What is hplc method

Table 2. The data tables for the HPLC trials used for generating the calibration curves. HPLC is a widely-used technique in the separation and detection for many applications. It is ideal for non-volatile compounds, as gas chromatography GC requires that the samples are in their gas phase.

Non-volatile compounds include sugars, vitamins, drugs, and metabolites. Also, it is non-destructive, which allows each component to be collected for further analysis such as mass spectrometry.

The mobile phases are practically unlimited, which allows changes to the polarity of pH to achieve better resolution. The use of gradient mobile phases allows for these changes during the actual trials. There has been concern over the possible health issues that may be associated with the artificial sweetener aspartame. Current product labeling does not show the amount of these components inside of the diet beverages. This method allows for quantifying these amounts, along with the caffeine and benzoate.

Other applications include determining the amounts of pesticides in water; determining the amount of acetaminophen or ibuprofen in pain reliever tablets; determining whether there are performance-enhancing drugs present in the bloodstream of athletes; or simply determining the presence of drugs in a crime lab. While the concentrations of these samples, and often the identity of the components, can be readily determined, the one limitation is that several samples could have close to identical retention times, resulting in co-eluting.

Analytical Chemistry. To learn more about our GDPR policies click here. If you want more info regarding data storage, please contact gdpr jove. Your access has now expired. Provide feedback to your librarian.

If you have any questions, please do not hesitate to reach out to our customer success team. Login processing Previous Video Next Video. Overview Source: Dr. Paul Bower - Purdue University High-performance liquid chromatography HPLC is an important analytical method commonly used to separate and quantify components of liquid samples.

Making the Mobile Phase Prepare the mobile phase by adding mL of acetonitrile to approximately 1. Carefully add 2. Dilute the solution to a total volume of 2. Add very slowly once the pH reaches 4. This should take around 50 drops to accomplish.

It is important to degas the mobile phase to avoid having a bubble, which could either cause a void in the stationary phase at the inlet of the column or work its way into the detector cell, causing instability with the UV absorbance. Creating the Component Solutions The three components that need to be made are caffeine 0. Add 0. Place this solution in a refrigerator to avoid decomposition during storage.

Making the 7 Standard Solutions The three components all have differing distribution coefficients, which affects how each interacts with both of the phases. Following the chart in Table 1 , pipet the proper amount of each component into a mL volumetric flask. Dilute each of the stock solutions to the mL mark on the volumetric flasks with mobile phase.

Pour each standard solution into labeled small vials in a sample rack. Store the racks of samples in a refrigerator, along with the remaining solutions in the mL volumetric flasks. This is high enough to allow all peaks to elute within 5 min and slow enough to allow for nice resolution.

Verify that the minimum and maximum pressure and the flow rate are set to the correct values on the front panel of the solvent delivery system the pump. Minimum pressure setting: psi this is to shut off the pump, if a leak occurs.

Maximum pressure setting: 4, psi this is to protect the pump from breaking, if a clog forms. Press "zero" on the detector's front panel in order to set the blank the blank is the pure mobile phase.

Start with the 3 single-component samples, which allows for identifying the peak of each component of interest. Verify that the data collection program is set to collect data for s, which allows enough time for all 3 peaks to elute through the detector. When ready to start the trial, rotate the injector handle to the inject position which injects the sample into the mobile phase and click "Start Trial" on the computer data collection program immediately.

For standards , only one of the three sequential peaks appear on the screen during the run Figure 1. Once s have passed, the data collection sends a prompt to save the data file. Save the data under a suitable file name e. Note the time in seconds for the peak of each trial, which is used in identifying that component.

Remove the syringe from the septum and repeat the process for each of the remaining working standards, using the same time per chromatogram as determined from the first run. Draw around 2 mL of the diet soda into a plastic syringe. Attach the filter tip to the syringe via Luer-Lok by twisting it in place. Push the liquid in the syringe through the filter and into a small glass vial. This gets rid of unwanted particulates that could potentially clog the separation column.

Calculations From the concentrations of the component solutions, calculate the concentration of all of the components in the standards, based upon the dilutions that were made for the 7 samples. After determining which peak corresponds to each component based upon the time it takes for each component to show their respective peak, enter these peak areas into a computer spreadsheet.

Create calibration curves of peak area vs. Determine the least-squares fit for each calibration curve. Calculate the concentration of each component in the diet sodas from the peak areas shown from the HPLC trials for the samples. Remember that the diet soda was diluted by a factor of 2 prior to injecting into the HPLC system. Based upon the results, calculate the milligrams of each component found in a oz can of soda.

HPLC is a highly versatile instrument, which is used in a wide range of analyses. Thanks for watching! From this set of experiments, it was determined that a oz can of these diet sodas contained the following amounts of each component: Diet Coke: Please enter your institutional email to check if you have access to this content.

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Adequate buffering capacity should be maintained when working closer to physiological conditions. Organic solvent: The organic solvent modifier is added to lower the polarity of the aqueous mobile phase. The lower the polarity of the mobile phase, the greater its eluting strength in reversed phase chromatography. Although a large variety of organic solvents can be used in reversed phase chromatography, in practice only a few are routinely employed. The two most widely used organic modifiers are acetonitrile and methanol, although acetonitrile is the more popular choice.

Isopropanol 2-propanol can be employed because of its strong eluting properties, but is limited by its high viscosity which results in lower column efficiencies and higher backpressures. Both acetonitrile and methanol are less viscous than isopropanol. All three solvents are essentially UV transparent. This is a crucial property for reversed phase chromatography since column elution is typically monitored using UV detectors. Acetonitrile is used almost exclusively when separating peptides.

Most peptides only absorb at low wavelengths in the ultra-violet spectrum typically less than nm and acetonitrile provides much lower background absorbance than other common solvents at low wavelengths. Ion suppression: The retention of peptides and proteins in reversed phase chromatography can be modified by mobile phase pH since these particular solutes contain ionisable groups. The degree of ionisation will depend on the pH of the mobile phase.

The stability of silica-based reversed phase media dictates that the operating pH of the mobile phase should be below pH 7. The amino groups contained in peptides and proteins are charged below pH 7. The carboxylic acid groups, however, are neutralised as the pH is decreased. The mobile phase used in reversed phase chromatography is generally prepared with strong acids such as trifluoroacetic acid TFA or ortho-phosphoric acid. These acids maintain a low pH environment and suppress the ionisation of the acidic groups in the solute molecules.

Varying the concentration of strong acid components in the mobile phase can change the ionisation of the solutes and, therefore, their retention behaviour. The major benefit of ion suppression in reversed phase chromatography is the elimination of mixed mode retention effects due to ionisable silanol groups remaining on the silica gel surface. The effect of mixed mode retention is increased retention times with significant peak broadening Figure 4. Peaks are broader and skewed, and retention time increases.

Reversed phase separations are most often performed at low pH values, generally between pH The low pH results in good solubility of the sample components and ion suppression, not only of acidic groups on the sample molecules, but also of residual silanol groups on the silica matrix. Acids such as trifluoroacetic acid, heptafluorobutyric acid and ortho-phosphoric acid in the concentration range of 0.

Note that phosphate buffers are not volatile. It is important to maintain the pH of the mobile phase in the range of 2. This is due to the fact that the siloxane linkage area cleaved below pH 2. Absorbance: An UV-visible detector is based on the principle of absorption of UV visible light from the effluent emerging out of the column and passed through a photocell placed in the radiation beam.

UV detector is generally suitable for gradient elution work. The mobile phase used should not interfere in the peak pattern of the desired compound hence it should not absorb at the detection wavelength employed Selectivity is affected by the surface chemistry of the reversed phase medium, the nature and composition of the mobile phase, and the gradient shape Figure 5. Both high column efficiency and good selectivity are important to overall resolution. However, changing the selectivity in a chromatographic experiment is easier than changing the efficiency.

Selectivity can be changed by changing easily modified conditions like mobile phase composition or gradient shape. Viscosity: Solvent of lowest possible viscosity should be used to minimize separation time. An added advantage of low viscosity is that high efficiency theoretical plate HETP values are usually lower than with solvents of higher viscosity, because mass transfer is faster. Viscosity should be less than 0. Temperature: Temperature can have a profound effect on reversed phase chromatography, especially for low molecular weight solutes such as short peptides and oligonucleotides.

The viscosity of the mobile phase used in reversed phase chromatography decreases with increasing column temperature. Since mass transport of solute between the mobile phase and the stationary phase is a diffusion-controlled process, decreasing solvent viscosity generally leads to more efficient mass transfer and, therefore, higher resolution.

Increasing the temperature of a reversed phase column is particularly effective for low molecular weight solutes since they are suitably stable at the elevated temperatures 8. However, among these the five dominant detectors used in LC analysis are the electrical conductivity detector, the fluorescence detector, the refractive index detector, mass spectrometry detector and the UV detector fixed and variable wavelength.

The detector selected should be chosen depending upon some characteristic property of the analyte like UV absorbance, fluorescence, conductance, oxidation, reduction, etc. Characteristics that are to be fulfilled by a detector to be used in HPLC determination are:. RP-HPLC is probably the most universal, most sensitive analytical procedure and is unique in that it easily copes with multi-component mixtures.

While developing the analytical methods for pharmaceuticals by RP-HPLC, must have good practical understanding of chromatographic separation to know how it varies with the sample and with varying experimental conditions in order to achieve optimum separation. To develop a HPLC method effectively, most of the effort should be spent in method development and optimization as this will improve the final method performance. Int J Pharm Sci Res.

Article Information Sr No: 9. Download: Cited By: The linearity of a test procedure is its ability within a given range to produce results that are directly proportional to the concentration of analyte in the sample. The range is the interval between the upper and lower levels of the analyte that have been determined with precision, accuracy and linearity using the method as written.

ICH guidelines specify a minimum of five concentration levels, along with certain minimum specified ranges. Acceptability of linearity data is often judged by examining the correlation coefficient and y-intercept of the linear regression line for the response versus concentration plot.

The regression coefficient r. The per cent relative standard deviation RSD , intercept and slope should be calculated. In the present study, linearity was studied in the concentration range 0.

The demonstration coefficient r 2 obtained for the regression line demonstrates the excellent relationship between peak area and concentration of progesterone. A method is said to be accurate if it gives the correct numerical answer for the analyte. The method should be able to determine whether the material in question conforms to its specification for example, it should be able to supply the exact amount of substance present. However, the exact amount present is unknown, which is why a test method is used to estimate the accuracy.

Furthermore, it is rare that the results of several replicate tests all give the same answer, so the mean or average value is taken as the estimate of the accurate answer. Some analysts adopt a more practical attitude to accuracy, which is expressed in terms of error. The absolute error is the difference between the observed and the expected concentrations of the analyte. Percentage accuracy can be defined in terms of the percentage difference between the expected and the observed concentrations Equation 1.

Percentage accuracy tends to be lower at the lower end of the calibration curve. The term accuracy is usually applied to quantitative methods but it may also be applied to methods such as limit tests. Accuracy is usually determined by measuring a known amount of standard material under a variety of conditions but preferably in the formulation, bulk material or intermediate product to ensure that other components do not interfere with the analytical method.

The per cent recovery should then be calculated. To document accuracy, ICH guidelines regarding methodology recommend collecting data from a minimum of nine determinations across a minimum of three concentration levels covering the specified range for example, three concentrations, three replicates each.

Developing a separation method for HPLC involves demonstrating specificity, which is the ability of the method to accurately measure the analyte response in the presence of all potential sample components. The response of the analyte in test mixtures containing the analyte and all potential sample components placebo formulation, synthesis intermediates, excipients, degradation products and process impurities is compared with the response of a solution containing only the analyte.

The resulting mixtures are then analysed, and the analyte peak is evaluated for peak purity and resolution from the nearest eluting peak. Once acceptable resolution is obtained for the analyte and potential sample components, the chromatographic parameters, such as column type, mobile phase composition, flow rate and detection mode, are considered set. An example of specificity criterion for an assay method is that the analyte peak will have baseline chromatographic resolution of at least 2.

In this study, a weight of sample placebo equivalent to the amount present in a sample solution preparation was injected to demonstrate the absence of interference with progesterone elution Figure 4. Precision means that all measurements of an analyte should be very close together. A useful criterion is the relative standard deviation RSD or coefficient of variation CV , which is an indication of the imprecision of the system Equation 2.

According to the ICH, 2 precision should be performed at two different levels - repeatability and intermediate precision. Repeatability is an indication of how easy it is for an operator in a laboratory to obtain the same result for the same batch of material using the same method at different times using the same equipment and reagents.

Intermediate precision results from variations such as different days, analysts and equipment. In determining intermediate precision, experimental design should be employed so that the effects if any of the individual variables can be monitored. In this study, the precision of the method repeatability was investigated by performing six determinations of the same batch of product.

The resulting data are provided in Table V, which show that the repeatability precision obtained by one operator in one laboratory was 0. The limit of detection LOD is defined as the lowest concentration of an analyte in a sample that can be detected, not quantified. It is expressed as a concentration at a specified signal:noise ratio,. The limit of quantitation LOQ is defined as the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method.

The ICH has recommended a signal:noise ratio The standard deviation of the response can be determined based on the standard deviation of the blank, on the residual standard deviation of the regression line, or the standard deviation of y-intercepts of regression lines. The method used to determine LOD and LOQ should be documented and supported, and an appropriate number of samples should be analysed at the limit to validate the level. Validation of sample and standard solution preparation may be divided into sections, each of which can be validated.

These include extraction; recovery efficiency; dilution process when appropriate; and addition of internal standards when appropriate. Although extraction processes do not actually affect the measuring stage they are of critical importance to the analytical test method as a whole.

The extraction process must be able to recover the analyte from the product; it must not lose for example, by oxidation or hydrolysis any of the analyte in subsequent stages, and must produce extraction replicates with high precision.

For example, during analysis of an ester prodrug the extraction process involves the use of strongly alkaline or acid solutions, it may cause some of the prodrug to be hydrolysed and, therefore, give false results.

Reference substances should be prepared so that they do not lose any of their potency. Thus it is necessary to validate that the method will give reliable reference solutions that have not been deactivated by weighing so little that an error is produced; adsorption onto containers; decomposition by light; and decomposition by the solvent. If the reference is to be made up from a stock solution then it must be validated that the stock solution does not degrade during storage.

Reagent preparation should be validated to ensure that the method is reliable and will not give rise to incorrect solutions, concentrations and pH values.

Samples and standards should be tested during a period of at least 24 h depending on intended use , and component quantitation should be determined by comparison with freshly prepared standards. For the assay method, the sample solutions, standard solutions and HPLC mobile phase should be stable for 24 h under defined storage conditions.

In the present study, the stabilities of progesterone sample and standard solutions were investigated. Test solutions of progesterone were prepared and chromatographed initially and after 24 h. The stability of progesterone and the mobile phase were calculated by comparing area response and area per cent of two standards with time.

Standard and sample solutions stored in a capped volumetric flask on a lab bench under normal lighting conditions for 24 h were shown to be stable with no significant change in progesterone concentration during this period Table VII. Robustness measures the capacity of an analytical method to remain unaffected by small but deliberate variations in method parameters.