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Spring 5-1999 Analytical SFE: Optimization and Applications Sohita Patel Seton Hall University

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Recommended Citation Patel, Sohita, "Analytical SFE: Optimization and Applications" (1999). Seton Hall University Dissertations and Theses (ETDs). 1256. https://scholarship.shu.edu/dissertations/1256 Analytical SFE: Optimization and Applications

by

Sohita Patel

Dissertation Submitted to the Department ofChemistry of Seton Hall University in partial fulfillment ofthe requirements for the degree ofDoctor of Philosophy.

May, 1999 We certify that we have read this thesis and that in our opinion it is adequate in scientific scope and quality as a dissertation for the degree ofDoctor ofPhilosophy.

APPROVED

Nichola . Snow, Ph. D. Research Mentor

Joseph T Maloy, Ph.D. Member e Dissertation Committee

u,..rv'___ / c:-­ John

Richard D. Sheardy, Ph.D.

Chairman, Department ofCheTnH' ITru Dedication

To my parents, Manu and Shakuntla Patel. This work is also dedicated to

my sister, Mohita Patel and brother, Dhaval Patel.

iii Acknowledgements

I would like to thank my mentor~ Dr. Nicholas H. Snow, for his support, advice, guidance, and willingness to let me work in his lab. Thank you Dr. Snow.

I also like to thank. my parents who have supported me through out my carrier as student.

I know this has not been a short period oftime. Thank you for your encouragement~ foresighted vision and wisdom, support and motivation.

I would also like to thank members of my research group for their help, especially Cindy

Mleziva Blake, who has proof read almost everything I have written for my thesis.

Last but not least, I would like to thank is Dr. Issam El-Achkar. You have taught me a lot more then just math. Thank you for your support, advice and guidance. You are the one, who has helped me to tackle every problem that came upon during graduate school.

I wonder ifI would have made through graduate school without your help. Thank you so much.

Finally, I would like to thank the Seton Hall University Research Council, and Isco Inc. for providing the materials and instruments needed for the completion ofmy research.

Many thanks to all ofyoul

iv Contents Page

Dedication iii

Acknowledgement iv

Contents v

List ofFigures V111

List ofTables Xl

Chapter 1 - Historical 1

Chapter 2 - Basic Principles and Introduction of 17 Supercritical Fluid Extraction (SFE)

I. Introduction 17 II. Properties ofPhase Diagram 18 m. Definition ofSupercritical Fluid 23 IV. Selection ofthe Supercritical Fluid 24 V. Principles of SFE 28 VI. The practice ofSFE 29 VII. Effects ofModifiers 30 VIII. Instrumentation 31 A. Pump 34 B. Extraction vessels 35 C. Restrictors 35 1. Linear 37 a. Fused silica capillary 38 b. Stainless steel capillary 38 D. Collection Systems 38 1. Cryogenic trapping in an empty container 39 2. Deposition in a liquid solvent 40 3. Retention on a solid surface 41 IX. Factors effecting the extraction recovery of SFE 42 A. Spiking procedures 42 1. Fresh 42 2. Aged 42 3. Suspended 43 4. Supercritical 43 B. Sample size 44 C. Temperature and Pressure 44 D. Sample matrix effects in SFE 47 1. Physical matrix effects 48 2. Chemical changes in the sample matrix 48

v X. Impact of matrix on extraction kinetics 48 A Pawliszyns'model 49

Chapter 3 - Optimization ofconditions in 60 Supercritical Fluid Extraction

I. Goal 60

II. Experimental 60 A. Reagents 61 B. Sample preparation 61 C. SFE conditions 61 D. GC conditions 63

III. Results and Discussion 64 A Non-polar analytes 67 1. Eicosane spiked onto ODS 67 2. Eicosane spiked onto silica gel 70

B. Polar acidic analytes 72 1. 0: - naphthol spiked onto ODS 72 2. 0: - naphthol spiked onto silica gel 75

C. Polar basic analytes 77 1. Propanolol spiked onto ODS 77 2. Propanolol spiked onto silica gel 78

IV. Conclusion 89

Chapter 4 - Determination ofCrude Fat in Food Products by 90 Supercritical Fluid Extraction and Gravimetric Analysis

I. Introduction 90 n. Experimental 90 Ill. Results and Discussion 92 IV. Conclusion 97

Chapter 5 - Supercritical Fluid Extraction ofPeppermint Oil 98 from Toothpaste.

VI I. Introduction. 98 II. Experimental. 99 III. Result and Discussion. 102 N. Conclusion. 113

Chapter 6- Extraction of Sulfur Containing Compounds using SFE 117

I. Introduction. 117 II. Experimental. 118 III. Result and Discussion. 120 IV. Conclusion. 125

Chapter 7- Conclusion. 133

References. 136

VB Figures

Figures page

Figure 1-1 - An example schematic ofthe 950 MDS-2000 3 instrument.

Figure 1-2 - A schematic of solid phase microextraction 5 device.

Figure 1-3 - A schematic ofa gas chromatograph 10

Figure 1-4 - A conceptual scheme for vapor phase analysis 14 by mass spectrometer.

Figure 2-1 Phase diagram ofcarbon dioxide. 19

Figure 2-2 Clausius-Clapeyron plots for the vapor pressures 22 ofthe solid and liquid phases ofa pure substances.

Figure 2-3 Schematic diagram of an off-line supercritical 32 fluid extraction apparatus.

Figure 2-4 A schematic ofthe ISCO extraction vessel 36

Figure 2-5 Effect oftemperature and pressure on the 46 Hildbrand solubility parameter for supercritical carbon dioxide.

Figure 2-6 Theoretical curve ofpercentage extraction versus 53 time.

Figure 2-7 Theoretical curve for the hot-ball model. 56

Figure 2-8 Theoretical curves for SFE, including solvation 58 effects, for spherical matrix particles.

Figure 3-1 - A typical chromatogram. 65

Figure 3-2 Theoretical dynamic extraction profile ofan 66 analyte from a solid matrix.

Figure 3-3 Extraction recovery ofeicosane spiked on ODS 69 at a constant temperature.

viii Figure 3-4 - Extraction recovery ofeicosane spiked on silica 71 gel at a constant temperature.

Figure 3-5 - Extraction recovery ofa-naphthol spiked on ODS 74 at a constant temperature.

Figure 3-6 - Extraction recovery ofa-naphthol spiked on 76 silica gel at a constant temperature.

Figure 3-7 - Extraction recovery ofpropranolol spiked on 79 ODS at a constant temperature.

Figure 3-8 - Extraction recovery ofpropranolol spiked on 81 silica gel at a constant temperature.

Figure 3-9 - Extraction recovery ofeicosane spiked on ODS 83 at a constant pressure.

Figure 3-10 - Extraction recovery ofeicosane spiked on silica 84 gel at a constant pressure.

Figure 3-11 - Extraction recovery ofa-naphthol spiked on ODS 85 at a constant pressure.

Figure 3-12 - Extraction recovery ofa-naphthol spiked on 86 silica gel at a constant pressure.

Figure 4-1 - Fat recovery from Snickers bar versus carbon 94 dioxide extraction volume.

Figure 5-1 - Calibration curve for menthoL 103 Figure 5-2 - An example total ionchromatogram ofpeppermint oil 104 standard.

Figure 5-2A - Mass spectrum ofpeak at RT - 6.6 minutes. 105

Figure 5-2B - Matching library spectrum ofthe peak in Figure 5-2A. 105

Figure 5-3 - An example chromatogram ofthe extract ofbrand 106 1 toothpaste.

Figure 5-3A - Mass spectrum ofthe peak at RT -7.6 minutes. 108

Figure 5-3B - Matching library spectrum ofthe peak in Figure 5-3A. 108

ix Figure 5-3C - Mass spectrum ofthe peak at RT - 8.0 minutes. 109

Figure 5-3D - Matching library spectrum ofthe peak in Figure 5-3C. 109

Figure 5-3E - Mass spectrum ofthe peak at RT - 8.2 minutes. 110

Figure 5-3F - Matching library spectrum ofthe peak in Figure 5-3E. 110

Figure 5-4 - An example chromatogram ofbrand 2 toothpaste. 111

Figure 5-4A - Mass spectrum ofthe peak at RT - 6.3 minutes. 112

Figure 5-4B - Matching library spectrum ofthe peak in Figure 5-4A. 112

Figure 5-5 - An example chromatogram ofthe brand 3 and 4. 114 toothpaste.

Figure 5-5A - Mass spectrum ofthe peak at RT - 7.6 minutes. 115

Figure 5-5B - Matching library spectrum ofthe peak in Figure 5-5A. 115

Figure 6-1 - A chromatogram ofextract ofgarlic clove using SFE. 123

Figure 6-2 - Mass spectra ofAllyl methyl sulfide. 126

Figure 6-3(A) - Mass spectrum ofAllyl sulfide. 127

Figure 6-3(B) - Matching library spectrum ofthe peak in Figure 6-3A. 127

Figure 6-4(A) - Mass spectrum ofMethyl propenyl disulfide. 128

Figure 6-4(B) - Matching library spectrum ofthe peak in Figure 6-4A. 128

Figure 6-5(A) - Mass spectrum ofDiallyl disulfide. 129

Figure 6-5(B) - Matching library spectrum ofthe peak in Figure 6-5A. 129

Figure 6-6 - Mass spectum of3-vinyl-1,2-dithiin. 130

Figure 6-7 - Mass spectum of2-vinyl-1 ,3-dithiin. 131

Figure 6-8(A) - Mass spectrum ofAllyl trisulfide. 132

Figure 6-8(B) - Matching library spectrum ofthe peak in Figure 6-8A. 132

x Tables

Tables Page

Table 2-1 - Properties ofsupercritical fluids vs. gases 25 and liquids.

Table 2-2 - Physical parameters ofcommonly used 26 supercritical fluids.

Table 3-1 Characteristics ofthe analytes used. 62

Table 4-1 - Typical results offat extraction from candy 93 bar.

Table 4-2 - Compiled results from crude fat extraction. 95

Table 5-1 - Conditions and parameters used for GCIMS. 101 Table 5-2 - Recovery ofmenthol from various toothpaste. 116 Table 6-1 - Sulfur containing compounds extracted by Deruaz 122 et at using headspace GC.

Table 6-2 - Seven compounds detected from the extracts ofthe 124 garlic clove using SFE.

xi Chapter 1

Historical

There are three major steps in the analysis of any given sample: sample preparation, chemical reaction or separation, and detection. Over the past few decades, the technology for the latter two steps ofthe analysis process has advanced tremendously compared to the sample preparation methods. This step is still very time consuming and prone to inaccurate results. (1) The sample preparation techniques used today are primarily the same ones used many years ago: liquid-liquid extraction, liquid-solid extraction, and soxhlet extraction, etc., with very little or no modification. This can be accredited in part to the slow acceptance of newer methods, despite their many advantages. These newer methods include Solid Phase Microextraction (SPME),

Microwave Assisted Solvent Extraction and Supercritical Fluid Extraction (SFE). These methods are described in detail below.

As mentioned above, there have been many extraction methods developed for preparing samples for further analysis. In developing any method there are several factors that must be considered. These include speed of analysis, cost, and type of results. Extracts should be ready for instrumental analysis without a further concentration step or purification step. Also, as an environmental concern, the extraction should generate little or no waste. The classical techniques for extractions, such as sonication and soxhlet extractions, do not meet these standards.(2,3)

1 Microwave assisted solvent extraction was used first in the 1986 by Ganzler et a1 using a simple household microwave oven.(4) Recently, commercially available microwave ovens have been introduced into analytical laboratories. Figure 1-1 is an example schematic of the Microwave assisted extraction instrument used by Lopez­

Avilla et al. for their studies.(5)

Microwave energy was explored as means of rapidly extracting analytes without degrading the entire sample. The knowledge of microwave energy as well as general applications to analytical and environmental chemistry was reviewed by Zlotorzynski. (6)

In the microwave extraction of a solid sample matrix, the sample is saturated with a common organic solvent or organic/aqueous solvent mixture and irradiated for a short period oftime. After cooling, the supernatant liquid is decanted and the matrix is rinsed several times. The combined extract is centrifuged, concentrated, or purified as necessary prior to analysis. Solvents lacking a dipole moment do not efficiently absorb microwave energy. Therefore, at least 10% polar solvent is generally required. The absorption of the energy by the solvent results in the disruption of weak hydrogen bonds, improved solvent penetration into the matrix, and enhanced solvation.(7) Since its beginning, this technique has been developed by the invention of the microwave instruments specifically aimed to furnish a temperature-regulated environment.

Microwave assisted solvent extraction can be used as an alternative method to traditional liquid solvent extraction methods. (8-13)

2 ----, T mperature probe

vessel water flnS~ - system ~ -+--printer or PC

inle fitting Figure 1-1- Schematic diagram ofthe temperature/pressure control system for the l\iIDS-2000 microwave system (adapted li"om reference 5)

3 Another recently developed extraction method that can be used for sample preparation is Solid Phase Microex:traction (SPME), a schematic of which is shown in

Figure 1-2.(14) This method, developed in 1989(14), is solventless and sensitive. It can be used for environmental, clinical and food samples.(14) It is composed of two processes. First, is the partitioning of the analytes between the matrix and the fiber coating and the second is to desorb the concentrated analytes into an analysis instrument.

In the first step, the coated fiber is exposed to the sample and the target analytes are extracted from the sample matrix into the coating. In the second step, the fiber with concentrated analytes is then transferred to an instrument for desorption and quantitation.

A technique very similar to supercritical fluid extraction (SFE) is accelerated solvent extraction (ASE). Conventional liquid solvents are used at elevated temperature and pressure for the extraction of solid or liquid matrices. Because of the elevated parameters, solute capacities, diffusivities, and extraction kinetics are increased, thereby greatly reducing extraction time and solvent volumes. However, ASE is still evolving as a sample preparation technique and should not be considered mature at all, both in regard to instrumentation and developed applications. Despite the elevated temperatures and pressures, ASE has never been used with a supercritical fluid as a solvent.

Fundamentally, SFE is advantageous as a sample preparation technique because it uses a supercritical fluid as the solvent. The strength of SFE resides in the fact that physical instrumental parameters can be changed to alter chemical interactions directly, unlike any other extraction technique. Thus, selectivity is the key when it comes to SFE.

4 Phmger

---Adjustable needle guide

1----Septum piercing needle

1---- Coated SPrdE fused silica fib er

Figure 1-2 - A schematic of Solid Phase l\I.Iicroextraction device (Rec1raw:n. from reterellCe II)

5 SFE a1so possesses a unique feature in that the extraction effluent can be directly channeled into an analytical chromatograph such as a Gas Chromatography (GC), High performance Liquid Chromatography (HPLC) or Supercritical Fluid Chromatography

(SFC). No other liquid extraction method can be directly coupled to an analytical chromatograph.

The use of supercritical fluids for performing analytical scale extractions is a recent development, while the occurrence of supercritical fluid phenomena can be documented to the early 19th century. The phenomenon of the 'critical state' has been known since 1822 when Cagnaird de la Tour noted (15) a lack of discontinuity when passing between the gaseous and liquid states. Andrews' 1869 study (16) on carbon dioxide is considered to be the first systematic study of a gas-liquid critical point, although it has been claimed (17) that the general idea ofthe 'critical state' was arrived at independently by Mendeleeff in 1861. Later, in 1879 and 1880, Hannay and Hogarth

(18-20) published the first account of the enhanced solvating properties of supercritica1 fluids.

Although, the solvent properties of supercritical fluid are well known, the extraction potential of these fluids remained unrecognized for many years, although the phenomena of 'retrograde condensation' of solutes from compressed gas solutions was recognized very early in the field of petroleum engineering.(21) The solvent properties of liquified gases were also explored throughout the 1940s and 50s, and the pioneering

6 research ofFrancis resulted in the compilation of an extensive collection oftemary phase diagrams for liquid C02 with organic and inorganic compounds as solutes. In these studies, Francis estimated the solubilities of 261 compounds in near-critical C02. (22)

However, it was not until the appearance of a key patent by Zosel (23) that the potential of supercritical fluid extraction as a processing technique became apparent. Since this time, there has been a steady growth in the number of applications of supercritical fluid extraction in the chemical engineering field.

Analytical SFE differs very much from preparative-scale SFE in that the quantities of solutes dissolved in the supercritical fluid are quite low compared to levels normally encountered in preparative SFE. In addition, the apparatus used to perform the extractions is miniaturized compared to any preparative-scale extractor. This, in part, is due to the frequent coupling of analytical SFE with chromatographic instrumentation in what has become known as 'on-line SFE.' By contrast, SFE may also be used independently of any other analytical technique, and in this 'off-line' mode, may approach a scale that is equivalent to bench-scale preparative units. (24)

From a historical perspective, it is somewhat difficult to precisely identify a specific study or individual that can be credited with inventing analytical SFE. This is partly due to the simultaneous development of SFE in several technical disciplines, as well as the lack of definition as to what really constitutes 'analytical SFE'. However, there is little doubt that the efforts of Stahl and Schilz in 1976 (25) to combine SFE with thin-layer chromatography demonstrated the considerable potential of the technique for analytical studies.

7 In the 1980's much progress in supercritical fluid technology occurred. For example, supercritical fluids have found widespread use in extractions (l6~ 19), chromatography (20~23), chemical reaction process (24,25) and oil recovery (26). Most recently, they have been used as a solvent for carrying out enzyme based reactions. (21'.

Unfortunately, although supercritical fluids are used effectively in myriad areas, there is still a lack of detailed understanding of fundamental processes that govern these peculiar solvents.

In an effort to overcome this weakness, significant efforts have been devoted to determining the fundamental aspects of solute-solute, solute-fluid, and solute-cosolvent interactions in supercritical fluids and supercritical processes (28-58). Some of these efforts have used optical spectroscopy (30,47,56,57) and others have used chromatographic methods as a tool to probe the aforementioned interactions. Many of these experimental studies have been advanced by theoretical calculations and modeling.

(48-55) A general conclusion from these studies is that there is local density augmentation (ie. solvent clustering) about the solute near the critical point. In addition, it has been suggested that there are enhanced solute-solute interactions near the critical point. (44,56,57)

In summary, supercritical fluid extraction is a sample preparation technique in which a sample is made ready to be analyzed by an analytical instrument. In our case,

GC was used to analyze the extracts from SFE with different detectors, namely flame ionization and mass spectrometery.

8 Gas Chromatography (GC)

Chromatography was first employed by Ramsey in 1905 to separate mixtures of gases and vapors.(58) In 1906, Tswett obtained discrete colored bands ofplant pigments on a chromatographic column, which he called chromatography.(59) The translation of chromatography literary means color writing. However, chromatography as we know it today has advanced tremendously and uses many kinds of detectors to detect the separation. In 1952, Martin and James introduced gas-liquid chromatography.(60-61) A schematic ofa GC is shown in Figure 1-3. Since then, there has been tremendous growth in this technique. One can say that besides HPLC, GC is one of the most widely used instruments for chromatography.

Chromatography is a physical method of separation in which the sample components to be separated are distributed between the two phases, stationary phase and a mobile phase. This definition sets two basic criteria for a chromatographic separation.

That is, there are two phases present, which are in contact with each other and that one of these phases is stationary while the other is moving. The chromatographic separation occurs as a result of repeated sorption-desorption events during the movement of the analytes. The separation is due to differences in the distribution ofthe individual sample components between the two phases. This distribution may be based on partitioning between the two phases or adsorption-desorption process.

In gas chromatography, the sample is volatilized and then carried by the mobile phase into the column where the separation process takes place. At the end ofthe

9 pressure regulator

oven detector data system colwnn

camer gas supply

Figure 1-3 - A schematic of a gas chromatograph

10 column, the analytes will emerge more or less separated by time. They are then detected and the detector signal is recorded. It is also possible for the separated analytes to merge directly into another instrwnent, for example, a mass spectrometer or in:frared spectrophotometer for further analysis. These types of set ups are called hyphenated techniques.

Compooeots ofa Gas Chromatograph

GC consists of three interlaced functions. 1. Sample introduction 2. Separation

3. Detection. These functions are carried out by the sample introduction system, a column and a detector. Along with them, a GC utilizes a cylinder of carrier gas, a controller that regulates the flow and pressure and a recorder to record the signal output.

The role of the sample introduction system or inlet is to vaporize the liquid sample and to allow the carrier gas to push the vapor in the form of a concentrated plug, onto the beginning ofthe column with a minimum time lapse.

The separation column contains the stationary phase, which can be either an adsorbent or a liquid distributed in the form of a thin film on the surface of a solid support. The solid support may consist of porous particles packed into the column tube in a packed column, but the liquid stationary phase may also be distributed on the inner wall of the column tubing in an open-tubular or a capillary column. In the case of the . capillary tube, the wall may be smooth, without having its physical characteristics changed or it may be modified by building up or depositing a porous layer on the original tube wall. This in turn is coated with the liquid phase and is called porous layer open­

11 tubular columns. The separation column is placed in a temperature-controlled oven

This is either maintained at a constant temperature during analysis, called isothermal analysis or its temperature is increased with time, called temperature programmed analysis.

The separated components emerge from the column along with the carrier gas and are detected by continuously monitoring some physical or chemical property of the effluent. A number ofdetectors are available to do this task. Some ofthe most common ones are the thennal conductivity and the flame ionization detectors. The thermal conductivity detector measures the differences in the thermal conductivity of the column effluent versus that of the pure gas. The flame ionization detector breaks down the sample components into ions in a hydrogen flame, which are collected to form signals.

Mass Spectrometry

Mass Spectrometry began when J. J. Thomson, in 1907, built a parabola mass spectroscope to support his work on positive ray analysis. (62) His student named F. W.

Aston used this technology of mass spectrometry to discover that many elements have naturally occurring stable isotopes. (63) As an organic chemist, he continued to use mass spectrometry to discover and characterize isotopes ofother elements. During the process, he got frustrated with the background ions that arose from the lubricants and other fluids associated with the vacuum system. A petroleum chemist pointed out that these types of background signals were related to structural characteristic of hydrocarbons and other organic molecules, which led to the invention oforganic mass spectrometry. (64-65) 12 Today, mass spectrometry is a micro-analytical technique requiring picomoles of sample to obtain characteristic information regarding the molecular weight and sometimes the structure of the analyte. (66) In all cases, some form of energy is transferred to the analyte molecules to induce ionization. In the classical technique of electron impact ionization, some of the initial molecular ions of the analyte "explode" into a variety offragment ions. These resulting fragments ofthe molecular ion along with the molecular ions produce a mass spectrum. In theory, the mass spectrum of each compound is unique and can be used as a chemical ''fingerprint'' to identify the analyte.

A summary ofthe entire process ofanalysis by classical mass spectrometry to the formation of a bar graph mass spectrum that is often seen in literature is illustrated in

Figure 1-4.

Here, M represents molecules of a pure compound in the gas phase. For analysis by classical electron ionization (EI) or chemical ionization (CI), the sample must have a vapor pressure greater than 10.2 mm Hg, because molecules of the sample must migrate by diffusion from the inlet system into the ionization chamber. Samples may be introduced into the mass spectrometer using a direct probe for pure solids or volatile liquids. Analytes purified by separation techniques such as GC, HPLC, Capillary

Electropherosis (CE) can enter the mass spectrometer as the separation takes place. As the neutral molecules randomly diffuse throughout the ion source, only a few hundreds of a percent ofthem are ionized. 13 M --t>

ad.serptlex ofeEess eJLeI'lY

Figure 1-4 - A conceptual scheme for vapor phase analysis by mass spectrometer.

14 The most common ionization process for gas phase analysis, EI, transfers energy to the neutral molecule in the vapor state, giving it sufficient energy to eject one of its own electrons and there by having a residual positive charge. This process can be dissipated through fragmentation ofcertain chemical bonds. Cleavage ofchemical bonds leads to the production of fragment ions whose mass equal to the sum of the atomic masses of the group of atoms retaining the positive charge during the fragmentation process. It is important to keep in mind that not all of the ~ ions decompose into fragment ions. For compounds producing a relatively stable ~, an intense molecular ion peak will be recorded because the ~ tends to resist fragmentation. Usually, however, most ofthe ~ ions decompose into fragment ions, and in these cases the mass spectrum contains only a small peak for the ~ ion. Various combinations ofthe above described processes are the basis ofthe chemical fmgerprint for a given compound.

The next step is to analyze all the ions in the ionization chamber according to the mass-to-charge ratio (mlz). Ions have an electrical charge that permits them to be controlled by various electric fields; they are separated by their mlz values in a mass analyzer. There are several types of mass analyzers: magnetic, transmission quadrupole, quadrupole ion trap, magnetic ion trap, and time-of-flight analyzer. Regardless of the analyzer used, the final resuh is the same. The ions are analyzed according to their abundance along mlz scale. During the process of data recording the data can be arranged in a bar-graph format.

15 As described in this chapter, SFE is one of the newer extraction technique for sample preparation. This method ofextraction has to be combined with an analyzer and a detector for further analysis. We chose to analyze our extracts from the supercritical fluid extraction by either GC or GCIMS.

16 Chapter 2

Introduction

In the last several decades, there have been tremendous advances in instrumental techniques, especially in chromatography, and spectroscopy. (67) Most instrumental techniques require extensive sample preparation prior to analysis. In common practice, an extraction is performed before the analyte can be placed into the instrument. To get to this point, however, most analysts are still using the old and time consuming methods of extraction such as solid-liquid extraction and soxhlet extraction. An ideal extraction method should be rapid, straight forward, inexpensive, yield quantitative recovery of the analytes without loss, yield a sample that is ready for analysis without further steps and generate little or no waste. Unfortunately, traditional extraction methods, such as liquid­ solid, liquid-liquid, and soxhlet extraction, (68-70) do not always meet these goals. These methods are often very lengthy, and involve several steps to prepare the sample for analysis. Hence, there has been the motivation for the development of Supercritical Fluid

Extraction (SFE). SFE is becoming an important tool in analytical science and has seen rapid development in the past decade. Reports in analytical SFE have been published since the mid 1980's (71). On and off-line techniques are in use and much work on SFE has been done by engineers on a larger technical scale. (72-74)

Supercritical fluids have been used in the extraction of several classes of analytes such as polyaromatic hydrocarbons (75-77), essential oils (78), natural products (79,80).

17 pesticide residues (81-86), hydrocarbons (87), synthesis (88,89), derivatization (90-93), metal extraction (94), aqueous samples. (95)

In order to understand how a supercritical fluid can be used for extractions, physiochemical properties that describe a chemical substance or mixture should be understood. For example, density, boiling point, and dielectric constant can all be used to characterize a particu1ar species or system as a solid, liquid, or gas. However, if a substance is heated and maintained above its critical temperature it becomes impossible to liquify it with pressure. (96) When pressure is applied to this system a single phase forms that exhibits unique physiochemical properties. (96-109) This single phase is termed as supercritical fluid. As the name implies, supercritical fluid is a substance that is above its critical temperature and pressure. Critical temperature (Tc) is the highest temperature at which a liquid phase may coexist in equilibrium with a separate vapor phase. The Critical

Point (C) is a point on a phase diagram where phase become indistinguishable.

Properties of a Phase Diagram

A phase diagram is used to describe the behavior of a substance at different temperature and pressures. This can be described visually through the description of a phase diagram ofa pure substance, shown in Figure 2-1. (110) If liquid (I) and a gas (g)

are in equilibrium and temperature and pressure are increased toward Tc, the liquid becomes less dense due to thermal expansion and the gas becomes more dense due to the pressure increase. At the critical point, C, the densities ofthe two phases become identical

18 Figure 2-1 - Phase Diagram for Carbon Dioxide

120 I I I For CO2 100 : Superc:rltlclll OuId I region T = 31DC I C I 80 CrtUclll Point I ______Pc = 73 atIn ,aiii Pc .; latin = 14.7pd II • SoBd Liquid t

20

0 -80 60

Temperature, DC

19 and there is no longer a distinction between the gas and the liquid; i.e., the substance becomes a 'supercritical fluid'. The critical point is characterized by its two coordinates on the diagram; the critical temperature Tc, and pressure Pc. The region of interest for

SFE is the region above Tc and Pc where densities, solubilities and other properties are intermediate between those oftypical gases and liquids.

The vapor pressure of the pure liquid is also critical to understand. There are misconceptions about relationship of vapor pressure, Pvap, of a pure liquid with temperature. An assumption that the ideal gas law governs the temperature dependence of vapor pressure and that the dependence is linear is fulse. In fact, when the temperature increases, the vapor pressure increases in an exponential manner. This can be further illustrated by examining the partitioning behavior along the gas/liquid line.

A vapor phase consisting ofN1 molecules at low temperature is considered. As the temperature of these molecules increase, the pressure increases in a linear fashion in accordance with the ideal gas law. However, as higher temperatures are approached, a larger fraction of molecules in the liquid phase acquire the necessary kinetic energy to escape the potential well of the liquid phase. This results in an increase in the number of molecules in the vapor phase by quantity N2. The total number of molecules in the vapor phase is now Nl + N2. The total increase in the pressure is now proportional to the sum of Nl + N2 as a result of the temperature increase. Based on this rationale, Pvap, increases in an exponential manner as higher temperatures are approached. This is also

20 demonstrated by the Clausius - Clapeyron equation (1), where the plot of In Pvap vs. Iff is linear.

InP =-AH + AS (1) Yap -ym -m RT R

When vapor is in equilibrium with solid, the temperature dependence of the pressure of vapor in the equilibrium with solid is very similar to that of the vapor pressure of the liquid, as described above. The only difference is that the enthalpy and entropy terms must be appropriate for sublimation, as in the following:

InPvap = -AHg + ASg (2) RT R

A plot of In Pvap vs. Iff for both the liquid - vapor equilibria on the same set of axes has an appearance ofFigure 2-2.

The straight line for the solid has both a more negative slope and a more positive intercept than that for the liquid. This must be true due to the following.

A Hsub > A Hvap & A Ssub > A Svap (3)

That is the enthalpy ofsublimation emplicitly includes the enthalpies ofboth fusion and vaporization, which are both greater then zero. Furthermore, the solid phase is more ordered than the liquid phase which account for the relative entropies.

21 ASsub

ASvap

In P ABnp

llquld

1011.41 1rr FIgm'e l-l - Clausius - Clapeyron plots for the vapor pressures ofthe sold and lquld phases ofa pure substance.

22 Because the magnitudes ofthe slope and intercept of the solid line must exceed those for the liquid line, the line must cross at some temperature T = T t. Above temperature T t , the solid will convert to liquid, and below Tt liquid will change to solid.

Now, the In Pvap vs. UT plots can be transJated to Pvap vs. T. First, In Pvap for the solid is less than In Pvap for the liquid; at large 1fT, Pvap for the solid is less than Pvap for the liquid at low temperature. Second, because the CJausius - CJapeyron plot for the solid has the steeper slope, the vapor pressure of the solid rises more rapidly with temperature than that for the liquid. Third, the plots ofPvap vs. T for solid and liquid are not linear, but rather have upward curvature. Lastly, these plots will cross each other at the temperature Tt. This crossover point is the discontinuity in the plot, at the triple point.

This is a resuh of the solid - vapor equilibrium and liquid - vapor equih'brium curves intersecting at a point on which both curves are lying simuhaneously. Therefore, all three phases coexist, consequently, this point is called a triple point.

Definition of Supercritical Fluid

Again, a supercritical fluid is a substance that is above its critical temperature and pressure. Every substance has a point in its gaseous state where, ifthe pressure is raised while the temperature is held constant above its critical temperature, it will exhibit properties ofboth gas and liquid. This means that, it is dense like a liquid, but like a gas, has low surface tension and transport properties. Its liquid like density reflects the solvating power; its diffusion coefficient is higher then liquid, which aids the transport of the analyte and results in shorter experiment duration and greater efficiency. Changing the

23 temperature and pressure within the supercritical state changes the density. Supercritical fluids posses dissolving powers similar to liquids. To illustrate how supercritical fluids compare to gases and liquids, Table 2-1 shows some of the more important physiochemical properties ofeach. The combined gas like properties ofmass transfer and liquid-like solvating characteristics of supercritical fluids, and along with other advantages descnbed earlier, has raised an interest in use ofSFE as an analytical technique. (111)

Selection of the Supercritical Fluid

The solvent strength of supercritical fluids approaches that of liquid solvents only as the density ofthe supercritical fluid is increased. The maximum solubility ofan organic compound is usually higher in a liquid solvent than in a supercritical fluid. Although, supercritical fluids do not have any advantages over liquid solvents in terms of solvating powers, other supercritical fluid characteristics demonstrate the potential for SFE to approach the idealized goals for analytical extraction. There are many substances used as supercritical fluids such as freons, nitrous oxide, ammonia, ethane, water, carbon dioxide, etc. with carbon dioxide being the most commonly used fluid. Table 2-2 shows various supercritical fluids, which have been used in performing SFE and SFC and their critical properties. Fluids with various polarities and solvating capabilities are available, providing one with variety ofchoices ofthe fluid for SFE.

24 Table 2-1 - General Properties of Supercritical Fluids vs. Gases and Liquids

Gas SF Liquid

3 3 Density (g/cm ) 10- 0.1-1 1

Diff. Coef(cm2/s) 10-1 10-3 -10-4 < 10-5

Viscosity (g/cm *s ) 10-4 10-3 -10-4 10-2

25 Table 2-2 - Physical parameters ofcommonly used supereritieal fluids

Tc (>C) Tb eC) Pc (atm) pc (g em,3)

CO2 31.3 -78.3 72.9 0.47

N20 36.5 -88.3 72.5 0.45

NH3 132.5 -33.2 112.5 0.24

Ethane 32.2 -88.4 48.2 0.20

Propane 96.8 -46.9 42.4 0.22

Butane 152.0 -0.4 37.5 0.23

CChF2 111.5 -29.6 40.2 0.56

CHCJF2 96.1 -40.6 49.1 0.52

H2O 374.2 100.2 218.3

26 Nevertheless, the choice frequently depends on practical considerations. Many of the listed fluids would not be suitable for practical extractions due to their unfavorable physical properties, costs, or reactivities. For example, ethylene, which exhibits a subambient critical temperature, has been widely investigated in the laboratory as an extractant.(112) However, its flammability limits its application in many analytical problems. Conversely, most polar fluids have high critical temperatures, which can prove destructive to both the analyte and the extraction system.(112) The isoelectronic analogue of CO2, N20 has been shown to be a useful extracting fluid; however, it exhibits a high reactivity toward many compounds and can cause dangerous physiological effects. Other fluids like fluoroform (HCF)), are unique in their ability to solubilize basic solutes through intermolecular hydrogen bonding in the supercritical fluid state, but the high cost and legal difficulties of the fluid limit its use for SFE.(I13) As a result, the majority of SFE investigations have used supercritical CO2 because of its availability, but low toxicity and cost, its reasonable critical parameters, operator safety and its ability to solvate a broad range oflow and moderate polarity organics and minimal environmental impact.

In addition, supercritical CO2 has Lewis base characteristics, induced dipole interactions that allow it to solvate numerous compounds ranging in polarity from non­ polar to moderately polar. Furthermore, CO2 provides an extraction environment free from molecular oxygen, thereby limiting potential oxidation ofthe extracted solutes. (112)

27 Principles of Extraction

There are three interrelated factors that influence the extraction recovery of a given extraction method: solubility, diffusion, and matrix.

1. solubility - ofthe solute in the solvent

2. matrix - sample containing the analyte ofinterest, whether solid or liquid.

3. diffusion - ability of the solvent to diffuse through the matrix in order to extract the

analyte(s).

A triangle that interconnects these three influential factors can be drawn.

Solubility ~ Matrix Diffusion

As SFE is one of the extraction methods, this rationale can be applied to supercritical fluid extractions. That is, for an extraction to be successful, the solute must be fairly soluble in the supercritical fluid. This factor is very important at the beginning of the extraction, when extraction is occurring at a higher rate.

The solute must be transported sufficiently rapidly by diffusion from the interior of the matrix in which it is contained. The diffusion process may be normal diffusion of the solute, or it may involve diffusion of the supercritical fluid into the matrix and perhaps subsequent replacement of solute by fluid molecules on surface sites. Often, the precise process will not be known, but fortunately it can be successfully modeled as diffusion and

28 given an effective di:tfusion coefficient. Therefore, in the discussion below, di:tfusion is used to describe all possible processes for transport of the solute out of the matrix. The time scale for di:tfusion depends on the di:tfusion coefficient and shape and dimensions of the matrix or matrix particles.

The third factor is that ofthe matrix (other than its effect on di:tfusion). Although, in many cases SFE will extract all ofa particular compound in a sample, in some cases not all of a compound is extractable, the rest is locked into the structure ofthe matrix, or too strongly bound to its surface.

The practice of SFE

As described in the previous section, carbon dioxide is a common supercritical fluid of choice. In general, when using carbon dioxide as a solvent, at a constant temperature, extraction at a lower pressure will favor less polar analytes, and extraction at higher pressure will favor higher molecular weight analytes.(71) This characteristic allows an extraction to be optimized for a particular compound class by simply changing the pressure and temperature. The effect of pressure and temperature is described in more detail in the later section, variables affecting SFE - temperature and pressure.

Nevertheless, poor recoveries associated with certain analyte/matrix combinations are often seen, and indicate that a suitable supercritical fluid must not only be able to solvate analytes of interest, but must also possess properties that allow it to interact with the analyte and the matrix to efficiently partition the analyte into the bulk supercritical fluid.

(71) For fairly polar analytes, however, one would think to use fluids with higher solvent

29 strength, but use of these fluids are limited by practical difficulties as described in the earlier section. Due to these difficulties, often, the extraction ofthe polar compounds are done using small amounts ofmodifiers such as methanol or water.

Effects of Modifiers

To improve the extraction recovery of an analyte with pure C02, modified fluids have been used to increase extraction efficiencies, Modifiers can either increase the solvation power of a supercritical fluid by interacting with the analyte and thus increase the solubility ofthe target analyte or interact with active sites on the sample matrix:, which can help CO2 to efficiently extract the analyte. (114.115) The most common modifier used in SFE is methanol because of its high solvent polarity, which can greatly increases the polarity of CO2• However, several other modifiers such as water, organic amines and acids, aromatic compounds, and other organic solvents are used as well Usually the usage of modifier improves the extraction efficiency. However, the effect of modifier depends on kind of modifier used, target analyte, and sample matrix. There are several modifier mechanisms that are postulated.

1. Interaction ofpolar modifiers with the active sites ofthe matrix.

2. Solute-modifier interactions that form stable species which preferentially

interact with mobile phase.

3. Short range cluster formation between the modifier and the mobile phase.

4. Decrease in the interfacial tension between the mobile phase and the stationary

phase.

5. Solubility ofthe analyte in the extraction m.ectia. (115)

30 6. Active sites are covered and prevents readsorption or partitioning of the

analyte back onto the matrix active sites. (116)

7. Interaction with the analyte/matrix complex and lower the activation energy

barrier ofdesorption. (117)

Recently, a different approach has been used to increase the extraction efficiency using pure CO2. That is, raising the extraction temperature while using pure C02. As described later in the chapter, at higher pressure and temperature, the solvent strength of the carbon dioxide is increased. This helps to increase the extraction recovery. However, when extraction temperatures are in the neighborhood of the vapor pressure of the compound of interest, the extraction recovery is significantly increased. This can be attnbuted to the vaporization of the compound at the temperature and pressure, which increases its mass transport of it. In another words, the solubility of the analyte is influenced not only by the density ofthe C02, but also by the vapor pressure ofthe target analyte. And solubility of the compound of interest is not significantly influenced by the density ofCO2 compared to the vapor pressure ofthe analyte.

Instrumentation

A basic schematic of an SFE instrument is shown in Figure 2-3 via schematic diagram. There are several parts ofthe SFE system that facilitate the extraction.

31 pump

cooled pumphead -,--".. ater bath L...--4---lo,--I '-----+-----' fluid system collection vial extraction vessel

Figure 2- 3 - A schematic diagram of an ofT-line sup ercritical fluid extraction apparatus.

32 These include a source offluid, a fluid delivery module, an extraction cell, a back pressure

regulating device and a collector for trapping the extract after supercritical fluid

extraction.

A pump is used to supply a known pressure ofthe extraction fluid to the extraction

vessel. which is placed in a heater to maintain the vessel at a constant temperature above

the critical temperature. During the extraction, the soluble analytes are partitioned. from

the bulk sample matrix into the supercritical fluid, then swept through a flow restrictor

into a collection device containing a small amount oforganic solvent, which is normally at

room temperature. The fluids used are usually gases at room temperature and pressure

and are vented from the collection vial while the analytes are retained. As the fluid passes through the restrictor, the fluid cools and expands to a gas phase at atmospheric pressure.

(118,119) The restrictor is either fixed or variable in diameter and is required to allow

fluid flow while allowing supercritical conditions to be maintained in the extraction celL

SFE can be performed in either a dynamic or static mode. For dynamic SFE, the

supercritical fluid is constantly mnning through the extraction cell, and a flow restrictor is

used to maintain pressure in the extraction vessel and allow the supercritical fluid to

depressurize into the collection vessel In this mode, extraction efficiencies are greater,

equilibration time is :taster; however, more fluid is required. Static SFE is performed by

pressurizing the cell and extracting the sample with no additional flow of supercritical

fluid. After a set

33 period of time, a valve is opened to allow the analytes to be swept into the collection vessel In this mode, opposite to dynamic mode, the extraction efficiencies are lower, equilibration is slower; however, not as much solvent is needed.

Details about each of the component that make up the SFE system are given below.

Source of fluid and Pumps

The fluid is usually supplied in high-pressure gas cylinders, which are equipped with inductor tubes, ifthe fluid is to be pumped into the SFE apparatus. This permits fluid delivery devices to operate more efficiently by assuring adequate liquification of gas in the pump cylinder or head without the need for external circulating cooling baths. In some cases, the fluid tank pressure has been found to be sufficient for performing SFE of trace levels oftoxicants from various sample matrices. (118) There are plenty of fluid delivery devices ranging from high pressure diaphragm compressors, to gas booster pumps, or reciprocating piston pumps, and syringe pumps. Each device has its own merits, but there are some general principles worth considering in selecting the fluid delivery module.

Many of the pumps used for delivering the fluid in analytical SFE are modified high performance liquid chromatography (HPLC) pumps and require an external cooling source to assure liquification of the fluid. Such cooling is critical to the performance of plunger based pumps to avoid cavitation due to the introduction of a two phase fluid mixture at the pump head. Syringe delivery pumps that are used for both SFE and SFC, also require a source ofcoolant for effective operation.

34 Extraction Vessels

There are many types of extraction cells used for the many manufactured instrument. However, the basic utility is same. Below is the description ofthe extraction cell used in our experiments. They are vessels that are compatible with the ISCO extractors (Lincoln, NE).

The sample cartridge consists of a metal body, two filter frits, and upper and lower endcaps as can be seen Figure 2-4. (121) The filter frits drop into the endcaps, which are then screwed onto the metal body, which contains the sample. Interchangeable cartridges are available with 0.5 mL, 2.5 mL and 10.0 mL interval volumes. The outside diameter of the filter frit is comprised of an o-ring, which forms a seal when the endcaps are screwed onto the sample cartridge. The filter frit diameter ring varies depending on the size ofthe cartridge.

The 10 m1 cartridge uses the filter frit with a diameter of 5/8" while the 2.5 mL and 0.5 mL cartridges use filter frits with 3/8" diameter.

Restrictors

Devices for regulating the extraction pressure on the extractor cell include narrow capillary tubing, back pressure regulators, or micro metering valves having adjustable flow orifices. The selection of the device is governed by fluid flow rate desired through the extraction cell and upstream pressure desired in the extractor. The use of capillary tubing as a rate limiting outlet requires that the analyst adjust the flow rate through the 35 ~ @

/ ,10.0 cartridge / /body cartridge filter hit b~. ® 5/8 inch diameter Cartridge filter /@(0.5mlcartridge fiter element frit 318 inch body diameter filter element " ~/ ® ~ ~

Figure 2- 4 - A schematic: of the ISeQ extraction vessel (adopted from the ISCQ manual, referente 121 )

36 extraction cell by varying the length or internal diameter of the capillary tubing. Such a procedure is difficuh compared to using a micro metering valve or a accurate back­ pressure regulator in conjunction with a precise fluid delivery system. (122) Another viable option is to use a micrometering valve for flow control into the extraction cell and a back-pressure regulator at the exit ofthe cell. The primary functions of an SFE restrictor or extraction pressure regulator are to control flow or pressure ofthe SFE solvent, usually

CO2 and to efficiently deposit the extracted analytes in a collection solvent or trap. Linear and variable restrictors are commonly used.

1. Linear restrictor: This type of restrictor is composed of a simple length of capillary

tubing that limits the flow of CO2 as it depressurizes to the ambient pressure of the

collection trap. Wrth a linear restrictor, the pump must maintain a constant set

pressure while the restrictor inner diameter and length determine the flow rate. Fused

silica capillary, typically 0.375 mm outer diameter and 20 to 50 J.UD inner diameter has

been widely used for restrictors in SFE as well as SFC due to its low cost and

availability. One drawback is that the fused silica becomes brittle and easily fractures

after exposure to carbon dioxide modified with polar solvents such as methanol.

Furthermore, temperature and pressure drops within an unheated restrictor at lower

flow rates tend to cause plugging when samples contain a large amount of extractable

materi.a4 because all the extracts are not flushed out into the collection vial. Plugging

problems have prompted development of various methods to counteract the Joule­

Thompson cooling ofexpanding CO2 by heating the restrictor.

37 There are two types oflinear restrictors that are commonly used.

A Fused silica capillary: Fused silica capillary tubing makes inexpensive restrictors that work well for many non-modifier SFE applications. Usually, 30-35 cm lengths of 50 f.Ul1 inner diameter capillary are used to establish flow rates of 1 - 2.5 mL/min at pressures under 7500 psi. When the system pressure is between 7500 and 10,000 ps~ a 30 em length of 40 f.Ul1 inner diameter capillary gives a suitable flow rate of 1 - 1.5 mL/min.

Fused silica can however, be limited with use of certain modifiers, as it can react with alcohol groups on the silica

B. Stainless steel capillary: These types of restrictors are appropriate for use with all

modifiers. They are generally available in nominal flow rates of 1, 2, 10, and

20mLImin. at 5000 psi.

2. Heated variable restrictor: this type of restrictor contains a temperature regulated

"cover" over the restrictor that allows the analyst to control the temperature of the restrictor to prevent clogging. Therefore, the name heated restrictor. Variable restrictor refers to its variable diameter. In another words, an analyst can manipulate the diameter ofthe restrictor in order to control the flow-rate.

Analyte collection systems in SFE

Restrictor plugging and poor analyte collection are two problems repeatedly spoken of that limit the reliability of SFE. Restrictor plugging may result from depressurization of the extraction fluid across a length of tubing, causing deposition of

38 analytes or matrix components on the restrictor walls, or to the frosting of the restrictor tip because of the Joule-Thomson cooling of the expanding CO2. Samples containing water are especially troublesome because of the low solubility of water in carbon dioxide and ice accumulation at temperature of 0 °c or lower. Heating of the restrictor or the collection vessel is sometimes used, but at the risk of poorer analyte collection. The need to isolate the extract after SFE has fostered a number of ingenious collection schemes.

These methods of analyte collection include cryogenic trapping in an empty container, deposition in a liquid solvent, or retention on a solid surface. These methods are described below.

1. Cxyogenic tnmping in an empty container: One ofthe first collection methods used for SFE was cryogenic trapping. (123) The basis of this method is to use very low temperatures to decrease the volatility of analytes. However, the results obtained were not especially promising. Burford et al. extracted polycyclic aromatic hydrocarbons

(PAHs) from XAD-2 resins and collected them in 100 ml volumetric flask. (124) When the flask was cooled to OoC but left open to the surrounding air, recoveries were less then

8%. Upon cooling to liquid nitrogen temperatures and sealing the flask, leaving only a small vent tube for exiting carbon dioxide, recoveries improved to about 80%. The increased recoveries were ascribed to the decrease in C02 aerosol formation that was purging the analytes from the flask. In another study by the same author, PAHs were collected in empty vials that were not cooled preceding the usage. Even then the analyte recoveries did not exceed 40%, although the vials were rapidly cooled by expanding CO2•

Overall, cryogenic trapping in an empty container is not practicable unless the analytes are

39 extremely nonvolatile and the collection vessel is cooled with liquid nitrogen before use, and therefore is not commonly used.

2. Deposition in a liquid solvent: One of the simplest collection methods that is found to produce high yields, is to place the tip of the restrictor directly into a few milliliters of liquid solvent. However, there are several problems associated with this technique. First, exiting CO2 can cause aerosol formation and subsequent purging and evaporation of the solvent and analytes. (125) The collection solvent can also spatter due to the release of the high pressure. This can be reduced by keeping the supercritical fluid flow rate low, generally less then hnl/min., and by cooling the collection solvent. Small volumes of solvent must sometimes be added as collection is proceeding to keep an adequate volume in the collection vial. Second, the analytes may not efficiently partition from the extraction fluid into the collection solvent. Solvent identity, volume or height of the collection vial, and temperature were found to be important parameters. (126) A greater solvent height in the collection vial was shown to be more important than volume, because the solvent analyte interaction time is longer before the gaseous CO2 escapes the vial. Langenfeld et al. used very volatile compounds for an experiment that showed that the collection efficiency was increased by 11 % when 2.5 mL of solvent volume was 22 mm high compared to 8 mm high. Third, heating the restrictor may be necessary to avert plugging.

The restrictor can be placed inside a stainless steel tube and wrapped with heating tape to a few centimeters short ofthe tip or placed inside an insulating aluminum block. Finally, yields are highest when the collection solvent was allowed to cool form the expanding

40 CO2to temperature as low as -40 °c. Cooling increased the solvent viscosity, decreasing the size ofthe CO2 bubbles, and reducing aerosol fonnation.

3. Retention on a solid surface: The first class ofsolid surfaces used an inert material such as stainless steel beads or silanized glass beads. (127-131) Analytes are trapped by deposition on the cryogenically cooled surface. The material is then rinsed with a few milliliters of solvent to recover the analytes. Glass beads were capable of trapping

hydrocarbons C IO to C40 with 75 to 95% yields at a temperature of 45°C. Trapping was not affected by increasing the flow rate to 5 mL/min. However, volatile analytes were recovered at only 10% yield from stainless steel beads following an extraction with C02.

The second class of solid surfaces utilizes a sorbent material such as silica, octadecyl siloxane (ODS), or other chromatographic stationary phase material. (128-132)

These traps utilize cryogenic cooling and analyte adsorption to specific sites on the surface. The traps can then be rinsed, potentially selectively for the analytes of interest, by appropriate choice ofthe elution solvent. A wide variety ofsorbents are available for use.

Both classes of traps work well for extractions with C~. However, when modifiers are used, the analytes may be rinsed from the trap by the modifier as the extraction proceeds. By elevating the trap temperature near the boiling point of the solvent, the solvent can be evaporated while the analytes are adsorbed. However, heating the sorbent eliminates cryogenic cooling, making it more difficult to efficiently trap volatile analytes.

41 In summary, the collection method appropriate for a given application is dependent on the analyte volatility, the need for a heated restrictor. the presence ofcosolvents in the extraction fluid, and the possibility of a selective extract by varying the solid surface.

Regardless ofthe method chosen, prudent consideration of the parameters involved, such as solvent or solid surface identity and trapping temperature, is essential so that poor collection efficiency does not Iimit the extraction results. (133-135)

Factors effecting the extraction recovery of SFE

In our experiments, effects of temperature, pressure and time of extraction for various analytes are shown. Our goal was to assess the utility of the spiked samples as standards and also to understand the relationship of the fi1ctors affecting the extraction efficiency. So, in order to simulate the role of the native matrix, in our experiments, the matrices were prepared by spiking the analyte. The spiking procedure may sound easy and straightforward, but there are several different procedures to do this task.

Spiking procedures

1. Fresh mike: The sample is placed into an extraction cell and the spiking analyte is injected onto the top of the sample so that the spiked analytes must be eluted through the entire sample during SFE. Once the analytes are spiked onto the matrix, the extraction cell is closed immediately to prevent the loss ofvolatile compounds.

2. Aged mike: This procedure is same as a fresh spike, except that once the analytes are spiked, the top ofthe extraction cell is left open for a given period oftime.

42 3. Suspended spike: Here the sample is placed in a container containing a solution of the spiked analyte. This mixture is then thoroughly mixed and left open to evaporate the solvent for a given period of time. Once the solvent is evaporated the sample is transferred into an extraction cell for extraction.

4. Supercritical §Pike: This method was developed by the Burford et al. in an attempt to improve the spiking process. Here the sample is placed in an extraction cell, and then the analyte is injected into the center ofthe sample. The cell is sealed immediately and placed into SFE. The cell is pressurized for a certain period of time followed by a dynamic extraction.

Burford et al. used PAHs to compare the extraction rates of spiked vs. native analytes from heterogeneous environmental sample. It was shown in their study that spiking the sample did not change the native P AH extraction rates based on the fact that the extraction curves for the native P AHs were the same for both spiked and unspiked samples.

Experimental Variables Affecting SFE

Several experimental variables must be considered and optimized for SFE to be successful including the choice of supercritica1 fluid, pressure and temperature, extraction time, sample size, the method used to collect the extracted analytes, and the equipment needed.

(136-140)

43 Sample size

Sample size is must be taken into consideration. Although SFE has been used to extract samples size ranging from less then one gram to hundreds of grams, samples less then 10 grams have been most commonly used. Large samples require larger amounts of supercritical fluids for quantitative extraction, which can make quantitative trapping ofthe extracts more difficult. Since, SFE is most often conducted using fluids that are gases at room temperature, the success ofthe trapping method depends on recovery ofthe analytes from the expanded gas flow upon depressurization. For example, 1 mL/min flow of supercritical C02 will result in a gas flow of- 500 mlImin. So as a rule, collection ofthe analytes is better at lower flow rates, up to ImLl min, especially when the analyte is volatile. At this rate, assuming that a typical sample has a void volume of- 30%, passing

10 void volumes of extraction fluid through 1, 10, 100 gram of samples would require about 3, 30, 300 min, respectively. These flow considerations indicate that to reduce extraction times, SFE of smaller samples is preferred unless larger samples are necessary to ensure sample homogeneity or to obtain sufficient sensitivity for trace analytes. (71)

Temperature and Pressure

Selecting the pressure and temperature conditions for SFE is of critical importance. Because SFE was developed by engineers for process engineering, very often the SFE conditions are based on pressure and temperature conditions where the target analytes have their highest solubility in the supercritical fluid. This condition can be approximated if the solubility parameters of the analytes are known and if certain correlations are used. Such a relation has been proposed by Giddings (71)

44 o == 1.25 pc1l2 (p/pl) (4)

where, 0 == Hildebrand solubility parameter

Pc critical pressure, p = density ofSF,

p1 =density offluid in liquid state

The effect oftemperature and pressure on the Hilderbrand solubility parameter for supercritical carbon dioxide is shown in Figure 2-5. These correlations are very useful when the target analytes represent a large percentage ofthe bulk sample, because they are mostly concerned with maximum solubility. However, they become less useful when the target analyte is present at trace levels. For these samples, dissolving the maximum amount ofthe target analyte in supercritical fluid is not a concern, because the analyte only needs to be soluble enough in the fluid so it can be transported out of the extraction vessel.

Again, solubility data and correlations provide very useful information for determining the initial SFE conditions. However, determining optimal conditions for trace level components has been mostly empirical for many reasons. First, analytical SFE often involves the recovery of a complex variety of analytes, instead of one target analyte. In these types ofcases, extraction must be optimized for several compounds, which can

45 10.0,.....=:------..., Pressure (atm)

2180

~ 730 1"""1 ('I")-e ~ 440 !j ---0 ... • B • 290 CII • i • iii 2.0 • r:t. • • i • 1 • G'!I • • • 1.0 • • • 100 :-TC 0.7 • • 80 • • O.5~·----~-----~--~------~----~--~ 20 10 100

Temperature (OC)

Figure 2-5 - meet or temperature and presSllft on the IOldebnmd solubUlty parameter ror supercritical. cmoD dloDde (adapted from rererence 71)

46 complicate the prediction of the optimal extraction condition. Second, solubility considerations address only part ofthe extraction problem. Due to the dependence of the extraction of an analyte on its distribution between the supercritical fluid and the sorptive sites on the sample matrix, the ability of the supercritical fluid to compete with the analytes for the sorptive sites may be more important than solubility considerations for detenninjng optimal extraction conditions. The importance of the competition for active sites between the analytes and the supercritical fluid has received very little attention in the development of SFE technique. Understanding the mechanism. and interaction among the matrix surface, the analytes, the supercritical fluid, as well as the kinetics that control these interactions, is very important to the design ofthe SFE methods.

Sample matrix effects in SFE

The nature ofthe sample matrix can have a profound effect on the results that are obtained with SFE. Unfortunately, knowledge of analyte solubilities in supercritical fluids does not always allow a prediction to be made as to the effectiveness of SFE for extracting analytes from a particular matrix. (140) Extraction of real sample matrices, such as, soils or biological tissues, should be carried out experimentally, rather than depending on theory or results obtained on neat analytes. Snyder, et al. have shown in their studies that the geometric size of the matrix particles can influence the speed and completeness with which a SFE can be conducted. (141)

47 Physical matrix effects

The physical morphology ofthe substrate undergoing SFE can have a pronounced influence on the efficiency of the extraction and the rate at which it is conducted. In general, the smaller the particle size of the substrate, the more rapid and complete the extraction will be. This effect is largely due to the shorter internal diffusional path lengths over which the extracted solutes must travel to reach the bulk fluid phase. (142)

Chemical changes in the sample matrix

The chemical composition of the sample matrix can have either an enhancing or retarding effect on the results that are obtained with SFE. One of the major parameters that influence the composition ofthe supercritical fluid extract is the moisture level in the sample matrix. A good example for this effect is shown by the work of Roselius et at. It is shown that aroma oils from tobacco products are preferentially removed in the absence of moisture, while the presence of water is required for the extraction of alkaloids from the tobacco matrix. (143)

Impact of matrix on extraction kinetics

The rate of removal of a solute from a matrix using a SFE is a function of its solubility in the fluid media and the rate of mass transport ofthe solute out of the sample matrix. Rate limiting kinetics can adversely impact on the rapid extraction of an analyte despite its favorable solubility characteristics in the supercritical fluid. This situation often

48 occW'S when the target analyte must be isolated from a complex sample matrix, such as a sorbent or a soil (144)

Pawliszyn has developed a theoretical model for the extraction process. (145,146)

He suggests that ifthe rate-limiting step of SFE is defined, then the optimization process would require modification in the appropriate extraction parameters to reduce these effects. And ifthe sample characteristics are known, then the parameters can be adjusted before starting the experiment.

This model assumes that a matrix particle consists of an organic layer on an impermeable core, and the analyte is adsorbed onto the core surtace. The extraction process can then be modeled by considering several basic steps. That is, to collect the analyte, the compound must first be desorbed from the surtace, then it must difiUse through the organic part ofthe matrix to reach the matrix fluid interface. At this point, the analyte must be solvated by the supercritical fluid, and then it must difiUse through the static fluid present in the porous matrix to reach the flowing bulk of fluid for transport through the interstitial pores ofthe matrix and eventually exit from the vesseL Pawliszyn designed a kinetic model based on the equations developed by chromatographers and engineers to investigate mass transport through porous media. He used the relationship that is used in chromatography to describe the contnbutions of each of the mass transfer steps to the height equivalent to a theoretical plate (lffiTP). The approach of summation of the relevant individual components for the overall performance is used to develop a modeL This is described by the term H.

49 H hRK + hDC + hDP + hED + hLD (5)

The effect ofthe slow desorption kinetics on the elution profile can be described by

hRK=~ (6) (l+k)2(1 +ko) kd

where k= partition ratio at a given condition

kd = dissociation constant ofthe analyte- matrix complex

ko = ratio 0fintraparticuiate void volume to the interstitial void space, here it is expressed as

(7)

where Ei = intraparticuiate pores

Ee = interstitial porosity

J.le = interstitial linear velocity, where J.le is

J.le = u ( 1+ ko) (8)

where u = Lito = chromatographic linear velocity

L = length ofvessel

to = gas hold up time

The contribution ofthe analyte in the swollen solid part ofthe matrix is expressed by

(9)

where de = thickness ofthe matrix component permeable to the analyte

Dc = diffusion coefficient ofthe analyte in matrix

50 In porous matrices, analyte resists to transfer into the fluid. This is taken into account by

the following expression

hDp = e ( kO + k + k ko) de2J.le (10)

where e = tortuosity factor and

Dp = diffusion coefficient ofthe analyte in the material filling the pores

which in most cases is supercritical fluid, SF. Therefore, Dp= Df= diffusion coefficient of the analyte in the fluid

Also, in the flowing bulk ofthe fluid the analyte goes through

(1) Eddy diffusion

hED=2ADp (11)

where A = structural parameter & for spherical particle it is 1

(2) Longitudinal diffusion

hLD=YMDf (12)

where YM = obstruction factor

Ifthe rate-determining step is intraparticle diffusion, then the rate of extraction will be a function of the particle size of the sample matrix. It should be recognized that some matrices when exposed to supercritical fluid would swell, thereby facilitating the mass transport ofthe analyte from within a sample matrix. 51 Based on Pawliszyn's model the mass extracted versus time curve obtained for an unretained, well solvated analyte (1c=O), the elution profile is initially increasing with constant slope, indicating a steady flux of analyte from the vessel, then levels off exponentially.

In some cases, when a large amount of analyte is present in the matrix, saturation ofthe fluid by the solute can occur. When the amount ofthe analyte exceeds the solvation capacity ofthe small volume offluid present in the extraction vessel, then this will result in longer extraction time.

For SFE to be ofuse to the analytical chemist, it must be quantitative. In order to do this a procedure needs to be developed for each application on the basis of a good understanding of the extraction process. Part of this process involves understanding the characteristic kinetics of SFE. Extraction by supercritical fluid is never complete in finite time. It is relatively rapid initially, but there then follows a long tail in the curve of percentage extracted versus time, as shown in Figure 2-6. (147)

As discussed earlier, efficiency of an extraction and extraction curve can be influenced by three interrelated factors. 1. Solubility 2. Matrix 3. Diftbsion. For an extraction to be successful, the solute must be fairly soluble in supercritica1 fluid. The solute must be transported sufficiently rapidly by diftbsion from the interior of the matrix in which it is incorporated. Some authors have discussed the extraction controlled by

52 Supercrltical Fluid Extraction

100 r--~::::====-=====m::::;;-,

o

Time

Figure 2-6 - Theoretical C1U'Ye ofpercentage extraction versus time (adapted fi'om reference 148)

53 diffusion out ofthe matrix based on theoretical models for continuous extraction unlimited by solubility. (148)

For an extraction ofa matrix in a continuous flow of flui~ which is fast enough for the concentration of a particular solute to be well below its solubility limit, the rate­ determining process is, therefore, the rate of diffusion out ofthe matrix. Of course, most ofthe practical examples of extraction are complex, but it is found that simple models can account for the main feature and lead to methods oftreatment for the resuhs of SFE. For simple theoretical models we can assume an effective diffusion coefficient, D, and a particular geometry for the matrix and solve the appropriate differential equation (the

Fourier equation) with assumed boundary conditions. These conditions include the compounds to be initially uniformly distributed within the matrix and that as soon as extraction begins, the concentrations of compound at the matrix surface is zero

(corresponding to no solubility limitation).

The solutions ofthe Fourier equation for various geometries are given by CarIsaw and Jaeger (149) in the context of heat conduction (where the same equation applies) and also by Crank (ISO), who has translated Carlslaw and Jaeger's equations into diffusion notation. Let's divide the geometries into two categories. Those with sphere geometry are applied to extraction of spherical particles. And others include irregularly shaped particles. A solution for a sphere can be described as the "hot ball model" because of the analogy of the mathematical solutions. A solution for hot spherical object being dropped into cold water is explained by the following(151). If the mass of solute in the matrix is

54 1llo initially, and m after a given time, a plot ofln (mlmo) versus time bas the form given in

Figure 2-7. It is characterized by a relatively rapid fall on to a linear portion, which corresponds to an extraction 'tail'. The physical explanation if the form of the curve is that the initial portion is extraction, primarily out of the outer parts of the sphere, which establishes a smooth concentration profile across each particle, peaking at the center and falling to zero at the surface. When this bas happened, the extraction yield becomes an exponential decay. The curve is characterized by two parameters: a characteristic time, tc, and the intercept ofthe linear portion, -I, which bas value of-D.5 for a sphere. The slope of the linear portion is -lltc and the linear portion appears to begin at approximately 0.5 tc. tc is theoretically related to the effective diffusion coefficient out ofthe matrix, D, and the radius ofthe sphere, r, by the equation

(1)

Usually the effective value of D is not known, altough its order of magnitude can be estimated. For systems of interest to SFE, D will be between 1 for oils and 4 for solids orders of magnitude below this value. The above equation shows a squared dependence on r and rationalizes the common sense rule that for rapid extraction, matrix particle must be small. This may be achieved for solids by crushing or grinding and for liquids by coating on a finely divided substrate, spraying or mechanical agitation. For solid matrix particle with r ofthe order of 0.1 mm, typical values oftc are between 10 and 100 min.

55 --.

.ei

TIme

Figure 2-7 - Theoretical c1II"Ye for the hot-baD modeL (adaptedfrom reference 151)

56 Experimental curve for "hot-ball" SFE model differ from the theoretical curves in two respects. First, the intercept is greater. In general, the value of! is thought to depend on the particle shape and size distribution and also the distribution of solute within the matrix particles. For a model system the spheres of the same size, with uniform solute concentration, It is 0.5. For real systems, values of approximately 2 are common and prediction ofthe values is not really possible. Thus, usually values ofic: and I can only be obtained by experiment. A continuous dynamic extraction, followed by treatment of the data by the methods described, is therefore an important preliminary study in designing a routine quantitative analytical procedure. Secondly, the curve does not full as steeply from zero, and this is thought to be due to the effect ofsolubility limitation.

One can also comment on the effects of solubility limitation on continuous extraction. So far it has been assumed that the solubility of the solute in the SF is essentially infinite and diffusion out ofthe matrix has controlled the rate ofextraction. To illustrate the interaction between solubility and matrix diffusion considerations, extraction ofa solute from a stationary matrix in a cell by steady flow ofthe fluid is shown in Figure

2-8.

Here the flow rates are slow enough that, at the beginning of the extraction, the concentration of the solute in the fluid is an appreciable fraction of its solubility, which varies with pressure. In this figure, curve 1 is at the lowest pressure, curve 5 at the highest and curve 6 is that for infinite solubility... The effect of solubility limitation is to reduce the high rate at the beginning ofthe extraction and also to reduce the slope ofthe

57 '1'­ Figure 2-8 - TheoreUeal eurve for SFE, blelD.dInM lolvaUon ell'eets, for spherleal matrb: partleles (adapted rrom rererence 151)

58 linear portion, with the extent of these effects increasing as the pressure falls and the solubility decreases".(151)

When performing a quantitative extraction, methods are developed and validated based on recovery of sample from a spiked matrix. Many debate the use of either a homogeneous or heterogeneous sample matrix when developing these analytical methods.

(152)

While determining the extraction efficiency for a homogeneous matrix can be a valid method, many believe that only a heterogeneous matrix would provide a more accurate representation of the native form of an analyte composite. This is because in a homogeneous matrix analytes interact with a uniform system of active sites. This is contrary to the heterogeneous system in which many different active sites can interact with the component of interest. The latter is a much better representation of what is typically found in nature, especially when considering such matrices as soil samples, sludge material, etc. Generally, spiked analytes may still be less retained on a prepared matrix

(heterogeneous or homogeneous) when compare to those recovered from native samples.

The overall effect would be a possible overestimated of recovery efficiency in comparison to the native sample, regardless ofmatrix used. Yet even with this possibility, many still use SFE to study the interactions which occur between analyte, matrix and supercritical fluid, with or without modifier. ( 152)

59 Chapter 3

Optimization of Conditions for Trace Analysis in Supercritical Fluid Extraction

Goal

The objective of our experiments was to determine the effects of temperature,

pressure and time of extraction for various analytes. Combination of three

temperature and pressure were used to this study: Temperatures: 35°C, 70 DC, and

150°C and pressures: 1500 psi, 5000 psi, and 8000 psi. Also, three types of analytes

were used to see the effects of various analytes on the extraction efficiency. These

included eicosane (a non-polar analyte), a-naphthol (acidic polar analyte) and

propranolol (a basic polar analyte). The extraction efficiency of these analytes were

determined from two matrices (s.p.). These two matrices were octadecyl silica (a non­

polar matrix) and silica gel (a polar matrix). Our goal was to assess the utility of the

spiked samples as standards and also to understand the relationship of the factors

affecting the extraction efficiency. The matrices were prepared by spiking the analyte

in order to simulate the role of the native matrix. The resulting extracts were then

analyzed by Gas Chromatograph-Flame Ionization Detector (GCIFID).

Experimental Conditions

Off-line SFE was employed to extract analytes of different polarity. Two types of matrices were used in order to portray interactions, which appear in a heterogeneous

60 system. Silica gel represented a IXllar matrix and octadecyl silica (ODS) represented a non-lXllar sample matrix. Table 3-1 illustrates the characteristics of each analyte employed in this study.(153,154)

Reagents: The following reagents and chemicals were used: eicosane, a.-naphthol,

Propranolol, and silica gel ordered from Aldrich at Milwaukee, WI. Octadecyl silica was obtained from Whatman, Inc., Clifton, NJ. Ethanol was obtained from Pharmco,

Brookfield, CT, and iso-octane was obtained from Fisher (Fairlawn, NJ). Also, SFC grade C(h was bought from AGL Welding Supply, Co. at Clifton, NJ.

Sample preparation

A 1.5 gram aliquot of either silica gel or octadecyl silica, HPLC column packing, was spiked with 1 mL of 1 ppt (parts per thousand) solution of the analyte. The solution was made by weighing out 1 gram of solute on an analytical balance and dissolving it in a 1 mL of solvent. (The solution was usually made in aliquots of 10-15 milliliters). The solvent of choice for eicosane was iso-octane and for naphthol and propranolol the solvent was ethanol The spiked sample was left open to air dry or the solvent was evaporated in the air. Once it was evaporated, it was shaken manually with a spatula for

1-2 minutes to achieve homogeneity and then placed into an extraction cell.

Supercritical Fluid Extraction

An Isco model 100DM syringe pump was filled with SFC grade CO2 (AGL Welding

Supply Co., Inc.), which was then pressurized to 1500 ps~ 5000 psi or 8000 psi. 61 Table 3-1 - Characteristics ofthe Analytes Used.

Analyte Characteristics Eicosane Non-polar Alpha-naphthol Polar & Weakly Acidic Propanolol Polar & Basic

62 Approximately 1.5 grams of sample was placed inside an extraction cell that was equipped with an Isco Model SFX 2-10 extractor. The cell was then placed into the extractor and pressurized by opening the inlet valve and letting CO2 enter. Once the cell was pressurized, the outlet valve was opened to flush the extraction cell with SF CO2•

The flow rate of the SF, at 1 m1Jrnin, was controlled by ~ 25 cm long stainless steel capillary, normally used for high flow rates (10 m1Jmin restrictor). The extracted analytes were collected by placing the outlet end of the restrictor into a centrifuge tube containing - 2.5 mL ofsolvent (either ethanol or iso-octane). As the solvent evaporated, it was added to the collection vial to maintain the solvent between 1-2 mL. The extraction cell temperatures were regulated by the extractor at 35, 70, 150°C.

The extract was collected after 5 minutes, then keeping the same sample in the extractor; the solvent vial was replaced with a fresh solvent vial. Again the extract was collected for

5 more minutes. Addition of these two extracts gives the total extraction time of 10 minutes. In this manner, a fresh solvent vial was placed to obtain extraction results at 5,

10,20,30, and 60 minutes. Each extract was then transferred to a 2 mL volumetric flask and diluted to volume. This solution was then placed into a 2 mL GC autosampler vial and was analyzed by GCIFID. Below are the conditions at which the GCIFID was operated.

GC conditions

GC analysis was performed on Hewlett Packard 5890 gas chromatograph with flame ionization detector. 3 I.d ofthe sample was placed onto the column using a Hewlett

63 Packard 7673A auto sampler, which was controlled by Hewlett Packard 7673 controller.

A HP-l column was employed for analysis with the dimensions of 12 M X 0.25mm X

0.25 J.lIIl. Helium was used as the carrier gas at a constant pressure of 15 psi. The inlet temperature was kept at 250°C. The temperature program was started at 175 °c which was held for 1 minute. It was then ramped at 5 °C/min to a final temperature of210°C.

The detector temperature was set t0280 0c. The resuhing chromatographs were collected on a HP 3393A integrator. Each sample was injected six times for reproducibility. The standard deviation for these injections were about 10% or less. Each sample at a given condition was extracted twice. The average ofthese data was calculated and the resuhing data were used to plot the graphs. Extraction time vs. extraction recoveries were used to plot the graphs.

Resultsl Discussion All GC analyses were optimized to obtain the shortest run time. Figure

3-1 shows a typical chromatogram for an injection of a.-naphthol spiked onto silica gel and extracted at 150°C and 8000 psi for 10 minutes. After obtaining all the chromatograms at various temperature and pressure at various times, graphs were sketched to show the efficiency at a given temperature and pressure. For example, Figure

3-3 shows the extraction efficiency of extracting eicosane spiked from ODS at a temperature of35°C and pressures of 1500, 5000 and 8000 psi. These curves in most of the graphs are linear in the beginning and then levels off: This is to be expected as will be described later and shown in Figure 3-2.(157) There are also some curves that deviate from the expected profile. These are described below.

64 Figure 3-1 A typical chromatogram

Peak at RT 0.466 = solvent

Peak at RT 1.610 =a-naphthol or the analyte

65 Amount of analyte present

3

Time

Figure 3-2 - Theoretical dynamic extraction profile of an analyte from a solid matrix. (adapted from reference 157)

66 The purpose was to study the extent of interaction between the analyte, matrix, and supercritical fluid by evaluating sample recovery at different extraction temperatures and pressures. As discussed previously in chapter 2, the recoveries are better than anticipated due to the fact that the extraction rates of spiked samples exceed those of native sample matrices. Nevertheless, even the extraction procedure of a spiked sample will fall victim to the effects of varying temperature and pressure during the supercritical fluid extraction process.

Non-polar anaMe

Eicosane anglyte spiked onto ODS mIItrix

The structure ofeicosane is shown below.

eicosane

The recoveries of eicosane spiked onto ODS are illustrated in Figures 3-3a-c. In each of these Figures, temperature was held constant as the pressure was varied. The results shown in Figure 3-3a are at a temperature of3S °C. Here the extraction recoveries at 1S00 psi and SOOO psi show high solubility while the recovery at 8000 psi is lower, possibly arising from recovery losses due to carbon dioxide eluting very rapidly from the restrictor during collection. This can be explained by theorizing that at this temperature,

C(h is relatively non-polar. This can also be seen in Figure 2-S. The solubility parameter is higher at lower pressure and temperature. Based on this it can be said that.

67 as the pressure increases, so do the polarizing capabilities of C02. Therefore, the non­

polar matrix should retain a non-polar substance such as eicosane more strongly as the supercritical fluid increases in polarity.

Figure 3-3b depicts the resuhs of the recoveries of eicosane from ODS at a temperature of 70°C. In this figure the extraction is slower. At 5000 psi and 8000 psi the recoveries are similar while recoveries at 1500 psi are much lower due to the lower solubility parameter value, as seen in Figure 2-5, which leads to lower solubility of eicosane at that condition. Again, the solvent strength of the supercritical fluid is expected to increase, as do the pressures employed during the extraction, therefore decreasing the recoveries as the pressures increase. Figure 3-3b also shows deviations from the anticipated recovery prediction. The vapor pressure of eicosane at 8000 psi forces it to evaporate from the matrix. This can be explained by the fact that at higher temperature, the vapor pressure of a given substance is higher. Ibis will make recoveries at this pressure higher than expected. Otherwise, the solubility of the non-polar analyte in the supercritica1 fluid increases as the pressure decreases.

In Figure 3-3c, variations ofanalyte recoveries are again depicted. Again, at 150

°c, the solubility parameter in Figure 2-5 is much lower compared to the other temperature used. Therefure, at 8000 psi, a lower recovery ofthe eicosane from the non­ polar matrix is expected. As illustrated in Figure 3-3c, recoveries at 8000 psi and 5000 psi are higher then at 1500 psi This can be again be attributed to the vapor pressure of eicosane at this temperature.

68 Figure 3-3a-c: Eicosane on ODS (non-polar analyte on non-polar s.p.)

Constant Temperature

Efficiency at 'Mfc

1500 ____ 5000 .3.. -+­ .-.lr-8OOO

O.-----~---r------+-~ o 10 20 30 40 50 eo llme

Efflclency at 70'C 100

80 -,-'500 .-.-8000-~ I';it. : 20

o ro 20 ~ ~ ~ 80 Time

100

O~--__-----­ __-­ ______~ o 10 20 30 60 TIIM

69 However, the recovery at 8000 psi is lower than at 5000 psi. This is not the case and can perhaps be attributed to the loss ofthe analyte during collection.

Overal~ Figures 3-3a-c portray optimum extraction recovery conditions at 1500 psi and 35°C. At lower temperature and pressure, the density of carbon dioxide is low and therefore more suitable to extract non-polar compounds such as eicosane from a non­ polar matrix (ODS).

Eicosane anaiyte spiked on to silica gel matrix

The recoveries of eicosane spiked onto silica gel are shown in Figures 3-4a-c.

Here eicosane is expected to be more weakly bonded to silica than to ODS. In each of these figures, temperature was held constant as the pressure was varied. The results shown in Figure 3-4a are at a temperature of 35°C. As the solubility parameter states

(Figure 2-5), as pressure is increased, so do the polarizing capabilities ofC02. Usually, a polar matrix will not retain a non-polar substance, but, at lower extraction pressures, the supercritical fluid will have a greater capability for extracting the non-polar analyte.

Figure 3-4a illustrates high recoveries and rapid extractions regardless of the pressures used.

Figure 3-4b depicts the recoveries of eicosane from silica gel at a temperature of

70°C. Here, recoveries at 1500 and 5000 psi remain high, however, at 8000 psi recovery is significantly depressed. This again can be explained by Figure 2-5, that the solubility parameter is higher at this pressure, and by losses during trapping.

70 Figure 3-4a-c: Eicosane on Silica gel (non-polar analyte on polar s.p.)

Constant Temperature

El'llclency atWe

100 t' 80 ___5!XXl j : ...... -earo l '$. 8 1D 0 0 1D 40 60 TIme

Efficiency at lO'C

100

80

60 I 40 "I 20

0 0 20 40 60 Time

Efficiency at 1 SO·C

100

80 1-4-1500 I 60 1_5000· ...... 8000 I 40 "I 20

0 0 20 40 60 Time

71 In Figure 3-4c, variations ofanalyte recoveries are illustrated at an extraction temperature of 150°C. Based on the Hildebrand solubility parameter in Figure 2-5, carbon dioxide has a lower solubility parameter value at this temperature, which would lead us to believe that extraction recoveries would be lower at this temperature and indeed this is the case when compared to 35 and 70°C. Also, the recoveries at 5000 and 8000 psi are greater then at 1500 psi This can be attributed to the vapor pressure of eicosane, and the lower recoveries at 8000 psi compared to 5000 psi, can be due to the loss of the analyte during the collection because ofthe rapid elution ofcarbon dioxide.

When comparing the sample matrix for the recovery of eicosane, the polar silica gel was highly efficient (Figures 3-4a-c) in comparison to the non-polar ODS (Figures 3­

3a-c). This is expected because a non-polar substance such as eicosane would interact much stronger with a non-polar, ODS, matrix through constant hydrophobic interactions.

Weaker van der Waal interactions exist when a polar matrix is used.

Polar acidic anaiyte ~ a-Naphthol

a-Nqphthol spiked onto ODS ItUIIrix

The recoveries of a-naphthol spiked onto ODS are shown in Figures 3-5a-c. In each of these Figures, temperature was held constant as the pressure was varied. a-Naphthol is a polar and weakly acidic substance. The structure ofa-Naphthol is shown below.

72 OH

Naphthol

The resuhs shown in Figure 3-5a are at a temperature of35 °C. As pressure is incre~ so is the solubility parameter of CO2 (see figure 2-5). Therefore, extraction recoveries should be higher for polar analytes such as a-naphthol because the supercritical fluid is more capable of solvating the polar analytes as pressure is increased. Generally, in this figure it can be seen that kinetics are interesting and a-naphthol is soluble at all pressures.

Figure 3-5b depicts the results of the recoveries of a-naphthol from ODS at a temperature of 70 °C. Again, the solubility of the supercritical fluid is expected to increase as does the pressures employed during the extraction (see Figure 2-5). Figure 3­

5b exemplifies the relationship anticipated between increasing pressure and extraction efficiency for polar analytes. In this figure, recoveries at 8000 psi are highest followed by recoveries at 5000 psi And there is almost no recovery at 1500 psi In Figure 3-5c, variations of analyte recoveries are again depicted. At 1500 ps~ a increased recovery is seen due to the analyte vapor pressure. The efficiencies of5000 psi and 8000 psi are high and practically identical at the higher temperature (150 °C). 73 Figure 3-5a-c: a-Naphthol on ODS (acidic polar analyte on non-polar s.p.)

Constant Temperature

Efficiency at 35°C

100 80 1"""-1500 I 60 i_ 5OOO I 40 i-,lr-8000 ~ 20 0 0 20 40 80 Time

Efficiency at 70°C

100 ,..------., l;' 80 •...... -1500 60 1_5000 j 40 -,lr-8000 -;It. 20 O~~~_.--~-=~====~ o 20 40 60 Time

Efficiency at 150 ·c

100

80 -'-1500 80 _5000 I 40 ~80001 'ItIl 20

0 0 10 20 30 40 50 80 Time

74 In this case, vapor pressure of the analyte and slight solvation overcome hydrogen bonding to the matrix.

a-Naphthol spiked onto silica gel matrir

The recoveries ofa-naphthol spiked onto silica gel are illustrated in Figures 3-6a­ c. In each ofthese Figures, temperature was held constant as the pressure was varied. a­ naphthol is a polar and weakly acidic substance. The results shown in Figure 3-6a are at a temperature of 35°C. Looking at Figure 2-5, we can see that solvent strength of the carbon dioxide increases with the increase in pressure. Therefore, extraction recoveries should be higher for polar analytes such as a-naphthol as the pressure increases. Since the matrix in this case is also polar, both the matrix and supercritical fluid will compete for the a-naphthol Figure 3-6a illustrates an increase in recoveries as higher pressures are used and almost no recovery ofthe analyte at the low pressure of 1500 psi. It can be assumed that at 1500 psi, the supercritical fluid was not capable of solvating the a­ naphthol and breaking the hydrogen bonding and it therefore remained in the matrix. In this figure, it can also be seen that the analyte is soluble at 5000 psi and 8000 psi, but, the recoveries are lower due to hydrogen bonding on stationary phase.

Figure 3-6b depicts the results ofthe recoveries ofa-naphthol from silica gel at a temperature of70°C. Here, the relationship anticipated between increasing pressure and extraction efficiency for polar analytes exemplified. As in Figure 3-68, there are almost no recoveries ofa-naphthol at 1500 psi. Again, in this figure, as in the Figure 3-68, the

75 Figure 3-6a-c: a-Naphthol on Silica gel (acidic polar analyte on polar s.p.)

~ 'I l Constant Temperature I I Efflclency at 35'c

t ro~------~ :1 60 ,_5000,-+-1500 S!-'-8000, I #20 ~ 10

O~~~-+--~--__-­ __~ o 10 20 30 40 50 60 I j Time

1 Efficiency at 70 °e I 100 eo 15al. eo _5tXIO. I 40 [3---.-ecm I it. I 20

1l I 0 j 0 20 40 TIme I i I Efficiency at 110°C t j 100 80 1 l!', -+-1500 I j 60 _5000 I 40 ---'-8000 # 20 0 I 0 20 40 60 t Time I

1 76 I analyte is soluble at 5000 and 8000 psi but bas lower recoveries due to the losses in

collection.

In Figure 3-6c, variations of analyte recoveries are again depicted. The solvent

strength of the supercritical fluid is expected to increase with the pressures employed

during the extraction process. Figure 3-6c shows the same relationships as Figures 3-6a

and 3-6b pertaining to recoveries and pressure increments. However, the major

difference in Figure 3-6c is the substantial recoveries shown at 1500 psi. At this

condition, there is significant recovery due to the vapor pressure ofthe analyte.

Overa14 the recoveries of the a -naphthol (Figures 3-6a-c) from the silica gel

matrix are much lower than those observed when implementing the non-polar ODS

matrix (Figures 3-5a-c).

Polar basic anaMe ~ Propranolol

Propranolol spiked onto an ODSmatrix

The recoveries of propranolol spiked onto ODS are illustrated in Figures 3-7a-c.

In each of these Figures, temperature was held constant as the pressure was varied.

Propranolol is a polar and basic substance. The structure of this compound is shown

below.

77 Propranolol

The resuhs shown in Figure 3-7a are at a temperature of 35°C. Extraction recoveries are expected to be higher for polar analytes such as propranolol because the supercritical fluid is more capable of solvating the polar analytes as pressure is increased.

Figure 3-7a illustrates no increase in recoveries as higher pressures are used. Overall, there is practically no recovery ofthe analyte from the non-polar matrix even at pressures as high as 8000 psi. Figure 3-7b depicts the results of the recoveries of propranolol from

ODS at a temperature of 70°C. Results shown are similar to those discussed for Figure

3-7a above. In Figure 3·7c, variations of analyte recoveries are again depicted. At all conditions there is practically no recoveries of the analyte. This is due to strong binding ofthe analyte via electrostatic and acidlbase interactions with the stationary phase.

Over all, no recovery is observed regardless of the pressure and temperature employed in the extraction.

Polar basic IIIUIlvte spiked onto a polqr IIUlIrix

The recoveries ofpropranolol spiked onto silica gel are illustrated in Figures 3-8a-c. In each ofthese Figures, temperature was held constant as the pressure was varied. Relative to eicosane propranolol is a polar and basic substance.

78 Figure: 3-7a-c: Propranolol on ODS (basic-polar analyte on non-polar s.p.)

Constant Temperature

Effflclency at 35 'e

100

80 eo I-+-35/1500 _35/5000 I 40 i--"-35/8000. '# 20 0 0 20 40 eo Time

Et'llcleonay at 70 ·e

100

80

80

I 40 --­ 7018000 · '# 20

0 0 10 20 30 40 50 80 Time

Emclency at 150'e

100

ao

80 I ...... ,50/1500 I ! _15015000 40 I """"'-'5011000 I '# 20

0 0 10 20 30 40 50 to Time

79 The resuhs shown in Figure 3-8a, are at a temperature of 35°C. At this

temperature, CO2 is re1atively non-polar. Extraction recoveries should be higher for polar analytes such as propranolol because the supercritical fluid is more capable of solvating the polar analytes as pressure is increased. Figure 3-8a illustrates an increase in recoveries as 5000 psi This can be an artifact. Nevertheless, there is practically no recovery ofthe propranolol at either 1500 psi or 8000 psi. Figure 3-8b shows the results of the recoveries of Propranolol form silica gel at a constant temperature of 70°C. In

Figure 3-8c, variations ofanalyte recoveries are again depicted. However, once again, no recovery is seen regardless ofthe pressure employed in the extraction.

The recoveries of this compound are much lower then expected when comparing this molecule with a-naphthol. This is likely due to the polar, basic properties of propranolol that can penetrate deeply into the CIS si1anized silica sorbent to interact with the silica matrix. Molecules such as propranolol which consist ofhydrophilic ''head'' and a hydrophobic "tail", the head to surface position is preferable to the opposite one. In this type ofsituation, both polar and hydrophobic interactions between the solute and the sorbent can occur. This theory was proposed by Marko et aL, who expJained that both mechanisms cause different elution abilities of organic liquids towards the basic analyte.

The polar interactions between basic solutes and the sorbent can be broken only by a liquid with strong proton acceptor properties.(155)

80 Figure: 3-8a-c: Propranolol on silica gel (basic polar analyte on polar s.p.)

Constant Temperature

Elfcl.ncy at 35°C 100

80 ~ 60 ! -+-35/1500 -35/5000 J 40 -.trr- 3518000 # 20

0 ~ 0 20 40 80 Time

Efficiency at 70°c

100 80 70 eo 1--+-__7015000"500 I 40 --.-7018000 '# 20 0 -- - - 0 20 40 eo Time

Efficiency at 150 °c

100

80 1"""-15G'1500 ED 1---15G'SXXl I 4) 1-.-15018OCX) i '# 2D

0 0 10 2D 3J 4J 9) eo TImI

81 Effect of extraction temperature at constant pressure

The sets of data shown in Figures 3-3 through 3-6 can be rearranged to see the effects of the recovery at constant pressure and varying the temperature. Figure 3-9 and

3-10 show a-naphthol spiked onto ODS and silica gel at constant pressure. The recoveries are as aspected when looking at Figure 2-5, expect, the recovery at 35°C and

1500 psi ofa -naphthol on ODS shows higher recovery then both at 70°C and 150°C.

The extraction recovery of the eicosane from both ODS and silica ge~ on the other hand show highest recovery at 35°C. However, at 70 °C and 150°C sometimes the recovery of eicosane is higher at then at as can be seen in Figures 3-11 and 3-12. This trend can be explained by the phenomena of vapor pressure of the anaiyte. The boiling point of eicosane at atmospheric pressure is at 220°C. We can say that the vapor pressure of the eicosane is significantly increased at the pressure of interest to vaporize the molecule, which aids in increasing recovery.

This data can be compared to the Hot-ball model described earlier. First, let's assume that ODS used in these experiments is sphere. Similar effect is observed in extraction curves for SFE based on this model for eicosane spiked onto ODS and silica gel in Figures 3-9 & 3-10. In this figure, there are mainly two types of curves. One where the curve levels off after 5 minutes and one where the slope is very steep until 5 minutes and then gradually levels off. In the former curve, where the recoveries are

100% after 5 minutes, the limiting step is the solubility and only solubility.

82 Figure 3-9a-c: a-Naphthol on ODS (acidic polar analyte on non-polar s.p.)

Constant Pressure

Efficiency at 1500 pal

120 -r------, 100 -+-35c _7Oc I : """"-150c ~ 40 20 o~~~~~~~==~=l o 10 20 30 40 50 80 Time

Efficiency at 5000 pal

100

80 -+-35c 60 _7Oc I 40 """"-150c ~ 20

0 0 10 20 30 40 80 Tim.

Efficiency at 8000 pal

100

80 35c 80 _7Oc I 40 E""""-15Oc ~ 20

0 0 20 40 80 Tim.

83 Figure 3-10a-c: a-Naphthol on Silica gel (acidic polar analyte on polar s.p.)

Constant Pressure

r:1m CI: 40 - .,. aJ v: ­ o _ o 10

Efficiency at aooo psi

100

I:.,. 2.0

o~----~----~--~ o eo

Efftclency at 8000 pal

100 80 -+-35c • _10c I : --.-150c 20

20 40 eo TII'M

84 Figure 3-11a-c: Eicosane on ODS (non-polar analyte on non-polar s.p.)

Constant Pressure

Efftclency at 1!OO psi I;100~~=====+======~ 'it 20 UA~~____------~ o~----~-----+----~ o 20 40 60 Time

100 15=9:==::=====1 80 -+-35c 60 _70c I 40 -'-150c 20

O __----~------~----~ o 20 40 60 Tim.

Emclency at 1000 psi

100 80 -'-35c 80 _70c I 40 --'-150c 20

O .....-==::===---+-----~----___1 o 20 40 60 -rime

8S Figure 3-12a-c: Eicosane on Silica gel (non-polar analyte on polar s.p.)

Constant Pressure

Efficiency at 1500 psi

100 nFi~~~~~~~=i IE '# 20 0~----~----4-----~ o 20 40 60 11me

Emciency at 5000 psi

100 ~ 1: 1 v~ l: l:

_7Oc i : -.1r-1Slc '# ~ 20 0 0 10 20 30 40 50 ED T1me

100 5'10 35c. _7Oc J: -'-15Oc. '# 2D ~ o~------~----~ o 2D TIma

86 In case where the recoveries are not 100010 after 5 minutes and the curve is leveled off: this means that the energy required for desorption of the analyte is much greater then what is available at the given condition. In the cases where the curve is steep until 5 minutes then gradually levels off, the first portion can be attributed to the solubility limitation while the second portion can be said to be limited by diffusion. The reason why the experimental curves are not similar to the theoretical curve is because the extraction ofeicosane occurs fairly rapidly and no further extraction recovery is achieved with increasing time.

There were two instances where unexpected behavior occurred. In figures 3-3 and 3-4 the increased temperature played an important role in increasing the vapor pressure of the analyte. As predicted by Figure 2-5, solubility decreases with temperature. Variation is seen due to significant vapor pressure ofthe analyte. That is, at increasing temperature the solubility of the eicosane in carbon dioxide is increased, which conflicts with the Figure 2-5.

Another instance where non-ideal behavior occurred is when there was practically no extraction ofpropranolol from either ODS or silica gel This behavior is non-ideal in a sense that there was no extraction when sample was spiked onto the matrix. Ideally there will be a complete extraction in the time required to pass a few void volumes. As described earlier, this is due to the fact that propranolol is polar basic analyte and can penetrate deeply into the C 18 silanized silica sorbent to interact with the silica matrix. In addition, propranolol can form hydrogen bond with the silica gel matrix.

87 Extraction Profiles

It is interesting to examine several specific cases of SFE that exhibit rate limiting phenomena. For example, the extraction of solutes from solid matrix. enclosed in a tubular vessel using a supercritical fluid closely parallels the kinetics observed in liquid extraction as shown in Figure 3-2.(156) The initial portion of the extraction curve is linear, indicating that quasi-equilibrium conditions are governing the partition of the solute into the mobile dense fluid phase. After a finite time, the yield curve starts to become convex with respect to the time axis as the extraction experiences a transition from equilibrium to diffusion controlled kinetics. In the final stage of the extraction, the kinetics is dominated by diffusive mechanisms, which may be quite complex, depending on the morphology of the substrate being extracted. For the case of the solid substrate, factors such as the degree of swelling of the substrate can have a profound effect on the yield curve. (156,157)

However, as can be seen, the results presented in Figures 3-3 and 3-4 exhibit several types of extraction recovery profiles. If it was solubility in question then the recoveries would constantly increase as the sample is extracted over a longer period of time. In some instances, however, a sigmoidal curve is observed, suggesting that the solubility could be the limiting step and/or the extraction kinetics is much slower. For example, this behavior is observed for eicosane spiked onto ODS extracted at 70°C and

8000 psi.

88 In most of the cases, however, the recoveries are different. The extraction recovery has a typical expected theoretical profile. That is, the initial extraction occurs rather quickly, which would indicate that in this time period the extraction depends on the solubility of the analyte in supercritical fluid. Then, the curve reaches a flat plateau, where diffusion limited extraction occurs. That is, the analyte has limited or slower mobility in the matrix.

For those curves, which have similar profiles but different recoveries at different conditions for the same analyte-matrix sample, it can be said that those extractions are controlled by enthalpy and so much so that, even after extraction for longer periods of time, the recoveries are not increased. This can be seen in Figure 3-5, where, both at 35 and 70 °C the curves of 1500 psi is the same, but with different recoveries.

Conclusion

Spiked samples can be used to study the efficiency of SFE extractions. To achieve higher extraction recovery, the supercritical fluid must be able to solvate the analyte as well as interact with the matrix. Also, vapor pressure of the analyte is very important parameter to take into consideration for optimization. Spiked samples are commonly believed to have no retention and to have complete extraction in the time that is required to pass few void volumes of the fluid. Our spiked samples using matrices approximating native matrices, as was shown, do not follow expected recoveries in the given period of time. They exhibited behaviors more like those for native samples.

Thus, spiked samples that accurately simulate native samples can be made for SFE.

89 Chapter 4

Determination of Crude Fat in Food Product by Supercritical Fluid Extraction and Gravimetric Analysis

Introduction

Analysis ofthe fat content of food is a major concern for the food industry as food labeling regulations and the resulting analytical requirements become increasingly strict.

The percentage of fat by weight is now reported on the label of almost all packaged food products sold in the United States. Typically, fats are removed from the complex food matrix by a combination of acid hydrolysis and solvent extraction (158), a labor and solvent-intensive process. Because of their hydrophobic nature, fats are suitable for extraction by nonpolar carbon dioxide. The literature shows many examples of acid hydrolysis and SFE that are used for the removal of crude fats from the food matrix.

There is an especially thorough comparison of two methods shown by Huang, et al. (159) and an SFE study by King et al. (160). Our purpose is to show that SFE can be used in place ofthe typically, labor and solvent intensive extraction methods. We have chosen to illistrate this by extracting fat from a candy bar (Snickers).

Experimental

All supercritical fluid extractions were performed on an SFX-2-10 extractor with syringe pump (Isco. Inc., Lincoln, NE) and a 10 mL/min stainless steel restrictor. Carbon dioxide was SFC/SFE grade with 1500 psi helium headspace and dip tube (MG

Industries, Allentown, PA). Alternatively, refrigeration grade (AGL, Linden, NJ), which 90 is much less expensive was used. When the refrigeration-grade C02 was used, the syringe pump was cooled to 10°C using a cold water circulator. Dynamic extractions were conducted at 8000 psi and 80 °C. All instrument components are connected using high pressure compression fittings ofthe same type employed in HPLC.

Candy bar samples (Snickers, M & M Mars, Co., Hackettstown, NJ) were obtained from local vending machines and the extraction was performed wearing gloves or with tongs. Approximately 1 g of candy was accurately weighed (to the nearest milligram) on an analytical balance (Mettler, Toledo, OR) and mixed loosely with a few grams ofCelite (Fisher). This mixture was placed into a 10 mL stainless steel extraction vessel (Isco) and the remaining vessel volume was filled with Celite. The assembly was weighed. The vessel was then placed into the extractor and extracted with the full syringe pump volume of supercritical carbon dioxide. The volume (approximately 70 mL) of pressurized carbon dioxide was recorded. After each extraction, the vessel was removed from the extractor, allowed to cool to room temperature, and reweighed. This extraction and weighing procedure was repeated until a nearly constant vessel assembly weight was obtained from the vessel. The percentage offat was then calculated from the difference in weight of the vessel assembly before and after extraction. Blank samples containing only Celite showed a loss ofapproximately 3 mg per pump volume ofcarbon dioxide under the above conditions. Mass calculations were adjusted for this loss. At the conclusion ofthe experiment, the extraction vessel was cleaned throughly with water and allowed to dry. It is noted that the waste from this experiment, a mixture of Celite and candy, is not hazardous.

91 Result and Discussion

Results from a typical candy bar analysis are shown in Table 4-1.

Figure 4-1 shows a plot of the extraction recovery versus the volume of carbon dioxide passed through the extraction vessel. Data points were obtained by reweighing the extraction vessel assembly after each full pump volume of carbon dioxide has been passed through. Immediately, it is seen that, as with any analytical extraction, extraction time has a profound etIect on recovery. Although the graph shows recovery plotted against carbon dioxide volume, which is the quantity easily measured, this is easily converted to extraction time using the known restrictor flow rate. The complete extraction requires 400-700 mL ofcarbon dioxide, or 40 -70 min at 10 mLlmin.

It is immediately noted that a complete extraction requires that several full syringe pump volumes (- 5 ) of carbon dioxide be used. This shows that the kinetic mctors may atIect extraction recoveries, along with partitioning. It is seen that nearly 100% recovery of the fat is obtained after about 400 mL of supercritical carbon dioxide has passed through the extraction vessel. To ensure complete extraction, several more pump volumes of carbon dioxide were passsed through the sample. A finely divided film of fatty material was observed coating the walls ofthe collection vessel and the entire laboratory took the odor of the chocolate and caramel. The results from 11 groups of students are summarized in

Table 4-2. It is seen that the mean percentage ofmts found in the sample was 21% with a

95% confidence interval of ± 2%. If the package labeling of 21 % is taken as the population mean it is seen that the analytical method is in agreement with the "literature", as the population mean falls within the confidence interval. It is noted that values ranged between 17% and 26% for individual analysis. This variation may be due to the

92 Table 4-1 - Resuhs ofFat Extraction from Candy Bar

Trial Total CO2 Total Mass Percentage of Percentage Fat

Volume Extracted Candy Bar Recovery

(mL) (mg) Removed

1 77 111 11.0 50

2 153 140 13.8 66

3 225 176 17.4 83

4 295 209 20.7 99

5 365 224 22.2 106

6 436 237 23.4 111

7 505 239 23.6 112

8 575 239 23.6 112

9 649 240 23.7 113

10 723 240 23.7 113

Total mass ofcandy: 1.011 g; mass ofcandy. vessel and celite assembly: 101.380 g; mass ofassembly following 700 mL CO2 extraction: 101.109 g. expected fat percentage: 21%)

93 Figure 4-1 - Fat Recovery from Snickers Bar Versus Carbon Dioxide Extraction Volume.

Fat Recovery versus Carbon Dioxide Volume

120 • • • • • 100 • ~ • at ~ 80 • i 60 • ..I: at • ~ 40 at Go 20 0 0 200 400 600 800 Carbon Dioxide Volume

94 Table 4-2 - Compiled Results from Crude Fat Extraction

Group Number of Mass of Fat Percentage of Percentage Fat

Extractions Removed (mg) Candy Bar Recovery

Removed

1 9 183 16.5 79

2 7 182 87

3 6 279 .8 123

4 6 234 23.2 110

5 4 228 24.3 115

6 4 244 24.3 115

7 4 204 20.4 97

8 4 189 18.5 88

9 5 212 120.6 98

10 6 185 18.5 88

11 10 240 23.7 113

Average -----­ 216 21.2 101

Standard --_..._­ 31 3.1 14

Deviation

95 heterogeneous nature of the samples. For example, some samples contained an entire peanut, while others did not. Peanuts as we know are relatively higher in fat content then the rest of the ingredients in the candy bar. This provides a realistic illustration of the sampling problems that may befall a real-world analytical method. There is also variation in the final volume ofcarbon dioxide used in the extractions, but this showed no correlation to the final percentage offat extracted, probably also due to the heterogeneous nature ofthe samples and ofstudent lab technique.

For each extraction, the percentage recovery is calculated as the ratio of the total crude fat removed from the sample to the total amount offat expected to be present. It is noted, as above, that several of the recoveries are greater than 100%, again a result of sample inhomogeneity or variable lab techniques. The percentage recovery gives a value for the efficiency ofthe extraction and shows that few extractions achieve 100% recovery or total efficiency, especially those carried out in a single step.

Qualitative examination of the data indicates that this extraction follows similar kinetics to those observed by Langenfeld et aL (161) and calculated by Pawliszyn (162) for analytes adsorbed onto soils. It is seen, therefore, that the removal of materials of interest from a complex matrix involves various forms oftransport, including diffusion through the matrix to its surface, transfer across the boundary between the matrix and the extraction fluid, and diffusion within the extraction fluid. It is also noted that many analytes will partition between the fluid and matrix more than once, indicating that analyte transport in SFE from this complex matrix is combination of a chromatography­ like elution and desorption from the matrix.

96 Conclusions

The use of supercritical fluid extraction, a modern instrumental technique, can be used to extract fat from a given matrix, in this case a Snickers candy bar. Specifically, the crude fat content a Snickers candy bar was determined to be 21 + 2%, consistent with the package labeling. It was also demonstrated that SFE is a straightforward and relatively inexpensive technique.

97 Chapter S

Supercritical Fluid Extraction of Peppermint on From Toothpaste

Introduction

It is very important to validate the ingredients in the products before they are sold.

Thus, it is very important to make sure that the product contains the correct amount of each compound as claimed on the label The goal of this work was to efficiently extract the peppennint flavor ingredients from toothpastes. The typical ingredients in the average toothpaste include about 75% humectant and water, 20010 abrasive (sand or powder calcium), 1-2% foaming and flavoring agents, 1-2% pH buffers, 1-1.5% coloring agents, binders, hand opacifiers, 0.1-0.3% fluoride (either stannous fluoride for sodium fluoride or mono fluoride phosphate.(l63) Extracting peppermint oil by the traditional extraction method of steam distilJation and Soxhlet extraction has several disadvantages including the heat instability of the essential oils and the tact that only voJatile components can be isolated.(l64,165)

In the effort to reduce the solvent waste and decrease the analysis time, in this experiment, various brands oftoothpastes were purchased and were analyzed for voJatile oil using supercritical fluid extraction. This was done using an external standard calibration curve using peppennint oil as the standard. Peppennint is one ofthe members from the mint fumily.(166) Other familiar ones include spearmint, water mint. pennyroyal and horsemint. The voJatile oil of peppennint oil is the main reason for its

98 popular use. This oil is typically found in concentrations of 0.3%-0.4%, but may be as high as 1.5%. Menthol is the main active constituent, followed by menthone and menthyl acetate, and, additionally there are more than 40 different compounds in the oil.{l67)

Other constituents include tannins, flavonoids, tocopherols, cartenoids, betaine, choline, azulenes, and rosmarinic acid and bitter principle (168,169) All these compounds give peppermint a pungent or spicy taste with cooling and drying energy.{l68)

Equipment:

All toothpaste were obtained from the bookstore at Seton Hall University.

SFE System: The extraction system consisted ofan SFX - 2 - 10 extractor with syringe pump. (Isco, Inc, Lincoln, NE) and a 10 mLlmin stainless steel restrictor. Isco standard lOml extraction thimbles were used in these experiments. Carbon dioxide used for this experiment was refrigeration grade (AGL, Linden, NJ). The syringe pump was cooled to

10°C using cold water-bath circulation system. By using this grade ofcarbon dioxide the cost of analyzing the sample is less expensive then the SFC/SFE grade with helium headspace and a dip tube. The extraction was done by the method described in the sample preparation section.

Collection system: The collection equipment included a centrifuge tube containing a solvent, which was placed in a waterbath at 50°c. The water in the waterbath was stirred with the magnetic bar and a stirring plate to ensure temperature ic; controlled and even flow of the water around the centrifuge tube. This enables the restrictor from clogging

99 when carbon dioxide depressurizes.

Analysis system: Separation and quantitative analysis of the SFE extracts were performed on a Shimadzu GC-MS system (equipped with mass spectra library

NIST62.Lffi) using HP-5 column with dimensions of 30 m X 0.25 mm X 0.25 IJl11. 1 fJI was injected manually using 10 fJI syringe. Table 5-1 shows the conditions and parameters used for the GC-MS.

Sample preparation

About I g of the toothpaste was weighed out using Mettler balance. To this

approximately 10-11 g of sodium sulfate (Na2S04) was added to 'dry' the sample. This mixture was then thoroughly mixed and placed in an extraction vial. The vial was not completely filled (- % full) to allow the sample to swell without clogging the restrictor.

This cell was placed into the sample holder ofthe SFE and extracted at 35 °c and constant flow-rate of 0.5 mIimin. The eluent was collected in few milliliters of ethanol placed in a collection vial. The collection vial was immersed in a waterbath at 40 °c to prevent the restrictor from clogging. (when C02 depressurizes, it cools the restnctor and the solvent in which the carbon dioxide depressurizes.) After the extraction was complete the extracts were diluted to 2 mL, then 1 to 10 mL. From this, it was diluted further to the ratio of 1 to 10 mL. (total dilution factor 200 in most cases). The resulting solution was analyzed by GCIMS (Sbirnadzu SC-17 A) for quantitation. An external calibration curve was prepared for determining the concentration ofthe sample (5 ppm to 200 ppm).

100 Table 5-1 - Conditions and parameters used for GeIMS.

Injection temperature 250°C Initial temperature 80°C Initial time 1 min Rate 10°C/min Final temperature 150°C Final time 1 min Detector temperature 280°C Pressure 100 kPa

101 Results and Discussion

The matrix of toothpaste is very soft solid and can melt very easily at high temperature, roughly above 50°C. Therefore, it is very important to ensure that enough drying reagent such as sodium sulfate is used to "solidify" the matrix. If not properly done, this can cause the clogging of the restrictor. This matrix contains a "foamy" material, as do all toothpastes, which might swell when supercritical carbon dioxide is passed through it. Thus, this requires a large enough void volume in the cell for the matrix to swell.

The calibration curve for the peppermint oil standard is shown in Figure 5-1. The peppermint oil, which was used for the standard gave several peaks in a chromatograph.

The chromatograph of the standard is shown in Figure 5-2. The cyclohexanol, 5 methyl-

2-(1-methylethyl) peak at around retention time 6.6 minutes is the peak of interest menthol. It was chosen for preparing the calibration curve. Other peaks in this chromatogram are due to the isomers of menthone ( l-menthone and d-menthone) at retention times of 6.3 and 6.5 minutes. Menthone is perfumery agent and has minty flavor.

Chromatograms from extraction of brand 1 toothpaste contained several peaks at fairly high concentrations that did not appear in great quantity in the standards as shown in Figure 5-3. These peaks eluted at retention times of 7.6, 8.0, and 8.2 minutes. This corresponds to 2-methyl-5- (l-methylethenyl) 2-cyclohexene-l-one; 3 phenyI2-propenal, and I-methoxy-4-(2-propenyl)benzene, respectively.

102 Figure 5-1 Calibration curve of menthol

1.E+08 -,------, 8.E+07 y = 416386x ~= 0.999 e«s 6.E+07 c( 4.E+07 2.E+07

O. E+OO ~:.....---,----..,....._--___,__------l o 50 100 150 200 Concentration (ppm)

103 Figure 5-2 - An example total ion chromatogram of peppermint oil standard

ablDldance

2 5 II Time (mio)

104 Figure 5-1A Mass spedrum of peak at RT - 6.6 minutes abundance

56 12

'21 41 ,. tI. I

Figure 5-1B - Its matching library spectrum

abundance

"

Menthol

'1 55

121 1.I

mlz --->

105 Figure 5-3 - An example chromatogram of the extracts of Brand 1 toothpaste

~------~------~------~------~1rnI~~~J1' abundance

nc

4 ,

Time (min)

106 The conunon names for these peaks eluting at 7.6, 8.0 and 8.2 minutes are carvone, which has spearmint or peppennint flavor, cinnamaldehyde, which has a flavor of cinnamon or burning aromatic taste as the name implies, and anisole. which is another compound that is used in perfumery. Figure 5·3A shows the mass spectra of the peak eluting at 7.6 minutes, corresponding to carvone and below it, is its matching library spectra in Figure 5·3B. Figure 5·3C shows the mass spectra ofcinnamaldehyde, the peak eluting at retention time of about 8.0 minutes. Below it, is the mass spectrum from library in Figure 5-3D. Similarly, Figure 5-3E shows the mass spectrum of the peak eluting 8.2 minutes, corresponding to anisole and below is its matching library spectra, which helps us to identifY and confirm the peaks.

An example of the extraction of volatile oil from brand 2 toothpaste is shown in

Figure 5-4. This figure shows the most abundant peak as the menthol peak and another peak at a approximate retention time of 6.56 min. corresponding to 5-methyl-2-(l­ methylethyl)-cyclohexanone. A conunon name for this peak is menthone, which is an oxidized form ofmenthol. An example mass spectrum ofthis menthone peak, eluting at retention time of approximately 6.3 minutes is shown in Figure 5-4A and its matching library spectra is shown in Figure 5-4B.

107 Figure 5-3A - Mass spectunn of the peak at RT - 7.6 minutes I abundance 1.

54

41

,. _17.1 jJJ ~j] 161 ,.,.,.. 221 ''11 4J4 58 ,. ,Ii ., mlz--> -­ • • - -- Figure 5-3B - rts matching library spectrum abundance

l-methyl-5-(l-methylethenyl-l-cylobexene-l-one

m/z-->

108 Figure 5-3C - Mass spet:turm or the peak at RT - 8.0 minutes abundance ," ,.

51 ."

I J62 _ i ,... 120m 161'1n _ j I so ,. mlz-> Figure 5-3D - Its maUhinc library spectmm

abundance

,.

."

5'

mlz--> -

109 Figure 5-3E - Mass spectrum Dr the peak at RT - 8.2 minutes HI abundance

111

131 17 1111 " 51

J. ~~IJ. ~ '59 176 2» .1 341 4Id 50 100 .. Z50 310 .. mlz-> • - - • - Figure 5-3F - Its matching library speetrum

abundanee 1- metho.l}"4(2-propenyl)-benzene

12'

51

mlz-->

110 Figure 5-4 - An example chromatogram of brand 2 toothpaste

• abundance

:s , , • Time (min)

III Figure 5-4A - Mass spectmm of the peak at RT - 6.3 minutes 1"1 abundance

41 ,. 5!1 " ",

114 IS

,III It .1 tr ,,, ,,, 2211 zsa !41 50 ,ao ,50 - 300 J50 mlz-> - • ­ Figure 5-4B - Its matching library spectrum

abundance meotilooe •

41

,,,

'14

mlz--> -

112 In Figure 5-5, an example chromatogram of the extract of brand 3 and 4 toothpaste, two peaks are eluted. First, at the retention time of approximately 6.6 minutes and second at the retention time of 7.6 minutes. These peaks are indentified as

2-methyl-5-(l methyl ethenyl)-2-cyclohexen-l-one, or commonly known as carvone, as shown in Figure 5-5A with its matching library in Figure 5-5B. The abundance of the carvone peak is greater then the menthol peak, nevertheless, the menthol peak was used to quantitate the peppermint oil and in turn menthol in the toothpaste. This probably gives less then actual recovery.

The recovery ofthe menthol in all the toothpaste used is shown in Table 5-2. As can be seen, the menthol concentration in brand 1 and 2 toothpastes are about 6-7 part per million. In brand 3 and 4, the menthol concentration was determined to be about 3 parts per million. Again, as described earlier, this low recovery ofthe menthol in brands 3 and

4 can be attributed to the fact that there might be different formulation in preparing different brands, therefore, different flavoring reagents.

Conclusion:

Supercritical fluid extraction is a fast, straightforward and inexpensive technique for extracting menthol or peppermint oil from toothpaste. This technique can be used to monitor other volatile oils. Due to the usage of supercritical carbon dioxide in supercritical fluid extraction, the extraction procedure is very easy; that is, very few steps are necessary to complete the extraction. Not only it is easy; it is also fast because supercritical carbon dioxide can reach into the pore ofthe solid matrix easier then liquid.

This also eliminated the dilution ofthe sample and heled to concentrate the sample.

113 Figure 5-5 - An example chromatogram of brand 3 and 4 toothpastes

nc abundance

"t'IC

I

Time (min)

114 Figure 5-5A - Mass spedrum of the peak at RT - 7.6 minutes abundance 11­ 54 12

I I

! 41 i I " I 1SO I 135 ii I I ilo. 011 .L IJjIT ~ IN ''II 202 2ZI 247 261 29' 321 361. SO tilo ,~ zOo z50 DD mI.z-> - Figure 5-58 - Its matching library spectrum abundance

2-methyl-5-(metbylethenyl- l-cyclohexen-l- one ,.

350 mlz->

115 Table 5-2 -Menthol extracted from various toothpastes.

Brand conc.(ppm) Brand 1 6.3 Brand 2 7.2 Brand 3 3.0 Brand 4 3.3

116 Chapter 6

Isolation and Identification of Sulfur-containing Molecules from Garlic

Introduction

Garlic (allium sativum) has been known since time immemorial for its medicinal pharmacological properties and characteristic flavor.{170-173) The chemical composition of garlic has been studied by many scientists along with its pharmacological properties.

(174-181) Traditional uses include antibiotic and fungicidal properties ofgarlic (182-189) and recently it has been proposed to have additional medicinal properties including a propensity to reduce platelet aggregation, decrease blood lipid concentrations and reduce cancer risk. (174-178,190-194) These pharmacological properties are associated with the presence ofsulfur containing compounds, which are the main components ofgarlic.

Numerous sulfur-containing molecules are present in garlic cloves, alline being the most important one. The characteristic pungent order of garlic is obtained after the reaction of the enzyme alliinase, which converts alline into allicine, when the garlic cloves are crushed. (195-202) Allicine is very unstable molecule chemically and is rapidly converted into a series of numerous other odorous sulfur containing molecules.

These sulfur containing volatile molecule include compounds such as diallyl disulfide, diallyl sulfide, diallyl trlsulfide.(170)

117 There are several noticeable fragment ions characteristic of the sulfur containing compounds. These characteristic ions can aid in determining the parent compound. Allyl has rnIz = 39 and 41, allyl sulfide has rnIz = 73, allyl disulfide has rnIz 105, and mono-, di-, trisulfides have rnIz = 47, 79, III respectively.

In this study, the feasibility of using supercritical fluid extraction (SFE) to extract this complex mixture of volatile compounds including the various mono-, di-, and trisulfides qualitatively from the garlic cloves is investigated. The reason why only sulfur containing polar compounds are chosen is to see if SFE can extract polar compounds.

The extraction efficiency of non-polar compounds using SFE was already shown in the earlier chapter (ch-4).

Experimental

Sample preparation

Garlic clove- Garlic was peeled and sliced so that it would fit into an extraction vial.

(only 1-2 g of the garlic was used). The garlic (- 1 g) was then mixed with sodium sulfate (-11-12 g) and placed into an extraction cell. The purpose ofsodium sulfate was to act as a drying reagent. (to absorb the moisture from the garlic) This mixture was then placed in an extraction vial for extraction.

SFE Instrument and conditions

An Isco mode 100DM syringe pump was filled with SFC grade CO2 (AGL Welding

Supply Co., Inc.), which was then pressurized to 8000 psi. Approximately 1-2 grams of

118 sample was placed inside an extraction cell that was equipped with an Isco Model SFX 2­

10 extractor. The cell was then placed into the extractor and pressurized by opening the inlet valve and letting CO2 to enter. Once the cell was pressurized, the outlet valve was opened to flush the extraction cell with SF CO2• The flow rate of the SF, at 1 mL/min, was controlled by - 25 cm long stainless steel capillary, normally used for high flow rates

(10 mL/min restrictor). The extracted analytes were collected by placing the outlet end of the restrictor into a centrifuge tube containing - 2.5 mL of chloroform solvent.

Chloroform was the solvent ofchoice because all ofthe extracts appeared to be soluble in the solvent. As the solvent evaporated, it was added to the collection vial to maintain the solvent level between 1~2 mL. The extraction cell temperature was regulated by the extractor at 35°C. The sample was extracted for 30 minutes at a rate of 1mL/min. After completion the extraction the extracts were analyzed by GCIMS.

GCIMS Instrument and Conditions

The analysis was done on HP 5890 g~ chromatograph with 5972 mass selective detector.

1,.11 ofthe sample was placed onto the column by manual injection. A HP-5, 5% PH ME

Siloxane column was employed for analysis with the dimensions of 30 m X 0.25 mm X

0.25~. Helium was used as the carrier gas at a constant pressure of 15 psi. The injector temperature was kept at 250 °C. The temperature program was started at 40 oC, which was held for 1 minute. It was then ramped at 10 0C/min to a temperature of250

0C and the second temperature program was from 250 0C at 20 0c/min to 300 0C. The detector temperature was set t0280 0C.

119 Mass specta were interpreted by first comparing with the library spectra to get some idea and then looking at the M+ ions that are characteristic ofthe sulfur containing compounds as they were stated in the introduction.

Result and Discussion

As it was presented in the chapter 1, non-polar compounds are known to exhibit higher extraction recovery at lower pressure and temperature and polar compounds are known to show higher recoveries at higher pressure and temperature. Our compounds of interest are polar, nevertheless, lower extraction temperature was used. This is because the garlic gets "cooked" at higher temperature and so the efficiency ofextraction is lost.

Using lower temperature can be compensated by longer extraction time and so extraction time of 30 minutes at ImL/min was used. Another thing to keep in mind is that, these conditions were chosen to provide a qualitative resuh only. For quantitative results, a plot of extraction time versus extraction time can be developed and the most efficient time can be used to quantitate the extracts. Again, our goal was to show that supercritical fluid extraction could indeed be used to extract the polar sulfur containing compounds from garlic.

There are several peaks that were observed in the chromatogram obtained from the extracts of garlic with supercritical fluid extraction. Since our main focus is on the extraction ofsulfur containing compounds, only peaks of interest are pointed out.

120 Deruaz et aL has shown that there are several sulfur-containing compounds that are present in a garlic clove.(171) Some of them are shown in Table 6-1. They showed the extraction of these compounds by coupling headspace gas chromatography to atomic emission spectrometric detection and mass spectrometry.

Headspace sampling is a gas-phase extraction method. It utilizes an equilibrium established between the volatile components of a sample (solid or liquid) and the surrounding gas phase in a sealed heat controlled vessel. At certain intervals aliquots of the sample is withdrawn from the gas phase and injected in the gas chromatograph for analysis.

The most obvious difference between the headspace sampling and SFE is that

SFE is off-line vs. headspace. Other differences can be seen by description given in chapter 2 about SFE and a brief description given above about headspace.

Figure 6-1 shows the chromatogram of the extract of the garlic clove using supercritical fluid extraction.

The compounds listed in Table 6-2 are from the peaks in the chromatogram shown in Figure 6-1. This chromatogram is from the extract of the garlic using supercritical fluid extraction. Also, Figures 6-2 to 6-8 shows the mass spectra ofthe peak

121 Table 6-1 - Sulfur containing compounds extracted by Deruaz et al. using headspace

GC.(reference 171)

Allyl methyl sulfide

Methyl disulfide

allyl sulfide

allyl methyl disulfide

methyl trans-propenyl disulfide

dimethyl trisulfide

dipropenyl disulfide

allyl propenyl disulfide

allyl disulfide

allyl methyl trisulfide

3-vinyl-l,2-dithiin

2-vinyl-l,3-dithiin

allyl trisulfide

122 Abundance 1500000

1000000

500000

o~~~~~~~~~~~~~~~~~~~~~~~~~~~ TIme-> 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

Figure 6-1 - A chromatogram ofextacts ofgarlic clove usiDg SFE

123 described in Table 6-2. Here there are total of seven peaks. The compounds allyl methyl disulfide and methyl trans-propenyl disulfide are structural isomers which could not be separated by the analyzing apparatus and therefore a single peak. Similarly, dipropenyl disulfide, allyl propenyl disulfide and allyl disulfide are structural isomers and as a result produce a single peak.

Mass spectra were used to identify each of these peaks. Figures 6-2 to 6-8 show the mass spectra used to ascertain the identity of each peaks along with library match of some of the compounds has identified the peaks as described in Table 6-1. The library spectra give a starting point to identify the peak and the ion fragments from the mass spectra aids in identifying the compounds with certainty. Some of the molecular ion fragments for sulfur containing compounds are given in the introduction.

Conelusion

Supercritical fluid extraction's ability to extract polar compounds such as compounds containing sulfur molecules was shown. A total of seven sulfur containing compounds were extracted form crush garlic by using supercritical fluid extraction. As it was intended in the goal, it was shown that SFE could be used to extract relatively polar compounds such as the compounds extracted in this application.

125 undance (3.210 ain) 4

8000

6000 3 4000 88

2000 59

O~~rr~~~~~~~~+r~~~~~~~~~+rrr~~~~ Iz--> 10 20 30 40 50 60 70 80 90 100 110

Fisure 6-2 - MIla specuum ofAllyl Methyl Sulfide

126 127 undance (4.346 min)

8000

73 6000

4000 99 114

2000 47

'1.--> 40 SO 60 10 80 90 ce 64133: l-Propeae, 3,3'-thiObia­

8000 73 6000 114 4000

99 2000 47 27

/'1.--> 50 80 90 100 110 120

Figure 6-3A-B - (A) Mass spectrum ofAllyl Sulfide (B) Its matcbiollibrary spectrum

128 (5.257 min)

aooc i 1 ; , 6000 ­

4000 ~

iii ~I Z--.> 130 ~undan{':'e I .' I .. l.

8000

73 6000

Fip1re 6-4A-B - (A) Mass spectrmn of Methyl propenyl disulftde (B) Its matching 1ibrary spectrmn

129 undance (7.990 tin) 1

8000

6000

4000 45 81 2000 146

O~~~~~~~~~~~~~TM~~~~~~~~TM~~~ &-->

8000

6000

4000 81 146 2000 45

O~~~~~~~+H~~~~~~~~~~~~~~~~~~~ /z-->

Figure 6-SA-B - (A) Mus spectrum or DiaUyl diJulfide (8) Its makbing spectrum

130 undance (9.741 ) 1 1 144

1000

1000 71 4045 77 4000

2000 58 85

O+r~~~~~~~~ z-->

Figure 6-6 - Mass spectrum of 3-vinyl-l,2-ditbiin

131 undance (10.155 ain)

8000

144 6000 45 111 4000 39 2000 9'1

O~~~~~~~~~~~~~~~~~~~~~~~~~~TT z-->

Figure 6-7 - Mass specturm of l-vinyl-l,3-ditbiin

132 undance (11.410.in) 41 1 1 3 8000 3 6000

4000

2000

41 8000

6000 113

4000 3~

2000 27 4 104 J i.1 S9 I 1.11 III I. • 91 LIt. II 137 146 l~lo 0 I 1 I I I I Iz--> 20 40 60 80 100 120 140 160 u!.o

Figure 6-8A.8 • (A) Mass spectnIm o(Allyl trisulfide (8) Its matching library spectnIm

133 Chapter 7

Conclusion

The over all objective of our experiments was to determine the effects of temperature, pressure and time of extraction for various analytes using supercritica1 fluid extraction.

The ultimate goal however was to assess the utility of the spiked samples as standards and also to understand the relationship ofthe factors affecting the extraction efficiency. It was shown in chapter 3 that spiked samples of eicosane, a-naphtho~ and propranolol could be used as a reference point. To achieve higher extraction recovery, the supercritica1 fluid must be able to solvate the analyte as well as interact with the matrix.

Also, vapor pressure ofthe analyte is very important parameter to take into consideration for optimization. Spiked samples are commonly believed to have no retention and to have complete extraction in the time that is required to pass few void volumes of the fluid. Our spiked samples using matrices approximating native matrices, as was shown, do not follow expected recoveries in the given period of time. They exhibited behaviors more like those for native samples. Thus, spiked samples that accurately simulate native samples can be made for SFE.

This characteristic of faster extraction rates and/or higher relative recoveries at higher extraction temperature suggests that multi mechanism for the release of the analytes studied. Changing the temperatures in the lower temperature range «100°C) result in increments in the release ofthe hydrocarbon (eicosane) from both matrix due to

133 enhanced diffusion rates and desorption kinetics. However, increased recoveries at elevated temperature (e.g. 150 °C) seem to be a result ofthermally induced desorption of the analyte from the sample matrix. The general conclusion of the study is that compound speciation within the sample has a major effect on SFE extractability and can be controlled by the extraction temperature.

In choosing the optimum conditions for extractions, both theoretical and practical considerations must be considered. The applications of both garlic and toothpaste showed that even though theoretically it would be wiser to use higher temperature, lower temperatures were used. This in due to the matrix involved in both cases. It can either plug the restrictor or can damage the instrument, which can result in less then expected efficiency. Even though, lower temperatures were used, supercritical fluid extraction was a viable method to extract the compound of interest in both applications.

In the chapter 5, menthol or peppermint oil was extracted from toothpaste. And in chapter 6 sulfurcontaining compounds were extracted from garlic cloves. Both of this methods showed that SFE can be used to extract volatile oils. Due to the usage of supercritical carbon dioxide in supercritical fluid extraction, the extraction procedure is very easy; that is, very few steps are necessary to complete the extraction. Not only it is easy; it is also fast because supercritical carbon dioxide can reach into the pore of the solid matrix easier then liquid. This also eliminated the dilution ofthe sample and helped to concentrate the sample.

134 Also, in this thesis (chapter 4) crude fat from Snickers bar was extracted. It was evident that supercritical fluid extraction, a modem instrumental technique, can be used to extract fat from a given matrix, in our case a Snickers candy bar. The crude fat content in a Snickers candy bar was determined to be 21 + 2%, consistent with the package labeling.

In conclusion, this thesis showed that supercritical fluid extraction can be used for extraction variety of compounds, and also that the spiked samples can be used as standards.

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