Chromatography. Gas Chromatography

Size: px
Start display at page:

Download "Chromatography. Gas Chromatography"

Transcription

1 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 between two immiscible phases viz. a miscible phase and an immiscible phase. The sample is dissolved in one phase, called the mobile phase. This phase can be a gas (gas chromatography) or liquid (liquid chromatography). The mobile phase is then passed through the other phase, called the stationary phase. This phase can be a gas or liquid but is typically a solid and is packed into a column. The stationary phase is made up of materials for which the mobile phase has differing affinities. As the mixture dissolved in the mobile phase interacts with the stationary phase, the components of the mixture separate and are eluted from the column. Each component can then be collected for analysis and quantification. The figure alongside shows a simple chromatographic separation process of a component ( mixture. The time taken for a compound to elute and be detected by the detector is called the retention time (R t ). Generally a longer column will allow for better separation as the compounds interact more with the stationary phase. There are two main types of chromatography that we will be looking at viz. gas chromatography and liquid chromatography which are used for volatile and non-volatile compounds respectively. Gas Chromatography In gas chromatography, the molecules partition between a gaseous mobile phase (carrier gas) and a stationary phase. The sample is injected into an injector port where it is volatilised and transported through the column by the carrier gas. Separation occurs through a process of adsorption onto the solid stationary phase or dissolving in the liquid stationary phase. The samples must be volatile and thermally stable over the period of the analysis. 1

2 ( The diagram shows a simple schematic of a typical gas chromatograph. The carrier gas is an inert gas e.g. nitrogen, argon, helium. The carrier gas should not interact with the sample in any way other than to transport the sample through the column. The carrier gas must be maintained at the appropriate flow rate to ensure that there is sufficient time for the sample to interact with the stationary phase (if the flow rate is too high, the sample will not separate effectively). The carrier gas flows through an injector port through which the sample is introduced into the instrument. A small volume (1 10 µl) of sample which has been dissolved in a volatile solvent is injected via an injector port onto the column. There are two modes in which an injector can operate; split and split-less mode (see more detail after columns). The injector port is at a higher temperature (~ 50 C higher) than the boiling point of the least volatile component in the sample. This ensures that the sample enters the column (i.e. stationary phase) as a vapour. The volume injected onto the column depends on the type of column being used. The column is housed in an oven to ensure that the temperature is controlled and to ensure that the sample remains in the gas phase. The column is at a lower temperature than the injector port, this may cause some components of your sample to condense. There are two different types of column; a packed column and a capillary column. A packed column has small support particles (finely ground diatomaceous earth) which may be coated with a non-volatile liquid. The columns can range in length up to 10 m and have an internal diameter that is between 1-5 mm. Packed Column Capillary columns are more commonly used in modern chromatography. The columns can range between m and have a significantly smaller diameter ( mm). The longer column length generally means that the capillary column show better separation than the packed column. A capillary column is a fused silica column that has been coated on the outside with a polymer which

3 improves its strength and flexibility. In a capillary column; the stationary phase is coated directly onto the walls of a column and not packed to fill up the column. There are 3 different types of capillary columns. The wall coated open tubular column (WCOT) is the most commonly used and has a liquid stationary phase film coated onto the column walls. Support coated open tubular (SCOT) columns have a thin layer of solid particles which are then coated with a liquid. This results in more separation efficiency than WCOT columns because of the higher surface area of the column. The inner surface of porous layer open tubular (PLOT) columns have an embedded layer of porous materials (alumina, molecular sieves), with the film thicker than WCOT and SCOT columns which more effectively separates gases and highly volatile compounds. Because of the differences in the structure of the two different types of columns, generally packed columns can tolerate a larger analyte volume than the capillary columns. This means that when a sample is injected onto the column via the injector port, the same volumes cannot be used for packed and capillary columns. A too high analyte concentration would result in the active sites of the column being blocked with analyte and some analyte then being un-retained by the column. To prevent continually diluting samples, an injector port can be used in two modes; split and split-less mode. Split mode is typically used when a capillary column is used and a fraction of the amount injected is automatically diverted to waste with a small amount entering the column. In split-less mode, the entire injected sample enters the column. So, when the sample is in split mode, it is injected into the injector port which is heated and it volatilises, it is carried through the injector port by the carrier gas and can either enter the column or be carried through the split valve (see diagram). When a mixture enters the column as a gas some of the analyte components may condense onto the stationary phase, some components may dissolve in the liquid film coating the surface of the stationary phase, or it may remain in the gas phase and be transported through the column without interacting with the stationary phase (i.e. be unretained this compound(s) will elute first). 3

4 The compounds that have a boiling point that is higher than the oven temperature will condense at the beginning of the column. However, what follows is a cycle of evaporation and condensation of those compounds along the length of the column, thus causing separation from other molecules with different boiling points. Molecules with a high boiling point will therefore spend a longer time on the column that those with lower boiling points. Other molecules will interact with the stationary phase depending on their solubility. The more soluble compounds will absorb onto the column more than the less soluble compounds and thus be retained for longer. The temperature of the column also plays a role in the elution of molecules. Low column temperatures will mean that the analytes spend a longer time on the column and while this leads to good separation, you will have long analysis time and peaks may not be sharp. High column temperatures will result in shorter analysis time, but if the temperature is too high then molecules may not interact sufficiently with the stationary phase. The elution of compounds is therefore dependent on a number of different factors which affect the interaction of molecules with the stationary phase; gas flow, rates of diffusion, boiling point and solubility with the stationary phase. Temperature Programming (to add) As the compounds elute from the column, a detector converts the analyte concentration into an electrical signal which can be used for quantification. There are a number of different detectors used in GC; the flame ionisation detector is commonly used for the analysis of most organic molecules. Essentially, the carrier gas and hydrogen are mixed and burned. As an organic compound elutes from the column and enter the detector, the compound will burn and produce electrons and ions. These collect at electrodes and a flow of current occurs which is detected as the signal. The output from the detector is recorded as a series of peaks, each one representing a different analyte. Gas chromatography is used for both qualitative and quantitative analysis. Compounds can be identified by matching their retention times to those of known standards. The higher the concentration of your analyte, the more ions it will generate creating a bigger signal; thus allowing for quantification. Quantification is carried out using the area under the analyte peak since this area is proportional to the concentration. 4

5 Other detectors that are widely used are the electron capture detector which is selective towards Halides, nitrates, nitriles, peroxides, anhydrides, organometallics). The thermal conductivity detector is used for most compounds since it measured thermal conductivity and it is known as a universal detector. One of the biggest sources of error in GC analysis is the injection of the sample. Because the sample injection volume is so small, any error made has a large impact. Using an internal standard allows for correction of injection errors. A known concentration of a compound is added to the samples and standards to be analysed. In this way any errors in the injection will be reflected in both the analyte and the internal standard. Because the initial concentration of the internal standard is known, correction to the data can be made using the ratio of the peak areas of the standard and sample. Gas chromatography is suited to organic compounds that are volatile. However, non-volatile compounds may be analysed if they are derivatised to produce volatile products. An example of this is the analysis of fatty acids. Fatty acids can be derivatised to form fatty acid methyl esters by the process of esterification. The methyl esters are volatile and can then be analysed by GC. Liquid Chromatography Liquid chromatography is a separation technique where the components of the sample are separated based on their affinity for a liquid mobile phase. The most widely used type of liquid chromatography is called high performance liquid chromatography (HPLC). There are a number of different types of HPLC and they are based on how separation occurs; adsorption chromatography, partition chromatography, ion-exchange chromatography and size exclusion chromatography. In adsorption chromatography, analytes are separated based on the degree of adsorption to the stationary phase which is made up of fine particles of silica or alumina. Generally non-polar compounds can be eluted and identified by adjusting the strength of the mobile phase. Partition chromatography is one of the most commonly used modes of separation and can be used for non-ionic compounds and low molecular weight compounds. A solid (e.g. silica) is coated with a thin film of stationary phase. The analytes interact with the mobile phase and equilibrate between the two. Partition chromatography can occur either in normal phase or reverse phase mode. In normal phase, the silica packing material is coated with polar/hydrophilic functional groups. Here, polar compounds will be retained on the column for 5

6 longer and are eluted using non-polar mobile phases e.g. hexane, methylene chloride. The less polar compounds elute faster (i.e. have shorter retention times). Thus the separation is based on polarity and compounds elute in order of increasing polarity. Reverse phase chromatography is the most popular modern mode of separation. Here the stationary phase is non-polar, with hydrophobic, long chain alkanes such as n-octyldecyl (C 18 ), n-decyl (C 8 ) chains and phenyl groups being the most common bonding phase onto silica particles. The mobile phase is a polar solvent such as water, methanol or acetonitrile. Reverse phase chromatography is the opposite of normal phase. The example below shows a three component mixture separated by normal and reverse phase chromatography. Note how the order of elution changes. A summary of the different response to a mixture using the different phases is presented in the table. Ion exchange (to be added) Size Exclusion Chromatography (to be added) High Performance Liquid Chromatography (HPLC) Particle size of the column packing will increase the separation efficiency. The smaller the particles, the larger the surface area for the components of the mixture to interact. This results in faster equilibrium between the mobile and stationary phases. A high pressure is used to force the mobile phase through the column to prevent issues arising from capillary action. Hence the instrumentation used for liquid chromatography is called HPLC. The brief schematic shows the process of HPLC. The mobile phase is typically a high grade, solvent with polarity that is dependent on the 6

7 molecules you are trying to separate. The solvent should be de-gassed and free of bubbles which would interfere with separation in the column. The pump forces the mobile phase and sample through the column at high pressure. As with GC, the flow rate of the mobile phase has an impact on separation and elution of the analytes. There are two different ways to enhance separation using the polarity of the mobile phase: isocratic and gradient elution. In isocratic elution, a solvent of mixture of solvents making up the mobile phase is pumped at a constant rate through the column. In gradient elution, the composition of the mobile phase is changed during the analysis. This change in polarity gradually will allow compounds to separate efficiently. The sample is introduced into the mobile phase using an injection valve. This valve is very different from the GC injection port; the sample is loaded into an injection loop. When the loop is set to load, sample can be injected into the sample loop. When the loop is switched to inject, an appropriate volume of the sample is transferred directly onto the column. This means that there are no injection errors that affect your analysis (like with GC). When the sample enters the column separation of the compounds occurs depending on the type of column present. As each compound elutes the signal is detected and plotted as a series of peaks. The retention times for each compound may be matched to standards thus allowing for qualitative analysis. The signal intensity is proportional to the concentration of the analyte and can therefore be quantified. The detector on the HPLC varies according to the type of compound being detected. In this module you will use one detector, a UV detector which you have already come across in Spectroscopy. 7

8 Some Key Equations and Definitions for Chromatography Peak Parameters t R total retention time - time between injection of sample and emergence of the peak maximum t M mobile phase hold-up time - time taken for a non-retained substance to emerge from the column t' R adjusted retention time - average time spent in the stationary phase = t R - t M k capacity factor (retention factor) = t' t R M average timespent instationaryphase averagetimespent inmobilephase no.molesinsp no.molesinmp V K V S M K K distribution constant = concentration in stationary phase concentration in mobile phase phase ratio (only applicable to liquid stationary phase) = V V M S volume of mobile phasein column volume of stationary phasein column w ½ peak width at half the peak height (the half-width w ½ or w h ) w b peak width at the base (note: w b = 1.70 x w ½ ) 8

9 Column Efficiency N plate number (number of theoretical plates) = t 5.55 w R 1 t 16 w R b H plate height (height equivalent to a theoretical plate - HETP) = N L (where L = length of the column) Separation selectivity coefficient (separation factor) = t' t' R R1 k k 1 K K 1 R S peak resolution found experimentally by: R S t w R b1 t w R1 b related to other parameters by: R N k S 4 1 k 9

Gas Chromatography (GC)! Environmental Organic Chemistry CEE-PUBH Analysis Topic 5

Gas 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 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

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

Gas Chromatography. Presented By Mr. Venkateswarlu Mpharm KTPC

Gas 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 information

2401 Gas (liquid) Chromatography

2401 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 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

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

Open Column Chromatography, GC, TLC, and HPLC

Open 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 information

Volatile organic compounds (VOCs):

Volatile organic compounds (VOCs): Volatile organic compounds (VOCs): Organic chemicals with a high vapour pressure at room temperature. High vapour pressure results from a low boiling point. The World Health Organization (WHO) defined

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

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

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

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

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

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

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

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

Gas Chromatography. Introduction

Gas 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 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

Skoog/Holler/Crouch Chapter 26 Principles of Instrumental Analysis, 6th ed. CHAPTER 26

Skoog/Holler/Crouch Chapter 26 Principles of Instrumental Analysis, 6th ed. CHAPTER 26 Skoog/Holler/Crouch Chapter 26 Principles of Instrumental Analysis, 6th ed. Instructor s Manual CHAPTE 26 26-1. (a) Elution is a process in which species are washed through a chromatographic column by

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

What is Chromatography?

What is Chromatography? What is Chromatography? Chromatography is a physico-chemical process that belongs to fractionation methods same as distillation, crystallization or fractionated extraction. It is believed that the separation

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

Gas Chromatography. Chromatography Laboratory Course. Dr. Christian Jungnickel Chromatography Course GC September 2005

Gas Chromatography. Chromatography Laboratory Course. Dr. Christian Jungnickel Chromatography Course GC September 2005 Gas Chromatography Chromatography Laboratory Course The laboratory course experiments General Aim: Gain general experience using a GC Constant Injection technique Temperature variations Qualitative and

More information

Course CHEM Chromatography

Course CHEM Chromatography Course CHEM 340 - Chromatography - Chromatographic Methods o Gas Chromatography (GC) o High performance Liquid Chromatography (HPLC) Terms Stationary phase A fixed place either in a column or on a planer

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

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

Gas Chromatography (GC)

Gas 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 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

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

CH 2252 Instrumental Methods of Analysis Unit V Gas Chromatography. M. Subramanian

CH 2252 Instrumental Methods of Analysis Unit V  Gas Chromatography.  M. Subramanian CH 2252 Instrumental Methods of Analysis Unit V Gas Chromatography M. Subramanian Assistant Professor Department of Chemical Engineering Sri Sivasubramaniya Nadar College of Engineering Kalavakkam 603

More information

Introduction to Gas Chromatography

Introduction to Gas Chromatography Introduction to Gas Chromatography 31-1 Objectives To know what is chromatography To understand the mechanism of compound separation To know the basic of gas chromatography system 31-2 Chromatography Definition

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

Experiment 6 Simple and Fractional Distillation

Experiment 6 Simple and Fractional Distillation Experiment 6 Simple and Fractional Distillation Vapor Pressure vs Temperature of Water Vapor Pressure vs Temperature of Water 25 Vapor Pressure vs Temperature of Water 25 Vapor Pressure (kpa) (kpa) 2 2

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

Chromatographic 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 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 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 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

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

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

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

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

7 INSTRUMENTAL CHROMATOGRAPHY

7 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 information

Harris: Quantitative Chemical Analysis, Eight Edition CHAPTER 23: GAS CHROMATOGRAPHY

Harris: 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 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

Chromatographie Methods

Chromatographie Methods Chromatographie Methods Fifth Edition A. BRAITHWAITE Department of Physical Sciences Nottingham Trent University and F. J. SMITH Department of Chemistry and Chemical Engineering University of Paisley BLACKIE

More information

GUIDELINES FOR THE DESIGN OF CHROMATOGRAPHIC ANALYTICAL METHODS INTENDED FOR CIPAC COLLABORATIVE STUDY

GUIDELINES 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 information

Chapter 31 Gas Chromatography. Carrier Gas System

Chapter 31 Gas Chromatography. Carrier Gas System Chapter 31 Gas Chromatography GAS-LIQUID CHROMATOGRAPHY In gas chromatography, the components of a vaporized sample are fractionated as a consequence of being partitioned between a mobile gaseous phase

More information

Gas Chromatography notes

Gas Chromatography notes Gas Chromatography notes 1. Here is some terminology related to this experiment: Chromatography is a technique in which compounds to be separated are distributed between a mobile phase and a stationary

More information

Chemistry Instrumental Analysis Lecture 27. Chem 4631

Chemistry 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 information

GAS CHROMATOGRAPHY. Mobile phase is a gas! Stationary phase could be anything but a gas

GAS CHROMATOGRAPHY. Mobile phase is a gas! Stationary phase could be anything but a gas GAS CHROMATOGRAPHY Mobile phase is a gas! Stationary phase could be anything but a gas Gas Chromatography (GC) GC is currently one of the most popular methods for separating and analyzing compounds. This

More information

Speakers. Moderator. John V Hinshaw GC Dept. Dean CHROMacademy. Tony Taylor Technical Director CHROMacademy. Dave Walsh Editor In Chief LCGC Magazine

Speakers. Moderator. John V Hinshaw GC Dept. Dean CHROMacademy. Tony Taylor Technical Director CHROMacademy. Dave Walsh Editor In Chief LCGC Magazine Webcast Notes Type your questions in the Submit Question box, located below the slide window You can enlarge the slide window at any time by clicking on the Enlarge Slides button, located below the presentation

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

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

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

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

Chemistry Gas Chromatography: Separation of Volatile Organics

Chemistry 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 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

Remember - Ions are more soluble in water than in organic solvents. - Neutrals are more soluble in organic solvents than in water.

Remember - Ions are more soluble in water than in organic solvents. - Neutrals are more soluble in organic solvents than in water. IN-CLASS PROBLEMS SEPARATION SCIENCE CROMATOGRAPHY UNIT Thomas Wenzel, Bates College In-class Problem Set - Extraction 1. Devise a way to separate the materials in the following sample by performing an

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

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

Introduction to Capillary GC. Page 1. Agilent Restricted February 2, 2011

Introduction to Capillary GC. Page 1. Agilent Restricted February 2, 2011 ?? Kβ? Page 1 Typical GC System Gas supply Injector Detector Data handling GAS Column Oven Page 2 CARRIER GAS Carries the solutes down the column Selection and velocity influences efficiency and retention

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

GC Instruments. GC Instruments - Sample Introduction

GC Instruments. GC Instruments - Sample Introduction 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

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

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

Introduction to Chromatographic Separations (Chapter 1) Many determinations involve separation followed by analysis chromatography electrophoresis

Introduction to Chromatographic Separations (Chapter 1) Many determinations involve separation followed by analysis chromatography electrophoresis Introduction to Chromatographic Separations (Chapter 1) Many determinations involve separation followed by analysis chromatography electrophoresis Chromatography: sample transported by mobile phase electrostatic

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

Gas chromatography. Advantages of GC. Disadvantages of GC

Gas 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 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

LC III: HPLC. Originally referred to as High-Pressure Liquid Chromatography. Now more commonly called High Performance Liquid Chromatography

LC 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 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

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

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

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

Gas Chromatography. Rosa Yu, David Reckhow CEE772 Instrumental Methods in Environmental Analysis CEE 772 #16 2

Gas Chromatography. Rosa Yu, David Reckhow CEE772 Instrumental Methods in Environmental Analysis CEE 772 #16 2 Print version Gas Chromatography Rosa Yu, David Reckhow CEE772 Instrumental Methods in Environmental Analysis CEE 772 #16 1 Contents The primary components to a GC system 1. Carrier Gas System (including

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

CHEM340 Tutorial 4: Chromatography

CHEM340 Tutorial 4: Chromatography CHEM340 Tutorial 4: Chromatography 1. The data in the table below was obtained from a chromatogram obtained with a 10 cm liquid chromatography column. Under the conditions used, the compound uracil is

More information

Biochemistry. Biochemical Techniques. 12 Gas Liquid Chromatography

Biochemistry. Biochemical Techniques. 12 Gas Liquid Chromatography Description of Module Subject Name Paper Name 12 Module Name/Title 12 Gas - liquid Chromatography 1. Objectives 1.1 To understand principle of Gas Liquid Chromatography 1.2 To explain the different components

More information

Theory and Instrumentation of GC. Chromatographic Parameters

Theory and Instrumentation of GC. Chromatographic Parameters Theory and Instrumentation of GC Chromatographic Parameters i Wherever you see this symbol, it is important to access the on-line course as there is interactive material that cannot be fully shown in this

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

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

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

ERT320 BIOSEPARATION ENGINEERING CHROMATOGRAPHY

ERT320 BIOSEPARATION ENGINEERING CHROMATOGRAPHY ERT320 BIOSEPARATION ENGINEERING CHROMATOGRAPHY CHROMATOGRAPHY Week 9-10 Reading Assignment: Chapter 7. Bioseparations Science & Engineering, Harrison, R; Todd, P; Rudge, S.C and Petrides, D,P CHROMATOGRAPHY

More information

CEE 772: Instrumental Methods in Environmental Analysis

CEE 772: Instrumental Methods in Environmental Analysis Updated: 3 November 2014 Print version CEE 772: Instrumental Methods in Environmental Analysis Lecture #14 Chromatography: Theory (Skoog, Chapt. 26, pp.674-693) (Harris, Chapt. 23) (641-664) David Reckhow

More information

An Introduction to Gas Chromatography Mass Spectrometry

An Introduction to Gas Chromatography Mass Spectrometry An Introduction to Gas Chromatography Mass Spectrometry Dr Kersti Karu email: kersti.karu@ucl.ac.uk Office number: Room LG11 Recommended Textbooks:- Analytical Chemistry, G. D. Christian, P. K. Dasgupta,

More information

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

Analytical Technologies in Biotechnology Dr. Ashwani K. Sharma Department of Biotechnology Indian Institute of Technology, Roorkee Analytical Technologies in Biotechnology Dr. Ashwani K. Sharma Department of Biotechnology Indian Institute of Technology, Roorkee Module - 3 Chromatographic methods Lecture - 2 Basic Concepts in Chromatography

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

Volumetric Analysis. Quantitative analysis answers the second question

Volumetric 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 information

Chapter 11 Conventional Gas Chromatography

Chapter 11 Conventional Gas Chromatography Chapter 11 Conventional Gas Chromatography Gas Chromatography GC is the first instrumental chromatographic method developed commercially It is relatively easy to introduce a stable flow and pressure for

More information

637. Thiamethoxam. HPLC method

637. Thiamethoxam. HPLC method 637. Thiamethoxam HPLC method CIPAC Collaborative Trial according to CIPAC Information Sheet N o 293 Dr. Sven Adolph Syngenta Crop Protection Münchwilen AG CH-4333 Münchwilen Switzerland May 212 page 1

More information

Introduction and Principles of Gas Chromatography

Introduction and Principles of Gas Chromatography Introduction and Principles of Gas Chromatography Jaap de Zeeuw Restek, Middelburg, The Netherlands Jaap.dezeeuw@restek.com Definition and Uses of Gas Chromatography GC Components and Types of Columns

More information

Lab 3 Guide: Gas Chromatography (GC) (Sept 8-14)

Lab 3 Guide: Gas Chromatography (GC) (Sept 8-14) Lab 3 Guide: Gas Chromatography (GC) (Sept 8-14) How GC works The Basic Idea Gas chromatography (GC) is mainly used for the qualitative analysis of samples: it answers the question What chemicals are present

More information

An Advanced Base Deactivated Capillary Column for analysis of Volatile amines Ammonia and Alcohols.

An Advanced Base Deactivated Capillary Column for analysis of Volatile amines Ammonia and Alcohols. An Advanced Base Deactivated Capillary Column for analysis of Volatile amines Ammonia and Alcohols. Jaap de Zeeuw, Ron Stricek and Gary Stidsen Restek Corp Bellefonte, USA To analyze basic compounds at

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

The Effects of Carrier Gas Viscosity and Diffusion on Column Efficiency in Capillary Gas Chromatography

The Effects of Carrier Gas Viscosity and Diffusion on Column Efficiency in Capillary Gas Chromatography Page 1 of 5 Return The Effects of Carrier Gas Viscosity and Diffusion on Column Efficiency in Capillary Gas Chromatography Stephanye D. Armstrong and Harold M. McNair Department of Chemistry, Virginia

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

Ch24. Gas Chromatography (GC)

Ch24. Gas Chromatography (GC) Ch24. Gas Chromatography (GC) 24.1 What did they eat in the year 1000? From 13 C content of cholesterol in ancient bone 13 C : 1.1%, 12 C: 98.9% 13 C/ 12 C ratio types of plants Bones of 50 people in Barton-on-Humber

More information

Introduction to Capillary GC

Introduction to Capillary GC ?? Kβ? Page 1 Typical GC System Gas supply Injector Detector Data handling GAS Column Oven Page 2 CARRIER GAS Carries the solutes down the column Selection and velocity influences efficiency and retention

More information