Essay: Reverse Phase High Performance Liquid Chromatography

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LC 2010 Shimadzu, Japan
Chromatographic Conditions:
Mobile phase: 10 Mm monobasic phosphate pH 2.45 adjusted with O’phosphoric acid containing 0.1% V/V triethylamine and acetonitrile (70:30)
Absorption maxima: 227nm
Injection Volume: 20??l
Column: C-18, dimension 250 ?? 4.6 mm, 5 ?? from Phenomenex
Flow rate: 1.4ml/min.
Preparation of diluent: 0.1 N HCl and acetonitrile in the ratio7:3 (v/v) was mixed to prepare the diluent.
Preparation of standard stock solution: 10mg drug was accurately weighed and put into 10ml volumetric flask containing 5 ml of diluent and sonicated for 10 mins. Then the volume was adjusted with more diluent up to the mark.
Preparation of sample solutions: Sample solutions of different conc. from 0.05, 0.1., 0.2, 0.3, 0.4, 0.5 ??g/ml were prepared from above stock solution and diluted with mobile phase.
Parameters of Validation
Linearity and Range
The linearity of an analytical method is its ability to elicit test results which are directly proportional to analyte concentration in samples within a given range. The range of an analytical method is the interval between upper and lower analyte concentration in sample including these concentrations for which it has been established that the analytical method has a suitable level of linearity, accuracy and precision. To establish the linearity and range of proposed methods, various aliquots of standard solution of drug were prepared from stock solution and analyzed.
Sample solutions of drug with different conc. from 0.25-2.5??g/ml were analyzed by UV spectrophotometer using methanol as blank at 245 nm (15) as good linearity was observed at this wavelength and their absorbance noted.
Sample solutions of drug with different conc. from 0.05, 0.1., 0.2, 0.3, 0.4, 0.5 ??g/ml were analyzed by HPLC and their area measured. Absorption maxima 227nm was used for analysis as it gave sharp peak as compared to 245nm. It was not possible to get separate peak at 245 nm. The chromatogram obtained is shown in Figure 2.
Precision studies were carried out to establish the repeatability and reproducibility of proposed methods by using six replicates of same concentration of sample solution.
Repeatability was determined by preparing six replicates of same concentration (1 ??g/ml and 0.5??g/ml in UV Spectroscopy and HPLC method respectively) of sample and their absorbance/area measured.
Reproducibility: Intraday precision study was carried out by preparing drug solution of same concentration (1 ??g/ml and 0.5??g/ml in UV Spectroscopy and HPLC method respectively) and analyzing it at three different times in a day. The same procedure was followed for three different days to determine interday precision. The results were reported as %RSD.
Accuracy of a method is the degree to which observed results correspond to true value of analyte in the sample. The determination was done at three different levels (80%, 100%, and 120% of sample concentration) .Three samples of each level were prepared and total 9 determinations done as per ICH guidelines. The samples were analyzed and their absorbance/area measured and results indicated as % RSD.
Robustness/ Ruggedness
Robustness/ Ruggedness of an analytical procedure is a measure of is its ability to remain unaffected by small changes in method parameters and provide an indication of its reliability.
Ruggedness was determined by carrying out analysis by two different analysts at the same operational and environmental conditions. The respective absorbance or area noted and results indicated as % RSD
To check the robustness of proposed methods: analysis was carried out at two different temperatures, room temperature and at 18??C and respective absorbance measured (UV Spectroscopy method).
Similarly analysis was carried out at three different wavelengths 224nm, 227nm and 230nm and different flow rates and respective area measured (HPLC method).
The results were indicated as %RSD
Limit of detection (LOD) is the lowest amount of analyte in the sample that can be detected. Limit of quantification (LOQ) is the lowest amount of analyte in sample that can be quantitatively determined by suitable precision and accuracy. LOQ and LOD were determined using the following equation LOQ-10?? /S, LOD-3.3?? /S where ?? is the standard deviation of response and S slope of related calibration curve.
Results and Discussion
Linearity and Range
Drug shows linearity in the concentration range of 0.25-2.5??g/ml and 0.05-0.5??g/ml by UV Spectroscopy and HPLC methods respectively. The linearity data is given in Table 1. Linearity of data was found by the plot of concentration verses absorbance (UV Spectroscopy method) and concentration verses area measured (HPLC method). The standard calibration curves obtained for both methods are given in Figure 3 and 4. Correlation coefficient obtained from standard curve is 0.996 and 0.995 by UV Spectroscopy and HPLC method respectively.
Table 1: Linearity data of Imiquimod
Method Concentration
(??g/ml) Absorbance/Area Statistical Analysis
Spectroscopy in methanol 0.25 0.0853??0.003 Intercept = 0.013
Slope = 0.334
Straight line equation
y = 0.334x – 0.013
Regression coefficient
R2 = 0.996
0.50 0.157??0.0052
0.75 0.238??0.0062
1.00 0.3173??0.0066
1.25 0.399??0.0043
1.50 0.4653??0.027
1.75 0.5656??0.0061
2.50 0.8436??0.022
HPLC 0.05 33697.9 ?? 55.08 Intercept = 24502
Slope = 14686
Straight line equation
y = 14586x + 24502
Regression coefficient
R2 = 0.995
0.1 37247.16 ?? 24.58
0.2 55267.56 ?? 51.20
0.3 66490 ??228.14
0.4 82654.06 ?? 119.79
0.5 99303.56 ?? 75.16
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