Theory and Instrumentation of GC. Sampling Techniques

Transcription

1 Theory and Instrumentation of GC Sampling Techniques i Wherever you see this symbol, it is important to access the on-line course as there is interactive material that cannot be fully shown in this reference manual.

2 Aims and Objectives Aims and Objectives Aims To investigate the various sampling techniques available for GC analysis To give a detailed description of manual injection and injection troubleshooting To describe the use of Autosamplers to increase sample capacity, accuracy and precision To describe the principles, primary operating parameters and practical implementation of a series of sampling techniques Objectives At the end of this Section you should be able to: Choose the relevant technique for the introduction of a wide range of sample types and presentations Describe the practical implementation of various sample introduction methods Demonstrate a good understanding of problems that occur in GC injection and describe how these might be practically overcome Explain the general principles of a series of sampling techniques Identify the major parameters for each of these techniques which require optimisation and describe optimisation strategies

3 Content Sampling Techniques Overview 4 Gas Sampling 4 Liquid Sampling 4 Solid Sampling 5 Manual Injection 6 Important Syringe Variables 6 Loading the syringe -Adequate washing and flushing of a syringe 7 Optimised Injection Routine 7 Cold Needle Technique 8 Hot Needle Technique 9 Air Gap Technique 10 Solvent Flush Technique 12 Automatic Liquid Sampling 12 Precision 14 Carry over 14 Gas sampling Devices 15 Gas sampling valves 17 Two position valve application 10 port valve 18 Pressure problems 19 Further Applications 20 Purge & Trap Autosamplers (a) 20 Stage 1 Wet Purge 22 Stage 2 Dry Purge 23 Stage 3 Desorb Preheat 23 Stage 4 Desorb 24 Stage 5 Thermal Bake 26 Purge & Trap Autosamplers (b) 26 Trap materials 27 Application notes 29 Thermal Desorption Autosamplers 29 Tow Stage Thermal Desorption 31 Tow Stage Desorption with Cold Trapping 32 Thermal Desorption Autosamplers Important parameters 33 Tube length and Internal Diameter 33 Predesorption checks 33 Tube heating 34 Gas Flows 34 Refocusing (single or two-stage desorption) 34 Sample Splitting 35 TD Sorbent selection and applications 35 Solid Phase Microextraction (SPME) 36 Extraction/Desorption Procedure 37 Important SPME Parameters 38 Fibre Characteristics 38 Immersion Sampling 38 Sample Factors 39 Headspace Sampling 39 Sample agitation 39 Inlet Liner Dimensions 39 Fibre Chemistry 40 Headspace Sampling (HS) 41 Crawford Scientific 2

4 Headspace Autosamplers 43 Inlet (Liner) 44 Flow 45 Start temperature 45 GC Cycle time 45 Equilibration Time 45 Temperatures 46 Injection Time and Volumes 46 Pressure (Pressurization Time, Loop Filling Time) 46 Vial (Sample amount) 46 Loop 47 Headspace Calibration and Quantitation 47 Calibration with Pure Sample Matrix 48 Calibration by the Mixing Method 48 Toluene peak 48 MHE 48 Crawford Scientific 3

5 Sampling Techniques Overview Perhaps the most difficult step in any GC analysis is the sample introduction. Solid and Liquid samples need to be converted to the gas phase, and then transported onto the column. Samples already in the gas phase need to be efficiently diverted or transported into the GC column avoiding any potential condensation en-route. There are many devices available for the introduction of various samples types into the GC and these will be studied in detail within this section. The primary aim with all sampling techniques is to ensure a representative and homogeneous aliquot of the sample under investigation is delivered to the GC column. In the main syringes are used to introduce liquid samples into the GC inlet. However, there are also many different instrument types to allow sampling of difficult matrices and to achieve pre-concentration of the analyte with respect to other matrix components. Gas Sampling Gas sampling methods generally require the entire sample to be in the gas phase at the conditions under which sampling occurs. If the samples are mixtures of gases and liquids, either heat and/or pressure are usually employed top ensure that the entire sample is a gas. A variety of techniques exist for gas sampling including, canisters, gas tights bags, gas tight syringes and valves. All of these are capable of introducing samples in to the GC inlet in a representative fashion when used correctly. The gas sample may be directly introduced into the GC column (when using certain valve types), however it is more usual to introduce the sample via a conventional GC inlet such as a Split/Splitless inlet. Headspace instrumentation is often used to sample the headspace gas above either a liquid or solid sample. All of the above techniques will be covered in more detail in this section. Liquid Sampling The traditional method of sampling liquids is by syringe, which can be used manually or in conjunction with an automatic liquid sampler. It is typical for a small aliquot of a volatile liquid sample to be introduced into a vaporising inlet where the sample is rapidly heated and transferred into the gas phase and subsequently swept on the GC column. Results are improved where the liquid used is volatile, however, less volatile liquids may also be sampled by syringe and special GC inlets such as cool-on-column (COC) or Programmed Thermal Vaporisation (PTV) can be used to overcome liquid volatility issues. Crawford Scientific 4

6 Syringe Most volatile liquids have large expansion coefficients and as such, care should be taken when using vaporising inlets such as the split/splitless device, not to overload the inlet. When using vaporising inlets care should also be taken not to overheat the sample during the volatilisation process, otherwise thermal degradation of the analyte might occur. Solid Sampling It is much less commonplace for solid sampling to be used in conjunction with GC analysis. By its nature, Gas Chromatography deals with the analysis of volatile species. GC solid sampling Solid samples are best handled by first dissolving them in a suitable volatile liquid and then injecting the solution using a syringe. The exception to this includes the categories of analysis in which volatile species are entrained in solid matrices. In these types of analysis it is usual to drive the analyte from the solid by first increasing the surface area of the solid (perhaps by micronisation) followed by subsequent strong heating. The evolved gases are then either sampled from the headspace around the sample (headspace analysis), or all evolved gases are swept into the GC column (programmed thermal vaporising analysis). Solid sampling options will also be covered in this section. Crawford Scientific 5

7 Manual Injection The most rudimentary method for liquid sample introduction into the GC inlet is to use a syringe to manually inject into the inlet usually a vaporising device such as a split/splitless inlet. The liquid is vaporised within the inlet via heating and part or all of the resulting gas swept into the GC column. The number and variety of different syringe types available is bewildering but there are several basic features that are important regardless of the style or manufacturer of the syringe, including: Syringe volume. Fixed or removable needle. Needle Outside diameter (OD). Needle point Style. There are several important concepts in Manual Injection of liquid samples that must be considered. These are mainly related to how representative the ultimate gas phase products are of the original sample. For example, it is possible for higher boiling (less volatile) compounds to condense on the needle when it is being withdrawn and cause relative losses of these compounds, therefore several specialist techniques are available for manual injection. It is important that the same volume of liquid is introduced each time into the GC inlet and that the volume is appropriate for the inlet internal dimension. Important Syringe Variables Syringe Volume: You chose a syringe so that the amount being injected is no smaller than 10% of the syringe nominal volume. That is no less than 0.5µL for a 5mL syringe and no less than 1µL for a 10µL syringe. The will keep quantitative errors to a minimum. Needle style (Fixed or removable): The fixed style of needle is preferred for experienced operators, autosampler injections and for applications requiring very low detection limits. These syringes contain needle and stainless steel fixing that is cemented onto the bottom of the glass syringe barrel and have the lowest dead volume of all syringe types. Fixed needle designs also show the lowest degree of carryover as the space between the barrel and needle is filled with cement. Removable needles are recommended for use with inexperienced operators or where the risk of needle bending is higher. The syringe has a screw thread or Luer fixing at the end of the barrel onto which a housing with an interchangeable needle can be attached. These needles are more economic for use where needle bending can be common but have lower accuracy and precision. Crawford Scientific 6

8 Needle Outside (OD) and Inside Diameter (ID): The inside diameter should be selected to ensure minimal dead volume without compromising the ability of the syringe to draw samples of normal viscosity. Medium to high viscosity samples should be diluted or a larger ID needle may be required. To reduce the possibility of bending, choose the widest available needle outside diameter suitable for the application. Autosampler syringes with 0.63mm OD needles, should be selected for all applications except oncolumn injection. Table 1. Typical needle Internal/External diameter combinations. Gauge Nominal OD (mm) Nominal ID (mm) 26s / / s s Needle Point Style: The cone shaped needle style has been developed for repeated use with an auotsampler device which will impact the inlet septum in the same place during each injection and can part the septum during the injection to reduce coring or splitting of septa. Bevelled point needles are recommended for manual injection as they will reduce septum damage in the situation where the septum is not pierced in exactly the same spot each time. Loading the syringe -Adequate washing and flushing of a syringe A syringe should be flushed with approximately 5-10 times its total capacity to eliminate carryover between samples. This is achieved by repeatedly drawing and expelling solvent/sample from the syringe. To avoid contaminating the sample, the first 2-3 washes should be discarded to waste. Small air bubbles can be removed by repeatedly drawing sample into the barrel and rapidly expelling the sample while keeping the needle tip immerse in the solution. Turning the syringe barrel upright while expelling the sample may also help remove bubbles. Optimised Injection Routine 1. Immerse the needle tip into the solution to be sampled. 2. Withdraw the plunger to aspire one syringe volume of liquid. 3. Remove the needle from the solution. 4. Expel the contents of the syringe into a waste vessel. 5. Repeat steps Immerse the needle into the liquid again. Take up and expel the sample at least 5 times, being careful to keep the needle tip submerged. 7. Fill the syringe past the intended delivery volume mark by at least 4mm. Crawford Scientific 7

9 8. Carefully position the plunger to the intended volume mark holding the syringe at eye level to avoid parallax error. Decide where you intend to stop the plunger tip whether at the front or back edge (or middle) of the calibration etching on the syringe barrel. For all measurements, use the position you decide on to set the plunger. 9. Check the liquid volume for and microbubbles. If you find any, repeat steps Draw the plunger back to draw at least 1µL of air into the syringe and to empty the needle 11. Hold the syringe with the calibration marks facing you. Place your forearms on the front corners of the GC top to maintain the syringe orientation. This puts the needle bevel in the same position injection after injection and reduces coring the septum. 12. Insert the needle through the septum to its full needle depth. 13. Hold the syringe vertical and allow the needle to heat up for about 5 seconds. Keep this time as constant as possible once you determine how long you want to use. This allows the needle to be at a constant temperature for each injection and minimizes sample discrimination. 14. Make the injection rapidly and smoothly by depressing the plunger. 15. Leave the needle in place for at least seconds. As long as you leave the needle in place at least 15 seconds, precision is enhanced. 16. Withdraw the syringe cleanly. These steps should give precision on the order <1% RSD on a repeated basis. Care should be taken using the hot needle injection technique with compounds that are thermally labile in the presence of a metal needle (eg. Organochlorine Pesticides). Compounds like these may decompose during the injection process. Cold Needle Technique There are two basic techniques for manual injection. The first technique is known as Cold Needle and mimics an autosampler injection performing a very fast injection, with minimal dwell time of the needle inside the hot injector, and very fast withdrawal after the liquid expulsion. The method is known as the cold needle technique, because the needle does not have time to heat up in the injector. Since the needle is not allowed time to become hot, the technique may result in liquid remaining in the needle at the end of the process unless a plunger in needle type syringe is used. The volume of sample solution dispensed using the cold needle technique will match the volume reading on the syringe barrel. Another potential problem with this technique relates to the analysis of less volatile compounds. Analytes in the gas phase encountering the cold needle in the injection port may condense onto the outside of the needle which is subsequently withdrawn from the injection port. This may cause the loss of less volatile sample components relative to more volatile components. This phenomenon, known as MassDiscrimination. Crawford Scientific 8

10 i Cold needle technique Hot Needle Technique The second method for manual injection, known as the Hot Needle technique, involves leaving the needle inside the injector for 5 seconds, or more, so that the needle heats up, before expelling the sample solution. This technique is often suggested when the sample contains very high boiling point compounds and one suspects that mass discrimination might occur. The principle is that the hot needle will help speed up the process of vaporisation, since the sample will start to vaporise as it travels through the needle. The hot needle will ensure that no sample solution remains inside the needle after the plunger is depressed and the higher boiling analyte does not condense onto the needle surface hence significantly reducing discrimination processes. The key difference between the hot and cold needle techniques is the dispensed volume. The cold needle technique dispenses the volume measured in the barrel. The hot needle technique will dispense the volume contained in the barrel, and in the needle. In the cold needle technique, the dispensed volume when the plunger is withdrawn to the 1µl mark is 1µL. In the hot needle technique the dispensed volume is 1.5µL as the needle generally Crawford Scientific 9

11 contains about 0.5µL of liquid, depending of course on needle length and internal diameter. This is acceptable providing the volume drawn into the barrel is consistent. However, if, for example, 2.0µL is injected for a standard and 1.0µL is injected for a sample, it is not acceptable to double the peak area of the sample and then extrapolate from the calibration curve. This is because the actual injected volume is 2.5µL for the standard and 1.5µL for the sample i.e., 2.5 / 1.5, rather than 2/1. i Hot needle technique Air Gap Technique The air gap method can be used in conjunction with either the hot or cold injection technique, but gives the most noticeable improvement when used with the cold needle technique. The sequence of events is outlined below. Crawford Scientific 10

12 Fill the syringe to above the required volume mark and then expel excess sample back to the required volume mark. Withdraw the liquid fully into the barrel so that an air gap can be clearly seen (use twice the volume of the liquid injected as a rule of thumb). Quickly introduce the needle into the GC inlet and immediately depress plunger. Quickly withdraw the needle from the inlet. i Air gap The benefit of using the air-gap technique is that whilst the air is passing through the needle the needle itself will be heating. When the sample liquid passes through the needle it will have warmed considerably and so the problems with discrimination of high boiling materials are significantly reduced or even eliminated. The benefits of the Air Gap technique are: No loss of volatiles before GC injection. Improved quantitative reproducibility. Complete sample transfer. Crawford Scientific 11

13 Solvent Flush Technique The solvent flush technique can be used when analysing polar compounds, which potentially might adsorb to the inside of the glass or needle. A small amount of solvent is withdrawn into the syringe followed by air and then followed by the sample. The aim is for the solvent to follow the sample through the syringe flow path and remove any adsorbed material on the inside surface of the barrel and needle. Potentially a different solvent could be used from the sample solvent e.g. a more polar solvent. Remember though to be aware that if the total volume injected into the liner is too large, and is in excess of the volume capacity of the liner, flashback can occur. Plunger in Needle Syringe: In this syringe design the plunger travels through the needle to help expel very small volumes. Usually used for volumes between 0.5 and 0.1 ml all liquid is expelled from both the barrel and needle for improved precision. Automatic Liquid Sampling Perhaps the most popular method of liquid sample introduction in Modern Gas Chromatography is the Automatic Liquid Sampling Device or Autosampler. These devices mimic the manual injection process and use automated syringe mechanisms to aspirate the sample and inject into Split/Splitless, Cool-on Column or Large Volume GC inlets. The autosampler is conventioanlly mounted on top of the GC, over the inlet. Most modern auotsampler devices use a syringe whose plunger is fitted to a stepper motor drive mechanism that allows very accurate sampling volume. Most samplers are also equipped with wash bottles to allow automated washing of the syringe before and after injection to reduce sample carryover. Perhaps the biggest advantage of the autosampler device is that it allows unattended operation and when combined with a sample tray can be used to inject literally hundreds of samples in overnight analytical campaigns. Crawford Scientific 12

14 Agilent 6890 GC with autosampler tray More sophisticated autosamplers use a fast injection technique that overcomes many of the discrimination and needle fractionation processes that were described in the topics on manual injection. By inserting the needle, pressing the plunger and removing the needle from the inlet with a cycle time of less than one second, the expelled sampled is in liquid form. As the needle has no time to heat, and due to the rapid expulsion of the sample, the problems due to loss of volatile material through needle fractionation or adsorption of the higher boiling compounds onto the needle inner surface is overcome. Further, because the needle is very rapidly withdrawn, there is no opportunity for less volatile analytes to condense onto the outer surface of the needle, so reducing or eliminating analyte mass discrimination. Crawford Scientific 13

15 Precision Autosamplers deliver an accurate and reproducible amount of sample to the GC inlet every time. In the example above a 1ml injection of C10 to C40 paraffin s in hexane is compared using manual and autosampler techniques. Automated versus manual injection It is clear that using the autosampler gives much less discrimination towards higher boiling (later eluting) compounds than the manual injection technique. Without fast injection the sample introduced is much richer in low boiling components because of fractional distillation that occurs needle. Not only does residual sample in the needle enter the inlet, but the low boiling components boil off first. This is known as needle fractionation or discrimination. Carry over Sample Carryover is the presence of peaks from an earlier injection in the present analysis. Autosamplers can use solvent washes and sample washes to minimise carryover. Care should be taken to ensure that the wash solvent used is compatible with the analyte / sample matrix for most effective washing. Crawford Scientific 14

16 Carryover reduction The example above shows the reduction in carryover possible by introducing a wash step into the autosampler program. For sample washes (discarding the wash each time) will typically reduce carryover to less than 10ppm although the number and type of wash required will depend on: The acceptable amount of carryover The viscosity and solubility of the analyte The viscosity and volatility of the sample solvent The wash volume The degree of wear in the syringe barrel Gas sampling Devices Gas sampling for GC can be achieved using either off-line or on-line devices. Off-line devices allow the collection of a gas sample using a cylinder, syringe or bag with subsequent introduction to the GC via a suitable inlet such as a split/splitless or packed inlet. Gas tight syringes, Tedlar bags and cylinders may all be used to collect gas samples from the site of interest prior to introduction using a gas tight syringe or specialist introduction device. Some examples and applications are given opposite. On-line sampling devices usually consist of heated valve units which are teed into the gas supply with a diversion valve to allow an aliquot of the gas to be sampled to the GC. Gas sampling valves usually give improved accuracy and precision over other gas sampling devices. It is important to note it is a requirement that the whole sample be in the gas phase prior to introduction into the GC and for this purpose, it is usual to heat the sampling device whenever there is a possibility of gas condensation. Heating gas sampling valves usually improves the quantitative accuracy of the analysis. The sampling loop pressure is of importance for accurate quantitative analysis. Any differences between the gas pressure in the sampling loop and the carrier gas pressure can result in large baseline shifts and the possibility of poor peak shape and quantitation. To avoid these problems the gas sample in the loop may be pressurised or alternatively Crawford Scientific 15

17 the GC carrier can be operated in constant pressure mode rather than the constant flow mode. Operating in constant pressure mode reduces the pressure differences and improves peak shape at the point of sample injection. Tedlar bags used for at-site gas sampling. Gas tight syringe can be used for sample transfer to the GC inlet. Gas tight syringe used for gaseous sample introduction. Syringes may have to be heated to avoid sample condensation. Heated gas sampling valve mounted on the upper casing of a GC. Canister sampling device. The sample gas is released from the canister at a controlled flow rate and volume into the sample inlet. Crawford Scientific 16

18 Gas sampling valves Gas sampling valves can automatically sample gas from one or multiple gas streams. Valves can also be used to bypass, vent and backflush pre-columns and many very complex applications exist some typical applications can be viewed below. In this simple application, a refinery gas stream is diverted to a sampling valve mounted onto the chassis of the GC. The valve is rotated to the load position and the gas stream is used to fill the sample loop. The loop is overfilled many times to flush any contaminants. Once the loop is filled, the valve is turned and the gas sample is transferred to a split/splitless inlet using a heated transfer line. There are several factors that require optimisation using Gas Sampling Valves and these typically include: Sample Loop Size, Split Flow Rate and Oven Temperature Program. These factors will determine the accuracy and peak shape quality of the analysis with sample loop size and split flow being most important in determining the peak shape. It should also be noted that and differential between the gas pressure in the sample loop and that of the carrier at the start of the analysis can bring about large baseline position shifts and problematical peak shapes. Plumbing diagram for a 6-port, 2 position gas sampling valve application for refinery gas analysis Separation of 12 refinery gas components using a gas sampling valve Crawford Scientific 17

19 Table 2. Analysis conditions. Item Information S/S inlet 175 o C, 30:1 split ratio typical Valve Gas sampling valve, 6-port Valve temperature 80 o C Sample loop 0.1mL typical Column flow (He) 5mL/min Column Plot alumina M 50m 0.53mm 0.25mm Oven 40 o C (2min) to 140 o C (5min), 4 o C/min Detector FID, 300 o C H 2 35mL/min Air 350mL/min Make up 26mL/min Two position valve application 10 port valve This is ideal for fixed gas-from-co 2 analysis where no "high boilers" are present. Column 1 is packed with a porous polymer and Column 2 with molecular sieve. The sample loop is loaded in Position A. When the valve is switched, the loop contents are sent onto Column 1. As the inorganic gases and methane leave Column 1 and enter Column 2, the valve is returned to Position A, reversing the column sequence. CO 2 now leaves Column 1, becoming the first peak. The inorganics and methane are separated by the mole sieve and pass through the porous polymer column to the detector. Position A: Plumbing diagram for a 10-port valve Crawford Scientific 18

20 Position B: Plumbing diagram for a 10-port valve Pressure problems The left hand example above clearly illustrates the problems often encountered when gas sampling loop and initial carrier gas pressure are mismatched. If the carrier gas pressure is higher than the loop pressure when the valve is switched, the gas sample in the loop will bleed into the inlet giving rise to problems with baseline shifts and analyte peak shape. For this reason it is often necessary to ensure the gas loop is under pressure prior to the injection, or the carrier gas is pressure programmed to ensure a low initial pressure to allow fast transfer of the sample from the loop and avoid changes in carrier flow to ensure a stable baseline position. Chromatogram comparison (with and without pressure control) Crawford Scientific 19

21 Further Applications Analysis of permanent gases and methane Combustible gas effluent monitoring Dual Channel Refinery Gas Analysis Flexible gas sampling solutions Natural gas analysis with single column Natural Gas and NGLiquid Analysis Oxygenated compounds in gasoline Refinery gas applications Refinery gas analysis Trace sulfur in beverage CO 2. Volatile Sulfur in Natural Gas Purge & Trap Autosamplers (a) Purge and Trap techniques are often referred to as Dynamic Headspace analysis. Unlike Static Headspace analysis, Purge and Trap techniques do not rely on establishing an equilibrium between volatile species in the sample matrix and a fixed volume of headspace gas and therefore are more sensitive than static techniques. Further, the use of a purge gas passing through the sample matrix overcomes many of the sample matrix problems encountered with Static Headspace Techniques. Samples containing Volatile Organic Compounds (VOC s) are introduced into a purge vessel and a flow of inert gas is passed through the sample at a constant flow rate for a fixed time. Volatile compounds are purged from the sample and transferred to and concentrated on an adsorbent trap. After the purging process is complete, the trap is rapidly heated and backflushed with carrier gas to desorb and transfer the analytes to the GC column. The nature of the materials in the Trap are of great importance and several different adsorbents exist to optimise the adsorption characteristics towards various analyte species. This subject is discussed in the next topic. Purge and Trap analysis has several variables that need to be considered when developing and optimising analytical conditions. Crawford Scientific 20

22 i Purge mode i Desorb mode Crawford Scientific 21

23 Stage 1 Wet Purge The purge gas (usually helium) is sparged through the sample. The purge gas dissolves volatile sample components and carries them from the sample towards the adsorbent trap. Helium is used as it shows very good solubility towards dissolved VOC components but is itself insoluble in most liquids and solids. The purge gas exits the instrument via the purge vent where flow rate can be measured. Typical purge flow rates are 30-50mL/min and purge times 10-15min. During the purge mode, the desorb (carrier) gas is directed onto the column. Three types of purge vessels (i.e., spargers) commonly are used in purge and trap systems. i Frit spargers are used for most water samples. The frit creates many small bubbles that travel through the sample to increase purging efficiency. Fritless spargers are used for samples that have high particulate content, or for industrial wastewater samples that may foam. They create fewer bubbles, which decreases purging efficiency but eliminates plugged frits and reduces foaming problems. Needle spargers are used when purging soil, sludge or solid samples. A narrow gauge needle is inserted into the sample and used to release a small stream of purge gas. The two common sizes of spargers are 5mL and 25mL. Three types of purge vessels (i.e., spargers) commonly are used in purge and trap systems. Frit spargers are used for most water samples. The frit creates many small bubbles that travel through the sample to increase purging efficiency. Fritless spargers are used for samples that have high particulate content, or for industrial wastewater samples that may foam. They create fewer bubbles, which decreases purging efficiency but eliminates plugged frits and reduces foaming problems. Needle spargers are used when purging soil, sludge or solid samples. A narrow gauge needle is inserted into the sample and used to release a small stream of purge gas. The two common sizes of spargers are 5mL and 25 ml. Crawford Scientific 22

24 Stage 2 Dry Purge During the wet purge a large amount of water may be transferred to the adsorbent trap. Various manufacturers overcome this problem in different ways including the addition of an extra hydrophilic trap between the sample and the adsorbent. The most common way to remove water from the trap is to purge the trap with an inert gas whilst the sparger is bypassed this is usually achieved by means of an extra switching valve. It is important to remove water from the sample prior to desorption as it can cause loss of sensitivity, reduced column lifetime and spurious peak shape in the final chromatogram. Typical dry purge times are between 1 and 4 mins. It should be noted that only traps using hydrophobic material can be used with a dry purge mode. During the dry purge mode, the desorb (carrier) gas is directed onto the column Important i It should be noted that only traps using hydrophobic material can be used with a dry purge mode. During the dry purge mosde, the desorb (carrier) gas is directed onto the column. Stage 3 Desorb Preheat Typical dry purge times are between 1 and 4 mins. Once the analytes are adsorbed onto the trap and water has been removed the purge gas is stopped and the trap is heated to 5 o C below the desorb temperature of the trap material being used. This step acts to pre-concentrate (or focus) the analytes into a small band on the adsorbent material to ensure efficient transfer onto the GC column. Without this step the chromatographic peaks would tail badly. Crawford Scientific 23

25 i During the preheat mode, the desorb (carrier) gas is directed onto the column. Stage 4 Desorb At the end of the preheat period the valve is switched and the carrier gas used to backflush the trap into the GC inlet. The flow rate and desorb temperature are vitally important and will depend upon the application, adsorbent type used and GC inlet type. Having a high desorb flow rate (>20mL/min.) is beneficial in terms of resulting peak shape, however this flow rate is incompatible with most capillary GC columns. To overcome this problem, a split flow is used to reduce gas flow to the GC column. It is important to balance the need for sensitivity with good peak shape and the relationship between desorb flow rate and split flow is optimised empirically. It is also possible to desorb the trap more slowly so that flow rates are compatible with capillary GC columns, and to re-focus the broad analyte band on the top of the GC using cryofocussing. This is a technique in which liquid nitrogen or CO 2 is blown around the top of the GC column in order to super cool it and cause condensation of the volatile analytes onto the stationary phase. Crawford Scientific 24

26 i Typical desorb times range from 2-4 minutes at temperatures between 180 and 250 o C. Desorb flow rates will vary depending upon application type but will typically lie in the range ml/min. EPA method 8020, RTX-5SIl M S column, 40m 0.45mm 1.5μm Where: 1. benzene 2. α, α, α-trifluorotoluene (SS) 3. toluene 4. ethylbenzene 5. m-xylene 6. p-xylene 7. O-xylene Analysis conditions. (Temperature Program) Carrier gas 9 ml/min (constant pressure) GC Finnigan 9001 Detector FID Oven temp 40 o C (hold for 2 min) 85 o C (hold for 1 min) (4 o C/min) 225 o C (hold for 2 min) (40 o C/min) Crawford Scientific 25

27 Stage 5 Thermal Bake After the desorb step the trap is baked, with gas flow, to remove any remaining sample components and contaminants from the trap in preparation for its next use. This step generally lasts 6-10 minutes; typical temperatures are C above the desorb temperature. To prevent damage to the adsorbent materials, do not exceed the maximum temperature of the trap. i Purge & Trap Autosamplers (b) Adsorbent materials are used to trap the VOCs that have been purged from the sample. The adsorbent must be able to retain compounds during the entire purging sequence and then rapidly release them during the desorption step. Each adsorbent has a unique trapping capability for a specific class or classes of compounds. Therefore, a trap may have several different beds of adsorbents. The weakest adsorbent material is placed at the inlet end of the trap, then the next strongest adsorbent, and so on. The more volatile compounds pass through the weaker adsorbents and are retained by the stronger adsorbents, while the less volatile compounds are retained on the weaker adsorbents and never reach the stronger adsorbents (from which they would be difficult to desorb). Crawford Scientific 26

28 The method shows the determination of low-level volatiles in solid and water by USEPA. Method 5035 using model 4552 Water/Soil Autosampler. The chromatrograms show typical BTEX standards. Typical application from O I Analytical (college Station, Texas, US). Table 3. Method. OI Analytical Model 4560 Purge and Trap Water Method Soil Method Trap # 10 (Tenax, silica gel, carbon molecular sieve Purge time and temp. 11 min, 15 o C 11 min, 15 o C Desorb time and temp. 4 min, 180 o C 4 min, 180 o C Bake time and temp. 10 min, 190 o C 10 min, 190 o C Infra-Sparge temp. 40 o C (NA) Sample inlet temp. 40 o C (NA) Water management ON ON Valve temp. 100 o C 100 o C Transfer line temp. 100 o C 100 o C Total cycle time 25 min 25 min Trap materials Adsorbent materials are used to trap the VOCs that have been purged from the sample. The adsorbent must be able to retain compounds during the entire purging sequence and then rapidly release them during the desorption step. Each adsorbent has a unique trapping capability for a specific class or classes of compounds. Therefore, a trap may have several different beds of adsorbents. The weakest adsorbent material is placed at Crawford Scientific 27

29 the inlet end of the trap, then the next strongest adsorbent, and so on. The more volatile compounds pass through the weaker adsorbents and are retained by the stronger adsorbents, while the less volatile compounds are retained on the weaker adsorbents and never reach the stronger adsorbents (from which they would be difficult to desorb). Tenax Tenax adsorbent is excellent for trapping nonpolar compounds and is hydrophobic so it does not retain water; however, it does have some disadvantages. Very volatile compounds are not retained well and must be trapped on a stronger adsorbent material. In addition, polar compounds like alcohols are poorly retained on this adsorbent. Tenax adsorbent also has limited thermal stability; the 2,6-diphenyleneoxide polymer thermally decomposes into toluene, benzene, and other aromatics. The particles melt together and permanently adhere to the trap; this then restricts carrier gas flow. As the adsorbent degrades, there often is a loss in response for brominated compounds. There are two grades of Tenax adsorbent used as a trapping material: Tenax GC and Tenax TA (Trapping Agent) adsorbents. Common background contaminants in Tenax GC adsorbent include benzene and toluene. Tenax TA adsorbent is a purer form and is more commonly recommended for thermal desorption applications. Silica Gel Silica Gel (surface area: m 2 /g): Silica gel is a stronger adsorbent than Tenax adsorbent. Silica gel is commonly used in conjunction with Tenax adsorbent as a trap for volatile organic pollutants. It is an excellent trapping material for polar and highly volatile compounds that are gases at room temperature; however, silica gel is extremely hydrophilic and will retain large amounts of water. Be aware that if a trap contains silica gel, dry purging will not reduce the water content. Carbon Black Graphitized Carbon Black or Carbopack Adsorbent (surface area: m 2 /g): Graphitized carbon black (GCB) is an alternative to Tenax adsorbent. GCB is available in many pore sizes and is effective in trapping volatile organics in the same range as Tenax adsorbent. GCB is hydrophobic and has excellent thermal stability, making it ideal for purge and trap techniques. Highly volatile compounds are not retained well on GCB and must be trapped on stronger adsorbent materials such as carbon molecular sieves. Carbon Molecular Sieves Carbon Molecular Sieves (surface area: m 2 /g): Carbon molecular sieves such as Carbosieve -SIII are alternatives to silica gel and charcoal. High surface areas make these materials ideal for trapping highly volatile compounds. They are commonly used in series after GCB because they retain compounds that break through the GCB. Carbon molecular sieves are hydrophobic and have excellent thermal stability. Carboxen Adsorbent (surface area: 1200m 2 /g) Carboxen Adsorbent (surface area: 1200m 2 /g): Carboxen adsorbent is a strong adsorbent designed to be used as the innermost adsorbent bed in the trap. This material traps Freon compounds, permanent gases, and light hydrocarbons. It has characteristics very similar to those of Carbosieve S-III packing material. Carboxen adsorbent is stable to temperatures of 300 C. Its only shortcoming is the adsorption of CO 2, which can interfere with early-eluting compounds, 2 Carboxen and Carboxen are similar materials. Crawford Scientific 28

30 Application notes Flavour Components Sulphur in Beer US EPA 5035 US EPA VOC in Water US EPA GC-MS Oxygenates from Water Optimising VOC Analysis Thermal Desorption Autosamplers Thermal Desorption techniques are an alternative to more conventional sample preparation techniques such as liquid extraction. The technique is well suited to analysis of trace level volatile organic compounds in a variety of real world matrices. There are many practical applications for this techniques ranging from QA/QC analysis to Environmental and Occupational Hygiene analyses. The technique is relatively simple and involves controlled heating of the sample in an inert gas stream (often the GC carrier gas) to extract target compounds into the vapour phase via dynamic gas extraction. The extracted volatiles are then transferred directly to the GC column. Thermal Desorption typically offers a 1000 fold enhancement in sensitivity, with little sample preparation requirements and importantly little interference from solvents which is particularly beneficial for GC-MS users. A schematic of a typical Thermal Desorption instrument is shown in which the combination of heat and the gas stream are used to extract volatile organics retained in a solid sample matrix or on a sorbent bed. Crawford Scientific 29

31 GC equipment with Split/Splitless injection port Application areas Industrial hygiene - monitoring the personal exposure of workers to toxic chemicals in the workplace Environmental monitoring for -Ambient urban air / Indoor air / Emissions from building materials and related consumer products / Tracer gas tests of building ventilation Materials QA/QC - residual solvents, residual monomer, taint, etc Flavour and fragrance - odour profiling, shelf life, etc Organic composition - water-based paints, syrups, medicinal ointments, adhesives, etc Accelerants in fire debris Biogenic emissions - plant volatiles, insect pheremones, body odour, VOC profiles from moulds/fungi/bacteria, etc Volatiles in soil and water Organic artifacts on silicon wafers and related electronic components Thermal desorption is unsuitable for the analysis of Inorganic gases except N 2 O and SF 6 Compounds that are too unstable for conventional GC analysis Compounds less volatile than n-c40, dinonyl phthalate or 6-ring polyaromatic hydrocarbons (PAHs) Organics in a sample matrix where the temperatures required for quantitative thermal desorption would cause severe degradation of the matrix itself Underivatised formaldehyde Crawford Scientific 30

32 Single-stage Desorption During the thermal desorption process, heat and a flow of inert gas are used to extract volatile and semivolatile organics retained in a sample matrix or on a sorbent bed. The analytes desorb into the gas stream and are ultimately transferred to the analyser. Although compounds can be transferred directly from the original sample to the analyser in one thermal desorption step, this simple, single-stage approach has limited practical application. The elution volume required for complete extraction of typical 100 mg - 1 g samples is too large giving poor analytical resolution and relatively low sensitivity. Tow Stage Thermal Desorption In order to improve the efficiency of peaks within the chromatogram thermal desorbers are usually manufactured as two-stage instruments - i.e. they contain a focusing mechanism for concentrating analytes desorbed from the sample tube before releasing them into the analytical system in as small a volume of vapour as possible. Two basic types of refocusing mechanism are used: Capillary cryofocusing Cold trapping GC equipment with Split/Splitless injection port and capillary Cryo-trap Crawford Scientific 31

33 Capillary cryofocusing Capillary cryofocusing does produce excellent, capillary-compatible chromatography, but it can be extremely costly in terms of liquid cryogen consumption. More importantly, such systems are prone to blocking with ice during the desorption of humid samples. Analytically, this spells disaster as thermal desorption is a dynamic process. Any blockage or restriction of the desorption gas flow has a significant impact on the efficiency of the process. Tow Stage Desorption with Cold Trapping Refocusing on a small electrically-cooled sorbent trap, which is then heated rapidly to desorb 99% of analytes in the first few seconds, is invariably the technique of choice for thermal desorption. Such systems have been shown to quantitatively retain analytes as volatile as C2 hydrocarbons while at the same time being able to desorb fast enough to produce uncompromised, high resolution capillary chromatography with low, or even zero, split ratio. There is the obvious benefit of eliminating costly liquid cryogen and little risk of blocking a typical 2 mm internal diameter secondary cold trap with ice. Small sorbent cold traps (typical I.D. 2 mm) are the best refocusing option for most thermal desorption applications. Desorbed at 40 o C/second or more, peak widths are in the order of 2 seconds wide at half height without additional focusing on the GC column. Risk of ice blockage is minimal and the moderate cooling temperatures required may be obtained with electrical (Peltier) coolers rather than liquid cryogen. Backflush configurations of this type of trap (i.e. those in which the direction of gas flow used during trap desorption is the opposite to that used during focusing) are extremely versatile. Packed with one or two appropriate sorbents and typically operating at just below 0 o C, they have been shown to quantitatively retain compounds as volatile as methyl chloride and ethane4,5 from low volumes of air. Primary (trap) desorption Crawford Scientific 32

34 Secondary (trap) desorption Thermal Desorption Autosamplers Important parameters Obviously, instrument operating parameters will vary from manufacturer to manufacturer. However, there are several generic steps to a Thermal Desorption method should be considered and the guidelines below should enable a reasonable start to method development or optimisation: Tube length and Internal Diameter Tube length and internal diameter is often restricted by the dimensions of the desorption oven, however various tube lengths and internal diameters are available all with inherent advantages and disadvantages. A conventional ¼-inch (6.4 mm) O.D. by 3.5-inch (89 mm) long desorption tubes, with 5 mm I.D. for stainless steel and 4 mm I.D. for glass, have become adopted as industry standard dimensions. These tubes have the added advantage of adsorbing/desorbing efficiently over the range ml/min which coincides perfectly with typical environmental / occupational hygiene requirements and the operating range of most pumps. Any sorbent within the range mesh can be used. Predesorption checks 1. Leak test: Once in position in the desorber flow path, each tube must be pressure tested, without heat or carrier gas flow, to ensure there are no leaks. Thermal desorption is usually a one shot process. If a leak develops, that analysis will be invalidated and the sample cannot be repeated. 2. Carrier gas purge: Each tube must be purged thoroughly with carrier gas to remove air before heat is applied. The presence of even the smallest traces of oxygen will result in sorbent and possible analyte oxidation, generating artifacts and compromising data quality. It is also advantageous if the tube purge time can be user selected - Some sorbents, notably molecular sieves, retain oxygen to some extent and it may Crawford Scientific 33

35 take several minutes to completely purge the tube, especially at lower gas flows. The cold trap should be in-line throughout the carrier gas purge to retain any ultra-volatile analytes desorbed from the tube prematurely. Tubes containing carbonised molecular sieves should ideally be purged for 15 minutes with a carrier gas flow of ml/min to complete eliminate oxygen before heat is applied. Tube heating A good minimum operating range for the desorption oven is 80 to 300 C for 1 to 100 minutes. This will allow quantitative desorption of compounds ranging in boiling point up to and above n-c30 providing other system parameters are selected appropriately. Gas Flows The system must be set up to use at least 10 ml/min flow through the tube during primary (tube) desorption and at least 2 ml/min flow through the cold trap during secondary (trap) desorption for efficient desorption. Use much faster desorption flows through the hot tube (>50 ml/min) and at least 10 ml/min through the cold trap when analysing high boilers (>n-c20.) Note that the lower the flow through the cold trap during primary desorption, the more efficient its retention of target analytes. At least 2 ml/min must be used to desorb the trap. All of this flow can be directed to the GC analytical column or to a combination of column and split vent. The flow through the cold trap should not normally be allowed to exceed 50 ml/min during tube desorption or 75 ml/min during trap desorption. Refocusing (single or two-stage desorption) The issue of number of desorption stages and how secondary/tertiary focusing, if required, should be achieved, is critical to successful thermal desorption. Most samples do require refocusing. Analytes retained on sorbent or loaded into tubes with an I.D. above 2 mm, simply cannot be desorbed quickly enough to produce capillary GCcompatible peaks. Cryofocusing onto coated or uncoated capillary tubing cooled using liquid cryogen coolant is one of the most commonly used refocusing methods for TD. It produces good quality capillary chromatography from the secondary desorption stage and is used successfully with many other GC introduction technologies - both to enhance sensitivity and improve peak shape. The principle drawback is that of ice blocking the sample flow path during the desorption of humid samples. Thermal desorption is a dynamic process and any restriction of the gas flow reduces its efficiency. The volatility range of capillary cryofocusing devices is also limited; with very volatile compounds requiring expensive packed capillaries and risk of aerosol formation with high boilers. Small sorbent cold traps (typical I.D. 2 mm) are the best refocusing option for most thermal desorption applications. Desorbed at 40 deg C/second or more, peak widths are in the order of 2 seconds wide at half height without additional focusing on the GC column Risk of ice blockage is minimal and the moderate cooling temperatures required may be obtained with electrical (Peltier) coolers rather than liquid cryogen. Packed with one or two appropriate sorbents and typically operating at just below 0 C, they have been shown to quantitatively retain compounds as volatile as methyl chloride and ethane4,5 from low volumes of air. They also work well with high boilers (3 and 4-ring PAHs, dioctylphthalate, PCB-52, etc.) and can be used for wide boiling range mixtures. Crawford Scientific 34

36 The chromatogram shown was carried out isothermally at 100 C to eliminate any on-column focusing effects. Minimal band broadening is observed. This optimizes detection limits for trace level environmental monitoring applications. A sensible minimum parameter range for sorbent cold traps is 0 to 30 C at the focusing temperature, 200 to 300 C for desorption with a user selectable hold time of 1 to 10 minutes at the top temperature. Sample Splitting Although trace level environmental samples benefit from splitless transfer to a capillary column as described above, routine occupational hygiene or materials QA/QC applications are more likely to require a high split ratio in order to avoid overloading the analytical column. Some solid samples contain 10 even 20% volatiles making it difficult to introduce less than a milligram of analyte to the desorber. Typical occupational hygiene samples may also be high: A 2.5 L air sample containing 50 ppm toluene for example contains nearly 500mg analyte. A single split point, offering split ratios up to 100/200:1 depending on column flow, is thus essential for most thermal desorption users. Single splitters are typically configured on the outlet to the secondary trap and should be fully adjustable by the user. It should also be possible to turn the split off completely if required. The availability of a secondary split point, on the inlet to the cold trap, greatly enhances system versatility. TD Sorbent selection and applications Selection of the correct sorbent or series of sorbents for the analytes of interest is one of the most important factors when developing a valid and robust thermal desorption method. The choice of sorbent principally depends upon the volatility (specifically the vapour pressure) of the analyte concerned. In short, the sorbent or series of sorbents selected must quantitatively retain the compounds of interest from the volume of air / gas sampled and must then release those compounds as efficiently as possible when heat is applied and the flow of (desorption) gas reversed. As vapour pressure data is not always readily available, a useful Rule of Thumb is to use the boiling point of the component as a guide to its volatility. In general, the more volatile the analyte to be trapped, the stronger the sorbent must be. Crawford Scientific 35

37 Selection of the correct sorbent or series of sorbents for the analytes of interest is one of the most important factors when developing a valid and robust thermal desorption method. There are many useful applications in the literature regarding Thermal Desorption autosamplers these links are to important literature applications and some general further reading about the technique: Agilent TD Overview.pdf Agilent Round the Clock Monitoring.pdf Perkin Elmer Automated TD.pdf Markes badge sampling.pdf Markes Basic introduction.pdf The following web-links also contains a host of useful information on Thermal Desorption sample introduction systems. A host of technical publications and applications from Markes International (UK): Some good technical notes from Scientific Instrument Services: An excellent set of application notes on TD from Perkin Elmer: summaries=on&catid=4 Solid Phase Microextraction (SPME) Solid phase microextraction (SPME) is a simple adsorption/desorption technique that uses a fibre coated in an adsorbent material to extract analytes from liquid matrices or headspace gas. SPME reduces the handling time, cost of equipment and amount of organic solvent required for analyte extraction compared to purge and trap, solid phase extraction (SPE) or headspace analysis. As a consequence of the reduced sample handling and need for preconcentration, SPME tends to give lower background signals (i.e better sensitivity) and depending on the choice of fibre coating, can be more highly selective than these other techniques. The SPME fibre is bonded to a stainless steel plunger housed inside a hollow needle. The fibre can be retracted into the needle, which is used to pierce septa. The fibre (usually around 1 cm in length) is then extended to expose the adsorbent to the liquid or headspace sample. The analytes adsorb onto the fibre and adsorption equilibrium is reached (usually within 2 to 30 minutes). The time to reach equilibrium can be altered with liquid agitation (stirring or vortexing), temperature, salt concentration, fibre immersion depth (liquid samples) and ph of the sample. Crawford Scientific 36

38 The fibre is then withdrawn into the holder and the outer needle used to pierce the septum of a split / splitless injector. The fibre is exposed in the inlet and analytes are desorbed and swept onto the GC column, where they may require focussing using cold trapping or low initial oven temperatures. The inlet temperature and time required for desorption will depend upon the analyte boiling point, fibre coating thickness and coating type. Extraction/Desorption Procedure Solid Phase Microextraction for GC is typically used to analyse volatile components in complicated matrices VOC s from water samples and flavours / odours from food and beverages are typical examples. The pdf documents below contain several key applications in solid phase microextraction GC. Extraction Desorption i Chlorinated pesticides by SPME.pdf Drinking water odours by SPME.pdf Flavour components of wine by SPME.pdf Pharmaceutical impurities by SPME.pdf Residual solvents by SPME.pdf VOC s from water by SPME.pdf Table 4. Performance comparison. Detection limits Presicion Expense Time (min) Solvent use Simplicity (%RSD) Purge & trap ppb 1-30 High 30 None No Headspace ppm Low 30 None Yes Liquid-Liquid 5-50 High mL Yes Extraction ppt Solid Phase 7-15 Medium 30 To 100mL Yes Extraction ppt SPME ppt <1-12 Low 5 None Yes Crawford Scientific 37

39 Important SPME Parameters SPME is an equilibrium technique the small amount of adsorbent used is unlikely to ever achieve exhaustive extraction of the analyte from the sample. Further, the partition coefficient of the adsorbent is high - ensuring that the analyte is very effectively extracted from the sample, achieving a notable concentration effect. Due to the equilibrium nature of the extraction technique it is vital to closely control the experimental parameters used for sample (and standard) extraction. Some of the most important parameters are explained below and illustrated opposite: Fibre Characteristics the chemistry (polarity) of the fibre coating and the geometry and porosity of the fibre itself will have a large influence on the extraction efficiency. Sampling Mode for volatile analytes Headspace sampling may be used, which greatly increases the selectivity of the method. For less volatile analytes, immersion sampling should be used. Sample Factors the sample matrix can be altered to render the analytes less soluble and so increase extraction efficiency. This usually involves the addition of a salt to alter the ionic strength or a ph adjustment to alter the extent of ionisation of the analyte. Sample Agitation extraction efficiency can be greatly increased by agitating the sample during extraction, however care must be taken to ensure agitation is consistent between samples. Liner Dimensions cryogenic cooling may be required to sharpen peaks from slowly desorbing analytes. Alternatively, small i.d. liners can be used to the same effect. Fibre Characteristics A thicker fibre coating will adsorb more analyte than a thin film. Thick fibre coatings are used to extract and retain highly volatile materials and transfer then to the GC inlet without quantitative loss. Thinner films are used for less volatile materials and have the advantage that they desorb much more efficiently leading to improved chromatographic peak shape. Table 5. Typical Coating Thickness vs Analyte Recovery Data. Analyte Relative Recovery (%) 100 µm 30 µm 7 µm Toluene 5 1 <1 Pyrene Immersion Sampling For large sample volumes the amount of analyte extracted is not related to sample volume, making SPME an ideal candidate for at-source sampling of, for example, lakes and ambient air, for qualitative analysis. There are two critical parameters for immersion sampling in SPME these are immersion depth and immersion time. Where quantitative analysis is required it is critical that these two parameters remain constant between standard solutions and sample solutions. Only in this way will accurate instrument calibration be possible as the technique is based on an equilibrium measurement. Empirically, the usual procedure is to allow the fibre to become saturated in order to obtain good analytical reproducibility. Crawford Scientific 38

40 Sample Factors Analytes that do not exhibit a vapour pressure (i.e. low volatility analytes) must be sampled by immersion sampling. To increase the extraction efficiency, salt may be added to the sample solution (typically 25-30% sodium chloride wt./vol.) to increase the ionic strength of the sample solution and reduce the solubility of the analytes. This is particularly recommended for trace analysis. The ph of the solution may also be adjusted to alter the ionisation state of the analyte this may also reduce the analyte solubility and increase the extraction efficiency. This approach may also increase the efficiency of headspace sampling Table 6. Effect of ph on signal intensity for selected compounds Analyte No salt Salt ph7 ph2 ph7 ph2 Phenol Nitrophenol Pentachlorophenol Headspace Sampling For a given sampling time immersion sampling is more sensitive than headspace sampling. However, gas phase kinetics dictate that the equilibrium is more rapidly obtained with headspace sampling. Using a combination of temperature and fibre polarity, headspace SPME techniques can be made much more selective than immersion techniques. To ensure highest sensitivity with Headspace SPME techniques the ration of liquid sample to headspace should be kept as high as is practically possible. Sample agitation Sample agitation enhances extraction and reduces extraction time, especially for higher molecular weight analytes with high diffusion coefficients. Inconsistent stirring causes poor precision and is worse than no stirring. Sonication promotes analyte adsorption, but can add heat to the sample. This might be beneficial for vaporizing the analytes for headspace extraction. Inlet Liner Dimensions Desorption of an analyte from an SPME fibre depends on the boiling point of the analyte, the thickness of the coating on the fibre, and the temperature of the injection port. Cryogenic cooling sometimes is required to focus slowly desorbed compounds at the inlet of the capillary column. Alternatively, an inlet liner with a narrow internal diameter (e.g., 0.75mm ID, compared to conventional 2mm ID liners) sharpens the peaks and often can eliminate the need for cooling. Crawford Scientific 39

41 Inlet linear dimension effect Where: 8. Chloromethane 9. Vinyl Chloride 10. Bromomethane 11. Chloroethane 12. Trichlorofluoromethane Analysis conditions. (Temperature Program) Fibre PDMS 100μm (Cat. No U) Column VOCOL, 60mm 0.25mm 1.5μm Oven 35 o C Carrier gas Helium, 40 cm/sec Inj 230 o C Fibre Chemistry The polarity of the analytes of interest should match that of the fibre used i.e. non-polar analytes are most efficiently extracted using non-polar fibres and vice versa. In SPME, because only a limited surface area of fibre is used (typically 1cm), the fibre coating is typically either totally non-polar or highly polar, to optimise extraction efficiency. The small differences in stationary phase polarity that are useful in GC columns (i.e. 0% phenyl vs 5% phenyl) will not produce acceptable selectivity differences in SPME. Benefit can be derived from the addition of an adsorbent material (i.e. Carbowax Polyethyleneglycol (PEG)), onto divinylbenzene polymer. The polymer increases the surface area and improves extraction efficiency of small polar molecules. The small pores in Carboxen make it particularly suitable for the extraction of small molecules. Crawford Scientific 40

42 Table 7. Effect of stationary phase on signal intensity for selected compounds Analyte Non polar Polar 85µm Non polar Polar Carboxen 100µm Polyacrylate 65µm 65µm particles/ PDMS PDMS/DVB Carbowax PDMS /DVB Methanol Ethanol Acetonitrile Isopropanol n-propanol Acetone Ethyl acetate Me- Propanone Headspace Sampling (HS) Headspace GC is used to determine volatile species released into an enclosed space above the sample (the headspace), by GC analysis of the headspace gas. The concentration of analytes in the headspace gas is proportional to the concentration in the original sample. Typical examples of headspace analysis include: Volatile Organic Compounds (VOC) from wastewater and contaminated land samples Residual solvents in packaging and pharmaceuticals Blood alcohol and toxicology screening Aroma components from food and beverages Diagnostic gas analysis from oils i Headspace analysis samples maybe solids or liquids and the volatile components of the sample are typically evolved into the headspace by heating the sample at a fixed temperature and for a fixed length of time in a vial of known volume. Like SPME, Headspace analysis is an equilibrium technique and not all of the analyte will evolve into the headspace gas. Therefore reproducibility of sample preparation is important and heating, agitation rate and the ratio of the sample volume (or weight) to the headspace gas volume must all be carefully controlled. The headspace gas is sampled manually using a heated gas tight syringe (to avoid analyte condensation) or using an automatic system employing a heated gas loop, which transfers the sample to the GC column via a heated transfer line. In each case a split/splitless injector is used to facilitate gas sample introduction into the GC. Crawford Scientific 41

43 The analyte will partition into the headspace gas until equilibrium is reached. Henrys Law dictates that the analyte concentration in the gas phase will increase rapidly at first slowing exponentially towards equilibrium. Daltons Law dictates that the relative concentrations of the individual components at equilibrium is the same as their relative concentrations in the sample prior to equilibration. That is a 2:1 ratio of analytes in the gas phase indicates a 2:1 ratio of analytes in the original sample. C G C k V O G / V L The re-arranged equation of mass balance above indicates that the concentration of analyte in the gas phase will depend upon the ratio of the volume of headspace gas to sample liquid (V G /V L ). In order to simplify the determination of analyte concentration in the original sample, this ratio is often kept at 1:1 with for instance 10mL of liquid sample being extracted in a 20 ml vial (i.e. 10ml of sample and 10 ml of headspace gas). Analyte solubility has a greater effect on headspace concentration (partition co-efficient) than analyte volatility. Table 8. Partition co-efficient for selected compounds Analyte Solvent K(25 o C) Toluene Decane ~ 3000 Toluene Water ~ 4 Ethanol Decane ~ 60 Ethanol Water ~ 5000 Ethanol Water with Na 2 SO 4 ~ 300 Larger partition co-efficient values indicate a lower concentration of analyte in the gas phase. Toluene is highly soluble in decane the partition coefficient is high and headspace toluene concentrations low. However, when extracting toluene from water, in which the analyte is much less soluble, the partition coeffcient is much lower and the headspace concentration much higher. Ethanol is highly soluble in water, however when the sample solution is saturated with salt its solubility decreases and the headspace concentration increases! Acrylonitrile is very much more volatile than 2-Ethylhexyl Acrylate, however its response in the chromatogram is very much lower. The response profile of the monomers in this water sample headspace extract, demonstrates that analyte solubility plays a more important part in headspace extraction efficiency. The highest extraction efficiency comes from the analyte with the lowest water solubility, not the highest volatility. Crawford Scientific 42

44 100 ppm monomers in water (equilibrated at 85 o C for 45 min, 30m 0.53mm 3.0mm, cyanopropylphenyl phase. Split 6:1 Headspace GC is typically used to analyse volatile components in complicated matrices VOC s from soild and water samples and ethanol content of blood being just two typical examples. The pdf documents below contain several key applications in Headspace GC: Ambient headspace analysis of polar volatiles in water.pdf Blood alcohol analysis.pdf Headspace of volatile priority pollutants from water.pdf Multiple headspace extraction of solids.pdf Residual solvents in pharamceuticals.pdf VOCs from water samples.pdf Headspace Autosamplers Headspace GC techniques are amenable to automation using gas sampling loops. Most commercially available systems operate on the cycle described below: Standby the sample is heated and agitated in a small oven enclosed within the instrument Pressurisation a concentric needle arrangement is used to introduce an inert gas into the vial headspace to increase the vapour pressure Loop filling / Venting the headspace gas is allowed to flow through a gas sampling loop to vent loop filling time and closed loop pressure equilibration times are of great importance Injection valves are altered to allow the carrier to flow through the gas loop and sweep the contents through an inert, heated transfer line into a split/splitless inlet and onto the GC column. A small split flow is often maintained to ensure efficient transfer onto the column and ensure sharp peaks. Cryogenic cold trapping / focussing of analytes at the column head is also possible to ensure good peak shape. Crawford Scientific 43

45 When using headspace auotsamplers there are several parameters that need to be optimised. Variables such as column flow, split flow and initial oven temperature may differ substantially from those used in conventional analysis. These parameters are discussed in greater detail opposite. i Headspace GC techniques automation by using gas sampling loops Table 9. Critical parameters for optimisation in headspace analysis GC Headspace Inlet (liner) Equilibration time Flow Temperatures Start temperature Injection times and volumes GC Cycle time Pressure (pressurization) Time (loop filling time) Vial (sample amount) Loop Inlet (Liner) As with SPME techniques, using a smaller internal diameter liner can considerabley sharpen peaks. This is particularly noticeable with trace level analytes. Inlet internal diameter effect Crawford Scientific 44

46 Flow Headspace GC analysis often requires a higher than conventional carrier gas flow (50-100mL/min.). Higher flow rates ensure tha the gas loop is emptied and the analytes transferred on to the column effciently. For this reason wider internal diameter column (0.53mm) are often used, as they create smaller back pressure at high flow. Start temperature Low initial oven temperatures are often used in Headspace GC to ensure that analytes are thermally focussed at the head of the analytical column. It is also possible to have cryo-trapping of analytes using conventional cryogenic column adapters as shown. GC Cycle time GC with a conventional cryogenic column adapter The GC cycle time must include the time required for headspace sample preparation (including sample incubation). Most modern instruments have communication between the headspace instrument and the GC to enable synchronisation. It is also possible to begin sample incubation whilst the GC separation of the previous sample is occurring, so reducing overall injection to injection cycle time. Equilibration Time As equilibration time is increased, the partition of the samples in the vapour phase rises, and reaches a plateau. The higher the equilibration temperature, the longer the equilibration time needs to be. It is not necessary to use over-long equilibration times, just long enough for the partitioning to equilibrate. Crawford Scientific 45

47 Example Temperatures See comments in equilibration time above. As the equlibration temperature increases the equilibration time increases. Injection Time and Volumes Increasing the injection volume at low transfer (carrier gas) flow rates can lead to peak broadening, even to loss of separation, especially for highly volatile compounds. Example Pressure (Pressurization Time, Loop Filling Time) The vial pressurisation is of great importance. Too high a vial pressure (usually adjusted by altering pressurisation time), will risk loss of analyte via the vial cap seal, septum needle seal or by overfilling the sample loop. Vial (Sample amount) Example Increasing the sample amount (relative to a fixed headspace volume) will bring an increasde in sensitivity. Note in the graphic though that the 15ml sample volume results in a smaller peak area than expecetd. This is mainly due to the equilibration time being too short for the increased sample volume sample volume and equilibration time are directly linked. Crawford Scientific 46

48 Toluene in water (oven temperature 80 o C, time 15 min, 20mL vial) Loop It is usually possible to change loop sizes to increase or decrease the volume of gas injected into the GC. Care should be taken with larger gas sample volumes to preserve peak efficiency during transfer to the GC column. Headspace Calibration and Quantitation Calibration can be problematical in Headspace GC analysis for both liquids and solids. Because the sample matrix plays an important role in the equilibration process, it is critical that the standards used for calibration also involve the same matrix. If a sample of pure matrix (i.e. containing no analyte) is available then this represents the best approach to calibration. The matrix may be spiked with varying amounts of analyte and a calibration curve constructed. If a sample of pure matrix is not available, it may be possible to exhaustively extract a sample containing a low analyte concentration in this way a pure matrix may be manufactured. It may also be possible to simulate a sample matrix by artificially combining the major components of the matrix. For example an alcoholic beverage matrix can be easily constructed as an aqueous solution of the alcohol at the correct concentration. It is also possible to calibrate using the method of standard additions. The actual sample is divided into several aliquots to which increasing amounts of the analyte are added. A plot of peak area against added amount will give the value for the analyte concentration in the original sample. Finally when calibrating for solid matrices, a similar technique to that described above may be used which is known as multiple headspace extraction. Calibration with Simulated Sample Matrix In this case, not all components of the sample matrix are available. If, however, only a few major components due to their high concentrations determine the properties of the sample matrix, the sample matrix for the calibration can be produced using these major components. Example: Headspace Analysis of aroma components in alcoholic drinks. For this, an aqueous-alcohol solution with the same alcohol concentration as the matrix can be used for calibration. Crawford Scientific 47

49 Calibration with Pure Sample Matrix If the sample matrix is known and available in pure form, the pure volatile substance can be simply mixed in a defined concentration, and this calibration sample used as an external standard. Examples: Headspace analysis of liquid, homogeneous sample matrices such as aqueous samples, mineral and edible oils, liquid polymers. Calibration by the Mixing Method In this procedure, two analyses must be made for each sample. In one analysis, a defined amount of the compound to be measured is added to the sample. The resulting peak is thus larger, and because the increase is proportional to the added amount, the original concentration can be determined. Toluene peak MHE In Multiple Headspace Extraction (MHE) Analysis repeated extractions of the sample are carried out under identical analytical; conditions. The analyte response will decrease with each subsequent extraction following an exponential decay pattern. Only after calibration (determination of detector response ratio) can the sample content be worked out from the area values. Crawford Scientific 48