Partial Modulation Method via Pulsed Flow Modulator for

Anal. Chem. 2004, 76, 6064-6076

Partial Modulation Method via Pulsed Flow Modulator for Comprehensive Two-Dimensional Gas Chromatography Huamin Cai* and Stanley D. Stearns

VICI Valco Instruments Co. Inc., P.O. Box 55603, Houston, Texas 77255

A partial modulation method by using a pulsed-flow modulator for comprehensive two-dimensional gas chromatography is proposed. The method is based on the fact that when a pulsed flow of inert gas is introduced into the conjunction between a primary and a secondary column, the concentration of analyte is disturbed, and a plug of higher or lower concentration is created. The plug, which forms a spike signal coupled to the primary GC signal, is then separated in a secondary column, creating a new dimension of GC information. The modulation is partial because only a fraction of the primary signal is modulated and converted into the secondary signal; the remaining primary signal stays unchanged. Therefore, this method yields a comprehensive two-dimensional chromatogram and a primary one-dimensional chromatogram in a single GC run. In this study, the modulation mode, modulation index, and modulation percentage are discussed and the reproducibility of peak areas and retention time are investigated. With a 5.8% modulation percentage and a primary peak half-width 1.7 times wider than the modulation time, the standard deviation for the peak areas are 0.15% for the primary and 0.78% for the secondary chromatograms. Chromatograms of laboratory-mixed hydrocarbons and of high-temperature fuel oil no. 6 standard are demonstrated. Comprehensive two-dimensional gas chromatography (GC×GC) is effective in analyzing complex samples because it has high peak capacity and because it can get more structural information by using primary and secondary columns of different polarities. To achieve a comprehensive two-dimensional GC×GC analysis, a modulator must be used to modulate the relatively broad primary dimension peak into a series of sharp peaks with a certain frequency. This series of sharp peaks passes though the secondary column to yield another dimension of information. Many different types of modulators have been developed. They can be roughly divided into two typessthermal modulators1-19 and valve switch modulators.20-24 Thermal modulators can be further divided into * To whom correspondence should be addressed. E-mail: [email protected]. (1) Phillips, J. B.; Liu, Z. U.S. Patent 5196039, 1993. (2) Liu, Z.; Phillips, J. B. J. Chromatogr. Sci. 1991, 29, 227-231. (3) de Geus, H. J. J. Chromatogr., A 1997, 767, 137-151. (4) Burger, B. V.; Snyman, T.; Burger, W. J. G.; van Rooyen, W. F. J. Sep. Sci. 2003, 26, 123-128. (5) Phillips, J. B.; Ledford, E. B. Field Anal. Chem. Technol. 1996, 1, 23-29.

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the resistively heated trap,1-4 heated sweeper,5-7 and cryogenic focus.8-19 Finally, the cryogenic focus method has two major different types: longitude movable trap8-13 and jet trap.14-19 Several articles have been published for reviewing and comparing these modulators.25-29 A group led by Philips did pioneering work in developing comprehensive two-dimensional chromatography with a thermal modulator. In 1991, Phillips and Liu1 reported a novel thermal modulator that uses two-stage thin-film resistant heaters. Many new versions of this type have been developed since. Further (6) Phillips, J. B.; Gaines, R. B.; Blomberg, J.; van der Wielen, F. W. M.; Dimandja, J. M.; Green, V.; Granger, J.; Patterson, D.; Racovalis, L.; de Geus, H. J.; de Boer, J.; Haglund, P.; Lipsky, J.; Sinha, V.; Ledford, E. B. J. High Resolut. Chromatogr. 1999, 22, 3-10. (7) de Geus, H.-J.; Schelvis, A.; de Boer, J.; Brinkman, U. A. Th. J. High Resolut. Chromatogr. 2000, 23, (3) 189-196. (8) Marriott, P. J.; Kinghorn, R. M. Anal. Chem. 1997, 69, 2582-2588. (9) Kinghorn, R. M.; Marriott, P. J. J. High Resolut. Chromatogr. 1998, 21, 620622. (10) Kinghorn, R. M.; Marriott, P. J. J. High Resolut. Chromatogr. 1999, 22, 235238. (11) Kinghorn, R. M.; Marriott, P. J.; Dawes P. A. J. High Resolut. Chromatogr. 2000, 23, 245-252. (12) Marriott, P. J.; Ong, R. C. Y.; Kinghorn, R. M.; Morrison P. D. J. Chromatogr., A 2000, 892, 15-28. (13) Marriott, P. J.; Kinghorn, R. M. J. Chromatogr., A 2000, 866, 203-212. (14) Ledford, E.. Jr.; Billesbach, C. J. High Resolut. Chromatogr. 2000, 23, 202204. (15) Beens, J.; Adahchour M.; Vreuls, R. J. J.; van Altena, K.; Brinkman, U. A. Th. J. Chromatogr., A 2001, 919, 127-132. (16) Beens, J.; Dalluge, J.; Adahchour M.; Vreuls, R. J. J.; Brinkman, U. A. Th. J. Microcolumn Sep. 2001, 13 (3), 134-140. (17) Hyo1tyla1inen, T.; Kallio, M.; Hartonen, K.; Jussila, M.; Palonen, S.; Riekkola, M.-L. Anal. Chem. 2002, 74, 4441-4446. (18) Pursch, M.; Eckerle, P.; Biel, J.; Streck, R.; Cortes, H.; Sun, K.; Winniford, B. J. Chromatogr., A 2003, 1019, 43-51. (19) Harynuk, J.; Go´recki, T. J. Chromatogr., A 2003, 1019, 53-63. (20) Bruckner, C. A.; Prazen, B. J.; Synovec, R. E. Anal. Chem. 1998, 70, 27962804. (21) Prazen, B. J.; Bruckner, C. A.; Synovec, R. E.; Kowalski, B. R. J. Microcolumn Sep. 1999, 11 (2), 97-107. (22) Seeley, J. V.; Kramp, F.; Hicks, C. J. Anal. Chem. 2000, 72, 4346-4352. (23) Sinha, A. E.; Johnson, K. J.; Prazen, B. J.; Lucas, S. V.; Fraga, C. G.; Synovec, R. E. J. Chromatogr., A 2002, 983 (1-2), 195-204. (24) Sinha, A. E.; Prazen B. J.; Fraga, C. G.; Synovec, R. E. J. Chromatogr., A 2003, 1019, 79-87. (25) Bertsch, W. J. High Resolut. Chromstogr. 1999, 22 (12), 647-665. (26) Phillips, J. B.; Beens, J. J. Chromatogr., A 1999, 856, 331-347. (27) Bertsch, W. J. High Resolut. Chromatogr, 2000, 23(3), 167-181. (28) Pursch, M.; Sun, K.; Winniford, B.; Cortes, H.; Weber, A.; McCabe, T.; Luong, J. Anal. Bioanal. Chem. 2002, 373, 356-367. (29) Kristenson, E. M.; Koryta´r, P.; Danielsson, C.; Kallio, M.; Brandt, M.; Ma¨kela¨, J.; Vreuls, R. J. J.; Beens, J.; Brinkman, U. A. Th. J. Chromatogr., A 2003, 1019, 65-77. 10.1021/ac0492463 CCC: $27.50

© 2004 American Chemical Society Published on Web 09/15/2004

Figure 1. Modulation processes of full modulation method (A), partial modulation with negative pulsed flow method (B), and partial modulation with positive pulsed flow method (vacancy chromatography method) (C).

research leading to a heated sweeper5-7 has been more successful. Both the thin-film resistant heater and the heated sweeper use a thick stationary phase to hold the primary effluent, periodically heating it to release and focus the fraction. A group led by Marriott8-13 succeeded in using a moving cryogenic trap as a modulator. The moving cryogenic trap cryogenically holds and focuses the primary effluent and releases it by instantly moving the trap away from the cooling zone. A jet cooling system14 reported by Ledford and Billesbach, and commercialized by Zoex Co., functions similarly to a moving cryogenic trap. However, it has no moving parts, switching from cryogen agent to hot air during the release period to quickly desorb the analyte. Almost all the thermal modulation methods enhance the GC signal tremendously. The development of high-speed diaphragm valves opens the possibility of using a valve as a GC×GC modulator, as successfully demonstrated by the Synovec group.20,21,23,24 When a valve is used as a modulator, the effluent from the primary column is fed into a sample loop and periodically injected into the secondary column. The valve switch modulator is very simple, has no breakthrough problems, and does not require liquid cryogensall major advantages. However, sensitivity is reduced, since only a fraction of the primary effluent is delivered to the secondary column and most is wasted. Seeley et al.22 developed a differential flow method that uses low primary and high secondary column flow rates. Their method lets as much as 80% of the primary effluent into the secondary column, consequently improving the sensitivity of the valve switch method. Almost all the modulators described use a full-modulation method, modulating the entire primary signal into the secondary signal. The valve switch modulator converts only a fraction of the primary signal to the secondary but is also usually categorized as

a full modulation method since it uses the injected portion to represent the sample through the whole modulation time, discarding the remains as waste. Recently30,31 we introduced a new partial modulation method via a pulsed flow modulator to achieve comprehensive two-dimensional GC separation. This method modulates only a part of the primary signal, so it has the following advantages: (1) the modulator is simple, with no sample volatility and oven temperature limits; (2) it can create much narrower secondary peaks; and (3) it gives both primary 1-D and comprehensive 2-D chromatograms in one GC run. However, the sensitivity of the method is not as high as that of full modulation method. In this paper we will discuss its implementation, characteristics, and performance. THEORY Partial Modulation Method. The partial modulation method is similar to the full modulation method, but with some key differences. Comparing the two methods is helpful in understanding the characteristics of the partial modulation method. Figure 1 shows flowcharts for these two processes with computergenerated data, where (A) is the full modulation method and (B) is the partial modulation method. Both processes start with two relatively broad peaks eluted from a primary column. For purposes of illustration, these two components are designed such that they can be partially separated in the primary column (a1) and (b1) and completely separated in the secondary column (a3) and (b3). In the full modulation process, the chemical signal of these two primary peaks is completely modulated into a series of sharp (30) Cai, H.; Stearns, S. D. Gulf Coast Conference, Galveston Island, TX, 2002; Abstr. 113. (31) Cai, H.; Stearns, S. D. Gulf Coast Conference, Galveston Island, TX, 2003; Abstr. 115.

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signals such as “spikes”, as in Figure 1 from (a1) to (a2). The amplitude of these “spikes” (the modulation envelope) follows the shape of the primary peaks, but at higher intensity. The higher amplitude of the “spike” as compared with the original primary signal is due to the modulator effectively “squeezing” all the primary chemical signals in the modulation time (usually a few seconds) into a narrow band of 10-100 mss∼100 times shorter. These squeezed sharp peaks are input into the secondary column for the secondary dimensional separation, as shown in Figure 1 from (a2) to (a3). Each “spike” in this series can be considered as one injection of the secondary dimension. If the “spike” contains two or more components, they could possibly be separated in the secondary dimension, shown in Figure 1 (a3). The final detector signal is then demodulated into a two-dimensional chromatogram presented in a contour plot format as in Figure 1 (a4). For the partial modulation method in Figure 1B, only a fraction of the primary signal is modulated into a secondary signal, with the remaining primary signal kept unchanged. The modulated sharp peaks are added to the primary signal and then fed into the secondary column. During the secondary dimensional separation, only the modulated signal, or “spikes” portion, goes through the separation process; the primary signal portion passes the secondary column with little effect, as shown in (b2) and (b3). Unlike in the full modulation method, the signal from the secondary column effluent (the detector signal) is a combination of the primary and secondary signals, which need to be separated. After the separation, we get two signalssprimary and secondary. The secondary signal is then demodulated into a 2-D chromatogram as in Figure 1 (b6), which is similar to the one generated by the full modulation method shown in (a4). So, the partial modulation method can yield a comprehensive 2-D chromatogram and a primary 1-D chromatogram simultaneously. Figure 1C shows a modulation process using a positive pulsed flow method, as opposed to the process in Figure 1B, which uses a negative pulsed flow to create a positive modulated signal. The modulator actually injects a series of pure carrier gas pulses into the secondary column and creates a series of negative peaks (c2). If the effluent from the primary column is a two-compound mixture, the negative peaks will be separated in the secondary column, resulting in two negative peaks that have the same retention characteristics as the positive peaks in the negative pulsed flow mode (c3). This GC process is actually vacancy chromatography, a technique first introduced by Zhukhovitsiski and Turkel’taub,32 who used it for process monitoring with a gas chromatograph. As in process B, after the detector signal separation, we get two signalssprimary and secondarysbut the secondary signal requires a reversal of the polarity. Compared with the full modulation method, the partial modulation method has the advantages of ease of implementation, narrower secondary peaks, and preservation of the primary signal during creation of the second dimension. The primary signal obtained by this method is real (not a reconstruction of the secondary signal), so it retains the resolution and accuracy of the primary dimension. However, because the secondary signal is coupled to the primary signal, an extra separation process is needed, and since the second-dimension signal has less intensity (32) Zhukhovitsiski, A. A.; Turkel’taub; Dokl. Acad. Nauk. USSR 1961 (143), 646.

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Figure 2. Schematic diagram of a pulsed flow modulator: (a) T connector, (b) pressure regulator, (c) cross connector, (d) pressure gauge, (e) needle valve, (f) fast solenoid switch valve, (g) module flow deliver line, (h) press-on Y connector. Refer to text for details.

than in the full modulation method, it has less sensitivity. In addition, the presence of primary effluent in the secondary dimension may influence the secondary retention time. These factors make the method unsuitable for a complex separation in the second dimension. The best application of the partial modulation method uses the primary signal to determine quantities, with the secondary signal used to identify class structure and to assist in determining quantities for those peaks that are not completely separated in the first dimension. Pulsed Flow Modulator. The pulsed flow modulator is one of the techniques for implementing the partial modulation method. The design is shown in Figure 2, with more details given in the Experimental Section. The function of the modulator is simple; it just injects a pulsed inert gas into the conjunction between the primary and secondary columns. The modulator can work in two modesspositive pulsed flow mode or negative pulsed flow mode. In the positive pulsed flow mode, the modulation gas flow stays low most of the time, with periodic high-flow pulses creating “spikes” subtracted from the primary signal. In the negative pulsed flow mode, the modulation gas flow is kept high most of the time, with periodic introduction of low-flow pulses to create “spikes” added to the primary signal. Figure 3 illustrates how the negative pulsed flow modulation works. Starting with Figure 3a, the effluent of the primary column mingles with the modulation gas and produces a relatively low concentration stream flowing into the secondary column. Then the flow rate of the modulation gas is quickly reduced for 10-30 ms, forming a negative flow pulse. During this brief time a relatively high concentration stream moves into the front of the secondary column as shown in Figure 3b. After the negative flow pulse, the flow rate of the modulation gas and the concentration in the stream flowing into the secondary column are restored to the previous level, isolating a high-concentration plug, indicated in Figure 3c. Pressure also plays a role in the formation of the local high-concentration plug. When the modulation gas flow is high, the pressure at the joint of the primary and secondary columns is also high. This pressure drops during the negative flow pulse, helping the sample move from the primary column to

Figure 3. Illustrations of the formation of a high-concentration sample plug in the conjunction between primary and secondary columns with a negative pulsed flow modulation. The pressure compression effects also contribute to the higher concentration of the plug. See text for details.

the secondary column. After the negative flow pulse, the pressure at that point increases again, compressing the high-concentration plug, which makes the concentration even higher, and pushing it into the secondary column. A positive pulsed flow mode has an opposite process, illustrated in Figure 3. Instead of a negative pulsed flow mode that creates a local higher concentration, the positive flow pulse creates a local lower concentration. This local lower concentration is formed when the pulsed inert gas injected into the stream flowing from the primary to the secondary column instantly dilutes the concentration of the stream, forming a sharp negative peak. Separation Conditions for the Secondary Dimension. The separation conditions for the secondary dimension differ from those for the full modulation method in that the mobile phase in the secondary dimension is not a pure carrier gas. Instead, it is instead a mixture of analyte and carrier gas, and their concentrations change. The presence of analyte in the mobile phase will influence the retention time in the second dimension, at least during points of high concentration such as when a solvent peak is eluted. However, from the chromatograms shown later in the paper, we believe that it does not have a significant effect, especially when the analyte concentration is within a workable range. In the positive pulsed flow modulation mode or the vacancy chromatography, the retention time would not be affected by the

presence of the analyte itself, but it might be affected by the presence of the other analytes. Modulation Index. The modulation index (modulation depth) is a measurement of the partial modulation method and is defined as the ratio of the secondary dimension signal or peak height over the primary dimension signal at the point just before the secondary column inlet. In other words, the modulation index is the height of the “spike” created by the pulsed flow modulator divided by the original primary signal. The “spike” could be positive (added) or negative (subtracted), depending on what pulsed flow mode is used. It is difficult to measure the signal directly at the juncture point, but it can be estimated from the recorded detector signal with the following equation:

Mindex ) Hs Ws1/2/PsTw

(1)

where Mindex is the modulation index, Hs is the secondary dimension peak height, Ws1/2 is the secondary dimension peak half-width, Ps is the primary signal at the secondary peak, and Tw is the modulation pulse width. Equation 1 assumes that the secondary peak area and primary signal height do not change after they pass through the secondary column, so the ratio of the secondary peak area to the area under this peak with the modulation pulse width is a constant that is Analytical Chemistry, Vol. 76, No. 20, October 15, 2004

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the modulation index, regardless of peak height and retention time in the second dimension. This equation is easy to use, but it is only suitable for a single component in the secondary dimension. If there are two or more components appearing in the secondary dimension, eq 1 should be modified to add all these peak areas together instead of just one. The other way to estimate the modulation index is to calculate the concentration differences between the high and low flow at the inlet of the secondary column. The real concentration at the modulation point is not easy to measure, but we can measure the flow rate difference and estimate the dilution factor to get the modulation index. For a negative pulse mode the equation can be written as follows:

Mindex )

C1 - C2 C1 Cp(Fcl/Flow) ) -1) -1) C2 C2 Cp(Fch/Fhigh) Fhigh Fcl - 1 (2) Flow Fch

where Mindex is the modulation index, as in eq 1, C1 is the concentration of analyte at the modulation point with the modulator at low flow position, C2 is the concentration of analyte at the modulation point with the modulator at high flow position, Cp is the concentration of analyte effluent from the primary column, Fhigh is the total flow rate measured at the end of the secondary column with the modulator at high flow position, Flow is the total flow rate measured at the end of the secondary column with the modulator at low flow position, Fch is the primary column flow with the modulator at high flow position, and Fcl is the primary column flow with the modulator at low flow position. In eq 2 the column flow ratio Fcl/Fch is not measurable, so the equation does not have much practical use. However, when Fcl/Fch is close to 1 (as with a long primary column producing high column inlet pressure, a short secondary column leading to low flow resistance, or both) or when Fhigh and Flow are close (low modulation index), eq 2 can be simplified to

Mindex ≈ Fhigh/Flow - 1

(3)

Note that this simplified equation always gives a negative error because the Fcl/Fch is always greater than 1 (Fcl > Fch), and the greater the Mindex, the bigger the error. However, this equation is useful for the initial setting of Mindex, because both flows are measurable. For a positive pulse mode, eqs 2 and 3 can be changed as follows:

Fch Cp Fhigh C1 - C2 C2 Flow Fch Mindex ) )1)1)1C1 C1 Fcl Fhigh Fcl Cp Flow (4) Mindex ≈ 1 - Flow/Fhigh

(5)

Since Fhigh is always greater than Flow and Fcl is always greater than Fch, the modulation index in eqs 4 and 5 will never exceed 6068

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Figure 4. Experiment setup of a comprehensive 2-D GC system with a pulsed flow modulator.

1. This means that, in the positive pulse flow mode, the intensity of the negative secondary signal will never be greater than that of the primary signal itself, which is obvious for vacancy chromatography. Modulation Percentage. Modulation percentage is another measurement useful in describing this method. It is defined as the percentage of the primary signal that is modulated and converted into the secondary signal or the percentage of the secondary peak area in the total (primary plus secondary) peak area. The full modulation method converts 100% of the primary signal into the secondary signal; the partial modulation method converts only a certain percentage of the primary into the secondary and leaves the rest unchanged. The conversion percentage can be calculated using the following equation, which is further simplified by using eq 1 with insignificant terms omitted:

M% )

HsWs1/2 × 100% ) HsWs1/2 + PsTp MindexTw MindexTw × 100% ≈ × 100% (6) MindexTw + Tp Tp

where M% is the modulation percentage, Tp is the modulation period time, and Mindex, Ws1/2, and Tw are the same as in eq 1. As shown in this equation, the percentage of modulation is proportional to both the modulation index and the flow pulse width and is inversely proportional to the flow pulse period. The M% can also be determined from measuring the peak area of the primary and secondary dimensions. EXPERIMENTAL SECTION Instruments. The pulsed flow partial modulation GC×GC system was set up on an Agilent 6890 gas chromatograph with flame ionization detection (FID) (Agilent Technologies, Wilmington, DE). The schematic of the GC×GC system is shown in Figure 4. In this setup, primary and secondary columns are both in the GC oven, connected by a press-on quartz Y connector. The third

port of the Y connector is connected to the pulsed flow modulator by a 40 cm long × 0.10 mm i.d. fused-silica tube. The inlet of the primary column is connected to an injector, and the secondary column exit is connected to the FID. Two different kinds of injectors were usedsa valve and a syringe. For C1-n-C6 hydrocarbons, injections were made with a six-port diaphragm valve with a 20-µL sample loop (DV22, VICI Valco Instruments Co. Inc., Houston, TX) and the oven temperature was set at 60 °C isothermal for 7.5 min. For the laboratory-prepared mixture, an autosampler (Agilent Technologies) injected 1-µL samples into a split/splitless injector in split mode with a split ratio of 100:1. The column oven temperature was held at 30 °C for 2 min and then increased to 220 °C at 8 °C /min, where it was held constant for 2 min. The detector was set at 230 °C and the injector at 200 °C. For the no. 6 fuel oil, the autosampler injected 1-µL samples into a split/splitless injector in pulsed splitless mode. The column oven temperature was held at 40 °C for 2 min and then increased to 330 °C at 10 °C /min, where it was held constant for 5 min. The detector was set at 300 °C and the injector at 250 °C. A PC running Chemstation rev. A.06.03 (Agilent Technologies) controlled the GC and collected the FID signal with a sampling rate of 200 points/s. All columns were obtained from VICI Gig Harbor Group (Gig Harbor, WA). For C1-n-C6 hydrocarbons and the laboratoryprepared mixture, the primary column was a VB-1 (100% dimethyl polysiloxane stationary phase) 30 m × 0.25 mm i.d. × 1.0 µm thickness, and the secondary column was a VB-50 (50% phenyl 50% dimethyl polysiloxane stationary phase) 2 m × 0.18 mm i.d. × 0.18 µm thickness. For no. 6 fuel oil, the primary column was a VB-NTZB (94% dimethyl polysiloxane-6% proprietary stationary phase) 30 m × 0.25 mm i.d. × 0.25 µm thickness, and the secondary column was a VB-35 (35% phenyl 65% dimethyl polysiloxane stationary phase) 2 m × 0.15 mm i.d. × 0.15 µm thickness. We selected a relatively large inner diameter for the secondary column because we used the negative pulsed flow mode for evaluating the method, which makes the secondary column flow rate much higher than the flow rate in the primary column. Chromatographic grade helium (Air Products and Chemicals. Inc., Allentown, PA) was used as the carrier gas. The helium was purified by a helium purifier (HP-1, VICI Valco Instruments Co. Inc.). Modulator. A homemade pulsed flow modulator was used in this study. A schematic diagram is shown in Figure 2. The helium carrier (or modulation) gas at the top of the diagram is split into two streams by the T connector (a) and then goes to two pressure regulators (b) (PR50A30Z1, VICI Valco Instruments Co. Inc.). One pressure regulator is adjusted for higher pressure output, so it can deliver higher carrier flow. The other regulator is set to keep a minimum flow through the delivery line (g) to prevent sample flow into the modulation valve. The settings of these two pressure regulators depend on the primary column flow, the size of the secondary column, and the modulation index. In this study, for a negative pulsed flow mode with a modulation index equal to ∼1, they were adjusted at 20.2 and 10 psi, respectively. Each carrier stream from the pressure regulators goes through a cross (X) connector to a three-way solenoid valve (f) (H010E1, Humphrey, Kalamazoo, MI). The cross also connects a pressure gauge (d) and a needle valve (e) (BNV1, VICI Valco Instruments Co. Inc.). The flow through the needle valve was set at ∼10 times higher

than the flow going into the solenoid valve, providing a vent to smooth the pressure change when the solenoid valve switches between high and low flow. The main component of the modulator is the fast solenoid valve, which performs high-speed switching between high and low flow. The solenoid valve is controlled by a digital valve sequence programmer (DVSP2, VICI Valco Instruments Co. Inc.), modified to reduce the adjustable step timing to as little as 10 ms. The delivery line (g), made from deactivated fused-silica tubing (40 cm long × 0.10 mm i.d.), delivers the modulated carrier gas to the press-on quartz Y connector (h) that also connects the primary and secondary columns. Note that the entire modulator, except for the delivery line and Y connector, is outside the GC oven and therefore not affected or limited by the GC oven temperature. Also note that the sample does not pass through the solenoid valve, avoiding any condensing, adsorption, and contamination. Data Processing. The detector signal is digitized and collected by Chemstation, which then exports it as a comma-separated value (.csv) formatted file. The file is transferred into Microsoft Excel (Microsoft Co. Redmond, WA) spread sheets, where a housecompiled program uses Excel macro and internal functions to separate the raw signal into primary and secondary signals. The secondary signal is demodulated and converted into a twodimensional matrix. The Excel Chart function is used to plot both the primary and the 2-D matrix data. The decoupling of primary and secondary signals from the raw detector signal involves the following steps: (1) detect secondary signals from the raw signal by using a differential equation and a deviation equation, (2) cut off the secondary signals and connected their bases with straight lines, (3) smooth the data by using a moving average to get the primary signal, and (4) subtract the primary signal from the raw detector signal to get the secondary signal. The demodulation process is a simple conversion of linear data into a 2-D matrix with a constant modulation time. Samples. Two gas samples and two liquid samples were used in this study. The gas samples were a 100 ppm C1-C6 n-paraffin standard mixture and a 1000 ppm C1-C6 n-paraffin standard mixture, both purchased from Scott Specialty Gases Co. (Plumsteadville, PA). A mixture of n-paraffin, aromatics, and phenols was prepared in our laboratory from mixtures of the groups purchased from PolyScience Co. (Niles, IL), diluted into pentane solvent (Sigma-Aldrich, St. Louise, MO). The concentration of each component is roughly 330 ppm. The fuel oil no. 6 standard (5000 µg/mL in methylene chloride) was purchased from Restek (Bellefonte, PA). RESULTS AND DISCUSSION Typical Chromatograms. A C1-C6 n-paraffin sample was chromatographed by the partial modulation method through the pulsed flow modulator. The results are shown in Figure 5, in which the following is given: (a) is an original detector signal for C1-C6 n-paraffin mixture; (b) is the same as (a), but with the same sample injected a second time at 1.43 min after the first run, so that the n-C5 peak of the second sample partially overlaps the n-C6 peak of the first sample; (c) is an expansion of the designated area of (b). The modulation conditions in Figure 5 are: negative mode, 30-ms pulse width, and 1-s period. Under these conditions, the modulation index is 1.3 and modulation percentage is 3.9%. The results show that, with the partial modulation method, the Analytical Chemistry, Vol. 76, No. 20, October 15, 2004

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Figure 5. Typical detector output signals of C1-C6 n-paraffin obtained by using partial modulation method though pulsed flow modulator. Where (a) is C1-C6 n-paraffin; (b) is the same sample but inject twice, the second one is made at 1.43 min after first injection, where C1-C6 come from first injection, and C1′-C6′ come from second injection; (c) is the expend part of the partially overlapped of C5′ and C6 in (b).

detector signal is an additive of the primary and the secondary signals as we expected. The secondary peaks in the chromatograms have good symmetry and are much narrower than the primary peaks. In Figure 5, the peak half-widths of n-C5 and n-C6 are 1.85 and 2.67 s, respectively, in the primary dimension. The corresponding secondary dimension peak half-widths are 41 and 51 ms, differing by a factor of 50. So they can be separated easily by either softerware or hardware. The secondary peak half-width for C1 in the example is even less, or ∼31 ms. The narrow secondary peak width of this method benefits from its directly flow modulation to the column stream, which is a faster process than thermal absorption/adsorption processes used by thermal modulators. If the primary column effluent is a mixture, it is 6070 Analytical Chemistry, Vol. 76, No. 20, October 15, 2004

possible to separate it in the secondary column as in the full modulation method, even though there are solutes continuously passing through the secondary column. Different Pulsed Flow Modulation Modes and High/Low Flow Ratios. Changing the pulsed flow mode (positive or negative) changes the way that the secondary signal is coupled to the primary signal, and changing the flow ratio (Fratio ) Fhigh/Flow) changes the modulation index Mindex. To investigate the effect of these changes, different modulation modes and high/low flow ratios were tested. The results are shown in Figure 6, in which (a1), (a2), and (a3) are generated with a negative pulsed flow modulation mode and (b1), (b2), and (b3) are generated with a positive pulsed flow modulation mode. The high/low flow ratio

Figure 6. Partially modulated chromatograms of n-C5′ and n-C6 obtained by using different pulsed flow modulation mode and high/low flow ratio. The n-C5′ and n-C6 peaks come from two different injections. The second injection time is chosen such that it lets second n-C5′ peak be partially overlapped with the first n-C6 peak. Only n-C5′ and n-C6 parts of each chromatogram are demonstrated.

(Fratio) and modulation indexes (Mindex) are given in each chromatogram. The modulation indexes are calculated from eq 1. As shown in Figure 6, when a negative pulsed flow mode is used, the secondary peaks are added to the primary signal and increase with the same direction as the primary signal. In contrast, when a positive pulsed flow mode is used, the secondary peaks are subtracted from the primary signal. Figure 6 also demonstrates that, for both modes, increasing the high/low flow ratio increases the peak height in the secondary dimension. However, an increase in flow ratio has less effect on the primary peaks, especially in the positive modulation mode. So we can control the high/low flow ratio to get a desired Mindex. Note that the Mindex in Figure 6 (b3) exceeds 1, which is not supposed to happen. The error comes from eq 1, which does not consider the band broadening due to the pressure change. The error is negligible with a low flow ratio but becomes serious with higher flow ratios, especially in the positive pulsed flow mode. Since the modulation index (Mindex) is the signal ratio of the secondary to the primary dimension, its value reflects the relative signal intensities of the primary and secondary signals. A low Mindex indicates a lower secondary signal (as compared to the primary signal), as shown in Figure 6 (a1) and (b1), and a high Mindex indicates a higher secondary signal, as shown in Figure 6 (a3) and (b3). However, as Figure 6 (b3) shows, in positive pulsed flow mode (vacancy chromatography) the negative peak heights are limited by the primary signal itself. A special situation is, when the Mindex is close to 1, the primary and secondary signals have similar intensities, as shown in Figure 6 (a2) and (b2). It is be

considered the best use of the partial modulation method because it not bias to any one. In most of our tests later, we used this condition for demonstration. The modulation percentages (M%) in Figure 6 are 1.3, 4.4, and 11% for (a1), (a2), and (a3), respectively, and 1.1, 3.5, and 6.0% for (b1), (b2), and (b3) calculated from eq 6. The numbers show that, with modulator parameters of 30-ms pulse width and 1-s modulation period, the M% is in the 1.3-11% rangesmuch lower than with the full modulation method. This is why the partial modulation method does not offer as high a degree of sensitivity enhancement as the full modulation method. In most applications, the primary and secondary signals are of equal intensity. Considering the noise level increase from the flow disturbance introduced by the modulator, actual sensitivity is even less than with onedimensional GC. This is generally not an important issue for 2-D GC, since many applications do not require high sensitivity. In those that do, sensitivity can be increased by techniques such as purge and trap, solid-phase microextraction, and cryogenic concentration. Note that in Figure 6 the primary retention times of C5′ and C6 increase with an increase in the modulation index. The reason for this is that a high modulation index needs a high modulation gas flow rate. This causes the pressure at the Y joint to increase, consequently reducing the primary column flow and lengthening the retention time. By the same reasoning, the separation of C5′ and C6 in the secondary dimension also gets worse, since the overall second-dimension flow rate increases. Also note that these factors have more effect in the negative pulsed flow mode Analytical Chemistry, Vol. 76, No. 20, October 15, 2004

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Figure 7. Illustration of the noise level of secondary dimension signal near a 3 ppm n-C5 and a 1000 ppm n-C6 component. The modulation mode is negative, modulation index is 1.3, and modulation period is 1 s.

[(a1)-(a3)] than in the positive pulsed flow mode [(b1) and (b2)], because the average total flow rate change in the negative mode [from 2.53 (a1) to 6.05 mL/min (a3)] is larger than that in the positive mode [from 1.39 (b1) to 1.53 mL/min (b3)]. Noise Level in the Secondary Dimension. It should be indicated that because the secondary signal is coupled to the primary signal, its noise level is not uniform. We found that the noise level in the secondary dimension increases as the primary signal increases. If we consider that the primary signal is essentially the secondary signal’s baseline, it is no surprise that a high baseline causes a high noise level. This situation is illustrated in Figure 7, which shows a secondary dimension signal of a 3 ppm n-C5 component (0.19 ng) situated near a 1000 ppm n-C6 component (77 ng). After the secondary signal is uncoupled from the primary signal, these two components are illustrated as two series of peaks until they are demodulated into a comprehensive 2-D chromatogram. The peak-to-peak noise level near 3 ppm n-C5 is ∼0.015 pAsthe same level as in the baselinesindicating an insignificant contribution to the noise level by the presence of the 3 ppm n-C5. However, the noise level in the n-hexane peaks is ∼0.18 pA, which is 12 times higher than that near the n-C5 peaks. If the n-C5 peaks move closer to the n-C6 peaks, the sensitivity for n-C5 will be significantly reduced. This is a major drawback of this method. However, this limitation only affects a complex second-dimension separation or a single second dimension run with a large concentration difference. Increasing the power of the primary separation reduces the effect of this limitation. Also note that in Figure 7 the presence of a 1000 ppm n-C6 component causes the noise level to increase by a factor of 12, and even with this noise level, it is possible to identify the 3 6072 Analytical Chemistry, Vol. 76, No. 20, October 15, 2004

ppm n-C5 component if it is separated from the n-C6 peak in the secondary dimension. Determining Quantities Using the Partial Modulation Method. A partially modulated chromatogram can be separated into two chromatogramssprimary and secondary. Both of them can be used for quantities, but they have different advantages. The primary signal provides more accuracy and is less influenced by modulation time, while the secondary signal makes it possible to determine quantities for those components that cannot be separated well in the first dimension. Of course, the best method for determining quantities would be one that uses all the information collected. That method would involve using both chromatogramssthe primary signal for basic quantities and the secondary signal as a guide for dividing the primary signal into its components. However, this method requires powerful software, and we do not intend to discuss it here in detail. The accuracy of the quantity determination using the partial modulation method has been investigated. In this investigation, a negative modulation mode with modulation index 1.3 is used. The test sample is a 100 ppm C1-n-C6 hydrocarbons mixture with a sample size of 20 µL. This sample was injected 10 times, and both the primary and the secondary peak areas have been recorded. The relative standard deviation (RSD) of these peak areas was calculated and used as a measure of the quantity determination precision. For comparison, the same sample was run with the same GC conditions using regular one-dimensional chromatography. All results are listed in Table 1, with the 1-D GC results at upper left, the primary signal results at upper right, the secondary signal results at lower left, and the results for the sum of primary and secondary signals at lower right. As these results

Table 1. C1-C6 n-Paraffin Peak Area Measured from 1-D and Partially Modulated 2-D Gas Chromatograms run no.

C1

C2

1 2 3 4 5 6 7 8 9 10 av RSD (%)

80.20 80.20 80.11 80.16 80.07 79.97 80.11 79.95 79.98 79.79 80.05 0.16

147.6 147.6 147.5 147.6 147.4 147.2 147.5 219.4 147.2 146.9 147.4 0.16

run no.

C1

1-D GC peak area (pA min) C3 C4 220.1 220.1 219.9 220.0 219.7 219.5 219.9 219.4 219.5 219.0 219.7 0.16

296.6 296.4 296.2 296.3 296.0 295.7 296.2 295.7 295.7 295.1 296.0 0.15

C5

C6

C1

359.5 359.2 359.0 359.3 358.8 358.3 359.0 358.3 358.5 357.6 358.8 0.16

438.3 437.7 437.6 437.6 437.1 436.7 437.6 436.5 436.9 436.1 437.2 0.15

75.30 76.61 77.70 75.47 75.07 76.18 77.61 77.16 75.65 75.22 76.20 1.33

2-D GC second-dimension peak area (pA min) C2 C3 C4 C5

2-D GC first-dimension peak area (pA min) C2 C3 C4 C5 139.7 142.8 142.4 137.5 138.8 142.0 142.8 141.2 137.8 138.8 140.4 1.49

C6

C1

C2

210.3 213.0 209.8 206.3 209.5 212.9 210.2 208.6 206.4 208.9 209.6 1.08

281.6 283.1 283.5 281.2 281.5 283.5 284.0 283.5 281.8 282.0 282.5 0.37

2-D GC total peak area (pA min) C3 C4

C6

342.3 343.0 342.4 342.5 342.6 343.7 342.7 342.6 342.9 343.7 342.8 0.15

418.2 419.4 418.1 417.6 418.0 419.1 418.2 418.3 418.5 420.4 418.6 0.20

C5

C6

1 2 3 4 5 6 7 8 9 10

5.80 4.57 3.50 5.57 5.98 5.13 3.58 3.93 5.46 6.07

9.54 6.60 7.04 11.75 10.54 7.61 6.60 8.25 11.67 11.00

12.23 9.75 12.97 16.13 13.06 10.19 12.60 14.18 16.37 14.37

18.19 17.07 16.44 18.57 18.22 17.02 16.22 16.73 18.35 18.83

21.59 21.35 21.52 21.18 21.15 21.05 21.39 21.39 21.30 21.42

26.09 25.91 26.01 25.92 25.70 26.07 26.02 25.71 25.92 25.67

81.10 81.18 81.21 81.03 81.05 81.31 81.19 81.09 81.11 81.29

149.3 149.4 149.4 149.3 149.3 149.6 149.4 149.4 149.5 149.8

222.6 222.8 222.7 222.4 222.5 223.1 222.8 222.8 222.8 223.3

299.8 300.1 300.0 299.7 299.7 300.5 300.2 300.2 300.1 300.8

363.9 364.3 364.0 363.6 363.8 364.7 364.1 364.0 364.2 365.1

444.3 445.3 444.1 443.5 443.7 445.1 444.2 444.0 444.4 446.0

av RSD (%)

4.96 20.01

9.06 23.05

13.18 16.63

17.56 5.48

21.33 0.78

25.90 0.61

81.15 0.12

149.4 0.11

222.8 0.11

300.1 0.12

364.2 0.13

444.5 0.18

show, the RSD from the 1-D GC holds very tightly around 0.16% for C1-C6, indicating a high precision for 1-D GC, which is not affected by the retention time (primary peak width). However, in 2-D GC, the RSDs for both primary and secondary signals increase as the carbon number decreases. For the primary signal, the RSD is 1.3% for C1 and 0.20% for C6; for the secondary signal, the RSD is 20% for C1 and 0.61% for C6. The increasing RSD for smaller carbon numbers is due to the narrower width of their primary peaks as compared to those of the larger carbon numbers. With a fixed modulation time, a narrow primary peak has less-modulated secondary peaks. This causes a larger deviation, since with the secondary signal randomly distributed on the primary signal, it could appear on the maximum primary peak height on one run but somewhere else on the next run. This factor affects the precision of the quantity determination more in the secondary signal than in the primary. As in the test for C1, the RSD is 1.3% for the primary peak and 20% for the secondary peak. If we add the two signals together, the additive signal has the same precision as 1-D GC and is not affected by the primary peak width, as the data in Table 1 show. Note that the peak area counts in the additive signals are similar to the 1-D peaks, but with a ∼1.5% increase. This increase is probably because the column flow is changed by the flow modulator, consequently affecting the FID response factor. To find the relationship between primary peak half-width and the standard deviation for the secondary peak area, the data in Table 1 are plotted and shown in Figure 8. In the plot, the x-axis is the primary peak half-width and the y-axis is the standard deviation of the secondary peak area. The secondary peak area given, if not otherwise specified, is the sum of the secondary peak areas for one compound and not just for one secondary peak. Data

Figure 8. Relationship between primary peak half-width and standard deviation of total secondary peak area with a fixed modulation time. The modulation mode is negative, modulation index is 1.3, and modulation period is 1 s.

used for plotting are listed in Figure 8. They are the results of testing C1-n-C6 compounds with the peak half-width from 0.79 to 3.15 s. Usually the peak half-width increases with the carbon number or the retention time; however, the narrowest peak in the test is C2, not C1 as would be expected. It is probably because the “C1 peak” is not pure methane but includes other impurities coeluting with methane that make the peak broaden. The number of second peaks listed in Figure 8 is the number of secondary peaks appearing on each primary peak. With a fixed modulation time, narrower primary peaks generate fewer secondary peaks, since there is less time for modulation. So with a one second modulation time, a C1 peak gets 2.5 peaks and a C6 peak gets 8, as listed in Figure 8. (The value of “2.5 peaks” does not mean Analytical Chemistry, Vol. 76, No. 20, October 15, 2004

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Figure 9. Chromatograms of alkane, aromatic, and phenol mixture obtained by using pulsed flow partial modulation method. The modulation index is ∼1.2 and the modulation period is 1 s.

that the secondary peak actually appears two and a half times; it means that the secondary peak sometimes appears twice on the primary peak and sometimes appears three times, depending on the location of these peaks.) Figure 8 clearly illustrates that the RSD of the secondary peak area decreases as the primary peak half-width increases. The decrease is not linear with peak halfwidth; it decreases quickly at the beginning, slows down, and finally tends toward a certain number. The relationship can be expressed by the power equation shown in Figure 8. With the equation, we can draw the following conclusions: (1) the minimum RSD of the secondary peak area is ∼0.58%, which is as precise as we can get using the secondary peak area for quantities; (2) when a primary peak half-width is equal to the modulation time (which is x ) 1, or 1 s in this case), the RSD of the secondary peak area is ∼8%; (3) if we want an RSD of a secondary peak area to be less than 1%, the ratio of the primary peak half-width over 6074

Analytical Chemistry, Vol. 76, No. 20, October 15, 2004

the modulation time must be greater than 1.75, to allow about five modulations on the primary peak. Separation Efficiency and Retention Time Reproducibility. During the partial modulation, the pulsed flow modulator disturbs both primary and secondary column flows and makes them pulsed rather than continuous. The flow rate measured from the secondary vent is an average of total flow. Since the column flow is pulsed, the question arises of whether the pulsed flow affects separation efficiency and retention time reproducibility. To answer this question, we tested a C1-n-C6 mixture using both 1-D and 2-D GC under the same GC conditions. Comparing the 1-D chromatogram of the test mixture with the primary chromatogram from the 2-D GC, we find that there is no significant difference in peak shape, peak half-width, and separation efficiency. For 1-D GC, the theoretical plate numbers of C2 and n-C6 are 126 000 and 90 000, respectively, and for 2-D, 127 000 and 91 000. So the pulsed column

Figure 10. Chromatograms of a fuel oil no. 6 standard obtained by using pulsed flow partial modulation method. The modulation index is from 0.96 to 2.9 and the modulation period is 2 s.

flow has no effect on primary separation efficiency. The seconddimension separation is inadequate to yield that conclusion; we would need to compare the results using a different modulation method. However, based on the symmetric secondary peaks and the separations in Figure 6, Figure 9 and Figure 10, we believe that the effect of the pulse flow on the secondary separation, if it exists at all, is limited. The precision of the retention time is listed in Table 2. The average RSD of the retention times is 0.074% for 1-D GC and 0.070% for 2-D GC, showing that both GC methods have the same level of retention time reproducibility. The reproducibility for second-dimension retention time cannot be directly measured because the modulator and data acquisition system are not synchronized, but by the evidence of the 2-D chromatogram in Figure 9 and Figure 10, it appears that the

precision of second-dimension retention time is good enough for the applicationssotherwise there would be no symmetric peaks in the contour plot. Oven Temperature. Since changing the oven temperature will change both the column flow rate and the modulation gas flow rate, it will change the modulation index and percentage. The direction of that change depends on the change ratio of these two gas flow rates. Generally speaking, since only a portion of the tubing delivering the modulation gas is inside the GC oven, the modulation gas flow is less subject to changes less than the column flow. So when the oven temperature increases, both gas flow rates decrease, but since the modulation gas flow decreases by a smaller percentage than the column gas flow, the modulation index increases slightly. However, if a constant column flow mode Analytical Chemistry, Vol. 76, No. 20, October 15, 2004

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Table 2. C1-C6 n-Paraffin Retention Time Measured from 1-D and 2-D Gas Chromatograph run no.

retention time of 1-D GC (min) C3 C4 C5

C1

C2

1 2 3 4 5 6 7 8 9 10

1.9180 1.9182 1.9163 1.9168 1.9197 1.9187 1.9198 1.9197 1.9168 1.9163

1.9886 1.9886 1.9867 1.9872 1.9902 1.9892 1.9904 1.9903 1.9872 1.9867

2.1501 2.1498 2.1481 2.1484 2.1515 2.1505 2.1520 2.1519 2.1484 2.1478

2.6059 2.6055 2.6037 2.6042 2.6071 2.6061 2.6083 2.6078 2.6037 2.6033

av RSD (%)

1.9180 0.075

1.9885 0.076

2.1498 0.075

2.6056 0.070

C1

3.7620 3.7610 3.7593 3.7597 3.7633 3.7615 3.7660 3.7647 3.7587 3.7571

6.6916 6.6840 6.6869 6.6886 6.6879 6.6895 6.6981 6.6949 6.6849 6.6814

1.8382 1.8390 1.8356 1.8356 1.8378 1.8385 1.8381 1.8383 1.8380 1.8389

1.9064 1.9059 1.9028 1.9035 1.9057 1.9053 1.9051 1.9055 1.9061 1.9073

2.0607 2.0599 2.0569 2.0595 2.0603 2.0598 2.0593 2.0600 2.0617 2.0625

2.4970 2.4987 2.4943 2.4940 2.4967 2.4980 2.4968 2.4960 2.4965 2.4976

3.6080 3.6044 3.6029 3.6035 3.6058 3.6081 3.6000 3.6023 3.6050 3.6075

6.4020 6.4046 6.4070 6.4062 6.4068 6.4025 6.4092 6.4120 6.4147 6.4173

3.7613 0.074

6.6888 0.075

1.8378 0.066

1.9054 0.070

2.0601 0.073

2.4965 0.060

3.6047 0.074

6.4082 0.079

is used, the situation is reversed. In that case, the column flow stays constant when the oven temperature increases, but the modulation gas flow decreases, making the modulation index decrease. For example, with a constant column flow mode, the modulation index is 2.9 at 80 °C, falling to 1.5 at 200 °C, and finally to 0.96 at 310 °Csabout a third of what it was at 80 °C. One could use programmed flow control for the modulation gas to keep the modulation index constant during oven temperature programming or to adjust the modulation index to a desired pattern. Chromatograms of a Hydrocarbon Mixture. A laboratoryprepared hydrocarbon mixture was run using the pulsed flow partially modulated method, in the negative pulsed flow mode with a modulation index of ∼1.2. The modulation time is 1 s. Because the polarity of the primary and secondary columns is different (VB-1 and VB-50), the 2-D separation is orthogonal. The first-dimension separation is mainly according to volatility, and the secondary dimension separation is mainly by polarity. Figure 9 shows these chromatograms, in which (a) is the primary dimension chromatogram and (b) is the comprehensive 2-D chromatogram. The mixture in Figure 9 contains 18 compounds within 3 groups: alkanes, aromatics, and phenols. As shown in Figure 9b, these three groups stay within three lines in comprehensive 2-D chromatogramsthe alkanes line, the aromatics line, and the phenols line. Each compound falls within its own line, indicating that the secondary column has a similar retention force to compounds in the same group. Obviously, the alkanes have the shortest retention time in the second dimension, followed by the aromatics, with the phenols having the longest retention time. This indicates that the alkanes have less interactive force to the secondary stationary phase than aromatics, and aromatics have less interactive force than phenols. The peak half-widths in the second dimension (in milliseconds) are as follows: alkanes, from 29 (n-hexane) to 39 (n-dodecane); aromatics, from 32 (benzene) to 38 (cymene); and phenols, from 38 (phenol) to 44 (p-ethylphenol). The wider phenol peaks are due to their longer retention time in the secondary dimension. The chromatograms in Figure 9 illustrate that with the partial modulation method only ∼5% of the total signal is modulated to create a comprehensive twodimensional chromatogram that gives compound structure information, leaving 95% of the primary signal to be used for highquality quantities. High-Temperature Fuel Oil. A petroleum sample is always a good candidate for testing a comprehensive two-dimensional 6076

primary retention time of 2-D GC (min) C2 C3 C4 C5

C6

Analytical Chemistry, Vol. 76, No. 20, October 15, 2004

C6

chromatograph because of its complexity and its well-established components. A fuel oil no. 6 standard was analyzed using the pulsed flow partial modulation method. The primary and secondary columns used in the analysis are VB-NTZB and VB-35. Just as in Figure 9, a negative pulsed flow mode was used. The modulation index ranges from 2.9 to 0.96, depending on oven temperature (constant column flow mode was used), and the modulation time was set at 2 s. Under these conditions, the modulation is between 8.6 and 2.9%. The resultant chromatograms are shown in Figure 10, in which Figure 10a is the primary dimension chromatogram and Figure 10b is its corresponding comprehensive two-dimensional chromatogram. Figure 10a shows a regular one-dimensional separation and profile of no. 6 fuel oil on a nonpolar column. In Figure 10b, a few hundred peaks have been found in the comprehensive two-dimensional chromatogram. We made no effort to identify each peak, but it can obviously be seen that there are different multiring areas. The chromatograms in Figure 10 demonstrate that, with the pulsed flow partial modulation method, just a small percentage of the total signal can be used to generate a comprehensive 2-D chromatogram. They also demonstrate that the modulator and the method are not limited by oven temperature considerations. In this experiment, we programmed the over temperature to as high as 330 °C. CONCLUSIONS The pulsed flow partial modulation method is a new method to achieve comprehensive two-dimensional chromatographic separation. This method creates both a primary one-dimensional chromatogram and a comprehensive two-dimensional chromatogram simultaneously. The one-dimensional chromatogram can be used for high-quality quantities and the comprehensive two-dimensional chromatogram, which is similar to those obtained by the full modulation method, can be used for both structure identification and quantities. By choosing different modulation indexes through changes in pulse flow amplitude, we can select different signal intensity ratios of secondary signal to primary signal. ACKNOWLEDGMENT The authors thank David Salge of Valco Instruments Co. Inc. for his assistance in preparing the manuscript. Received for review May 21, 2004. Accepted August 4, 2004. AC0492463