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Note 34: Selection Of Thermal Desorption and Cryo-Trap Parameters In the Analysis Of Teas

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By John J. Manura
1999

INTRODUCTION

The range of organics in a sample such as green and black teas can range from very light gases to very large organics. The techniques available to analyze these samples include headspace GC, Purge and Trap thermal desorption (P&T TD), direct thermal extraction (DTE), solvent extraction and newer techniques, as solid phase micro extraction (SPME) and supercritical fluid extraction (SFE). Proper selection of the analysis technique as well as optimization of the system parameters is required to achieve the desired results. One of the best techniques to analyze food products for flavors is the Purge and Trap System combined with cryo-focusing of the analytes at the front of the GC column before analysis. Through the selection of the purge and trap sampling temperature, GC and thermal desorption system parameters and cryo-trap parameters the range of volatile and non-volatile organics that are analyzed can be selected. In the following article, we are utilizing samples of green and black tea via the Purge and Trap Technique (P&T) with the Short Path Thermal Desoption System (SPDT) to study the P&T parameters, thermal desorption parameters and cyro-trapping parameters which can be adjusted to selectively analyze a range of analytes in these samples. Through the selection of these parameters, data can be obtained similar to what would be received via other techniques such as multidimensional chromatography.

Sampling Technique

Figure 1

Figure 1 - Comparison of GC Sampling and Sample Introduction Techniques

In order to study the volatile organics responsible the aroma and flavor in food products such as teas, a variety of GC sampling techniques can be utilized (Figure 1). These six major sample preparation and GC introduction techniques can be used for the analysis of volatiles and semi-volatiles in food products. Each of these techniques has their unique advantages and disadvantages in the analysis of volatiles and semi-volatiles in food products. The selection of the particular volatile extraction technique or GC injection technique is effected by a number of factors (Table I) which must be considered both for the selection of the sampling technique as well as in its setup and operation. These advantages and disadvantages are briefly described below.


Table I - Factors to Consider in the Selection of a Method for the Analysis of Volatiles in Food Products
  • Sample Matrix - Gas - Liquid - Solid
  • Amount of Sample Available for Analysis
  • Concentration of Volatiles in the Sample Matrix
  • Detection Limit of GC Detector
  • Amount of Water in the Sample
  • Solvents or Major Interfering Compounds
  • Thermal Stability of the Analytes in the Sample
  • Range of Volatiles and Semi-Volatiles in the Sample
  • Range of Volatiles and Semi-Volatiles Desired in Analysis Results
  • Number of Samples to be Analyzed
  • Sample Preparation Time
  • Cost of Analysis
  • Cost of and Disposal of Solvents

Headspace

Headspace is a technique which has been around for a long time. It is most suited for the analysis of the very light volatiles in samples that can be efficiently partitioned into the head space gas volume from the liquid or solid matrix sample. Higher boiling volatiles and semi-volatiles are not detectable with this technique. In addition, the sensitivity of the technique is limited, typically to concentrations in the ppm to ppt range. However, the technique is a preferred method for the analysis of gases and very light volatiles which can not be analyzed by other techniques such as P&T Thermal Desorption. The technique is also a preferred technique when sample automation is required and is also a used in screening of samples.

Solid Phase Micro Extraction

Solid Phase Micro Extraction (SPME) is currently a new and popular technique. It requires minimal accessory equipment (low cost) and is easy to use. Its major shortcomings are the lack of sensitivity (ppt) and the limited range of volatiles which can be analyzed. It works very well with the less volatiles organics such as PCB's and can be automated.

P&T TD

Purge and Trap (P&T) Thermal Desorption is also a very popular technique and is routinely used for the analysis of volatiles in environmental samples as well as food samples. Through the proper selection of adsorbent resins such as Tenax® TA, water can be eliminated from being introduced into the GC. This is important for the analysis of high water content samples such as food products. The Purge and Trap technique is more sensitive by at least a factor of 1000 over headspace techniques. Typical sensitivity is in the ppb range. By purging samples at higher temperatures, higher molecular weight compounds can be detected. However, the Purge and Trap technique requires more time for sample preparation and can not normally be automated (an automated version of the SPTD System is about 2 years away from production). In addition, very light volatiles and gases will not be trapped on the adsorbent resins and therefore will be missed in the analysis. The P&T technique has proven to be the optimal method for the analysis of flavors in food due to its high sensitivity and the ability to eliminate the water from the sample for analysis. Volatiles ranging from pentane through the terpenes can readily be analyzed with this technique.

Solvent Extraction

Solvent extraction has been routinely utilized for the analysis of flavors in foods. However, it has several limitations and disadvantages. Since the cost and disposal of solvents is dramatically increasing, other methods will be needed in the future to replace costly solvent extraction. Also, the technique is limited in the range of volatiles that can be analyzed with solvent extraction. During the evaporation or reduction steps in solvent extraction, all of the very volatile compounds and much of the volatile organics will be lost due to evaporation. Proper selection of solvents is required in order to achieve good extraction and separation of the analytes and a wide range of analytes may not be able to be analyzed in one analysis. However, for the analysis of the semivolatiles and non volatile organics, solvent extraction is probably the method of choice. Detection limits for the non-volatile organics are in the ppb range.

Super Critical Fluid Extraction

Super Critical Fluid Extraction (SFE) is also a very popular technique for the analysis of organics in food products. It is an appropriate technique for the analysis of the less volatile compounds, much like solvent extraction. It has limitations for the range of analytes that can be extracted simultaneously. This limits its usefulness for the analysis of food products with 200 or more volatile and semi-volatile compounds. However, if you are looking for a particular semi-volatile analyte or a narrow selection of analytes, this technique is preferable over solvent extraction. This technique can be automated which also makes it advantageous in many instances.

Direct Thermal Extraction

Direct Thermal Extraction (DTE) is a new technique which is unique to the SIS Short Path Thermal Desorption System. In this technique, volatiles and semi-volatiles can be thermally extracted directly from solid matrix samples without the use of any solvents or any other sample preparation. The advantages of this technique are that a wide range of volatiles and semi-volatiles can be analyzed and the high sensitivity of the technique (typically ppb ranges on samples less than 1.0 gram). Its main disadvantage is the extraction of water into the GC column which will form an ice plug. Most food products contain a high degree of water making this technique not practical. However, for dried food products such as spices and teas, this technique can be utilized. Since no sample preparation is required, the sampling time is small, just weigh the sample into the desorption tube and analyze it. Figure 2 compares the Purge and Trap technique with the Direct Thermal Extraction Technique of a sample of Green Tea. The DTE technique will detect lower molecular weight volatiles than the P&T technique because these volatiles would normally pass through the adsorbent resin in the P&T desorption tube. The DTE technique will also detect higher molecular weight volatiles, because they are extracted directly from the sample thermally at higher temperatures than can be achieved with P&T sampling. Lastly, the DTE extraction technique is more sensitive by at least a factor 10 to 100. In the example in Figure 2, the DTE sample was split 20:1 whereas the P&T sample was not split.

Figure 2

Figure 2 - Comparison Of the Direct Thermal Extraction Technique and Purge and Trap For Teas

Experimental

For most of the samples shown below, 200 milligrams of Black Tea was weighed into a 10.0 ml purge and trap tube and 5.0 ml of distilled water was added. The Purge and Trap Apparatus is described in Application Notes No. 18, 22, 25, 30, 31 and 33. The purge and trap vessel was heated to the temperatures listed below in a hot water bath, and the volatiles were extracted using high purity helium in the Purge and Trap system at a flow rate of 22.5 ml per minute and sampled for 20 minutes (450 ml total). The volatiles were purged onto a desorption tube containing 100 milligrams of Tenax TA which had been previously conditioned. A dry purge of 20 ml per minute was also used to prevent the condensation of water on the adsorbent resin. After sample collection, the desorption tube was purged at room temperature with an additional 100 ml of helium to remove the water from the Tenax resin on the trap.

The desorption tubes were then attached to the Scientific Instrument Services Short Path Thermal Desorption System and thermally desorbed at 250 degrees C, a desorption flow rate of 4 ml per minute for 5 minutes into the GC injection port. The GC injection port was maintained at 250 degrees C, and the volatiles were cryo-focused at the front of the GC column using the Scientific Instrument Services GC Cryo-Trap at a temperature of -70 degrees C. After the desorption process was complete, the GC Cryo-Trap was ballistically heated to 225 degrees C to release the trapped volatiles in a narrow band for chromatography. An H.P. 5890 Series II GC with Electron Pressure Control attached to the HP 5989A Engine Mass Spectrometer was used for the analysis and identification of the analytes. A J&W DB5-MS capillary column was used (60 meter x 0.32 mm x 0.25u film thickness) at a flow rate of 1.8 ml/min. The GC oven was programmed from 30 degrees to 250 degrees at 10 degrees per minute. The analytes were detected using the mass spec to scan the mass range from 25 to 400 daltons.

Results and Discussion

P&T Sampling Temperature

Figure 3

Figure 3 - Effect of Purge and Trap Sampling Temperature During Sample Collection

The higher the temperature of the Purge Trap, the higher the range of volatiles which will be extracted from the sample. This is shown in Figure 3. Two identical samples of black tea (200 milligrams in 5.0 ml of water) were purged with 450 ml of Helium at 50 degrees and 80 degrees C onto a Tenax Trap. After collection, the desorption tubes were purged off line with an additional 100 ml of dry helium to remove the water from the Tenax trap. The Tenax traps were then desorbed at 250 degrees into the GC, Cryo-Trapped at -70 degrees and then analyzed. As expected, a wider range of volatiles was extracted from the sample at the higher sampling temperature. However, the limitation of sampling is about 90 degrees C with this technique due to the water present in the sample matrix. Higher temperatures would saturate the adsorbent with water making it useless for trapping and sample analysis.

Purging To Remove Volatiles

Figure 4

Figure 4 - Effect Of Purging Tenax Trap To Remove Volatiles

Purging samples collected on thermal desorption resins can be used to remove unwanted lower molecular weight volatiles. The purging step after sample collection in Figure 3 was used to remove the water from the sample. Longer purge times or purging samples at higher temperatures can be used to selectively remove additional volatiles from the sample being analyzed. Figure # 4 (top chromatogram) shows the same samples of black tea collected on a Tenax trap. After collection, the desorption tube was attached to the thermal desorption system but NO needle was attached. The desorption system blocks were set to 150 degrees C and the desorption tube was inserted into the blocks, heated to 150 degrees and purged with 200 ml of helium. After this initial purge, a transfer line needle was attached to the desorption tube, the desorption tube blocks were reset to 250 degree C and the sample was analyzed using the thermal desorption system. The more volatile analytes are not present in this chromatogram, since they were eliminated in the 150 degree purging step. This technique is often used to simplify a chromatogram or more often to remove large components from the sample. For instance, when analyzing wine samples, the amount of ethanol is so great that it would produce an enormous broad GC peak that would interfere with the analysis. By selectively purging the Tenax Trap with helium, the ethanol can be eliminated and the analysis of the volatiles and flavors in wine can be easily accomplished.

Thermal Desorption Temperature

Figure 5

Figure 5 - Effect Of Desorption Temperature On Sample Analysis

The thermal desorption temperature can also be used to select the range of volatiles or semi-volatiles that will be thermally desorbed into the GC and detected. In the blacks teas shown in Figure 5, the same black teas were prepared as described previously and then desorbed at temperatures of 150 degrees and 250 degrees C. At the lower temperature, only the lower range of volatiles can be observed. The higher molecular weight analytes are still on the adsorbent resin. At the higher temperature, both the higher molecular weight volatiles and semi-volatiles are detected.

The combination of purging time to remove lower molecular weight volatiles and desorption temperature to observe higher molecular weight volatiles can be used in combination to selectively extract fractions from samples being analyzed via the Purge and Trap Thermal Desorption technique. Results similar to multidimensional chromatography are achievable by selecting these parameters for the particular analytes of interest.

Fractional Thermal Desorption

Figure 6

Figure 6 - Fractional Thermal Desorption Of Black Tea

It is also possible to sequentially desorb the same sample at consecutively higher temperatures. This is shown in Figure 6. The same sample of black tea was prepared as before. The sample was then desorbed at 150 degrees and analyzed. The same sample was then redesorbed at 200 degrees and analyzed again. Finally, the same sample was desorbed a third time at 250 degrees. At 150 degrees C, only the lower boilers were detected. At 200 degrees, the lower boilers were missing, since they were extracted for the previous chromatogram, and only the medium boilers were detected. However, at 250 degrees C, not only higher boilers were detected, but also many of the lower boiler reappeared. These lower boilers were mainly aldehydes. This is probably due to the thermal decomposition of the acids to their corresponding aldehyde at the higher desorption temperatures.

Cryo-Trap Temperature

Figure 7

Figure 7 - Effect of Cryo-Trap Temperatures Of Black Tea

Finally, the Cryo-Trap temperature can be selected to trap specific ranges of volatiles. The lower the temperature of the GC Cryo-Trap, the lower the range of volatiles that will be detected by the GC. In Figure 7, two samples of the same black tea were trapped at 0 degrees C and -70 degrees C. More than 12 additional volatiles are seen at the lower temperature which are not trapped at the higher temperature. At the higher temperature, the very light volatiles are not trapped and therefore are not seen in the chromatogram. They would be present as broad peaks in the early (first 5 minutes) parts of the total chromatogram. This technique is often used to eliminate low boiling volatiles from the sample being analyzed. For example, by trapping at +10 degrees C, no water will be trapped and therefore will not interfere with the analysis. However, when using this technique, it is important to set the cryo-trap time to at least 5.0 minutes after the sample has been collected. This long trapping time will give the GC column sufficient time to elute these compounds through the GC column so that they do not interfere with the subsequent sample analysis.

Conclusion


Table II - Parameters Which can be Adjusted to Select Analytes for Analysis

Temperature Parameter                Low Temperature                   High Temperature

-----------------------------    ------------------------------    -----------------------------

Purge and Trap Temp              Purge only low boilers            Purge low and High boilers

Post Purge of Desorption Tube    Remove water and low volatiles    Remove higher boilers     

Thermal Desorption Blocks        Extract low boilers only          Extract low and high boilers

Cryo-Trap Temperature            Trap Low and high boilers         Trap high boilers only


A variety of techniques are available for the sample collection and analysis of volatiles in food samples as well as other samples. One of the more popular techniques is the Purge and Trap Thermal Desorption technique in conjunction with the GC Cryo-Trap. By proper selection of the system parameters including sampling temperature, desorption tube purging, desorption temperature and cryo-trap temperature selective results can be obtained as listed in Table II. Interfering compounds can be eliminated from the chromatogram or analysis time can be shortened to analyze for specific analytes. In other cases where a wide range of volatiles is desired, the system parameters can be adjusted to analyze the majority if not all of the analytes of interest in the sample matrix. These parameters can be optimized to achieve analysis results similar to multidimensional analysis but at a much lower cost in both equipment and equipment expertise.

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