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GC Analysis
K. Jinno (Ed.), Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction Journal of Chromatography Library Series, Vol. 53 0 1992 Elsevier Science Publishers B.V. All rights reserved.
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Chapter 13
Introduction of Directly Coupled SFE / GC Analysis Tsuneaki Maeda Research Center, DKK CORPORATION,4-13-14, Kichijoji Kitamachi, Musashinoshi, Tokyo, Japan
Toshiyuk~Hoboa ‘Department of Chemistry, Tokyo Metropolitan University, 1-1, Minami Ohsawa, Hachioji-shi, Tokyo, Japan
INTRODUCTION Recently, supercritical fluid is widely used for industrial processing such as decaffenation of coffee beans, extraction of bitter essence from hop and so on. It is because it has unique phase properties for separation as well as extraction. Since supercritical fluid is much easily produced than before using the advanced technologies, it became much popular and created new fields like supercritical fluid chromatography (SFC) and supercritical fluid extraction (SFE). Application of these techniques is increasing in these days. The supercritical region is defined as the one above the critical temperature and critical pressure. This region gives a single phase and the substance in this region is called supercritical fluid. It is the forth state of the substance. One of the unique properties of the fluid, solubility, depends on the density which can be controlled by changing the pressure and temperature. The diffusion coefficient of the fluid lays in the midst of gas and liquid, which means that the mass transfer rate is faster in the fluid than liquid. Then if it is used for extraction in place of liquid, the extraction time could be made shorter. The low viscosity allows it to be used as the mobile phase for capillary and micropacked column SFC. These fundamental part of supercritical fluid techniques and applications of both SFE and SFC have been described in many literatures [l61. The analytical SFE is often compared with solvent extraction methods and between them there exist large differences. Actually they have many advantages and disadvantages. For analytical purposes, particularly for solid sample analysis, SFE is superior t o liquid extraction method, namely Soxhlet method, in speed, solvent waste problem, and so on. As the sample preparation method for chromatography, SFE is a relatively new method of choice. In this chapter, directly coupled SFE / GC analysis method which is one of so called hyphenated methods being used in the instrumental analysis is described. It has such a convenience that the sample preparation, separation and detection can be done in a straight line. Advantageously the technique can provide the followings; a) simplify the sample preparation procedure. b) make the analysis time shorter.
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c) minimize the contamination. d) maximize the use of capabilities of both methods. At the same time, however, the method requires the understanding of the both methods coupled. This chapter starts with the overview of SFE for GC,then describes on the chracteristics of GC and SFE, and finally on the on-line SFE / GC interface and its application. SFE FOR GC SFE has been used intensively and shown that it has a great capability in the sample preparation for GC as well as for other chromatographies, i.e, SFC, LC, IR, MS and so on. Many reviews pointed out the prospect of SFE and described opinions on the analytical scale SFE [7-121. Basically, there is almost no difference between off-line and on-line SFE. Limitation of On-line SFE I GC It is reasonable to think that the sample range applicable t o the on-line SFE / GC is limited by the fundamental circumstances of GC. Although supercritical fluid can dissolve liquid samples of high boiling point, GC can not analyze those samples which has low volatility and thermally unstable substances. In Figure 1, molecular ranges of substances which can be handled by the ordinary chromatographies and SFE are presented. Another aspect is that the solubility of a compound in the supercritical fluid can not exceed that in liquid of same material. It means that the compound that is hard to dissolve i n t o the liquid is harder to dissolve into the solvent in the supercritical fluid state. In addition, the higher molecular weight or polar compounds are difficult t o be dissolved i n t o the supercritical fluid as far as non-polar C02 or slightly polar NzO are used as the fluid material. Probably the limitation also comes from SFE side. One of the limitation from GC side can be overcome by using a high temperature capillary column such as aluminum claded fused silica capillary column with stable stationary phase. Nowadays, up to CIOO can be analyzed using these technically advanced systems [13-181. It is, however, still not enough to cover the whole range of SFE. It can be pointed out that unexpected or undesirable components extracted by a SFE will be carried into the GC system and disturb analysis. In case of an off-line SFE, further clean-up or pretreatment step can be employed t o eliminate such interferences [8]. Although online SFE / GC is a simple technique, aplicable sample is limited compared t o the offline SFE. As the result, on-line SFE / GC method requires suitable sample selection and appropriate setting of extraction conditions.
SFE as a sample preparation technique From the sample preparation point of view, SFE has two types of technique for controlling the solubility strength. One is to use a single solvent and control its strength by changing the pressure and temperature. The solubility,power of dissolution, in supercritical fluid is related to the density of the fluid which can be controlled by the temperature and pressure. Thereafter, using single solvent, selective extraction could be expected. Several off-line SFE studies reported the possibility of fractionation or class-selectiveextraction [8,12,19-221. These techniques are also useful for the on-line SFE. Various kinds of fluid materials have been investigated and compared each other. From the gas chromatographicpoint of view, SFE using
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Molecular weight range applicable to the on-line SFE / GC
LC(SEC)
SFC
GC
SFE 100
lo*
lo2
lo3
lo4
lo5
10 lo7 Molecular weight (daltons)
Figure 1. Molecular weight range applicable to the chromatography and SFE. liquefied gas is desirable. Further, intentional solvent-solute separation is not required because of its property; liquefied gas is evaporatively separated from the solute in the collection device by depressurization. Usually, carbon dioxide is the primary choice for the experiments, but it has no polarity. Therefore, many other fluid materials including NzO, CHF3, CHClF2, SF6, C2H6, i-CdH10are used as they show characteristic properties [7,23-261. The other technique is solvent polarity control method either by adding a polar solvent, which is called modifier or entrainer, or by changing solvent itself. An example for the latter case is from non polar C02 to slightly polar N20. As the modifier, methanol is commonly used. These techniques are applicable to the on-line SFE / GC, too. Basically, the role of SFE is just to dissolve the component in the sample matrix and transfer to the collection place. It is advisable to do the preliminary test and select the optimal solvent and extraction conditions using an off-line SFE before applying sample to the on-line SFE. Important thing to think about when the technique is chosen is what is the goal of analysis, whether it requires trace analysis or profile analysis. It is also important to get the information about sample matrix, properties, interferences, co-existing components and the concentration of analytes. Putting these information into consideration the best sample preparation procedure should be constructed. Basically, the process of construction is same as in other sample
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preparation technique.
Optimization of SFE conditions There are many factors to be considered depending on the goal of analysis, so as to achieve optimum extraction efficiency . If the goal is to get the profile or matrix composition of a sample, it is required to use the fluid at the maximum solubility. On the contrary, if the trace components in the sample are the analytes, it does not need to think about maximum solubility. In this case, as King [8] described, it is better t o choose the condition that can separate the analytes from the matrix without interference. The density of fluid increases with increasing pressure and the diffusivity increases with the temperature, which means that time saving extraction can be achieved at high temperature. These points are described by the reports showing the change of k at different temperatures [27-291. Supercritical fluid behaves like gas at high temperature and low density, and like liquid a t low temperature and high density. Next point is the elimination of the matrix effect. If a test gave a sufficient solubility and good recovery from standard solution,but poor recovery from actual sample, the interaction between targeted component and matrix must be considered. In this case, first the extraction time should be extended. Then, either the use of polar modifier o r change of the fluid to a more polar one such as N2O and Freon should be tried. Another way t o eliminate matrix effect is the addition of some other modifier which can break the interaction between solute and matrix. The selection of such a modifier should be done depending not on the solubility, but on the interaction. These are just rough guidelines to pursue the optimum SFE condition. Another approach to obtain good recovery of polar components is a derivatization of analytes in the fluid [30,311. Lastly, it is important t o remember that SFE is the time saving technique, but still requires time to dissolve the analytes. It can not dissolve the objectives in a moment. CONSTRUCTION of ON-LINE SFE / GC SYSTEM On-line SFE / GC construction is shown in Figure 2. There are three important parts. They are a solvent delivery pump, a SFE oven and a GC. The system is very simple to build up. The high pressure pump deliver the fluid at a higher pressure than the critical one and the extraction vessel is put in a oven to keep the temperature higher than the critical one. The examples of extraction vessel design are shown in Figure 3. An empty column or a guard column are commonly used for the vessel. The restrictor is connected after the SFE vessel t o maintain the extraction pressure high, and passing in it the pressure is reduced to meet the GC analysis. As a restrictor, fused silica capillary tubing or crimped tubing are usually used. The former is a linear restrictor and the latter is a tapered one. Extraction solvent is introduced into the GC as a gas and the solutes in the gas are concentrated in the inlet of the column during extraction. The most important part of on-line SFE / GC is the interface between SFE and GC.
SFE / GC INTERFACE There are many types of sample introduction system for capillary gas chro-
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matography developed aiming to get a good separation. These systems allow liquid or gaseous samples introduced into the GC in a narrow band. Selection of a system is dependent on the sample to be analyzed. But in case of SFE, special consideration different from the direct sample introduction technique in the GC will be required. The solvent commonly used in SFE is liquefied gas (CO2, N20, SF6 etc.), which changes to the gaseous state after depressurization. Therefore, the solvent is a gas when it is introduced into the interface of a on-line SFE / GC. The volume of extraction solvent will expand about a thousand times ( I d of liquid C02 expands to about 560ml CO2 gas). Besides, for SFE it requires a few minutes to finish in the case of a micro extractor is used. It must be considered that a large amount of gaseous solvent must be separated and removed from the solutes in the interface. It takes long time for the completion of a SFE when a micro scale extraction vessel is used. Therefore a cryogenic focusing technique should be applied t o prevent the band spreading. In this section, the interfaces so far reported are classified and overviewed. Direct injection system Figure 4. shows the schematic diagram of direct injection system [32-351. This type of system uses a heated tee piece joint to prevent the plugging. The end of the restrictor is put inside of the retention gap and the carrier gas enters coaxi-
Extraction oven Extraction vessel
Flow restrictor
Interface
1
I
I I
Caniergas
L
-Detector
.Column Liq. CO2
Solvent delivery system for SFE (High pressure pump)
Gas chromatograph
Figure 2. Schematic diagram of a typical on-line SFE / GC.
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ally along the restrictor. Whole solvent will flow through the column and whole extracted compounds are introduced into the column. The extracted compounds are focused in the retention gap. The retention gap also prevents the deposition of the non-volatile components in the column. During extraction, the carrier gas flow is stopped in order to get rid of the high back pressure effects. In this system, the flow rate of a SFE solvent is restricted by the capillary column and utilization of a micro extraction system is just recommended. Thus, when a vessel of large volume is used, intermittent introduction of the extractant, Like a heart cutting technique in multidimensional chromatography,should be employed [33,34]. Wright et al. [321
1/16"Tube 4to
restrictor
Ferrule 1/16"o.d.tube 1.Micro extraction vessel(
Il16"Tube
__+
q , , &---/
wlqy
Joint body
Nut
Extraction cell Ferrule lI4"o.d.tube 2. Semi micro extraction vesselb0.lml)
J o i n t body
Filter
&/16"Tube --+to restrictor Ferrule
-b
Filter Extraction cell Ferrule 1/4"o.d. or 318"o.d. tube 3. Conventional extraction vessel bO.5m.l) using empty column for liquid chromatography
t o restrictor
Ferrule
Figure 3. Examples of extraction vessels designed for on-line SFE /GC.
26 1
Extraction solvent inlet
Extraction solvent inlet
joint
+
Carrier gas inlet
-
ii
Retention gap
, I, I
o : C 0 2 , x : Light,
Figure 4. Direct injection system.
0
: Heavy
Figure 5. Direct cool on column injection system.
and Lohleit et al. [34] reported selective extraction method with some examples. Lohleit et al. [34] also investigated the performance of this system by comparing with the thermal desorption analysis. They showed that the former can analyze the higher molecules than the latter. This system might be applicable to the small volume extraction and wide range of boiling point samples.
Cool on column injection system This system is similar to the direct injection system but the depressurised extraction solvent is led out from the interface. A conventional cool on column injector was used after only taking out the septum [36-411. Figure 5. shows the schematic of this system. As the restrictor is inserted into the analytical column, the solutes are deposited in the column while it is cooled for a cryogenic focusing. The extracted components are retained in the liquid stationary phase and volatile components are vented with solvent. Since the restrictor is not heated in this system, plugging will be happened easily during extraction. Some modifications from on-line SFE / SFC t o on-line SFE / GC and another system similar t o the direct
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Extraction solvent inlet
v
Extraction solvent inlet
Extraction vessel
P-
Restrictor Septum
/
o O* O01
Column
Figure 6. Split I' splitless injection system.
/---I
Column i
Figure 7. Programmed temperature vaporizer injection system.
injection system using a tee piece with a vent line were reported [12,42-441. These systems might be applicable to the trace analysis where large amount of matrix is not coextracted.
Split I' splitless injection system This system is the simplest one that does not need any modification of a conventional split I' splitless injector [45-491. Figure 6. shows the schematic diagram of this system. The restrictor is just inserted through the septum during the extraction and withdrawn after it. The injector is heated t o protect a plugging. The column is initially cooled to focus the extracted components. The solvent is finally
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led to the split vent or septum purge vent. In this system, sample is splitted regardless of split or splitless mode, and the split ratio depends on the flow rate of extraction solvent. It means that the higher the extraction pressure is, the higher the split ratio is. This system is very easy to use but sample injection takes long time, and be resulted in a discrimination. This system is also similar t o the hot needle injection method where sample is injected very slowly. Though there is no maximum volume of extraction vessel, relatively high molecular weight compounds can not be introduced uniformly.
Programmed temperature vaporizer injection system For this system a conventional programmed temperature vaporizer injection system P"v)can be used without any modification [501. Figure 7. shows the schematic diagram of this system. The restrictor is just inserted through the septum during the extraction and withdrawn after it. The extraction solvent and the components with high vapor pressure exit from the solvent vent line. The injector is kept at sub-ambient temperature for trapping the extracted components inside the injector. At the end of the extraction the restrictor is withdrawn from the septum, the solvent vent line is closed and the programmed temperature operation of the injector is started. Then the whole trapped components kom the sample is introduced into the column and analysis is started. Houben et al. [501 described that since the extracted components are trapped by reduced vapor pressure, in this system, the complete trapping of the components is accomplished if they have boiling points at least 250°C higher than the trapping temperature. It seems that it has a chance t o have the plugging problem, because the restrictor is not heated. Similar range of samples to the cool on column injection system could be applicable. As for the volume of the extraction vessel, there is no restriction. Thermodesorption I cold-trap iltjection system This type of system is the modification of a thermodesorption I cold-trap injector [511. Figure 8. shows this system. In order to prevent plugging the restrictor is inserted into the heated inlet region, where usually an adsorbent-packed trap is placed. During the extraction the extracting solvent is wasted through the purge vent. The extracted components are trapped on the surface of the cold trap placed under the heated region. The property of this trapping system is the same as one in the PTV injection system. When the extraction is completed, the purge vent line is closed and the carrier gas line is opened with a rapid heating of the cold trap. The whole trapped compounds are led into the column. The original system [52] is developed for the volatile components. The maximum operating temperature of the cold trap region is 350°C. Since it is placed on the GC oven, relatively cool part exists between the cold trap and the analytical column. The semi-volatile components may deposit on this cool part. This system might be applicable to the volatile components. Miscellaneous irqiection system Some other systems were reported [53,541. The extracted sample is trapped in a loop which is connected to a 6 way port valve. After trapping, the loop is heated to introduce the extracted sample into the column. Figure 9 shows the schematic of this system. This system might be applcable to the volatile components.
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Extraction solvent inlet
Extraction solvent inlet
Extraction vessel
Restrictor
Carrier gas
d?
/
Valve
i
Column
Figure 8. Thermodesorption-cold trap injection system.
Figure 9. Loop injection system.
Conclusion Above mentioned studies suggest that a restrictor heating and a cryogenic focusing should be used during extraction. There are a number of variations of the focusing technique for the volatile sample introduction [55-581. Since the focusing temperature should be higher than the freezing point of SFE solvent, the cryogenic focusing using liquid nitrogen can't be applied. It's also important to understand the properties of a conventional GC injection system when a conventional injection system is used as the interface. There are many literatures on the capillary column GC sample injection system [59-691. It is recommended to refer those information before selecting the interface. The sample injection system is very important part of the capillary column GC. Though many systems are developed and applied, those words of which Sandra described in his book [591 are still alive; "Sample introduction in capillary gas chromatography has always been, and in fact still is, a problem. There is still no such thing as a universal injection system and there probably never will be." The situation about on-line SFE / GC interface is absolutely the same, as far as it is using capillary column.
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PRACTICE OF ON-LINE SFE / GC Most of the papers reported on the instrumentation and application of online SFE / GC have been referred in the previous section. Here, evaluation of practical on-line SFE / GC system using a conventional split / splitless injector will be presented on the basis of authors' experiments. The flow diagram of the system developed is shown in Figure 10 [701. A micro extraction vessel, approximately 110 pl, was used. An inner diameter of 20 pm and 10 cm long fused silica capillary
High pressure C02
\L
----------
6 way port
Extraction vessel
Septum purge vent Split / splitless injector Backpressure valve Column head pressure
Column oven i _____---__________--_____--
to FID S.V.; 3 way port solenoid valve Splitless injection; Carrier gas flow through N.C. to C.
Figure 10. Flow diagram of on-line SFE / GC system.
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Table 1 Anakytical GC conditions Column Purge vent flow Carrier gas Column head pressure Injector temperature Detector Detector temperature
Methyl silicone, 0.25mm i.d., 5m long, film thickness 0. lum (Quadrex) 28dmin He 0.7kg/cm2 300°C FID 300°C
(A)
yo 1
.o
I 15
I 10
15
I
c[
I 20
I
20
-
I
2.5
Time(mm)
I
10
I
15
I
20
I
25
I
30 Time(min
I
25 Time(mn)
Figure 11.Effect of cryogenic focusing on separation. (A) Without focusing, column oven temp. 100°C, (B) with focusing, column oven temp. lOO"C, (C) without focusing, column oven temp. 50°C, (D) with focusing, column oven temp. 50°C.
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(B) C 14
C 14
c20
C30
-
.5
20
25
C30
c20
30 Time(min)
1
15
I
20
I
25
I
30 Timebin)
Figure 12. Chromatograms obtained using different injection modes. Conventional split / splitless injector for GC was used; (A) Split injection mode, (B) splitless injection mode. tubing was used as a restrictor. The cryogenic focusing system is adapted inside the column oven t o focus the extracts in the front part of the column. Analytical conditions are presented in Table 1. Alkanes were used for the standard sample. A narrow-bore capillary column with thin film stationary liquid phase was used t o separate high molecular weight alkanes.
Cryogenic focusing The cryogenic focusing effect is shown in Figure 11. In this system, since the extracted components are trapped in the stationary liquid phase of the column, the cryogenic focusing depends on the oven temperature, film thickness of the liquid phase and focusing time. As the boiling points of target components are high enough, focussing can be effected at the top of the column, when the column oven is kept at near ambient temperature. The effect of focusing was reported elsewhere [38,41,711. Differences of split I splitless injection This system has a conventional split / splitless injector for capillary column GC, but its function is different from conventional use described in previous discussion. Figure 12 shows the chromatograms obtained using both split and splitless injections. Figure 12 (A) was obtained at the split ratio of 1/ 25 and (B) was obtained using the splitless injection. The chromatogram(B) shows the extracted components were escaped with extraction solvent even in the case of splitless mode. Extraction time and extraction pressure When the supercritical fluid has enough solubility for the sample components, two important factors should be considered, in order to complete the extraction successfully. The first is the sample transfer time and the second is the time to dissolve in the solvent. Figure 13 shows the recovery changes under various pressure and extraction time. Figure 13 (A) shows that at 200 atm., the fluid has
-
c20 c30 DI c40
g
3
5
3
5
10
15
10
15
Extraction time (min)
1.0
% W b
?
3
2
2
0.5
0.0
Extraction time(min)
figure 13.Effect of extraction time on relative recovery (peak height of each component after 15 min extraction = 1.00). Sample; C20, C30, C40 n-alkanes (50ng/ each component), Volume of extracion vessel 110~1,Extraction temperature 50°C, Extraction pressure; (A) 2OOatm, (B) 150atm. enough solubility but 3 minute extraction time is not enough because of the inunifonnity of the recoveries of each component. The reason is that the transfer time depends on the void volume of the extraction vessel. Figure 13 (B) shows the differences of dissolving time among the compounds tested. It shows the higher molecular weight hydrocarbons dissolved slowly. The extraction time required is related to the total volume of extraction solvent and the void volume of extraction system.
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Selective extraction In SFE, simple separation can be achieved by selecting the solubility. Figure 14 shows an example of selective extraction. It shows the C40 has low solubility at the extraction conditions of 100 atm., 80 "C and 15 minute. Figure 15 also shows the selective extraction. Figure 15 (B) shows the selective discrimination of molecules whose molecular weights are higher than C50. The large molecules required a longer time to dissolve in the extraction solvent than a smaller one. It was also slowly injected into the column, which caused discrimination. The class-selective analysis using different extraction pressure was demonstrated [32,34,39].
APPLICATION OF ON-LINE SFE / GC Typical applications of SFE / GC were listed in the reviews [6,7,711.They are roughly divided into followings; the pollutant from environmental solids [32,35polymer 37,47,48],the adsorbed compounds from sorbent resins [33,34,40,511, In additives [441,and the flavor o r fragrance from food products [38,39,41,43,54]. these applications, the most emphasized is the merit of analysis time saving. Compared to the thermal desorption method, on-line SFE / GC technique can recover the higher molecules without using high temperature [35,36,40,42,1. Figure
g 1.00 El
0
P Q)
k
4 Q)
4
2 c20 c30 c40
0
200/15/50
150/15/50 100/15/50 100/15/80
Extraction condition (at& time/ temp.)
Figure 14. Effect of extraction conditions (C20=1.00). Peak heights of alkanes extracted under various SFE conditions. Volume of extraction vessel 110~1,Sample; C20, C30,C40 n-alkanes (50ng / each component).
270
I
I
I
25
20
40 I Timebin)
I
35
30
!6 C30 c22 c20
j
C32 C36 C40 C44
*C50 \
I
'/ F"" ~
Figure 15. Selective &actionation of C12-C60 mixture. Column oven temperature profile; 100°C 2min up t o 380°C a t the rate of 10°C / min hold 15 min. Injector and detector temperature 380°C. SFE conditions; (A) 100atm, 8O"C, 15min, (B) 300atm, 50"C, 15min. (GC analysis start at 15 minutes). * Apparently discriminated components
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16 shows the chromatograms obtained using carbon dioxide as a carrier gas [72]. The separation is carried out under a low linear flow velocity of carrier gas on a narrow bore column. It shows that a high resolution is obtained using a dense gas. In this case, the extraction solvent (carbon dioxide) does not need to be replaced by the other carrier gas such as helium. Another example of on-line SFE / GC is shown in Figure 17. On-line SFE / GC is powerful system for the GC analysis of various kind of samples. It has a short history, but it will become an important technique for the sample introduction method in GC analysis. Separation techniques such as on-line SFC / GC [73] and on-line SFE / SFC / GC will also make GC widely applicable.
E h I
0
I
10
(B)
I
0
I
10
I
20
I
30
II I
20
I
30
I
40
Time(min)
I
40
Timebin)
Figure 16. Effect of mobile phase on on-line SFE / GC; (A) carbon dioxide, (B) helium. Sample; low density polyethylene, GC column inlet pressure 0.8atm, Column oven temperature profile; 50°C 2min up to 300°C a t the rate of 8°C / min. hold 15 min. Capillary column; 15m x 250pm i.d., methylsilicone O . l p m , Detector FTD, SFE conditions; 200atm, 50"C, lmin, cell volume 1lOpl.
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I/ 10
20
I
30 40 Time(min)
10
20
40 Time(min)
30
Figure 17. Application of on-line SFE / GC on tea samples; (A) tea 7.9mg, (B) green tea 15mg. Analytical conditions; Mobile phase : C02, Column oven temperature profile; 50°C 2min up to 300°C at the rate of 8"C/min hold 15 min. Capillary column; 15m x 250pm i.d. methylsilicone O.lpm, Detector; FID range lO"1, SFE conditions; 250atm 60°C lmin , cell volume 110~1,solvent CO2.
ACKNOWLEDGMENT We thanks Hiroko Tatematsu for the assistance of the study.
REFERENCES 1 J. M. L. Penninger, M. Random, M. A. McHugh, V. J. Krukonis (eds.),
Supercritical Fluid Techonlogy, Elsevier, Amsterdam, 1985. 2 K. P. Johnston, J. M. L. Penninger (eds.), Supercritical Fluid Science and Technology, American Chemical Society, Washington, DC, 1989. 3 B. A. Charpentier, M. R. Sevenants (eds.), Supercritical Fluid Extraction and Chromatography. Techniques and Applications,American Chmical Society, Washington, DC, 1988. 4 R. M. Smith (ed.), Supercritical Fluid Chromatography, The Royal Society of Chemistry, London, 1988. 5 C. M. White (ed.), Modern Supercritical Fluid Chromatography,Huthig, Heidelberg, 1988. 6 M. L. Lee, K. E. Markides (eds.),Analytical Supercritical Fluid Chromatography and Extraction, Chromatography Conferences, Inc., Utah, 1990. 7 S. B. Hawthrne, Anal. Chem., 62 (1990) 633A. 8 J. W. King, J. Chromatogr. Sci., 27 (1989)355.
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9 K. G. Furton, J. Rein, Anal. Chim. Acta, 236 (1990) 99. 10 S. K. Poole, T. A. Dean, J . W. Oudsema, C. F. Poole, Anal. Chim. Acta, 236 (1990) 3. 11 W. Pipkin, LC. GC., 10 (1992) 14. 12 M. R. Andersen, J. T. Swanson, N. L. Porter, B. E. Richter, J. Chromatogr. Sci., 27 (1989) 371. 13 S. R. Lipsky, M.L.Duffy, LC. GC., 4 (1986) 898. 14 S. R. Lipsky, M.L.Duffy, HRC & CC, 9 (1986) 376. 15 S. R. Lipsky, M.L.Duffy, HRC & CC, 9 (1986) 725. 16 Y. Takayama, T. Takeichi, S. Kawai, HRC, 11(1988) 732. 17 T. Welsch, U. Teichmann, HRC, 14 (1991) 153. 18 I. Hagglund, K. Janhk, L. Blomberg, A. BemgrAd, S.G. Claude, M. Lymann, R. Tabacchi, J. Chromatogr. Sci., 29 (1991) 396. 19 R. M. Campbell, M. L. Lee, Anal. Chem., 58 (1986) 2247. 20 M. Nishioka, D. G. Whiting, R. M. Campbell, M. L. Lee, Anal. Chem., 58 (1986) 2251. 2 1 K. S. Nam,S. Kapila, A. F. Vanders, P. K. Pun, Chemosphere, 20 (1990) 873. 22 D. M. Kassim, M. S. Hameed, Sep. Sci. and Tech., 24 (1990) 1427. 23 S. B. Hawthorne, D. J. Miller, Anal. Chem., 59 (1987) 1705. 24 B. W. Wright, C. W. Wright, R. W. Gale, R. D. Smith, Anal. Chem. 59 (1987) 38. 25 K. Sakaki, T. Yokochi, 0. Suzuki, T. Hakuta, JAOCS, 67 (1990) 553. 26 S. F. Y. Li, C. P. Ong, M. L. Lee, H. K. Lee, J. Chromatogr., 515 (1990) 515. 27 M. G. Randon, T. A. Nonis, Am. Lab. (1984) 17. 28 T. L. Chester, D. P. Innis, HRC & CC, 8 (1985) 561. 29 A. Wilsch, G. M. Schneider, J. Chromatogr. 357 (1986) 239. 30 J. W. Hills, H. H. Hill, Jr., T. Maeda, Anal. Chem. 63 (1991) 2152. 3 1 S. B. Hawthorne, C.J.Miller, D. E. Nivens. D. C. White, 64 (1992) 405. 32 B. W. Wright, S. R. Frye, D. G. McMinn, R. D. Smith, Anal. Chem., 59 (1987) 640. 33 M. Lohleit, K. Bachmann, J. Chromatogr. 505 (1990) 227. 34 M. Lohleit, R. Hillmann, K. Bachmann, Z. Anal. Chem., 339 (1991) 470. 35 R. F. Mauldin, J. M. Vienneau, E. L. Wehry, G. Manmantov, Talanta, 37 (1990) 1031. 36 S. B. Hawthorne, D. J. Miller, J. Chromatogr. Sci., 24 (1986) 258. 37 S. B. Hawthorne, D. J. Miller, J. Chromatogr., 403 (1987) 63. 38 S. B. Hawthorne, M. S. Krieger, D. J. Miller, Anal. Chem., 60 (1988) 472. 39 S. B. Hawthorne, D. J. Miller, M. S. Krieger, Z. Anal .Chem., 330 (1988) 211. 40 S. B. Hawthorne, D. J. Miller, Anal. Chem., 6 1 (1989) 736. 4 1 S. B. Hawthorne, D. J. Miller, M. S. Krieger, J . Chromatogr. Sci., 27 (1989) 347. 42 J. H. Raymer, G. R. Velez, J. Chromatogr. Sci., 29 (1991) 467. 43 B. J. Murphy, B. E. Richter, J. Microcol. Sep., 3 (1991) 59. 44 S. Schmidt, L. Blomberg, T. Wannnman, Chromatgraphia,28 (1989) 400. 45 J. M. Levy, J. P. Guzowski, W. E. Huhak, HRC&CC, 10 (1987) 337. 46 S. A. Liebman, E. J. Levy, S. Lurcott, S. O'Neil, J. Guthrie, T. Ryan, S. Yocklovich, J. Chromatogr. Sci., 27 (1989) 118. 47 J. M. Levy, R. A. Cavalier, T. N. Bosch, A. F. Rynaski, W. E. Huhak, J. Chromatogr. Sci., 27 (1989) 341. 48 S. B. Hawthorne, D. J. Miller, J. J . Langenfeld, J. Chromatogr. Sci., 28(1990)
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2.
49 J . M. Levy, E. Storozynsky, R. M. Ravey, HRC, 14 (1991) 661. 50 R. J. Houben, Hans-G. M. Janssen, P. A. Leclercq, J. A. Rijks, C. A. Cramers, HRC,13 (1990) 669. 51 M. W. F. Nielen, J. T. Sanderson, R. W. Frei, U. A. Th. Brinkman, J. Chromatogr., 474 (1989) 388. 52 H. T. Badings, C. de Jong, R. P. M. Dooper, HRC & CC, 8 (1985) 755. 53 K. Hartonen, M. Jussila, P. Manninen, Maja-L. Riekkola,J. Microcol. Sep., 4 (1992) 3. 54 F. I. Onuska, K. A. Terry, HRC, 12 (1989) 527. 55 K. Grob, A. Habich, J. Chromatogr., 321 (1985) 45. 56 J. F. Pankow, HRC & CC, 9 (1986) 18. 57 B. Kolb, B. Liebhardt, L. S. Ettre, Chromatographia, 21 (1986) 305. 58 Y. Yokouchi, Y. Ambe, T. Maeda, Anal. Sci., 2 (1986) 571. 59 P. Sandra (ed), Sample Introduction in Capillary Gas Chromatography Volume 1,Huethig, Heidelberg, 1985. 60 K. Grob (ed), Classical Split and Splitless Injection in Capillary GC, Huethig, Heidelberg, 1988. 61 K. J. Hyver (ed), High Resolution Gas Chromatography, Hewlett-Packard, Avondale, 1989. 62 R. L. Grob (ed), Modern Practice of Gas Chromatography, J o h n Wiley & Sons, New York, 1985. 63 J. V. Hinshaw, Jr., J. Chromatogr. Sci., 25 (1987) 49. 64 J . V. Hinshaw, J. Chromatogr. Sci., 26 (1988) 142. 65 M. Herraiz, G. Reglero, E. Loyola, T. Herraiz, HRC&CC, 10 (1987) 598. 66 K. Grob, Z. Li, HRC, 11(1988) 626. 67 A. Tipler, G. Johnson, HRC, 13 (1990) 365. 68 B. W. Hermann, L. M. Freed, M. Q. Thompson, R. J. Phillips, K. J. Klein, W. D. Snyder, HRC, 13 (1990) 361. 69 P .L. Wylie, R. J. Phillips, K. J . Klein, M. Q. Thompson, B. W. Hermann, HRC, 14 (1991) 649. 70 T. Maeda, H. Tatematsu, F. Morishita, Proceedings of the 14th International Symposium of Capillary Chromatography, Baltimore, U.S.A.,(1992). 71 I. L. Davies, M. W. Raynor, J. P. Kithinji, K. D. Bartle, P. T. Willams, G. E. Andrews, Anal. Chem., 60 (1988) 683A. 72 T. Maeda, H. Tatematsu, F. Morishita, Anal. Sci. 7 (1991) 219. 73 J . M. Levy, J. P .Guzowski, Z. Anal. Chem., 330 (1989) 207.
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Chapter 13
Introduction of Directly Coupled SFE / GC Analysis Tsuneaki Maeda Research Center, DKK CORPORATION,4-13-14, Kichijoji Kitamachi, Musashinoshi, Tokyo, Japan
Toshiyuk~Hoboa ‘Department of Chemistry, Tokyo Metropolitan University, 1-1, Minami Ohsawa, Hachioji-shi, Tokyo, Japan
INTRODUCTION Recently, supercritical fluid is widely used for industrial processing such as decaffenation of coffee beans, extraction of bitter essence from hop and so on. It is because it has unique phase properties for separation as well as extraction. Since supercritical fluid is much easily produced than before using the advanced technologies, it became much popular and created new fields like supercritical fluid chromatography (SFC) and supercritical fluid extraction (SFE). Application of these techniques is increasing in these days. The supercritical region is defined as the one above the critical temperature and critical pressure. This region gives a single phase and the substance in this region is called supercritical fluid. It is the forth state of the substance. One of the unique properties of the fluid, solubility, depends on the density which can be controlled by changing the pressure and temperature. The diffusion coefficient of the fluid lays in the midst of gas and liquid, which means that the mass transfer rate is faster in the fluid than liquid. Then if it is used for extraction in place of liquid, the extraction time could be made shorter. The low viscosity allows it to be used as the mobile phase for capillary and micropacked column SFC. These fundamental part of supercritical fluid techniques and applications of both SFE and SFC have been described in many literatures [l61. The analytical SFE is often compared with solvent extraction methods and between them there exist large differences. Actually they have many advantages and disadvantages. For analytical purposes, particularly for solid sample analysis, SFE is superior t o liquid extraction method, namely Soxhlet method, in speed, solvent waste problem, and so on. As the sample preparation method for chromatography, SFE is a relatively new method of choice. In this chapter, directly coupled SFE / GC analysis method which is one of so called hyphenated methods being used in the instrumental analysis is described. It has such a convenience that the sample preparation, separation and detection can be done in a straight line. Advantageously the technique can provide the followings; a) simplify the sample preparation procedure. b) make the analysis time shorter.
256
c) minimize the contamination. d) maximize the use of capabilities of both methods. At the same time, however, the method requires the understanding of the both methods coupled. This chapter starts with the overview of SFE for GC,then describes on the chracteristics of GC and SFE, and finally on the on-line SFE / GC interface and its application. SFE FOR GC SFE has been used intensively and shown that it has a great capability in the sample preparation for GC as well as for other chromatographies, i.e, SFC, LC, IR, MS and so on. Many reviews pointed out the prospect of SFE and described opinions on the analytical scale SFE [7-121. Basically, there is almost no difference between off-line and on-line SFE. Limitation of On-line SFE I GC It is reasonable to think that the sample range applicable t o the on-line SFE / GC is limited by the fundamental circumstances of GC. Although supercritical fluid can dissolve liquid samples of high boiling point, GC can not analyze those samples which has low volatility and thermally unstable substances. In Figure 1, molecular ranges of substances which can be handled by the ordinary chromatographies and SFE are presented. Another aspect is that the solubility of a compound in the supercritical fluid can not exceed that in liquid of same material. It means that the compound that is hard to dissolve i n t o the liquid is harder to dissolve into the solvent in the supercritical fluid state. In addition, the higher molecular weight or polar compounds are difficult t o be dissolved i n t o the supercritical fluid as far as non-polar C02 or slightly polar NzO are used as the fluid material. Probably the limitation also comes from SFE side. One of the limitation from GC side can be overcome by using a high temperature capillary column such as aluminum claded fused silica capillary column with stable stationary phase. Nowadays, up to CIOO can be analyzed using these technically advanced systems [13-181. It is, however, still not enough to cover the whole range of SFE. It can be pointed out that unexpected or undesirable components extracted by a SFE will be carried into the GC system and disturb analysis. In case of an off-line SFE, further clean-up or pretreatment step can be employed t o eliminate such interferences [8]. Although online SFE / GC is a simple technique, aplicable sample is limited compared t o the offline SFE. As the result, on-line SFE / GC method requires suitable sample selection and appropriate setting of extraction conditions.
SFE as a sample preparation technique From the sample preparation point of view, SFE has two types of technique for controlling the solubility strength. One is to use a single solvent and control its strength by changing the pressure and temperature. The solubility,power of dissolution, in supercritical fluid is related to the density of the fluid which can be controlled by the temperature and pressure. Thereafter, using single solvent, selective extraction could be expected. Several off-line SFE studies reported the possibility of fractionation or class-selectiveextraction [8,12,19-221. These techniques are also useful for the on-line SFE. Various kinds of fluid materials have been investigated and compared each other. From the gas chromatographicpoint of view, SFE using
257
Molecular weight range applicable to the on-line SFE / GC
LC(SEC)
SFC
GC
SFE 100
lo*
lo2
lo3
lo4
lo5
10 lo7 Molecular weight (daltons)
Figure 1. Molecular weight range applicable to the chromatography and SFE. liquefied gas is desirable. Further, intentional solvent-solute separation is not required because of its property; liquefied gas is evaporatively separated from the solute in the collection device by depressurization. Usually, carbon dioxide is the primary choice for the experiments, but it has no polarity. Therefore, many other fluid materials including NzO, CHF3, CHClF2, SF6, C2H6, i-CdH10are used as they show characteristic properties [7,23-261. The other technique is solvent polarity control method either by adding a polar solvent, which is called modifier or entrainer, or by changing solvent itself. An example for the latter case is from non polar C02 to slightly polar N20. As the modifier, methanol is commonly used. These techniques are applicable to the on-line SFE / GC, too. Basically, the role of SFE is just to dissolve the component in the sample matrix and transfer to the collection place. It is advisable to do the preliminary test and select the optimal solvent and extraction conditions using an off-line SFE before applying sample to the on-line SFE. Important thing to think about when the technique is chosen is what is the goal of analysis, whether it requires trace analysis or profile analysis. It is also important to get the information about sample matrix, properties, interferences, co-existing components and the concentration of analytes. Putting these information into consideration the best sample preparation procedure should be constructed. Basically, the process of construction is same as in other sample
258
preparation technique.
Optimization of SFE conditions There are many factors to be considered depending on the goal of analysis, so as to achieve optimum extraction efficiency . If the goal is to get the profile or matrix composition of a sample, it is required to use the fluid at the maximum solubility. On the contrary, if the trace components in the sample are the analytes, it does not need to think about maximum solubility. In this case, as King [8] described, it is better t o choose the condition that can separate the analytes from the matrix without interference. The density of fluid increases with increasing pressure and the diffusivity increases with the temperature, which means that time saving extraction can be achieved at high temperature. These points are described by the reports showing the change of k at different temperatures [27-291. Supercritical fluid behaves like gas at high temperature and low density, and like liquid a t low temperature and high density. Next point is the elimination of the matrix effect. If a test gave a sufficient solubility and good recovery from standard solution,but poor recovery from actual sample, the interaction between targeted component and matrix must be considered. In this case, first the extraction time should be extended. Then, either the use of polar modifier o r change of the fluid to a more polar one such as N2O and Freon should be tried. Another way t o eliminate matrix effect is the addition of some other modifier which can break the interaction between solute and matrix. The selection of such a modifier should be done depending not on the solubility, but on the interaction. These are just rough guidelines to pursue the optimum SFE condition. Another approach to obtain good recovery of polar components is a derivatization of analytes in the fluid [30,311. Lastly, it is important t o remember that SFE is the time saving technique, but still requires time to dissolve the analytes. It can not dissolve the objectives in a moment. CONSTRUCTION of ON-LINE SFE / GC SYSTEM On-line SFE / GC construction is shown in Figure 2. There are three important parts. They are a solvent delivery pump, a SFE oven and a GC. The system is very simple to build up. The high pressure pump deliver the fluid at a higher pressure than the critical one and the extraction vessel is put in a oven to keep the temperature higher than the critical one. The examples of extraction vessel design are shown in Figure 3. An empty column or a guard column are commonly used for the vessel. The restrictor is connected after the SFE vessel t o maintain the extraction pressure high, and passing in it the pressure is reduced to meet the GC analysis. As a restrictor, fused silica capillary tubing or crimped tubing are usually used. The former is a linear restrictor and the latter is a tapered one. Extraction solvent is introduced into the GC as a gas and the solutes in the gas are concentrated in the inlet of the column during extraction. The most important part of on-line SFE / GC is the interface between SFE and GC.
SFE / GC INTERFACE There are many types of sample introduction system for capillary gas chro-
259
matography developed aiming to get a good separation. These systems allow liquid or gaseous samples introduced into the GC in a narrow band. Selection of a system is dependent on the sample to be analyzed. But in case of SFE, special consideration different from the direct sample introduction technique in the GC will be required. The solvent commonly used in SFE is liquefied gas (CO2, N20, SF6 etc.), which changes to the gaseous state after depressurization. Therefore, the solvent is a gas when it is introduced into the interface of a on-line SFE / GC. The volume of extraction solvent will expand about a thousand times ( I d of liquid C02 expands to about 560ml CO2 gas). Besides, for SFE it requires a few minutes to finish in the case of a micro extractor is used. It must be considered that a large amount of gaseous solvent must be separated and removed from the solutes in the interface. It takes long time for the completion of a SFE when a micro scale extraction vessel is used. Therefore a cryogenic focusing technique should be applied t o prevent the band spreading. In this section, the interfaces so far reported are classified and overviewed. Direct injection system Figure 4. shows the schematic diagram of direct injection system [32-351. This type of system uses a heated tee piece joint to prevent the plugging. The end of the restrictor is put inside of the retention gap and the carrier gas enters coaxi-
Extraction oven Extraction vessel
Flow restrictor
Interface
1
I
I I
Caniergas
L
-Detector
.Column Liq. CO2
Solvent delivery system for SFE (High pressure pump)
Gas chromatograph
Figure 2. Schematic diagram of a typical on-line SFE / GC.
260
ally along the restrictor. Whole solvent will flow through the column and whole extracted compounds are introduced into the column. The extracted compounds are focused in the retention gap. The retention gap also prevents the deposition of the non-volatile components in the column. During extraction, the carrier gas flow is stopped in order to get rid of the high back pressure effects. In this system, the flow rate of a SFE solvent is restricted by the capillary column and utilization of a micro extraction system is just recommended. Thus, when a vessel of large volume is used, intermittent introduction of the extractant, Like a heart cutting technique in multidimensional chromatography,should be employed [33,34]. Wright et al. [321
1/16"Tube 4to
restrictor
Ferrule 1/16"o.d.tube 1.Micro extraction vessel(
Il16"Tube
__+
q , , &---/
wlqy
Joint body
Nut
Extraction cell Ferrule lI4"o.d.tube 2. Semi micro extraction vesselb0.lml)
J o i n t body
Filter
&/16"Tube --+to restrictor Ferrule
-b
Filter Extraction cell Ferrule 1/4"o.d. or 318"o.d. tube 3. Conventional extraction vessel bO.5m.l) using empty column for liquid chromatography
t o restrictor
Ferrule
Figure 3. Examples of extraction vessels designed for on-line SFE /GC.
26 1
Extraction solvent inlet
Extraction solvent inlet
joint
+
Carrier gas inlet
-
ii
Retention gap
, I, I
o : C 0 2 , x : Light,
Figure 4. Direct injection system.
0
: Heavy
Figure 5. Direct cool on column injection system.
and Lohleit et al. [34] reported selective extraction method with some examples. Lohleit et al. [34] also investigated the performance of this system by comparing with the thermal desorption analysis. They showed that the former can analyze the higher molecules than the latter. This system might be applicable to the small volume extraction and wide range of boiling point samples.
Cool on column injection system This system is similar to the direct injection system but the depressurised extraction solvent is led out from the interface. A conventional cool on column injector was used after only taking out the septum [36-411. Figure 5. shows the schematic of this system. As the restrictor is inserted into the analytical column, the solutes are deposited in the column while it is cooled for a cryogenic focusing. The extracted components are retained in the liquid stationary phase and volatile components are vented with solvent. Since the restrictor is not heated in this system, plugging will be happened easily during extraction. Some modifications from on-line SFE / SFC t o on-line SFE / GC and another system similar t o the direct
262
Extraction solvent inlet
v
Extraction solvent inlet
Extraction vessel
P-
Restrictor Septum
/
o O* O01
Column
Figure 6. Split I' splitless injection system.
/---I
Column i
Figure 7. Programmed temperature vaporizer injection system.
injection system using a tee piece with a vent line were reported [12,42-441. These systems might be applicable to the trace analysis where large amount of matrix is not coextracted.
Split I' splitless injection system This system is the simplest one that does not need any modification of a conventional split I' splitless injector [45-491. Figure 6. shows the schematic diagram of this system. The restrictor is just inserted through the septum during the extraction and withdrawn after it. The injector is heated t o protect a plugging. The column is initially cooled to focus the extracted components. The solvent is finally
263
led to the split vent or septum purge vent. In this system, sample is splitted regardless of split or splitless mode, and the split ratio depends on the flow rate of extraction solvent. It means that the higher the extraction pressure is, the higher the split ratio is. This system is very easy to use but sample injection takes long time, and be resulted in a discrimination. This system is also similar t o the hot needle injection method where sample is injected very slowly. Though there is no maximum volume of extraction vessel, relatively high molecular weight compounds can not be introduced uniformly.
Programmed temperature vaporizer injection system For this system a conventional programmed temperature vaporizer injection system P"v)can be used without any modification [501. Figure 7. shows the schematic diagram of this system. The restrictor is just inserted through the septum during the extraction and withdrawn after it. The extraction solvent and the components with high vapor pressure exit from the solvent vent line. The injector is kept at sub-ambient temperature for trapping the extracted components inside the injector. At the end of the extraction the restrictor is withdrawn from the septum, the solvent vent line is closed and the programmed temperature operation of the injector is started. Then the whole trapped components kom the sample is introduced into the column and analysis is started. Houben et al. [501 described that since the extracted components are trapped by reduced vapor pressure, in this system, the complete trapping of the components is accomplished if they have boiling points at least 250°C higher than the trapping temperature. It seems that it has a chance t o have the plugging problem, because the restrictor is not heated. Similar range of samples to the cool on column injection system could be applicable. As for the volume of the extraction vessel, there is no restriction. Thermodesorption I cold-trap iltjection system This type of system is the modification of a thermodesorption I cold-trap injector [511. Figure 8. shows this system. In order to prevent plugging the restrictor is inserted into the heated inlet region, where usually an adsorbent-packed trap is placed. During the extraction the extracting solvent is wasted through the purge vent. The extracted components are trapped on the surface of the cold trap placed under the heated region. The property of this trapping system is the same as one in the PTV injection system. When the extraction is completed, the purge vent line is closed and the carrier gas line is opened with a rapid heating of the cold trap. The whole trapped compounds are led into the column. The original system [52] is developed for the volatile components. The maximum operating temperature of the cold trap region is 350°C. Since it is placed on the GC oven, relatively cool part exists between the cold trap and the analytical column. The semi-volatile components may deposit on this cool part. This system might be applicable to the volatile components. Miscellaneous irqiection system Some other systems were reported [53,541. The extracted sample is trapped in a loop which is connected to a 6 way port valve. After trapping, the loop is heated to introduce the extracted sample into the column. Figure 9 shows the schematic of this system. This system might be applcable to the volatile components.
264
Extraction solvent inlet
Extraction solvent inlet
Extraction vessel
Restrictor
Carrier gas
d?
/
Valve
i
Column
Figure 8. Thermodesorption-cold trap injection system.
Figure 9. Loop injection system.
Conclusion Above mentioned studies suggest that a restrictor heating and a cryogenic focusing should be used during extraction. There are a number of variations of the focusing technique for the volatile sample introduction [55-581. Since the focusing temperature should be higher than the freezing point of SFE solvent, the cryogenic focusing using liquid nitrogen can't be applied. It's also important to understand the properties of a conventional GC injection system when a conventional injection system is used as the interface. There are many literatures on the capillary column GC sample injection system [59-691. It is recommended to refer those information before selecting the interface. The sample injection system is very important part of the capillary column GC. Though many systems are developed and applied, those words of which Sandra described in his book [591 are still alive; "Sample introduction in capillary gas chromatography has always been, and in fact still is, a problem. There is still no such thing as a universal injection system and there probably never will be." The situation about on-line SFE / GC interface is absolutely the same, as far as it is using capillary column.
265
PRACTICE OF ON-LINE SFE / GC Most of the papers reported on the instrumentation and application of online SFE / GC have been referred in the previous section. Here, evaluation of practical on-line SFE / GC system using a conventional split / splitless injector will be presented on the basis of authors' experiments. The flow diagram of the system developed is shown in Figure 10 [701. A micro extraction vessel, approximately 110 pl, was used. An inner diameter of 20 pm and 10 cm long fused silica capillary
High pressure C02
\L
----------
6 way port
Extraction vessel
Septum purge vent Split / splitless injector Backpressure valve Column head pressure
Column oven i _____---__________--_____--
to FID S.V.; 3 way port solenoid valve Splitless injection; Carrier gas flow through N.C. to C.
Figure 10. Flow diagram of on-line SFE / GC system.
266
Table 1 Anakytical GC conditions Column Purge vent flow Carrier gas Column head pressure Injector temperature Detector Detector temperature
Methyl silicone, 0.25mm i.d., 5m long, film thickness 0. lum (Quadrex) 28dmin He 0.7kg/cm2 300°C FID 300°C
(A)
yo 1
.o
I 15
I 10
15
I
c[
I 20
I
20
-
I
2.5
Time(mm)
I
10
I
15
I
20
I
25
I
30 Time(min
I
25 Time(mn)
Figure 11.Effect of cryogenic focusing on separation. (A) Without focusing, column oven temp. 100°C, (B) with focusing, column oven temp. lOO"C, (C) without focusing, column oven temp. 50°C, (D) with focusing, column oven temp. 50°C.
267
(B) C 14
C 14
c20
C30
-
.5
20
25
C30
c20
30 Time(min)
1
15
I
20
I
25
I
30 Timebin)
Figure 12. Chromatograms obtained using different injection modes. Conventional split / splitless injector for GC was used; (A) Split injection mode, (B) splitless injection mode. tubing was used as a restrictor. The cryogenic focusing system is adapted inside the column oven t o focus the extracts in the front part of the column. Analytical conditions are presented in Table 1. Alkanes were used for the standard sample. A narrow-bore capillary column with thin film stationary liquid phase was used t o separate high molecular weight alkanes.
Cryogenic focusing The cryogenic focusing effect is shown in Figure 11. In this system, since the extracted components are trapped in the stationary liquid phase of the column, the cryogenic focusing depends on the oven temperature, film thickness of the liquid phase and focusing time. As the boiling points of target components are high enough, focussing can be effected at the top of the column, when the column oven is kept at near ambient temperature. The effect of focusing was reported elsewhere [38,41,711. Differences of split I splitless injection This system has a conventional split / splitless injector for capillary column GC, but its function is different from conventional use described in previous discussion. Figure 12 shows the chromatograms obtained using both split and splitless injections. Figure 12 (A) was obtained at the split ratio of 1/ 25 and (B) was obtained using the splitless injection. The chromatogram(B) shows the extracted components were escaped with extraction solvent even in the case of splitless mode. Extraction time and extraction pressure When the supercritical fluid has enough solubility for the sample components, two important factors should be considered, in order to complete the extraction successfully. The first is the sample transfer time and the second is the time to dissolve in the solvent. Figure 13 shows the recovery changes under various pressure and extraction time. Figure 13 (A) shows that at 200 atm., the fluid has
-
c20 c30 DI c40
g
3
5
3
5
10
15
10
15
Extraction time (min)
1.0
% W b
?
3
2
2
0.5
0.0
Extraction time(min)
figure 13.Effect of extraction time on relative recovery (peak height of each component after 15 min extraction = 1.00). Sample; C20, C30, C40 n-alkanes (50ng/ each component), Volume of extracion vessel 110~1,Extraction temperature 50°C, Extraction pressure; (A) 2OOatm, (B) 150atm. enough solubility but 3 minute extraction time is not enough because of the inunifonnity of the recoveries of each component. The reason is that the transfer time depends on the void volume of the extraction vessel. Figure 13 (B) shows the differences of dissolving time among the compounds tested. It shows the higher molecular weight hydrocarbons dissolved slowly. The extraction time required is related to the total volume of extraction solvent and the void volume of extraction system.
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Selective extraction In SFE, simple separation can be achieved by selecting the solubility. Figure 14 shows an example of selective extraction. It shows the C40 has low solubility at the extraction conditions of 100 atm., 80 "C and 15 minute. Figure 15 also shows the selective extraction. Figure 15 (B) shows the selective discrimination of molecules whose molecular weights are higher than C50. The large molecules required a longer time to dissolve in the extraction solvent than a smaller one. It was also slowly injected into the column, which caused discrimination. The class-selective analysis using different extraction pressure was demonstrated [32,34,39].
APPLICATION OF ON-LINE SFE / GC Typical applications of SFE / GC were listed in the reviews [6,7,711.They are roughly divided into followings; the pollutant from environmental solids [32,35polymer 37,47,48],the adsorbed compounds from sorbent resins [33,34,40,511, In additives [441,and the flavor o r fragrance from food products [38,39,41,43,54]. these applications, the most emphasized is the merit of analysis time saving. Compared to the thermal desorption method, on-line SFE / GC technique can recover the higher molecules without using high temperature [35,36,40,42,1. Figure
g 1.00 El
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Figure 14. Effect of extraction conditions (C20=1.00). Peak heights of alkanes extracted under various SFE conditions. Volume of extraction vessel 110~1,Sample; C20, C30,C40 n-alkanes (50ng / each component).
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Figure 15. Selective &actionation of C12-C60 mixture. Column oven temperature profile; 100°C 2min up t o 380°C a t the rate of 10°C / min hold 15 min. Injector and detector temperature 380°C. SFE conditions; (A) 100atm, 8O"C, 15min, (B) 300atm, 50"C, 15min. (GC analysis start at 15 minutes). * Apparently discriminated components
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16 shows the chromatograms obtained using carbon dioxide as a carrier gas [72]. The separation is carried out under a low linear flow velocity of carrier gas on a narrow bore column. It shows that a high resolution is obtained using a dense gas. In this case, the extraction solvent (carbon dioxide) does not need to be replaced by the other carrier gas such as helium. Another example of on-line SFE / GC is shown in Figure 17. On-line SFE / GC is powerful system for the GC analysis of various kind of samples. It has a short history, but it will become an important technique for the sample introduction method in GC analysis. Separation techniques such as on-line SFC / GC [73] and on-line SFE / SFC / GC will also make GC widely applicable.
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Figure 16. Effect of mobile phase on on-line SFE / GC; (A) carbon dioxide, (B) helium. Sample; low density polyethylene, GC column inlet pressure 0.8atm, Column oven temperature profile; 50°C 2min up to 300°C a t the rate of 8°C / min. hold 15 min. Capillary column; 15m x 250pm i.d., methylsilicone O . l p m , Detector FTD, SFE conditions; 200atm, 50"C, lmin, cell volume 1lOpl.
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Figure 17. Application of on-line SFE / GC on tea samples; (A) tea 7.9mg, (B) green tea 15mg. Analytical conditions; Mobile phase : C02, Column oven temperature profile; 50°C 2min up to 300°C at the rate of 8"C/min hold 15 min. Capillary column; 15m x 250pm i.d. methylsilicone O.lpm, Detector; FID range lO"1, SFE conditions; 250atm 60°C lmin , cell volume 110~1,solvent CO2.
ACKNOWLEDGMENT We thanks Hiroko Tatematsu for the assistance of the study.
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