Voltammetry comprises a group of electroanalytical methods in which information about the analyte is derived from the measurement of current as a function of applied potential. It is based upon the measurement of a current that develops in an electrochemical cell under conditions of complete polarization. The potential of the polarizable working electrode serves as a driving force for the electrochemical reaction. The working electrode varies as the activity of the analyte changes. It is the electrode at which the analyte is oxidized or reduced.
The reference electrode provide a constant potential that does not change during the potential measurement (potential is known). The counter electrode is the electrode coupled to the working electrode but plays no part in determining the magnitude of the potential being measured.
The resulting current is known as “faradaic current” which obeys Faraday’s Law. The two factors that governs the current are the mass transport and charge transfer. Mass transfer is the rate of movement from the bulk of the solution to the electrode surface. Charge transfer is the rate of transfer from electrode to the solution species and vice versa.
Stripping involves deposition of the analyte in microelectrode. It involves electrodeposition, equilibration and stripping step. After time, the electrolysis is discontinued, stirring is stopped, and the deposited analyte is determined by voltammetric procedures. Stirring helps deposition of the analyte on the electrode. In anodic stripping, the electrode behaves as cathode during deposition step and an anode during stripping step, with the analyte being oxidized back to its original form. Cathodic stripping on the other hand, the electrode behaves as an anode during the deposition step and a cathode during stripping.
Theoretically, deposition is allowed to occur during a carefully measured period. The electrolysis period is determined by the sensitivity of the method ultimately employed for the completion of the analysis. The unknown sample was analyzed using stripping by standard addition technique.
Some applications of voltammetry are the determination of metal ion concentrations in water and kinetic studies of reactions specifically studies about oxidation and reduction processes in various media.
References:
Skoog, D.A., et al. 2004. Fundamentals of Analytical Chemistry. 8th ed. Singapore: Brooks/Cole.
Chemistry 137.1. 2006. Modern Analytical Chemistry Manual. Institute of Chemistry, College of Arts and Sciences, University of Philippines College Laguna.
Friday, May 21, 2010
Wednesday, May 19, 2010
The Effort and The Prize
Calibration Curve for the OI Analytical Total Organic Carbon Analyzer
I have been working with the OI Analytical Total Organic Carbon Analyzer for several months and last April 29, 2010 did I only get my ultimate goal: optimize it and produce a good calibration curve. Maam Agnes, my boss, my mentor, commented that the RF or response factor should have to be less that 0.5 but due to the age factor of the equipment, 0.5 is already acceptable.
Now my next goal for the TOC Analyzer is to make a quality control chart. I hope I can in the near future, while I still have my hands on it. ☺
Wednesday, May 5, 2010
THIN LAYER CHROMATOGRAPHY OF LIPIDS
Lipids are one of the major constituents of foods, and are important in our diet for a number of reasons. They are a major source of energy and provide essential lipid nutrients. Nevertheless, over-consumption of certain lipid components can be detrimental to our health, e.g. cholesterol and saturated fats. There are different types of lipids. They are fatty acids, triacylglycerols, gylcerophospholipids, sphingolipids, steroids and others which include waxes or terpenes.
Lipids contain a lot of calories in a small space. Since they are generally insoluble in polar substances such as water, they are stored in special ways in you body's cells. They can also function as structural components in the cell. Lipids are also used as hormones that play roles in regulating our Physiology (metabolism). Phospholipids are the major building blocks of cell membranes.
Different food samples can be analyzed using thin-layer chromatography in identifying the lipid groups composing them. Thin-layer chromatography supports the identity of a compound in a mixture by comparing the, retention factor (Rf) of the sample to the Rf of the standard.
During food analysis with TLC, different food samples are added with ethanol-ether solution so to extract the lipid components from the food. It undergoes centrifugation then the supernatant is left standing for several minutes to concentrate it. The TLC plate is spotted with the samples and is then placed inside the chamber with petroleum ether, diethyl ether and glacial acetic acid mixture as the solvent cum mobile phase. The TLC plate is then placed in another jar containg iodine crystals to develop the spots separated. Visualization using iodine crystals is semi-destructive since the iodine absorbs onto the spots though not permanent. This is the reason why pencil is needed to mark the spots before and after the separation stage.
Coomassie blue R can be also used for visualizations in lipid classification. When Coomassie blue binds to proteins in acid solution, it has an absorbance shift from 465 nm to 595 nm. The absorbance data can then be used in Beer's law to determine protein concentration and ultimately the actual amount of protein in a given solution.
The components, visible as separated spots, are identified by comparing the distances they have traveled with those of the known reference materials. The distance of the start line to the solvent front (=d) is measured together with the distance of center of the spot to the start line (=a). The distance the solvent moved is then divided by the distance the individual spot moved. The resulting ratio is called Rf-value. The value should be between 0.0 (spot did not moved from starting line) and 1.0 (spot moved with solvent front) and is unitless.
Once a TLC has been developed, staining is frequently necessary to aid in the visualization of the components of a reaction mixture. This is true primarily because most organic compounds are colorless. The staining of a TLC plate with iodine vapor is among the oldest methods for the visualization of organic compounds. It is based upon the observation that iodine has a high affinity for both unsaturated and aromatic compounds.
Other visualization techniques are:
1. Ultraviolet light
Good for visualizing any compounds which are UV-active, particularly those with extended conjugation, aromatic rings, etc. Spot(s) must be lightly traced with a pencil while visible, since when the UV light is removed, the spots disappear.
2. Potassium Permanganate
This particular stain is excellent for functional groups which are sensitive to oxidation. Alkenes and alkynes will appear readily on a TLC plate following immersion into the stain and will appear as a bright yellow spot on a bright purple background. Alcohols, amines, sulfides, mercaptans and other oxidizable functional groups may also be visualized, however it will be necessary to gently heat the TLC plate following immersion into the stain. These spots will appear as either yellow or light brown on a light purple or pink background. Again it would be advantageous to circle such spots following visualization as eventually the TLC will take on a light brown color upon standing for prolonged periods of time.
3. p- Ansaldehyde #2
A more specialized stain used for terpenes, cineoles, withanolides, acronycine, etc. As above, heating with a heat gun must be employed to effect visualization.
4. Phosphomolybdic acid stain
Phosphomolybdic acid stain is a good "universal" stain which is fairly sensitive to low concentrated solutions. It will stain most functional groups, however it does not distinguish between different functional groups based upon the coloration of the spots on the TLC plate. Most often, TLC's treated with this stain will appear as a light green color, while compounds of interest will appear as much darker green spots. It is necessary to heat TLC plates treated with this solution in order to activate the stain for visualization.
Other methods that can be used in lipid analysis are: a.) electrophoresis- is the movement of an electrically charged substance under the influence of an electric field. This movement is due to the Lorentz force, which may be related to fundamental electrical properties of the body under study and the ambient electrical conditions by the equation given below. F is the Lorentz force, q is the charge carried by the body, E is the electric field; b.) Supercritical Fluid Chromatography (SFC) -is a robust and easy-to-use form of normal phase chromatography ideally suited to the analysis and purification of low to moderate molecular weight, thermally labile molecules. It is especially suited to the separation of chiral compounds. Similar to high performance liquid chromatography (HPLC), SFC typically utilizes carbon dioxide as the mobile phase, therefore, the entire chromatographic flow path must be pressurized; c.) Reversed phase HPLC-the principles of the separation are well known, and the instrumentation is straightforward. The stationary phases used are almost exclusively of the octadecylsilyl ("ODS") type, with an octyl phase being recommended occasionally as an alternative. The mobile phase is either acetonitrile (mainly) or methanol containing some water. If free fatty acids are analysed, a little acetic acid can be added to ensure sharp peaks. These solvents are transparent to UV light at 205 to 210 nm, so UV detection at such wavelengths can be employed. However, much greater sensitivity is possible if phenacyl or related derivatives of fatty acids are prepared for detection at higher wavelengths. Then, the detector responds only to the ester moiety giving a quantitative molar response. Astonishing sensitivity is obtainable, down to femtomole levels, by using specific derivatives with fluorescence detection although quantification then presents problems; d.) chiral chromatography–is avariant of column chromatography, where the stationary phase is chiral instead of achiral. The enantiomers of the same compound then differ in affinity to the stationary phase, thus they exit the column at different times.
Gas Chromatography can only analyze volatile substances. Intact triacylglycerols and free fatty acids are not very volatile and are therefore difficult to analyze using GC. For this reason lipids are usually derivitized prior to analysis to increase their volatility. Triacylglycerols are first saponified which breaks them down to glycerol and free fatty acids, and are then methylated. Saponification reduces the molecular weight and methylation reduces the polarity, both of which increase the volatility of the lipids. The concentration of different volatile fatty acid methyl esters (FAMEs) present in the sample is then analyzed using GC. The FAMES are dissolved in a suitable organic solvent that is then injected into a GC injection chamber. The sample is heated in the injection chamber to volatilize the FAMES and then carried into the separating column by a heated carrier gas. As the FAMES pass through the column they are separated into a number of peaks based on differences in their molecular weights and polarities, which are quantified using a suitable detector. Determination of the total fatty acid profile allows one to calculate the type and concentration of fatty acids present in the original lipid sample.
Silver ion chromatography is based on a distinctive property of unsaturated organic compounds that has the ability to complex with transition metals, in this instance with silver. The complexes are of the charge-transfer type, like the unsaturated compound acts as an electron donor and the silver ion as an electron acceptor.
Mass spectrometry is also very useful in lipid analysis. Mass spectrometry is an analytical tool used for measuring the molecular mass of a sample. Mass spectrometers can be divided into three fundamental parts, namely the ionization source, the analyzer, and the detector. The sample has to be introduced into the ionization source of the instrument. Once inside the ionization source, the sample molecules are ionized, because ions are easier to manipulate than neutral molecules. These ions are extracted into the analyzer region of the mass spectrometer where they are separated according to their mass (m) -to-charge (z) ratios (m/z). The separated ions are detected and this signal sent to a data system where the m/z ratios are stored together with their relative abundance for presentation in the format of a m/z spectrum. The analyzer and detector of the mass spectrometer, and often the ionization source too, are maintained under high vacuum to give the ions a reasonable chance of traveling from one end of the instrument to the other without any hindrance from air molecules. The entire operation of the mass spectrometer, and often the sample introduction process also, is under complete data system control on modern mass spectrometers.
Different classes of lipids can be analyzed by different methods or techniques. Cholesterol content of plasma is determined by hydrolysis. Sterols can be analyzed by GC or reversed phase HPLC. Waxes on the other hand are analyzed by thin layer and HPLC.
I. References
Bettelheim, F.A. and March A. 1990, Introduction to Organic and Biochemistry, 3rd ed. Saunders College Publishing, Philadelphia, USA.
Hoover, R., W. Sosulski, and W. Waczkowski. 1989. ‘Efficiencies of Solvent Systems for Extraction of Legume Lipids’, Fat Science Technology, vol. 91, issue 6.
Sabularse, V.C., et. al. 2006, Biochemistry Lecture Booklet, Los Baños, Institute of Chemistry.
Stryer, L. 1995, Biochemistry, 4th ed., W.H. Freeman & Company, New York.
Zubay, G.L., Parson, W.W. and Vance, D.E. 1995, Principles of Biochemistry, W.C. Brown Publishers, Iowa.
Lipids contain a lot of calories in a small space. Since they are generally insoluble in polar substances such as water, they are stored in special ways in you body's cells. They can also function as structural components in the cell. Lipids are also used as hormones that play roles in regulating our Physiology (metabolism). Phospholipids are the major building blocks of cell membranes.
Different food samples can be analyzed using thin-layer chromatography in identifying the lipid groups composing them. Thin-layer chromatography supports the identity of a compound in a mixture by comparing the, retention factor (Rf) of the sample to the Rf of the standard.
During food analysis with TLC, different food samples are added with ethanol-ether solution so to extract the lipid components from the food. It undergoes centrifugation then the supernatant is left standing for several minutes to concentrate it. The TLC plate is spotted with the samples and is then placed inside the chamber with petroleum ether, diethyl ether and glacial acetic acid mixture as the solvent cum mobile phase. The TLC plate is then placed in another jar containg iodine crystals to develop the spots separated. Visualization using iodine crystals is semi-destructive since the iodine absorbs onto the spots though not permanent. This is the reason why pencil is needed to mark the spots before and after the separation stage.
Coomassie blue R can be also used for visualizations in lipid classification. When Coomassie blue binds to proteins in acid solution, it has an absorbance shift from 465 nm to 595 nm. The absorbance data can then be used in Beer's law to determine protein concentration and ultimately the actual amount of protein in a given solution.
The components, visible as separated spots, are identified by comparing the distances they have traveled with those of the known reference materials. The distance of the start line to the solvent front (=d) is measured together with the distance of center of the spot to the start line (=a). The distance the solvent moved is then divided by the distance the individual spot moved. The resulting ratio is called Rf-value. The value should be between 0.0 (spot did not moved from starting line) and 1.0 (spot moved with solvent front) and is unitless.
Once a TLC has been developed, staining is frequently necessary to aid in the visualization of the components of a reaction mixture. This is true primarily because most organic compounds are colorless. The staining of a TLC plate with iodine vapor is among the oldest methods for the visualization of organic compounds. It is based upon the observation that iodine has a high affinity for both unsaturated and aromatic compounds.
Other visualization techniques are:
1. Ultraviolet light
Good for visualizing any compounds which are UV-active, particularly those with extended conjugation, aromatic rings, etc. Spot(s) must be lightly traced with a pencil while visible, since when the UV light is removed, the spots disappear.
2. Potassium Permanganate
This particular stain is excellent for functional groups which are sensitive to oxidation. Alkenes and alkynes will appear readily on a TLC plate following immersion into the stain and will appear as a bright yellow spot on a bright purple background. Alcohols, amines, sulfides, mercaptans and other oxidizable functional groups may also be visualized, however it will be necessary to gently heat the TLC plate following immersion into the stain. These spots will appear as either yellow or light brown on a light purple or pink background. Again it would be advantageous to circle such spots following visualization as eventually the TLC will take on a light brown color upon standing for prolonged periods of time.
3. p- Ansaldehyde #2
A more specialized stain used for terpenes, cineoles, withanolides, acronycine, etc. As above, heating with a heat gun must be employed to effect visualization.
4. Phosphomolybdic acid stain
Phosphomolybdic acid stain is a good "universal" stain which is fairly sensitive to low concentrated solutions. It will stain most functional groups, however it does not distinguish between different functional groups based upon the coloration of the spots on the TLC plate. Most often, TLC's treated with this stain will appear as a light green color, while compounds of interest will appear as much darker green spots. It is necessary to heat TLC plates treated with this solution in order to activate the stain for visualization.
Other methods that can be used in lipid analysis are: a.) electrophoresis- is the movement of an electrically charged substance under the influence of an electric field. This movement is due to the Lorentz force, which may be related to fundamental electrical properties of the body under study and the ambient electrical conditions by the equation given below. F is the Lorentz force, q is the charge carried by the body, E is the electric field; b.) Supercritical Fluid Chromatography (SFC) -is a robust and easy-to-use form of normal phase chromatography ideally suited to the analysis and purification of low to moderate molecular weight, thermally labile molecules. It is especially suited to the separation of chiral compounds. Similar to high performance liquid chromatography (HPLC), SFC typically utilizes carbon dioxide as the mobile phase, therefore, the entire chromatographic flow path must be pressurized; c.) Reversed phase HPLC-the principles of the separation are well known, and the instrumentation is straightforward. The stationary phases used are almost exclusively of the octadecylsilyl ("ODS") type, with an octyl phase being recommended occasionally as an alternative. The mobile phase is either acetonitrile (mainly) or methanol containing some water. If free fatty acids are analysed, a little acetic acid can be added to ensure sharp peaks. These solvents are transparent to UV light at 205 to 210 nm, so UV detection at such wavelengths can be employed. However, much greater sensitivity is possible if phenacyl or related derivatives of fatty acids are prepared for detection at higher wavelengths. Then, the detector responds only to the ester moiety giving a quantitative molar response. Astonishing sensitivity is obtainable, down to femtomole levels, by using specific derivatives with fluorescence detection although quantification then presents problems; d.) chiral chromatography–is avariant of column chromatography, where the stationary phase is chiral instead of achiral. The enantiomers of the same compound then differ in affinity to the stationary phase, thus they exit the column at different times.
Gas Chromatography can only analyze volatile substances. Intact triacylglycerols and free fatty acids are not very volatile and are therefore difficult to analyze using GC. For this reason lipids are usually derivitized prior to analysis to increase their volatility. Triacylglycerols are first saponified which breaks them down to glycerol and free fatty acids, and are then methylated. Saponification reduces the molecular weight and methylation reduces the polarity, both of which increase the volatility of the lipids. The concentration of different volatile fatty acid methyl esters (FAMEs) present in the sample is then analyzed using GC. The FAMES are dissolved in a suitable organic solvent that is then injected into a GC injection chamber. The sample is heated in the injection chamber to volatilize the FAMES and then carried into the separating column by a heated carrier gas. As the FAMES pass through the column they are separated into a number of peaks based on differences in their molecular weights and polarities, which are quantified using a suitable detector. Determination of the total fatty acid profile allows one to calculate the type and concentration of fatty acids present in the original lipid sample.
Silver ion chromatography is based on a distinctive property of unsaturated organic compounds that has the ability to complex with transition metals, in this instance with silver. The complexes are of the charge-transfer type, like the unsaturated compound acts as an electron donor and the silver ion as an electron acceptor.
Mass spectrometry is also very useful in lipid analysis. Mass spectrometry is an analytical tool used for measuring the molecular mass of a sample. Mass spectrometers can be divided into three fundamental parts, namely the ionization source, the analyzer, and the detector. The sample has to be introduced into the ionization source of the instrument. Once inside the ionization source, the sample molecules are ionized, because ions are easier to manipulate than neutral molecules. These ions are extracted into the analyzer region of the mass spectrometer where they are separated according to their mass (m) -to-charge (z) ratios (m/z). The separated ions are detected and this signal sent to a data system where the m/z ratios are stored together with their relative abundance for presentation in the format of a m/z spectrum. The analyzer and detector of the mass spectrometer, and often the ionization source too, are maintained under high vacuum to give the ions a reasonable chance of traveling from one end of the instrument to the other without any hindrance from air molecules. The entire operation of the mass spectrometer, and often the sample introduction process also, is under complete data system control on modern mass spectrometers.
Different classes of lipids can be analyzed by different methods or techniques. Cholesterol content of plasma is determined by hydrolysis. Sterols can be analyzed by GC or reversed phase HPLC. Waxes on the other hand are analyzed by thin layer and HPLC.
I. References
Bettelheim, F.A. and March A. 1990, Introduction to Organic and Biochemistry, 3rd ed. Saunders College Publishing, Philadelphia, USA.
Hoover, R., W. Sosulski, and W. Waczkowski. 1989. ‘Efficiencies of Solvent Systems for Extraction of Legume Lipids’, Fat Science Technology, vol. 91, issue 6.
Sabularse, V.C., et. al. 2006, Biochemistry Lecture Booklet, Los Baños, Institute of Chemistry.
Stryer, L. 1995, Biochemistry, 4th ed., W.H. Freeman & Company, New York.
Zubay, G.L., Parson, W.W. and Vance, D.E. 1995, Principles of Biochemistry, W.C. Brown Publishers, Iowa.
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