When metal ions have been introduced into a reverse-phase column, the organicsolvents listed above are often ineffective. In those cases, the following mixturemay be useful: 0.05 M EDTAWater flush.
Relatively insensitive to flow fluctuations but sensitive totemperature fluctuations; non-destructive, cannot be used withgradient elution; solvents must be degassed to avoid bubbleformations; laser-based RI detectors offer highersensitivity.
Used to introduce chromophores into alcohols and amines, usingpyridine as the solvent; with silica gel as the stationaryphase, relatively low-viscosity, low-polarity solvents can beused for detection of digitalis glycosides by HPLC followingderivatization with p-nitrobenzoyl chloride.
Charged polar solutes (acid or base) can be efficiently extracted from polar media, including water and less polar solvents, using a specific mode of extraction known as ion-exchange extraction (IX) [5, 38]. In this case, the isolation mechanism is based on the high-energy electrostatic interaction between the charged functional groups of analytes and sorbent. Thus, the sorbent selection depends on the analyte charge. The cation exchange (CX) column extracts basic analytes (primary, secondary, tertiary, and quaternary amines). In contrast, the anion exchange (AX) column was used to isolate the acidic analytes (carboxylic acid, sulphonic acid, and phosphates). According to the ionic group bonded to the surface, AX and CX can be classified into weak and strong ion exchangers. Strong cation exchangers (SCX) involve a strong acidic functional group, such as an ionized sulfonic acid over the pH range. Weak cation exchange resin (WCX) functionalized with a negatively charged group at high pH and changed to neutral at low pH, like carboxylic acids. Strong anion exchangers (SAX) consist of fully ionized groups, such as quaternary ammonium groups, over the entire pH range. Weak anion exchangers (WAX) have primary, secondary, or tertiary amine moieties ionized at low pH but neutral at high pH (Table 2).
Mixed-mode SPE has become very popular in the last decade, exhibiting two or more mode interaction mechanisms such as hydrophobic and ion-exchange functional groups attached to the surface [3, 5]. A hydrophobic group can range from short-chain (i.e., C2 group with a high selectivity) to highly retentive (such as a C18 group). The IX functionalities can be CX, AX groups, or both in one sorbent. The mixed-mode approach is preferred due to the reproducible ease of binding a single functional group to the silica surface. In addition, different ratios of single active group sorbents can be mixed if other retention properties are necessary. The development of mixed-mode sorbents can provide clean extracts from highly complex interference. The eluent should include non-polar solvents with appropriate buffers, acid, or bases to elute analytes retained in the sorbent by hydrophobic and IX retentive interaction mechanisms.
The pH can be one of the most critical factors in controlling the retention and elution of the analyte. Best recoveries are obtained when the sample pH condition provides the analyte in the optimum state for interacting with the sorbent material [53]. The pH can be a source of many problems with different SPE retention mechanisms. In samples loaded in NP SPE, the pH is not usually an issue in the interactions because the elution solvents must be typically non-polar. In the case of the RP mechanism, there is no needs to adjust the sample pH if the analytes are neutral compounds, and retention strengthens under RP conditions. However, the pH of charged ionizable compounds should be adjusted to 2 units above or below the analyte pKa according to the charged group (basic or acidic compounds). So the solute becomes in a neutral state (uncharged compounds), as shown in Fig. 10. Neutralizing base exists at least two pH units above the analyte pKa.
The values in the table below except as noted have been extracted from online and hardbound compilations . Values for relative polarity, eluant strength, threshold limits and vapor pressure have been extracted from: Christian Reichardt, Solvents and Solvent Effects in Organic Chemistry, Wiley-VCH Publishers, 3rd ed., 2003. For Spectra of Solvents, jump to the bottom of this p For an Organic Chemistry Directory, see: .
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The tables below were posted (10/23/98) and revised (07/28/09) and updated (04/10/10) by Steve Murov, Professor Emeritus of Chemistry.
Separation of compounds is based on the competition of the solute and the mobile phase for binding sites on the stationary phase.[3] For instance, if normal-phase silica gel is used as the stationary phase, it can be considered polar. Given two compounds that differ in polarity, the more polar compound has a stronger interaction with the silica and is, therefore, better able to displace the mobile phase from the available binding sites. As a consequence, the less polar compound moves higher up the plate (resulting in a higher Rf value).[6] If the mobile phase is changed to a more polar solvent or mixture of solvents, it becomes better at binding to the polar plate and therefore displacing solutes from it, so all compounds on the TLC plate will move higher up the plate. It is commonly said that "strong" solvents (eluents) push the analyzed compounds up the plate, whereas "weak" eluents barely move them. The order of strength/weakness depends on the coating (stationary phase) of the TLC plate. For silica gel-coated TLC plates, the eluent strength increases in the following order: perfluoroalkane (weakest), hexane, pentane, carbon tetrachloride, benzene/toluene, dichloromethane, diethyl ether, ethyl acetate, acetonitrile, acetone, 2-propanol/n-butanol, water, methanol, triethylamine, acetic acid, formic acid (strongest). For C18-coated plates the order is reverse. In other words, when the stationary phase is polar and the mobile phase is nonpolar, the method is normal-phase as opposed to reverse-phase. This means that if a mixture of ethyl acetate and hexane as the mobile phase is used, adding more ethyl acetate results in higher Rf values for all compounds on the TLC plate. Changing the polarity of the mobile phase will normally not result in reversed order of running of the compounds on the TLC plate. An eluotropic series can be used as a guide in selecting a mobile phase. If a reversed order of running of the compounds is desired, an apolar stationary phase should be used, such as C18-functionalized silica.
The following table gives the expected carbon-13 (13C) chemical shifts, in ppm relative to tetramethylsilane, for various useful NMR solvents. In some solvents, slight changes can occur with change of concentration (Refs. 2-3). Column definitions for the table are as follows.
Several parameters which can affect the biphasic system were analyzed: contact time, volume of solvent, volume ratio, type of organic solvent, biomass amount and concentration of solvents, to extract the highest amount of lipids from microalgae. The results were optimized and up to 83.5% of lipid recovery yield and 94.6% of enhancement was successfully achieved. The results obtain from GC-FID were similar to the analysis of triglyceride lipid standard.
Microalgae consists of high lipid content and is very potential as a sustainable feedstock for commercial biofuel production. The lipid content in microalgae constitutes of more than 50% of the total composition in the biomass [1, 2]. They are listed as a third-generation of biomass which can convert carbon dioxide into lipids and biofuels through transesterification. The productivity of microalgae oil in liters per hectare of land is higher compared to palm oil and sunflower oil by more than 16-fold [3, 4]. Furthermore, microalgae can grow in abundance and emerge as a potential source for lipid extraction for biofuel generation to replace petroleum fuel [5]. Lipids are a class of organic compounds which comprises of fatty acids and glycerol in its natural form, commonly known as triglyceride. The microalgae cell contains various types of lipid, however, the lipid favored for biofuel production are neutral lipids. Phospholipids and glycolipids are polar lipids which belongs to the category of structural lipids mainly used for construction of cell wall membranes, such as being integrated with fiber and polysaccharide in the raw molecular compound in the cell wall membrane [6]. The extraction technique for lipid extraction commonly involves the use of organic solvents to dissolve lipids and further purification of the lipid content by evaporating the solvents [7]. The lipids from microalgae could be processed to produce cosmetic products, biofuel, nutraceuticals, and synthetic polymer [2, 8,9,10].
The conventional solvent extraction techniques using alcohol and organic solvents by Folch method or Bligh and Dyer method is a common practice for lipid extraction in a laboratory scale [11, 12]. The alcohol solvent is prone to attracting polar compounds as it contains hydroxyl group molecules (OH), while organic solvent is categorized as non-polar solvent which can dissolve lipids in the solution. The molecular structure of every alcohol contains similar carbon chain with non-polar characteristic due to the carbon and hydrogen both consisting of low electronegativity difference, and OH group with polar properties attached at the tail of the chains [13, 14]. Thus, alcohol has been categorized as a polar group of solvent due to the presence of hydroxyl functional group in the chains. Typical solvent extraction uses different type of solvents in the immiscible phase, where the distribution of ions and particle may occur according to the compatibility of the types of solvent to attract or release ions in the solution [15]. The conventional alcohol and organic solvent extraction does not create a significant cell disruption mechanism for the microalgae biomass. Pre-treatment processes such as mechanical or chemical method are needed to induce the cell wall membrane disruption before the lipid compounds may be released and readily dissolve into the organic solvent [16].
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