Lab Analysis: Reverse Phase Chromatography

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HPLC column can be categorized into two types
• Reverse Phase
• Normal Phase
3.4.1.1. Reverse Phase Chromatography Reverse Phase system has a non-polar stationary phase over which polar solvents are used as mobile phase; it is generally used for separation of polar species.
3.4.1.2. Normal phase chromatography Normal Phase system has a polar stationary phase over which non-polar solvents are used as mobile phase; it is generally used for separation of non-polar species.[38]. A detection system is coupled with HPLC to detect and quantify the constituents.
3.4.2. Method development HPLC is state of art equipment which can separate and detect constituents of For the HPLC analysis of a particular mixture, a system is to be developed. Developing
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The method for analysis was developed based on the constituents of our analyte.
3.4.2.1. Stationary Phase/Column Reverse Phase C-18 column was used. This C-18 column is the most common of all and is efficient for separation of polar species.
3.4.2.2. Mobile Phase Methanol, Acetonitrile, Water or any of their combination is generally reported to be compatible with Reverse Phase C-18 column, however other mobile phases can also be used [39]. Combination of Acetonitrile (ACN) and water is used in ration of 70:30 in this analysis.
3.4.3. Column length 25 cm column was selected because it gave better separation and delayed retention time for DPA, thus excluding the early peaks given with 10 cm column. Dimensions of the selected column were 25cm length and 5mm diameter.
3.4.4. Flow Rate Flow rate of the mobile phase was adjusted to be 1 ml/min as it gives clear peaks around a retention time of 5-7 minutes. Flow rate of 1.5ml/min gave peaks before 1 minute, as any peak before 1 minute is not recommended to be considered so the flow rate was decreased to 1ml/min. At flow rate of 1ml/min, although the peaks shifted in the time range of 3-4mins but the separation was not efficient and the peaks were partly overlapping. Flow rate was decreased to 0.5ml/min which gives clear separation and peaks in the time range of 4-5
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An intermediate value could have been selected to detect both the formic acid and methanol. However, both the compounds are polar compounds which show almost same retention time on C18 column [40]. It was also verified from the experimental results that both species give peaks around the same time of 4-5 minutes, so to avoid the confusion and overlapping of peaks, a wavelength of 250nm was selected where Formic acid gives high absorption and gives sharp peaks while methanol does not give any peak at 250nm. Around 4-5 minutes there will be a peak of Formic acid while any other peak will be an

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