{"id":3,"date":"2024-04-04T04:31:14","date_gmt":"2024-04-04T01:31:14","guid":{"rendered":"https:\/\/sisu.ut.ee\/heritage-analysis\/51-chromatography\/"},"modified":"2024-07-18T14:34:38","modified_gmt":"2024-07-18T11:34:38","slug":"51-chromatography","status":"publish","type":"page","link":"https:\/\/sisu.ut.ee\/heritage-analysis\/51-chromatography\/","title":{"rendered":"5.1. Chromatography"},"content":{"rendered":"<div style=\"height:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/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<p>Chromatography is one of the most powerful techniques in analytical chemistry, which is widely used for the analysis of different cultural heritage materials. In this course, only the most general theoretical aspects of chromatography will be presented.<\/p>\n\n\n\n<p>Chromatography is a technique to separate individual components (typically organic compounds)\u00a0 of a mixture based on their distribution or adsorption characteristics. The separation is achieved in a two-phase system: the<strong> mobile<\/strong> and <strong>stationary phase<\/strong>. The mobile phase flows through the stationary phase and carries components of the mixture with it. The components can move between the phases \u2013 from the mobile phase to the stationary phase and back to the mobile phase. The compounds that reside preferably in the mobile phase elute from the chromatographic system earlier and compounds that stay preferably in the stationary phase elute later. Chromatographic separation depends on different properties of the compounds, e.g. polarity (liquid and gas chromatography), boiling point\u00a0(gas chromatography) and ionic charge (liquid chromatography).\u00a0[1]<\/p>\n\n\n\n<p>In an analytical chromatographic system, the separated compounds are detected and represented as <strong>peaks <\/strong>on a<strong> chromatogram<\/strong>. A chromatogram starts (time = 0 min) at the moment when the sample is introduced into the chromatographic system and lasts until all the compounds are assumed to be eluted from the system. Each peak in a chromatogram is characterised by its<strong> retention time (<em>t<\/em><sub>R<\/sub>)<\/strong>, <strong>peak height <\/strong>and<strong>\u00a0peak area<\/strong>\u00a0(see Fig.1). Retention time is the time it takes for a compound to pass through the system under the set chromatographic conditions.<\/p>\n\n\n\n<p>In chromatography, identification (<strong>qualitative analysis<\/strong>) of compounds is done based on (1) retention time and possibly (2) additional information given by the detector (see below). For the <strong>quantitative analysis<\/strong>, the area (or sometimes height) of the peak is used \u2013 the higher it is, the higher the compound\u2019s content in the sample. For qualitative as well as quantitative analysis, the analysis of standard compounds is usually required. A standard compound (aka a standard substance, standard material, or a chemical standard) is the same compound as the analyte and is often obtained from a supplier with a known concentration and purity. [1]<span style=\"font-size: 11.6667px;\">\u00a0<\/span><\/p>\n<\/div>\n\n\n\n<p><\/p>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"664\" height=\"354\" src=\"https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/fig1_chrom.png\" alt=\"5.1 Chrom\" class=\"wp-image-43\" srcset=\"https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/fig1_chrom.png 664w, https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/fig1_chrom-300x160.png 300w\" sizes=\"auto, (max-width: 664px) 100vw, 664px\"><figcaption class=\"wp-element-caption\">Fig. 1.  Gas chromatogram of a mixture of organic compounds (different solvents).<\/figcaption><\/figure>\n\n\n\n<p><\/p>\n\n\n\n<p>In the following video the general aspects of chromatography are explained.<\/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>A short overview of gas and liquid chromatography is given below.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\"><span style=\"color: #b22222;\">1. Gas chromatography (GC)<\/span><\/h2>\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>Gas chromatography (GC) is considered to be the most <strong>efficient<\/strong>, <strong>sensitive<\/strong> and <strong>reliable<\/strong> method for the analysis of mixtures <strong>volatile compounds<\/strong>. GC is a type of chromatography where the mobile phase is a gas (mainly He, N<sub>2<\/sub> or H<sub>2<\/sub>). As the mobile phase carries the components through the stationary phase, it is also called the carrier gas. GC enables the <strong>qualitative<\/strong> and <strong>quantitative analysis<\/strong> of\u00a0compounds that are <strong>volatile<\/strong>, i.e. have <strong>boiling points below 500\u00b0C<\/strong>\u00a0and<strong>\u00a0<\/strong>are <strong>thermally stable<\/strong>. The separation in the GC system is mostly based on the difference in boiling points of the components. However, the components\u2019 chemical affinity for the stationary phase is also an important factor. [2,3]<\/p>\n\n\n\n<p>The sample can be introduced as <strong>liquid<\/strong> or <strong>gas<\/strong> into the GC. A solid or liquid sample can be dissolved in a solvent, such as diethyl ether, toluene, dichloromethane, etc. Water is usually not suitable as it can damage the column. It is also possible to directly inject and analyse gases or the vapour phase above the sample (<strong>headspace method<\/strong>). In the <strong>headspace method<\/strong>, a liquid or a solid containing the volatile analyte(s) is kept in a closed system (e.g. a vial) until an equilibrium between the two phases (gas vs liquid or gas vs solid) is <span lang=\"EN-US\">established<\/span>. Then the vapour phase is analysed by injecting it into the GC system. [2]<\/p>\n\n\n\n<p>Only about 10\u201220% of common organic compounds can be analysed by GC (boiling point below 500\u00b0C). Some less volatile compounds, can be made more volatile by <span lang=\"EN-US\"> <strong>derivatisation<\/strong> (e.g.,<strong> methylation, silylation<\/strong>)<\/span>. \u00a0<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 a process where a compound is chemically modified before the analysis. Derivatisation makes it possible to analyse many compounds that would otherwise not be GC amendable. Sometimes derivatisation is used to <span lang=\"EN-US\">improve the chromatographic separation.<\/span> Some examples of compounds requiring derivatisation include oils, polymers, sterols, waxes, and drugs.\u00a0[2,4]\u00a0<\/p>\n<\/div>\n\n\n\n<p class=\"has-text-align-center\"><\/p>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full is-resized\"><img loading=\"lazy\" decoding=\"async\" width=\"1198\" height=\"748\" src=\"https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/GC_Fig2.png\" alt=\"\" class=\"wp-image-871\" style=\"width:841px;height:auto\" srcset=\"https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/GC_Fig2.png 1198w, https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/GC_Fig2-300x187.png 300w, https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/GC_Fig2-1024x639.png 1024w, https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/GC_Fig2-768x480.png 768w\" sizes=\"auto, (max-width: 1198px) 100vw, 1198px\"><figcaption class=\"wp-element-caption\">Fig. 2. Overall scheme of a GC system.<\/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>The overall scheme for GC system is presented in Fig. 2.\u00a0For the analysis with GC, the sample (typically as a solution) is injected with a micro syringe manually or by the autosampler into the <strong>sample inlet<\/strong>. The inlet is a heated chamber where the compounds in the sample are evaporated and directed into a continuous flow of carrier gas.\u00a0The <strong>carrier gas<\/strong> (mobile phase), together with the gaseous sample, is directed through the <strong>column\u00a0<\/strong><span lang=\"EN-US\">(the tube which holds the stationary phase)<\/span>, where the chromatographic separation takes place.\u00a0<span lang=\"EN-US\">As explained above, those compounds that spend a smaller fraction of time in the mobile phase have longer retention time (toluene in Fig. 1) and compounds that spend a bigger fraction of time in the mobile phase have shorter retention times (ethanol in Fig. 1).<\/span> [2,3]<\/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>There are two main types of columns \u2013 <b><span lang=\"EN-US\">capillary <\/span><\/b><span lang=\"EN-US\">and <b>packed columns <\/b>(the latter are rarely used and will not be discussed in this course)<\/span>. In a <strong>capillary column<\/strong>, the stationary phase is applied as a thin layer on the internal surface of the column. The column itself is a very long <span lang=\"EN-US\">(typically 25\u201230 m but can be even 100 m)<\/span> and thin tube (inner diameter of 0.1-0.5 mm) made of fused silica. The <strong>stationary phase<\/strong> is a polymeric liquid (mainly polysiloxanes, polyethylene glycols), where\u00a0functional groups have often been added to vary the polarity of the stationary phase.[2]\u00a0In cultural heritage, mostly capillary columns are used. For example, a lot of analysis of paint binders <span lang=\"EN-US\">(oil and protein-based)<\/span>, waxes, natural resins, etc., are carried out with non-polar columns (for example, colums having a<span lang=\"EN-US\"> 5% diphenyl-95% dimethyl-polysiloxane stationary phase). <span style=\"font-size: 11.6667px;\">\u00a0<\/span><\/span>[4-6]<\/p>\n\n\n\n<p>The GC column is located in the <strong>column thermostat<\/strong> (also known as the <strong>oven<\/strong>), which enables accurate temperature control of the chromatographic process.\u00a0<span lang=\"EN-US\">Typically, temperatures in the range of 40 to 300<\/span><span lang=\"EN-US\">\u00b0<\/span><span lang=\"EN-US\">C are used for GC column. At lower temperatures, the retention times of compounds are longer and at higher temperatures, they are shorter.<\/span>\u00a0In most methods<span lang=\"EN-US\">, the temperature in the oven is increased during chromatographic analysis (often called the <b>run<\/b>).\u00a0<\/span><span lang=\"EN-US\">The \u201cschedule\u201d of the temperature change<\/span> is called a <strong>temperature program<\/strong>.\u00a0After the separated compounds exit the column, they are detected in a detector. [2]<\/p>\n\n\n\n<p><\/p>\n<\/div>\n<\/div>\n\n\n\n<p>The following two main <strong>detectors<\/strong>\u00a0are most often used for GC in the field of cultural heritage:<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li><p><em><strong>Mass spectrometric (MS) detector\u00a0<\/strong><\/em>is the most powerful and versatile GC detector. In the case of MS, the separated compounds are ionised (in <strong>Electron Ionisation (EI)<\/strong> or <strong>Chemical Ionisation (CI) sources<\/strong>, see section <a title=\"\" data-url=\"https:\/\/sisu.ut.ee\/heritage-analysis\/52-mass-spectrometry\" href=\"https:\/\/sisu.ut.ee\/heritage-analysis\/52-mass-spectrometry\" target=\"_blank\" rel=\"noopener\">5.2 Mass Spectrometry<\/a><span lang=\"EN-US\"> for more information<\/span>) and sorted by their mass-to-charge (<em>m\/z<\/em>) ratio. The mass spectrum\u00a0<span lang=\"EN-US\">e<\/span>nables identification of the separated compound by a) searching database of mass spectra or<span style=\"font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> by <\/span><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> b) analysing the standard substance of the compound. With the MS detector, it is possible to detect practically all compounds that fit the volatility criteria. The MS detector is also the most used GC detector for the analysis of cultural heritage samples.\u00a0[5,6]<\/span><\/p><\/li>\n\n\n\n<li><p><strong style=\"background-color: rgb(255, 255, 255); font-size: revert; text-align: var(--bs-body-text-align);\"><em>Flame ionisation detector (FID).\u00a0<\/em><\/strong><span style=\"background-color: rgb(255, 255, 255); font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">In FID, the compounds are burned and ionised in the hydrogen flame. FID detects only compounds, that contain at least one C-H or C-C bond, so it is suitable for the analysis of almost all\u00a0organic compounds, including components of paint binders, varnishes,\u00a0etc. FID is robust, highly sensitive and often used for quantitative analysis. For the identification, the standard substance of the compound with the same fixed chromatographic conditions must be analysed and retention times are compared. However, such identification is suitable only for relatively simple samples.<\/span><span style=\"background-color: rgb(255, 255, 255); font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align); font-size: 11.6667px;\">\u00a0<\/span><span style=\"background-color: rgb(255, 255, 255); font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">[2]<\/span><span style=\"background-color: rgb(255, 255, 255); font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> <\/span><\/p><\/li>\n<\/ul>\n\n\n\n<h2 class=\"wp-block-heading\"><span style=\"color: #b22222;\"><em>Sample introduction techniques<\/em><\/span><\/h2>\n\n\n\n<p>Two additional <span lang=\"EN-US\">sample introduction techniques<\/span> are widely used in cultural heritage analysis with a GC-MS system to enhance the analysis:\u00a0<strong>pyrolysis (Py-)GC-MS<\/strong> and <strong>solid phase microextraction (SPME) GC-MS<\/strong>.<\/p>\n\n\n\n<p>In <strong>Py-GC-MS<\/strong> the sample is heated up to 600-1000 \u00b0C in a pyrolizer that is connected to the injector port of a GC. This enables the analysis of compounds that do not evaporate <span lang=\"EN-US\">under usual GC sample inlet conditions (e.g., polymers)<\/span>. <span lang=\"EN-US\">In Py-GC-MS, t<\/span>he sample is decomposed, emitting smaller volatile molecules \u2013 pyrolysis products. Their analysis gives information about the initial sample. Py-GC-MS is especially useful for the analysis of various synthetic and\/or polymeric materials. [5,6]<\/p>\n\n\n\n<p><strong>SPME<\/strong> is a modified headspace method that is used with GC-MS to enable the analysis of volatile or semi-volatile organic compounds (VOCs and SVOCs, respectively), usually without sample alteration. The SPME is an equilibrium extraction technique based on the partition of the analyte(s) between <span lang=\"EN-US\">the vapour phase above the sample and a polymer covered fused silica fibre. <\/span> After\u00a0adsorption <span lang=\"EN-US\">of sample components on the fibre<\/span>, the fibre is inserted into the GC inlet, where the analytes are desorbed and directed to the column for separation. SPME GC-MS can provide information about the components of the sample and about their degradation products. [5,6]<\/p>\n\n\n\n<p><span lang=\"EN-US\">Despite different sample introduction methods and derivatisation approaches available, there is still a very large number of compounds that cannot be analysed with a GC. Then liquid chromatography can be of help.<\/span><\/p>\n\n\n\n<h2 class=\"wp-block-heading\"><span style=\"color: #b22222;\">2. Liquid chromatography (LC)<\/span><\/h2>\n\n\n\n<p>Liquid-chromatography (mostly <strong>high-performance liquid chromatography \u2013 HPLC<\/strong> or <strong>ultra-high-pressure liquid chromatography \u2013 UHPLC<\/strong>) is nowadays the most widely used separation technique. <span lang=\"EN-US\">The discussion below applies to both HPLC as well as UHPLC. <\/span>The simplified overall scheme of a HPLC is presented in Fig. 3.<\/p>\n\n\n\n<p class=\"has-text-align-center\"><\/p>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full is-resized\"><img loading=\"lazy\" decoding=\"async\" width=\"1055\" height=\"736\" src=\"https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/LC_Fig3.png\" alt=\"\" class=\"wp-image-874\" style=\"width:769px;height:auto\" srcset=\"https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/LC_Fig3.png 1055w, https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/LC_Fig3-300x209.png 300w, https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/LC_Fig3-1024x714.png 1024w, https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/LC_Fig3-768x536.png 768w\" sizes=\"auto, (max-width: 1055px) 100vw, 1055px\"><figcaption class=\"wp-element-caption\">Fig. 3. Overall scheme of an HPLC system.<\/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>The logic of LC is similar to GC \u2013 the compounds are carried through a stationary phase and separated according to their different properties. In LC, the <strong>mobile phase is a liquid <\/strong>\u2013<strong> eluent <\/strong>\u2013\u00a0that is pumped through the stationary phase. Compared to GC, LC depends on more different <span lang=\"EN-US\">interactions<\/span>: a) compound vs\u00a0eluent; b) compound vs\u00a0stationary phase;\u00a0 c) mobile phase vs\u00a0stationary phase and d) mutual interaction of the mobile phase molecules.\u00a0<span lang=\"EN-US\">Optimisation of different parameters, like mobile phase composition and pH, stationary phase type, eluent additives, etc. is very important for achieving good separation in LC and the suitable set of parameters depends on the type of material to be analysed. [1]<\/span><\/p>\n\n\n\n<p>One of the key aspects of\u00a0LC analysis is the optimisation of the mobile phase composition. This includes the selection of <strong>solvents<\/strong> <span lang=\"EN-US\">and pH (<strong>buffers<\/strong>) and their concentration<\/span>. The <strong>buffer<\/strong>\u00a0<strong>solution<\/strong> is a solution that helps to keep the pH of the mobile phase constant. This is important if\u00a0the analysed compounds have acidic or basic properties.\u00a0<span lang=\"EN-US\">If this is the case, then varying the buffer pH allows to change the polarity of the compounds and thereby their retention time and separation from each other.<\/span> [7]<\/p>\n\n\n\n<p><span lang=\"EN-US\">In simpler cases, LC separation is carried out isocratically \u2013 eluent composition does not change during the analysis.\u00a0<\/span>Nowadays, most LC instruments allow <strong>gradient elution<\/strong>. In this case,\u00a0eluent composition changes during the chromatographic run. This approach help\u00a0to improve the separation of components with similar properties and reduce the analysis time <span lang=\"EN-US\">of compounds with very different properties<\/span>.<span style=\"font-size: 11.6667px;\">\u00a0<\/span>[7]<\/p>\n\n\n\n<p>Other parameters that can be chosen are, for example, the <span lang=\"EN-US\">stationary phase type <\/span>particle diameter, the diameter and length of the column, the flow-rate of the eluent, etc.\u00a0<\/p>\n\n\n\n<p>After the separation in the column, the compounds are directed into the <strong>detector<\/strong>. Many different types of detectors can be used with LC depending on the properties of the components and information that is needed. Some of the most used detectors for LC are UV-Vis\/PDA, fluorescence, and mass-spectrometric detectors.<\/p>\n<\/div>\n\n\n\n<p><\/p>\n\n\n\n<h2 class=\"wp-block-heading\"><span style=\"color: #b22222;\"><em>Detectors for LC<\/em><\/span><\/h2>\n\n\n\n<ul class=\"wp-block-list\">\n<li> <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);\"> is probably the most common LC detector. A\u00a0<\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">photodiode array detector (PDA)<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> enables recording of the whole UV-Vis <\/span><span lang=\"EN-US\" style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">absorption\u00a0<\/span><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">spectrum from each <\/span><span lang=\"EN-US\" style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">chromatographic\u00a0<\/span><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">peak and\u00a0thereby <\/span><span lang=\"EN-US\" style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">provides some information<\/span><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> for component identification. For compound identification, standard material chromatograms obtained under the same conditions are needed. This detector typically embraces both the\u00a0<\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">visible range (400-780 nm)<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> \u2013 useful for detecting coloured compounds\u00a0\u2013 and the <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">UV range (190-400 nm)<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> \u2013\u00a0useful for detecting\u00a0\u00a0compounds containing double bonds (C=C), nitro groups (-NO<\/span><sub style=\"font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">2<\/sub><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">), halogens (-Br, -I) etc. UV-Vis\/PDA is not suitable for detecting compounds containing only single bonds and no heavy atoms, such as simple alcohols, ethers, etc. It is also not suitable for\u00a0trace analysis: only compounds with considerable concentration can be detected. In conservation, LC-UV-Vis\u00a0is used to analyse dyes,\u00a0lipids, binders, polysaccharides, etc. [6,8]<\/span> <\/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);\">Mass spectrometer (MS) <\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">as a detector is the most powerful detector available in LC. Mass spectrometry is a very sensitive technique (very low concentrations can be detected) and enables identifying compounds according to the <\/span><strong style=\"font-size: revert; text-align: var(--bs-body-text-align);\">mass-to-charge ratio (<em>m\/z<\/em>)<\/strong><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> of the ion corresponding to the analysed molecule,\u00a0as well as the\u00a0<\/span><em style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">m\/z<\/em><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\"> ratios of fragments formed from it. Mass spectrometry is more thoroughly explained in section <\/span><a href=\"https:\/\/sisu.ut.ee\/heritage-analysis\/52-mass-spectrometry\" target=\"_blank\" rel=\"noreferrer noopener\">5.2. Mass Spectrometry<\/a><span style=\"font-size: revert; font-weight: var(--bs-body-font-weight); text-align: var(--bs-body-text-align);\">. [6,8]<\/span> <\/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><p><strong>Fluorescence detector (FLD)<\/strong> <span lang=\"EN-US\">is suitable for detecting fluorescent compounds<\/span>. Fluorescence is not a universal phenomenon: most compounds do not fluoresce. <span lang=\"EN-US\">But f<\/span>luorescence is\u00a0observed for many dyes and some pigments, as well as\u00a0polycyclic aromatic hydrocarbons (PAHs) that can be found in bitumen, tars. Thus, fluorescence is a characteristic property of <span lang=\"EN-US\">limited number of materials a<\/span>nd makes their identification easier. The sensitivity of the detector varies widely, depending on\u00a0the detected compound(s). For compounds with intense fluorescence (e.g. the ones mentioned above), the sensitivity can be quite high.\u00a0FLD has specific niche applications and is much less used than UV-Vis\/PDA. [6,8]<\/p><\/li>\n<\/ul>\n\n\n\n<h2 class=\"wp-block-heading\"><span style=\"color: #b22222;\"><em>Types of LC<\/em><\/span><\/h2>\n\n\n\n<p>There are different possibilities for how to separate compounds in LC: by their polarity (<strong>reverse phase <\/strong>and<strong> normal phase chromatography<\/strong>), size (<strong>size exclusion chromatography<\/strong>) and <span lang=\"EN-US\">ion-ion interaction (<\/span><strong>ion chromatography<\/strong>). Each of these is a separate technique in the domain of LC.\u00a0These techniques are compared in Table 1.<\/p>\n\n\n\n<div class=\"wp-block-columns is-layout-flex wp-container-core-columns-is-layout-9d6595d7 wp-block-columns-is-layout-flex\">\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:100%\">\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>\u00a0Table 1. Comparison of liquid chromatographic techniques [6,7].<\/p>\n\n\n\n<div class=\"wp-block-group is-layout-constrained wp-block-group-is-layout-constrained\">\n<figure class=\"wp-block-table aligncenter has-small-font-size\"><table class=\"table table-hover\"><thead><tr><th class=\"has-text-align-center\" data-align=\"center\"><strong>Type of LC<\/strong><\/th><th class=\"has-text-align-center\" data-align=\"center\"><strong>Basic principle<\/strong><\/th><th class=\"has-text-align-center\" data-align=\"center\"><strong>Analysed compounds<\/strong><\/th><th class=\"has-text-align-center\" data-align=\"center\"><strong>Mobile phases (eluent)<\/strong><\/th><th class=\"has-text-align-center\" data-align=\"center\"><strong>Stationary phases<\/strong><\/th><\/tr><\/thead><tbody><tr><td class=\"has-text-align-center\" data-align=\"center\"><strong>Reversed Phase (RP)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\">The stationary phase is less polar than mobile phase \u2192 polar compounds are less retained (short retention time).<\/td><td class=\"has-text-align-center\" data-align=\"center\">Moderately polar (dyes or protein-based binders like animal glue, egg, casein) to polar compounds (resins, etc.)<\/td><td class=\"has-text-align-center\" data-align=\"center\">Polar solvent mixtures (water, methanol, acetonitrile, etc.)<\/td><td class=\"has-text-align-center\" data-align=\"center\">Silica gel particles modified with octadecyl (C18) or octyl (C8) groups, etc.<\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\"><strong>Normal Phase (NP)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\">The stationary phase is more polar than the mobile phase \u2192 polar compounds are more retained (long retention time).<\/td><td class=\"has-text-align-center\" data-align=\"center\">Rather non-polar (lipids, waxes, tars)<\/td><td class=\"has-text-align-center\" data-align=\"center\">Non-polar solvent mixtures (hexane, ethyl acetate, toluene, acetone, dichloromethane, etc.)<\/td><td class=\"has-text-align-center\" data-align=\"center\">Silica gel (bare) or modified with polar groups.<\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\"><strong>Size exclusion chromatography (SEC)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\">Separation is based on the size of the analyte. Bigger molecules have shorter retention times.<\/td><td class=\"has-text-align-center\" data-align=\"center\">Proteins, water-soluble polymers, oligo- and polysaccharides, etc.<\/td><td class=\"has-text-align-center\" data-align=\"center\">Solvent mixtures (tetrahydrofuran, chloroform, water, etc.)<\/td><td class=\"has-text-align-center\" data-align=\"center\">Silica gel or polymeric material with different pore sizes.<\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\"><strong>Ion chromatography<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\">Compounds are separated due to the ion exchange process with the stationary phase. Analytes with higher charge have longer retention times.<\/td><td class=\"has-text-align-center\" data-align=\"center\">Ionisable compounds, such as proteins, amino acids, etc.<\/td><td class=\"has-text-align-center\" data-align=\"center\">Aqueous buffers<\/td><td class=\"has-text-align-center\" data-align=\"center\">Polymer beads (styrene-divinylbenzene copolymer) modified with ionic functional groups (carboxylic acid, diethylaminopropyl, etc.)<\/td><\/tr><\/tbody><\/table><\/figure>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\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-69d96ce3dba42-heading\">\n            <button class=\"accordion-button collapsed\" type=\"button\" data-bs-toggle=\"collapse\" data-bs-target=\"#accordion-69d96ce3dba42-collapse\" aria-expanded=\"true\" aria-controls=\"accordion-69d96ce3dba42-collapse\"><span style=\"font-size: 16px;\"><strong>References<\/strong> (click here)<\/span><\/button>\n        <\/h2>\n        <div id=\"accordion-69d96ce3dba42-collapse\" class=\"accordion-collapse collapse\" aria-labelledby=\"accordion-69d96ce3dba42-heading\">\n            <div class=\"accordion-body\">\n<ol>\n<li>Heftmann, E. Chromatography. <em>Fundamentals and applications of chromatography and related differential migration methods \u2013 Part A: Fundamentals and techniques,<\/em> 6th ed.; Elsevier B. V.: Amsterdam, the Netherlands, 2004.<\/li>\n<li>H\u00fcbschmann, H.-J. <em>Handbook of GC\/MS: Fundamentals and Applications<\/em>, 2nd, Completely Revised and Updated Edition; WILEY-VCH Verlag GmbH &amp; Co. KGaA: Weinheim, Germany, 2009.<\/li>\n<li>McNair, H., M.; Miller, J. M. <em>Basic Gas Chromatography<\/em>, 2nd ed.; John Wiley &amp; Sons, Inc.: Hoboken, NJ, USA, 2009.<\/li>\n<li>Tammekivi, E. <em>Derivatization and Quantitative Gas-Chromatographic Analysis of Oils<\/em>, PhD thesis; University of Tartu Press: Tartu, Estonia, 2021.<\/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>Mazzeo, R. <em>Analytical Chemistry for Cultural Heritage<\/em>; Springer International Publishing: Switzerland, 2016.<\/li>\n<li>Niessen, W., M., A. <em>Liquid Chromatography-Mass Spectrometry<\/em>, 3rd ed.; CRC Press: USA, 2006.<\/li>\n<li>Peets, P. <em>Development of Instrumental Methods for the Analysis of Textile Fibres and Dyes<\/em>, PhD thesis; University of Tartu Press: Tartu, Estonia, 2020.<\/li>\n<\/ol>\n<p><\/p><\/div>\n        <\/div>\n        <\/div>\n    <\/div>\n\n\n\n<p>The slides used in the video can be downloaded from\u00a0here:<\/p>\n\n\n\n<div class=\"wp-block-group attached-files-group is-layout-constrained wp-block-group-is-layout-constrained\">\n<div class=\"wp-block-file\"><a id=\"wp-block-file--media-cbb05f2c-4633-403f-95b8-ddfa2f2aeb63\" href=\"https:\/\/sisu.ut.ee\/wp-content\/uploads\/sites\/285\/general_aspects_of_chromatography.pdf\" target=\"_blank\" rel=\"noreferrer noopener\">General_aspects_of_chromatography.pdf<\/a><\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>Chromatography is one of the most powerful techniques in analytical chemistry, which is widely used for the analysis of different cultural heritage materials. In this course, only the most general theoretical aspects of chromatography will be presented. Chromatography is a &#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-3","page","type-page","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"https:\/\/sisu.ut.ee\/heritage-analysis\/wp-json\/wp\/v2\/pages\/3","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=3"}],"version-history":[{"count":31,"href":"https:\/\/sisu.ut.ee\/heritage-analysis\/wp-json\/wp\/v2\/pages\/3\/revisions"}],"predecessor-version":[{"id":1149,"href":"https:\/\/sisu.ut.ee\/heritage-analysis\/wp-json\/wp\/v2\/pages\/3\/revisions\/1149"}],"wp:attachment":[{"href":"https:\/\/sisu.ut.ee\/heritage-analysis\/wp-json\/wp\/v2\/media?parent=3"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}