Mobile phase A consisted of water with 0. The gradient was as follows: 0. The flow rate was 0. An electronic valve actuator with a Rheodyne selector valve was used to divert LC flow to waste, at the first 1 minute, when no data acquisition was taking place. Samples were analyzed with an AB Sciex triple quadropole mass spectrometer equipped with Turbo Ionspray.
Analyst version 1. Nitrogen was used as the nebulizer, auxiliary, collision and curtain gases. Analytes were detected by tandem mass spectrometry using multiple reaction monitoring MRM with a dwell time of ms. Ten standards containing efavirenz concentrations of 1, 2. Drug free blank human plasma lots were used for bulk preparation of standards and QCs. Selectivity is ensured at LLOQ in 6 different lots of human plasma plus one pooled blank human plasma lots. The internal standard working solution was prepared by diluting the internal standard stock solution with 0. Amber glass vials were used for storing stock solutions.
The plate was vortex-mixed on 96 well plate-mixer at rpm for 10 minutes and centrifuged at rpm for 10 minutes. Method validation was performed according to guidelines set by the United States Food and Drug Administration FDA for bioanalytical method validation . This method was validated in terms of linearity, specificity, low limit of quantitation LLOQ , recovery, intra-day and inter-day accuracy and precision, carryover assessment, dilution integrity, matrix effect, and stability of the analyte during sample storage and processing procedures.
3 Analytical methodologies
Six sets of QCs were used during 3 core validation runs, for assessing intra-day and inter-day accuracy and precision. For stability studies and sample analysis only low, medium and high QCs are used for accepting the batch. For the evaluation of the linearity of the standard calibration curve, the analyses of efavirenz in plasma samples were performed on three independent days using fresh preparations.
Each calibration curve consisted of a double blank sample, a blank sample and ten calibrator concentrations. Another two double blank samples were analyzed immediately following the highest concentration standard in each run to monitor for carry-over of efavirenz or the internal standard. Each calibration curve was constructed by plotting the analyte to internal standard peak area ratio y against the analyte concentrations x.
The resulting a, b and c parameters were used to determine back-calculated concentrations, which were then statistically evaluated. Six different lots of blank efavirenz free plasma were evaluated with and without internal standard to assess the specificity of the method at LLOQ QC. Potential interference by antiretroviral drugs tenofovir, emtricitabine, and ribavirin concomitantly administered to the patients was also evaluated by spiking blank plasma at their therapeutic concentrations. Recovery was calculated by comparing the peak areas of efavirenz added into blank plasma and extracted using the protein precipitation procedure with those obtained from efavirenz spiked directly into diluted blank post extraction solvent at the four QC concentrations.
For all stability studies, freshly prepared and stability testing QC samples were evaluated by using a freshly prepared standard curve for the measurement. The concentrations obtained from all stability studies were compared to freshly prepared QC samples, and the percentage concentration deviation was calculated. Y represents the peak area ratio of the analyte to the internal standard and x is the concentration of the analyte. The mean correlation coefficient of the weighted calibration curve generated during the three day validation for efavirenz using various weighing are as shown below.
Representative chromatogram of blank, LLOQ, internal standard are shown in Figure 1 and a representative calibration curve is shown in Figure 2. Carryover was evaluated on each run during the validation. The carryover of efavirenz was 0. At the ten calibration standards, the inter-day precision ranged from 4. These data confirm that the present method has satisfactory accuracy, precision, and reproducibility for the quantification of efavirenz throughout a wide dynamic range.
For internal standard percent extraction recoveries was Data indicated that the extraction efficiency for efavirenz and internal standard using protein precipitation was satisfactory and was not concentration dependent. This is also consistent with previously reported results  , . Matrix effect can affect the reproducibility from the analyte or the internal standard of the assay.
The matrix effect or intensity of ion suppression or enhancement is caused by co-eluting matrix components. The matrix effect was tested at low QC concentration for six individual lots of blank plasma and pooled plasma utilizing previously reported method . Only minor differences were observed between the pure standards and the post-extracted spiked samples data not shown , illustrating that the HPLC separation conditions had little effect by background signal of plasma after simple protein precipitation clean up step.
The precision for bench-top 24 hours stability ranged from 4. A small amount of acetic acid is added to lower the pH of the mobile phase, which keeps the silanol in the stationary packing phase in an undissociated state. This reduces the adsorption peak from tailing, giving narrower peaks. Each group uses a set of the 7 vials containing different concentrations of the standard solutions Table 1. The first 3 are used to identify each peak, and the last 4 are for creating a calibration chart for each component.
Standards 1—3 are also used for the calibration chart. Table 1. Volumes of stock standards used to prepare the 7 provided working standards total volume of each standard is 50 mL. The three components that need to be made are caffeine 0. These concentrations, once diluted in the same fashion, put the standards at the levels found in the soda samples.
The three components all have differing distribution coefficients, which affects how each interacts with both of the phases. The larger the distribution coefficient, the more time the component spends in the stationary phase, resulting in longer retention times in reaching the detector. Figure 1. The chromatogram of the 3 components. From left to right, they are caffeine, aspartame, and benzoate.
This sufficiently gets rid of any gases in the samples. Figure 2. High-performance liquid chromatography, or HPLC, is a highly versatile technique that separates components of a liquid mixture based on their different interactions with a stationary phase. HPLC is an adaptation of column chromatography. In column chromatography, a column is packed with micro-scale beads called the stationary phase.
The stationary phase beads are functionalized with chemical groups that induce an interaction between the bead and the components of a mixture located in the liquid, or mobile phase. As the mixture flows through the column, the components interact with the stationary phase differently. In HPLC, column chromatography is performed at a higher flow rate, and therefore higher pressure, than classical column chromatography.
This enables the use of smaller stationary phase beads with a greater surface area to volume ratio, which greatly increases the interaction of the stationary phase and components in the mobile phase. This video will introduce the basics of the operation of HPLC by demonstrating the separation of components of various diet sodas. There are two types of HPLC used in the laboratory: analytical, and preparative. In analytical HPLC, the instrument is used to identify components of a small volume, and the analyzed sample is then discarded as waste.
In preparative HPLC, the instrument is used to purify a mixture and a desired amount of each component is collected in fractions. The HPLC instrumentation consists of a series of simple components. First, the mobile phase, held in solvent reservoirs, is pumped through the system by one or more pumps at a constant flow rate. The sample is injected into the mobile phase stream by the sample injector. The sample, diluted by the mobile phase, is then delivered to the HPLC column, where the components of the sample are separated.
The components are then analyzed by the detector, and either saved in fractions for later use, or transferred to a waste bottle. The HPLC column is the key component to the system. It is composed of a metal or plastic cylinder, packed with micro-scale beads of stationary phase, or chromatography resin. The sample mixture flows through the packed particle bed at a constant flow rate and each component interacts with the stationary phase as it flows by. The compounds interact with the stationary phase differently, and therefore travels down the length of the column to the detector at a different rate.
The time required for a component to exit the column, or elute, is called the retention time. The result is a plot of retention time vs. The retention time is used to identify the component. The peak size, specifically the area under the peak, is used to quantify the amount of the compound in the initial solution. The choice of stationary phase depends on the properties of the components in the sample mixture. The most commonly used stationary phase is silica beads, as they are an inert nonpolar material that forms micro-scale beads, and achieves sufficient packing density.
The most common type of HPLC is reversed-phase chromatography, which utilizes a hydrophobic stationary phase, typically silica beads with C18 chains bonded to the beads' surface. The components are eluted in order of decreasing polarity. The mobile phase used in reversed-phase chromatography is typically a mixture of water and an organic solvent, such as acetonitrile. Depending on the sample, the mobile phase can remain a constant ratio of water and organic solvent, known as isocratic mode. The mobile phase ratio can also be changed linearly or stepwise during the separation, to create a mobile phase gradient.
A gradient elution can prevent peak broadening of the less polar components, thereby improving the separation and shortening the elution time. In this experiment, HPLC will be used to separate and quantify three common components of diet soda. First, to prepare the mobile phase, add mL of acetonitrile to 1.
Then carefully add 2. Dilute the solution to a total volume of 2 L. The resulting solution should have a pH between 2. Adjust the pH to 4. Filter the mobile phase through a 0. It is important to degas the solution, as bubbles can cause voids in the stationary phase, or work their way to the detector cell and cause instability in measurements. Prepare three component solutions of caffeine, benzoate, and aspartame, which are three typical components of diet sodas.
These component solutions are then used to prepare the standard solutions that will be utilized to determine the unknowns.
Prepare mL of the caffeine and benzoate solutions. Prepare mL of the aspartame component solution. Store the solution in the refrigerator when not in use to avoid decomposition. Next, prepare 7 standard solutions, each with different concentrations of caffeine, benzoate, and aspartame. Pipet the proper amount of each component into a volumetric flask, and dilute to the mL mark with mobile phase. The first 3 solutions each contain one component, to enable peak identification. The other 4 solutions contain a range of concentrations of all 3 components, in order to correlate peak height to concentration.
Pour each standard solution into a labeled vial in a sample rack. Store the sample rack with samples and the remaining solutions in the refrigerator. First, set up the mobile phase and waste containers. Ensure that the waste lines are fed into a waste container, and are not recycling back into the mobile phase. Ensure that the inlet mobile phase line is fed into the mobile phase container. Verify that the flow rate of the mobile phase is set to 0. This flow rate will enable all components to elute within 5 min, but is slow enough to ensure resolution of individual peaks.
Next, verify the minimum and maximum pressures on the solvent delivery system. These settings shut the pump off in case of a leak or clog, respectively. Press "zero" on the detectors front panel, to set the blank. Then fill the syringe with that solution.
Evaluation of Analytical Chemical Methods for Detection of Estrogens in the Environment
Begin with the 3 single-component samples in order to identify the peak of each component. Next, manually inject the solution, by placing the injector handle in the load position. Verify that the data collection program is set to collect data for s, which allows for enough time for all 3 peaks to elute through the detector. Figure 5. Figure 6. Different types of columns can be applied for different fields.
Depending on the type of sample, some GC columns are better than the others. It produces fast run times with baseline resolution of key components in under 3 minutes. Moreover, it displays enhanced resolutions of ethanol and acetone peaks, which helps with determining the BAC levels.
High-Performance Liquid Chromatography (HPLC)
This particular column is known as Zebron-BAC and it made with polyimide coating on the outside and the inner layer is made of fused silica and the inner diameter ranges from. There are also many other Zebron brand columns designed for other purposes. Another example of a Zebron GC column is known as the Zebron-inferno. Its outer layer is coated with a special type of polyimide that is designed to withstand high temperatures. As shown in figure 6, it contains an extra layer inside.
Moreover, it is also used for acidic and basic samples. The detector is the device located at the end of the column which provides a quantitative measurement of the components of the mixture as they elute in combination with the carrier gas. In theory, any property of the gaseous mixture that is different from the carrier gas can be used as a detection method.
These detection properties fall into two categories: bulk properties and specific properties. Bulk properties, which are also known as general properties, are properties that both the carrier gas and analyte possess but to different degrees. Specific properties, such as detectors that measure nitrogen-phosphorous content, have limited applications but compensate for this by their increased sensitivity. Each detector has two main parts that when used together they serve as transducers to convert the detected property changes into an electrical signal that is recorded as a chromatogram.
The first part of the detector is the sensor which is placed as close the the column exit as possible in order to optimize detection. The second is the electronic equipment used to digitize the analog signal so that a computer may analyze the acquired chromatogram.
The sooner the analog signal is converted into a digital signal, the greater the signal-to-noise ratio becomes, as analog signal are easily susceptible to many types of interferences. An ideal GC detector is distinguished by several characteristics. The first requirement is adequate sensitivity to provide a high resolution signal for all components in the mixture.
This is clearly an idealized statement as such a sample would approach zero volume and the detector would need infinite sensitivity to detect it. In modern instruments, the sensitivities of the detectors are in the range of 10 -8 to 10 g of solute per second.
Furthermore, the quantity of sample must be reproducible and many columns will distort peaks if enough sample is not injected. An ideal column will also be chemically inert and and should not alter the sample in any way. In addition, such a column would have a short linear response time that is independent of flow rate and extends for several orders of magnitude. Moreover, the detector should be reliable, predictable and easy to operate.
Understandably, it is not possible for a detector meet all of these requirements. Mass Spectrometer MS detectors are most powerful of all gas chromatography detectors. When the sample exits the chromatography column, it is passed through a transfer line into the inlet of the mass spectrometer. The sample is then ionized and fragmented, typically by an electron-impact ion source. During this process, the sample is bombarded by energetic electrons which ionize the molecule by causing them to lose an electron due to electrostatic repulsion.
Further bombardment causes the ions to fragment.
Getting new data
Most ions are only singly charged. The Chromatogram will point out the retention times and the mass spectrometer will use the peaks to determine what kind of molecules are exist in the mixture. A simple quadrupole ion-trap consists of a hollow ring electrode with two grounded end-cap electrodes as seen in figure. Ions are allowed into the cavity through a grid in the upper end cap. Ions that are too heavy or too light are destabilized and their charge is neutralized upon collision with the ring electrode wall.
Emitted ions then strike an electron multiplier which converts the detected ions into an electrical signal. This electrical signal is then picked up by the computer through various programs. They are rugged, easy to use and can analyze the sample almost as quickly as it is eluted. The disadvantages of mass spectrometry detectors are the tendency for samples to thermally degrade before detection and the end result of obliterating all the sample by fragmentation.
Figure Arrangement of the poles in Quadrupole and Ion Trap Mass spectrometers. Flame ionization detectors FID are the most generally applicable and most widely used detectors. In a FID, the sample is directed at an air-hydrogen flame after exiting the column. At the high temperature of the air-hydrogen flame, the sample undergoes pyrolysis, or chemical decomposition through intense heating.
Pyrolized hydrocarbons release ions and electrons that carry current. A high-impedance picoammeter measures this current to monitor the sample's elution. These properties allow FID high sensitivity and low noise. The unit is both reliable and relatively easy to use. However, this technique does require flammable gas and also destroys the sample. Thermal conductivity detectors TCD were one the earliest detectors developed for use with gas chromatography. The TCD works by measuring the change in carrier gas thermal conductivity caused by the presence of the sample, which has a different thermal conductivity from that of the carrier gas.
Their design is relatively simple, and consists of an electrically heated source that is maintained at constant power. The temperature of the source depends upon the thermal conductivities of the surrounding gases. The source is usually a thin wire made of platinum, gold or. The resistance within the wire depends upon temperature, which is dependent upon the thermal conductivity of the gas. TCDs usually employ two detectors, one of which is used as the reference for the carrier gas and the other which monitors the thermal conductivity of the carrier gas and sample mixture.
Carrier gases such as helium and hydrogen has very high thermal conductivities so the addition of even a small amount of sample is readily detected. The advantages of TCDs are the ease and simplicity of use, the devices' broad application to inorganic and organic compounds, and the ability of the analyte to be collected after separation and detection. The greatest drawback of the TCD is the low sensitivity of the instrument in relation to other detection methods, in addition to flow rate and concentration dependency.
Schematic of thermal conductivity detection cell. Figure 13 represents a standard chromatogram produced by a TCD detector. In a standard chromatogram regardless of the type detector, the x-axis is the time and the y-axis is the abundance or the absorbance. From these chromatograms, retention times and the peak heights are determined and used to further investigate the chemical properties or the abundance of the samples. Electron-capture detectors ECD are highly selective detectors commonly used for detecting environmental samples as the device selectively detects organic compounds with moieties such as halogens, peroxides, quinones and nitro groups and gives little to no response for all other compounds.
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Therefore, this method is best suited in applications where traces quantities of chemicals such as pesticides are to be detected and other chromatographic methods are unfeasible. The simplest form of ECD involves gaseous electrons from a radioactive? As the analyte leaves the GC column, it is passed over this?
The electrons from the? In the absence of organic compounds, a constant standing current is maintained between two electrodes. With the addition of organic compounds with electronegative functional groups, the current decreases significantly as the functional groups capture the electrons. The advantages of ECDs are the high selectivity and sensitivity towards certain organic species with electronegative functional groups.
However, the detector has a limited signal range and is potentially dangerous owing to its radioactivity. In addition, the signal-to-noise ratio is limited by radioactive decay and the presence of O2 within the detector.
Related Selective Sample Handling and Detection in High-Performance Liquid Chromatography
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