The separation of two families of plastics additives. (Phenolic antioxidants and UV absorbers) has been achieved by high performance liquid chromatography.
Sequential Optimisation of the Separation of a Complex Mixture of Plastics Additives by HPLC with a Quaternary Gradient and a Dual Detection System E. Lesellier* / A. Tchapla L.E.T.I.A.M., IUT d'ORSAY, Plateau du Moulon, BP 127, 91403 Orsay Cedex, France
Key Words Column liquid chromatography Plastics additives Optimization Elution gradient Light-scattering detector
Summary The separation of two families of plastics additives (Phenolic antioxidants and UV absorbers) has been achieved by high performance liquid chromatography With a quaternary gradient. A methodology of separation based on a sequential optimization is described. After a Preliminary study of the effects of solvents on retention of compounds, the mobile phase is first chosen for each class of additives (Irganox and Tinuvin), then for the mixture of all the compounds and the separation is finally optimized. The importance of the column phase ratio is also reported. The use of two detectors, UV absorbance and light-scattering, enables all the COmpounds to be detected. The performance of the detectors has been compared and the effects of the nebulization temperature on the detection of low molecular mass compounds is reported.
Introduction Some phenolic antioxidants widely used in plastics, and UV absorbers derived from benzotriazole, are toxic. Thus, the toxicological protection of humans, requires the control of the presence of those additives in the Packaging of foodstuffs, medicinal or pharmaceutical Products to prevent their possible migration into food or blood [1]. SOme tracing methods are already in use to study, "in situ',, their presence or the degradation products of these compounds (radioactivity, ESR) [2, 3].
The extraction of these compounds can be difficult, particularly in the case of a complex matrix, and invariably involves the problem of the extraction recovery such as accidental degradation of the products during the extraction. Soxhlet extraction does not achieve extraction of all the high molecular mass compounds such as certain antioxidants [4] or P A H [5]. In comparison supercritical extraction seems to be more efficient [5, 6], non destructive, and, especially, faster [7]. Moreover this method of extraction presents the added advantage of being readily coupled to supercritical fluid chromatography [7-11]. These advantages explain why capillary SFC has undergone a rapid development for the analysis of plastics additives - due to its chromatographic properties (high efficiency), to the use of a universal detector (FID), and to the possibilities of coupling with mass spectrometry [4]. However, the contraints associated with the use of capillary columns, or to its coupling with mass spectrometry required the use of pure CO 2 as the mobile phase. Then the only means of varying eluent strength is to modify the CO 2 density, mainly by performing a gradient of pressure during the analysis. But, if this density modification has an effect on the eluent strength, it seems to only slightly modify the selectivity. Therefore, in the case of complex mixtures, the pressure gradient must be progressive enough to obtain the separation of both very dissimilar or similar compounds. This leads to analysis times of between 70 and 90 min [7, 12]. However, even in this case, coelution of some peaks is still noted [12]. Liquid chromatography has also been widely used for these analyses [13, 14, 15], but so far without resolving entirely the separation problem [15]. Nevertheless, the diversity of the eluent strengths which can be devised, and, more particularly, the various selectivities offered by the numerous solvents, do allow the optimization of some complex separations under gradient conditions with relatively short analysis times. 135
Chrornatographia Vol. 36, 1993 0009-5893/93
0135-09 $ 3.00/0
9 1993 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH
At the same time, the UV spectra of the compounds studied allows a choice of wavelength which avoids a drift of the baseline due to the gradient. The problem of drift is also observed in SFC due to the gradient of pressure with a F.I.D. detector. On the other hand, the detection of antioxidants which do not have an chromophore can be done by coupling capillary SFC with a mass spectrometer. The easier to use light-scattering detector is equally applicable for compounds with a molecular mass of more than 500 [16]. We describe in this paper the analytical steps employed in the separation of 21 phenolic antioxidants and Tinuvin included in plastics. The methodology developed for a more complex mixture than those actually encountered, will be useful for samples whose composition in additives is unknown, but which may contain any of the standard compounds which often, are associated for their synergistic effects. This methodology may also be adapted to the analysis of other compounds like the phosphitic antioxidants (e.g. TNPP), or nonyl phenol.
Experimental Chemicals The additives were kindly provided by Ciba-Geigy (Rueil Malmaison, France). Solvents were filtered though GFF 0.5 gm (Whatman, Springfield, Kent, UK). Water was prepared with an Elga system (Bucks, UK) (18 M ohms), all organic solvents were HPLC grade: acetonitrile (SDS, Vitry sur Seine, France), methanol (Carlo Erba, Rodano, Italy) and THF (Merck, Darmstadt, Germany).
Apparatus The liquid chromatograph used incorporated a quaternary pump (PU 4100, Unlearn, Cambridge, UK) and an injection valve (Rheodyne 7125, Cotati, CA, USA) with a 20 lxl sample loop. Spectra were collected with a PU 4121 diode array detector (Unicam, Cambridge, UK) connected to a PC compatible computer which was also used for the PU 6100 optimization software (Diamond) (Unlearn, Cambridge, UK). The detection of Irganox PS 800 and PS 802 was performed with a light scattering detector DDL21 (Cunow, Cergy, St Christophe, France). The nebulization gas was nitrogen (U grade) at a pressure of 1.8 bar. In our report on the study of the influence of the nebulization temperature vs the response factor of the light scattering detector, the results are expressed as the ratio of responses of the light scattering detector and UV detector which were coupled in-line. The quantity of the compound analysed being the same, the 136
UV detector response is used as an internal reference. However, because the absolute values of those ratios depend on the operating conditions of the two detectors, they cannot be compared. Only the change of this ratio is significant. On the other hand, for the comparison of sensitivities, the responses of the two detectors were measured for the same analysis, under the conditions for which the noise of the detectors was the same. Then, the ratio of the two responses, i.e. the relative sensitivity, may be compared between two compounds. The columns were the following: a LiChrospher 100 RP 18 e (250 x 4 mm, 5 gm, Merck, Darmstadt, Germany), an Ultrabase UB 225 (250 x 4.6 mm, 5 lxm, S.F.C.C., Eragny, France), and a Brownlee Spheri 5-ODS (250 • 4.6 mm, 5 gin, Applied Biosystem, Santa Clara, CA, USA). The compounds were injected in a dissolution solvent which was the closest to the mobile phase. The spectrum of every product, injected separately, was recorded to confirm the identification of compounds when mixtures were analysed. In the case of very similar spectra, some compounds were reinjected alone under the same analytical conditions as the previous mixture. The asymmetry factor of Tinuvin was measured at a height of 10 % of the chromatographic peak. The retention curves and the tables were performed by a Macintosh Classic II computer with the Cricket Graph and Mac Draw softwares. The development of the separation was performed first with phenolic antioxidants then, afterwards with the UV absorbers, by using the results obtained with the antioxidants family. Then, the optimization consisted of mixing the two families, and optimizing the separation of compounds which were eluting in the same elution area and which were not completely resolved. This optimization involved the mobile phase composition and also the choice of the column.
Results and Discussion 1 Separation by Families 1.1 Irganox Preliminary studies. Due to the numerous compounds and to their spectral similarities, we decided not to work with an optimization software. On the contrary, the composition of the starting solvent was chosen from the results of a preliminary optimization performed on six phenolic antioxidants with the PU 6100 optimization software (Unicam) [17]. This starting mobile phase was made up of four solvents: THF, water, acetonitrile, methanol, 45:15:20:20, (v/v/v/v). The compounds were injected separately onto a LiChropher 100 RP 18 e column to determine their order of elution. This analysis under isocratic conditions shows that the antioxidants could be classified into four groups according to their retention times (Table I). Chromatographia Vol. 36, 1993
Table I Variation of the capacity factor k' of Irganox vs. the percentage of THF in the eluent.
Compounds Irg 1222 lrg 1098 Irg 245
BHT Irg 1035 Irg 259 Irg 1010 Irg 1330 Irg 565 Irg 1076
Percentage of THF in the eluent 45 % 40 % 35 % 0.27 0.37 0.33 0.86 1.06 1.28 3.28 3.34 4.76 7.43
0.52 0.7 0.85 1.65 2.78 3.28 13.52 15.67 nd nd
0.71 1.30 1.64 nd nd nd nd nd nd nd
The first group is made up of Irganox 245, 1098 and 1222 for which the retention times are very close, the Second one of BHT, Irganox 1035 and 259, the third One of Irganox 1010 and 1330 and finally the fourth, formed by the compounds more strongly retained by the column, but whose separation is quite satisfactory: Irganox 565 and 1076. This result shows that an isocratic analysis will not achieve a correct separation within a reasonable retention time for all the solutes. Indeed, to separate the first three compounds one must decrease the eluent Strength. Such a step will inevitably lead to excessive retention of high molecular mass antioxidants. In these circumstances only gradient analysis is able to respond to these two opposite objectives. This modification could equally modify the absorbance of the mobile phase during the analysis. To avoid this Problem, we worked in the UV range at the wavelength of 280 nm which is the second absorption peak of the Phenolic antioxidants and at which Tinuvin also absorbs. Choice of the mobile phase. The first objective was to determine an adequate composition for the separation of the less retained compounds. Of the four commonly Used solvents T H F and water war the ones which show the more opposed eluent strengths (measured against the retention of Irganox) [17]. THF being a strong Solvent, is reduced in amount of decrease the eluent Strength of the mobile phase, and it is replaced by Water. In other respects, the simultaneous presence of methanol and acetonitrile in the mobile phase allows Specific interactions between the eluent and the solutes (hydrogen bond for methanol with the hydroxy group of phenol, n interaction for acetonitrile with the aronaatic nucleus), which favour the solubilisation of the COmpounds. Thus, for a better understanding of the effects of variation in mobile phase composition vs Separation, we have chosen to keep the percentage of the two solvents constant during this first analysis (20 % acetonitrile; 20 % methanol). Chrornatographia Vol. 36, 1993
Figure 1
Chromatogram of a mixture of three lrganoxes. 1 = Irganox 1222; 2 = Irganox 245; 3 = Irganox 1098. Column: LiChrospher 100 RP 18 e. Mobile phase: (THF, H20, CH3OH, CH3CN) (35/25/20/20; v/v/v/v). Ambient temperature; flow rate: 1.0 mi/min.
Table I shows the variation of retention times of compounds vs the decrease in T H F percentage in the mobile phase. For a composition of 40 % THF, only three components are still coeluting: Irganox 245, 1098 and 1222. We must also stress the fact that a variation of 5 % in THF (and a corresponding change of 5 % in water) leads to a trebling of the retention time for the antioxidants. This important increase must be reduced later in the analysis by an increase of the eluent strength. The first three Irganox compounds are separated in less than 6 min with 35% of T H F and 25% of water (Figure 1). This composition of the mobile phase has been selected to start the analysis, and the duration of this isocratic step has been fixed at 5 min, according to the retention times of those solutes and of the dead volume of the chromatographic system. Then, as previously discussed, the variation of THF/ water should be inverted, to accelerate the elution of the remaining antioxidants. Some trials ensured satisfactory conditions for the separation of the ten Irganox compounds studied (Figure 2a). When the analytical conditions are well defined, it is still possible, if the separation is not quite satisfactory, particularly when the column grows old, to increase the resolution by reducing the flow rate of the mobile phase (Figure 2b). 137
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, tb
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~
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Figure 3
b
C h r o m a t o g r a m of a m i x t u r e of 9 U V absorbers. 1 = Tinuvin 312; 2 = Tinuvin P; 3 = Tinuvin 234; 4 = C h i m a s o r b 81; 5 = Tinuvin 320; 6 = Tinuvin 350; 7 = Tinuvin 326; 8 = Tinuvin 328; 9 = Tinuvin 327. Analytical conditions as in .Figure 2.
O
1.2 T i n u v i n
O-JL~ i
IIIII
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Figure 2 C h r o m a t o g r a m of a m i x t u r e of ten phenolic antioxidants. 1 = Irganox 1222; 2 = Irganox 245; 3 = Irganox 1098; 4 = BHT; 5 = l r g a n o x 1035; 6 = Irganox 259; 7 = Irganox 1010; 8 = Irganox 1330; 9 = Irganox 565; l0 = Irganox 1076. Column: L i C h r o s p h e r 100 RP 18e. Mobile phase: T H F 35 THF 50 H20 25 H20 10 C H 3 O H 20 C H 3 O H 20 C H 3 C N 20 C H 3 C N 20 5 min ~ 35 min A m b i e n t t e m p e r a t u r e , a) flow rate: 1 ml/min; b) flow rate: 0.5 ml/ rain.
138
We have applied the previous gradient to the separation of nine Tinuvins, to assess whether the simultaneous separation of the two families studied was feasible with the optimized conditions of the Irganox separation (Figure 3). The elution order of Tinuvins P, 320, 328 and 350 is logically explained by the increase of the number of carbons linked to the aromatic ring. Likewise, the substitution of a methyl group (Tinuvin 320) by a more polar phenyl group (Tinuvin 324) favors a faster elution of the solute. On the other hand, the addition of a chlorinated group (Tinuvin 327 with regard to Tinuvin 320), which imparts a more polar character, results in an increase of the retention which is surprising in reversed phase chromatography. The chromatogram obtained shows a poor separation of Tinuvins 320, 350 and 326, and especially a tendency to splitting. Indeed, it seems that these compounds have a solubility even weaker in water than that of Irganox. This fact results in some partial precipitation and redissolution leading to the observed splitting. To avoid this undesirable effect, it is necessary to reduce the amount of water.
Chromatographia
V o l . 36, 1993
However, this decrease involves an increase of the eluent strength of the solvent which is detrimental to the separation of the compounds with low retention, and at the same time, we must reduce the percentage in THF. Thus, it is the amount of the acetonitrile/ methanol (50/50; v/v) mixture (less eluting than T H F but with a greater solubility than water) which will be increased. The isoeluting proportions of these solvents have been calculated from retention curves obtained With binary mixtures THF/water; THF/acetonitrile and THF/methanol [17]. Table II reports the evolution of the asymmetry factor measured on Tinuvin 234 as well as of the capacity factor of Tinuvin P vs the modification of the amount in Water in the mobile phase. Effectively, the decrease in the percentage in water in the eluent gives a symmetrical peak of Tinuvin, which gives a better separation of those families. We also observe a decrease in the retention time of the COmpounds for the quaternary mixture of solvents COmpared to those expected for the binary mixtures (Table II). This phenomenon has already been reported [17, 18] and shows the existence of specific interactions between the solvents in a quaternary mixture which are not predictable from results obtained on binary mixtures.
ticularly the Ultrabase UB 225 which was retained for the following work. 2 Separation by Elution Z o n e Previous analyses showed that the compounds with a similar retention are Irganox 245, 1098, 1222, the B H T and the Tinuvin 312 and P. These compounds were therefore chosen for use in subsequent work. The amount of T H F being maintained constant at 5 %, we varied the percentage of water and of the acetonitrile/methanol (50/50) mixture.
2
HOwever, under those conditions, the elution of lrganox becomes too fast. This leads to a more or less important coelution of Irganox 245, 1098, 1222 and t~HT, and to the eventual coelution of Tinuvin 312 and P when all the compounds are to be analysed together. Unfortunately, it seems impossible to totally suppress the THF, which is essential for a good solubilisation of the additives, and which would counterbalance this retention diminution. However, another means of increasing retention is to tnerease the solute/stationary phase interactions, which COuld be brought about by increasing the phase ratio. ~Ve have tested other columns in order to find one Whose phase ratio would be greater than that of the LiChropher 100 RP 18 e, but without diminishing the Selectivity of the solutes (Figure 4). To do that, we have ar~alysed the six less retained compounds on the LiChropher column, with a gradient which enabled the Separation of these solutes. The two columns tested (Brownlee and Ultrabase) are effectively more retentive than the LiChrospher, par-
Table 11 Asymmetryfactor (a.f) of Tinuvin 234 and capacity factor k' of Tinuvin P vs the percentage of water in the eluent. % water a.f Tin 234 k' Tin P
25
15
10
8
2.4
2.1
1.6
1.4
1.73
1.63
1.36
1.34
Chromatographia Vol. 36, 1993
a)
6
b) 5
c) 6
2
2'5
io
rnin
Figure 4 Chromatogram of a mixture of 6 phenolic antioxidants. 1 = Irganox 1222; 2 = Irganox 1098; 3 = Irganox 245; 4 = BHT; 5 = Irganox 1035; 6 = Irganox 259. Mobile phase as in Figure 2. a) column LiChrospher 100 RP 18 e. b) column Brownlee Spheri 5 0 D S . c) column Ultrabase UB 225.
139
retention
time
(minutes) 20n
15
10'
14
15
16
17
18
19
20
21
% water
Figure 5
Variation of the retention of six antioxidants and UV absorbers vs the percentage of water in the mobile phase. 9 = Irganox 1222; 9 = Tinuvin 312; O = Irganox 245; # = Tinuvin P; + = Irganox 1098; [3 = BHT. Column: Ultrabase UB 225. Ambient temperature; flow rate: 1.0 ml/min.
17
19
14
89
19
d lb
2b
K 3b
Figure 6
Separation of 19 additives of plastics. Column: Ultrabase UB 225. 1 = Irganox 1222; 2 = Tinuvin 312; 3 = lrganox 245; 4 = Tinuvin P; 5 = Irganox 1098; 6 = BHT; 7 = Irganox 1035; 8 = Tinuvin 234; 9 = Irganox 259; 10 = Chimasorb 81; U = Tinuvin 320; 12 = Tinuvin 350; 13 = Tinuvin 326; 14 = Irganox 1010; 15 = Tinuvin 328; 16 -- Tinuvin 327; 17 = Irganox 1330; 18 = lrganox 565; 19 = Irganox 1076. Mobile phase: THF 5 5 5 30 H20 18 10 0 0 CH3OH 38.5 42.5 47.5 35 CH3CN 38.5 42.5 47.5 35 Duration of steps (min) 5 1 4 1 4 15 140
rain
Figure 5 shows that the s e p a r a t i o n of I r g a n o x 1222, 245, Tinuvin 312 and Tinuvin P is o b t a i n e d o v e r a range with between 15 and 20 % of w a t e r in the mobile phase, and that at the limits of this range two couples of c o m p o u n d s will coelute in turn: I r g a n o x 245 and Tinuvin 312 at 15 % , I r g a n o x 245 with Tinuvin P at 20 %. T h e optimal separation, b a s e d u p o n a calculation of the m a x i m u m separation, is located b e t w e e n 17 and 18 % for these two couples. U n d e r these conditions, the last one of the four solutes, Tinuvin P, has a retention time n e a r 9.30 min. This composition s e e m s to be ideal to begin the analysis of a mixture of nineteen c o m p o u n d s . H o w e v e r , for B H T and for I r g a n o x 1098 which are eluted later, the separation is b e t t e r with a low p e r c e n t a g e of w a t e r in the mobile phase. : On account of the dead v o l u m e of the columns (2.6 ml) and of the difference in retention b e t w e e n Tinuvin P and I r g a n o x 1098, which reaches 5 min for 18 % of water, the change in the mobile phase composition must take place early enough in the analysis and be fast. In addition, the a m o u n t of w a t e r should be sufficient to permit a satisfactory s e p a r a t i o n of B H T and Irganox 1098, without being excessive. In the latter case, the retention would be diminished leading to coelution of the first four solutes. T h e first two mobile p h a s e c o m p o s i t i o n s described in Figure 6 r e s p o n d to those d e m a n d s shown on the corresponding c h r o m a t o g r a m , and p e r m i t the good separation of the six additives studied. U n d e r those conditions, the last c o m p o u n d was eluted in 11.30 min. Then, a rapid change is applied to suppress the presence of w a t e r in the mobile phase and to avoid the artificial splitting of Tinuvin. O n c e this step is done, a gradient in T H F - m e t h a n o l / a c e t o n i t r i l e is p e r f o r m e d to increase progressively the eluent strength, in o r d e r to force the o t h e r solutes through the column (Figure 6). T h e nineteen p r o d u c t s are efficiently s e p a r a t e d in 35 min. W e then applied those conditions to a mixture of antioxidants which can be e n c o u n t e r e d in industry. This mixture is c o m p o s e d of nonyl phenol, a precursor of the synthetic trinonylphenol (TNPP), B H T , Irganox 565 and 1010, and T N P P and Irgafos 168. A first analysis using the previous conditions showed a coelution b e t w e e n the B H T and the nonylphenol, and an excessive retention of T N P P . T h e eluent strength of the starting mixture was c o n s e q u e n t l y d e c r e a s e d with the increase of the a m o u n t of water, p e r m i t t e d in this case because of the lack of Tinuvin. T h e n , an gradual increase of the elution strength t o o k place before those which were in the previous gradient (10 min instead of 15 min), but whose slope and final p e r c e n t a g e are the s a m e (Figure 7). T h e six c o m p o u n d s are well resolved, and the analytical conditions quickly found f r o m the initial gradient. The two c o m p o u n d s , n o n y l p h e n o l and trinonylphenol present two p e a k s whose width is a b n o r m a l l y high, which leads us to s u p p o s e the p r e s e n c e of numeroUS Chromatographia Vol. 36,1993
1 6
lb
2b
3b
min
Figure 7
Chromatogram of a industrial mixture of six plastics additives. 1 = nonyl phenol; 2 = BHT; 3 = Irganox 1010; 4 = Irganox 565; 5 = Irgafos 168; 6 = TNPP. Mobile phase: THF 5 30 H20 20 0 CH3OH 37.5 35 CH3CN 37.5 35 Duration of steps (min) 10 I Column: Ultrabase UV 225.
13
17
18 19
20
21
16
14
L l
lO
I
20
3()
min
Figure 8
Chromatogram of a mixture of 21 plastics additives. 1 = Irganox 1222; 2 = Tinuvin 312; 3 = Irganox 245; 4 = Tinuvin P; 5 = Irganox 1098; 6 = lrganox 1035; 7 = Tinuvin 234; g = Irganox 259; 9 = Chimasorb 81; 10 = Tinuvin 320; U = Tinuvin 350; 12 = Tinuvin 326; 13 = Irganox 1010; 14 = Tinuvin 328; 15 = Tinuvin 327; 16 = Impurity of Irganox PS 800; 17 = Irganox 1330; 18 = Irganox 565; 19 = Irganox PS 800; 20 = Irganox 1076; 21 = Irganox PS 802. Analytical conditions as in Figure 6. Detection conditions of DDL: nebulization temperature = 35 ~ N 2 pressure: 1.8 bar.
Products of similar chemical structures which are Coeluting under these analytical conditions. This hyPothesis has been verified by mass spectrometry [19].
3 Coupled Detection UV-DDL A l i g h t - s c a t t e r i n g d e t e c t o r w a s c o u p l e d to t h e U V detector, Figure 8 shows the analysis of nineteen COmpounds d e t e c t a b l e b y U V a n d I r g a n o x PS 800 a n d PS 802. The two additional compounds eluted between Irganox 1330 a n d 564 f o r I r g a n o x PS 800, a n d a f t e r I r g a n o x 1076 for t h e I r g a n o x PS 802. It is n o t s u r p r i s i n g t h a t t h o s e Chrornatographia Vol. 36, 1993
t w o c o m p o u n d s a r e e l u t e d l a s t f r o m t h e c o l u m n in r e v e r s e d - p h a s e b e c a u s e t h e y h a v e o n e (1076) o r t w o (PS 802) h y d r o c a r b o n c h a i n s w i t h 18 c a r b o n a t o m s . O n e i m p u r i t y was also d e t e c t e d b e t w e e n T i n u v i n 327 a n d I r g a n o x 1010. C o m p l e m e n t a r y a n a l y s e s s h o w e d t h a t this i m p u r i t y was d u e to I r g a n o x PS 802. O n t h e o t h e r h a n d , t h e r e is a s i g n i f i c a n t d i f f e r e n c e in the response areas between the compounds, either with U V o r with D D L . T h u s , B H T w h i c h was d e t e c t e d by U V is n o t w i t h D D L . T h e d e t e c t i o n c o n d i t i o n s u s e d ( n e b u l i s a t i o n t e m p e r a t u r e o f 35 ~ do not enable v i s u a l i s a t i o n o f t h e d e g r a d a t i o n of t h e p r o d u c t . Its low m o l e c u l a r m a s s (206) c o u l d e x p l a i n this r e s u l t .
141
Ratio
H o w e v e r , s o m e studies h a v e b e e n p e r f o r m e d to study the nebulization t e m p e r a t u r e of the gas. T h e y show that a decrease in this t e m p e r a t u r e to 15 ~ increases the ratio of the two responses (Figure 9a). As the r e s p o n s e of the U V d e t e c t o r remains constant regardless of the nebulization t e m p e r a t u r e (Figure 9b), it is the response of the light-scattering d e t e c t o r which increases, by a factor of 30 for B H T .
0,8
0,6 ~
a)
0,4
T h e effect of the nebulization t e m p e r a t u r e has also b e e n studied for o t h e r c o m p o u n d s b e t w e e n 15 and 80 ~ (Figure 10). T h e increase of t e m p e r a t u r e is detrimental to the detection of I r g a n o x 1222 (M:356) and of Tinuvin P (M:225), whose m o l e c u l a r masses are the lowest in the family of c o m p o u n d s to which they belong. T h e variation of the nebulization t e m p e r a t u r e does not have a significant effect on the other compounds. Indeed, we have c o m p a r e d the inherent sensitivities of the two detectors. T h e results in T a b l e 1II show that the sensitivity of the U V d e t e c t o r is slightly higher than that of D D L for I r g a n o x (by a factor of 5 on average). For Tinuvin, whose m o l e c u l a r mass is generally lower than that of Irganox, the sensitivity of D D L m a y be decreased by a factor of 50. This difference d e p e n d s not only on the masses but also on the n u m b e r of c h r o m o p h o r e s that then have in the molecule. Indeed, although I r g a n o x 1330 and 1010 have the highest molecular masses, they are the ones for which the U V detection is the most important because they posses respectively 3 and 4 phenolic groups.
0,2
0,0 0
!
!
20
30
40
T~
Response
area
25000
20000
b)
15000
10000
5000 o
I
I
2O
30
40
T~ Figure 9
Conclusions
Detection of BHT vs the nebulization temperature. Mobile phase: THF/CH3OH/CH3CN; 30/35/35 (v/v/v). Column: Ultrabase UB 225; flow rate: 1 ml/min. a) Ratio of the response DDL/UV. b) UV response (considered as internal reference).
T h e different studies p e r f o r m e d in the course of this w o r k have shown that for a c o m p l e x mixture the elution strength should be modified during the analysis
Table I11 Comparison of the sensitivity of the UV and DDL (light-scattering) detectors for different
compounds studied. (a): Irganox; (b): Tinuvin Compounds
M
Irg 245 Irg 1222 Irg 565 Irg 1098 Irg 1035 Irg 1076 Irg 1010 Irg 1330
586 356 615 636 642 530 1176 732
Number of phenolic groups 2 1 2 2 2 1 4 3
Sensitivity ratio 3.0 3.2 4.9 4.3 5.0 2.9 5.6 8.7
(a)
(b)
142
Compounds
Tinuvin 234
Chimasorb 81
Tinuvin 312
Tinuvin P
M
446
326
312
225
Sensitivity ratio
8.7
19.7
23
53
Chromatographia Vol. 36, 1993
Ratio
T h e diversity of m o b i l e p h a s e s which can b e used in H P L C gives rise to a d e g r e e o f selectivity v a r i a t i o n which is not available in S F C with capillary c o l u m n s .
4 -
T h e c o u p l i n g of the light-scattering d e t e c t o r with the U V d e t e c t o r e n a b l e s all the p h e n o l i c a n t i o x i d a n t s and U V a b s o r b e r s to be d e t e c t e d .
Acknowledgment I.I
T h e a u t h o r s t h a n k Ciba G e i g y for p r o v i d i n g the standard compounds, Jean Bleton (L.E.T.I.A.M., IUT O r s a y ) for his w o r k in the m a s s s p e c t r o m e t r y analysis, M r L a n d r i e u ( M e r c k ) for the p r o v i d i n g of the LiC h r o s p h e r c o l u m n , S . F . C . C . - S h a n d o n for the U l t r a b a s e U B - 2 2 5 c o l u m n a n d T o u z a r t et M a t i g n o n for the Brownlee Spheri 5-ODS columns.
m
0
20
b'igure
3o
40~
50~ 60~ Temperature (~
70~
810
g0
10 Variation of the response ratio of some additives vs the nebulization temperature. Analytical conditions as in Figure 9. 9 ~- Irganox 245; 4, = Irganox 1222; O = Irganox 1010; ~ = Irganox 1030; 9 = Chimasorb 81; * = Tinuvin 234; [] = Tinuvin 312; + = Tinuvin p.
to o b t a i n a c o m p l e t e s e p a r a t i o n . I n d e e d , it s e e m s better, after a p r e l i m i n a r y study to m e a s u r e the relative retention of all the c o m p o u n d s , to aim for the ideal conditions of s e p a r a t i o n by g r o u p s of c o m p o u n d s Unresolved r a t h e r t h a n by t r e a t i n g all the p r o d u c t s simultaneously, On the o t h e r hand, it s e e m s t o o difficult to use a systematic i n v e s t i g a t i o n plan for d e t e r m i n i n g the chrom a t o g r a p h i c b e h a v i o u r of solutes w h e n they are n u m e r o u s , w h e n o n e is faced with the choice of possible ~ o b i l e p h a s e s in q u a t e r n a r y mixtures. An analytical step, b a s e d on a s e q u e n t i a l study and an Understanding of the v a r i a t i o n of solute r e t e n t i o n s , Seems to p r o v i d e a q u i c k e r a n s w e r to s o m e c o m p l e x Separations.
References [1] J. Botrel, in "Les Polymeres: Chimie et Reglementation des Emballages", Ed. Masson, Paris, 1982. [2] A.M. Riquet, O. Ackerman, A. Gaudemer, G. Pascal, Food Chem. 26, 271 (1987). [3] K. Figge, Fd. Cosmet. Toxieol. 10, 825 (1972). [4] D. Dilettato, P.J. Arpino, K. Nguyen, A. Bruchet, HRC/CC 14, 335 (1991). [5] S.B. Hawthorne, D.J. Miller, Anal. Chem. 59, 1705 (1987). [6] B. W. Wright, C. W. Wright, R.W. Gale, R.D. Smith, Anal. Chem. 59, 38 (1987). [7] 11. Daimon, Y. Hirata, Chromatographia 32, 549 (1991). [8] M. Ashraf-Khorassani, J.M. Levy, HRC/CC 13, 742 (1990). [9] M. Ashraf-Khorassani, D.S. Boyer, J.M. Levy, J. Chrom. Sei. 29, 517 (1991). [10] N.J. Cotton, K.D. Bartle, A.A. Clifford, S. Ashraf, R. Moulder, C.J. Dowle, HRC/CC 14, 164 (1991). [11] P.J. Arpino, D. Dilettato, K. Nguyen, A. Bruchet, HRC/CC 13, 5 (1990). [12] M.W. Raynor, K.D. Bartle, LL. Davies, A. Williams, A.A. Clifford, Anal. Chem. 60, 127 (1988). [13] J.D. Vargo, K.L. OIson, Anal. Chem. 57, 672 (1985). [14] J.D. Vargo, K.L. Olson, J. Chromatogr. 14, 503 (1991). [15] R.C. Nielson, J. Liq. Chromatogr. 353, 215 (1991). [16] M. Lafosse, M. Dreux, L. Morin-Allory, J. Chromatogr. 404, 95 (1987). [17] E. Lesellier, P. Saint Martin, A. Tchapla, LC-GC 5 (II), 38 (1992). [18] P.J. Schoenmakers, in "Optimization of chromatographic selectivity", J. Chromatogr. Library 53, 1986. [19] unpublished results. Received: Sep 30, 1992 Accepted: Nov 20, 1992
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