Laboratoire de Chimie Technique de / ' € c o l e Polytechnique F6dGrale de L ausanne Switzerland
An analytical method for the determination of hydroxyl content in poly(ethy1ene glycol) is presented and verified. It consists of substituting the hydroxyl group with a chromophoric siloxy group, purification of the silylated polymer, and a photometric determination of the chromophor concentration. The silanization of the primary hydroxyls with dimethylaminosilanes is shown to proceed quantitatively under very mild conditions and that the elimination of the excess reagent by precipitation is easy. The precision of the method is f4.5% (95% confidence level) down to 5 X mol kg-I and it is estimated to be mol kg-'. The method has applicable to a lower limit of about the same precision as the current standard acylation method but yields a 1000-fold gain in sensitivity.
Substitution of a functional group by a chromophor and the subsequent photometric determination of the chromophor concentration is a n efficient method for the quantitative analysis of small concentrations of t h e substituted group. Obviously, success depends on t h e availability of a selective reagent capable of quantitatively substituting t h e functional group of interest under mild conditions. T h e silanization of hydroxyl groups by monofunctional trimethylsilanes is a common technique in gas chromatography t o transform alcohols into t h e more volatile trimethylsiloxy derivatives. I n t h e present work, it is shown t h a t chromophor-containing silanes substitute quantitatively hydroxyl end groups in poly(ethy1ene glycols) in an analogous reaction permitting their 0003-2700/79/0351-0007$01 O O / O
analysis down to concentrations of IO4 mol kg-' (mol hydroxyl per kg polymer) while t h e lower limit with t h e procedures actually used is about 10 mol kg Current methods consist of a n acvlation step with an acid anhydride followed hv selective hydrolysis of t h e excess reagent and determination of the e x c i w acid by titration T h e hydroxvl content is then calculated with reference t o t h e titration of a blank Acetic anhydride is by far t h e most frequentlv used reagent ( 1 8 ) but phthalic anhydride (41, .i-nitrophthalic anhydride (.5) and pyromellitic dianhydride ( 6 ) have also been proposed. Other. non-acidimetric titration methods d s o imply t h e determination of t h e excew reagent ( 7 , 8) T h e result depends on a relatively Emall difference between titrations. hence t h e lower limit of measurable hydroxyl concentration IS ahout 0.1 mol kg '. T h e same is true for a n interesting method (9) which consists of t h e transformation of the hvdroxyl compounds into the chloroformates with phosgene. followed hv evaporation of the excess reagent and argentometric titration of t h e chloride content In our laboratory, t h e phthalic anhydride procedure was slightly improved bv modifying it to become a direct method ( 1 0 ) . T h e acylated polymei was isolated and purified by repeated precipitations and t h e acid phthalate monoester content was titrated directlv. T h e main difficulty was still t h e elimination of t h e excess reagent which seemed t o be strongly retained bv the pol-mer. The same phenomenon was observed by Kammerer a n d Grover ( 2 1 ) who found t h a t five precipitations were necessary t o purify t h e poly(ethy1ene glycol) acylated with henzalazoben7nic anhvdride a t 200 "C
'
S 1978 American Chemical Societv
8
ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
Repetition of this method in our laboratory gave erroneously high hydroxyl concentrations: probably at the high acylation temperature, t h e polymer chains were partly cleaved. In most cases t h e silylation of hydroxyl groups is a fast reaction proceeding at moderate temperatures. Apart from chlorosilane, hasic silylating agents such as disilaeane, silylalkylamines, silylamides, etc. are often used. T h i s type of reagent seemed suitable for t h e treatment of poly(ethy1ene glycols) in view of the stability of the polyether chain in a hasic milieu. Moreover, gas chromatographic d a t a of ethers on methylsilicones and of silanes on poly(et,hylene glycols) show that t h e interaction of silanes with ethers is negligible, t h u s suggest,ing t h a t there will he n o complex formation hetween silylating agent a n d t h e polyether. Silanes are very soluhle in m a n y organic solvents. An easy separation of t h e excess reagent was therefore expect.ed when purifying t h e polymer by precipitation. Accordingly we attempted to int,roduce a chromophoric silyl group into poly(ethy1ene glycols) by a silylation reaction followed by separation and purification of t h e silylated polymer b y precipit,ation, then proceeding to derive t h e hydroxyl concentration b y photometric measurements. EXPFRTMENTAI, General. The structure of all synthetized siibstances was confirmed by UV, TR, and 'H NMR spectra as well as by elemental analysis. The elemental analyses were made by W. Manser (Laboratory of Organic Chemistry at the Swiss Federal Institlite of Technology, Zurich). All melting points are uncorrected and the densities (corrected for vacuiim) are averages of at least two determinations. Materials. Poly(ethy1ene glycols) with two terminal hydroxyl groups of nominal molecular weights: 600, 1000, 2000, and 20000, pract. grade, were from Fluka AC, (Ruchs, Switzerland). Phenyldimethylchlorosilane (pract.), triphenylchlorosilane (piirnm). 1-bromonaphthalene (puriss.), methyl bromide (purum), htityllithium (pract; 1.5 M in hexane), dimethylamine (puriim), 1-nonanol, 5-nonanol, and 2-methyl-2-octanol (purum) were also from Fluka as well as the aluminum oxide for chromatography (type 507C, neutral, activity I) and the cation-exchange resin (Amberlite IR-120, strong acid, H+ form). The anion-exchange resin (Lewatite M P 7080, weak haw) was from Merck (Darmstadt, Germany). The solvents: diethylene glycol dimethyl ether, toluene, henzene, petroleum et,her (bp 30--46 "C), 1,2-dimethoxyethane, and cyclohexane (all purum, Fluka) were distilled over NaH before use. Spectroscopic grade pent,ane and ethanol were from Merck (Uvasol). A p p a r a t u s . For gas chromatographic analyses, a PackardBecker (Delft, Holland; model 419) research gas chromatograph was used, equipped with flame ionization detectors. The carrier gas was helium. The spectrographs used were: IR-Spectrometer (model 700, Perkin-Elmer), UV-Spect,rophotometer (model 635. Varian-Techtron), and 'H NMR instrument (model T-60, Varian). Poly(ethy1ene Glycols). Purification of the Poly(ethylene glycols) (Hydroxyl Terminated: H-PEG). A solution of 100 g of commercial practical grade polymer in 400 mL of 0.01 M NaOH was digested a t 40 "C for 24 h, then passed through two ionexchange columns, the first containing 50 mL of Amberlite-IR 120 (H+ form) and the second 50 mI, of T,ewat,ite M P 7080 (base form). After evaporation of the water, the residue was dissolved in 1000 mL of toluene and filtered on an aluminum oxide column (50 g). T h e agitated solution was then thermostated at the temperture indicated below and 5.0 1, of petroleum ether added dropwise. T h e slurry was cooled to 0 "C, allowed to stand for 15 min, and the precipitate was filtered. The temperature of precipitation was critical; i t was 0 "C for H-PEG of molecular weight 600 (H-PEG-600), 10 "C for H-PEG-1000. 20 "C for H-PEG-2000, and 30 "C for polymers of higher molecular weight. T h e purified product was dried at room temperature/O.l Torr. Dimethoxypoly(ethylenP glycols) (M-PEG)were prepared from the purified poly(ethy1ene glycols) following the Williamson ether synthesis (Synthesis and properties of t,hese substances as stationary phases for gas chromatography will he described in a forthcoming paper). Ry this procediire dimethoxypoly(ethy1ene
glycols) (M-PEG) of nominal molecular weights: 600, 1000, 2000, and 20000 were prepared, with a content of iinreacted hydroxyls 10 ' mol kg '. of about Silylating Agents. Starting materials were commercially availahle chlorosilanes with the exception of the l-napht,hyldimethyl derivative. This substance was prepared by the procedure of Ref. 1 2 modified according to Ref. 13. (Synthetic methods using Grignard componnds described in Ref. 14 and 15 provided us with only impure samples). 1-Nnl?hthyldimcfhylchloros~lane.A solution of 21.5 g of I-hromonaphthalene (0.104 mol) in 50 mT, of hexane was added t o a qnliition of 0.104 mol of butyllithiiim in 75 mT. of n-hexane. After 2 h, the precipitation of naphthyllithium was complete. The precipitate was filtered, washed with hexane, and dissolved in 300 mL of ether. This solution was added dropwise at room t,emperat,iire to 2fi.8 g (0.208 mol) of dimethyldichlorosilane dissolved in h0 mT, of n-hexane. After 2 h the solution was filtered and the volatiles were evporated a t room temperature/l2 Torr. Distillation of the residue gave (108-110 "(1/0.15 Torr): 17.1 g of I -naphthyldimethylchlorosilane;yield 76%; n z o=~ 1.59fi7; d2" = 1.121 g c m ~ (lit.: ' n2"" = 1.5951 ( 1 4 ) and 1.5957 ( 1 6 ) ; dz0, = 1.190 g cm ' ( 1 4 ) and l.lF)I ( 1 5 ) ) . Silornncn and Aminosilnncs. 1,,?-niphpnyl-1,7,S,S-tetrnmpthyldisilnzane (IA). In a flask a solution of 12.0 g of freshly distilled phengldimethylchlorosilane was prepared in 200 m1, of carbon tetrachloride and kept for 20 h at room temperatnre in an atmosphere of ammonia by introducing a slow current of dry ammonia over the solution. The solution was filtered to separate the ammoniiim chloride and the solvent evaporated at, atmospheric prwsure. On distillation, the residue first evolved NH3 which condensed in the liqriid nit,rogen cooled trap (Apparently, phenyldimet,hylsilylamine, I R , was formed in the reaction which dimerized on heating in vacuum). The main fraction distilled a t 110 ' ( 1 j O . l Torr: 7.1 g of colorless liquid; yield 71%; nZoD= 1 .*5882; d2" = 0.984 g cm (lit.: nZoD= 1.5460; dZo4= 0.978 and 0.987 g cm ' in Ref. I 6 and 17). Triphcnyl.cilylaminc (2R). In an analogous reaction, 15.0 g of triphenylchlorosilane and ammonia gave after reaction and evaporation of the solvent a residue which yielded after recrystallization from petroleum ether. 10.1 g pure triphenylsilylamine, 2R, of mp ,57-58 "C; yield 72% (lit.: 55-56 "C ( 1 8 ) and .59--60 "C (19)). DimPthslnminosilanex. &nerol Procedure. A IO'% solution of 1 equiv monochlorosilane in hexane (in benzene if necessary for solubility reasons) was stirred in a flask equipped with a reflux condenser cooled at --20"C. From a communicating flask 2.2 equiv of dimethylamine vapor was slowly introduced over the solution and the mixture kept at room temperature overnight. After filtration and evaporation of the solvent, the residue was distilled or crystallized. Typical yields: 75-9670. Phenyldimethyl(dimpthylamino)silane (1C): 58 g dimet,hylamine, 100 g phenyldimethylchlorosilane; 1000 mL hexane. Distillation of the raw product gave at 82--83 " C / l 5 Torr: 100 g pure dimethylaminosilane, IC; n ' " ~= 1.4975; d'" = 0.912 g cm-3; yield 9 5 7 ~ . Triphenyl(dimethylami~o),~ilanc ( 2 0 : 3.7 g dimethylamine; 11.0 g triphenylchlorosilane, 100 mL benzene. Crystallization of the raw product from petroleum ether gave: 8.5 g crystal powder of m p 78 "C; yield 7570 (lit.: mp 80-81 " C (20)). I-Naphthyldimcthyl(dimethy/amino)silnne(3C): 4.5 g dimethylamine, 11.I g 1-naphthyldimethylchlorosilane,100 mL hexane. Distillation of the raw prodiict gave at 89 "C/O.Ofi Torr: 7.8 g pure dimethylaminosilane, 3C: yield 68%; nZon= 1.5735; d'' = 1.001 g ~ m - ~ . UV S t a n d a r d s (lD-3D). About 5 g of dimethylaminosilane was heated with two equivalents of 2-methoxyethanol under conditions given in the "Recommended Procedure". Distillation of the resulting mixture gave the 2-methoxyethoxy derivative in nearly quantit,ative yield. The raw triphenyl(2'-methoxyethoxv)silane was further purified by crystallization from cyclohexane/hexane (1/4: v/v) at --lo "C. The physical data of the standards are summarized in Table I; for the IJV spectra see Figure 1. Kinetic Experiments. The silylating agent was dissolved in toluene to give a '2 M solution "A". Equimolar quantities of 1-nonanol, 5-nonanol, and 2-methyl-2-oct;lnol (1.44 g of each) were mixed with tridecane (1 .OO g) to give solution "R". Solution R
ANALYTICAL CHEMISTRY VOl -
__
-
51, NO 1, JANUARY 1979
9
-
Tahle 1. Physiral Data of R'., R"-,a n d R"'-(2'-methoxyethoxy)silanes~ R , R ' , R"
h p (*(?)/Torr
phenyldimethyl I n triphenyl 2 D 1-naphthyldimethvl 3 D
mp ("C)
70" 10.1 ._
d'" g r m
..
0 958
67-6gb
..
11l"iO 03
'
~ ( hmol )
n20D
267 ( 2 6 4 ) 1 06 x 1 0 ' ( 2 6 4 ) 5 .33 x 10' ( 2 8 2 5 )
14848
1.5582
1051
a T h e purity of t h e sample5 cnntrolled h y gas chromatographv was hetter than 99.8%
6 7 68 "c' in Ref 21 -. -
-
- .
_ _
.
(nm)
-
-
Tahle-IT. Reaction R a t e s i f DifferentSilyIating Agentsa with a Primary, Secondary, and Tertiary Al&hoI at Different Temperaturesa silylating agent" _- . _ . phenyldimethyl triphenvl triph enyl I / 2 ( -NH-) (disilazane) -NH, -N(CH, ), 2R 2c - __ __1 A - _k,, mol ( k , '1, min 1, ( L min mol ' ) k , min-' t , , 2 , min k, min ' 1 , ~,min
R'R"R" X tempera-, ture, " C
alcohol prim
60
0.002 -0
sec
tert prim
100
-
ser
tert prim sec tert
140
0.049 0.0032 -0 0.1 44 0.0054 -0
3.50
0.0087 0.00059 0 0,0096 0.001 6 0.00049
(115) ( 3 690)
0.034
20
0.000.5 -0
7 390
0.0038 --0
k , min
'
t , , , ,rnin
fast 0.37 0.001
14 217
0 1.8
700
4.8 128
8.1
0.086
(104) (627) (2026)
I-naphthyldimethvl - N(CH, ) 2 3c - -
182
a Silylating agent of the general formula: R ' R ' R"'SiX, 2 M-solution in toluene, molar ratio alcoholisilane, 1 / l o , alcohols prim, 1-nonanol, see, 5-nonanol; tert, 2-methyl-2-ortanol; iero-order reaction rate is designated by k , . & IOO W
After cooling to an appropriate temperature. the silvlated polvmer precipitated hv adding dropwise 50 mT, of low-hoiling petroleum ether to the stirred Goliltion (the temperature of the precipitation I S critical see under section "Purification of H-PEG' ) 4fter precipitation, the mivture is stirred a t 0 'C for 15 min. then filtered on a sintered glass filter iG3) and w a ~ h e dwith 5-mT portions of 20 mT, of petroleum ether The precipitat~I S redissolved on the filter in 5 mT, of warm 1.2-dimethoxyethane and precipitated a second time as ahove If the expected hvdroxvl content of the polymer is lower than 10 mol kg I , the precipitation Should he repeated a third time After the last precipitation. the polymer i s washed on the filter with 20 mL of ITvasol-grade pentane, dried for 5 h a t room tempertrire/O 1 Torr or i r - a stream of dry nitrogen A portion of the dry sample I C diwolved in ITvasoI-grade ethanol so as to yield an ahsorhance of 0 2 0 8 The ahsorbanre of the solution is measured vs ethanol as reference at 264 nm (= A) for the phenvldimethvlsilvl. 1, a n d the triphenvlsilvl. 2, a n d at 282 5 nm for the 1-naphthyldimethvlsil~l,3, derivative. If the sample concentration i s higher than t 0 g 1, (=- c ) , a n ethanolic iolrition of the pure polviethvlene glvcol) of ahoiit the same concentration must he used as a reference T h e molarit\ of the silvl groiips in the sollition. m-,], is given hv IS
1000
100
'
7
-
' f t
+
.
10
z-; 1 .
I
1
t
-
4
*
-
do
'
~
-
-
i
*
. .
.
. '
ZOO nm( 1 )
Figure 1. UV spectra in ethanol of phenyldimethyl(2'-methoxyethoxy)silane, 1, triphenyl(2'-rnethoxyethoxy)silane, 2, and l-naphthyldimethyl(2'-rnethoxyethoxy)silane, 3
was first chromatographed to determine the initial ratios, So,,,,, of the peak areas of the alcohols, nnlr, relative to that of tridecane, a T n , where S,,,,, = n,l,/n.rD. O n e m1, of sollition A Wac thermostated a t a given temperature and 120 PI, of solutinn R was added, corresponding to a molar ratio of 1 to 10 for each alcohol relative to the silylat,ing agent. Periodically, 10 p T , of the reaction mixture were withdrawn with a syringe and mixed with 10 .UT, of cold methanol. From the gas chromatogram of this mixture, at the time, t , were determined. the ratios of the peak areas, St,nlcr vs. time, t (min) gave T h e slope of the linear plot In (.5'o,al~/St,aic) the pseudo-first-order constants, h (min.'), summarized in Tahle
mCnI A / < , = r/EWpS,I
'
(1)
wh~rA e is the ahsorhance of the soliltion. c is the concentration of the silylated polvmer in g 1) ', t h 15 t h e molar ahwrptivitv of the sil\ilating agent, and F.WPs,,is the equivalent weight of the silblated polbmer which r a n be calculated hy
FWps,, = c t A / A
TI. Determination of the Hydroxyl Content of Poly(ethy1ene glycols). Recommended P r o c e d u r e . For polymers with expected equivalent weights higher then 1000 a sample of I g (0.5 g for lower equivalent weights) is dissolved in 5 mT, of dry I ,2dimethoxyethane. The dissoliition is omitted if the melting point of the polymer is less than 60 "C.T h e flask is purged with dry argon (or nitrogen) a n d three equivalents for each expected equivalent hydroxyl (hut not less than 200 mg) of dimethylaminosilnne is added. T h e homogeni~edmixtiire is kept at 60 O C for 30 rnin if 1 -naphthyldimethyl(dimethylRmino)silane, 3C, or phenyldimethyl(dimethylamino)silane, l C , is iised or ? h / 8 0 "C for triphenyl(dimethvlaminn)siiane, 2C. The sample i s then diluted at, room temperatiir~to 10 mT, with 1.2-dimethoxyethane.
mol T,
r; mol
'
(2)
In order to obtain the eqiiivalent height of the hvdroxvl ter mmated polvmer, EWPoH,the moleciilar weight of the ~ i l vgroiip, l hlWs,I,and that nf the ciihstitiited proton must be taken into accoiint F,WpnH
- cionsof Table 111 are sufficient. Under these conditions, secondary alcohols are also quantitatively silylated by the agents 1C, and 3C (tertiary alcohols t o a small extent). I t is interesting t o note that the reaction with the silazane, l A , could he catalyzed by a m monium chloride and the corresponding experimental conditions are also included in Table 111. For the analytical procedure, it is necessary to consider more closely t h e separation of the silylated polymer and t h e quantitative elimination of t h e excess silylating agent. Therefore, t h e procedure with 1-naphthyldimethyl(dimethy1amino)silane was applied under the conditions proposed in Table I11 t o four polymers, two of them hydroxyl terminated, t h e other two with methoxy end groups of unknown, but certainly very low, hydroxyl content. Two nominal molecular weights were chosen for both types of polymers, 600 and 20 000, representing extreme types regarding the diffi-
ANALYTICAL CHEMISTRY. VOL. 51, NO. 1. JANUARY 1979
11
Table 111. Conditions Proposed for Silylation of Poly(ethy1ene Glycols) with Agents of the general formula: R'R"R"'SiX silylating agent ____ -. -. -. phenyl1 -naphthylR'R"R"': phenyldimethyl triphenyl triphenyl dimethyl dimethyl x '/z(-NH-) -" 2 -N(CH 3 ) ? -N( CH ) 2 -N( CH 3 1 2 1A 2B 2c 1c 3c with 2 mg NH,CI proposed temperature, C 100 80 80 80 60 60 proposed time. h 4 4 2 2 0.5 0.5 MWa foundb 2123 2145 2189 2167 21 49 2123 %
a Molecular weights, MW, are obtained by applying the proposed method t o a polyethylene glycol of nominal molecular weight of 2000. b Confidence interval for one determination calculated from these results: A s s =- 6 6 corresponding t o 3.1% ( 9 5 % confidence level). Average of all determinations: 2149 i 27.
- _______________-- - - -_ - - - - - Table IV. Relative Errors for t h e Determination of Hydroxyl Content Using t h e 1-Naphthyl Derivative, 3C, as Silylating Agent
I__
H-PEG-
Ra Deviation about t h e average in column %
1
P
2000
a
+3.04 +2.62 - 0.76 ~-1.49 2.55 -0.86
b c 2
a
b
c V @OH
Age(%)
5.23 0.949 2.4
H-PEG2 OM +0.85 +1.71 -3.42 +3.42 -0.85 -1.71
M-PEG-600 --3.83 +2.55 ~0.43 +3.40 -1.70 -0.85
6.14 0.117 2.6
7.30 2.35 x 2.8
M-PEG-1000
M-PEG-2000
+ 1.09
+ 0.05
-0.22
+ 3.45
+ 0.83
--2.77 + 2.57 - 2.02 - 0.84 +3.01
-1.15
+ 2.58 -1.90 --0.40 2.60 5 . 3 6 x 10.' 1.7
M-PEG-2OM
-1.19 --3.12 ~-1.60 + 1.63
5.63 6.75 x 2.5
5.79 9.88 x 10.' 2.5
R , repetition of the procedure; P, repetition of the precipitation and of the photometric measurements; % O H , hydroxyl content, average of six determinations. The relative deviations about the average are given in percent of the average; V is the variance of the relative error; A q i is the confidence limit of the average. ___
__.
._~__
culties involved in t h e purification of t h e silylated polymers hv precipitation and filtrat,ion. For this operation, t h e best pair of solvents proved t o he 1.2-dimethoxyethane and low boiling petroleum ether. T h e sample7 were redissolved and precipitated repeatedly and each time a portion of t h e pre. cipitate was subjected t o photometric analysis. T h e results a r e plotted in Figure 2, expressed ac apparent hydroxyl content its a function of the number of precipitations and show that in t h e case of t h e hydroxyl terminated polymers a constant silyl concentration is attained after t h e first precipitation. Three precipitations were necessary however for the methylated polymers in order to obtain consistent results. C o n t r o l of t h e Recommended P r o c e d u r e . After having established the experimental conditions for the silylation and t h e separation of poly(ethy1ene glycol.), the analvtical procedure was subjected to a series of contrnls. In a series of experiments a purified. hydroxyl terminated poly(ethy1ene glycol) of nominal molecular weight of 2000 was silylated with different silylating agent. under the experimental conditions listed in Tahle 111. '!'he silyl content of the sample was determined after t h e ~ w t u : dprecipitation. T h e calculated molecular weighti. given in 'T'ahle 111. shnw a re!ative error of 3.1%. T h e determination of t h e molecular weight of this sample by vapor pressure osmometry gave a yalue of 21.7 X 10'. in excellent agreement with t h e aI7erage of t h e determinations by the v x i o u s iilylation procedures which was 121.5 f 0.3) x IO2. In another series of experiments, the hvdroxyl content of two hydroxyl terminated and four methoxylated pnly(ethy1ene glycols) was determined using I - n a p h t hyldimethyl(dimethy1amino)silane. Each polymer was silylated in two parallel experiments and the silylated polvmers were purified hy repeated precipitations following t h e Recommended Procedure. These pure samples gave 6 results for each polymer, corresponding t o the second. third. and fourth precipitation for the hydroxyl terminated p o l v m ~ r( P = CI.b.
-. . ..
-3
1:
..+ ~i
n-1
. .
*
. ... .
-.
.
.-. ..
M-PEG-BOO *
.
..
..
2
3
4
. M-PEG-ZOM
5
Figure 2. Apparent hydroxyl concentrations, moH,in different poly(ethyleneglycols) against n. the number of ptecipitations. Recommended Procedure with I-naphthyldimethyl(dimethylamino)silane
and c in Table J V ) and t o the third, fourth, and fifth precipitation for t h e methoxylated polymer (F' = a , b, and c in Table TV). For each polymer the deviations from the average were calculated and expressed as percentage of t h e average of the 6 results and the variances of these relative errors were calculated. T h e results, summarized in Table IV, demonstrate t h a t t h e relative errors are t h e same for polymers differing in hydroxyl content by a factor of 2OOO (Application of Fisher's test to t,he largest and smallest variance indicates t h a t the difference between them is not significant). Having shown t h a t t h e experimental domain is hoinoscedastic in relative errors. a n analysis of variance was applied t o the percentage deviations proving that no additional error was introduced by repeating the operations following the precipitation steps.
12
A N A L Y T I C A L CHEMISTRY, VOL. 5 1 , N O . 1, J A N U A R Y 1979
Table V. Determination of the Hydroxyl Content of a Methoxylated Poly(ethy1ene glycol) with Small Quantities of Hydroxyl Terminated Polymer Added. Molecular Weight of Both Polymers: 21.5 x l o 2 H-PEG a d d e d , wt%
hydroxyl content, mmol kg-' ______ ___
0.50
1.00 --___----_--I-.
added 0.0 4.7 9.3
found 4.7
9.3
14.8
calculated (4.7) 9.4 14.0
_______
_I____
From t h e results the confidence limit for one determination is calculated as 3~4.9%( a t t h e 9570 confidence level). This figure, compared with the confidence limit calculated from t h e results listed in Table TI1 (3.1 70),also shows that there is no significant difference in analytical results when using different silylating agents. W e had no other methods of high enough sensitivity to check the small figures found for the hydroxyl content in methylated poly(ethy1ene glycols). Therefore, in a third series of experiments we prepared mixtures of a methylated product of relatively high hydroxyl content (4.7 mmol kg I ) with a hydroxyl terminated polymer of the same molecular weight and analyzed these samples after silylation with l - n a p h thyldimethyl(dimethy1amino)silane. T h e results reported in Table V show t h a t t h e values found experimentally are in satisfactory agreement with those calculated as the sum of t h e hydroxyl concentration in t h e methylated product and t h a t of the hydroxyl terminated additive. In a final series of experiments, we examined the reaction of t h e 1-naphthyldimethyl(dimethy1amino)silane with a few substances containing other functional groups which could interfere with t h e hydroxyl determination. A ketone (3pentanone) and an ester (hexyl acetate) failed t o react with the reagent. On t h e other hand, an acid (hexanoic acid) reacted quantitatively t o give the corresponding silyl ester
acylation titration method. However, since the relative error remains essentially the same for very low hydroxyl concentrations, it can he used successfully in these cases where a titration method would completely fail. T h e lower detection limit can he estimated a t about 10 mol kg considering that a 570 solution of t h e 1-naphthylsiloxy derivative of such a polymer would yield a n absorbance of 0.03. In conclusion, t h e silylation by dimethylaminosilanes is quantitative even a t very low hydroxyl concentrations and proceeds under mild conditions. T h e remarkable ease of the reaction suggests the possibility of its application t o the derivatization of nonchromophoric alcohols for UV detection in liquid chromatography.
',
LITERATURE CITED J. S. Fritz and G. M. Schenk, Anal. Chem., 31, 1606 (1959).
G. M. Schenk and J. S. Fritz, Anal. Chem., 32, 987 (1960). R. S. Stetzler and C. F. Smullin, Anal. Chem., 34, 194 (1962). P. G. Eiving and B. Warchowsky. Anal. Chem.. 19, 1006 (1947). J. A . Fioria, J. W. Dobratz, and J. H. McClure, Anal. Chem., 36, 2053 (1964). S. Siggia. J. G. Hanna. and R . Culrno, Anal. Chem., 33, 900 (1961). D. H. Reed, F. E. CritchfieM, and D. K. Elder, Anal. Chem., 35, 571 (1963). L. Maros, J. Perl, and M. Szaklcs-Pint&, Ann. Univ. Sci. Budap. Robndo fijtvos Nominafae, Sect. Chim., 7, 37 (1965). D . G. Bush, L. J. Kunzelsauer. and S.M. Merrill, Anal. Chem., 35, 1250 (1963). H. P. Keller, Doctoral Thesis No. 165, Ecole Poiytechnique F6d6rale de Lausanne, 1973. H. Kamrnerer and P. N. Grover, Makromol. Chem.. 99, 49 (1966). H. Gilman and C. G. Brannen, J . A m . Chem. SOC.,73, 4640 (1951). M. F. Shostakovskij and Kh. I. Kondratev, Izv. Akad. Nauk. SSSR, Otd. Khim. Nauk., 1956, 967. M. F. Shostakovskii and Kh. I . Kondratev. Izv. Akad. Nauk. SSSR. otd. Khim. Nauk., 1957, 319. A . D. Petrov and T. I . Chernysheva, Izv. Akad. Nauk. SSSR, Otd. Khim. Nauk., 1951, 820 D. Ya. Zhinkin, G. N. Mal'nova, and Zh. V. Gorisiavskaya. Zh. Obshch. Khim., 38, 2800 (1968). N. S. Nametkin, V. M. Vdovin, and E. D. Babich, Khim. Gsterotsikl. Soedin., 1967, 146. C. A . Kraus and R. Rosen, J Am. Chem. Soc , 47, 2746 (1925). R . Fessenden, J . Org. Chem., 25, 2191 (1960). H. Gilman, 6.Hofferth, H. W. Melvin. and G. E. Dunn. J . A m . Chem. Soc., 72, 5767 (1950). H. Gilrnan. G. E. Dunn, H. Hartzfeld, and A. G. Smith. J . Am. Chem. Soc., 77, 1287 (1955).
CONCLUSIONS T h e fact t h a t t h e relative error i5 independent of the hydroxyl content suggests t h a t the precision of t h e method is limited mainly by t h e error of t h e photometric measurement. Therefore, in t h e cases of polymers of high hydroxyl content, the precision may even be inferior t o t h a t of t h e
RECEIVED for review July 24, 1978. Accepted September 18, 1978. This work is a report on p a r t of a project supported by t h e Fonds National Suisse de la Recherche Scientifique.
Separation and Determination of Nanogram Amounts of Inorganic Tin and Methyltin Compounds in the Environment Robert S. Braman' and Michael A. Tompkins Department of Chemistry, University of South Florida, Tampa, Florida 33620
Analytical methods have been developed for the determination of trace amounts of inorganic tin and methyltin compounds. Tin compounds in aqueous solution at pH 6.5 are converted to the corresponding volatile hydride, SnH,, CH,SnH,, (CH,),SnH,, and (CH,),SnH, by reaction with sodium borohydride. These are scrubbed from solution, cryogenically trapped on a U-tube, and separated upon warming. Detection limits are approximately 0.01 ng as Sn when using a hydrogen-rich, hydrogen-air flame emission type detector (SnH band). Parts-per-trillionconcentrations of methyltin compounds were found in a variety of natural waters and in human urine. 0003-2700/79/0351-0012$01.00/0
There is snhstantial interest in the possihle existence of biomethylated compounds of tin. Wood, Ridley, and Dizikes ( I ) have studied the tin hiomethylation process and Nelson, McClain, and Colwell (2) have identified the biological formation of methyltin compounds.
