Note 19: A New Programmable Cryo-Cooling/Heating Trap for the Cryo-Focusing of Volatiles and Semi-Volatiles at the Head of GC Capillary Columns

By John J. Manura , Chris Baker and John Manos
1999

INTRODUCTION

The Cryo-Trap consists of a small heating and cooling chamber which surrounds the front 5 inches of the GC capillary column. This unit is installed inside the GC column oven just under the GC injection port to permit the trapping of volatiles and semi-volatiles either on column or on a guard column in front of the capillary column. A separate digital dual temperature range controller permits the accurate temperature setting and regulation of both the heating and cooling temperatures of the GC Cryo-Trap. The system can be used either manually to switch between the cooling and heating cycles or can be operated automatically via an input signal from a controlling device, as the S.I.S. Short Path Thermal Desorption System or GC signal switch. This new GC Cryo-Trap was originally designed for use in conjunction with the S.I.S. Short Path Thermal Desorption System, but has additional applications, as the GC headspace analysis. This will be demonstrated. Normally, in order to cryo trap volatiles at the head of the GC column, the entire GC oven is cryo cooled using liquid C02 at temperatures that cannot go below -40 degrees C. With the new GC Cryo-Trap, volatiles can be trapped at temperatures down to -70 degrees C at the head of the GC column using less than CO2 of the C02 required to cool the entire GC oven. This results in more efficient trapping of the volatiles on column as well as substantial cost savings in C02. To release the volatiles from the Cryo-Trap, a heater coil inside the Cryo-Trap rapidly heats the capillary column to temperatures up to 400 degrees C. The released organics are subsequently temperature programmed through the GC column.

Theory of Operation

Figure # 1 - GC Cryo-Trap

The GC Cryo-Trap consists of a small heating/cooling chamber which is 3/4" in diameter and 5" long (Figure # 1). The GC capillary freely passes through the center of this chamber via a 1/16" O.D. stainless steel capillary guide tube. Capillary columns or guard columns up to megabore sizes (0.53 mm I.D.) can pass through this stainless steel capillary guide tube. Around the stainless steel capillary guide tube, a heating coil is wound to provide for the rapid heating of the capillary guide tube and GC capillary guard column. A thermocouple is attached to the stainless steel capillary guide tube to provide the signal to regulate the heating and cooling temperatures and to display the temperature on the digital temperature display of the Electronics Control . Liquid C02 for the cooling of the Cryo-Trap is introduced into the top inlet via an electrically controlled valve. The bottom outlet vents the dispersed C02 into the GC oven, or optionally it can be vented external to the GC via appropriate plumbing.

Figure # 2 - GC Cryo-Trap in GC Oven

The Cryo-Trap mounts on the side of the oven wall using a specially designed mounting bracket in the H.P. 5890 series gas chromatographs (Figure # 2). On the Varian and other model gas chromatographs, the Cryo-Trap mounts to the injection port column fitting with the clamp provided. No drilling or other GC modifications are required for the installation of the GC Cryo-Trap in any GC oven. Power requirements are 110 VAC, 3 amp max. An external supply of Liquid C02 is required for the cooling operation.

Figure # 2b - GC Cryo-Trap Electronics Control Module

Both heating and cooling of the Cryo-Trap are controlled by the Cryo-Trap Electronics Control provided with the system. The heating and cooling temperatures are set via the digital temperature controller (Figure # 2b). The Electronics Control can be used to manually switch between cooling and heating or optionally can be controlled automatically via an input signal provided by the GC operating system or by a controlling system, as the Short Path Thermal Desorption System. This input switches the system from the normal cooling cycle to the heating cycle when the chromatography is ready to begin. Cryo-Cooling temperatures down to -70 deg C can be set via the controller using liquid C02 as the cooling gas. Liquid Nitrogen can not be used with this system. A new model is now available for cooling with liquid nitrogen at temperatures down to 180 deg. C (Model 961). Heating temperatures, to remove the trapped volatiles from the trap, of up to 400 degrees C are achievable at a ramp rate greater than 800 degrees C per minute. This provides more than sufficient heating to release both the volatiles and semi-volatiles from the trap efficiently with sharp and narrow peak shapes.

The GC Cryo-Trap was originally designed to be used with the S.I.S. Short Path Thermal Desorption System. New software for the Desorption System permits the remote 2 connector on the back of the Short Path Thermal Desorption System Model TD-2 to be used for the automatic control of the Cryo-Trap. This will automatically switch the Cryo-Trap to begin the cooling cycle, when the initial gas purge step is begun on the Thermal Desorption system. When the Desorption process is complete, the Thermal Desorption system will activate the Cryo-Trap to switch from the cooling cycle to the heating cycle and begin the GC oven temperature program. Optionally, an input signal from a GC system program or other relay switching signal that can provide for a switch closure at the appropriate sequence of events in the analysis of your samples can be utilized to control the operation of the GC Cryo-Trap.

The new S.I.S. Short Path Thermal Desorption System incorporates the electronics in the desorption system controller. Therefore, a separate controller is not required when using the TD-3 thermal desorption system. All other desorption systems and headspace injectors will require the electronics temperature controller described above.

Selection of Cryo-Trap Cooling Temperature

Figure # 3 - Trapping Efficiency of Hydrocarbons On the GC Cryo-Trap As a Function of Temperature

The cooling temperature of the Cryo-Trap can be set at any value from GC oven temperature down to -70 deg C for the Model 951 GC Cryo-Trap or down to 180 deg for the model 961 GC Cryo-Trap. This temperature depends on the requirements of the user and the range of compounds that need to be cryo-trapped and subsequently chromatographed. Figure # 3 shows the efficiency of the GC Cryo-Trap for the trapping of a series of the straight chain hydrocarbons pentane (C-5) through nonane (C-9). For this analysis, a guard column was used inside the Cryo-Trap which consisted of a DB-5 megabore column, 0.53 mm I.D. x 150 mm long x 5.0 u film thickness. The GC column consisted of a 0.32 mm I.D. x 60 meter x 0.25 u film thickness DB-5-MS (J&W) capillary column. At room temperature (20 deg. C), only nonane (C9) is effectively trapped on the guard column. The lower molecular weight hydrocarbons pass through the guard column without being retained. As the temperature of the Cryo-Trap is sequentially lowered, additional lower boiling hydrocarbons are trapped on the guard column inside the Cryo-Trap. At the lowest temperature (-70 deg. C), pentane (C-5) is effectively trapped on the Cryo-Trap guard column and subsequently chromatographed. The resulting GC peak has excellent resolution and symmetrical peak shape. By operating the Cryo-Trap at -70 deg C, compounds such as pentane with melting points of -100 deg C can be effectively trapped on the guard column. Using this thick film megabore guard column at -70 deg C we have been able to accurately quantify acetone, ethyl acetate, methylene chloride and chloroform in pharmaceuticals using the Cryo-Trap in conjunction with the S.I.S. Short Path Thermal Desorption system. The resolution of the eluted peaks is very sharp. In particular the early eluting peaks are sharper than could be achieved via direct injection of these compounds.

Selection of Guard Columns

The selection of the Guard column that passes through the Cryo-Trap depends on the requirements of the user. The purpose of the guard column is to trap the volatiles and semi-volatiles on the inner surface of this column inside the Cryo-Trap using liquid C02 as the cooling fluid, and then to rapidly release these organics when the guard column is heated. Normally the guard column should not extend more than 20 mm beyond the end of the Cryo-Trap. This is particularly important when thick film guard columns are used, otherwise peak broadening will occur. A low dead volume connectors used to join the guard column to the GC capillary column (Figure # 2).

For higher boilers such as the semi-volatiles, a blank deactivated guard column provides for the optimum resolution of analysis. This will provide for trapping based on the melting points of the compounds of interest. Compounds that are trapped on the uncoated fused silica guard columns are rapidly released when the guard column is heated causing the eluted compounds to adsorb on the front of the capillary column in a very narrow band. This results in the highest resolution of chromatographic peaks. The use of megabore (0.53 mm I.D.) columns will minimize the occurrence of water plugs and permit the analysis of larger sample sizes. However, the uncoated guard columns will not trap compounds with melting points below the cryotrapping temperature which may limit their usefulness for many of the volatile organics.

For most volatiles, the capillary column itself can be used in place of the guard column by passing the capillary column straight through the Cryo-Trap and into the GC injection port. This type of connection has the advantage that no additional fittings such as capillary GC unions are required (therefore less chance for leaks to develop). However, use of thin film capillary columns may not adequately trap some of the very volatile organics. If microbore capillary columns are utilized for analysis, there are advantages to using megabore guard columns due to their ability to handle larger sample sizes and minimize the formation of water plugs.

Figure # 4 - Effect of Guard Columns On the Analysis of Hydrocarbons Using the GC Cryo-Trap

In order to trap the more volatile organics, guard columns with thicker liquid phases perform the best. Figure # 4 demonstrates this improvement in performance with increasing thickness of liquid phase coatings. When the same series of straight chain hydrocarbons are analyzed as in the cryo cooling study above, the uncoated deactivated fused silica guard column traps hydrocarbons down to heptane (C-7). By trapping on a 0.25 u film thickness DB-5 column, hydrocarbons down to hexane (C6) can be trapped using the Cryo-Trap. When the thick film guard column (2.5 u film thickness DB-VRX) is used in the Cryo-Trap, pentane (C-5) is effectively trapped on the guard column. The same results were obtained with a 5.0 u film thickness DB-5 megabore guard column. While providing for the efficient trapping of volatile organics, this thick film guard column may not be useful if higher molecular weight compounds are to be analyzed. These higher molecular weight compounds may prove difficult to remove from the thick film guard columns at the upper temperature limits of the liquid phase. As a result, peak shape of the higher boilers will suffer if thick film guard columns are utilized. For the analysis of semi-volatiles (such as the PNA'S), uncoated deactivated fused silica guard columns are normally used. For even more volatile organics, as gases, micropacked megabore columns could also be used as guard columns. However, these columns have limitations in the range of compounds that can be desorbed off the packing as well as the upper temperature limit of the packing.

Guard columns from microbore through megabore are usable inside the GC Cryo-Trap. Megabore guard columns are normally recommended due to their larger surface area which permits the trapping of larger sample sizes with higher water content. A slight loss in resolution may be evident when megabore guard columns are used in the GC Cryo-Trap. In contrast, the microbore guard columns will provide for slightly higher resolution but are more susceptible to the formation of ice plugs if the samples analyzed contain any appreciable levels of water.

Thermal Desorption Applications

Figure 5 - Comparison of Cryo-Trap Temperatures. Purge & Trap of 200 mg Black Tea at 80 deg. C, 450 ml Purge Onto Tenax® TA, Desorb at 250 Degrees C Into GC

Figure # 5 5 demonstrates the usefulness of the Cryo-Trap to trap the volatiles from a sample of Black Tea. The sample was prepared by purging the volatiles from 200 milligrams of black tea in 5.0 ml of water at 80 deg. C. The volatiles were trapped on a Tenax TA trap and then thermally desorbed into the GC injection port at a desorption temperature of 250 deg. C for 5.0 minutes. A 5.0 u film thickness megabore DB-5 column was used as the guard column and a 60 meter by 0.32 mm I.D. by 0.25 u film thickness DB-5-MS capillary column was used for the chromatographic analysis. The GC was programmed from 30 deg to 250 deg. at 3 deg./minute. In the top chart, the Cryo-Trap was set to 0 degrees C to trap the volatiles. Volatiles down to Methyl-Isobutyl ketone were trapped. When the Cryo-Trap was set to -70 degrees C, more than eight additional volatiles including acetone were trapped and identified which were not trapped at the higher Cryo-Trap temperature. In many instances, the detection and identification of these lighter volatiles are important. These lighter volatiles can be easily analyzed utilizing the GC Cryo-Trap. Analysis by other methods, as liquid extraction, would produce poor sensitivity of these compounds due to sample loss via evaporation during sample extraction and concentration. The thermal desorption process in conjunction with the GC Cryo-Trap proves useful for the isolation and chromatography of these volatile compounds.

Figure # 6 - Purge & Trap of 0.3 ul of Men's Cologne, Purge Onto Tenax TA, Desorb at 250 Degrees Into GC

An example of a Purge and Trap Thermal Desorption analysis of Men's Cologne is demonstrated in Figure # 6. For this sample 0.3 ul of the cologne was injected onto a Tenax TA desorption trap via the S.I.S. Direct Injection Adaptor. The Tenax Trap was then purged off line with 200 ml of Helium at room temperature (25 degrees C) to remove the water and ethanol from the sample. Since water and ethanol comprise the majority of this sample, it is advantageous to purge or remove these materials from the sample. This will result in better chromatography and resolution of the resultant peaks in the chromatogram. The Tenax Trap was then transferred to the Thermal Desorption system and thermally desorbed at 260 degrees for 5.0 minutes into the GC injection port. The GC Cryo-Trap was maintained at -20 degrees C during the desorption process after which it was heated to 200 degrees C. An uncoated deactivated megabore fused silica guard column was used in the Cryo-Trap and the chromatography was accomplished on a 60 meter by 0.32 mm I.D. by 0.25 u film thickness DB-5-MS capillary column. The GC column was programmed from 60 deg to 280 deg at 3 deg./min. A large number of terpenoid type compounds indicative of plant or flower extracts were detected and identified in this sample. In addition, several alcohols, aliphatic hydrocarbons, aromatics and phthalates were also identified among the more than 200 peaks in the chromatogram. This sample demonstrates the usefulness and versatility of the combination of thermal desorption and the GC Cryo-Trap for the analysis of liquid samples.

Figure # 7 - Direct Thermal Extraction of Penicillin at 150'C

We have previously utilized the Short Path Thermal Desorption System for the analysis of residual solvents in pharmaceutical and other solid materials. The use of the GC Cryo-Trap has extended the range of compounds that can be detected and identified using this accessory. Previously, we utilized liquid C02 to cool the entire GC oven. This technique was limited to cooling the oven to -40 deg. C. Attempts to cool the oven colder used excessive amounts of C02. However, the GC Cryo-Trap can routinely trap volatiles at -70 deg. C utilizing only 10% of the liquid C02 used to cool the entire GC oven. An example of the detection and analysis of residual solvents in the solid pharmaceutical Penicillin, is exhibited in Figure # 7. For this analysis, 64 milligrams of the crushed penicillin was inserted in the desorption tube on top of a glass wool plug. The sample was placed in the Thermal Desorption system and thermally extracted at 150 deg. C into the GC injection port. The GC Cryo-Trap was maintained at -70 deg C during the desorption process after which it was heated to 200 deg C. The guard column was a 5.0 u film thickness DB5 megabore column. The analysis was performed on a 60 meter by 0.32 mm I.D. by 0.25 u film thickness DB-5-MS capillary column programmed from 30 deg. to 250 deg. at 3 deg./min. In addition to the compounds previously observed when the entire oven was cooled, we were now able to detect and identify trace levels of acetone and 2-methylpropanol. This was attributable to the lower trapping temperatures achieved by the GC Cryo-Trap.

In many cases, it is advantageous to operate the Cryo-Trap at higher temperatures. This is useful when samples for analysis contain high quantities of water. It is also useful in order not to trap the dilution or sample solvent on the guard column. By maintaining the guard column and the capillary analysis column above the melting points of the solvent, these organics will pass through the columns without being retained during the desorption process, while the higher molecular weight compounds will be trapped on the guard column for subsequent analysis. The GC Cryo-Trap can maintain the guard column temperature at any temperature from the column temperature itself down to -70 degrees C.

Headspace GC Analysis

Figure # 8 - GC Headspace Analysis of Gasoline Vapor via GC Cryo-Trap. Gasoline Vapor In Air at 0.01 ul/ml. Inject 1.0 ml of Headspace Air Into GC Capillary Column Over 15 sec. Cryo-Trap At -70 deg. C For 2. 0 min Then Heat Cryo Trap to 200 deg. C and Then Chromatograph.

The GC Cryo-Trap can also be used with GC headspace techniques. In many GC headspace methods, as Forensic Arson testing, 1.0 ml of air is injected directly onto a 2 mm to 4 mm I.D. packed column. The large diameter packed GC columns are capable of handling this large volume sample size, since the normal column flow is 15 to 60 ml/min. However, with GC microbore capillary columns which operate at flow rates from 0.5 to 2.0 ml/min, the injection of 1.0 ml of air sample would result in very broad tailing chromatographic peaks due to the large sample volume in relation to the capillary column flow. By using the GC Cryo-Trap set to -70 deg. C and injecting 1.0 ml of air into the GC injection port, the volatiles are trapped on the guard column inside of the Cryo-Trap. No matter how large the sample or the time of injection, the organic compounds are trapped effectively in the guard column. Figure #8 demonstrates the analysis of a reference arson sample. The sample was prepared by injecting 1.0 ul of gasoline into a 100 ml clean glass flask with PTFE sealed cap. The flask was heated to 60 C for 5.0 minutes. This provides for a concentration of gasoline of 0.01 ul/ml in air. One (1.0) milliliter of air from the flask was injected into the GC injection port slowly over about a 15 second time interval using a 3 cc plastic disposable syringe. The GC was operated in the splitless mode and the sample was cryo trapped in the Cryo-Trap at -70 deg. C for 2.0 minutes. This allowed sufficient time to trap the organics from this large sample volume in the Cryo-Trap. A 5.0 u film thickness DB-5 megabore guard column was used in the Cryo-Trap and a 60 meter by 0.32 mm I.D. by 0.25 u film thickness DB-5-MS capillary column was used for the analysis. After the injection and cryo-trapping step was complete, the Cryo-Trap was heated to 200 deg. C and the GC programmed from 30 deg. to 100 deg. C at 300 deg./min. The resulting chromatogram exhibits a wide range of volatile hydrocarbons and aromatics. Several light volatiles including butane and pentane produced highly resolved and symmetrical GC peaks. This technique could be applied to other headspace techniques to permit the use of microbore columns and extend the usefulness of this technique.

Figure # 9 - 2.0 ml of Air Containing Tobacco Smoke (15 minutes after burning) Injected Into GC and Trapped on Cryo-Trap at -70 deg C for 2.0 Minutes. GC Temperature Program from 30 to 250 deg. C at 3 deg/min. Direct Thermal Extraction of Penicillin At 150'C

A second sample of the headspace analysis utilizing the GC Cryo-Trap is shown in Figure # 9. For this analysis, smoke from a burning cigarette was permitted to collect in a 100 ml flask which was then sealed. The sample was allowed to set for 15 minutes to permit the particulate matter to settle out of the gas phase, after which 2.0 ml of the air sample were removed from the flask using a 3.0 ml plastic disposable syringe. This gas sample was then injected into the GC injection port over about 10 seconds and trapped on the GC Cryo-Trap at -70 deg. C. The guard column and capillary column and conditions are identical to the gasoline in air sample analyzed above. The major peak in the chromatogram was nicotine. From the analysis of several samples of tobacco smoke, it was determined that the ratio of nicotine to the other volatiles present in the cigarette decreased with time. In addition to nicotine, a large number of aromatics and other organics were identified in the cigarette smoke.

Conclusion

The GC Cryo-Trap has proven to be a useful technique for the analysis of volatiles and semi-volatiles via Thermal Desorption and Headspace GC techniques. These volatile organics are trapped in a narrow band in a capillary guard column in the GC Cryo-Trap at the head of the GC column for subsequent GC analysis. This reduces the use of C02 by more than 90% as compared to cooling the entire GC oven. In addition, cooling temperatures down to -70 degrees C are routinely utilized with the GC Cryo-Trap. The GC Cryo-Trap interfaces with the S.I.S. Short Path Thermal Desorption System and other relay switch controllers for the automatic control and operation of the Cryo-Trap. The GC Cryo-Trap permits the trapping and analysis of volatile organics via several GC introduction techniques and analysis methods to permit the analysis of compounds that would have been otherwise difficult to analyze.