1.4 STANDARD OPERATING PROCEDURE (SOP)
1. Before running the sample, the purge valve which located on pump module was opened by loosen it. This is for removal of any residues or contaminants inside the HPLC tubing. The HPLC purged for 30 minutes.
2. After that, tightened up the purge valve.
3. In the CHEMSTATION, the method set up in the METHOD parameter.
4. From the METHOD menu, Edit Entire Method selected.
5. Parameters which are injection volume, mobile phase, flow rate, temperature and detectors were set up.
6. After finish setting up the method, the method saved and named.
7. Next, for setting up the samples information the Run Sequences selected. Then in the sample tray, the sample vial was placed.
8. After setting up the HPLC method and sample information, Switch Pump On clicked to initiate the pump.
9. When all parts are in ready condition, the injection of sample conducted.
10. For data analysis, chromatogram showing the retention time and area were obtained.
11. After finish analysing the sample, before off the module the HPLC was flushed.
1.5 PRECAUTION STEPS
One of the precaution steps for HLPC equipment is to correct solvent preparation. This is to minimize amounts of times used for errors like troubleshooting spurious peaks, baseline noise and others. In addition, make sure all the reagents and solvents at their highest quality. This is because HPLC grade contain no impurities to produce spurious peaks in a chromatogram baseline. Also, make sure all buffers in a fresh preparation and ensured the buffer pH unaffected by prolonged storage and that there is no microbial growth present. Furthermore, make sure to filter all HPLC solvents by using 0.45 µm filters before using. This is to prevent from blockages of any particulate matter. Then, next precautions need to take after the filtration process is to store the solvents in a covered reservoir. This is to prevent it from contamination with dust.
Another precaution needed to take before the freshly prepare mobile phase pumped around the HPLC system is degassed the mobile phase thoroughly for removal of all dissolved gasses. For example, air bubbles which is a system that can create high-pressure system. Thus, the system having problems such as the system become unstable and a spurious peaks. Also to prevent cross contamination the filters should be kept clean whenever they are not used by storing them in a solution of 50% acetonitrile / 50 % water to microbial growth for stopping dust and dirt from embedding the filter pores. The solvent lines should be kept clean, growth-free and do not have a sharp bends or creases in them. As for the solvent reservoirs, it should be placed higher than the pump inlet manifold inside the instrument. Be careful, not to use highly acidic or basic solvents unless HPLC system and column have been engineered to accommodate them. This is to prevent damaging due to extreme pH conditions on certain components which are seals, plungers and other. Another precaution needed t take is never allowed a HPLC column or system to stand with water or buffer in it for an extended period of time. Always flush it with a solvent mix which contains a minimum of 20% organic in water to inhibit growth.
Also, make sure the buffer soluble for the proposed of washing or storing phase. If the buffer not soluble, the steps should be taken are to flush the system with a solvent mix that is highly aqueous to remove the buffer from the system and column. After that, change it to the proposed wash or storage solvent mix. Before attempting any solvent change, make sure the solvent already in the system and column is compatible with the new solvent. Buffer transparency is a variable that should be measured prior to buffer use, as it will vary with salt concentration. When choosing a buffer and additive mixture a care should be taken to make sure the solution of the two does not produce a solid salt which lead to system contamination. Buffers should always be flushed from the analytical column and instrument after use to avoid salts being deposited on delicate frits and other parts. To make sure HPLC performed successfully and has improved the column performance, a care should be taken on the column efficiency, capacity factor and other components which are it should be measured at the start and end of the clean-up procedure.
Furthermore, make sure that no buffers or samples are present on the column and that the solvent used prior to the clean-up is miscible with the first wash solvent. After the clean-up, ensure that the test mobile phase is compatible with the last solvent in the column. Another important precaution is to make sure the HPLC system does not have built-in extra dead volume, because this will increase the band spreading value. Lastly but not least, run a test for a column that has not been used recently or is old to make sure that it is working properly and has not been contaminated.
1.6 APPLICATIONS
HPLC Applications in identification of olive oils or to detection the addition of other oils, such as hazelnut oil, to olive oil. One method of determining the quality or authenticity of olive oil is by HPLC analysis of the carotene content and lutein content. HPLC equipment detect triglycerides for the identification of adulteration, characterize and classify cultivars such as varieties of cultivated plants brought by selective bleeding. For example, methods were established to characterise and classify 10 almond cultivars (Aparicio & Aparicio-Ruiz, 2000).
In addition, an HPLC application is in detection of soy proteins in bovine milk. If there are presence or addition of soy proteins in bovine milk may be adulterated. For example, HPLC method is available to detect soy proteins in unheated milk. Various methods have also been developed to detect the presence of bovine, ovine, and caprine (goat) proteins in mixtures of milk or cheeses (Nollet, 2004).
Furthermore, HPLC used in separation and analysis of chemical and biological compounds that are non-volatile and thermally unstable which are in pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol), in salts like sodium chloride and potassium phosphate, in proteins like egg white or blood protein, in chemicals like polymers (e.g. polystyrene, polyethylene) ,hydrocarbons like asphalt or motor oil, natural products such as ginseng, herbal medicines, plant extracts and in thermally unstable compounds such as trinitrotoluene (TNT) and enzymes. HPLC also used for qualitative and quantitative analysis.
Another application of HPLC is in chemistry and biochemistry research analyzing complex mixtures, purifying chemical compounds, and developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation.