Evaluation of Bonded-Phase Extraction Techniques Using a Statistical

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 26, 2016 | http://pubs.acs.org Publication Date: December 15, 1986 | doi: 10.1021/ba-1987-0214.ch017

17 Evaluation of Bonded-Phase Extraction Techniques Using a Statistical Factorial Experimental Design R. E. Hannah, V. L. Cunningham, and J. P. McGough Smith Kline & French Laboratories, Philadelphia, PA 19101 Isolation techniques using bonded-phase silicas (bonded-phase extraction) were evaluated as an alternative to liquid-liquid extraction methods. The relative importance of four variables on extraction efficiencies for bonded-phase isolation techniques was evaluated by using a statistical 2 factorial experimental design (4 variables at 2 levels). Extraction efficiencies were based on percent recoveries for a 27-component synthetic test mixture containing a variety of organic compounds typical of those likely to be found in samples of interest. The experimental variables that were identified and included in the design were sample pH, nonpolar solid-phase extraction strength, polar-phase extraction strength, and conditioning solvent concentration. This 2 extraction factorial design allowed more information to be obtained with relatively few runs by varying several parameters at once. The application of statistical methods permitted the evaluation of data quality and also the determination of variable interactions. 4

4

ENVIRONMENTAL PROCESS ANALYSIS requires the characterization of

chemical process and waste streams in order to evaluate their environmental abuse potential and treatability characteristics. An integral part of this analysis, as well as environmental fate determinations, is the isolation of organic compounds and metabolic products from very complex matrices such as waste water effluents, process streams, biological reactors, and fermentation broths. Generally, the organics involved are fairly polar, water-soluble compounds that must be ex0065-2393/87/0214/0359$06.00/0 © 1987 American Chemical Society

Suffet and Malaiyandi; Organic Pollutants in Water Advances in Chemistry; American Chemical Society: Washington, DC, 1986.


360

ORGANIC POLLUTANTS IN WATER

tracted f r o m aqueous solutions. Although l i q u i d - l i q u i d extraction methods usually work w e l l for nonpolar organics, they are not always selective enough to separate the compounds of interest from all matrix interferences and frequently produce poor extraction efficiencies for more polar analytes. Bonded silicas are n o w widely used i n chromatographic separation techniques. Disposable extraction columns containing these materials are capable of extracting organic compounds f r o m various sample matrices very efficiently. Extraction systems using bonded-phase silicas have been successfully applied to a w i d e range of sample preparation problems involving biological and biomedical studies (1-7); petrochemical analysis (8); food and cosmetics analysis (9-12); and environmental analysis involving raw water, drinking water, and waste water matrices (13-16). In most of these applications, the goal is to isolate a specific compound or class of compounds f r o m the sample. M o r e recently, especially i n the environmental area, solid-phase extraction techniques are being applied to much broader compound groups such as priority pollutants (15) and hazardous organic compounds (16). W i t h these broader applications i n m i n d , an experimental design was set up to evaluate bonded-phase extraction techniques as general survey methods for organics in aqueous matrices.

Factorial Experimental Design In a factorial experiment, a fixed number of levels are selected for each of a number of variables. F o r a full factorial, experiments that consist of all possible combinations that can be formed f r o m the different factors and their levels are then performed. This approach allows the investigator to study several factors and examine their interactions simultaneously. T h e object is to obtain a broad picture of the effects of the selected experimental variables and detect major trends that can determine more promising directions for further experimentation. A d vantages of a factorial design over single-factor experiments are (1) more than one factor can be varied at a time to allow the examination of interaction effects and (2) the use of all experimental runs in evaluating an effect increases the efficiency of the experiment and provides more complete information. In this study, a factorial experiment was set up to evaluate the effects of four variables at two levels on extraction efficiencies b y using bonded-phase isolation techniques and a 27-component synthetic test mixture. T h e compounds studied and the respective mass ions used for quantitation are given in the box. T h e compounds i n the mix vary greatly in water solubility and volatility and, in general, represent a w i d e class of organic compounds typical of those present in environmental samples. T o maximize solute recoveries, the procedure was



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361

Test Mixture Components and Quantitation Mass Ions Compound Mass Ion Isooctane 99 Acetone 43 Tetrahydrofuran 42 Ethyl acetate 61 Methyl ethyl ketone 72 Isopropyl alcohol 45 Ethanol 45 Methylene chloride 84 Methyl isobutyl ketone 100 Tetrachloroethylene 164 Toluene 65 Dodecane 85 Cyclohexanone 55 Dimethylformamide 73 Cyclohexanol 57 1,2-Dichlorobenzene 146 1-Octanol 84 Dimethyl sulfoxide 63 1,3,5-Trichlorobenzene 180 1,2,4-Trichlorobenzene 180 Nitrobenzene 123 Phenol 94 4-Methylphenol 108 1,2,3,4-Tetrahy droisoquinoline 132 2.4- Dichlorophenol 162 2.5- Dichlorophenol 162 4-Chlorophenol 128

designed to use two extraction column types of different polarities connected in series. The first column or primary column was a nonpolar phase, and the sorbent for the second column was more polar in nature. Also, in half the experimental runs, the samples were fortified with 500 ppm of methanol. The hydrophobic nonpolar phases required conditioning with methanol prior to sample loading to wet the phase surface. This facilitated contact between the aqueous sample matrix and the hydrophobic phase surface and maximized extraction efficiency. Methanol, acting as a bridge solvent, was added directly to the sample prior to extraction to determine what additional effect, if any, it had on extraction efficiency. Four experimental variables were selected: sample p H , primary column type, secondary column type, and methanol concentration. By using each of the four variables at two levels, the complete arrangement of experimental runs became a 2 X 2 X 2 X 2 o r 2 factorial design requiring 16 runs. Table I represents the design matrix; the high and 4


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Table I. 2* Factorial Design Matrix Variable Levels

b

Test Run Number


0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

pH

+ —

+

—

+

—

+

—

+ + —

+ —

+

CI

C2

BS

-

-

-

+ +

— —

— — — — —

— —

+ + — —

+ + — —

+

+ + + +

— — — —

+ + + +

+ + + + + + + +

Test runs were made in random order to eliminate possible bias. p H : low level ( - ) 2.0, high level (+) 8.0; C I nonpolar phase extraction type: low level (—) C18, high level (+) C8; C2 polar phase extraction type: low level (—) cyano, high level (+) diol; BS bridge solvent concentration in sample: low level (—) 0 ppm, high level (+) 500 ppm.

fl b

l o w levels of each of the variables are coded as plus and minus signs. F o r the quantitative variables—pH and methanol concentration—a plus sign represents the high level. F o r the qualitative variables—primary column type and secondary column type—it does not matter which level is associated with the plus sign, as long as the designations are consistent. Table II represents the design matrix with the variable levels uncoded.

Experimental Chemicals and Standard Solutions. Cyclohexanone, cyclohexanol, 1,3,5trichlorobenzene, 1,2,4-trichlorobenzene, phenol, 4-methylphenol, 4-chlorophenol, 1,2,3,4-tetrahydroisoquinoline, 1-chlorohexane, 1-chlorododecane, and 1-chlorooctadecane were obtained from Aldrich. Acetone, tetrahydrofuran, ethyl acetate, toluene, dimethyl sulfoxide, and methanol were obtained from J. T. Baker. Distilled-in-glass isooctane, methylene chloride, ethyl ether, and pentane were obtained from Burdick and Jackson. Analytical standard kits from Analabs provided methyl ethyl ketone, isopropyl alcohol, ethanol, methyl isobutyl ketone, tetrachloroethylene, dodecane, dimethylformamide, 1,2dichlorobenzene, 1-octanol, nitrobenzene, 2,4-dichlorophenol, and 2,5dichlorophenol. All chemicals obtained from the vendors were of the highest purity available and were used without further purification. High-purity water


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363

Bonded-Phase Extraction Techniques Table II. 2* Factorial Design Matrix

Test Run Number

pH

Cl

C2

BS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8

C18 C18 C8 C8 C18 C18 C8 C8 C18 C18 C8 C8 C18 C18 C8 C8

cyano cyano cyano cyano diol diol diol diol cyano cyano cyano cyano diol diol diol diol

0 0 0 0 0 0 0 0 500 500 500 500 500 500 500 500


0

0 b

Test runs were made in random order to eliminate possible bias. See Table I for description of variable levels.

was obtained by passing deionized carbon-filtered water supplied by the in-house system through a second system (Hydro Ultra Water Systems) contain ing mixed-bed, high-capacity, ion-exchange cartridges and an activated carbon cartridge. The water then passed through a final carbon polishing filter and was ultrafiltered with a 0.2-μτη filter. Individual stock solutions of the test compounds were prepared in methanol at a concentration of 50 mg/mL. A standard test mixture was prepared by adding 100-^L aliquots of each of the individual stock solutions to 500 mL of ultrapure water to give a concentration of 10 ppm per component. Samples requiring the addition of 500 ppm of methanol (conditioning solvent) were prepared by adding 6.3 μία of methanol to 10-mL aliquots of the aqueous standard mix just prior to extraction. The pH of the samples was adjusted with either 6 M HC1 (for pH 2 samples) or 6 M NaOH (for pH 8 samples). To separate ionic strength effects from pH effects, the ionic strength of the samples was held constant. Gas chromatographic-mass spectrometric (GC-MS) calibration standard mixes for quantitation were prepared in ethyl ether at concentrations of 20, 50, and 75 ppm. Internal standard spiking solution containing 1-chlorohexane, 1-chlorododecane, and 1-chlorooctadecane was prepared from individual stock solutions in methanol of each component. Two hundred microliters of each solution were added to ethyl ether and diluted to 2 mL. Forty microliters of this internal standard mix was added to the column extracts before diluting to 2 mL to yield a final concentration of 100 ppm per internal standard component. Equipment and Instrumentation. Solid-phase extraction columns were obtained from J. T. Baker. Octadecyl (C18) and octyl (C8) 1-mL low-displace ment columns were used as the primary extraction columns. Cyano and diol


364



3-mL columns were used as the secondary extraction columns. A Finnigan 1020 GC-MS was used to analyze the column extracts. The autoquantitation software available with this system was used to generate calibration curves and perform the necessary calculations. Procedures. S O R B E N T C O N D I T I O N I N G . To facilitate partitioning of organics in aqueous solution onto hydrophobic nonpolar sorbents, the sorbents must first be conditioned with methanol to increase their wettability. This solvation of the solid phase is necessary to provide efficient and reproducible extractions. The conditioning process was carried out according to the instruc tions accompanying the extraction columns. SAMPLE LOADING. Prior to sample loading, the conditioned primary and secondary columns were connected in series by using column adapter connec tors (J. T. Baker). The sorbents in each column were not allowed to dry out. A 10-mL aliquot of the 10-ppm aqueous standard mixture adjusted according to the treatment number of the design matrix (Table II) was loaded onto the column at a rate of 1.0-1.5 mL/min under an air pressure of 10 lb/in. applied at the top of the column. Air pressure was used instead of a vacuum to minimize losses of the more volatile components. 2

SORBENT ELUTION. The loaded extraction columns were disconnected and eluted with solvents individually. The elution procedure was as follows: First, 1 column bed volume of pesticide-grade pentane was added to the sorbent to displace any residual water. Then, 3 column bed volumes of ethyl ether were added. The eluting solvents were collected in a 2-mL volumetric flask. The eluates from the primary and secondary columns were combined in the volumetric flask. The internal standard solution was added, and the final extract volume was adjusted to 2 mL. Air pressure was again used to push the solvents through the sorbents to avoid volatility losses.

Analytical Methods. All of the sorbent extracts were analyzed by using GC-MS. The solutions were chromatographed on a 30-m X 0.32-mm i.d. Supelcowax 10 capillary column with a film thickness of 1 μπι (Supelco). The Supelcowax 10 is a Carbowax PEG 20 M bonded-phase capillary column. The instrument conditions were as follows: injector, 250 °C; separator oven, 250 °C; column over initial, 40 °C programmed to 250 °C at 6 °C/min; linear velocity of helium carrier gas, 35 cm/s at 40 °C; column head pressure, 6 lb/in. ; mass range, 33-333 amu scanned in 1-s intervals. Two-microliter aliquots were injected in the split mode at a 10-to-l split ratio. A typical chromatogram appears in Figure 1. Quantitative results were produced for each compound on the basis of in ternal standard method calculations. A three-point calibration curve was gene rated for each compound by using peak areas of a quantitation ion extracted from the mass spectrum of the compound. The ion was selected on the basis of it being a uniquely characteristic mass of the compound. The use of extracted ion quantitation produces more accurate results than total ion-current quantita tion in cases in which two or more components are not completely resolved chromatographically. This situation is generally the case in complex mixture analysis. The quantitation ions selected for each of the compounds in the mix are listed in the box. The Supelcowax 10 column has the ability to accept water injections. Direct aqueous injections of the 10-ppm standard water mix were performed to verify 2


Suffet and Malaiyandi; Organic Pollutants in Water Advances in Chemistry; American Chemical Society: Washington, DC, 1986. 1-CL C«,

— Thlq.

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Nitrobenzene

1,2,4 T C B

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366


the integrity of the sample solutions prior to extraction. A chromatogram of an aqueous injection of the mixture appears in Figure 2. Except for a base line upset at approximately 8 min, all features of the chromatogram appear very much like the ethyl ether standard mixture chromatogram in Figure 1.


Results and Discussion T h e percent recoveries of 20 compounds f r o m all 16 experimental runs are listed in Table III. T h e results for the seven remaining compounds in the mixture are not listed because either they were not recovered at all i n any of the extractions (dimethylformamide and dimethyl sul foxide), or because determinate system errors were discovered i n the experimental protocol a n d rendered the data for those compounds unreliable. M a i n effects were calculated to determine the influence of each variable o n the extraction efficiency for each compound. A n effect is the change in response for a variable as you move f r o m the l o w to high level of that variable over all conditions of the other variables. The main effect of a variable is the difference between the average of the high-level responses and the average of the low-level responses. A cubic representation of responses for methyl isobutyl ketone is illustrated schematically i n Figure 3. T h e values at each vertex of the cube represent the response (percent recovery) measured at that combination of variable levels. The values followed b y a minus sign represent the response when variable Β (primary column type) was l o w (C18). The values followed b y a plus sign represent the response when variable Β was high (C8). Schematically, the change i n responses i n going f r o m the l o w to high level of variable D (methanol concentration) can be determined b y comparing the response values on the bottom plane of the cube (low) to the top plane of the cube (high). Likewise, the effect of changing variable A (pH) is determined b y comparing the left and right faces of the cube, and the effect of variable C (secondary column type) is determined b y comparing the responses on the front and back faces of the cube. T o manually calculate the effects of a 2 factorial design for 20 different compounds w o u l d be quite time-consuming. Fortunately, more rapid methods can be used, one being Yate's algorithm (17). This algorithm is applied to the responses after they have been arranged in standard order. A factorial design is in standard order when, as in Table I, the first column consists of alternating minus and plus signs, the second column consists of successive pairs of minus and plus signs, the third column consists of alternating sets of four, etc. B y using the Yate's algorithm contained i n R S I software (Bolt, Beranek, and Newman) mounted on a V A X system (Digital), analysis tables were generated for each compound. Table I V contains the results for p-cresol. C o l u m n 0 4


HANNAH ET AL.

Bonded-Phase Extraction Techniques


17.


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Evaluation of Bonded-Phase Extraction Techniques Using a Statistical

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