Table of Contents
How do you prepare a solution for HPLC?
Prepare 5 mmol/l Sodium 1-hexanesulfonate, 20 mmol/l phosphate aqueous solution (Dissolve 10 ml of sodium 1-hex- anesulfonate (0.5 M solution) (Product No. 31532-06) 2.31g of phosphoric acid (Purity: 85\%), (Product No. 08964-92) in distilled water to make 1L solution.).
How do you prepare a sample for HPLC analysis?
* The sample should be dissolved in the eluent to be used and filtrated with 0.45μm disposable filter. Then, it should be injected into HPLC system about 10 to 50μL. * Avoid overloading.
How do you prepare a standard solution for a calibration curve HPLC?
To prepare the standards, pipette the required amount in the volumetric flask. Then fill the flask to the line with solvent, and mix. Continue making the standards by pipetting from the stock solution and diluting. For a good calibration curve, at least 5 concentrations are needed.
What is stock solution for HPLC?
In chemistry, a stock solution is a large volume of common reagent, such as hydrochloric acid or sodium hydroxide, at a standardized concentration. In biochemistry, the term is often used to refer to a concentrated solution, from which one can dilute into a working concentration of solution.
How do you calibrate an HPLC instrument?
CHECKPOINT: LEAKAGE TEST (BY PRESSURE DROP)
- Ensure that, the instrument is ready for calibration and Start-up procedure is followed.
- Place inlet tubing of the Pump into the Water HPLC grade through a suction filter.
- Allow the mobile phase to flow for about 5 min.
- Block Pump outlet with the block screw.
Why phosphate buffer is used in HPLC?
Since the retention of ionizable compounds is very sensitive to the mobile phase pH, it is necessary to control the pH of the mobile phase by the addition of a buffer. Phosphate and acetate are particularly useful buffers because they can be used at wavelengths below 220 nm.
What kind of samples can be analyzed using HPLC?
While urine is the most common medium for analyzing drug concentrations, blood serum is the sample collected for most medical analyses with HPLC. Other methods of detection of molecules that are useful for clinical studies have been tested against HPLC, namely immunoassays.
How much sample is needed for HPLC?
More than 0.5 mL would be safe to be used with 2 mL vial. In my experiments, the volume is 300 uL and I use total recovery vials instead of the normal 2 mL ones.
How do you make stock solutions?
A stock solution is prepared by weighing out an appropriate portion of a pure solid or by measuring out an appropriate volume of a pure liquid, placing it in a suitable flask, and diluting to a known volume. Exactly how one measure’s the reagent depends on the desired concentration unit.
Why caffeine is used in HPLC calibration?
Caffeine is convenient because it is not volatile, readily available, safe, has strong UV adsorption if you are using UV detector, and does not retain too long on RP columns (faster calibration).
What is high performance liquid chromatography (HPLC)?
An Introduction to High Performance Liquid Chromatography. High Performance Liquid Chromatography, or HPLC, is the most common analytical separation tool and is used in many aspects of drug manufacture and research. HPLC is used for: 1. Qualitative and quantitative analysis of unknown mixtures – determining what is there, and how much. 2.
How should I filter my HPLC solvents before use?
All HPLC solvents should be filtered through a 0.45 µm filter before use. This removes any particulate matter that may cause blockages.
What equipment do I need to run an HPLC assay?
Most assays require a standard 10-25 cm x 4.6 mm C18 column, HPLC grade methanol or acetonitrile as the organic solvent, buffer components, a pH meter and the appropriate reference standard. Samples must be syringe filtered. The DPAL can provide technical advice and funding assistance.
What are the advantages of reference wavelength HPLC?
The benefit of this method is that the signals are all acquired using the same time base (unlike Method #1). Using the concept of Method # 2 described above, many HPLC manufactures added a software feature known as a the ‘Reference Wavelength’ to their systems.