Anal. Chem. 1990, 62, 1397-1402 (15) Sentell, K. B.; Barnes, K. W.; Dorsey, J. G. J. Chromatogr. 1988,
1397
(26) Korenaga, T.; Shen, F.; Yoshida, H.; Takahashi, T.; Stewart, K. K. Anal. Chlm. Acta 1988, 214, 97-105. (27) Foley, J. P.; Dorsey, J. G. Chromatographla 1984, 18, 503-511. (28) Long, G. L; Wlnefordner, J. D. Anal. Chem. 1983, 55, 712A-724A. (29) Reijn, J. M.; Poppe, H.; Van der Linden, W. E. Anal. Chem. 1984, 56,
455, 95-104.
(16) Snyder, L. R. High-Performance Liquid Chromatography, Advances and Perspectives, Volume 1·, Horvath, C„ Ed.; Academic Press: New York, 1980; pp 207-316. (17) Brooks, S. H.; Left, D. V.; Hernández Torres, . A.; Dorsey, J. G. Anal. Chem. 1988, 60, 2737-2744. (18) Brooks, S. H.; Williams, R. N.; Dorsey, J. G. Anal. Lett. 1988, 21,
943-948.
(30) Wada, H.; Hiraoka, S.; Yuchi, A.; Nakagawa, G. Anal. Chlm. Acta 1986, 179, 181-188. (31) Linares, P.; Luque de Castro, D. M.; Valcárcel, M. Anal. Chem. 1985,
583-598.
(19) Brooks, S. H.; Dorsey, J. G. Anal. Chlm. Acta 1990, 229, 35-46. (20) Deelder, R. S.; «roll, M. G. F.; Beeren, A. J. B.; Van den Berg, J. . M. J. Chromatogr. 1978, 149, 669-682. (21) Tijssen, R. Anal. Chlm. Acta 1980, 114, 71-89. (22) Reijn, J. M.; Van der Linden, W. E.; Poppe, H. Anal. Chlm. Acta 1981, 123, 229-237. (23) Reijn, J. M.; Van der Linden, W. E.; Poppe, H. Anal. Chim. Acta 1981, 126, 1-13. (24) Engelhardt, H.; Neue, U. D. Chromatographla 1982, 15, 403-408. (25) Selavka, C. M.; Jiao, K.; Krull, I. S. Anal. Chem. 1987, 59,
57, 2101-2106.
(32) Ruzicka, J.; Stewart, J. W. B. Anal. Chlm. Acta 1975, 79 , 79-91. (33) Ruzicka, J.; Stewart, J. W. B.; Zagatto, E. A. Anal. Chlm. Acta 1976,
81, 387-396.
(34) Hungerford, J. M.; Christian, G. D. Anal. Chim. Acta 1987, 200, 1-19.
Received for review November 17, 1989. Accepted March 23, 1990. The authors are grateful for support of this work by NSF CHE-8704403.
2221-2224.
Characterization of Formaldehyde and Formaldehyde-Releasing Preservatives by Combined Reversed-Phase Cation-Exchange High-Performance Liquid Chromatography with Postcolumn Derivatization Using Nash’s Reagent William Russell Summers Buckman Laboratories International, Inc., P.O. Box 8305, Memphis, Tennessee 38108-0305
standpoint, formaldehyde exhibits substantial antimicrobial activity; however, concerns regarding its carcinogenicity have led to the use of alternative preservative compounds. Interestingly, many of these compounds are synthesized by using formaldehyde as a precursor and undergo decomposition on varying time scales, thereby releasing formaldehyde to the formulation in which they are incorporated. For some of these compounds, formaldehyde release may, in fact, constitute the primary mode of biological activity (1). The chemical characterization of this class of preservatives, i.e., formaldehyde-releasers, is the subject of this report. Formaldehyde has been determined by many different techniques during the last several decades, some of which have evolved sufficiently to be utilized by modern analytical instrumentation. An approach common to most of these techniques is the reliance on derivatization reactions which produce highly absorbent or fluorescent derivatives; this is due to the low molar absorbance of formaldehyde. Some of the earliest work relevant to this project was undertaken by Nash (2), who exploited the Hantzsch reaction, in which formaldehyde reacts with 2,4-pentanedione in the presence of ammonium salts to form, 3,5-diacetyl-l,4-dihydrolutidine, equation 1. In 1963, Belman demonstrated that this deriv-
A method has been developed for the characterization of that class of preservative compounds that release formaldehyde as their primary mode of biological activity. This method Incorporates a derivatization step specific to molecular methylol functionalities and formaldehyde following a separation of the preservative compounds by high-performance liquid chromatography (HPLC). The derivative thus produced displays a characteristic absorbance at 410 nm and fluorescence at 510 nm. The study compounds, which typically exhibit no characteristic absorbance or fluorescence, are thereby rendered detectable by either of these phenomena. The postcolumn approach used in this study circumvents the labor-intensive step of batch derivatization and, more Importantly, It allows for the speclatlon of Individual preservative compounds In a mixture containing not only other preservatives but also free formaldehyde. The study compounds range In polarity from essentially nonpolar to cationic, l.e. quaternary ammonium salts. The HPLC separation step was therefore configured In a mixed mode, progressing from a typical reversed-phase eluant profile to a cation exchange eluant profile via a ternary gradient. The method detection limit Is ca. 400 ppb and the linear dynamic range Is 2 orders of magnitude. The relative standard deviation is around 0.5%, and accuracy Is 100%, within precision.
I
*3
INTRODUCTION
atization chemistry could be monitored quantitatively by fluorometry (3). Wilson (4, 5) reported that this reaction was specific for formaldehyde in the presence of n-butyraldehyde, citronellal, dodecaldehyde, piperonal, benzaldehyde, paraldehyde, and other aldehyde-releasing preservatives, e.g., di-
Many substances used in a variety of applications incorporate preservative compounds to inhibit microbial growth and the degradation it causes. Typical products that fall into this category might include water-based formulations of paints, cosmetics, and personal care products. From an efficacy 0003-2700/90/0362-1397602.50/0
©
1990 American Chemical Society
1398
·
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990 h2coh
Table I. Eluant Program for Nonionic and Quaternary Ammonium Preservatives %
eluant water
acetonitrile DAP-HCl
10.0 min
min
0.0 min
5.0 min
5.1 min
70 30
70 30
60 30
60 30
70 30
0
0
10
10
0
10.1
Germall-115 (I) H2COH
methoxane (which releases acetaldehyde). This study also demonstrated that the Hantzsch reaction proceeded with unit efficiency for Germall-115, one of the compounds studied in the current project. The derivatization efficiency for Dowicil-200 (a stereoisomer of Dowicil-75, also one of the study compounds) was reported to be 100% by Wisneski (6). Other derivatization reagents used for the determination of formaldehyde include chromotropic acid (7) and 2,4-dinitrophenylhydrazine (8). The latter reagent is arguably one of the more widely studied derivatization procedures used in the identification of organic compounds. A shortcoming to these procedures (including those based on Nash’s reagent) is that they are usually carried out in a “batch” mode, i.e., prior to a separation step. The difficulty with this approach becomes significant when a mixture of formaldehyde and formaldehyde-releasing compounds is derivatized. The derivatization chemistry is specific only to formaldehyde and the methylol functional group—not to the preservative compounds themselves. Therefore the analytical response measured via derivatization cannot be unambiguously attributed to a specific source of formaldehyde. In the case of 2,4-dinitrophenylhydrazine, the derivative itself cannot be resolved from the starting reagent, since there is no change in the wavelength of maximum absorbance. Separation of the derivative is usually accomplished by HPLC (9), although this still does not specify the source of the formaldehyde derivative. An elegant but little used solution to this difficulty is to configure the derivatization step after a separation step, so that the various sources of formaldehyde that may be present in a mixture are resolved prior to their derivatization. The Nash derivatization chemistry is a logical candidate for this approach, since the precursors are all transparent to detection by absorbance or fluorescence, whereas the derivative absorbs strongly at 410 nm and fluoresces at 510 nm. This provides for ideal leverage of the signal-to-noise ratio. Stack and Davis (10) have described just such an approach for the characterization of Quaternium-15 (a stereoisomer of Dowicil-75), which is a formaldehyde-releasing quaternary ammonium salt. Their methodology, in fact, provided much of the groundwork for the technique reported herein. The impetus for this project arose from a need to characterize a newly developed preservative with respect to free formaldehyde content. The preservative itself was synthesized by using formaldehyde as a starting component; hence it was imperative that the methodology applied be specific as to the source of formaldehyde. Postcolumn derivatization using Nash’s reagent following separation via HPLC was therefore chosen as the analytical technique. Several significant refinements were made to the Stack and Davis procedure in order to update it and extend its scope to include as broad a class of analytes as possible. First, the eluant was changed. The isocratic acetonitrile-ethanol-water eluant was replaced by a ternary gradient eluant profile of acetonitrile-waterdiaminopropionic acid in order to elute both nonpolar and cationic species in a single run. This refinement provided for the resolution of a mixture of three of the more prominent formaldehyde-releasing preservatives in use today: Germall-115 (I), Glydant (II), and Dowicil-75 (III). Second, the
Dowicil-75 (IH)
300-ft capillary reaction coil bathed in a constant 66 °C water was replaced by a pair of packed-bed reaction coils housed in a solid-state heating block. The base-line width of the Dowicil peak shown in the Stack and Davis paper is around 15 min. The low dead-volume (750 µL) of the postcolumn reaction coils used in this study reduced this peak width to less than 1 min and significantly shortened the analysis time. Finally, the linear dynamic range reported by Stack and Davis was 20:1, using fluorescence detection. A linear dynamic range of 100:1 was obtained in this study by using absorbance detection.
bath
EXPERIMENTAL
SECTION
The apparatus utilized in this study consisted of a Dionex Model 4000i high-pressure liquid chromatography system with UV-vis absorbance detectors, conductivity detectors, Ionpac membrane reactors, postcolumn heater and mixing coils, and associated peripheral components. Although this system is typically operated in the ion exchange mode, no modification was made to the liquid flow path to accommodate the reversed-phase column and eluants. A polymeric reversed-phase column (the Hamilton PRP-1,4.6 X 150 mm, 5 µ diameter spheres) was used throughout. The 50-µ injection loop supplied with the instrument
was
used as received.
Elution of nonionic preservatives can be accomplished using a 70/30 water/acetonitrile isocratic eluant profile with a flow rate of 0.5 mL/min. Preservatives containing a quaternary ammonium functionality were retained and precipitated on the column with this eluant profile, rather than being eluted in the void volume as expected. This was evidenced by a significant increase in column back pressure. In order to elute all species of interest, a ternary gradient program was utilized as specified in Table I, with a flow rate of 0.5 mL/min. This program essentially parks the Dowicil-75 on the column until all nonionics are eluted and then drives it off with a step change in the ionic strength of the eluant. The DAP-HC1 reagent (diaminopropionic acid monohydrochloride) is the standard monovalent cation eluant recommended by Dionex, only doubled in concentration. Although the duration of the gradient program is only about 10 min, reequilibration of the column limits the frequency of sample injection to 30 min-1.
The column effluent was input to a Dionex Ionpac Membrane Reactor (P/N 037689). This device allows the continuous addition of the derivatizing reagent from a pressurized reservoir (Dionex P/N 037053) into the effluent stream through a semipermeable membrane; in essence, it is an enhanced mixing tee. The reagent/effluent stream was then directed through either one or both
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990 Reagent Delivery Module
Nash's Reagent
Reactor
Eluant Manifold
·
1399
Pump
Column
Reaction Coil
Figure
1.
Retention Time
Schematic of the fluid flow path used in this study.
of the reaction coils (Dionex P/N 039349) housed in a solid-state heating block (Dionex P/N 039348). The void volume of each reaction coil is 750 mL. The temperature of the heating block is switch-selectable to 40, 60, 80,100, and 130 °C. The derivative thus synthesized was detected by monitoring the absorbance of the heater effluent at 410 nm. For preservatives that display characteristic absorbances, a detector placed between the column exit and the point at which the derivatization reagent is introduced provides for an extra degree of specificity in identifying the compounds of interest. Figure 1 is a schematic of the fluid flow path used in this study. Preparation of Nash’s Reagent. Nash’s reagent was prepared by dissolving 154 g of ammonium acetate, 3 mL of glacial acetic acid, and 2 mL of 2,4-pentanedione (99%) in 1 L of water. This results in a solution that is 2 M in ammonium acetate and 0.02 M in 2,4-pentanedione, with a pH of 6.0. The rate of use of this reagent resulted in its being prepared weekly; from an efficacy standpoint the necessity of doing this was not ascertained, although this timetable is consistent with the lifetime of Nash’s reagent typically reported in the literature. Preparation of Formaldehyde Standards. Formaldehyde standards were prepared by serial dilution of 37% USP formaldehyde as received from Mallinckrodt. Final concentrations utilized were 92.5, 37.0, 27.75,18.5,9.25,3.7,0.74,0.37, 0.185, and 0.037 ppm. Preparation of Samples. Glydant is a product of Analabs, Inc., North Haven, CT. It is available as a 55% aqueous solution of the active ingredient l,3-(dihydroxymethyl)-5,5-dimethylhydantoin. A 55 ppm sample was prepared by serial dilution of the product as received in two steps of 10 mL/1000 mL. Fresh preparations were made prior to each data acquisition session in order to avoid potential complications arising from instability. Germall-115 is a product of Sutton Laboratories, Roselle, NJ. It is sold as a solid consisting of imidazolidinylurea (N,N"-
methylenebis [IV '-[3 (hydroxy methyl)- 2,5-dioxo-4-
imidazolidinyljurea]). This molecule contains 2 mol of formaldehyde per mole of active ingredient. Dissolution of 100 mg/L resulted in chromatographic data similar in scale to that observed for the other study compounds. Dowicil-75 is a product of Dow, Inc., Midland, MI. It is available as a 67.5% active solid formulation of trcms-l-(3chloroallyl)-3,5,7-triaza-l-azoniaadamantane chloride. The cis isomer is sold as Dowicil-200, a 94% active formulation. Dowicil-200 is known in the cosmetic industry as Quatemium 15. Since this compound possesses 6 mol of formaldehyde per mol of active ingredient, a 10 mg/L solution is adequate to result in a peak of similar intensity to that obtained for the other study compounds. Preparation of Eluants. Fisher Optima grade acetonitrile was used as received from the manufacturer. The water used for this study was obtained from a Bamstead Nanopure water purifier system and was submicrometer filtered. The conductivity was typically 17.5 mfl. The DAP-HC1 eluant was prepared by dissolving 0.14 g of diaminopropionic acid monohydrochloride (99%, Aldrich) per liter of water and adding 4.15 mL of concentrated HC1. This results in a solution that is 50 mM in HC1 and 5 mM in DAP. Data Acquisition. Data were acquired from the 1.0-V fullscale output of the absorbance detector using an IBM PC/XT
(
min
)
Figure 2. Chromatogram of an aqueous mixture of GermalH 15 (100 ppm formulation), Glydant (55 ppm active ingredient), and Dowicll-75 (10 ppm formulation). Abscissa values were obtained by measuring the absorbance of the DDL derivative at 410 nm.
loaded with Interactive Microware’s (State College, PA) Chromatochart-PC, version 2.0. Input sensitivity was 1.0-V full-scale. The data acquisition rate was 1.25 Hz. Peak integration was performed by using the integration routine resident in the software.
RESULTS AND DISCUSSION the chromatogram of an aqueous mixture of
2 is
Figure Germall-115, Glydant, and Dowicil-75. These data illustrate the resolution of the three study compounds in the presence of free formaldehyde and in the presence of each other. The resolution depicted in this plot represents a compromise among the objectives of maximizing base-line separation and minimizing analysis time, band broadening, and reequilibration time. A constant organic modifier content of 30% provided for the optimum resolution per the above criteria, although the acetonitrile content can be increased to 40% for an isocratic eluant profile if no quaternary ammonium compounds are to be eluted. The step increase in the ionic strength of the eluant profile at 5.1 min elapsed time (table I) resulted in the optimum focusing of the Dowicil-75 peak while preventing the reequilibration time from becoming inordinately long. With the resolution aspects of the analysis having been established, it then remained to optimize the detection of the resolved species. The method of detection employed in this study involved the nonselective derivatization of molecular methylol functional groups and free formaldehyde following selective separation of these moieties via HPLC. The postcolumn chemistry proceeds according to the Hantzsch reaction, Eq 1, and is based on the formaldehyde characterization work of Nash (2). The 3,5-diacetyl-l,4-dihydrolutidine (DDL) reaction product exhibits characteristic absorption at 410 nm and fluorescence at 510 nm. The fact that the precursors to the DDL reaction product are transparent at 410 nm (resulting in an absorbance signal riding on theoretically zero background) coupled with the lack of a fluorescence detector suggested the use of absorbance monitoring of the DDL derivative in this case. Sensitivity and dynamic range benefits traditionally attributed to fluorescence spectrometry recommend this detection technique as a viable alternative as well. In order to conform to the extant literature, Nash’s reagent was used without modification in this study. The variables amenable to evaluation were therefore limited to reaction time and temperature. Figure 3 depicts the analytical response, gauged in terms of chromatographic peak area, as a function of reaction time and temperature for a 28 ppm formaldehyde standard. The reaction time was addressed in this system by the number of reaction coils installed in the heating block, which can house
1400
·
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990 Thousands ,----
600
----
•
.0 +
500 '
400
•
C
1
300
Formaldehyde
/
Twiee-thru
Glydant
¡I
Once-thru
0.5-
/
200 100
!-
0
0
10
40
30
20
50
Temperature
60 (
80
70
90
degrees C
100
110
10
120
Retention Time ( min
)
Figure 3. Peak area of 28 ppm HCHO standard as a function of reaction time (vis. the number of reaction colls) and temperature.
)
Figure 5. Chromatogram of 55 ppm Glydant active ingredient.
1.0
-
.04
Formaldehyde 0.5 r
Dowícil-75
,
0.5Germall-1
1
5
'
0
0
J--------1---,---------0
0------- -------i---------10
5
10
5
Retention Time
Retention Time
(
min
15
15
)
(
min
)
Figure 6. Chromatogram of 10 ppm Dowicil-75 as received.
Figure 4. Chromatogram of 100 ppm Germall-115 formulation. to
up to two coils. The two curves shown in Figure 3 therefore correspond to routing the column effluent through one reaction coil and through two reaction coils connected in series. The reaction temperature was controlled via the temperature of the heating block from ambient to 100 degrees °C (although the heating block has a setting for 130 °C, this proved to be above the upper limit of the Tefzel tubing connecting the heating block to the detector). These data indicate that maximum sensitivity in the postcolumn derivatization scheme is attained when two reaction coils are used in series at 100 °C. These data also indicate that the derivatization reaction has reached completion under these conditions. A final conclusion to be drawn from these data is that the variability based on reaction time and temperature will be minimized by operating at 100 °C with two reaction coils, since this regime lies on a plateau in the response trend. Returning to Figure 2, it can be seen that the detection of formaldehyde-releasing compounds by derivatization of their molecular formaldehyde is sensitive to their molar formaldehyde content—rather than to the concentration of the preservative compound, per se. All other things being equal, Dowicil-75, which contains 6 mol of latent formaldehyde, is detected at much lower levels than is either Germall-115 or Glydant, both of which contain 2 mol of latent formaldehyde. However, an examination of the chromatograms of the in-
dividual preservative compounds demonstrates that their relative stability differences render the foregoing analysis significantly less conclusive. The chromatogram of a 100 mg/L aqueous solution of Germall-115 is shown in Figure 4. These data indicate that the majority of this preservative molecule has liberated its formaldehyde in the time elapsed between dissolution and analysis (ca. 10 min). Glydant, Figure 5, also exhibits a rel-
——--------
I
C
x··
-1-
Dowicil
Glydant
B
d
F
L
1
x^
:
Í
-
·:
-I-
-
0
—
O
H
O
i
0.1 1------------1------------J---0 5
10
15
20
25
30
Time (hours)
Figure 7. Ratio of the formaldehyde derived from the intact preservative molecule to free formaldehyde as a function of time elapsed since dissolution.
atively high free formaldehyde content; however, since it is formulated as a liquid, it cannot be inferred that this free formaldehyde is necessarily attributable to molecular decomposition. It may be due to residual precursor formaldehyde from the synthesis of this product. Dowicil-75 has retained its latent formaldehyde over this time scale, Figure 6. Although these data do indicate trends in relative stabilites among these compounds, these trends are strictly valid only in the physical and chemical environment encountered by the sample during preparation and analysis. These conditions may or may not be typical of the environments in which these compounds are actually used. Figure 7 is a plot of the ratio of the molecular formaldehyde peak area to the free formaldehyde peak area as a function of time. Here and throughout this paper, the reference to the
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
·
1401
Table II. Calibration Data for Formaldehyde Standards HCHO
peak area,
concn,
arbitrary units
ppm
0.1
1
100
10
HCHO Concentration
(
ppm
220000
7.0
0.05
0.037
224705 222426 224709
224000
0.6
0.05
398878 403469 395160
399000
1.0
0.05
24211 24410 24693
24400
1.0
1.0
91890 92090 91820
91900
0.1
2.0
217980 218010
217900
0.1
2.0
442110 441580 443180
442300
0.2
2.0
668600 671900 671100
671000
0.3
2.0
926302
924000
0.2
2.0
3000583 2 999346 3017496
3010000
0.3
2.0
0.37
Figure 8. Peak area of the DDL derivative as a function of formaldehyde concentration.
formaldehyde peak area is meant to connote the peak area of the DDL derivative, since this is the moiety that was actually being monitored. These data were generated by using an automated sample loader to inject samples of Dowicil-75 and Glydant repeatedly over approximately 24 h (due to the low initial abundance of molecular formaldehyde observed for Germall-115, this compound was not included in the decay study). These data illustrate the utility of this methodology in assessing the time-dependent chemical stability of the study compounds without resorting to indirect microbiological efficacy testing methods. With the experiment configured in this fashion, the chemical effects of the analysis are held constant over time; the cause for the change in the ratio of molecular (or bound) formaldehyde to free formaldehyde with time must therefore be attributable to intrinsic instabilities associated with the individual preservative molecules themselves. The trends illustrated in Figure 7 depict a higher initial latent preservative capacity for Dowicil-75 compared to that for Glydant, which rapidly decays to a level below that for Glydant. The latent activity for Glydant is initially low in a relative sense (60% of the theoretical formaldehyde content is present as free formaldehyde, 40% as molecular formaldehyde), but it maintains this relationship at a fairly constant level over the time frame of the decay study. The linear dynamic range of this method is illustrated in Figure 8. This is a plot of formaldehyde peak area as a function of concentration for the entire set of working calibration standards. The raw data used in constructing this plot are compiled in Table II. Prior to plotting, peak area values were normalized to 2.0 absorbance units full scale. The dotted line serves to guide the eye to those concentration values at which the analytical response deviates from linearity. The data points plotted are averages of three observations at each concentration; 95% confidence intervals are not of sufficient magnitude to appear on the scale chosen for this plot (typical within-run shot-to-shot relative standard deviation is 0.5%). The solid line is the linear least-squares fit to the 0.37-37 ppm data. The correlation coefficient of this trend line is 0.9996 within the stipulated range of concentrations, indicating a linear dynamic range of 2 decades. Deviation from linearity on the low concentration end of this plot was due to a base-line perturbation of the formaldehyde region of the chromatogram associated with mixing noise in the membrane reactor. Although more precise adjustment of the flow rate of the derivatization reagent (via the pressure on its reservoir) might have alleviated this problem somewhat, efforts beyond those taken approached the point of diminishing returns. Deviation from linearity on the high concentration end of this plot was due to exceeding the dynamic range of the absorbance detector itself. Absorbance readings for the higher concentration formaldehyde standards were
rel std dev, % AUFS
230038 236324 206380
1000
)
peak area
0.0185
0.185 0.01
av
3.7
9.25
18.5
27.8
37.0
217 590
923415 922 507
92.5
acquired on the 2.0 absorbance units full scale (AUFS) setting. At a reading of 2.0 absorbance units, 99% of the light interrogating the sample cell is absorbed. Quantification of the absorbance signal is therefore tenuous at this level. However, the positive deviation of the detector response was unexpected. Ostensibly the absorbance signal was smeared out in time at the output extreme of 1 V (the signal for the 93 ppm formaldehyde standard was definitely clipped at this level). Theoretically, high-end deviation is negative in this type of measurement, and indeed this was the case when peak heights were
plotted
as a
function of concentration.
CONCLUSION A method for characterizing preservatives that release formaldehyde as their primary mode of biological activity has been developed. This method has been validated via application to several of the more prominent preservative compounds in use today. The scope of the method encompasses the range from nonionic compounds to those displaying a quaternary ammonium cationic nature. The precision and linear dynamic range are typical for HPLC techniques in general. Although the accuracy of this method was not explicitly evaluated in this study, the derivatization chemistry was shown to proceed to completion under the conditions employed. This fact, coupled with previous work demonstrating that the derivatization chemistry proceeds with unit efficiency, implies comparable efficiency for this method as well. The incorporation of the derivatization step in a postcolumn mode imparts several benefits. Those compounds that exhibit characteristic absorbance or fluorescence can be detected prior to derivatization, thereby providing for an additional degree
1402
Anal. Chem. 1990, 62, 1402-1407
of analytical specificity. This approach is also more amenable to automation than are those approaches that utilize a batch mode derivatization step either before separation via HPLC or independent of the separation step. Additionally, by removal of manual derivatization from the analysis procedure, the precision of the analysis should be improved. Finally, the postcolumn approach to derivatization is unique in that it provides for the resolution of several formaldehyde-releasing preservatives in a mixture containing free formaldehyde. Although this method has been applied to the characterization of several different types of analyte species, the determination of these and other related species in end-product formulations will require additional effort. Presumably this effort would entail extraction of the preservative from the formulation and some form of filtration. These steps, coupled with matrix interferences, may or may not compromise the utility of this method. Experience in the author’s laboratory with proprietary compositions of matter has in fact shown this methodology to be quite robust.
Water in Oil Microemulsions Chromatography
as
Registry No. I, 6440-58-0; II, 39236-46-9; formaldehyde, 50-00-0.
III,
4080-31-3;
LITERATURE CITED (1) Sondossi, M.; Rossmoore, H. W.; Wireman, J. W. J. Ind. Microbiol. (2) (3) (4) (5)
1986, 1, 87-96. Nash, T. Blochem. J. 1953, 55, 416-421. Belman, S. Anal. Chim. Acta 1963, 29, 120-126. Wilson, C. H. J. Soc. Cosmet. Chem. 1974, 25, 67-71. Sheppard, E. P.; Wilson, C. H. J. Soc. Cosmet. Chem. 1974, 25,
655-666.
(6) Wisneski, . H. J. Assoc. Off. Anal. Chem. 1980, 63 (4), 864-868. (7) Sawicki, E.; Hauser, T. R.; McPherson, S. Anal. Chem. 1962, 34,
1460-1464.
L; Fuson, R. C,; Curtin, D. Y.; Morrill, T. C. The Systematic Identification of Organic Compounds, 6th ed.; Wiley: New York, 1980; pp 162-163. (9) Kuwata, K.; Uebori, M.; Yamasaki, H. J. Chromatogr. Sci. 1979, 17, 264-268. (10) Stack, A. R.; Davis, . M. J. Assoc. Off. Anal. Chem. 1984, 67(1), 13-15. (8) Shriner, R.
Received for review December 15,1989. Accepted March 2, 1990.
Mobile Phase in Liquid
Alain Berthod,* Odile Nicolas, and Maurice Porthault Laboratoire des Sciences Analytiques, UA CNRS 435, Universite de Lyon
diagram of water-sodium diethylhexyl sulfosucclnate or Aerosol OT (AOT)-heptane was determined. L2 liquid systems containing up to 45 % water and 42 % AOT were used as mobile phase In an unbonded silica column. The capacity of two test solutes, 4-nltrobenzoic acid and 4factors, nltrophenol, were measured for about 50 different L2 compositions. It was found that the k' evolutions were very dependent upon the physicochemical microemulsion structure described by using the parameter *fw/A0T, the water/AOT molar ratio. It was shown that drastic changes In chromatographic parameter occurred at Afw/A0T < 10, conditions under which most of the water molecules are tightly bound to the surfactant polar head and sodium Ion. Up to 50% decrease of the column dead-volume was observed when the L2 system water content Increased. The column permeability was changing at low water content mobile phase. These phenomena were related to AOT adsorption onto the silica stationary phase. At 4fw/A0T > 10, the AOT adsorption and the column permeability were constant and dependent upon the AOT concentration. However the test solute retention were still evolving significantly.
The use of ordered liquid systems in chemical analysis is becoming more popular (1). Most ordered liquid systems contain surfactant molecules. They present some inhomogeneous character at the microscopic down to molecular level. The polarity of contiguous “microdomains” is very different. Author to whom all correspondence should be sent. 0003-2700/90/0362-1402S02.50/0
69622 Villeurbanne cedex, France
Such systems include micellar solutions, emulsions, and microemulsions (2). Micellar solutions are liquid systems containing water and surfactant molecules. Emulsions are made of water, oil, and surfactant. One phase, for example the oil phase, is dispersed in the other and stabilized by surfactant molecules. Microemulsions contain oil, water, a surfactant, and, most often a medium chain alcohol acting as a cosurfactant. Microemulsions form spontaneously and are very stable with time. The physicochemical structure of microemulsions is still under investigation. However, it is possible to use the LI, L2, and bicontinuous classification (3). A LI microemulsion is an oil in water system. Oil microdroplets, enclosed in a surfactant-cosurfactant layer, are dispersed in an aqueous continuous phase. L2 microemulsions are water in oil systems. In a bicontinuous structure, it is not possible to tell which phase is dispersed in which (Figure 1) (4). The use of micellar solutions as mobile phases in reversed-phase liquid chromatography was first reported by Armstrong and Henry 10 years ago (5). Micellar liquid chromatography (MLC) has been the subject of numerous review articles (1, 6-8). In most MLC applications, normal micellar solutions, i.e. aqueous solutions of various surfactant, were used as the mobile phase. Few reports have described the use of reversed micelles or L2 microemulsion mobile phases in which an oil, hexane, cyclohexane, heptane, or isooctane, is the continuous phase (Figure 1). Armstrong used cyclohexane-Aerosol OT (AOT)-water L2 microemulsions as mobile phases in thin-layer chromatography (9). Nucleosides, such as adenosine, cytidine, guanosine, and uridine were separated on reversed-phase thin-layer plates and L2 cyclohexane microemulsions. Dorsey was the first to use reversed micellar phases for normal-phase chromatography
L2 microemulsions are water In oil systems. The mass phase
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1990 American Chemical Society
