expert analytical testing

Extractables and Leachables Extraction Techniques

Second Edition

A collection of articles designed to help improve your knowledge and skills. TABLE OF CONTENTS

P.03 INTRODUCTION

P.04 SONICATION

P.05 REFLUX

P.07 SOXHLET

P.10 SEALED VESSEL

P.11 PRESSURISED SOLVENT EXTRACTION

P.14 MICROWAVE ASSISTED EXTRACTION

P.16 SUPERCRITICAL FLUID EXTRACTION (SFE)

P.19 OVEN EXTRACTION

P.22 LIQUID EXTRACTION SURFACE ANALYSIS (LESA)

P.24 DIRECT ANALYSIS IN REAL-TIME (DART) & DESORPTION ELECTROSPRAY IONISATION (DESI)

P.27 HEADSPACE

P.29 THERMAL DESORPTION

P.30 SUMMARY

P.31 CONCLUSION INTRODUCTION

In extractable and leachable studies there are a range of extraction techniques that can be used to either produce a solution for further analytical study or directly analyse the materials. A selection of the most common extraction techniques are listed in Table 1. Each of the techniques will then be discussed, detailing what is involved to set up the equipment, the advantages and disadvantages as well as some of the limitations of each technique.

The primary aim of any extractable study is the acceptance or rejection of a given material. The acceptance or rejection can only be achieved with knowledge. This can be achieved by a number of ways, including; understanding of the materials likely composition, manufacturers information and the definitive testing. In all aspects of extractable testing it is important to remember that the extraction is not so vigorous as to deform or degrade the material which would likely produce extractables that will not be observed as leachables. Extractables are potential leachables, it is the leachables that the patient is exposed to and are of toxicological concern. There is a sweet spot for extractable studies in that they should be aggressive enough to produce worst case leachables but not so harsh as to still allow for a correlation between extractables and leachables.

There is no single extraction technique that can provide all the information needed for an extraction study and so multiple techniques are typically used.

Table 1 Potential extraction techniques:

Technique Solution based Complexity

Sonication Yes Low Re ux Yes Low Soxhlet Yes Low Sealed vessel e.g. Autoclave Yes Medium Pressurised Solvent Extraction e.g. ASE™ Yes High Microwave Assisted Extraction Yes High Supercritical uid extraction Yes High Oven Yes Low Direct analysis e.g. DART, DESI No High Liquid Extraction Surface Analysis E.g. LESA No High Headspace Either Low Thermal desorption Either High Dynamic headspace Either High

Extractables and Leachables Extraction Techniques: Volume I Page 3 SONICATIONSONICATIONSONICATIONSONICATION

SONICATION

Sonication is one of the simplest extraction techniques in terms of equipment, solvent selection and sample preparation. A known weight of material is placed in a container with a known volume of solvent. The ratio of sample-to-solvent and the overall amounts of material required are dependent upon: method requirements; analytical limits of detection; and the sample volumes required for testing. For example, standard metal analyses by inductively couple plasma will require far more solvent volume (typically 10-20 mL) compared with gas chromatography (GC) or high-performance liquid chromatography (HPLC) analyses (≤1 mL).

The sample is placed in the sonic bath and sonicated for the prescribed time (or over a range of times) until asymptotic levels are reached. Potential issues can arise with sonication duration due to the relative efficiencies of sonic baths. One sonic bath can be more efficient than another in terms of energy supplied and temperature increase of the solution. The degree of temperature increase of the sample (which can have a significant influence on the degree of extraction) can vary significantly from machine to machine.

The choice of solvent and the analyte being extracted can have a large impact on the efficiency of this technique[1-3] . In general, the more volatile the solvent, the greater is the efficiency of extraction when compared with other extraction techniques such as reflux. Sonication results using a low-boiling-point solvent such as dichloromethane (DCM) (boiling point 40 °C) will more closely match those achieved with an extraction technique such as reflux than if a solvent such as isopropanol (boiling point 82 °C) is used. This could be due to the kinetics involved because the reactions will be occurring at similar temperatures with DCM compared with the wide differential with isopropanol.

1. Saim, J.R. Dean, P. Abdullah and Z. Zakaria, Journal of Chromatography A, 1997, 791, 361. 2. R. Banjoo and P.K. Nelson, Journal of Chromatography A, 2005, 1066, 9. 3. F. Guerin, Journal of Environmental Monitoring, 1999, 1, 1, 63.

Extractables and Leachables Extraction Techniques: Volume I Page 4 reflux

REFLUX

Reflux is another simple technique in which the sample is placed in a flask containing the solvent with a condenser fitted on top of the flask. The flask is heated for a set time, allowed to cool and then the solution can be analysed. The fundamentals of the extraction are around the boiling point of the solvent which makes the technique universal.

With any setup using a condenser, the efficiency of the condenser can be important. Each condenser has a potential cooling capacity and efficiency. Different types of condenser exist, from the basic ‘Liebig condenser’. To the more complex ‘Graham condenser’ which has a spiral coil running the length of the condenser.

a) b) c)

Vapour Trail Water Trail

Graham condenser, with three possible configurations: a) cooling with jacket, b) cooling with spiral and c) combined jacket and spiral cooling. [1]

Extractables and Leachables Extraction Techniques: Volume I Page 5 There are two basic configurations for a Graham condenser. In the first, the spiral contains the coolant, and the condensation takes place on the outside of the spiral. This configuration maximises flow capacity because vapors can flow over and around the spiral.

In the second configuration, the jacket tube contains the coolant, and condensation takes place inside the spiral. This configuration maximises collected condensate because all the vapors must flow through the entire length of the spiral, thereby having prolonged contact with the coolant. Other condensers such as the Allihn condenser and ‘Friedrich condenser’ are available.

For reflux extraction and also for any other type of extraction, the correct sample-to-solvent ratio should be used. This is to make sure that a solution is obtained that spans a suitable concentration range e.g. too concentrated or dilute, as well as consideration of the amount of solution required for analyses and the required limit of detection based on the Analytical Evaluation Threshold (AET).

Heating is commenced and the material extracted at the boiling point of the solvent. The boiling point of the solvent is the key controlling factor with this technique, along with the duration of extraction. Extraction should continue until asymptotic levels or exhaustive conditions are reached, depending on the guidelines that are being followed.

Reflux may not be suitable if the material to be extracted is particularly thermally labile because it is exposed to complete heating of the solvent. This potential thermal impact can be exacerbated by the material sinking and being in contacted with the bottom of the flask and potentially be exposed to higher temperatures. Reflux extraction does have the advantage that any solvents can be used including mixtures.

Other related techniques that also heat the sample in a solvent such as microwave-assisted extraction (MAE) or pressurised solvent extraction (PSE) will be discussed in more detail in following weeks.

1. Update on Undertaking Extractable and Leachable testing A Feilden ISBN 978-1-84735-455-6

Extractables and Leachables Extraction Techniques: Volume I Page 6 S XHLET

SOXHLET

Soxhlet extraction is probably the most common technique used for the extraction of materials from solid samples, and has been used for more than a century. It was originally used for the determination of fat in milk [1]. As such, all other extraction techniques are generally compared with Soxhlet extraction.

The sample is typically placed in a thimble (usually made from cellulose) but doesn’t need to be and the solvent is placed in a flask below the thimble. The solvent is heated and recondenses into the thimble. Once the liquid reaches a defined level, it is siphoned back to the flask, transporting the extracted species back into the bulk liquid. The solvent is then distilled again, thereby delivering pure solvent to the material being extracted. The number of cycles (‘turnovers’) can affect the extraction. Hence, a minimum number is normally expected and this varies from material to material and species to species being extracted.

The advantage of Soxhlet is the repeated delivery of fresh pure distilled solvent to the sample, allowing continual shifting of the transfer equilibrium. It is also a very simple system requiring a heater, condenser and inexpensive glassware. Soxhlet has one advantage over reflux in that fine-particulate matrices can be used because these are contained in the extraction thimble, resulting in only extracted analytes in solution and is more amenable to potentially thermally challenging samples.

A standard Soxhlet apparatus can use large solvent volumes, a potential disadvantage to the technique. However, automated Soxhlet systems are available that offer additional functionality, such as warm Soxhlet, hot extraction and continuous extraction. Several solvent-extraction systems based on the Soxhlet device allow fast and safe extraction from materials. Some of this include the ‘Soxtec Systems’ from FOSS. These are automated or semi-automated analysers which extract lipids rapidly and accurately. These instruments carry out boiling, rinsing and solvent recovery. Similarly, Soxtherm extractors from Gerhardt GmbH have been developed to reduce extraction times. The sample to be analysed is weighed into cellulose thimbles and inserted in the extraction device. The Büchi Extraction System UniversalExtractor E-800 is an automated system which can be used to carry out an extraction according to the original Soxhlet principle. Four different extraction methods are possible without making changes to the unit: Soxhlet standard, Soxhlet warm, hot extraction, and continuous extraction Basically, the system has an inert-gas supply to avoid oxidation during extraction and to accelerate the evaporation and drying process even with high-boiling-point solvents (up to 150 °C).

Extractables and Leachables Extraction Techniques: Volume I Page 7 Solvent selection is important with Soxhlet extraction. Take for example an acidified or basified solvent. Only the pure solvent would be distilled to be in contact with the sample. However, the species extracted would be in prolonged contact with the adjusted solvent (probably under exaggerated conditions). These exaggerated conditions would be produced by the distillation of water leaving an increased concentration of the modify agent (acid or base). So a more acidic or alkali solution would be present which could increase the chance of the extracted analytes reacting at this higher of lower pH

If possible the extracting solvent used in an extraction study should be the solvent used in the product. An extreme example of this would be trying to use the solvent in a pressurised metered-dose inhaler that uses a hydrofluoroalkane (HFA) propellant. The solvent (typically HFA 227 or HFA 134a, but could move to HFa 152) is a hydrofluorocarbon with a boiling point of less than –15 °C for HFA 227 or ‑26 °C for HFA 134a.

The HFA can be added to a Soxhlet apparatus to run a sub-ambient Soxhlet in which the cooling of the solvent needs to be quite extreme because the solvent needs to be cooled to at least –30 °C (hence the term ‘sub-ambient’). The cold temperature is required to condense the propellant to allow continued extraction. This can be achieved several approaches, the simplest being the use of a dry-ice and acetone condenser.

Condenser

Extraction Chamber

Vapour Siphon Arm

Boiling Flask

Extraction Solvent

Soxhlet apparatus Sub-ambient Soxhlet apparatus with a dry-ice/acetone condenser

Extractables and Leachables Extraction Techniques: Volume I Page 8 Several challenges affect the use of sub-ambient Soxhlet apparatus. The dominant factor is the kinetics because the extraction is being carried out at –20 °C. A common rule of thumb is that for every 10 °C change in temperature the rate of reaction doubles. Hence, sub-ambient Soxhlet under the same conditions will be (very approximately) 20-times slower than Soxhlet with DCM as the solvent simply due to the kinetics of the reaction. This would require a 20-day extraction compared with a typical 24-h extraction, and for this duration it is becoming closer to a leachable study. To combat this, the surface area of the material can be increased, typically requiring cryomilling. Secondly, by maximising the sample-to-solvent loading, the amount extracted is maximised. In addition, running the extraction for much longer than one working day is extremely challenging. A dry-ice/acetone condenser needs to be periodically recharged with additional dry ice and acetone. A re-circulating condenser operating at –30 °C requires a specialist solvent to work (such as ethylene glycol or a fluorocarbon).

1. Soxhlet, Die Gewichtsanalytische Bestimmung des Milchfettes, Polytechnisches J. (Dingler’s), 1879, 232, 461.

Extractables and Leachables Extraction Techniques: Volume I Page 9 SealedVessel

SEALED VESSEL

Sealed vessels are another very simple technique; the sample is placed in the solvent again with appropriate levels of sample and solvent to achieve appropriate method limits of detection. This important area of sample to solvent ratio will be discussed in a future blog.

A sealed vessel in its simplest form is an oven, which can be simply static or with agitation using an orbital shaker as an example. A way of putting more energy into the extraction process can be achieved by using an autoclave that uses high pressure steam for heating, which allows the solvent in the sealed vessel to increase significantly without solvent loss.

Other pressurised systems such as microwave assisted extraction or pressurised solvent extraction will be discussed in more detail in later blogs.

Sealed vessel or autoclave extraction is predominately used with aqueous media, whether with pure water or pH adjusted water. Typical conditions involve temperatures at 121 °C with various extraction times [1].

The conditions in USP <381> Elastomeric Closures for Injections is as follows: ‘Heat in an autoclave so that a temperature of 121 ± 2 °C is reached within 20 to 30 minutes, and maintain this temperature for 30 minutes. Cool to room temperature over a period of about 30 minutes’.

The use of organic solvents is much less common, but examples do exist with the use of alcohols [2] and these tend to be very specialist applications.

If an organic solvent was to be used then general conditions of heating with the solvent at 10 °C below the boiling point of the solvent are recommended.[3]

1. D. R. Jenke, E. K. Chessa and G Jakubowskia International Journal of Pharmaceutics Volume 108, Issue 1, 25 July 1994, Pages 1-9 2. C.B. Muchmore, J.W. Chen, A.C. Kent and K.E. Tempelmeyer, American Chemical Society, Division. Fuel Chemisty. Preprints, 30(2) (1985) 24-34. 3. D. R. Jenke, J Castner et al PDA J Pharm Sci and Tech 2013, 67 448-511

Extractables and Leachables Extraction Techniques: Volume I Page 10 Pressurised Solvent Extraction

PRESSURISED SOLVENT EXTRACTION

Pressurised solvent extraction (PSE) or Accelerated solvent extraction (ASE) [trademark of Dionex and ThermoFisher Scientific] was introduced in the mid 1990s as an alternative to other extraction methods discussed previously such as Soxhlet or sonication for solid samples. With PSE the sample is placed in a stainless steel cell through which a solvent is passed utilising high temperatures and pressures. Two common forms of the PSE apparatus exist, one from Dionex is shown in Figure 1, with variations in cell extraction size and solvent selection. Another pressurised solvent extraction system, the speed extraktor is available from Buchi, see Figure 2.

Figure 1. ASE apparatus. [1] Figure 2. SpeedExtraktor E916 from Buchi.

The elevated temperature and pressure typically leads to significantly higher capacity of the extraction solvent to dissolve the target analytes, improves the rate of mass transport and effectiveness of sample wetting and matrix penetration, which improves the desorption of analytes from active sites on, and within, the sample particles. This can result in reduced solvent volumes and shorter extraction durations.

However, pressurised solvent extractors do suffer from a number of drawbacks. Firstly, the sample size has to be reduced e.g. cut up or milled to allow it to be added to the pressure vessel. This is not an issue for environmental samples such as soils and why PSE is often used with environmental samples [2]. Due to the efficient nature of the extraction there is a chance that the extraction could be too vigorous and produce extractables that will not be found as leachables.

Extractables and Leachables Extraction Techniques: Volume I Page 11 ASE may be conducted in two ways: (1) Dynamic ASE, where the solvent is continuously pumped through the extraction cell. (2) Static ASE, where the extraction cell is filled with solvent, pressurised for a specified time and then drained to the collection vial.

It is also possible to combine the two or to perform multiple extraction cycles. The majority of ASE applications (about 75%) have been performed in the static extraction mode. Such systems can reach temperatures up to 200 °C and pressures up to 200 bars and can accommodate cells of various volumes, ASE has a significant advantage over a number of extraction techniques in that binary solvent mixtures can be used either using one weak solvent to carry out pre-extraction with a weak solvent to remove some interfering (less strongly sorbed) compounds before the extraction of the compounds of interest [3,4]. The extract complexity may also be reduced by using a sequential extraction procedure with solvents of increasing solvent strength [5]. Such procedures may also be used to assess the strength of matrix–analyte interactions [6]. A recent paper on the utilisation of ASE in the extractable study of single use systems [7] has shown the speed at which an extraction study can take place.

The selectivity of the extraction or leaching process may be further enhanced through the addition of a matrix retainer to the extraction cell. Use of alumina as a fat retainer was already suggested in 1996 in a Dionex application note [8]. Since then, many other adsorbents have been used for the same purpose, for example, Florisil, silica gel and diol- and cyanopropyl-silica [9].

The Buchi apparatus differs from the ASE apparatus in that it is a parallel system where up to 6 samples can be analysed at the same time rather than in series. One has higher throughput, the other higher flexibility. The ASE system can be left running for extened periods with changes to the solvents used, whilst the Buchi system process up to 6 samples at a time. The Buchi SpeedExtraktor analysis is shown in Figure 3. The solvent mixture is transferred into separate extraction cells by a pump at high pressure (<150 bar) and heated (<200 °C). The extracts are collected in vials to either be concentrated or directly for analysis.

Figure 3. Extraction approach for the Buchi Speed Extraktor.

Extractables and Leachables Extraction Techniques: Volume I Page 12 1. A Feilden., Update on Undertaking Extractable and Leachable testing ISBN 978-1-84735-455-6 2. P. Vazquez-Roig, Y. Picó, Pressurized liquid extraction of organic contaminants in environmental and food samples, Trac. Trends Anal. Chem. 71 (2015) 55–64 3. J. McKiernan ., J. Anal. At. Spectrom., 14(4), 607–613 (1999). 4. M. Papagiannopoulos and A. Mellenthin, J. Chromatogr. A, 976(1–2), 345–348 (2002). 5. M. Bergknut , Environ. Toxicol. Chem., 23(8), 1861–1866 (2004). 6. H. Schroder, J. Chromatogr. A, 1020(1), 131–151 (2003). 7. N Dorival-Garcia et Al Talanta Volume 219, 1 November 2020, 121198 8. Dionex application note ASE 322, Dionex Corp., Sunnyvale, California, USA (1996). 9. C. Huie, Anal. Bioanal. Chem., 373(1–2), 23–30 (2002).

Extractables and Leachables Extraction Techniques: Volume I Page 13 Microwave Assisted Extraction

MICROWAVE EXTRACTION

Microwave extraction can be performed in a very similar way to pressurised solvent extraction. In microwave extraction, the extraction typically occurs in one of two systems: Focused microwave-assisted extraction (FMAE), where the extraction occurs in an open vessel under atmospheric pressure or pressurised microwave-assisted extraction (PMAE) is a closed pressurised system and it is this approach that is very similar to pressurised solvent extraction. It is rare for a microwave extraction to be utilised as the extraction technique for a controlled extraction study but is more likely to be used as part of routine control where the material is well known and the speed advantages, and low solvent volumes can be utilised effectively.

FMAE is less common but it has been applied to certain examples as well as specific extractions such as PAHs [1] [2] [3] [4], PCBs, pesticides [5] [6], organometallic compounds [7] [8], and dioxins or furans [9] from spiked, real, and reference material samples. PMAE is far more common but does suffer from a number of disadvantages in terms of analysis time as it takes time to make sure the extraction vessels can cope with the extraction pressure and are suitably sealed. It can also take a considerable amount of time for the vessels to cool to room temperature before the pressure is reduced and they can be opened. For routine testing the low solvent volumes, short extraction times and the ability to do multiple samples at once overcome the disadvantages. Examples exist of its use in the following fields: the extractions of environmental pollutants, such as hydrocarbons (HCs) [10], PAHs [11] [12], organochlorine pesticides (OCPs) [13] [14], polychlorinated biphenyls (PCBs) [15], dioxins or furans, triazines and alkyl or aryl phosphates, in soil, sediment and sludge sample matrices.

The primary concern with microwave heating is the ability of the solvent to absorb the microwaves e.g., dielectric polarisation of the solvent must be possible. This heating efficiency depends on the dielectric constant (g) of the solvent and in most cases is proportional to the polarity of the solvent. Examples of the dielectric constant for common extractable solvents are in Table 1.

Solvent Dielectric Constant (g) Solvent Loss Tangent (2.45 GHz at 20 °C) Hexane 1.89 at 20 °C Hexane 0.020

Dichloromethane 8.93 at 25 °C Dichloromethane 0.042

Methanol 33.0 at 20 °C Methanol 0.659

Water 80.1 at 20 °C Water 0.123

Table 1. Dielectic constant of common solvents. Table 2. Microwave loss tangents of common extraction solvents

In addition to the solvents ability to absorb microwave energy, its ability to convert the energy to heat is important. The efficiency of the conversion of microwave energy to heat is given by the dielectric loss factor or loss tangent. Loss tangents of common solvents are shown in Table 2. More polar solvents are typically used in microwave extraction.

Extractables and Leachables Extraction Techniques: Volume I Page 14 Therefore, the overall efficiency of heating using microwave energy is usually expressed by the dissipation factor which is the ratio of the dielectric loss factor and the dielectric constant of the involved matrix [16]. Concerning the overall efficiency, these data show a large difference between various solvents and, moreover, that methanol has been shown to be more favourable than water despite its lower dielectric constant [17]. Standard setups suffer from that only one solvent mix can be used at a time since the rate of heating can and does vary quite dramatically. Other possible issues are that the material to be extracted can be altered. It can be termed as ‘“accelerations of chemical transformations in a microwave field that cannot be achieved or duplicated by conventional heating’ [18]. A typical example would be the additional curing of rubber with microwaves. Whether this happens in standard extraction conditions has not been proved one way or another but will depend on a number of factors, including temperature, as well as possibly solvent and extraction duration. Another factor to consider with microwave heating is that for some solvents the average temperature of the solvent can be considerably higher than the atmospheric boiling point because the microwave power is dissipated over the whole volume of the solvent again potentially degrading or altering the material.

Commercial systems include the safety systems required of a microwave which are;

• No handling of pressurised vessels • Closed rotor with protection lid • Stable oven cavity with resealing safety door • Pressure increase control • Controlled pressure limits for each vessel type • 70 bar safety disk (overpressure tolerance)

1. Y.Y. Shu and T.L. Lai, Journal of Chromatography A 927 (2001) 131. 2. S. Dupeyron, P.M. Dudermel and D. Couturier, Analusis 25 (1997) 286. 3. M. Letellier, H. Budzinski, P. Garrigues and S. Wise, Spectroscopy 13 (1996/1997) 71. 4. L.E. Garcia-Ayuso, J.L. Luque-Garcia and M.D.L. de Castro, Analytical Chemistry. 72 (2000) 3627. 5. O. Zuloaga, N. Etxebarria, L.A. Fernandes and J.M. Madariaga, Journal of High Resolution Chromatography. 23 (2000) 681 6. C.F. Cao, Z. Wang, L. Urruty, J.J. Pommier and M. Montury, Journal of Agricultural and Food Chemistry. 49 (2001) 5092 7. J. Szpunar, V.O. Schmitt, R. Lobinski and J.L. Monod, Journal Analytical Atomic Spectrometry. 11 (1996) 193. 8. I.R. Pereiro, V.O. Schmitt, J. Szpunar, O.F.X. Donard, R. Lobinski, Analytical Chemistry. 68 (1996) 4135. 9. E. Eljarrat, J. Caixach, J. Rivera, Chemosphere 36 (1998) 2359 10. A. Pastor, E. Vazquez, R. Ciscar and M. de la Guardia, Analytica Chimica Acta 344 (1997) 241. 11. R.C. Lao, Y.Y. Shu, J. Holmes and C. Chiu, Microchemical Journal 53 (1996) 99. 12. V. Lopez-Avila, R. Young and W.F. Beckert, Analytical Chemistry. 66 (1994) 1097. 13. K. Li, J.M.R. Belanger, M.P. Llompart, R.D. Turpin, R. Singhvi and J.R.J. Pare, Spectroscopy 13 (1996/1997) 1. 14. I. Silgoner, R. Krska, E. Lombas, O. Gans, E. Rosenberg and M. Grasserbauer. Journal of Analytical Chemistry. 362 (1998) 120. 15. G. Dupont, C. Delteil, V. Camel and A. Bermond, Analyst 124 16. A. Zlotorzynski, Critical Reviews in Analytical Chemistry, 25, 43 (1995). 17. C. Molins, E. A. Hogendoorn, H. A. G. Heusinkveld , D. C. van Harten, R van Zoonen and R. A. Baumann Chromatographia, 43, 527-532 (1996) 18. C Oliver Kappe, D Dallinger and S, S, Murphree Practical Microwave synthesis for Organic Chemists.Whiley 2008

Extractables and Leachables Extraction Techniques: Volume I Page 15 Supercritical uid extraction

SUPERCRITICAL FLUID EXTRACTION (SFE)

Supercritical fluid extraction (SFE) and Supercritical fluid chromatography (SFC) are very closely linked. SFE/SFC utilizes extreme conditions of temperature and pressure in such a way that the mobile phase remains as a supercritical fluid, as can be seen in Figure 1. Supercritical fluids possess unique properties, intermediate between those of gas and liquids. These depend on the pressure, temperature and composition of the fluid. In particular, their viscosity is lower than that of liquids, and the diffusion coefficients are higher, allowing more efficient extractions. In addition, the density (and therefore the solvent power of the fluid) may be adjusted by varying both the pressure and the temperature, affording the opportunity of theoretically performing highly selective extractions. This is where SFC is used in large scale applications. Table 1 shows a range of large scale industrial applications.

Area Material Purpose

Liquid Phase Supercritical Area Food Coffee beans Recovery of furans and pyrazine types [2] Tea Decaffeinated [3] Pc Critical Point Hop Hop extract Solid Phase Pressure

Triple Point Red pepper Spicy extract, pigment

Gas Phase Fish meal Fish oil

Pharmaceutical Natural products Medicinal ingredients, bioactive Natural Temperature Tc Pressure - Temperature of a pure substance products (where the subscript c denotes the critical conditions) Chemical Electrical/ Degreasing electronic parts

Figure 1. Phase diagram showing the supercritical area [1]. Table 1. Large scale industrial applications. Super critical fluid extraction.

Extractables and Leachables Extraction Techniques: Volume I Page 16 The basic equipment is very similar to a standard HPLC system, with the following exceptions/additions:

• A source of CO2 (commonly a tank) • The ability to regenerate the CO2 – i.e. remove the organic modifier prior to re-circulating or re-depositing in the tank • A backpressure restrictor placed after the analytical column/extraction cell

Organic Modifiers

Figure 2. Example of SFC/SFE apparatus.

The SFE may be carried out in either static or dynamic mode. The pressure in the system is maintained by means of a restrictor (either fixed or variable, the latter making the pressure independent of the flow rate). At the end of the restrictor, the fluid is depressurized and the extracted analytes are trapped in an organic solvent or on a solid phase filled cartridge (from which the analytes are later eluted with a small volume of organic solvent).

Due to the numerous parameters affecting the extraction efficiencies, SFE affords a high degree of selectivity and it is this reason that supercritical fluid extraction has a wide range of industrial-scale uses. However, on the other hand, this makes the optimisation quite tedious and difficult in practice and maybe prevent all potential species being extracted.

The parameters to consider are linked to the extraction parameters inside the cell, to the nature of the solutes or to the nature of the matrix. The important parameters in SFE are both the pressure and temperature inside the cell. A pressure increase leads to a higher fluid density, thus increasing the solubility. The inverse is observed with the temperature; however, increasing the temperature may enhance the solubility of volatile analytes. In addition, higher temperatures may be required to overcome solute–matrix interactions, as observed for the extraction of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins from environmental matrices.

Extractables and Leachables Extraction Techniques: Volume I Page 17 The polarity of compounds is the most significant factor to be considered when working with SFE. Pure CO2 efficiently extracts non-polar to low polarity compounds. For polar solutes, a modifier is added to enhance the extraction. For very polar and ionic compounds, the modifier may be a complexing agent, an ion-pair reagent or a derivatisation reagent. As an example, the addition of tetrabutylammonium enabled the extraction of anionic surfactants from sewage sludge to be performed. The addition of the modifier directly to the matrix (prior to the extraction) may help in disrupting the analyte–matrix interactions; however, it requires that a static extraction be performed first, to avoid sweeping the modifier out of the cell. In cases where the analytes do not readily derivatise, the addition of a derivatisation reagent may still be useful as it can react with the active sites of the matrix, thus enhancing the extraction, as has already been observed during the extraction of PAHs from urban dust. [4]

The users of SFE must be aware of the fact that the addition of a modifier to CO2 presents severe drawbacks, due to the technical factors, and so it should be avoided or minimized whenever possible. The presence of the modifier changes the values of the critical pressure and temperature, so that too high a modifier content may result in a temperature lower than the critical value, resulting in a subcritical state, with higher viscosity and lower diffusion coefficients than the supercritical state; in this case, the technique is commonly called enhanced-fluidity liquid extraction (EFLE). In addition, as the modifier enhances the solvating power of the fluid, it reduces the extraction selectivity as more matrix materials or non-target analytes are co- extracted. Finally, the modifier condenses upon depressurization, which may result in elution of the retained compounds when a solid trap is used as the collection device, since then it may act somewhat like a chromatographic device. The nature of the matrix (water content, percentage of organic carbon, humic/fulvic materials, etc.) and its physical characteristics (such as porosity or particle size) are of prime importance for the success of an extraction,9 as with other extraction techniques. Milling the matrix is recommended, to limit the diffusion step inside the matrix and to increase the surface area, which increases the rate of extraction when it is limited by matrix effects. Also, the addition of a drying agent (such as sodium sulfate) may prevent the plugging of the restrictor by ice in the presence of humid matrices. Caution must also be taken when filling the vessel to ensure a homogeneous bed of material (to prevent channelling) and to take into account possible swelling of the matrix (such as polymers) upon introduction of supercritical CO2. In addition, very fine particles may be swept out of the cell by the fluid and result in plugging and mechanical transfer problems. Finally, a sorbent may be added in the cell to retain matrix material and increase the selectivity of the extraction.

1. A Feilden., Update on Undertaking Extractable and Leachable testing ISBN 978-1-84735-455-6 2. Hurtado-Benavides, A.; Dorado, D.A.; Sánchez-Camargo, A.D.P. Study of the fatty acid profile and the aroma composition of oil obtained from roasted colombian coffee beans by supercritical fluid extraction. J. Supercrit. Fluids 2016, 113, 44–52. 3. Nunes da Ponte, M. Chapter 1 supercritical fluids in natural product and biomass processing—An introduction. In High Pressure Technologies in Biomass Conversion; The Royal Society of Chemistry: Cambridge, UK, 2017; pp. 1–8. 4. B. A. Benner, Jr, Analytical Chemistry., 1998, 70 (21), pp 4594–4601

Extractables and Leachables Extraction Techniques: Volume I Page 18 Oven Extraction

OVEN EXTRACTION

Oven extraction is another very simple extraction technique in which the sample is placed in a suitable container or even the sample itself along with a solvent and then place in an oven for a period of time. It can either be placed in a static environment or far more commonly in a dynamic way through shaking/agitation [1]. It is this approach of elevated temperatures and agitation that the BioPhorum published in their study plan for single-use systems (40ºC and 50 rpm on an orbital shaker) [2]

Oven extraction has a particular advantage in the size and scale of the extraction, it can go from a few microlitres to litres (the automotive industry has ovens that are used to look for volatile organic compounds of complete cars [3].

The complexity of an oven extraction can be increased by increasing the level of automation. This can start at simultaneous extraction and analysis to a whole range of additional activities e.g. centrifugation. Two systems exist that can carry out simultaneous extraction and analysis for volatile and semi-volatile/non-volatile species. This simultaneous extraction and analysis can be a tremendous time-saver and hence can also maximise equipment utilisation and minimise analyst involvement. As with all extraction techniques, the principle is reliant on the use of appropriate temperature, time and solvent conditions. Direct analysis of the very volatile species after heating e.g. by headspace gas chromatography will not be discussed in this article.

For the volatile species, the automated extraction and analysis process is as follows. A vial is loaded with an appropriate amount of material (weight) and the vial capped and crimped. The vial is then loaded onto the equipment. The remainder of the process can be fully automated, as shown in the following example. A known volume of solvent is added by the instrument, containing an appropriate internal standard. The vial is automatically transferred to the heating agitator, where it is shaken at elevated temperatures for a set time. See Figure 2 for the apparatus. After the set time, a sample is taken automatically and injected directly into the GC with mass spectrometry (MS) detection and/or flame ionisation detection. The apparatus can also add a range of solvents during the analysis, allowing for prolonged unattended operation. This standard analysis can allow for the usual identification and quantification to take place. The time and the temperature in the agitator can be varied to produce asymptotic extraction levels. The asymptotic levels can be readily plotted in graphs.

The equipment shown in Figure 2 is Gerstel MPS 2 dual-rail multipurpose autosampler fitted on top of an Agilent 7890GC,5975B MSD. The solution was supplied and configured by Anatune Limited. One rail is equipped with a 1 mL syringe to address liquid handling for the sample preparation whilst on the other rail a 10-µl syringe allows injection of the freshly prepared sample onto the GC-MS. The automated sample preparation was performed using the following objects: Solvent Filling Station, Agitator, wash station and 10mL/20mL vial trays. The solution offers a vial capacity of up to 240 vials for long unattended experiments. The system is controlled with Gerstel Maestro software with full integration into an Agilent MSD Chemstation producing

Extractables and Leachables Extraction Techniques: Volume I Page 19 a single sequence table. For a more simplified system, a single rail can be used but, in this case, the solvent containing an internal standard would have to be added manually. Since this work was done there have been advances with the hardware allowing an increased range of activities in sample preparation which is outside the scope of this article.

Figure 1. Shaking apparatus Agilent 7890GC, 5975B MSD and Gerstel MPS 2 dual-rail system.

Example data generated using the shaking apparatus is shown in Figure 2. This was generated using the prescribed extraction duration with the agitator set to 10 °C below the boiling point of the solvent. It shows that DCM is the most effective solvent at extracting compounds and that asymptotic levels are reached after ~30 min. In this case, the optimum extraction time for DCM is very close to the GC analysis time. This allows maximum analysis to be undertaken in the minimum time if the extraction is carried out while the analysis of the previous samples is carried out.

Figure 2. Asymptotic plot showing the levels of a given species with each solvent against time (presented previously [i])

Extractables and Leachables Extraction Techniques: Volume I Page 20 The shaking system described above could also be connected to a HPLC inlet system. For such a system, careful considerations must be made when deciding whether an extraction solvent could be used with a reverse-phase (aqueous-based) chromatographic setup. For example, hexane is not water-soluble. In addition, if the system were to be used for HPLC sample preparation, then it would take up a considerable amount of laboratory space.

For the semi-volatile-to-non-volatile species, a similar mechanical extraction system exists: the I‑Chem explorer. An example of the Agilent set up is shown in Figure 3. But can also be used with Waters and Shimadzu [4].

Figure 3. The I-Chem explorer.

The sample, with solvent, is heated together with agitation for appropriate times, similar to the previous system. By sampling at prescribed intervals, asymptotic extraction conditions can be determined. Sample agitation is achieved by the use of a magnetic flea. The vial can be heated up to 150 ºC and with stirring up to 1500 rpm. After heating, the sample is automatically analysed by liquid chromatography with suitable detectors, such as ultraviolet and/or MS. This technique does suffer from several drawbacks. As discussed previously, solvents such as DCM and hexane cannot be easily used with this system because they are not miscible with typical HPLC mobile phases. The solvent also has to be added before the analysis/heating being started, so accurate timings of asymptotic levels may not be feasible. However, as long as these can be produced reproducibly, this should not be an issue.

1. A Feilden., Update on Undertaking Extractable and Leachable testing ISBN 978-1-84735-455-6 2. BioPhorum best practise guide https://www.biophorum.com/wp-content/uploads/Best-practices-guide-for-extractables- testing-April-2020.pdf accessed August 2020 3. ISO 12219-1:2012(E) Interior air in Road Vehicles-part 1: The Whole Vehicle Testing Environmental Chamber-specification and Method for the determination of volatile Organic Compounds inside the Vehicle 4. https://reactionanalytics.com/products/ accessed August 2020

Extractables and Leachables Extraction Techniques: Volume I Page 21 Liquid Extraction Surface Analysis

LIQUID EXTRACTION SURFACE ANALYSIS (LESA)

Direct Analysis

Extraction techniques utilising solvent in direct contact with materials have been discussed in previous pages.

Next we will cover extraction techniques that rely on direct analysis of the container closure system/device. If we define direct analysis as an approach in which the extraction of an analyte from a material and/or a solution cannot be separated from the actual analysis, then there are an ever-increasing number of analytical techniques that fit this description. The classical techniques for direct analysis would be infrared and Raman spectroscopy. However, these techniques, whilst very useful, do not alter the material being tested and as such are not carrying out an extraction. These will not be discussed further save for that they could be used non-destructively to identify and quantify extraction solutions, as well as identifying materials so as to assist with the identification of likely extractables.

The direct analysis techniques can be divided into two subgroups:

1. Those that analyse what is on the surface or a few nm into the material. 2. Those that facilitate the extraction and analysis of species from within the material being tested.

Liquid Extraction Surface Analysis (LESA)

As the title suggests this is a surface technique where extractables are analysed based on what is present on or near the surface. The LESA process brings the extraction solvent from a pipette tip into contact with the surface of a sample held in the sample plate of an Advion TriVersa NanoMate. The analyte is extracted from the surface [1]. The solvent is then retracted back into the pipette tip and sprayed through the ESI chip in the normal manner, see Figure 1. There are a number of advantages and disadvantages to this approach. The advantages can include:

• Mapping of a surface of a material can be accomplished by taking the analysis at numerous points and producing an extractable map. This could help identify defects. • By having and using a very small sample volume high sensitivity can be achieved. • Extraction and analysis can be very fast. • Could be used as a rapid confirmatory tool.

Extractables and Leachables Extraction Techniques: Volume I Page 22 The disadvantages can include:

• It may need to be assumed that the surface is homogeneous. • Complex solutions may be produced as there is no separation and so an idea of what could be present would be needed.

As with all extraction techniques, it is important to decide on what questions the study is trying to answer and choose the most appropriate extraction technique.

Figure 1 The LESA process (schematic [2] )

1. V. Kertesz and G.J. Van Berkel, Journal of Mass Spectrometry, 2010, 45, 3, 252 2. A Feilden., Update on Undertaking Extractable and Leachable testing ISBN 978-1-84735-455-6

Extractables and Leachables Extraction Techniques: Volume I Page 23 DART & DESI

DIRECT ANALYSIS IN REAL-TIME (DART) AND DESORPTION ELECTROSPRAY IONISATION (DESI)

Continuing on forms of direct analysis in this article we take a look at direct analysis in real-time (DART) and desorption electrospray ionisation (DESI). DART & DESI are the most common direct analysis techniques [1][2]. Whilst these direct analysis techniques may seem to be solventless systems there is a degree of ablation/excitation of the surface using either a solvent or an excited gas to allow for the analysis of species on or near the surface of a material. A list of possible ambient ionisation techniques and their year of introduction is given in Table 1 at the bottom of the article. As can be seen from the table there is a wide and very varied list of techniques with some showing more relevance to the analysis of materials. Only two of the most commonly used techniques will be discussed in more detail.

DESI involves the spray of a charged microdroplets from a pneumatically-assisted electrospray needle, as per standard electrospray ionisation (ESI) mass spectrometry. The spray is directed towards the surface of object, where it impacts the surface, desorbing the analytes into the gas phase where it is ionised and subsequently sampled by the mass spectrometer. A number of factors can affect the analyte response and selectivity, such as capillary tip to sample and sample to collector distances as well as angles of incidence.

Figure 1 – Example of DESI set up [32].

DART relies on the formation of a plasma discharge in a heated helium gas stream to give atmospheric pressure chemical ionisation (APCI). The helium atoms react with water molecules via chemical ionisation processes and subsequent downstream ionisation of the sample occurs by thermal desorption into the hot gas stream and then into the mass spectrometer. For more details on the ionisation techniques see Ambient ionisation mass spectrometry: current understanding of mechanistic theory; analytical performance and application areas (Daniel J Weston Analyst 2010, 135 p 661-668) [33]. In general DART is used in fit for purpose applications as it is more geometrically independent when compared to a technique like DESI. DART has been used to identify common stabilisers used in polypropylene [34] Other surface analytical techniques such as EDX or TOF-SIMs will not be discussed but they too have their potential niche area of analysis.

Extractables and Leachables Extraction Techniques: Volume I Page 24 References 1. R. B. Cody, J. A. Laramee and H. D. Durst, Anal. Chem., 2005, 77, 2297–2302 2. Z. Takats, J. M. Wiseman, B. Gologan and R. G. Cooks, Science, 2004, 306, 471–473. 3. A Feilden., Update on Undertaking Extractable and Leachable testing ISBN 978-1-84735-455-6 4. M. J. Ford and G. J. Van Berkel, Rapid Commun. Mass Spectrom., 2004, 18, 1303–1309. 5. R. B. Cody, J. A. Laramee and H. D. Durst, Anal. Chem., 2005, 77, 2297–2302. 6. C. N. McEwen, R. G. McKay and B. S. Larsen, Anal. Chem., 2005, 77, 7826–7831. 7. M. Z. Huang, H. J. Hsu, C. I. Wu, S. Y. Lin, Y. L. Ma, T. L. Cheng and J. Shiea, Rapid Commun. Mass Spectrom., 2007, 21, 1767–1775. 8. I. F. Shieh, C. Y. Lee and J. Shiea, J. Proteome Res., 2005, 4, 606. 9. Z. Takats, I. Cotte-Rodriguez, N. Talaty, H. Chen and R. G. Cooks, Chem. Commun., 2005, 1950–1952. 10. J. S. Sampson, A. M. Hawkridge and D. C. Muddiman, J. Am. Soc. Mass Spectrom., 2006, 17, 1712–1716. 11. Z. Takats,N. Czuczy,M. Katona and R. Skoumal, Proceedings of the 54thASMS Conference on Mass Spectrometry and Allied Topics, 2006. 12. H. Chen, A. Venter and R. G. Cooks, Chem. Commun., 2006, 2042– 2044. 13. R. Haddad, R. Sparrapan and M. N. Eberlin, Rapid Commun. Mass Spectrom., 2006, 20, 2901–2905. 14. H. Chen, Z. Ouyang and R. G. Cooks, Angew. Chem., Int. Ed., 2006, 45, 3656. 15. W. C. Wetzel, F. J. Andrade, J. A. C. Broekaert and G. M. Hieftje, J. Anal. At. Spectrom., 2006, 21, 750–756. 16. L. V. Ratcliffe, F. J. Rutten, D. A. Barrett, T. Whitmore, D. Seymour, C. Greenwood, Y. Aranda-Gonzalvo, S. Robinson and M. McCoustra, Anal. Chem., 2007, 79, 6094–6101. 17. N. Na, M. Zhao, S. Zhang, C. Yang and X. Zhang, J. Am. Soc. Mass Spectrom., 2007, 18, 1859–1862. 18. H. Chen, A. Wortmann and R. Zenobi, J. Mass Spectrom., 2007, 42, 1123–1135. 19. J. Wu, C. S. Hughes, P. Picard, S. Letarte, M. Gaudreault, J. Levesque, D. A. Nicoll-Griffith and K. P. Bateman, Anal. Chem., 2007, 79, 4657–4665. 20. P. Nemes and A. Vertes, Anal. Chem., 2007, 79, 8098–8106. 21. ] M. Haapala, J. Pol, V. Saarela, V. Arvola, T. Kotiaho, R. A. Ketola, S. Franssila, T. J. Kauppila and R. Kostiainen, Anal. Chem., 2007, 79, 7867–7872. 22. Y. H. Rezenom, J. Dong and K. K. Murray, Analyst, 2008, 133, 226–232. 23. F. Andrade, J. Shelley, W. Wetzel, M. Webb, G. Gamez, S. Ray and G. Hieftje, Anal. Chem., 2008, 80, 2654–2663. 24. R. Haddad, R. Sparrapan, T. Kotiaho and M. N. Eberlin, Anal. Chem., 2008, 80, 898–903. 25. R. B. Dixon, J. S. Sampson, A. M. Hawkridge and D. C. Muddiman, Anal. Chem., 2008, 80, 5266–5271. 26. J. T. Shelley, S. J. Ray and G. M. Hieftje, Anal. Chem., 2008, 80, 8308–8313. 27. J. D. Harper, N. A. Charipar, C. C. Mulligan, X. Zhang, R. G. Cooks and Z. Ouyang, Anal. Chem., 2008, 80, 9097–9104. 28. L. Nyadong, A. S. Galhena and F. M. Fernandez, Anal. Chem., 2009, 81, 7788–7794. 29. G. J. Van Berkel, V. Kertesz and R. C. King, Anal. Chem., 2009, 81, 7096–7101. 30. S. Crotti and P. Traldi, Comb. Chem. High Throughput Screening, 2009, 12, 125–136. 31. A. N. Martin, G. R. Farquar, P. T. Steele, A. D. Jones and M. Frank, Anal.Chem., 2009, 81, 9336–9342. 32. Z. Takats et al. Science 2004 306,471-473 33. Daniel J Weston Analyst 2010, 135 p 661-668 34. Haunschmidt M et Al Analyst 2010 135 p 80-85)

Extractables and Leachables Extraction Techniques: Volume I Page 25 Technique Acronym Year of Introduction

Desorption electrospray ionisation DESI 2004

Surface sampling probe SSP [4] 2004

Direct analysis in real time DART [5] 2005

Atmospheric solids analysis probe ASAP [6] 2005

Electrospray laser desorption ionization ELDI [7] 2005

Fused droplet electrospray ionization FD-ESI [8] 2005

Direct atmospheric pressure chemical ionization DAPCI [9] 2005

Matrix-assisted laser desorption electrospray ionization MALDESI [10] 2006

Jet desorption electrospray ionization JeDI [11] 2006

Extractive electrospray sonization EESI [12] 2006

Desorption sonic spray ionization DeSSI [13] 2006

Atmospheric pressure thermal desorption ionization APTDI [14] 2006

Helium atmospheric pressure glow discharge ionization HAPGDI [15] 2006

Plasma-assisted desorption ionization PADI [16] 2007

Dielectric barrier desorption ionization DBDI [17] 2007

Neutral desorption extractive electrospray ionization ND-EESI [18] 2007

Laser diode thermal desorption LDTD [19] 2007

Laser ablation electrospray ionization LAESI [20] 2007

Desorption atmospheric pressure photo-ionization DAPPI [21] 2007

Infra red laser ablation electrospray ionization IR-LAESI [22] 2008

Flowing atmospheric-pressure afterglow FAPA [23] 2008

Easy ambient sonic spray ionization EASI [24] 2008

Remote analyte sampling transport and ionization relay RASTIR [25] 2008

Laser ablation flowing atmospheric-pressure afterglow LA-FAPA [26] 2008

Low temperature plasma LTP [27] 2008

Desorption electrospray metastable-induced ionization DEMI [28] 2009

Liquid micro-junction surface sampling probe/electrospray ionization LMJ-SSP/ESI [29] 2009

Surface activated chemical ionization SACI [30] 2009

Single particle aerosol mass spectrometry SPAMS [31] 2009

Table 1: A list of possible ambient ionisation techniques

Extractables and Leachables Extraction Techniques: Volume I Page 26 HEADSPACE HEADSPACE

Continuing on forms of direct analysis from DART & DESI, let’s look at headspace as an extraction technique.

Headspace is the classical technique that can extract the sample without the need for a solvent. As such, only volatile-to-semi- volatile species are extracted. Headspace is a means of introducing the volatile components from a liquid or solid sample into a gas chromatograph for analysis when it is difficult or impossible to inject the actual sample. Also, if very volatile species are present and an extracting solvent would interfere with the analysis, headspace offers an analytical methodology. The basic principle is well documented[1] and as such this article will only cover the most basic principles. The basic principle is as follows: The solid, liquid or gaseous sample is placed in a vial and sealed. The vial is heated, allowing the volatile components to escape out of the sample to form a gaseous headspace. After a set time, the headspace gas is extracted from the vial and injected into a gas chromatograph for analysis. Headspace theory is based around the tendency of a material to go into the gas phase, producing the partition coefficient, K. K is defined as CL divided by CG (where CL is the concentration of the analyte in the condensed phase and CG is the concentration of the analyte in the gas phase). This is shown schematically in Figure 1. Reducing K will increase the sensitivity of the Headspace analysis. Methods to reduce K are listed below.

• Addition of mineral salts to the matrix (sodium sulphate is common) • Addition of another liquid to the matrix • Increasing the temperature • Agitation of the sample

Figure 1 Headspace extraction (schematic)

Extractables and Leachables Extraction Techniques: Volume I Page 27 For extractables, the key factors are temperature and time, especially for solid samples. The time to diffusion t is related to the diffusion coefficient D and the diffusion path length t= d2/2D

Ideally, the temperature should be above the glass transition temperature of the material to enhance the rate of species diffusing through a material. In general, Headspace is a simple, reliable and easily automated technique which allows for the analysis of very volatile species without potential interference from an extracting solvent. It does not, however, provide as-low detection limits as other Headspace techniques such as solid-phase microextraction (SPME), in-tube extraction, or dynamic headspace (DHS). How these related techniques work and their advantages will be discussed in subsequent blogs.

[1] Kolb and L.S.Ettre in Static Headspace Gas Chromatography, Wiley-VCH, Inc., New York, NY, USA, 1997.

Extractables and Leachables Extraction Techniques: Volume I Page 28 Thermal Desorption

THERMAL DESORPTION

Thermal desorption is another gas chromatography sample introduction technique and is well suited to trace level analysis of volatile organic compounds. In this technique, a known volume of sample air is passed through a trap comprising a tube containing a powdered adsorbent such as charcoal, tenax etc. Contaminants in the air are trapped in the adsorbent bed. After collection of the sample, the trap is then inserted into the carrier gas path at the inlet to the chromatograph and rapidly heated to release the analytes into the column. Figure 1 shows the thermal desorption unit along with a selection of desorption tubes. However, for the analysis of solid samples, the material to be analysed is placed in the tube over which a gas is passed while the tube is heated. In thermal desorption, the partition can be a dynamic process in which the analyte goes from the gas state and is then moved to the inlet of a gas chromatograph where they are cryofocused (by Peltier cooling, liquid CO2 or liquid N2 [in increasing order of cooling efficiency]). Once the analyte is moved away from the solid sample, the partition coefficient favours the transfer of the analyte away from the solid sample into the gaseous state[1]. With Headspace, the process is typically static, where once the equilibrium coefficient is reached no further analyte is extracted.

In general, thermal desorption can be more sensitive than standard headspace but care must be taken in selecting the correct sorbent to make sure it has the suitable capacity and the ability to trap the required analytes.

Figure 1. Thermal desorption apparatus along with a selection of desorption tubes. Reproduced with permission from Marks International Limited

1. Zweiben and A.J. Shaw, PDA Journal of Pharmaceutical Science and Technology, 2009, 63, 353.

Extractables and Leachables Extraction Techniques: Volume I Page 29 SUMMARY

The range of solvent extraction techniques have been discussed in some detail previously. There is no perfect extraction technique that should be used. Each of them has a number of advantages and disadvantages and depending on solvent choice and the end goal, different techniques will be preferred. There should always be a solventless system to enable the extraction and analysis of very volatile species without the potential. Different extraction techniques were considered for the following situation: extraction requires the use a polar, semi-polar and non-polar solvents on a material that has not been extracted before with the goal of achieving asymptotic extraction. It has also been assumed that the sample can be easily loaded into the extraction vessel. Each technique has been given a relative score, the more green the better.

Equipment Development Sample Range of Sample Technique Running cost Speed Flexibility Simplicity Efficiency cost time preparation solvents degradation

Sonication

Reflux

Soxhlet

Sealed Vessel

Pressurised Solvent Extraction Microwave Assisted Extraction Supercritical Fluid Extraction

Oven

N/A Direct Analysis

N/A LESA

N/A Headspace

N/A Thermal Desorption

N/A Dynamic Headspace

• Equipment cost: Purchase price of equipment including validation e.g. temperature mapping of ovens • Running cost: Including cost of solvents (some have high solvent usage) • Speed: How quickly can the extraction be completed • Flexibility: Can a wide range of samples be extracted • Development time: How long does it take to develop a suitable method • Simplicity: How easy is it to use and set up • Sample preparation: How much effort is required to get a sample ready for extraction • Range of solvents: Are there any restrictions in solvents/mixtures that can be used • Efficiency: How much can be extracted included range of extractables • Sample degradation: The likelihood to deform or degrade the material being extracted

Extractables and Leachables Extraction Techniques: Volume I Page 30 CONCLUSION

My personal preference would be Soxhlet or reflux for the proposed situation because of the combination of the efficiency, speed and ease of use and the ability to achieve asymptotic levels relatively easily. I would also include one of headspace/ thermal desorption/dynamic headspace with the decision being around operational complexity vs sensitivity.

If the material was well known and there was an end goal of process control with high throughput then the choice of extraction equipment would be pressurised solvent extraction/ microwave extraction. If the actual use was relatively short term and or the materials were likely to degrade then the use of an oven to carry out the extraction would be appropriate. Also if the material to be tested was large and cutting the sample would be difficult/ not desirable then the only appropriate technique would be an oven.

It is important to always think about the end goal of the study and use the appropriate extraction technique, duration, temperature, solvent and stoichiometry to enable the analytical chemist to achieve the required level of sensitivity based on the analytical evaluation threshold (AET).

ABOUT THE AUTHOR

Dr Andrew Feilden is the European E&L Strategic Director at Hall Analytical. He joined Hall in November 2019 where he is a technical expert in the field of E&L testing undertaking commercial, operational and technical thought leadership activities. He has presented on the field of extractables and leachables in over 16 countries world wide. Andrew has written a number of papers and publications and is the inventor of 2 patents. He has a degree and D Phil from the University of York, is a Fellow of the Royal Society of Chemistry and was a Scientific advisor to IPAC-RS and ex-cochair of ELSIE

CONTACT US

expert analytical testing Waterside Court 1 Crewe Road Wythenshawe Manchester M23 9BE UK phone: +44 (0)161 286 7889 email: [email protected] web: www.hallanalytical.com

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