{"id":5,"date":"2024-04-04T04:31:15","date_gmt":"2024-04-04T01:31:15","guid":{"rendered":"https:\/\/sisu.ut.ee\/heritage-analysis\/61-chromatography\/"},"modified":"2024-07-18T14:33:07","modified_gmt":"2024-07-18T11:33:07","slug":"61-chromatography","status":"publish","type":"page","link":"https:\/\/sisu.ut.ee\/heritage-analysis\/61-chromatography\/","title":{"rendered":"6.1. Chromatography"},"content":{"rendered":"<div style=\"height:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p>In this section, practical aspects and analysis with GC-MS\/ FID and HPLC with different detectors are discussed, and examples are given.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\"><span style=\"color: #b22222;\">Analysis with GC-MS and GC-FID methods<\/span><\/h2>\n\n\n\n<p><strong>Gas chromatography (GC)<\/strong> is a widely used technique for the analysis of organic compounds of cultural heritage materials. The main detectors used for GC are <strong>mass<\/strong> <strong>spectrometric (MS)<\/strong> detector and <strong>Flame ionisation detector (FID)<\/strong> (more discussed in <a title=\"\" data-url=\"https:\/\/sisu.ut.ee\/heritage-analysis\/51-chromatography\" href=\"https:\/\/sisu.ut.ee\/heritage-analysis\/51-chromatography\" target=\"_blank\" rel=\"noopener\">5.1 Chromatography<\/a>) that both enable qualitative and quantitative analysis. For the analysis of cultural heritage materials, <strong>GC-MS<\/strong> is the preferred technique because it enables a more reliable identification of the compounds present in the sample: a characteristic mass spectrum for every chromatographic peak is obtained. <strong>GC-FID<\/strong> lacks such a possibility: identifying is only possible when standards are used and identification is based on retention time (<em>t<\/em><sub>R<\/sub>). Identification by retention time only is not very reliable as many compounds can have similar retention times.<\/p>\n\n\n\n<p><strong>The overall steps for GC-MS (and GC-FID) analysis:<\/strong><\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>Sample preparation<\/strong>: depends on the material. Typically, it includes dissolving the sample or extracting the compounds with a suitable solvent (e.g. with hexane, toluene, etc.). During the sample preparation,<span class=\"file media-element file-os-files-link\" data-file_info=\"%7B%22fid%22:63438,%22view_mode%22:%22os_files_link%22,%22type%22:%22media%22%7D\"> <strong>derivatisation<\/strong>\u00a0<\/span>(<a href=\"https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/6.1_derivatisation.pdf\" target=\"_blank\" rel=\"noreferrer noopener\">CLICK HERE<\/a> to read more about it) is often used to increase the volatility of the analytes and to enhance the chromatographic separation. With the multi-component mixtures, additional procedures may be needed, e.g. dilution, centrifugation, filtration of the solution, etc. Pyrolysis and SPME are special cases of sample preparation and their use is described below.\n<ul class=\"wp-block-list\">\n<li><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">In the case of <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">quantitative analysis<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">, an internal standard of known concentration is usually added to the sample solution. Internal standard is a compound that is chemically similar but not identical to the analyte(s).<\/span> <span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">Also, calibration solutions containing the derivatives of the components of interest and internal standards must be made.<\/span> <\/li>\n<\/ul>\n<\/li>\n<\/ul>\n\n\n\n<div style=\"height:0px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>Chromatographic analysis<\/strong> <strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">of  the samples using the GC-MS (or GC-FID) instrument<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">:<\/span>\n<ul class=\"wp-block-list\">\n<li><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">Choosing a suitable method<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">: Using a <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\"><em>pre-existing method<\/em><\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"><em> <\/em>developed for a specific material type requires minimal (if any) parameter adjustments before analysis. <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\"><em>Development of a new measurement method<\/em><\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> includes selecting a suitable column, adjusting\u00a0the parameters, such as temperature program, injection volume, split ratio, etc. Method development is usually based on the analysis of standard materials.<\/span> <\/li>\n\n\n\n<li><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">Injection of samples<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> can be done manually with a micro syringe (one-by-one) or with an autosampler (if the instrument is equipped with it). The autosampler injects the sample(s) automatically according to the<em> <\/em><\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\"><em>measurement series (sequence)<\/em> <\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">set by the operator.<\/span> <\/li>\n\n\n\n<li><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">Useful tips<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">: Before the GC analysis, it may be beneficial to heat the column at a high temperature to clean the system. This should also be done after the analysis of a complex (real-life) samples.<\/span> <span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">In the case of samples with unknown composition, it is advised to add <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\"><em>blank injection(s)<\/em> <\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">(e.g., injecting only the solvent) after the sample injection to make sure that the system is clean.\u00a0 Also, when starting work, it is useful to make a blank injection to verify that the system is clean.<\/span> <\/li>\n<\/ul>\n<\/li>\n<\/ul>\n\n\n\n<div style=\"height:0px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>Interpretation of the results<\/strong>: For <strong>GC-FID<\/strong> the identification of the<strong> peaks on the chromatogram<\/strong> is based on the comparison of retention times of standard materials with the peaks obtained from the sample. In the <strong>GC-MS<\/strong> analysis beside <strong>chromatogram<\/strong> also <strong>mass spectra<\/strong> can be obtained \u2013 to each peak on the chromatogram corresponds also an averaged mass spectrum (see Fig. 1). For GC-MS, comprehensive commercial MS libraries are available that simplify the identification of the detected compounds.\n<ul class=\"wp-block-list\">\n<li><p>The identification of the analysed material is usually based on the presence of <strong><em>specific biomarkers<\/em><\/strong> (e.g., abietic acid, dehydroabietic acid, and other resin acids for colophony resin), <strong><em>examining the molecular profile<\/em><\/strong> (e.g., the simultaneous presence of certain fatty acids, alcohols, and saturated hydrocarbons for beeswax) or by <strong><em>quantifying characteristic compounds<\/em><\/strong> (e.g., proteinaceous materials can be identified based on the quantities of specific amino acids).<\/p> <\/li>\n<\/ul>\n<\/li>\n<\/ul>\n\n\n\n<p>In the following video, Dr Eliise Tammekivi demonstrates the usage of GC-MS and GC-FID in more detail.<\/p>\n\n\n\n<p><\/p><div class=\"ratio ratio-16x9 mb-3\"><div class=\"video-placeholder-wrapper video-placeholder-wrapper--16x9\">\n\t\t\t    <div class=\"video-placeholder d-flex justify-content-center align-items-center\">\n\t\t\t        <div class=\"overlay text-white p-2 w-100 text-center d-block justify-content-center align-items-center\">\n\t\t\t            <div>To view third-party content, please accept cookies.<\/div>\n\t\t\t            <button class=\"btn btn-secondary btn-sm mt-1 consent-change\">Change consent<\/button>\n\t\t\t        <\/div>\n\t\t\t    <\/div>\n\t\t\t<\/div>\n<\/div>\n\n\n\n<p>In the video, as an example, the analysis of <strong><em>fresh linseed oil for determining its fatty acid composition<\/em><\/strong> was carried out.\u00a0To make the analysis with GC possible, the oil components (triacylglycerides aka TAGs) were hydrolysed and then <strong><em>derivatised with TMTFTH<\/em> <\/strong>(aka <strong><em>Meth-Prep II<\/em><\/strong>) reagent that produced a <strong><em>mixture of methylated fatty acids<\/em><\/strong>. These compounds are (differently from TAGs) sufficiently volatile for GC analysis. After the chromatographic run a chromatogram was obtained, where the compounds in the mixture were successfully separated and then identified (see Fig. 1). [1]<\/p>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"960\" height=\"526\" src=\"https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/6.1_fig1.jpg\" alt=\"6.1 Figure 1.png\" class=\"wp-image-55\" title=\"6.1_fig1.jpg\" srcset=\"https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/6.1_fig1.jpg 960w, https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/6.1_fig1-300x164.jpg 300w, https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/6.1_fig1-768x421.jpg 768w\" sizes=\"auto, (max-width: 960px) 100vw, 960px\"><figcaption class=\"wp-element-caption\">Fig. 1. Example of GC-MS analysis. Chromatogram of methylated fatty acids of linseed oil and internal standard (hexadecane). The separated compounds are represented as peaks on the chromatogram, which can be identified by their retention times. For each peak (an example is given for methyl palmitate peak eluting at 14.82 min), a mass spectrum can be obtained and compared to the mass spectra provided by the MS library. If the match is good (often over 90-95%), then with a high probability, it can be said that the peak corresponds to the match (in this case, methyl palmitate). The peak areas (or heights) given by the software can be used to construct the calibration curve for the quantitative analysis.<\/figcaption><\/figure>\n\n\n\n<div class=\"wp-block-group is-vertical is-layout-flex wp-container-core-group-is-layout-8cf370e7 wp-block-group-is-layout-flex\">\n<p class=\"has-text-align-left\">There are two sampling techniques that are commonly used in the field of cultural heritage with the GC-MS system: <strong>pyrolysis (Py) GC-MS<\/strong> and <strong>solid-phase microextraction (SPME) GC-MS<\/strong>.\u00a0<\/p>\n\n\n\n<p class=\"has-text-align-left\"><strong>Py-GC-MS<\/strong> is probably one of the most valuable tools for the analysis of various types of cultural heritage materials. In pyrolysis higher temperature (commonly over 600\u00b0C)\u00a0 can be used to desorb and pyrolyze the analysed compounds <strong><em>without extensive sample preparation<\/em><\/strong>. For cultural heritage materials, pyrolysis is often performed in the presence of a <strong><em>derivatisation reagent<\/em><\/strong> (thermally assisted derivatisation) to decrease the polarity of the compounds and thereby increase the volatility of the formed fragments, and to improve the separation of the analytes. Although pyrolysis essentially destroys the original material, the obtained pyrolysis products contain molecular markers that are used for the identification of the original material. [2]\u00a0<\/p>\n\n\n\n<p><strong>SPME GC-MS<\/strong> can be used for a limited selection of materials. It is <strong><em>only used for the analysis of volatile or semi-volatile organic compounds<\/em><\/strong> (<strong>VOCs<\/strong> and <strong>SVOCs<\/strong>, respectively) coming from the object, like acetic acid, styrene, monoterpenes, alkanes, etc. Such compounds can be emitted by artefacts as a result of decomposition (ageing). The volatile compounds adsorbed in the SPME sampler can be injected into a GC using the same SPME sampler, <strong><em>without additional sample pre-treatment<\/em><\/strong>.<strong>\u00a0<\/strong>[2,3]\u00a0<\/p>\n\n\n\n<p>Table 1 summarises some of the GC-MS\/FID, Py-GC-MS, and SPME GC-MS possibilities for the analysis of different cultural heritage materials.<\/p>\n<\/div>\n\n\n\n<p><\/p>\n\n\n\n<p><\/p>\n\n\n\n<div class=\"wp-block-group is-vertical is-layout-flex wp-container-core-group-is-layout-8cf370e7 wp-block-group-is-layout-flex\">\n<p>Table 1. Properties of GC-MS, Py-GC-MS, and SPME GC-MS for the analysis of cultural heritage materials [1\u20134].<\/p>\n\n\n\n<figure class=\"wp-block-table has-small-font-size\"><table class=\"table table-hover\"><thead><tr><th><\/th><th><strong>GC-MS\/FID<\/strong><\/th><th><strong>Py-GC-MS<\/strong><\/th><th><strong>SPME GC-MS<\/strong><\/th><\/tr><\/thead><tbody><tr><td><strong>Analysed materials<\/strong><\/td><td>Mostly natural (oils, waxes, resinous materials, dyes, proteins, polysaccharides) and some synthetic (e.g. alkyd) materials<\/td><td>Natural materials (oils, waxes, resinous materials, dyes, proteins, polysaccharides, lacquers), synthetic materials and polymers (alkyd, acrylics, vinyl resins, etc.)<\/td><td>VOCs, SVOCs, and volatile degradation products of natural resins, lacquers, some synthetic polymers (e.g., cellulose acetate, polyvinyl acetate), cellulose (e.g., paper, wood), oils, and waxes<\/td><\/tr><tr><td><strong>Sample preparation<\/strong><\/td><td>\u2212 <strong>Sample size:<\/strong> usually 0.1-20 mg.<br>\u2212 Sample dissolved\/extracted with organic solvent (methanol, ethanol, toluene, hexane, dichloromethane, etc.). NB! Water cannot be used.<br>\u2212 <strong>Derivatisation is almost always required<\/strong> \u2192 choice of derivatisation reagent depends on sample type, pre-treatment, aim of analysis, etc. <strong><em>Main reagents:<\/em><\/strong> TMAH, TMTFTH aka Meth-Prep II, BSTFA, BSA.<\/td><td>\u2212 <strong>Sample size:<\/strong> normally 0.1-1 mg.<br>\u2212 <strong>Sample preparation is not needed<\/strong>!<br>\u2212 Sample placed into the pyrolyzer (heated chamber). If needed, derivatisation reagent (e.g., TMAH, HMDS) can be added.<\/td><td>\u2212 <strong>Sample size:<\/strong> larger than for GC-MS\/FID and Py-GC-MS \u2192 only volatile compounds are analysed.<br>\u2212 <strong>Sample preparation is not needed!<\/strong><br>\u2212 <strong>Derivatisation is not needed!<\/strong><br>\u2212 SPME fibre held near analysis object or sample for specific time (depends on analytes, size of object, etc.) to collect organic analytes. Fibre inserted to the GC injector where analytes are desorbed at high temperature and carried to the GC column.<\/td><\/tr><tr><td><strong>Advantages<\/strong><\/td><td>\u2212 Possible to analyse aged, oxidised, degraded materials.<br>\u2212 Suitable for quantitative analysis (sample amount of at least 10 mg is often needed for reliable quantification).<br>\u2212 Interpretation based on reference libraries.<br>\u2212 Additional device is not needed for sample treatment (e.g,. pyrolyzer).<br>\u2212 Gives more info than SPME-GC-MS.<br>\u2212 The results are easier to interpret compared to Py-GC-MS.<\/td><td>\u2212 Capable of analysing essentially any organic material (e.g., natural and synthetic materials, polymers, etc.).<br>\u2212 Sample pre-treatment is not needed.<br>For some polymers derivtisation is not needed.<br>\u2212 Possible to analyse aged, oxidised, degraded materials.<br>\u2212 Possible to analyse very small samples.<\/td><td>\u2212 Easy interpretation \u2013 only volatiles compounds are analysed.<br>\u2212 Non-destructive.<br>\u2212 Sample pre-treatment, incl. derivatisation is not needed.<br>\u2212 Possible to analyse aged, oxidised, degraded materials.<br>\u2212 Possible to monitor the condition of objects (e.g., in museums).<\/td><\/tr><tr><td><strong>Disadvantages<\/strong><\/td><td>\u2212 Comprehensive sample preparation, e.g., hydrolysis, extraction, derivatisation, etc.<br>\u2212 Not suitable for the analysis of most synthetic polymers.<br>\u2212 Destructive method.<br>Fragmenting technique \u2192 many peaks in the chromatogram make interpretation complicated<\/td><td>\u2212 Complex interpretation \u2192 very fragmenting technique.<br>\u2212 Destructive method.<br>\u2212 Side reactions, e.g., isomerisation of double bonds, etc. can occur.<br>\u2212 Non-quantitative method due to the pyrolysis side reactions.<\/td><td>\u2212 Limited to volatile\/semi-volatile analytes.<br>\u2212 Requires more sample than GC-MS\/FID or Py-GC-MS.<br>\u2212 Possible to quantify volatiles near the object, but not in the object<\/td><\/tr><\/tbody><\/table><\/figure>\n<\/div>\n\n\n\n<p><\/p>\n\n\n\n<p>In conclusion, the choice of the GC column, detector, and method (parameters) depend on the analysable material and question in hand. If the components of the sample are sufficiently volatile (or can be made volatile by derivatisation), then GC-MS is the preferred method. When the sample contains polymers (incl. aged cultural heritage materials) that cannot be processed into suitable derivatives, then Py-GC-MS is the preferred approach. Both of these methods are destructive. SPME GC-MS is a non-destructive method that does not require sample preparation but is only suited for the analysis of VOCs and SVOCs of the object.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\"><span style=\"color: #b22222;\">Analysis with HPLC using different detectors<\/span><\/h2>\n\n\n\n<p><strong>Liquid chromatography (LC)<\/strong> is another separation technique that is widely used for the analysis of cultural heritage materials. With LC, qualitative and quantitative analysis can be carried out. Different materials require a different LC approach and occasionally quite extensive sample preparation. In this course, only the most common LC techniques have been discussed and\u00a0the main focus being on <strong>reversed phase (RP) LC<\/strong>.<\/p>\n\n\n\n<p>In LC, <strong>only solutions can be analysed<\/strong>. The sample must a) be dissolved in a suitable solvent or solvent mixture or b) it must be possible to extract the compound(s) from the sample into solution. Aged materials, such as paints, varnishes, etc. need usually a <strong>multi-step solvent extraction and\/or derivatisation<\/strong> before they can be analysed with LC.<\/p>\n\n\n\n<p><strong>Overall steps during HPLC analysis:<\/strong><\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>Sample preparation:<\/strong>\n<ul class=\"wp-block-list\">\n<li><strong>Dissolving or extracting the sample <\/strong>with a suitable solvent\/solvent mixture (choice of solvents depends on the material (see Table 2). Sample amount needed for LC analysis is <em><strong>roughly 1 mg of sample per 1 ml of solvent(s)<\/strong><\/em>. With MS detector, the amount of sample can be lower than 1 mg (MS detectors are very sensitive).\u00a0Optional additional procedures with the sample solution include dilution, derivatisation, adding internal standards (only needed for quantitative analysis), etc.<\/li>\n\n\n\n<li><strong>Filtering or centrifuging of the sample solution. <\/strong><em><strong>NB! This is a very important step in LC analysis and must not be skipped!<\/strong><\/em><strong>\u00a0<\/strong>The sample solution must not contain any solid particles to avoid contamination and damage to\u00a0the LC system and column. For filtering, dense syringe filters are used. If the amount of sample solution is &lt;50 \u03bcl, filtration is not the best solution because some of the solution will always remain in the filter. In that case, the solution is centrifuged and the clear supernatant is used for analysis.<\/li>\n\n\n\n<li><strong>Preparation of additional standard solutions for analysis<\/strong>:\n<ul class=\"wp-block-list\">\n<li><strong><em>Calibration standard solutions for quantitative analysis<\/em><\/strong> \u2013 solutions containing known different concentrations of compounds of interest.<\/li>\n\n\n\n<li><strong><em>Reference standard solutions for qualitative analysis<\/em><\/strong>\u00a0\u2013 solutions containing pure (certified) compounds. Reference standard solutions are measured under the same conditions as analysed sample and these results will help to identify and verify retention times and mass spectra of individual sample components.<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n\n\n\n<div style=\"height:0px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>Measurement of samples:<\/strong>\n<ul class=\"wp-block-list\">\n<li><strong>Choosing a suitable LC method<\/strong>: using a\u00a0<strong>pre-existing method<\/strong>\u00a0developed for the specific material type requires minimal, if any, parameter adjustments before analysis.\u00a0<em><strong>Development of a new method<\/strong><\/em>\u00a0involves selection of suitable solvent(s) and column; optimisation of LC parameters, such as flow rate, injection volume, eluent composition, etc. Method development is based on the analysis of reference materials.\u00a0<em><strong>Most time-consuming and difficult step!<\/strong><\/em><\/li>\n\n\n\n<li><strong>Injection of samples<\/strong>: done manually or by an autosampler (more common nowadays) according to the set sequence.<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n\n\n\n<div style=\"height:0px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>Useful tips for analysis (instrument maintenance)<\/strong>: compared to GC, LC system needs more maintenance and has more parameters that need to be monitored\/checked.\n<ul class=\"wp-block-list\">\n<li>For analysis, the <strong>LC system must be clean, have no leakages, blockages, or air trapped in the system (bubbles)<\/strong>. To ensure this, the selected solvent(s) are run through the system and the pressure is monitored before injecting any samples. The instrument is ready for analysis when the system is conditioned with the solvents selected for the analysis, the system pressure and detector signal are <span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">stable.<\/span> <\/li>\n\n\n\n<li><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">It is advised to add <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">blank injections<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> to the sequence (injection of only solvent or no sample at all) e.g., after each sample \u2013 this helps to keep the system clean and prevents carryover of compounds from one sample to the next.<\/span> <\/li>\n\n\n\n<li><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">At the end of the analysis it is important to <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">clean the system by rinsing with suitable solvent(s)<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">.<\/span> <\/li>\n\n\n\n<li><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">To help protect and prolong the lifetime of the column, a <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">guard column<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> is used. It is a short column (usually 1 cm) with the same functionality as the main column. For UHPLC instruments, using an <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">in-line filter before the column<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> (or before the guard column) is advised, also. This will stop the solid particles that have entered the system and thus, prevents damage to the main column and instrument.<\/span> <\/li>\n<\/ul>\n<\/li>\n<\/ul>\n\n\n\n<div style=\"height:0px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">Interpretation of results<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">: for <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">qualitative analysis<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> retention times of peaks on the chromatogram belonging to the sample compounds are compared to retention times of the standard compounds. For <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">quantitative analysis<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">, a calibration graph is compiled (concentration of the compound vs peak area\/height on the chromatogram) and from there the concentration for the compound can be calculated.\u00a0In addition, different detectors can provide additional information about the sample compounds, e.g., with <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">UV-VIS\/PDA detector <\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">\u00a0obtained UV-Vis absorbance spectra can give information whether a compound is coloured; <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">mass spectrometric (MS) detector<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> gives information about the mass, isotopic pattern, and chemical composition of the compounds, etc.<\/span> <\/li>\n<\/ul>\n\n\n\n<div style=\"height:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p>In the video below, Dr Pilleriin Peets\u00a0gives a brief overview of how to carry out analysis with an HPLC system with different detectors on the basis of dye samples. In general, the analysis steps are similar to the analysis of other materials.<\/p>\n\n\n\n<p><\/p>\n\n\n\n<p><\/p><div class=\"ratio ratio-16x9 mb-3\"><div class=\"video-placeholder-wrapper video-placeholder-wrapper--16x9\">\n\t\t\t    <div class=\"video-placeholder d-flex justify-content-center align-items-center\">\n\t\t\t        <div class=\"overlay text-white p-2 w-100 text-center d-block justify-content-center align-items-center\">\n\t\t\t            <div>To view third-party content, please accept cookies.<\/div>\n\t\t\t            <button class=\"btn btn-secondary btn-sm mt-1 consent-change\">Change consent<\/button>\n\t\t\t        <\/div>\n\t\t\t    <\/div>\n\t\t\t<\/div>\n<\/div>\n\n\n\n<div class=\"wp-block-group is-vertical is-layout-flex wp-container-core-group-is-layout-8cf370e7 wp-block-group-is-layout-flex\">\n<div style=\"height:0px\" aria-hidden=\"true\" class=\"wp-block-spacer wp-container-content-42c95ffb\"><\/div>\n\n\n\n<p>As explained in section <a title=\"\" data-url=\"https:\/\/sisu.ut.ee\/heritage-analysis\/51-chromatography\" href=\"https:\/\/sisu.ut.ee\/heritage-analysis\/51-chromatography\" target=\"_blank\" rel=\"noopener\">5.1 Chromatography<\/a> different <strong>LC approaches<\/strong> are in use \u2013 <strong>reversed phase chromatography (RP)<\/strong>, <strong>normal phase chromatography (NP)<\/strong>, <strong>size exclusion chromatography (SEC)<\/strong>, etc. <strong>RP <\/strong>is by far the most used LC technique and has been commonly applied for the analysis of a variety of cultural heritage materials. Also, <strong>SEC<\/strong> has been frequently used for cultural heritage materials, mainly for monitoring changes, degradation, and ageing of lipids, varnishes, polymers, etc. Up to now, <strong>NP chromatography<\/strong> has found limited use for the analysis of cultural heritage materials. <\/p>\n\n\n\n<p>Table 2 presents an overview of the analysis of different materials with LC, focusing currently on the more common LC techniques.\u00a0<\/p>\n<\/div>\n\n\n\n<p><\/p>\n\n\n\n<div class=\"wp-block-group is-vertical is-layout-flex wp-container-core-group-is-layout-8cf370e7 wp-block-group-is-layout-flex\">\n<p>Table 2. Analysis of different materials using more common LC techniques [2,5\u20137].<\/p>\n\n\n\n<figure class=\"wp-block-table aligncenter has-small-font-size\"><table class=\"table table-hover\"><thead><tr><th><\/th><th><strong>LC separation technique<\/strong><\/th><th><strong>Sample preparation<\/strong><\/th><th><strong>Comments<\/strong><\/th><\/tr><\/thead><tbody><tr><td><strong>Textile dyes<\/strong> [8]<\/td><td><strong>RP LC with C18 column<\/strong> \u2192 water mixed with methanol\/acetonitrile as eluent system.<\/td><td>\u2212 <strong>Dye extracted with solvent(s):<\/strong><br><strong>*direct dyes<\/strong> (some natural dyes e.g., safflower; synthetic azo dyes): MeOH, EDTA, DMSO;<br><strong>*mordant dyes<\/strong> (most natural dyes, e.g., madder): HCl diluted with H2O, MeOH;<br><strong>*vat dyes<\/strong> (e.g., indigo): DMF, DMSO, pyridine.<br>\u2212 <strong>Multi-step extraction<\/strong> used, if needed.<br>\u2212 Sample solvent(s) evaporated and extract re-dissolved in mobile phase.<\/td><td>\u2212 Different dyes need different LC methods \u2192 optimisation of LC conditions!<br>\u2212 <strong>Suitable detectors:<\/strong> UV-Vis\/PDA, MS(\/MS); FLD is suitable only for some specific dyes (e.g., logwood).<br>\u2212 Identification based on <em>t<\/em><sub>R<\/sub> and spectral data of reference compounds.<\/td><\/tr><tr><td><strong>Oils, fats, waxes (incl. archaeological residues, alkyd resins, etc.)<\/strong> [7]<\/td><td><strong>RP LC with C18 column<\/strong> \u2192 isopropanol mixed with methanol, acetonitrile vs ethanol as mobile phase. <br><strong>NP LC<\/strong> is rarely applied and needs further testing.<\/td><td>\u2212 <strong>Applies for RP LC:<\/strong> sample dissolved\/ extracted with suitable solvent(s):<br><strong>*oils, waxes, alkyd resins:<\/strong> hexane, tetrahydrofuran, n-heptane<br><strong>*fats:<\/strong> tetrahydrofuran, n-heptane, hexane, chloroform\/methanol mixture.<br>\u2212 Hydrolysis, derivatisation, and\/or microwave extraction can be used (e.g., for aged samples).<br>\u2212 Sample solvent(s) evaporated and extract re-dissolved in mobile phase.<\/td><td>\u2212 Fresh and modern materials can mostly be analysed. [9]<br>\u2212 Analysis is problematic with aged and small samples \u2192 Py-GC-MS or direct MS are preferred.<br>\u2212 Mostly MS(\/MS) detector is used, UV-Vis\/PDA is also suitable.<br>\u2212 Identification and quantification are based on the analysis of TAGs in case of oils and fats.<\/td><\/tr><tr><td><strong>Protein paint binders (egg yolk, egg white, also animal glues) <\/strong>[10]<\/td><td><strong>RP LC with C18 column<\/strong> \u2192 acetonitrile mixed with water as mobile phase.<br><strong>SEC<\/strong> is used to monitor aging and changes in materials. [7]<\/td><td>\u2212 Extraction with ammonia, NaOH.<br>\u2212 Enzymatic hydrolysis (with e.g., trypsin).<br>\u2212 Drying and re-dissolving of sample in acidified water (or another suitable solvent).<br>\u2212 <strong>Derivatisation<\/strong> with o-phthalaldehyde, etc. when using FLD and phenylisothiocyanate, etc. when using UV-Vis detector [11].<\/td><td>\u2212 <strong>Suitable detectors:<\/strong> MS(\/MS), UV-Vis\/PDA, FLD.<br>\u2212 Identification and quantification based on the analysis of amino acids \u2192 database needed.<\/td><\/tr><tr><td><strong>Natural resins (colophony, dammar, shellac, etc.) <\/strong>[12]<\/td><td><strong>RP LC with C18 column<\/strong> \u2192 acetonitrile\/methanol mixed with water as mobile phase.<br><strong>NP LC<\/strong> is rarely applied and needs further testing.<br><strong>SEC <\/strong>is used to monitor degradation, changes in resinous materials.<\/td><td>Applies for RP LC:<br>\u2212 Sample dissolved\/extracted with methanol, isopropanol, ethanol, etc.<br>\u2212 An ultrasonic bath (heated) is used if needed.<\/td><td>\u2212 <strong>Suitable detectors:<\/strong> UV-Vis\/PDA, MS(\/MS).<br>\u2212 Identification based on standard\/reference data.<\/td><\/tr><tr><td><strong>Polysaccharides (wood, paper pulp, gums, etc.)<\/strong> [7]<\/td><td><strong>SEC: <\/strong>gel with controlled pore size as stationary phase, usually tetrahydrofuran as mobile phase.<br><strong>RP\/NP not suitable.<\/strong><\/td><td>Tetrahydrofuran for dissolving and eluting of sample.<\/td><td>\u2212 <strong>Suitable detectors:<\/strong> UV-Vis\/PDA, more specific detectors (e.g. refractive index detector (RID)).<br>\u2212 Identification based on molecular weight distribution.<br>\u2212 SEC suitable to monitor changes in materials (e.g. degradation of paper and wood, gums, etc.).<\/td><\/tr><tr><td><strong>Synthetic polymers (PVA, acrylics, etc.)<\/strong> [6,7]<\/td><td><strong>SEC:<\/strong> gel with controlled pore size as stationary phase, usually tetrahydrofuran as mobile phase.<\/td><td>Tetrahydrofuran for dissolving and eluting of sample.<\/td><td>\u2212 <strong>Suitable detectors:<\/strong> UV-Vis\/PDA, also more specific detectors, like refractive index detector (RID) are used.<br>\u2212 Possible to study molecular weight distribution \u2192 describes the composition, decomposition state, age, etc.<br>\u2212 Identification is based on standard\/reference data.<\/td><\/tr><\/tbody><\/table><\/figure>\n<\/div>\n\n\n\n<p><\/p>\n\n\n\n<p>As mentioned above, LC systems with different detectors give different type of information. In Fig. 2, an example of an analysis result of cochineal extract obtained with LC instrument coupled with UV-VIS\/PDA and MS detectors is presented. This means that in addition to the chromatogram (Fig. 2a) also in case of UV-VIS\/PDA, the UV-Vis spectrum (Fig. 2b) and with MS detector a mass spectrum (Fig. 2c) corresponding to a specific chromatographic peak can be obtained.<\/p>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"788\" height=\"591\" src=\"https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/6.1_figure2.jpg\" alt=\"6.1_Fig2\" class=\"wp-image-56\" title=\"6.1_figure2.jpg\" srcset=\"https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/6.1_figure2.jpg 788w, https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/6.1_figure2-300x225.jpg 300w, https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/6.1_figure2-768x576.jpg 768w\" sizes=\"auto, (max-width: 788px) 100vw, 788px\"><figcaption class=\"wp-element-caption\">Fig. 2. (a) Chromatogram obtained from cochineal extract analysis; (b) the UV-Vis spectrum and (c) MS spectrum corresponding to the most intensive chromatographic peak. The analysis of the chromatogram, UV-Vis and MS spectra confirm the presence of carminic acid with a corresponding UV-Vis absorbance near 490 nm and ion signal at <em>m\/z<\/em> 491 (obtained in negative mode).<\/figcaption><\/figure>\n\n\n\n<p class=\"has-text-align-center\"><\/p>\n\n\n<p><\/p><div class=\"accordion mb-3\">\n        <div class=\"accordion-item accordion-item--white\">\n        <h2 class=\"accordion-header\" id=\"accordion-69d951355f96f-heading\">\n            <button class=\"accordion-button collapsed\" type=\"button\" data-bs-toggle=\"collapse\" data-bs-target=\"#accordion-69d951355f96f-collapse\" aria-expanded=\"true\" aria-controls=\"accordion-69d951355f96f-collapse\"><span style=\"font-size: 16px;\"><strong>References<\/strong> (click here)<\/span><\/button>\n        <\/h2>\n        <div id=\"accordion-69d951355f96f-collapse\" class=\"accordion-collapse collapse\" aria-labelledby=\"accordion-69d951355f96f-heading\">\n            <div class=\"accordion-body\">\n<ol>\n<li>Tammekivi, E.; Vahur, S.; Keki\u0161ev, O.; van der Werf, I. D.; Toom, L.; Herodes, K.; Leito, I. Comparison of Derivatization Methods for the Quantitative Gas Chromatographic Analysis of Oils. <em>Anal. Methods<\/em>. <strong>2019<\/strong>, <em>11<\/em> (28), 3514\u20133522. https:\/\/doi.org\/10.1039\/C9AY00954J.<\/li>\n<li>Colombini, M. P.; Modugno, F. <em>Organic Mass Spectrometry in Art and Archaeology<\/em>; John Wiley &amp; Sons, Ltd.: Chichester, UK, 2009.<\/li>\n<li>Bonaduce, I.; Ribechini, E.; Modugno, F.; Colombini, M. P. Analytical Approaches Based on Gas Chromatography Mass Spectrometry (GC\/MS) to Study Organic Materials in Artworks and Archaeological Objects. <em>Top. Curr. Chem.<\/em><strong> 2016<\/strong>, <em>374<\/em> (1), 6, 1-37. https:\/\/doi.org\/10.1007\/s41061-015-0007-x.<\/li>\n<li>H\u00fcbschmann, H.-J. <em>Handbook of GC\/MS. Fundamentals and Applications<\/em>, 2nd ed.; Wiley-VCH Verlag GmbH &amp; Co. KGaA: Weinheim, Germany, 2009.<\/li>\n<li>Stuart, B. H. <em>Analytical Techniques in Materials Conservation<\/em>; John Wiley &amp; Sons, Ltd.: Chichester, UK, 2007.<\/li>\n<li>Mazzeo, R. <em>Analytical Chemistry for Cultural Heritage<\/em>; Springer International Publishing: Switzerland, 2016.<\/li>\n<li>Degano, I.; La Nasa, J. Trends in High Performance Liquid Chromatography for Cultural Heritage. <em>Top. Curr. Chem.<\/em><strong> 2016<\/strong>, <em>374<\/em> (2), 20, 1-20. https:\/\/doi.org\/10.1007\/s41061-016-0020-8.<\/li>\n<li>Peets, P.; Vahur, S.; Kruve, A.; Haljasorg, T.; Herodes, K.; Pagano, T.; Leito, I. Instrumental Techniques in the Analysis of Natural Red Textile Dyes. <em>J. Cult. Herit.<\/em> <strong>2020<\/strong>, <em>42<\/em>, 19\u201327. https:\/\/doi.org\/10.1016\/j.culher.2019.09.002.<\/li>\n<li>La Nasa, J.; Zanaboni, M.; Uldanck, D.; Degano, I.; Modugno, F.; Kutzke, H.; Tveit, E. S.; Topalova-Casadiego, B.; Colombini, M. P. Novel Application of Liquid Chromatography\/Mass Spectrometry for the Characterization of Drying Oils in Art: Elucidation on the Composition of Original Paint Materials Used by Edvard Munch (1863\u20131944). <em>Anal. Chim. Acta<\/em>. <strong>2015<\/strong>,<em> 896<\/em>, 177\u2013189. https:\/\/doi.org\/10.1016\/j.aca.2015.09.023.<\/li>\n<li>Vinciguerra, R.; De Chiaro, A.; Pucci, P.; Marino, G.; Birolo, L. Proteomic Strategies for Cultural Heritage: From Bones to Paintings. <em>Microchem. J.<\/em> <strong>2016<\/strong>, <em>126<\/em>, 341\u2013348. https:\/\/doi.org\/10.1016\/j.microc.2015.12.024.<\/li>\n<li>Prikryl, P.; Havl\u00ed\u010dkov\u00e1, L.; Pac\u00e1kov\u00e1, V.; Hradilov\u00e1, J.; \u0160tul\u00edk, K.; Hofta, P. An Evaluation of GC-MS and HPLC-FD Methods for Analysis of Protein Binders in Paintings. <em>J. Sep. Sci.<\/em> <strong>2006<\/strong>, <em>29<\/em> (17), 2653\u20132663. https:\/\/doi.org\/10.1002\/jssc.200600171.<\/li>\n<li>van der Doelen, G. A.; van den Berg, K. J.; Boon, J. J.; Shibayama, N.; de la Rie, E. R.; Genuit, W. J. L. Analysis of Fresh Triterpenoid Resins and Aged Triterpenoid Varnishes by High-Performance Liquid Chromatography\u2013Atmospheric Pressure Chemical Ionisation (Tandem) Mass Spectrometry. <em>J. Chromatogr. A.<\/em> <strong>1998<\/strong>, <em>809<\/em> (1\u20132), 21\u201337.\u00a0<a title=\"\" href=\"https:\/\/doi.org\/10.1016\/S0021-9673(98)00186-1\" data-url=\"https:\/\/doi.org\/10.1016\/S0021-9673(98)00186-1\">https:\/\/doi.org\/10.1016\/S0021-9673(98)00186-1<\/a>.<\/li>\n<\/ol>\n<p><\/p><\/div>\n        <\/div>\n        <\/div>\n    <\/div>\n","protected":false},"excerpt":{"rendered":"<p>In this section, practical aspects and analysis with GC-MS\/ FID and HPLC with different detectors are discussed, and examples are given. Analysis with GC-MS and GC-FID methods Gas chromatography (GC) is a widely used technique for the analysis of organic &#8230;<\/p>\n","protected":false},"author":151,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"inline_featured_image":false,"footnotes":""},"class_list":["post-5","page","type-page","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"https:\/\/sisu.ut.ee\/heritage-analysis\/wp-json\/wp\/v2\/pages\/5","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/sisu.ut.ee\/heritage-analysis\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/sisu.ut.ee\/heritage-analysis\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/sisu.ut.ee\/heritage-analysis\/wp-json\/wp\/v2\/users\/151"}],"replies":[{"embeddable":true,"href":"https:\/\/sisu.ut.ee\/heritage-analysis\/wp-json\/wp\/v2\/comments?post=5"}],"version-history":[{"count":30,"href":"https:\/\/sisu.ut.ee\/heritage-analysis\/wp-json\/wp\/v2\/pages\/5\/revisions"}],"predecessor-version":[{"id":1147,"href":"https:\/\/sisu.ut.ee\/heritage-analysis\/wp-json\/wp\/v2\/pages\/5\/revisions\/1147"}],"wp:attachment":[{"href":"https:\/\/sisu.ut.ee\/heritage-analysis\/wp-json\/wp\/v2\/media?parent=5"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}