HIGH PERFORMANCE LIQUID CHROMATOGRAPHY INSTRUMENTATION. Mrs. G. Aruna Mpharm (PhD) Dept. of PA & QA KTPC
|
|
- Aron Dean
- 5 years ago
- Views:
Transcription
1 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY INSTRUMENTATION Mrs. G. Aruna Mpharm (PhD) Dept. of PA & QA KTPC 1
2 HPLC INSTRUMENTATION CONSIST OF Solvent Reservoir( hplc solvent reservoir systems) Pumps Pre Guard Column Sample injection system Columns Detector Recorder and integrators 2
3 3
4 4
5 HPLC SOLVENT RESERVOIR SYSTEMS 5
6 HPLC SOLVENT RESERVOIR SYSTEMS These are the glass bottles use to store the mobile phase. The mobile phase is pumped under pressure from one or several reservoirs and flows through the column at a constant rate. Desirable feature in the solvent delivery system is the capability for generating a solvent gradient. 6
7 HPLC SOLVENT RESERVOIR SYSTEMS Degasser is needed to remove dissolved air by subjecting the mobile phase under vacuum, distillation, spurging with fine spray of an inert gas at lower solubility such as Argon and Helium or by heating and ultrasonic stirring. Filtration is needed to eliminate suspended particles and organic impurities. 7
8 PUMPS Pass mobile phase through column at high pressure and at controlled flow rate. Performance of pump directly affects the Rt, reproducibility, detector sensitivity 8
9 PUMPS IDEAL CHARCETRISTIC OF A PUMP Non corrosive and compatible with solvent. Provide High pressure to push mobile phase Provide constant flow rate to mobile phase. Easy to change for one mobile phase to another. 9
10 PUMPS Should have reproducible flow rate and independent of column back pressure. Should not leak. High pressure generated by pump should not lead to an explosion. It should be easy to dismantle and repair. 10
11 TYPE OF PUMP USED IN HPLC Reciprocating pump Displacement pump Pneumatic pump 11
12 RECIPROCATING PUMP 12
13 RECIPROCATING PUMP WORKING Contains reciprocating piston that moves back and forth in hydraulic chamber. By the movement of piston solvent flow into the column under high pressure. When piston moves backward inlet valve open while exit valve closes. This result in mobile phase being 13 drawn into the main chamber (cylinder).
14 RECIPROCATING PUMP When the piston moves to the front the inlet valve closes and the exit valve opens. The reduction in volume in main chamber due to forward motion of piston result in mobile phase moving out of the exit valve under high pressure. DISADVENTAGE Pulsed flow which must be damped as they produce a base line noise on the chromatogram 14
15 ADVANTAGES RECIPROCATING PUMP Generate high output pressure (upto10000 poise). Ready adaptability to gradient elusion. Provide constant flow rate. Pressure generated is so high that any back pressure generated in the column due to higher viscosity of stationary phase can be easily overcome. 15
16 DISPLACEMENT PUMP / SYRINGE PUMP 16
17 DISPLACEMENT PUMP / SYRINGE PUMP WORKING Works on the principle of positive solvent pressure. Consist of screw or plunger which revolves continuously driven by motor. Rotatory motion provides continuous movement of the mobile phase which is propelled by the revolving screw at greater speed and pushes solvent through small needle like outlet. Consist of large syringe like chamber of capacity ml.
18 DISPLACEMENT PUMP / SYRINGE PUMP ADVANTAGES Flow is pulse free. Provide high pressure upto atm. Independent of column back pressure and viscosity of solvent. Simple operation. DISADVENTAGE Limited solvent capacity Gradient elution is not easy. 18
19 PNEUMATIC PUMP WORKING Pressure from a gas cylinder delivered through a large piston drivers the mobile phase. Pressure on the solvent is proportional to the ratio of piston usually 50: 1. 19
20 PNEUMATIC PUMP The driving air is applied, piston moves, inlet closes & outlet open pushing mobile phase to the column. A lower pressure gas source of 1-10 atm can be used to generate high liquid pressure.( atm ) About 70 ml of the mobile phase is pumped from every stroke. 20
21 PNEUMATIC PUMP ADVANTAGES Pulse free flow. Generates high pressure. DISADVANTAGES Limited volume capacity (70 ml ) Pressure output and flow rate depends on the viscosity and column back pressure. Gradient elusion is not possible. 21
22 Pulse damper Important damping methods include A triple headed pump: - two heads in different stages of filling as the third is pumping. A tube with an air space or a flexible bellows or tube: - where a gas (air space) or a flexible metal vessel takes up some of the solution energy. When pump refills, this energy is released and a smooth pressure pulsation result. A restrictor: - I this method, a 25 cm length of 4 mm bore.stainless steel tubing. Packed with 20µm glass 22 beds, is placed between the pump and the column.
23 Sample injection system Septum injectors Stop flow septumless injection. Rheodyne injector / loop valve type. 23
24 Sample injection system Septum injection port. Syringe is used to inject the sample through a self sealing inert septum directly into the mobile phase. Drawback: - leaching effect of the mobile phase with the septum resulting in the formation of ghost peaks. Stop flow septumless injection. Flow of mobile phase through the column is stopped for a while. Syringe is used to inject the sample. Drawback: formation of ghost peak. 24
25 Sample injection system Rheodyne injector / loop valve type. Sophisticated modern method with good precision. Sample is introduced in the column without causing interruption to mobile phase flow. Volume of sample ranges between 2 µl to over 100 µl. 25
26 Sample injection system Operation of sample loop. sampling mode Injection mode. Sample is loaded at atmospheric pressure into an external loop in the micro volume sampling valve, and subsequently injected into the mobile phase by suitable rotation of the valve. Micro volume sampling valve operation of a Sampling loop. 26
27 27
28 COLUMN Made up of stainless steel or heavy glass to withstand the pressure. The columns are usually long (10 30 cm) narrow tubes. Contains stationary phase at particle diameters of 25 µm or less. The interior of column should be smooth and uniform. Column end fitting are designed to have a zero void volume. 28
29 CLASSIFACTION OF CLOUMN BASED ON APPLICATION column Main column Guard column Analytical column Preparative column Standard column Narrow bore Short fast column Micro preparative Preparative column Macro preparative 29
30 CLASSIFACTION OF COLUMN ON THE BASES OF COMPONENTS BONDED PHASE COLUMN COLUMN WHERE LIQUID IS INPERMAGNETED ON SOLID INERT SUPPORT 30
31 STANDARD COLUMN Internal diameter 4 5 mm and length cm. Size of stationary phase is 3 5 µm in diameter. Used for the estimation of drugs, metabolites, pharmaceutical preparation and body fluids like plasma. NARROW BORE COLUMN Internal diameter is 2 4 mm. ( signal is increased 4 times ) Require high pressure to propel mobile phase. Used for the high resolution analytical work of compounds with very high Rt. 31
32 SHORT FAST COLUMN Length of column is 3 6 cm. Used for the substances which have good affinity towards the stationery phase. Analysis time is also less (1-4 min for gradient elusion & sec for isocratic elusion). PREPARATIVE COLUMN Used for analytical separation i.e. to isolate or purify sample in the range of mg form complex mixture. Length cm Internal diameter 6 mm or more. 32
33 Preparative column are of three type : Micro preparative or semi preparative column Modified version of analytical column Uses same packaging and meant for purifying sample less then 100 mg. Preparative column Inner diameter 25 mm. Stationary phase diameter µm 33
34 Macro Preparative Column Column length cm Inner diameter 600 mm 34
35 GUARD COLUMN They are placed anterior to the separating column. Serve as a protective factor that prolongs the life and usefulness of the column. They are dependable column designed to filter or remove Particles that clog the separation column. Compounds and ions that could ultimately cause baseline drift, decrease resolution, decrease sensitivity and create false peaks. 35
36 Bonded phase column Here the molecules, comprising the stationary phase i.e. the surface of the silica particles, are covalently bonded to a silica based support particles. The most popular bonded phase,siloxanes, are formed by heating the silica particles in dilute acid for the day so as to generate the reactive Silonol group. 36
37 OH OH OH ו ו ו - Si O Si - O - Si I I I 37
38 Silonal group is the treated with organochlorosilane. CH3 CH3 ו ו OH + Cl Si R - Si O Si R + HCl Si CH3 CH3 These bonded phases are stable between the ph range 2 9 and upto temperature of 80º C. Bonded phase is made with a linear C 18 hydrocarbon, also know as ODS (octadecyl silane) bonded phase. Used in pharmaceutical analysis or separation of less polar 38 components.
39 An alkyl nitrile column or cyano column which has 12 carbon atoms with the last atom appearing as a nitrile group (CN).moderately polar column. Amino alkyl bonded phase column which is normally C 8, last C atom bearing NH2 group. Polar column. Use full in separation of CHO, peptides, amino acids. Advantages Can withstand high pressure exerted by mobile phase. Life of column is more. No bleeding effect Disadvantages Very expensive Manually can not be fabricated 39
40 COLUMN WHERE LIQUID IS INPERMAGNETED ON SOLID INERT SUPPORT. These are not use widely now days. stationary phase dose not have the strength to stay in the column on account of the physical forces exerted by the mobile phase at very high pressure. Amount of loading on inner support is minimum stationary phase starts bleeding out of the column and can cause resistance to mass transfer. 40
41 METHOD OF PACKING Depends on the mechanical strength of stationary phase. Particle size of the stationary phase. Particles of greater then 20 µm dry packing Particles of lesser then 20 µm slurry packing / wet packing. DRY PACKING Particle size greater then 20 µm filled into vertical clamped column in small quantity. Deposition is done by tapping or vibrating the column. Column is unclamped and the tapped on the firm surface to obtain dense and reproducible packing. 41
42 WET / SLURRY PACKING Particle size with diameter less then 20 µm can only be placed wet as a suspension. Suspension should be stable, it should not sediment, and agglomentation should be avoided. 42
43 43
44 DETECTORS Based on the application, the detectors can be classified into Bulk property detectors Solute property detectors. Response time should be least (ten times less than the peak width of the solute in time units. 44
45 BULK PROPERTY DETECTORS Compare an over all change in physical property of mobile phase with or without an eluting solute. These types of detectors tend to be relatively insensitive and require temperature control. e.g. Refractive index detector. SOLUTE PROPERTY DETECTORS They respond to a physical property of the solute that is not exhibited by the pure mobile phase. These detectors are more sensitive, detect the sample in nanograms quantity. e.g. uv visible detector, electrochemical detector, fluorescence detector. 45
46 ULTRAVIOLET VISIBLE DETECTOR UV detectors are the most commonly used detector. They measure the ability of a sample to absorb light. This can be accomplished at one or several wavelengths. A variable wavelength UV detector, capable of monitoring from 190 to nm will be found suitable for the detection of the majority of samples. 46
47 Mobile phase from the column is passed through a flow cell held in the radiation beam of uv / visible spectrophotometer. Selective in nature, detect only those solutes that absorb uv/ visible radiation e.g. alkenes, aromatic compounds and compound having multiple bonds between C and O, N or S. BASICALLY TWO TYPES OF ABSORBANCE DETECTORS ARE AVAILABLE fixed wavelength detector variable wavelength detector 47
48 Fixed wavelength detector HPLC detectors which do not allow changing the wavelength of the radiation called fixedwavelength detectors. 48
49 Fixed wavelength detector Low-pressure mercury lamp emits very intense light at nm. By filtering out all other emitted wavelengths, utilize only 254 nm line to provide stable, highly sensitive detectors capable of measuring subnanogram quantities of any components which contains aromatic ring. The 254 nm was chosen since the most intense line of mercury lamp is 254 nm, and most of UV absorbing compounds have some absorbance at 254 nm. 49
50 VARIABLE-WAVELENGTH DETECTORS Detectors which allow the selection of the operating wavelength called variable wavelength detectors. 50
51 VARIABLE-WAVELENGTH DETECTORS Sensitivity for any absorptive component by selecting an appropriate wavelength individual sample components have high absorptivity at different wavelengths and thus, operation at a single wavelength would reduce the system's sensitivity Depending on the sophistication of the detector, wavelength change is done manually or programmed on a time basis into the memory of the system. 51
52 FLOURIMETRIC DETECTORS Very sensitive, but very selective. It is possible to detect even a presence of a single analyte molecule in the flow-cell. Fluorescence occurs when compounds having specific functional groups are excited by shorter wavelength energy and emit higher wavelength radiation. 52
53 FLOURIMETRIC DETECTORS Fluorescence is often collected at right angle to excitation beam. Only one sixth of fluorescence is collected. If concave mirror is placed around the sample cell abut 75 % of the emission is collected. With all sample cells, scattered radiation from the excitation source is selectively removed with cur off or band pass filters placed before photomultiplier tube. 53
54 Refractive Index Detector/ Differential refractometer The detection principle involves measuring of the change in refractive index of the column effluent passing through the flow-cell. It responds to any solute whose refractive index is significantly different from that of the mobile phase. Principle: it is based on two principles. Deflection ( deflection type refractometer) Reflection (reflection type refractometer) 54
55 Deflection type refractometer. Measure the deflection of a beam of a monochromatic light by double prism. Eluent passes through one half of prism & pure mobile phase to other half known as reference compartment. Reference and sample compartment are separated by diagonal glass divider. Auto zero is used to set, out put signal to zero when mobile phase is in both the compartments. 55
56 Deflection type refractometer. Tungsten lamp provides beam of light collimated through lens and passes through Eluent and reference compartment. Reflected by the mirror through the same compartment again. The beam of light is focused on a beam splitter before passing into the photo detector. 56
57 Deflection type refractometer. Refractive index of the mobile phase is changed due to the presence of solute, the beam from the sample compartment is deflected which produces the change signal that is proportional to the concentration of solute. Advantages Wide range of linearity. Covers entire refractive index range. 57
58 Reflection type refractometer Measure change in % of reflected light at glass liquid interface as the reflective index of liquid changes. Based on the Fresnels law of reflection which states The amount of liquid reflected at a glass- liquid interface varies with the angle of incidence and the refractive index of the liquid 58
59 working: Reflection type refractometer Two collimated beams from the projector (light source & lens) illuminate the reference and sample cell. Cells are formed of Teflon gasket, which is clamped between the cell prism and a stainless steel reflecting back plate. 59
60 As the light of beam is transmitted through the cell interfaces, it passes through the liquid film and impinges on the surface of the reflecting back plate. Diffused, reflected light appears as two spots and passes through the lens and detected by photo detector. The ratio of the reflected light to transmitted light is function of refractive index of the two liquid, the illumination of the cell back plate is direct measure of the refractive index of the liquid in each chamber 60
61 Electrochemical Detector/ Amperometric detector It is based on the measurements of the current resulting from an oxidation/reduction reaction of the analyte at a suitable electrode. The level of the current is directly proportional to the analyte concentration 61
62 Recorder and integrators Recorders are used to record the response obtained from the detector after amplification. They record the baseline and all the peaks obtained, with respect to time. Retention time for all the peaks can be calculated. Integrators are improved versions of recorder with data processing capabilities. They can record the individual peaks with retention time height and width of peak, peak area, etc. 62
63 References Instrumental method of analysis (seventh edition ) Willard merritt and dean settle page no Instrumental method of chemical analysis B.K.Sharma page no-, 58,59. Pharmaceutical drug analysis -2nd edition Ashutosh kar page no ,459,466 Pharmaceutical analysis vol-2 Dr.A.V.kasture page no 52,53. 63
64 Thank you 64
Chemistry Instrumental Analysis Lecture 31. Chem 4631
Chemistry 4631 Instrumental Analysis Lecture 31 High Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) Solvent Delivery
More informationBiochemistry. Biochemical Techniques HPLC
Description of Module Subject Name Paper Name 12 Module Name/Title 13 1. Objectives 1.1. To understand the basic concept and principle of 1.2. To understand the components and techniques of 1.3. To know
More informationHigh Pressure/Performance Liquid Chromatography (HPLC)
High Pressure/Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the
More informationHigh Performance Liquid Chromatography
High Performance Liquid Chromatography What is HPLC? It is a separation technique that involves: Injection of small volume of liquid sample Into a tube packed with a tiny particles (stationary phase).
More informationHPLC Workshop 16 June 2009 What does this do? Chromatography Theory Review Several chromatographic techniques Even though each method utilizes different techniques to separate compounds, the principles
More informationHigh Performance Liquid Chromatography
Updated: 3 November 2014 Print version High Performance Liquid Chromatography David Reckhow CEE 772 #18 1 HPLC System David Reckhow CEE 772 #18 2 Instrument Basics PUMP INJECTION POINT DETECTOR COLUMN
More informationHigh Performance Liquid Chromatography
Updated: 3 November 2014 Print version High Performance Liquid Chromatography David Reckhow CEE 772 #18 1 HPLC System David Reckhow CEE 772 #18 2 1 Instrument Basics PUMP INJECTION POINT DETECTOR COLUMN
More informationHPLC. High Performance Liquid Chromatography (HPLC) Harris Chapter 25
High Performance Liquid Chromatography (HPLC) Harris Chapter 25 12/1/2005 Chem 253 - Chapter 25 1 HPLC Separation of nonvolatile or thermally unstable compounds. If the analyte/sample can be found to be
More informationLC III: HPLC. Originally referred to as High-Pressure Liquid Chromatography. Now more commonly called High Performance Liquid Chromatography
LC III: HPLC What is HPLC? Originally referred to as High-Pressure Liquid Chromatography Now more commonly called High Performance Liquid Chromatography In general: The instrument controlled version of
More informationHPLC Praktikum Skript
HPLC Praktikum Skript Assistants: Gianluca Bartolomeo HCI D330, 3 46 68, bartolomeo@org.chem.ethz.ch Sahar Ghiasikhou HCI E330, 2 29 29, ghiasikhou@org.chem.ethz.ch 1. Introduction In chromatographic techniques,
More informationHigh Performance Liquid Chromatography
STANDARDBASE techniques: High Performance Liquid Chromatography Drenthe College, The Netherlands 1. Introduction HPLC. High Performance Liquid Chromatography High Performance Liquid Chromatography (HPLC)
More information2501 High Performance Liquid Chromatography
2501 High Performance Liquid Chromatography High Performance Liquid Chromatography Scheme Chp25:: 1 High Performance Liquid Chromatography Components of HPLC High Performance Liquid Chromatography Scheme
More informationPRINCIPLES AND APPLICATION OF CHROMATOGRAPHY. Dr. P. Jayachandra Reddy Mpharm PhD Principal & professor KTPC
PRINCIPLES AND APPLICATION OF CHROMATOGRAPHY Dr. P. Jayachandra Reddy Mpharm PhD Principal & professor KTPC CHROMATOGRAPHY Laboratory technique for the Separation of mixtures Chroma -"color" and graphein
More informationBasic principles of HPLC
Introduction to the theory of HPLC HPLC (High Performance Liquid Chromatography) depends on interaction of sample analytes with the stationary phase (packing) and the mobile phase to effect a separation.
More informationChromatography. Gas Chromatography
Chromatography Chromatography is essentially the separation of a mixture into its component parts for qualitative and quantitative analysis. The basis of separation is the partitioning of the analyte mixture
More informationChapter content. Reference
Chapter 7 HPLC Instrumental Analysis Rezaul Karim Environmental Science and Technology Jessore University of Science and Technology Chapter content Liquid Chromatography (LC); Scope; Principles Instrumentation;
More informationChem 230, Fall, 2014 Homework Set # 3 Short Answer SOLUTIONS
Chem 230, Fall, 2014 Homework Set # 3 Short Answer SOLUTIONS 1. List two advantages of temperature programming in GC. a) Allows separation of solutes with widely varying retention factors in a reasonable
More informationCHROMATOGRAPHY. The term "chromatography" is derived from the original use of this method for separating yellow and green plant pigments.
CHROMATOGRAPHY The term "chromatography" is derived from the original use of this method for separating yellow and green plant pigments. THEORY OF CHROMATOGRAPHY: Separation of two sample components in
More informationChemistry 3200 High Performance Liquid Chromatography: Quantitative Determination of Headache Tablets
Chemistry 3200 High Performance Liquid Chromatography: Quantitative Determination of Headache Tablets Liquid chromatography was developed by Tswett in early 1900 s and was shown to be a powerful separation
More informationInstrumental Chemical Analysis
L2 Page1 Instrumental Chemical Analysis Chromatography (General aspects of chromatography) Dr. Ahmad Najjar Philadelphia University Faculty of Pharmacy Department of Pharmaceutical Sciences 2 nd semester,
More informationCh.28 HPLC. Basic types of Liquid Chromatography Partition (LLC) Adsorption (LSC) Ion Exchange (IC) Size Exclusion (SEC or Gel Chromatography)
Ch.28 HPLC 28.1 Basic types of Liquid Chromatography Partition (LLC) Adsorption (LSC) Ion Exchange (IC) Size Exclusion (SEC or Gel Chromatography) High Performance (Pressure) LC Glass column st.steel (high
More informationChapter 1. Chromatography. Abdul Muttaleb Jaber
Chapter 1 Chromatography Abdul Muttaleb Jaber What is Chromatography? Chromatography is a physico-chemical process that belongs to fractionation methods same as distillation, crystallization or fractionated
More informationVolumetric Analysis. Quantitative analysis answers the second question
Volumetric Analysis Volumetric analysis is a form of quantitative analysis involving the measuring of volumes of reacting solutions, it involves the use of titrations. When buying food we often have two
More informationLUMEFANTRINUM LUMEFANTRINE
July 2008 LUMEFANTRINE: Final text for addition to The International Pharmacopoeia (July 2008) This monograph was adopted at the Forty-second WHO Expert Committee on Specifications for Pharmaceutical Preparations
More informationDetermination of Caffeine by HPLC
Determination of Caffeine by HPLC Introduction It was a long history before real high performance liquid chromatography (HPLC) had evolved. The very first indication of a chromatographic separation was
More informationChromatographic Methods of Analysis Section: 5 Gas Chromatography (GC) Prof. Tarek A. Fayed
Chromatographic Methods of Analysis Section: 5 Gas Chromatography (GC) Prof. Tarek A. Fayed Gas Chromatography (GC) In gas chromatography, the sample is vaporized and injected onto the head of a chromatographic
More informationCork Institute of Technology. Summer 2005 Instrumental Analysis (Time: 3 Hours) Section A
Cork Institute of Technology Higher Certificate in Science in Applied Biology Award (National Certificate in Science in Applied Biology Award) Answer FIVE questions; answer Section A, TWO questions from
More informationChemistry Gas Chromatography: Separation of Volatile Organics
Chemistry 3200 Gas chromatography (GC) is an instrumental method for separating volatile compounds in a mixture. A small sample of the mixture is injected onto one end of a column housed in an oven. The
More informationNEVIRAPINE ORAL SUSPENSION Final text for addition to The International Pharmacopoeia (February 2009)
February 2009. NEVIRAPINE ORAL SUSPENSION Final text for addition to The International Pharmacopoeia (February 2009) This monograph was adopted at the Forty-third WHO Expert Committee on Specifications
More informationChromatography. Mrs. D. MEENA MPharm PA & QA KTPC
Chromatography Mrs. D. MEENA MPharm PA & QA KTPC INTRODUCTION ANALYTICAL TECHNIQUES Analytical chemistry involves separating, identifying and determining the relative amount of the components in a sample
More informationGas Chromatography. Presented By Mr. Venkateswarlu Mpharm KTPC
Gas Chromatography Gas Chromatography Presented By Mr. Venkateswarlu Mpharm KTPC What is Gas Chromatography? It is also known as Gas-Liquid Chromatography (GLC) GAS CHROMATOGRAPHY Separation of gaseous
More informationChromTech HPLC System
ChromTech HPLC System High-prssure Gradient System (A) High-pressure pump: 2 pcs UV/VIS detector: 1 piece Gradient control module: 1 unit High-pressure gradient mixer: 1 piece Single channel chromatography
More informationChromatography & instrumentation in Organic Chemistry
Chromatography & instrumentation in Organic Chemistry What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify
More information2401 Gas (liquid) Chromatography
2401 Gas (liquid) Chromatography Chromatography Scheme Gas chromatography - specifically gas-liquid chromatography - involves a sample being vaporized and injected onto the head of the chromatographic
More informationLuminescence transitions. Fluorescence spectroscopy
Luminescence transitions Fluorescence spectroscopy Advantages: High sensitivity (single molecule detection!) Measuring increment in signal against a dark (zero) background Emission is proportional to excitation
More informationLiquid storage: Holds the solvent which is going to act as the mobile phase. Pump: Pushes the solvent through to the column at high pressure.
High performance liquid chromatography (HPLC) is a much more sensitive and useful technique than paper and thin layer chromatography. The instrument used for HPLC is called a high performance liquid chromatograph.
More informationChapter 27: Gas Chromatography
Chapter 27: Gas Chromatography Gas Chromatography Mobile phase (carrier gas): gas (He, N 2, H 2 ) - do not interact with analytes - only transport the analyte through the column Analyte: volatile liquid
More informationARTEMETHER AND LUMEFANTRINE ORAL SUSPENSION:Final text for addition to The International Pharmacopoeia (November 2008)
November 2008 ` ARTEMETER AND LUMEFANTRINE RAL SUSPENSIN:Final text for addition to The International Pharmacopoeia (November 2008) Category. Antimalarial. Storage. Artemether and lumefantrine oral suspension
More informationChapter 27: Gas Chromatography. Principles Instrumentation Detectors Columns and Stationary Phases Applications
Chapter 27: Gas Chromatography Principles Instrumentation Detectors Columns and Stationary Phases Applications GC-MS Schematic Interface less critical for capillary columns Several types of Mass Specs
More informationInstrumental Analysis II Course Code: CH3109. Chromatographic &Thermal Methods of Analysis Part 1: General Introduction. Prof. Tarek A.
Instrumental Analysis II Course Code: CH3109 Chromatographic &Thermal Methods of Analysis Part 1: General Introduction Prof. Tarek A. Fayed What is chemical analysis? Qualitative analysis (1) Chemical
More informationInstitute for Chemical Education, Fun With Chemistry; Vol. 1, Sarquis, Mickey and Sarquis, Gerry, Ed.; University of Wisconsin Madison, 1991,
EXPERIIMENT #7 LIIQUIID CHROMATOGRAPHY References: Bidlingmeyer, B. A.; Warren Jr., F. V. An Inexpensive Experiment for the Introduction of High Performance Liquid Chromatography J. Chem. Educ. 1984, 61,
More informationSpectroscopy. Page 1 of 8 L.Pillay (2012)
Spectroscopy Electromagnetic radiation is widely used in analytical chemistry. The identification and quantification of samples using electromagnetic radiation (light) is called spectroscopy. Light has
More informationTransferring HPLC methods from the HP 1090 Series to the Agilent 1100 Series HPLC system
Transferring HPLC methods from the HP 1090 Series to the Agilent 1100 Series HPLC system Technical Note Introduction The HP 1090 Series HPLC system, introduced in 1983, is regarded to be an outstanding
More informationFigure 1. Structures for Vitamin B2 and Vitamin B1.
CH 461 & CH 461H F 18 Name Experiment 2C Integrated Laboratory Experiment DETERMINATION OF RIBOFLAVIN: A COMPARISON OF TECHNIQUES PART C. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY The purpose of this experiment
More informationChromatographic Analysis
Chromatographic Analysis Distribution of Analytes between Phases An analyte is in equilibrium between the two phases [S 1 ] [S 2 ] (in phase 1) (in phase 2) AS [S2 ] K 2 A S [S1 ] 1 AS, A 1 S Activity
More informationARTEMETHER AND LUMEFANTRINE TABLETS: Final text for addition to The International Pharmacopoeia (July 2008)
July 2008 ARTEMETER AND LUMEFANTRINE TABLETS: Final text for addition to The International Pharmacopoeia (July 2008) This monograph was adopted at the Forty-second W Expert Committee on Specifications
More informationDATES: LAB: Liquid Chromatography Separation of Grape Kool-Aid
NAME: AP CHEMISTRY DATES: LAB: Liquid Chromatography Separation of Grape Kool-Aid PURPOSE There are a number of analytical techniques used to separate components of a mixture, or solution. They include
More informationSpectroscopy and Chromatography
Spectroscopy and Chromatography Introduction Visible light is one very small part of the electromagnetic spectrum. The different properties of the various types of radiation depend upon their wavelength.
More informationChemistry Instrumental Analysis Lecture 27. Chem 4631
Chemistry 4631 Instrumental Analysis Lecture 27 Gas Chromatography Introduction GC covers all chromatographic methods in which the mobile phase is gas. It may involve either a solid stationary phase (GSC)
More information7 INSTRUMENTAL CHROMATOGRAPHY
7 INSTRUMENTAL CHROMATOGRAPHY 7.1 Introduction There are two forms of chromatography, very widely used in analytical laboratories, which rely on electronic control of the process and detection of the species.
More informationThe Low-Temperature Evaporative Light-Scattering Detector (LT-ELSD)
The Low-Temperature Evaporative Light-Scattering Detector (LT-ELSD) Basically all compounds which are less volatile than the mobile phase can be detected. Detection is based on a Universal property of
More informationPhysical Separations and Chromatography
Lab #5A & B: Physical Separations and Chromatography Individual Objectives: At the end of these experiments you should be able to: Ø Distinguish between Rf and tr; chromatograph and chromatogram; adsorption
More informationPacked Column for Ultra-Fast Reversed-Phase Liquid Chromatography, TSKgel Super-ODS. Table of Contents
No. 089 SEPARATION REPORT Packed Column for Ultra-Fast Reversed-Phase Liquid Chromatography, TSKgel Super-ODS Table of Contents 1. Introduction 1 2. Column Specification 1 3. Features of Packing Materials
More informationGas Chromatography (GC)! Environmental Organic Chemistry CEE-PUBH Analysis Topic 5
Gas Chromatography (GC)! Environmental Organic Chemistry CEE-PUBH 5730-6730 Analysis Topic 5 Chromatography! Group of separation techniques based on partitioning (mobile phase/stationary phase). Two immiscible
More informationFall 2012 Due In Class Friday, Oct. 19. Complete the following on separate paper. Show your work and clearly identify your answers.
CHEM 322 Name Fall 2012 Due In Class Friday, Oct. 19 Complete the following on separate paper. Show your work and clearly identify your answers. General Separations 1. Describe the relative contributions
More informationChromatography and its applications
Chromatography and its applications Reference Introduction to chromatography theory and practice Instrumental methods of chemical analysis by H.Kaur INTRODUCTION There are many methods which have been
More informationION CHROMATOGRAPHY SYSTEM S 150
ION CHROMATOGRAPHY SYSTEM S 150 WATER ANALYSIS ENVIRONMENTAL ANALYSIS ANION & CATION ANALYSIS ION CHROMATOGRAPHY IIon Chromatography is an analytical separation technique based on ionic interactions. Dissolved
More informationFigure 1. Structures for Vitamin B2 and Vitamin B1.
CH 461 & CH 461H F 15 Name Experiment 2C Integrated Laboratory Experiment DETERMINATION OF RIBOFLAVIN: A COMPARISON OF TECHNIQUES PART C. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY The purpose of this experiment
More informationChromatography. Chromatography is a combination of two words; * Chromo Meaning color * Graphy representation of something on paper (writing)
Chromatography Chromatography is a combination of two words; * Chromo Meaning color * Graphy representation of something on paper (writing) Invention of Chromatography Mikhail Tswett invented chromatography
More informationHarris: Quantitative Chemical Analysis, Eight Edition CHAPTER 23: GAS CHROMATOGRAPHY
Harris: Quantitative Chemical Analysis, Eight Edition CHAPTER 23: GAS CHROMATOGRAPHY Chapter 23. Gas Chromatography What did they eat in the year 1,000? GC of Cholesterol and other lipids extracted from
More informationDetermination of Polymer Modifier in Asphalt
Standard Method of Test for Determination of Polymer Modifier in Asphalt AASHTO Designation: T xxx-xx (2005) 1. SCOPE 1.1. This method of test is used to determine the polymer content of an asphalt sample.
More informationHigh Performance Liquid Chromatography (HPLC)
High Performance Liquid Chromatography (HPLC) Ahmad Aqel Ifseisi Assistant Professor of Analytical Chemistry College of Science, Department of Chemistry King Saud University P.O. Box 2455 Riyadh 11451
More informationQuestions on Instrumental Methods of Analysis
Questions on Instrumental Methods of Analysis 1. Which one of the following techniques can be used for the detection in a liquid chromatograph? a. Ultraviolet absorbance or refractive index measurement.
More informationLC Technical Information
LC Technical Information Method Transfer to Accucore.6 μm Columns Containing solid core particles, which are engineered to a diameter of.6μm and a very narrow particle size distribution; Accucore HPLC
More informationLiquid Chromatography
Liquid Chromatography 1. Introduction and Column Packing Material 2. Retention Mechanisms in Liquid Chromatography 3. Method Development 4. Column Preparation 5. General Instrumental aspects 6. Detectors
More informationH 3 CO H 3 CO S CH 3
FENITROTHION 35 H 3 CO P H 3 CO S O CH 3 NO 2 ISO common name Chemical name Fenitrothion O,O-Dimethyl O-4-nitro-m-tolyl phosphorothioate (IUPAC) O,O-Dimethyl O-(3-methyl-4-nitrophenyl)- phosphorothioate
More informationChemistry 311: Instrumental Analysis Topic 4: Basic Chromatography. Chemistry 311: Instrumental Analysis Topic 4: Basic Chromatography
Introductory Theory, Basic Components, Qualitative and Quantitative applications. HPLC, GC, Ion Chromatography. Rouessac Ch. 1-7 Winter 2011 Page 1 Chromatography: The separation of analytes based on differences
More informationMass Spectrometry in MCAL
Mass Spectrometry in MCAL Two systems: GC-MS, LC-MS GC seperates small, volatile, non-polar material MS is detection devise (Agilent 320-MS TQ Mass Spectrometer) Full scan monitoring SIM single ion monitoring
More informationHPLC Background Chem 250 F 2008 Page 1 of 24
HPLC Background Chem 250 F 2008 Page 1 of 24 Outline: General and descriptive aspects of chromatographic retention and separation: phenomenological k, efficiency, selectivity. Quantitative description
More informationExperiment 1: Thin Layer Chromatography
Experiment 1: Thin Layer Chromatography Part A: understanding R f values Part B: R f values & solvent polarity Part C: R f values & compound functionality Part D: identification of commercial food dye
More informationCYCLOSERINE Final text for addition to The International Pharmacopoeia. (November 2008) CYCLOSERINUM CYCLOSERINE
December 2008 CYCLOSERINE Final text for addition to The International Pharmacopoeia (November 2008) This monograph was adopted at the Forty-third WHO Expert Committee on Specifications for Pharmaceutical
More informationPure Chromatography Consumables Pure flexibility. Pure specialization. Pure convenience.
Pure Chromatography Consumables Pure flexibility. Pure specialization. Pure convenience. Pure Consumables More focus on your application The Pure consumable portfolio offers an unrivaled range of products
More informationLIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC/MS) Presented by: Dr. T. Nageswara Rao M.Pharm PhD KTPC
LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC/MS) Presented by: Dr. T. Nageswara Rao M.Pharm PhD KTPC INTRODUCTION Principle: LC/MS is a technique that combines physical separation capabilities of liquid
More informationGC Instruments. GC Instruments - Columns
GC Instruments 1 Fairly simple instrumentation Maintaining constant average pressure is important! Pressure controls flow rate T influences retention (k ) Flow rate monitoring Changing flow rate changes
More informationDetermination of Volatile Substances Proof of Food Adulteration
ANALYSIS OF FOOD AND NATURAL PRODUCTS LABORATORY EXERCISE Determination of Volatile Substances Proof of Food Adulteration (method: gas chromatography with mass spectrometric detection) Exercise guarantor:
More informationChem 321 Name Answer Key D. Miller
1. For a reversed-phase chromatography experiment, it is noted that the retention time of an analyte decreases as the percent of acetonitrile (CH 3 CN) increases in a CH 3 CN/H 2 O mobile phase. Explain
More informationGUIDELINES FOR THE DESIGN OF CHROMATOGRAPHIC ANALYTICAL METHODS INTENDED FOR CIPAC COLLABORATIVE STUDY
Page 1 of 13 CIPAC/4105/R GUIDELINES FOR THE DESIGN OF CHROMATOGRAPHIC ANALYTICAL METHODS INTENDED FOR CIPAC COLLABORATIVE STUDY Prepared for CIPAC by Dr M J Tandy*, P M Clarke and B White (UK) The rapid
More informationGas Chromatography (GC)
Gas Chromatography (GC) Ahmad Aqel Ifseisi Assistant Professor of Analytical Chemistry College of Science, Department of Chemistry King Saud University P.O. Box 2455 Riyadh 11541 Saudi Arabia Office: AA53
More informationHigh Performance Liquid Chromatography (HPLC) of Three Analgesics
High Performance Liquid Chromatography (HPLC) of Three Analgesics OVERVIEW/THEORY Real-life samples are often complex mixtures of many substances (e.g., sea water contains many salts and other impurities
More informationLECTURE 2. Advanced Separation Science Techniques Present and Future Separation Tools
LECTURE 2 Advanced Separation Science Techniques Present and Future Separation Tools Jack Henion, Ph.D. Emeritus Professor, Analytical Toxicology Cornell University Ithaca, NY 14850 Lecture 2, Page 1 Contents
More informationChromatographic Methods of Analysis Section 2: Planar Chromatography. Prof. Tarek A. Fayed
Chromatographic Methods of Analysis Section 2: Planar Chromatography Prof. Tarek A. Fayed Planar chromatography includes two types: 1- Thin Layer Chromatography (TLC). 2- Paper Chromatography (PC). Thin
More informationAbstract: An minimalist overview of chromatography for the person who would conduct chromatographic experiments, but not design experiments.
Chromatography Primer Abstract: An minimalist overview of chromatography for the person who would conduct chromatographic experiments, but not design experiments. At its heart, chromatography is a technique
More information10/2/2008. hc λ. νλ =c. proportional to frequency. Energy is inversely proportional to wavelength And is directly proportional to wavenumber
CH217 Fundamentals of Analytical Chemistry Module Leader: Dr. Alison Willows Electromagnetic spectrum Properties of electromagnetic radiation Many properties of electromagnetic radiation can be described
More informationTEPZZ A_T EP A1 (19) (11) EP A1 (12) EUROPEAN PATENT APPLICATION. (51) Int Cl.:
(19) TEPZZ 6 6697A_T (11) EP 2 626 697 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 14.08.2013 Bulletin 2013/33 (1) Int Cl.: G01N 30/32 (2006.01) G01N 30/20 (2006.01) (21) Application
More informationIntroduction to Chromatographic Separations
Introduction to Chromatographic Separations Analysis of complex samples usually involves previous separation prior to compound determination. Two main separation methods instrumentation are available:
More informationOpen Column Chromatography, GC, TLC, and HPLC
Open Column Chromatography, GC, TLC, and HPLC Murphy, B. (2017). Introduction to Chromatography: Lecture 1. Lecture presented at PHAR 423 Lecture in UIC College of Pharmacy, Chicago. USES OF CHROMATOGRAPHY
More informationTECHNICAL TEMEPHOS. 1. Specification. Full specification WHO/SIT/19.R4 Revised 10 December Description
WHO/SIT/19.R4 TEMEPHOS TECHNICAL TECHNICAL TEMEPHOS 1. Specification 1.1 Description Full specification WHO/SIT/19.R4 Revised 10 December 1999 The material shall consist of temephos together with related
More informationCfE Higher Chemistry. Unit 3: Chemistry in Society. Chemical Analysis as part of quality control
CfE Higher Chemistry Unit 3: Chemistry in Society Chemical Analysis as part of quality control 06/12/2017 Composition and purity 06/12/2017 Learning Outcomes : I can explain the basic principle of how
More informationAtomization. In Flame Emission
FLAME SPECTROSCOPY The concentration of an element in a solution is determined by measuring the absorption, emission or fluorescence of electromagnetic by its monatomic particles in gaseous state in the
More informationGas Chromatography. Introduction
Gas Chromatography Introduction 1.) Gas Chromatography Mobile phase (carrier gas) is a gas - Usually N 2, He, Ar and maybe H 2 - Mobile phase in liquid chromatography is a liquid Requires analyte to be
More informationFigure 1 Enlargement of Powdered Activated Carbon by microscope.
Introduction The following is a description of how the Powdered Activated Carbon (PAC) dosing system operates. Firstly an overview of how the Semi Dry Reactor and the purpose of carbon injection is described.
More informationPrinciples of Gas- Chromatography (GC)
Principles of Gas- Chromatography (GC) Mohammed N. Sabir January 2017 10-Jan-17 1 GC is a chromatographic technique utilizes gas as the mobile phase which is usually an inert gas (Hydrogen, Helium, Nitrogen
More informationGas chromatography. Advantages of GC. Disadvantages of GC
Advantages of GC Gas chromatography Fast analysis, typically minutes Effi cient, providing high resolution Sensitive, easily detecting ppm and often ppb Nondestructive, making possible on - line coupling;
More informationFundamentals of Mass Spectrometry. Fundamentals of Mass Spectrometry. Learning Objective. Proteomics
Mass spectrometry (MS) is the technique for protein identification and analysis by production of charged molecular species in vacuum, and their separation by magnetic and electric fields based on mass
More informationfor Acclaim Mixed-Mode HILIC-1 Column
for Acclaim Mixed-Mode HILIC-1 Column Product Manual for ACCLAIM Mixed-Mode HILIC-1 Page 1 of 17 Product Manual for ACCLAIM Mixed-Mode HILIC-1 Column 5µm, 4.6 x 250mm, P/N 066844 5µm, 4.6 x 150mm, P/N
More information2. a) R N and L N so R L or L R 2.
1. Use the formulae on the Some Key Equations and Definitions for Chromatography sheet. a) 0.74 (remember that w b = 1.70 x w ½ ) b) 5 c) 0.893 (α always refers to two adjacent peaks) d) 1.0x10 3 e) 0.1
More informationNew Product Update. HPLC Columns
New Product Update HPLC Columns ZORBAX MicroBore HPLC Columns ZORBAX Extend-C18 Columns ZORBAX Bonus-RP Columns Inertsil ODS-2 Cartridge Columns ZORBAX Carbohydrate Analysis Columns ZORBAX Eclipse dsdna
More informationChromatography- Separation of mixtures CHEM 212. What is solvent extraction and what is it commonly used for?
Chromatography- Separation of mixtures CHEM 212 What is solvent extraction and what is it commonly used for? How does solvent extraction work? Write the partitioning coefficient for the following reaction:
More informationMEDROXYPROGESTERONE INJECTION
September 2011 RESTRICTED DRAFT PROPOSAL FOR The International Pharmacopoeia MEDROXYPROGESTERONE INJECTION (September 2011) DRAFT FOR COMMENT Should you have any comments on the attached draft proposal,
More informationWhat type of samples are common? Time spent on different operations during LC analyses. Number of samples? Aims. Sources of error. Sample preparation
What type of samples are common? Sample preparation 1 2 Number of samples? Time spent on different operations during LC analyses 3 4 Sources of error Aims Sample has to be representative Sample has to
More information