HPLC Mass Spectrometry Assay Lecture PDF

HPLC(High Performance Liquid chromatography):mass spectrometry assay LEARNING OUTCOME Principle of chromatography HPLC LEARNING Types of chromatic separations Application of HPLC OUTCO...

HPLC(High Performance Liquid chromatography):mass spectrometry assay LEARNING OUTCOME Principle of chromatography HPLC LEARNING Types of chromatic separations Application of HPLC OUTCOMES Mass spectrometry LC-MS (Liquid chromatography Mass Spectrometry) Quantitative QC - Module 7 2 CHROMATOGRAPHY Chromatography is a technique which separates components in a mixture due to the differing time taken for each component to travel through a stationary phase when carried through it by a mobile phase. CHROMATOGRAPHY Basically, all chromatographic systems consists of two phases phases: Mobile phase: liquid or gaseous and flows over or through the stationary phase Stationary phase: solid, liquid or a solid/liquid mixture which is immobilized CHROMATOGRAPHY TERMS Analyte Substance that is to be separated during chromatography Immobilized phase Stationary phase which is immobilized on the support particles or on the inner wall of the column tubing Mobile phase Phase which moves in a definite direction. (liquid/gas). Consists of the sample being separated/ analyzed and the solvent that moves the sample through the column. Effluent Mobile phase leaving the column. DIFFERENT TYPES OF CHROMATOGRAPHY METHODS Paper chromatography Liquid chromatography Gas chromatography High performance liquid chromatography Paper chromatography HIGH PERFORMANCE LIQUID CHROMATOGRAPHY HPLC is an extension of conventional liquid chromatography. Powerful tool in analytical techniques Columns are tightly packed, and the eluent is forced through the column under high pressure(up to 5,000 psi) by a pump. Allows to use a very smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing through it. Allows a much better separation of the components of the mixture.  HPLC TECHNIQUE Utilizes liquid mobile phase to separate the mixture. Analytes are first dissolved in a solvent then through the column under high pressure of up to 400 atm. Mixture is resolved into its components in the column. The total separation time is often 5 or 10 minutes rather than hours or even days required for some separations by gravity flow with the larger systems.  Pump Injector Column Detector  A pump forces the mobile phase through the column at a much greater velocity than gravity- flow columns. Pumps are designed in order to maintain a stable flow rate, avoiding pulsations even when the composition of the mobile phase varies flow range – 0.01-10 ml/min Injectors are divided into 2 types: Valve type It is connected to the column by means of a pipe and consists of valves which can be opened and closed. it is used more often. Syringe type It is directly connected to the column and the sample is delivered with the aid of a syringe Inject the liquid sample under high pressure. Produce minimum band broadening. Produce minimized possible flow disturbances. Volume must be small (10-50 uL).  Smooth-bore stainless steel or heavy-walled glass tubing. Hundreds of packed columns differing in size and packing are available from manufacture. E.g. Column packing vary in size (3 to 20 um) with the smaller particles used mostly for analytical separations and the larger ones for preparative separation. DETECTOR HPLC detectors monitor the elute as it leaves the column. Produce an electronic signal proportional to the concentration of each separated component Crucial in trace analysis High sensitivity Fast response Insensitive to changes in type of solvent, flow rate and temp. DETECTORS USED IN HPLC ng nanogram p-gran = = pg Type Principle Detection Comments limit Spectro- Measure