Chromatography Part 2 PDF

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Summary

Liquid Chromatography (LC)- Based on the distribution of solutes between the liquid MP and the SP which contains small particles “packed” into a tube (column). - The most common LC in clinical laboratories is High Pressure Liquid Chromatography (HPLC). Liquid on its own will not flow quickly through...

Description

MLSC-3111, Clinical Biochemistry II Liquid Chromatography (LC) -

Based on the distribution of solutes between the liquid MP and the SP which contains small particles “packed” into a tube (column). The most common LC in clinical laboratories is High Pressure Liquid Chromatography (HPLC). Liquid on its own will not flow quickly through a column. High Pressure Liquid Chromatography (HPLC), requires a high pressure to facilitate the flow of the liquid MP through the packed column. The MP is pumped through the SP. HPLC can use any of the previously described models of separation by using different types of particles packed into the column.

HPLC Components -

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MP, the mobile phase is a liquid contained in a bottle or flask. Pump, provides pressure to pump liquid through. Aspirates MP from solvent reservoir and delivers a reproducible, constant flow through the column. The flow rate can be adjusted by adjusting the pump speed. Sample Injector, to inject the sample into the system. Introduces sample into the path of the MP. Requires a small syringe is used to inject sample into a fixed loop injector. o Fixed Loop Injector, a programmable device that allows variable and precise volume injection. Column, for adsorption. Uses packing material encased in stainless steel columns, unlike gas chromatography. The packing can be very small, so the tightly packed particles only let high pressure liquid pass through. The stainless-steel casing serves to contain the high pressure. The inner diameter ranges from 2 um to 2.0 mm. Length ranges from 30250 mm. Detector, to detect components exiting the column. Uses UV or UV/VIS detectors, which are the same as absorbance detectors in spectrophotometry. Requires the use of a flow cell. o Flow Cell, a clear glass cell that allows the detector to detect changes as the MP and components flow through it. Recorder, records the signal detected by the detector to make the chromatogram. Waste, liquid MP must have a waste receptacle for when it exits the column, unlike like gaseous MPs.

Normal vs Reverse Phase Chromatography -

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Normal Phase Chromatography, the original type of chromatography. Based upon polarity since the SP is highly polar, even more polar than the MP. The SP will attract polar components more than the MP will. The MP is an organic solvent that is slightly polar. Its goal is to remove polar components from the column so they elute out. Elution Order, the highest polar components elute later than low/non-polar ones as they are more attracted to the SP.

MLSC-3111, Clinical Biochemistry II -

However, the MP is made of expensive and toxic/dangerous organic solvents. Reverse Phase, basically the opposite of normal phase. Also based upon polarity, but the MP is highly polar, even more polar than the SP. Polar solutes will move with the MP and will elute quicker. Non-polar solutes interact with the SP and are retained. The MP has organic solvents such as acetonitrile added to it, which will then remove non-polar components. This is the most common form of HPLC in the lab since all the aqueous samples we get in the lab flow easily in the polar MP solvent.

Types of HPLC Elution -

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Isocratic Elution, the same MP strength is pumped through the column throughout the run. Gradient Elution, differing MP strength is pumped through the column during the run. At an original MP solvent strength, fast solutes elute quickly, and slow solutes are retained. At higher MP strengths, the slow solutes will elute faster. Gradient elution allows for faster chromatography time without having fast solutes elute off the chromatographs with the MP. Some MPs will react better with certain components, giving them better resolution. The gradient elution allows for the transitioning/changing of MP fluid part way through the run. Gas chromatography has a similar feature, expect it uses different gas temperatures instead of different pump rates/solvents.

Principles of Identification and Quantitation -

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Recorder, records the detector signal versus the time it was recorded to create a chromatogram. The location of the peak allows us to identify the component. The size of the peak allows us to quantitate the component. Retention Time (tR), the time interval between the time of injection of a component and the detection of the component. This gives us the time at which the compound has eluted out of a column and describes the time the component remains in the SP. The peak is used to detect the time. Components in the sample mixture will have different retention times based upon their affinities to the SP and MP. Retention time is unique to a solute under given reproducible chromatographic conditions and therefor, can be used to identify the presence of that solute in the mixture. Dead Time, the time required for the MP to pass through the column and the detector. Standards with known concentration of each component are used for validation. The time it takes for the standard to reach the detector is the expected retention time for its respective analyte. A retention time window is usually used to account for slight variation in elution times in components.

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Qualitative Analysis, retention time is used to identify a compound by telling us if it is positive or negative for being in solution. However, retention time does not prove identity as other unknown compounds may elute within the same retention time. MP Flow Rate, flow rates that are too high will have falsely lowered retention times. Flow rates that are too low will cause falsely increased retention times. Anything that affects the flow rate will affect retention time. Maintenance includes checking flow rate routinely. If peaks do not elute when expected, we cannot identify the components properly. Quantitative Analysis, can be done two ways. o Measuring the Peak Height, if the peaks are very sharp. o Measuring the Peak Area, multiply the peak height by its width. The quantitation area relates to how much component is present. The width can be measured in two places. o The Width at Half-Height o The Width at Baseline

Calibration -

External Calibration, a series of standards of differing concentrations are run. Their results are plotted onto a graph to form a standard curve. Internal Standardization, requires the use of an internal standard (IS), which needs to be mixed into the sample before we inject the sample. o Internal STD, compound with similar chemical structure as the analyte but not normally found in an unknown sample. Whatever happens to the sample components also happens to the IS. Corrects for any loss of analyte during injection or extraction. o Correcting for IS, the software detects peak size for each sample peak and for the IS peak. o Response Ratio, calculation performed for each sample peak (sample peak/IS peak). The ratio is therefor independent of the volume of pretreated sample since the volumes cancel out mathematically. This corrects for chromatographic differences in the system between injections since the ratio will not change when the solute and the IS are in the same conditions. o During external calibration, the system plots response ratios of the peak size of each solute to the peak size of the internal standard. Each sample peak concentration is determined by Cunk= (Response Ratiounk/Response RatioIS) x CSTD.

MLSC-3111, Clinical Biochemistry II

Factors Affecting Chromatography -

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Peak Separation, depends on two factors. The difference in the retention of solutes and the narrowness of the solute peak width. Resolution, describes the degree of separation between two eluting components by chromatography. It is the ability of a column to discriminate (resolve) chromatographic peaks between two solutes. It is based upon the distance and width of peaks. Resolution Factor can be quantitated to understand how reliable our separation is. The formula for resolution is , where is the difference in peak elution times and is the average width of peaks at the baseline. A low resolution factor (> 0.40) means very little separation between peaks. Referred to as co-elution of two components. A resolution greater than 1.25 is sufficient for good quantitation. By determining the resolution we can know if our calculated analyte concentrations can be trusted.

Factors Affecting Resolution -

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Column Length and Diameter, smaller diameters lead to better resolution and longer elution times. Temperature, too high temperatures will cause the peaks to be too close together. Flow Rate, too high flow rates result in peaks being too close together. Sample Size, large volumes cause a loss of resolution and degradation of peak sample. Resolution depends on the interaction of retention factor and column efficiency. Efficiency, the ability of a column to separate a solute as a single peak having a narrow width. High efficiency is directly proportional to the sharpness of the peaks. Efficiency is affected by column length, particle size and component diffusion due to multiple flow paths. Molecules disperse during chromatography which broadens peaks and diminishes efficiency. Separation can be achieved by controlling and manipulating these interactions. Multiple Flow Paths (Eddy Diffusion), solute molecules in the MP will take different random paths through the packed stationary phase column, causing a broadening of peaks. The greater the number of packing particles, the greater the difference in “path length” between travelling molecules. More packing particles causes greater band broadening, decreased efficiency and poorer resolution. Diffusion of molecules increases proportionally with the time molecules spend in the column. Increased column time causes decreased efficiency. Natural Diffusion, causes symmetrical peaks (Gaussian Peaks). Non-Uniform Diffusion, causes asymmetrical peaks. Leads to trailing peaks where broadening only occurs on one side of the peak. Caused by non-uniform particle sizes or column packing.

MLSC-3111, Clinical Biochemistry II

Peak Detection -

Detectors, monitor the eluate leaving the column by producing an electrical signal proportional to each separated component concentration.

Thermal Conductivity Detector (TCD) -

Used for gas chromatography. The compound carried in the gas MP increases thermal conductivity of the gas. The detector will measure the change in thermal conductance as the MP/components pass through it to plot it on a chromatograph. Generally used in combination with helium gas systems (the preferred carrier gas). This detector is not as sensitive as other detectors and has trouble picking minute levels of components. However, it does not require a flame.

Flame Ionization Detector (FID) -

Uses a flame (from hydrogen gas and compressed air) to combust components (analytes) in the carrier gas (SP). The carrier gas flows into the hydrogen flame with the components. The FID contains a collector electrode that is placed above the flame. A flame jet and the collector electrode have opposite charges. Therefor, as the sample combusts, ions form and move toward the charged collector electrode. The current produced by flow of ions is proportional to concentration of ions from the combusted components and is plotted. Disadvantage is that it uses a flame and compressed hydrogen gas. You also have to light the flame....