sample preparation in hplc

how to prepare sample n types

1. SAMPLE PREPARTAION IN HPLC M.PAVAN KUMAR Dept of pharmaceutical analysis

2. SAMPLE PREPARTAION IN HPLC Introduction : Sample preparation is an essential part of HPLC analysis intended to provide a reproducible and homogenous solution that is suitable for injection on to the column . Aim of sample preparation : Should be free of interferences; will not damage the column; compatible with HPLC method .

3. Types of sample: Sample matrices can be classified as organic and inorganic . Further divided into: solids, semisolids (gels, creams and suspensions), liquids and gases. Gaseous samples are usually by GC but sample which is labile and thermally unstable or prone to adsorb are better by HPLC. Compared to gaseous or solids, liquids are much easier to prepare for HPLC. Preliminary processing of solids and semisolids, reducing sample particle size is required. It is homogenous allowing more sampling with greater accuracy and precision. Dissolves faster and easier, because of greater surface area. Methods to reduce particle size are blending, chopping, cutting and grinding, milling etc.

4. Drying a sample drying the sample to constant weight is usually necessary for reliable assay. Filtration: Particles should be removed prior to injection because of their adverse effect on column HPLC filters in the range of 0.25 to 2 µm. Sample pretreatment for liquid samples : Liquid-liquid extraction: based on Nernst distribution it is useful for separating analytes from interferences by partitioning the sample between immiscible liquids or phases . Solid phase extraction : It is similar to liquid-liquid extraction where as LLE in one stage separation process/SPE is chromatographic procedure has more advantages than LLE. More complete extraction of analyte; More efficient separation of interferences from analyte; Reduced organic solvent consumption; Easier collection of total analyte fraction; More convenient; removal of particulates; more easily automated.

5. Membrane separation : Membranes are made from synthetic organic polymers, cellulose or glass fibers. Filtration and SPE with discs represent the major applications of membrane for sample preparation. Sample pretreatment for solid samples : A sample must be in a liquid state prior to HPLC analysis some insoluble solid contain soluble analytes such as additives in a solid polymer, fats in food and poly aromatic hydrocarbons in soil. Traditional extraction methods : N o one solvent extraction technique can be used for all samples there are certain methods for pretreatment of solids mainly used are soxhlet extraction and leaching.

6. Newer extractions : Supercritical fluid extraction; Microwave assisted solvent extraction; Accelerated solvent extraction; Column switching : Column switching is a powerful technique for the separation and cleans up of complex multi-component samples. Main objective is to maximize the injection of analyte band onto column and minimizing the interfering compounds . Principle : It can be carried out either manually or automatically but mostly are automated. The simplest system uses a single pump with a three or four part valve placed between column1 and column 2. The column position allows mobile phase from column 1 to bypass column 2.

7. Derivatization : Derivatization involves a chemical reaction between an analyte and a reagent to change the chemical and physical properties of an analyte . The four main uses of derivatization in HPLC are : Improve detectability, Change the molecular structure or polarity of analyte for better chromatography, Change the matrix for better separation and Stabilize a sensitive analyte . Detectability : D erivatization in HPLC mainly for analyte detectability; the two most common types of derivatization - The addition of a chromphore or flurophore . Allows detection of analyte that can’t be detected in its normal form or to increase its sensitivity . Detection can be made by UV detection, Fluorescence detection, Pre and post column derivatization, Chiral analysis by derivatization.

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