Experiment Objectives
\nBy experiments, learn the basic principles of chromatographic analysis and the basic operation of the instrument, and understand the process of data analysis.
\nLearn about chromatographic quantitative methods and compare the advantages and disadvantages of different methods.<\/p>\n
HPLC Introduction
\nHigh speed: HPLC uses high-pressure infusion equipment, the flow rate is greatly increased, and the analysis speed is extremely fast, only a few minutes generally; while the classical method depends on gravity feeding, it takes several hours to complete an analysis.
\nEfficient: The filler particles are very fine and regular, the coating on the stationary phase is uniform, and the mass transfer resistance is small, so the column efficiency is very high. It can complete the separation of hundreds of materials within minutes.
\nHigh sensitivity: The detector is extremely sensitive: UV- 10-9g, fluorescence detector- 10- 11g.<\/p>\n
Comparison HPLC with GC<\/p>\n
Object and scope of analysis: GC analysis is limited to gases and low-boiling stable compounds, which account for only 20% of the total organic matter; HPLC can analyze high-boiling, high-molecular-weight stable or unstable compounds that account for 80% of the total organic matter.<\/p>\n
The choice of mobile phase: GC uses limited optional kinds of “inert” gases as the mobile phase, carry only, small effect on components. HPLC using the mobile phase is a mixture of liquid or various liquids, there are many kinds of optional mobile phase. In addition to its role as a carrier, it can also interact with the components and compete with the effect of stationary phase, that is, the contribution of mobile phase is much more for separation, and the separation can be controlled and improved by the solvent.<\/p>\n
Operating temperature: GC requires high temperatures; HPLC is usually performed at room temperature.
\nApplication fields
\nDue to its high sensitivity, quantitative accuracy, and suitable for analysis of non- volatile and thermally unstable components, HPLC separation analysis is extremely versatile in industrial and scientific research, especially in biology and medicine. For example, analysis of amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, drugs, polysaccharides, polymers, pesticides, sub-antibiotics, cholesterol, and metal organic are mostly done by HPLC.<\/p>\n
HPLC consists of Pump system, injection system, separation system, detector and data processing system.<\/p>\n
High-pressure infusion system
\n1) Mobile phase bottle: 1-2L glass bottle with solvent filter (Ni alloy), its pores are approximately 2 \u03bcm, preventing particulates from pumping.
\n2) Degasser: ultrasonic degassing or vacuum heating degassing. The solvent passes through the degassing membrane in the degasser, and the relatively small molecular weight gas is removed from the solvent through the membrane.<\/p>\n
3) High-pressure pump: The requirements for the infusion pump: good sealing, stable flow without influx, wide adjustable range, and corrosion resistance.<\/p>\n
Experiment<\/p>\n
Experimental conditions
\n1)Inject 10\u03bcL mix standard solutions (methyl paraben, propyl paraben, and butyl paraben) in different ratio mobile phases of methanol:water = 90:10, 80:20, and 70:30 to test, record retention time and the resolution between the various components, with the resolution greater than 1.5 and the shortest retention time as the principle of selecting the best mobile phase matching ratio.
\nNote: Each time you change the mobile phase, you need to wait about 5 minutes to balance the chromatographic system (both signal and pressure lines are balanced).
\n2) Inject 10 \u03bcL mix standard solutions (methyl paraben, propyl paraben, and butyl paraben) at different mobile phase flow rates of 0.8, 1.0, and 1.5 mL\/min and optimal mobile phase ratios injection analysis, the same principle selected the best mobile phase flow rate.<\/p>\n
Qualitative analysis
\nUnder the optimal experimental conditions, inject 10 \u03bcL each of methyl paraben, propyl paraben, and butyl paraben and the retention time of each peak is recorded. Compared with the result obtained in step 2).<\/p>\n
Quantitative analysis
\nCalibration curve
\nUnder the optimal experimental conditions, 2, 5, 8, 10, 15, and 20 \u03bcL of the mixed standard sample was injected and analyzed, and the standard curve is established by injection volume and peak area.
\nIt is required that the correlation coefficient of this curve is greater than 0.99. If it is not satisfied, retesting can be performed on experimental points that deviate significantly.<\/p>\n
Unknown sample analysis
\nInject 10 \u03bcL unknown sample to test, quantitative analysis by calibration curve. After analysis, wash the HPLC system with pure methanol about 10min, then shut off workstation and power switch.<\/p>\n
Questions
\nBased on the experimental data, discuss the differences in analysis methods between gas chromatography and liquid chromatography.
\nExplore the application value of HPLC analysis.
\nWhether the UV detector is suitable for detecting all organic compound? Why?
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