Open Column Chromatography, GC, TLC, and HPLC

Size: px
Start display at page:

Download "Open Column Chromatography, GC, TLC, and HPLC"

Transcription

1 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 AND SPECTROSCOPY Chromatography = process of separating compounds to isolate a specific molecule Spectroscopy = identifies a specific molecule Starting with a mixture of unknowns, you can use chromatography and a reference standard to try and identify substances Starting with a mixture of compounds, can use chromatography to separate and isolate a molecule and then spectroscopy to identify this molecule If retrieving a molecule from a biological system, use spectroscopy to localize the molecule and deduce a mechanism of action CHROMATOGRAPHY Utilizes a stationary phase and a mobile phase to separate compounds in a mixture Most common mechanisms of separation: o Partition method utilizes like-dissolves-like theory o Adsorption interaction at a surface (stationary phase) with certain compounds, while others get pulled out with the stationary phase Interactions are relatively weak, including dipole-dipole, dipole-induced dipole, and induced dipole- induced dipole o Exclusion porous stationary phase separates molecules by size Liquid chromatography (LC) vs. Gas chromatography (GC) o LC = solution is passed through a system with an adsorbent stationary phase and a liquid mobile phase. Solutes in the solution will separate based on if they are adsorbent to the stationary phase or not. Mobile phase passes through the column using pressure or just gravity Utilizes differing POLARITIES to separate these solutes Utilizes SOLUBILITY of the solute to keep in mobile phase o GC = gas passes through a system with an adsorbent stationary phase and a gaseous mobile phase. Volatiles in the gas will separate based on if they are adsorbent to the stationary phase or not. Mobile phase passes through column using pressure Utilizes differing POLARITIES to separate these volatiles Utilizes VOLATILITY of the gas to keep in mobile phase

2 Column can be glass or metal o Metal used for gas and high performance liquid chromatography o Glass used for column chromatography Stationary phase o Composed of organic molecules fused to particles (usually silica) o In normal phase chromatography the stationary phase is POLAR and HYDROPHILIC Groups = -OH, -NH2, CN, etc o In reverse phase chromatography the stationary phase is NON-POLAR and HYDROPHOBIC Groups = C8, C18, phenyl, phenyl-hexyl, etc Mobile phase o Liquid that is composed of different chemicals to alter polarity o In normal phase chromatography the mobile phase is relatively (in comparison to stationary phase) NON-POLAR and HYDROPHOBIC Groups = isopropanol > ethyl acetate > chloroform > dichloromethane > hexanes (starting from most hydrophobic to least) o In reverse phase chromatography the mobile phase is relatively (in comparison to stationary phase) POLAR and HYDROPHILIC Groups = water > acetonitrile > methanol (starting from most hydrophilic to least) o Important note** - the strength of the solvent (mobile phase) is based on how closely it resembles the stationary phase. The more closely it resembles the stationary phase, the stronger it is (b/c can compete with stationary phase for solutes) o Flows: Isocratic flow same composition throughout the entire duration of the separation Step gradient flow isocratic steps (gets progressively more polar or nonpolar with each addition of solvent) Gradient flow almost the same as step-gradient flow except this continues progressively over time rather than in steps (needs a machine to be accomplished, so HPLC or GC) Band widening occurs as one analyte separates from another throughout the column o If different bands overlap (broadening is too slow) won t be able to separate the analytes o To improve band separation, may need a stronger solvent (mobile phase)

3 o **Important if you want to use a stronger solvent to increase band separation, do not go TOO much stronger. If needed something to be more polar, for example, for a normal phase chromatography, still wouldn t want to use an actual polar solvent. Just need something slightly more polar (but still relatively non-polar in comparison to the stationary phase) than the current solvent THIN LAYER CHROMATOGRAPHY Stationary phase (usually silica gel) coats a plate, and is placed in a container with the mobile phase (liquid) The mobile phase travels up the plate and solutes bind to the stationary phase depending on relative polarity The plate can be visualized with various methods in order to see where different analytes have bound to the stationary phase COLUMN CHROMATOGRAPHY Different column types: o Open/gravity column = pack column with solid stationary phase and let gravity pull down the mobile phase o Flash column = uses pressure from the top to push the mobile phase down o Vacuum liquid column = uses vacuum at the bottom of the column to pull the mobile phase down

4 Solid phase extraction column = comes pre-packed with stationary phase o Usually used to quickly run a solution through and get rid of unwanted material before running through an HPLC column o Is disposable Small particle size gives better resolution, but would need more pressure to pull mobile phase down HPLC High Performance Liquid Chromatography uses moderate to high pressure to push solvent through the column Ultra Performance Liquid Chromatograph uses extremely high pressures to push solvent through the column with very small particles in the stationary phase As column length increases, resolution increases o There is more time for the solvent to be in contact with the stationary phase As column particle size decreases, resolution increases o There are more chances for the solvent to interact with the stationary phase (more particles) COMPONENTS OF HPLC INSTRUMENTATION Injection port to inject the analyte Mobile phase Controller to set parameters of the experiment Pumps hooked up to the solvent to push it through Column UV detector to identify the samples Degasser use to get rid of bubbles in the solvent Autosampler allows you to run multiple samples in the same sequence SCALES FOR HPLC Preparative o For larger sample sizes > 5 mg o Use flow rate > 10mL/min Semi-preparative o For sample sizes > mg o Use flow rate > 1-3 ml/min Analytical o For smaller sample sizes > mg o Use flow rate > ml/min

5

6 THE CHROMATOGRAM Gives you a readout for what s in your sample o X-axis is time o Y-axis is absorbance Can compare your readout with reference compounds to try and identify the analyte o MUST be using the same system in order to compare (same stationary phase, same mobile phase) Can use to isolate pure compounds GEL PERMEATION/ SIZE EXCLUSION CHROMATOGRAPHY Particles in the stationary phase has holes in them so that smaller solutes in the mobile phase will get trapped there and larger molecules will bypass the stationary phase Used for protein separations o If you know the MW of the protein you want to isolate, can determine which size pores you will need in your stationary phase AFFINITY CHROMATOGRAPHY Stationary phase is made custom to bind a particular analyte

7

8

9

HPLC Background Chem 250 F 2008 Page 1 of 24

HPLC 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 information

Chromatography. Gas Chromatography

Chromatography. 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 information

High Performance Liquid Chromatography

High 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 information

Instrumental Chemical Analysis

Instrumental 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 information

High Performance Liquid Chromatography

High 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 information

Introduction to Chromatography

Introduction to Chromatography Introduction to Chromatography Dr. Sana Mustafa Assistant Professor Department of Chemistry, Federal Urdu University of Arts, Science & Technology, Karachi. What is Chromatography? Derived from the Greek

More information

Chromatography- 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? 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 information

Downstream Processing Prof. Mukesh Doble Department Of Biotechnology Indian Institute of Technology, Madras. Lecture - 33 HPLC

Downstream Processing Prof. Mukesh Doble Department Of Biotechnology Indian Institute of Technology, Madras. Lecture - 33 HPLC Downstream Processing Prof. Mukesh Doble Department Of Biotechnology Indian Institute of Technology, Madras Lecture - 33 HPLC Today, we are going to talk about the HPLC. HPLC is an analytical tool, which

More information

Abstract: An minimalist overview of chromatography for the person who would conduct chromatographic experiments, but not design experiments.

Abstract: 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 information

HPLC. High Performance Liquid Chromatography (HPLC) Harris Chapter 25

HPLC. 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 information

Chapter content. Reference

Chapter 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 information

Gas Chromatography. Vaporization of sample Gas-solid Physical absorption Gas-liquid Liquid immobilized on inert solid

Gas Chromatography. Vaporization of sample Gas-solid Physical absorption Gas-liquid Liquid immobilized on inert solid Gas Chromatography Vaporization of sample Gas-solid Physical absorption Gas-liquid Liquid immobilized on inert solid Principles Instrumentation Applications 18-1 Retention Volumes Volumes rather than times

More information

Course goals: Course goals: Lecture 1 A brief introduction to chromatography. AM Quality parameters and optimization in Chromatography

Course goals: Course goals: Lecture 1 A brief introduction to chromatography. AM Quality parameters and optimization in Chromatography Emqal module: M0925 - Quality parameters and optimization in is a separation technique used for quantification of mixtures of analytes Svein.mjos@kj.uib.no Exercises and lectures can be found at www.chrombox.org/emq

More information

High Pressure/Performance Liquid Chromatography (HPLC)

High 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 information

Chromatography. Mrs. D. MEENA MPharm PA & QA KTPC

Chromatography. 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 information

Chromatography Outline

Chromatography Outline Chem 2001 Summer 2004 Outline What is? The Chromatogram Optimization of Column Performance Why Do Bands Spread? Gas High-Performance Liquid Ion-Exchange 2 What is? In chromatography, separation is achieved

More information

https://www.chemicool.com/definition/chromatography.html

https://www.chemicool.com/definition/chromatography.html CHROMATOGRAPHY 1 Chromatography - a physical method of mixture separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while

More information

DEFINITION CHROMATOGRAPHY

DEFINITION CHROMATOGRAPHY Chromatography DEFINITION CHROMATOGRAPHY The separation of a mixture by distribution of its components between a mobile and stationary phase over time mobile phase = solvent stationary phase = column packing

More information

Chromatography & instrumentation in Organic Chemistry

Chromatography & 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 information

High Performance Liquid Chromatography

High 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 information

High Performance Liquid Chromatography

High 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 information

penta-hilic UHPLC COLUMNS

penta-hilic UHPLC COLUMNS penta-hilic UHPLC COLUMNS penta-hilic Highly retentive, proprietary penta-hydroxy-ligand Excellent peak shape for polar compounds with a variety of functional groups: acids, bases, zwitterions strong and

More information

Liquid storage: Holds the solvent which is going to act as the mobile phase. Pump: Pushes the solvent through to the column at high pressure.

Liquid 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 information

Instrumental 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. 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 information

Introduction to Chromatographic Separations

Introduction 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 information

CHEMISTRY Unit 3, Area of Study 1: Chemical Analysis

CHEMISTRY Unit 3, Area of Study 1: Chemical Analysis Watch this lesson online: https://edrolo.com.au/vce/subjects/chemistry/vce-chemistry/aos-1-chemical-analysis/chromatography-hplc-glc/column-chromatography/#watch CHEMISTRY Unit 3, Area of Study 1: Chemical

More information

HPLC 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 information

Chromatographic Analysis

Chromatographic 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 information

Chromatographic Separation

Chromatographic Separation What is? is the ability to separate molecules using partitioning characteristics of molecule to remain in a stationary phase versus a mobile phase. Once a molecule is separated from the mixture, it can

More information

Chromatography. 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) 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 information

Luminescence transitions. Fluorescence spectroscopy

Luminescence 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 information

Chromatography: Thin-Layer Chromatography (TLC) & Column Chromatography

Chromatography: Thin-Layer Chromatography (TLC) & Column Chromatography Chromatography: Thin-Layer Chromatography (TLC) & Column Chromatography Part 1, p. 184: Separation of spinach pigments by TLC. (4 th Ed. P. 180) Part 2, p. 192: Separation of Fluorene and Fluorenone by

More information

Chapter 23 Introduction to Analytical Separations

Chapter 23 Introduction to Analytical Separations Chapter 23 Introduction to Analytical Separations Homework Due Monday April 24 Problems 23-1, 23-2, 23-7, 23-15, 23-27, 23-29, 23-32 Analytical Separations: Universal approach to analyzing complex mixtures

More information

Experiment 1: Thin Layer Chromatography

Experiment 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 information

Column Liquid Chromatography Experiment Adapted for Use in Secondary Schools

Column Liquid Chromatography Experiment Adapted for Use in Secondary Schools Column Liquid Chromatography Experiment Adapted for Use in Secondary Schools Mark Langella WISTA The most modern and sophisticated methods of separating mixtures that the organic chemist has available

More information

PRINCIPLES 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 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 information

Chromatography. writing in color

Chromatography. writing in color Chromatography writing in color Outlines of Lecture Chromatographic analysis» Principles and theory.» Definition.» Mechanism.» Types of chromatography.» Uses of Chromatography. In 1906 Mikhail Tswett used

More information

Analytical Chemistry

Analytical Chemistry Analytical Chemistry Chromatographic Separations KAM021 2016 Dr. A. Jesorka, 6112, aldo@chalmers.se Introduction to Chromatographic Separations Theory of Separations -Chromatography Terms Summary: Chromatography

More information

Gel Permeation Chromatography - GPC

Gel Permeation Chromatography - GPC Isolation and Separation Methods J. Poustka, VŠCHT Praha, ÚAPV 2014, http://web.vscht.cz/poustkaj Gel Permeation Chromatography - GPC Separation and clean-up method Group separation of compounds with similar

More information

Chemistry Instrumental Analysis Lecture 28. Chem 4631

Chemistry Instrumental Analysis Lecture 28. Chem 4631 Chemistry 4631 Instrumental Analysis Lecture 28 High Performance Liquid Chromatography () Instrumentation Normal Phase Chromatography Normal Phase - a polar stationary phase with a less polar mobile phase.

More information

LC and LC/MS Column Selection Flow Chart

LC and LC/MS Column Selection Flow Chart LC and LC/MS Column Selection Flow Chart To use the column selection diagram below, simply follow the path for your analyte and mobile phase. At the far right, follow your final column selection to the

More information

Packings for HPLC. Packings for HPLC

Packings for HPLC. Packings for HPLC Summary of packings for HPLC In analytical HPLC, packings with particle sizes of 3 to 10 µm are preferred. For preparative separation tasks, also particles with diameters larger than 10 µm are applied.

More information

Liquid Chromatography

Liquid 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 information

Ch.28 HPLC. Basic types of Liquid Chromatography Partition (LLC) Adsorption (LSC) Ion Exchange (IC) Size Exclusion (SEC or Gel Chromatography)

Ch.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 information

Chapter 26. An Introduction to Chromatographic Separations. Chromatography

Chapter 26. An Introduction to Chromatographic Separations. Chromatography Chapter 26 An Introduction to Chromatographic Separations Chromatography 1 Chromatography-Model as Extraction Chromatography-Model as Extraction 2 Chromatography Planar Chromatography-Types paper chromatography

More information

Chem 230, Fall, 2014 Homework Set # 3 Short Answer SOLUTIONS

Chem 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 information

Chemistry 3200 High Performance Liquid Chromatography: Quantitative Determination of Headache Tablets

Chemistry 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 information

LEARNING OBJECTIVES CHEM 212: SEPARATION SCIENCE CHROMATOGRAPHY UNIT. Thomas Wenzel, Bates College. In-class Problem Set Extraction.

LEARNING OBJECTIVES CHEM 212: SEPARATION SCIENCE CHROMATOGRAPHY UNIT. Thomas Wenzel, Bates College. In-class Problem Set Extraction. LEARNING OBJECTIVES CHEM 212: SEPARATION SCIENCE CHROMATOGRAPHY UNIT Thomas Wenzel, Bates College In-class Problem Set Extraction Problem #1 1. Devise a scheme to be able to isolate organic acids, bases

More information

Chromatography. What is Chromatography?

Chromatography. What is Chromatography? Chromatography What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. Mixture

More information

CHROMATOGRAPHY AND MASS SPECTROMETER

CHROMATOGRAPHY AND MASS SPECTROMETER 22 CHROMATOGRAPHY AND MASS SPECTROMETER 22.1 INTRODUCTION We know that the biochemistry or biological chemistry deals with the study of molecules present in organisms. These molecules are called as biomolecules

More information

CHROMATOGRAPHY. 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. 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 information

Chapter 27: Gas Chromatography. Principles Instrumentation Detectors Columns and Stationary Phases Applications

Chapter 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 information

CHEM 429 / 529 Chemical Separation Techniques

CHEM 429 / 529 Chemical Separation Techniques CHEM 429 / 529 Chemical Separation Techniques Robert E. Synovec, Professor Department of Chemistry University of Washington Lecture 1 Course Introduction Goal Chromatography and Related Techniques Obtain

More information

penta-hilic UHPLC COLUMNS

penta-hilic UHPLC COLUMNS penta-hilic UHPLC COLUMNS Highly retentive, proprietary penta-hydroxy-ligand penta-hilic Excellent peak shape for polar compounds with a variety of functional groups: acids, bases, zwitterions strong and

More information

Partitioning. Separation is based on the analyte s relative solubility between two liquid phases or a liquid and solid.

Partitioning. Separation is based on the analyte s relative solubility between two liquid phases or a liquid and solid. Chromatography Various techniques for the separation of complex mixtures that rely on the differential affinities of substances for a gas or liquid mobile medium and for a stationary adsorbing medium through

More information

Chromatographic Methods: Basics, Advanced HPLC Methods

Chromatographic Methods: Basics, Advanced HPLC Methods Chromatographic Methods: Basics, Advanced HPLC Methods Hendrik Küpper, Advanced Course on Bioinorganic Chemistry & Biophysics of Plants, summer semester 2018 Chromatography: Basics Chromatography a physical

More information

Disadvantage: Destructive Technique once analyzed by GC, the sample is lost

Disadvantage: Destructive Technique once analyzed by GC, the sample is lost Gas Chromatography Like other methods of chromatography, a partitioning of molecules must occur between the stationary phase and the mobile phases in order to achieve separation. This is the same equilibrium

More information

M > ACN > > THF

M > ACN > > THF Method Development in HPLC Dr. Amitha Hewavitharana School of Pharmacy University of Queensland Method Development in HPLC References: D A Skoog Principles of instrumental analysis, 3 rd Edition Chapters

More information

Chapter 27: Gas Chromatography

Chapter 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 information

Principles of Gas- Chromatography (GC)

Principles 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 information

Chemistry Instrumental Analysis Lecture 31. Chem 4631

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 information

for Acclaim Mixed-Mode HILIC-1 Column

for 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 information

CHAPTER 6 GAS CHROMATOGRAPHY

CHAPTER 6 GAS CHROMATOGRAPHY CHAPTER 6 GAS CHROMATOGRAPHY Expected Outcomes Explain the principles of gas chromatography Able to state the function of each components of GC instrumentation Able to state the applications of GC 6.1

More information

Biochemistry. Biochemical Techniques HPLC

Biochemistry. 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 information

Experiment UPHPLC: Separation and Quantification of Components in Diet Soft Drinks

Experiment UPHPLC: Separation and Quantification of Components in Diet Soft Drinks Experiment UPHPLC: Separation and Quantification of Components in Diet Soft Drinks bjective: The purpose of this experiment is to quantify the caffeine content of a diet soda sample using Ultra-High Performance

More information

2. a) R N and L N so R L or L R 2.

2. 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 information

Basic principles of HPLC

Basic 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 information

Chapter 1. Chromatography. Abdul Muttaleb Jaber

Chapter 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 information

PAPER AND THIN LAYER CHROMATOGRAPHY (TLC)

PAPER AND THIN LAYER CHROMATOGRAPHY (TLC) PAPER AND THIN LAYER CHROMATOGRAPHY (TLC) Objectives Understand the principle of Paper and Thin Layer Chromatography (TLC). Diagnose two samples of urine for Phenylketonuria and Cystinuria, using paper

More information

Chromatography and other Separation Methods

Chromatography and other Separation Methods Chromatography and other Separation Methods Probably the most powerful class of modern analytical methods for analyzing mixture of components---and even for detecting a single component in a complex mixture!

More information

LECTURE 2. Advanced Separation Science Techniques Present and Future Separation Tools

LECTURE 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 information

Introduction to biochemical practicals. Vladimíra Kvasnicová

Introduction to biochemical practicals. Vladimíra Kvasnicová Introduction to biochemical practicals Vladimíra Kvasnicová arrangement of practicals laboratory safety regulations laboratory equipment dealing with automatic pipette instructions: http://vyuka.lf3.cuni.cz/

More information

Chromatography and Functional Group Analysis

Chromatography and Functional Group Analysis Chromatography Chromatography separates individual substances from a mixture. - to find out how many components there are - to match the components with known reference materials - to use additional analytical

More information

For Chromatography, you might want to remember Polar Dissolves More, not like dissolves like.

For Chromatography, you might want to remember Polar Dissolves More, not like dissolves like. Chromatography In General Separation of compounds based on the polarity of the compounds being separated Two potential phases for a compound to eist in: mobile (liquid or gas) and stationary Partitioning

More information

Chromatography Lab # 4

Chromatography Lab # 4 Chromatography Lab # 4 Chromatography is a method for separating mixtures based on differences in the speed at which they migrate over or through a stationary phase which means that a complex mixture will

More information

Chromatography 1 of 26 Boardworks Ltd 2016

Chromatography 1 of 26 Boardworks Ltd 2016 Chromatography 1 of 26 Boardworks Ltd 2016 Chromatography 2 of 26 Boardworks Ltd 2016 What is chromatography? 3 of 26 Boardworks Ltd 2016 Different instrumental methods can be used to analyse and identify

More information

Physical Separations and Chromatography

Physical 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 information

INSTITUTE OF MEDICAL BIOCHEMISTRY FIRST FACULTY OF MEDICINE, CHARLES UNIVERSITY IN PRAGUE. Chromatography. in biochemistry

INSTITUTE OF MEDICAL BIOCHEMISTRY FIRST FACULTY OF MEDICINE, CHARLES UNIVERSITY IN PRAGUE. Chromatography. in biochemistry INSTITUTE OF MEDICAL BIOCHEMISTRY FIRST FACULTY OF MEDICINE, CHARLES UNIVERSITY IN PRAGUE Chromatography in biochemistry Prof. RNDr. Věra Pacáková, CSc. Faculty of Natural Sciences, Charles University

More information

HPLC Praktikum Skript

HPLC 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 information

Polymer analysis by GPC-SEC. Technical Note. Introduction

Polymer analysis by GPC-SEC. Technical Note. Introduction Polymer analysis by GPC-SEC Technical Note Introduction Gel Permeation Chromatography (GPC), also referred to as Size Exclusion Chromatography (SEC) is a mode of liquid chromatography in which the components

More information

Lab.2. Thin layer chromatography

Lab.2. Thin layer chromatography Key words: Separation techniques, compounds and their physicochemical properties (molecular volume/size, polarity, molecular interactions), mobile phase, stationary phase, liquid chromatography, thin layer

More information

Analytical Technologies in Biotechnology Prof. Dr. Ashwani K. Sharma Department of Biotechnology Indian Institute of Technology, Roorkee

Analytical Technologies in Biotechnology Prof. Dr. Ashwani K. Sharma Department of Biotechnology Indian Institute of Technology, Roorkee Analytical Technologies in Biotechnology Prof. Dr. Ashwani K. Sharma Department of Biotechnology Indian Institute of Technology, Roorkee Module - 3 Chromatographic Method Lecture - 1 Introduction and Basic

More information

LC Technical Information

LC 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 information

LIQUID CHROMATOGRAPHY

LIQUID CHROMATOGRAPHY LIQUID CHROMATOGRAPHY RECENT TECHNIQUES HPLC High Performance Liquid Chromatography RRLC Rapid Resolution Liquid Chromatography UPLC Ultra Performance Liquid Chromatography UHPLC Ultra High Pressure Liquid

More information

Shodex TM ODP2 HP series columns

Shodex TM ODP2 HP series columns HPLC Columns Shodex TM ODP2 HP series columns Better retention of highly polar substances Technical notebook No. 6 Contents 1. Introduction 1-1. Specifications 1-2. Eluent Compatibility of ODP2 HP Series

More information

Separation Techniques and Extraction methods of Phytochemicals

Separation Techniques and Extraction methods of Phytochemicals Separation Techniques and Extraction methods of Phytochemicals Introduction herbal medicines and the products derived from them are traditionally used for their benefits in prevention and treatment of

More information

LUMEFANTRINUM LUMEFANTRINE

LUMEFANTRINUM 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 information

CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY

CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY Theme 06. CHROMATOGRAPHY (I): CHROMATOGRAPHY ELEMENTARY BASES 1599-ENVIRONMENTAL ANALYTIC METHODS Grade in Environmental Sciences Course 2013-14 Second Semester Professors: Miguel A Sogorb (msogorb@umh.es)

More information

HPLC. GRATE Chromatography Lab Course. Dr. Johannes Ranke. September 2003

HPLC. GRATE Chromatography Lab Course. Dr. Johannes Ranke. September 2003 HPLC GRATE Chromatography Lab Course Dr. Johannes Ranke Organisation The groups Start at 9:00 am End at 18:00 pm at the latest Friday, 19th we will finish at 2:00 pm Thursday, 11th: Lecture at 08:15 am

More information

Liquid Chromatography

Liquid 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 information

not to be republished NCERT THE technique of chromatography is vastly used for the separation, Chromatography UNIT-5 EXPERIMENT 5.

not to be republished NCERT THE technique of chromatography is vastly used for the separation, Chromatography UNIT-5 EXPERIMENT 5. UNIT-5 Chromatography THE technique of chromatography is vastly used for the separation, purification and identification of compounds. According to IUPAC, chromatography is a physical method of separation

More information

GC Instruments. GC Instruments - Columns

GC 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 information

[S016. CHROMATOGRAPHY]

[S016. CHROMATOGRAPHY] Phyto-Analysis Sheet Number : 16 Prof. Dr. Talal Aburjai Page 1 of 9 How to read the chromatogram? Comes from any automated chromatography. The chromatograms show the 0 t (t m ) which indicates the solvent

More information

Institute for Chemical Education, Fun With Chemistry; Vol. 1, Sarquis, Mickey and Sarquis, Gerry, Ed.; University of Wisconsin Madison, 1991,

Institute 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 information

CHROMATOGRAPHY, AND MASS SPECTRAL. FRACTIONS OF Lasianthus

CHROMATOGRAPHY, AND MASS SPECTRAL. FRACTIONS OF Lasianthus f^ l^ltt^^p^^* V^ COLUMN CHROMATOGRAPHY, HPLC AND MASS SPECTRAL ANALYSIS OF SOME FRACTIONS OF Lasianthus lucldus Biume 8.1 Column Chromatography 8.2 HPLC 8.3 Preparatory TLC 8.4 Mass Spectral Analysis

More information

Spectroscopy and Chromatography

Spectroscopy 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 information

Minimizing Solvent Impact on Purification of Nitrogencontaining

Minimizing Solvent Impact on Purification of Nitrogencontaining Minimizing Solvent Impact on Purification of Nitrogencontaining Compounds J. Liu and P. C. Rahn Biotage Discovery Chemistry Group US 1725 Discovery Drive Charlottesville, VA 22911 USA 1 Abstract This paper

More information

Lab 10 Guide: Column Chromatography (Nov 3 9)

Lab 10 Guide: Column Chromatography (Nov 3 9) Lab 10 Guide: Column Chromatography (Nov 3 9) Column Chromatography/ Isolation of Caffeine from Tea, Exp. 7B, pages 67-72 in Taber After an organic reaction it s common to get a mixture of products. Usually

More information

2501 High Performance Liquid Chromatography

2501 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 information

CHAPTER CHROMATOGRAPHIC METHODS OF SEPARATIONS

CHAPTER CHROMATOGRAPHIC METHODS OF SEPARATIONS Islamic University in Madinah Department of Chemistry CHAPTER - ----- CHROMATOGRAPHIC METHODS OF SEPARATIONS Prepared By Dr. Khalid Ahmad Shadid Chemistry Department Islamic University in Madinah TRADITIONAL

More information