Symptoms that you are having a problem with your filament:
During GCMS operation, the sequence run suddenly stopped. Upon checking the ChemStation logbook, this is what I found:
ACQ5975 'Unrecovable mass spec fault: 8’
ACQ5975 There is no emission current at runtime 16.93.
6890 Not ready: F Inlet pressure 9kPa at runtime 0.20
6890 Not ready: Host system at runtime 1.16
Manual Mode Run Method Error – Consult Logbook
Manual Mode Instrument Error, refer to logbook
Also, to investigate more, I run the autotune which eventually tune action stopped with a message: “There is no emission current”
Chemstation Help is very helpful. It provided tips on how to resolve the problem.
I grabbed the chance that I could to shut down the computer and clean everything so as to eliminate other possible sources of the error and other issues that may arise during analysis.
The inlet was cleaned, while the column was detached from the injector and detector, and a new liner and septa was installed. Also, the FID was cleaned of char that has accumulated in the assembly.
Again, I started the GCMS using the usual protocol and performed the autotune. The tune action has stopped and the same error appeared in the display panel of the software. There is no emission current.
Solution:
1. Go to View, click Vacuum and Control View.
2. Go to Parameters, click Manual Tune. On filament, change entry 1 to 2 (depends on which filament you are currently using)
3. Click OK.
4. Click File, save tune parameters as atune.u (filename for autotune).
Perform autotune and tune evaluation. If results are not yet acceptable, continue baking out the system until autotune and tune evaluation results are within acceptable limits.
Make sure that the busted filament will be replaced soon. At least you have a spare filament when emergencies like this comes up.
Thursday, January 19, 2012
Wednesday, October 5, 2011
all is well :)
i had been lazy writing/posting some chemical stuff for a while that's why this blog had been growing some moss and organisms particular to stagnation.
anyways, despite the [pseudo]writer's writers' block, this year had been motivatingly dynamic, well, at least, chemistry-wise. :)
well, i almost got started with my masters in soil science, but, fortunately, i underwent a very drastic career turn. i am now endowed to aroma in rice and my job also calls for my contribution in the quality evaluation program in rice. everything is exciting that i almost got no time to write my adventures in chemistry.
last may, i started with my new group doing research on rice grain quality. t'was a sudden shift from environmental aspect of rice research to the 'more direct' aspect, i.e. cereal chemistry rice chemistry.
i was also given the chance to continue some aroma research on rice and to plant aromatic lines in our experiment station's quarantine plot.
last august, i was able to participate in the 3rd INQR Symposium. See http://irri.org/news-events/irri-news/thailand-international-network-for-quality-rice-meets-in-bangkok. meeting and interacting with other scientists and getting to know what cool stuff they are doing is simply amazing! every member of the network was generous enough to share their knowledge and humble enough to listen to comments and other ideas on how they could improve their experiments.
well, so far, i had been learning more stuff from my colleagues on gas chromatography, laboratory management, rice chemistry and more more on rice.
anyways, despite the [pseudo]writer's writers' block, this year had been motivatingly dynamic, well, at least, chemistry-wise. :)
well, i almost got started with my masters in soil science, but, fortunately, i underwent a very drastic career turn. i am now endowed to aroma in rice and my job also calls for my contribution in the quality evaluation program in rice. everything is exciting that i almost got no time to write my adventures in chemistry.
last may, i started with my new group doing research on rice grain quality. t'was a sudden shift from environmental aspect of rice research to the 'more direct' aspect, i.e. cereal chemistry rice chemistry.
i was also given the chance to continue some aroma research on rice and to plant aromatic lines in our experiment station's quarantine plot.
last august, i was able to participate in the 3rd INQR Symposium. See http://irri.org/news-events/irri-news/thailand-international-network-for-quality-rice-meets-in-bangkok. meeting and interacting with other scientists and getting to know what cool stuff they are doing is simply amazing! every member of the network was generous enough to share their knowledge and humble enough to listen to comments and other ideas on how they could improve their experiments.
well, so far, i had been learning more stuff from my colleagues on gas chromatography, laboratory management, rice chemistry and more more on rice.
Thursday, June 23, 2011
Baseline problem with FID on Agilent 6890GC
It has been more than a month since I started with this new instrument -
Agilent 6890 Gas chromatograph.
Parts were cleaned and at the same time familiarized. Extraction procedures were repeatedly done for the analyses of the fragrant chemical in rice and yet, I still couldn’t find the peak on my samples.
The precious peaks were only seen on the reference material which was from 2ppm 2-AP Japan in ethanol and in-house standard 2-AP pandan (Yes, 2-acetyl-1-pyroline can be extracted from pandan leaves with dichloromethane).
Why couldn’t I find them in the obviously fragrant samples that I have??
Since the GC was started to be used again last May, the baseline was already observed to be at ~ 90. For an optimum setting, baseline should not be more than 20pa. I only have 2 suspicions as sources of this problem:
1. Since I couldn’t detect 2-AP from my samples, it could be possible that I may have committed an error on the extraction itself.
2. It could also be possible that the problem lies within the GC system.
Over all, a high baseline is a result of the following factors:
1. Gas supply problems and inefficient oxygen trap (for helium) and moisture trap (for compressed air)
2. Poor or bleeding column
3. Electrical current leakage
4. Poor flame stability and contaminated FID jet assembly
One by one, these factors were evaluated.
1. Checking on the gas traps, those were indeed old and needed replacements. They have been used since 2006. :)) Furthermore, one is really not sure.
2. The old column (HP-5MS) was baked; the baseline was still the same. A new column (HP-5) was installed and conditioned. Baseline was still at ~70-90pa. It could be possible that the column is really not the problem. [Changing the column meant that the GC also has to have some changes on its settings but these changes are automatic, so no worries on that part.]
3. leakages on the fid set up was evaluated using the procedures described by Agilent. Flame was first extinguished and the level of current observed at the baseline is evaluated. As expected, the signal (for baseline) gradually went down to nil. This is a good sign that the fid doesn’t have any "stray" electric current which may have caused the high baseline. During this evaluation, the fid was still at its operating temperature at 300deg C where as the column and injector temperatures were not at their usual set points.
4. At this point, the column and the carrier gases are eliminated as the culprits. The column was detached to the detector where as the latter was fitted with a no-hole ferrule. The FID was then again ignited, allowed to stabilize and then the baseline was again checked for any significantly unusual change. Still, we are all in vain.
The guy from the technical support had already given us the go signal, if we are that brave, we could bypass the moisture and oxygen traps and let the gases flow directly from the tanks to the instrument. Hmm, at this stage, we won’t do anything that may cause more damage to the GC.
The only safest thing left to do is dismantle the FID assembly and check for any contamination.
And so we did. The fid jet was indeed charry. So the finest sandpaper was used to scratch-off all the char from the jet.
The result made us all happy. The baseline had decreased to 60 and now we are trying to condition it so that we could have a more stable baseline.
As of yesterday, we are now getting peaks from the standards as well as from my samples. This only goes to show that there was no error in preparing the samples just that the fid needs a lot of care and maintenance these days.
Agilent 6890 Gas chromatograph.
Parts were cleaned and at the same time familiarized. Extraction procedures were repeatedly done for the analyses of the fragrant chemical in rice and yet, I still couldn’t find the peak on my samples.
The precious peaks were only seen on the reference material which was from 2ppm 2-AP Japan in ethanol and in-house standard 2-AP pandan (Yes, 2-acetyl-1-pyroline can be extracted from pandan leaves with dichloromethane).
Why couldn’t I find them in the obviously fragrant samples that I have??
Since the GC was started to be used again last May, the baseline was already observed to be at ~ 90. For an optimum setting, baseline should not be more than 20pa. I only have 2 suspicions as sources of this problem:
1. Since I couldn’t detect 2-AP from my samples, it could be possible that I may have committed an error on the extraction itself.
2. It could also be possible that the problem lies within the GC system.
Over all, a high baseline is a result of the following factors:
1. Gas supply problems and inefficient oxygen trap (for helium) and moisture trap (for compressed air)
2. Poor or bleeding column
3. Electrical current leakage
4. Poor flame stability and contaminated FID jet assembly
One by one, these factors were evaluated.
1. Checking on the gas traps, those were indeed old and needed replacements. They have been used since 2006. :)) Furthermore, one is really not sure.
2. The old column (HP-5MS) was baked; the baseline was still the same. A new column (HP-5) was installed and conditioned. Baseline was still at ~70-90pa. It could be possible that the column is really not the problem. [Changing the column meant that the GC also has to have some changes on its settings but these changes are automatic, so no worries on that part.]
3. leakages on the fid set up was evaluated using the procedures described by Agilent. Flame was first extinguished and the level of current observed at the baseline is evaluated. As expected, the signal (for baseline) gradually went down to nil. This is a good sign that the fid doesn’t have any "stray" electric current which may have caused the high baseline. During this evaluation, the fid was still at its operating temperature at 300deg C where as the column and injector temperatures were not at their usual set points.
4. At this point, the column and the carrier gases are eliminated as the culprits. The column was detached to the detector where as the latter was fitted with a no-hole ferrule. The FID was then again ignited, allowed to stabilize and then the baseline was again checked for any significantly unusual change. Still, we are all in vain.
The guy from the technical support had already given us the go signal, if we are that brave, we could bypass the moisture and oxygen traps and let the gases flow directly from the tanks to the instrument. Hmm, at this stage, we won’t do anything that may cause more damage to the GC.
The only safest thing left to do is dismantle the FID assembly and check for any contamination.
And so we did. The fid jet was indeed charry. So the finest sandpaper was used to scratch-off all the char from the jet.
The result made us all happy. The baseline had decreased to 60 and now we are trying to condition it so that we could have a more stable baseline.
As of yesterday, we are now getting peaks from the standards as well as from my samples. This only goes to show that there was no error in preparing the samples just that the fid needs a lot of care and maintenance these days.
Friday, May 21, 2010
ANODIC STRIPPING VOLTAMMETRY (ASV)
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.
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.
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.
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Thursday, December 10, 2009
toxicity and mercury
The environment is composed of indistinguishably numerous compounds that can be either classified as organic or inorganic materials. These compounds are constantly redistributed along the different constituents of the biosphere as well as in the hemisphere.
Figure 1. Biogeochemical processes. (http://www.chemgapedia.de/vsengine /media/vsc/en/ch/16/uc/images/biogeochem.jpg)
Mercury poisoning is not so prevalent nowadays. However, its toxicity should also be noted such that it can enter our biological pathways and upset them causing harmful consequences. Therefore there is a necessity to identify how the mercury poison human beings. Identifying the locations and level of exposure would increase proper precautions on the handling of such compounds. This would help scientists and laboratory technicians come up with ways of handling such compounds.
Figure 1. Biogeochemical processes. (http://www.chemgapedia.de/vsengine /media/vsc/en/ch/16/uc/images/biogeochem.jpg)
However, these continuous and persistent redistributions or cycles of these compounds may also have noxious effects to organisms specifically to human beings. In addition to that, the entry of human activities to the naturally-occurring cycles may increase the risk of toxicity of compounds in the environment. A good example of compounds posing risks to organisms is mercury.
Mercury is said to be a naturally-existing element which may occur in its elemental, organic or inorganic forms. Normally, it is found in measurable quantities in the environment. However, once it exceeds the normal levels and bioaccumulates in living tissues, harmful and detrimental effects are expected to transpire.
Figure 2. Mercury in different forms posing risks to humans. (http://www.greenfacts.org/)
Mercury poisoning is not so prevalent nowadays. However, its toxicity should also be noted such that it can enter our biological pathways and upset them causing harmful consequences. Therefore there is a necessity to identify how the mercury poison human beings. Identifying the locations and level of exposure would increase proper precautions on the handling of such compounds. This would help scientists and laboratory technicians come up with ways of handling such compounds.
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