Chromatography

T. Trimpe 2006 http://sciencespot.net/

What is chromatography?
From Wikipedia ... Chromatography (from Greek word for chromos for colour) is the collective term for a family of laboratory techniques for the separation of mixtures. It involves passing a mixture which contains the analyte through a stationary phase, which separates it from other molecules in the mixture and allows it to be isolated. Which means ... Chromatography is the physical separation of a mixture into its individual components. We can use chromatography to separate the components of inks and dyes, such as those found in pens, markers, clothing, and even candy shells. Chromatography can also be used to separate the colored pigments in plants or used to determine the chemical composition of many substances.

http://members.shaw.ca/vict/chemistry_test3.htm

Derived from the Greek word Chroma meaning colour and graphy means writing. Chromatography is a technique for separating mixtures into there component in order to analyze ,identify ,purify and quantify the mixtures or components

Chromatography

Separate

Analyze Identify Purify Quantify Components

Mixture

Uses for Chromatography

Chromatography is used by scientists to: Analyze examine a mixture, its components,
and their relations to one another

Identify determine the identity of a mixture or

components based on known components

Purify separate components in order to isolate

one of interest for further study

Quantify determine the amount of the a mixture

and/or the components present in the sample

Chromatography
Chromatography separate molecules using partitioning characteristics of molecule to remain in a stationary phase versus a mobile phase

Brief History of Chromatography

1903 Tswett, a Russian botanist coined the term chromatography. He passed plant tissue extracts through a chalk column to separate pigments by differential adsorption chromatogrpahy 1931 Richard Kuhn used chromatography to separate isomers of polyene pigments; this is the first known acceptance of chromatographic methods

http://www.chemgeo.uni-hd.de/texte/kuhn.html

Micheal Tswett invented the chromatography in 1901 during his research on plant pigments. He used the technique to separate various plant pigments such as chlorophyll, xanthophylls, Carotenoids.

Chromatography

History of the Main techniques

1938 Thin Layer chromatography by Russian scientist N.A Izamailov and M.S Shraiber 1941 Liquid-Liquid partition chromatography developed by Archer John, Porter Martin and Richard Laurence Millington Synge 1944 Paper Chromatography one of the most important methods in the development of biotechnology 1945 Gas Chromatography 1st analytical gas-solid (adsorption) chromatography developed by Fritz Prior 1950 Gas Liquid Chromatography by Martin and Anthony James; Martin won the Nobel Prize in 1952

British chemist Archer John Porter Martin, corecipient, with Richard L. M. Synge, of the 1952 Nobel Prize in chemistry, "for their invention of partition chromatography."

History of the Main Techniques

1966 HPLC named by Csaba Horvath, but didnt become a popular method until 1970s 1950s Ion-Exchange chromatography declassified this technique 1930s Affinity Chromatography was developed for the study of enzymes and other proteins

library.thinkquest.org

Examples of Chromatography
Liquid Chromatography
Used to identify unknown plant pigments & other compounds.

Thin-Layer Chromatography
Uses thin plastic or glass trays to identify the composition of pigments, chemicals, and other unknown substances.

Gas Chromatography
Used to determine the chemical composition of unknown substances, such as the different compounds in gasoline shown by each separate peak in the graph below.

Paper Chromatography
Can be used to separate the components of inks, dyes, plant compounds (chlorophyll), make-up, and many other substances

Terminology
Elution
- washing of the mixture

Stationary Phase
-

Eluent
- additional solvents used for elution

Mobile Phase
- fluid carrying the mixture of analytes

Effluent
- exiting fluid stream

Residency
- time spent on column

Paper Chromatography Lab

Obtain the supplies youll need.
1 large beaker (or plastic cup) 1 small beaker (or plastic cup) filled with water 4 pieces of filter paper 4 black markers for testing 4 small pieces of masking tape Pencil (to attach to the top of the filter paper) Permanent marker Timer
Pencil Filter Paper Tape Label with marker

Ink Mark

Write the pen number on a piece of masking tape with a permanent marker and place it at the top of the strip. Choose one of the testing markers and draw a thick line near the bottom of the filter paper - about inch from the bottom. Pour a small amount of water into the large cup and then hang the paper strip in the cup. Make sure the ink line does not touch the water only the bottom of the filter paper. Allow the water to move up the paper for 5 minutes and then remove the strip from the water. Hang it on the side of the table to dry. Follow these directions to test the other pens.

Illustration of Chromatography
Stationary Phase

Separation

Mobile Phase

Mixture
Component s Blue Black Red Yellow Affinity to Stationary Phase
---------------  

Components
Affinity to Mobile Phase
Insoluble in Mobile Phase           

Principles of Paper Chromatography

Capillary Action the movement of liquid within the spaces
of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity.

Solubility the degree to which a material (solute) dissolves

into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents. Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.

Paper Chromatography Experiment

Preparing the Isopropanol Solutions

Prepare 15 ml of the following isopropanol solutions in appropriately labeled beakers: - 0%, 5%, 10%, 20%, 50%, and 100%

Preparing the Chromatography Strips

Cut 6 strips of filter paper Draw a line 1 cm above the bottom edge of the strip with the pencil Label each strip with its corresponding solution Place a spot from each pen on your starting line

Developing the Chromatograms

Place the strips in the beakers Make sure the solution does not come above your start line Keep the beakers covered Let strips develop until the ascending solution front is about 2 cm from the top of the strip Remove the strips and let them dry

Developing the Chromatograms

Developing the Chromatograms

Observing the Chromatograms

0%

20%

50%

70%

100%

Concentration of Isopropanol

Black Dye
1. Dyes separated purple and black 2. Not soluble in low concentrations of isopropanol 3. Partially soluble in concentrations of isopropanol >20%

0%

20%

50%

70%

100%

Concentration of Isopropanol

Blue Dye
1. Dye separated blue 2. Not very soluble in low concentrations of isopropanol 3. Completely soluble in high concentrations of isopropanol

0%

20%

50%

70%

100%

Concentration of Isopropanol

Green Dye
1. Dye separated blue and yellow 2. Blue Soluble in concentrations of isopropanol >20% 3. Yellow Soluble in concentrations of isopropanol >0%

0%

20%

50%

70%

100%

Concentration of Isopropanol

Alternative Experiments

Alternative Experiments

Alternative Experiments

THIN LAYER CHROMATOGRAPHY

Thin layer chromatography is done exactly as it says using a thin, uniform layer of silica gel. The silica gel is the stationary phase. The mobile phase is a suitable liquid solvent or mixture of solvents.

Silica gel is a form of silicon

dioxide (silica). The silicon atoms are joined via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are attached to OH groups.

Plate preparation
Mix the absorbent :- water & Silica gel Spread a thin layer of absorbent on an unreactive hard surface Glass, plastic, thick aluminium. Heat in oven at 110C for 30 minutes to activate and dry the plate

TLC Procedure
Place a small amount of solvent in a beaker In pencil, draw a straight line across the plate about 1 cm from the end of the plate Place a drop of sample solution on the line
BEFORE

Visualization
Destructive visualization Spray plate with H2SO4, and then bake in the oven at 110C for 15-20 minutes. Compound is destroyed but all spots will be visible Non-destructive visualization By using UV light

TLC Procedure

Application

Used in the quantization of plasma Lipids and Fatty Acids: Neutral lipids and free fatty acids were extracted from plasma and separated on TLC plates.

Column Chromatography
The stationary phase (column packing) in the column is very polar! Polar compounds are going to be attracted to the polar column packing by hydrogen bonding or dipole-dipole attractions. Polar compounds are going to move slowly! Non-polar compounds are going to come off the column first, while the polar compounds are going to come off the column last. Usually, one starts will a less polar solvent to remove the less polar compounds, and then you slowly increase the polarity of the solvent to remove the more polar compounds.

Principles of Separation on a column

Principles of Separation

Principles of Separation

Principles of Separation

Principles of Separation

Principles of Separation

Principles of Separation

REMEMBER
The stationary phase is POLAR The more polar component interacts more strongly with the stationary phase The more polar component moves more slowly. The non-polar component moves more rapidly.

Reverse phase chromatography

Silica is alkylated with long chain hydrocarbon groups, using 18 carbons long. This is usually referred to as C-18 silica.
CH3 CH3 CH2 CH2 CH3 CH3 O Si O O Si O SiCH3)3 O Si O O Si O O SiCH3)3 O Si O O Si O O Si O O O Si O O

17
Si O Si

CH3 CH3

17
Si O Si

SiCH3)3

O O Si O O

O O Si

O O

O O

Reverse Phase column chromatography

The stationary phase (column packing) is now NON-POLAR Non-polar compounds will move more slowly because they are attracted to the column packing. The more polar component moves more quickly down the column. Polar solvents, such as water and methanol are used in reverse phase chromatography Used mainly in columns, such as HPLC

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

PRINCIPLE

High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster .

HOW DOES HPLC WORK?

HPLC system
Solvent Reservoir Degasser Solvent Delivery System (Pump) Injector

Column &oven Detectors Recorder (Data Collection)

Components
Solvent Reservoir Usually one or more glass or stainless steel reservoirs each of which contains 200-1000 ml of solvent
Isocratic elution - single solvent separation technique Gradient elution - 2 or more solvents, varied during separation

Gradient system
Isocratic system Fixed (un-changeable) mixing ratio during analysis Gradient system Changeable mixing ratio during analysis
HPGE (High Pressure Gradient) LPGE (Low Pressure Gradient)

Aim of gradient - problems in isocratic mode in isocratic mode

Methanol / water = 6 / 4

Long analysis time

Methanol / water = 8 / 2

Poor separations

(Column : ODS type)

Aim of gradient - solution Gradual change of the mixing ratio during analysis
95% Methanol concentration in mobile phase

30%

Short analysis time & Excellent separation

Degasser
Problems caused by dissolved air(O2, N2)in mobile phase Unstable delivery in pump Bigger noise and large baseline-drift in detector cell  In order to avoid causing the problems, mobile phase should be degassed. vacuum pumping systems distillation system a system for heating and stirring the solvents sparging system - bubbles an inert gas of low solubility through the solvent

Column
straight, 15 to 150 cm in length; 2 to 3 mm i.d. packing - silica gel, alumina, Celite

Types of Detector
UV/Visible - Fixed wavelength - variable wavelength Photo Diode Array Refractive index Fluorescence Evaporative light scattering Conductivity Electrochemical

Refractive Index Detector

INJECTION OF THE SAMPLE: Injection of the sample is entirely automated. RETENTION TIME: The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound. Different compounds have different Retention times. The retention time will vary depending on: the pressure used the exact composition of the solvent the nature of the stationary phase (size & compositition) the temperature of the column

INTERPRETATION :
The output will be recorded as a series of peaks. Each peak represents a compound in the mixture. The area under the peak is proportional to the amount of any particular compound which has passed the detector.

GAS-LIQUID CHROMATOGRAPHY

Principle
In gas-liquid chromatography, the mobile phase is a gas such as helium and the stationary phase is a high boiling point liquid absorbed onto a solid. How fast a particular compound travels through the machine will depend on how much of its time is spent moving with the gas as opposed to being attached to the liquid in some way.

How does GLC work?

Retention time
The time taken for a particular compound to travel
through the column to the detector is known as its retention time. compounds have different retention times depends upon: The boiling point of the compound. The solubility in the liquid phase.

The temperature of the column.

INTERPRETATION :
The output will be recorded as a series of peaks. Each peak represents a compound in the mixture. The area under the peak is proportional to the amount of any particular compound which has passed the detector.

AREA OF THE PEAK

If two different substance has passed through. Compound absorbs less UV light have smaller area & which absorbs more UV light have larger area of its peak.

Study Overview

Conclusion

Intensive Insulin Therapy in Surgical ICU

Ion-Exchange Chromatography
Usually employed with HPLC Ions are charged molecules - cation positively charged ion - anion negatively charged ion These ions do not separate smoothly under the traditional methods of the liquid and mobile phases of chromatography Requires alteration methods of either the mobile phase or stationary phase are required - mobile phase suppresses the ionic nature of the analyte - stationary phase incorporate ions of the opposite charge to attract and retain analyte

How do you get those columns to work

There is a glass column coated with a resin polymer The resin is either positively charged (an acid) or negatively charged (a base) An analyte will have ions opposite of the resins charge eluting off the ion of interest

Resin
Common resins are copolymerized styrenes vinylic and aromatic functional groups styrene derivative and divinylbenzene

Creates better stability due to crosslinking of the benzene rings Creates a swelling within the polymer affecting the porosity while taking in the mobile phase liquid Aromatic substitution reaction makes these polymers ideal for charged functional groups

Cation Exchange resins

The functional group in cation exchange resins are usually acids Sulfonic acids SO3H (strong acid resin) are added to the resin by sulfonation reactions Res-(SO3H) + M+ Res-(SO3M) + H+ Carboxylic acid COOH (weak acid resin) Res-COOH + M+ Res-COOM + H+ With both the strong and the weak acid exchange sites an acidic Hydrogen is attached to a functional group chemically bound to the resin Cation exchange is good for removing metal ions from an aqueous solution

Anion Exchange Resins

The functional groups added to the resin is similar to cation resins but are basic instead of acidic Quaternary ammonium a strong base -- CH2N(CH3)3+OH CH2N(CH3)3+OH- + B Res-CH2N(CH3)3+Cl- + OH-

Polyalky amine a weak base -- NH(-R)2+OHNH(-R)2+OH- + BRes-NH(-R)2+B- +OH-

To Affinity and Beyond

The rate of ion exchange is controlled by the law of mass action. At equal concentration the greater affinity molecule will control the cation exchange resins in the acid form. However if a much higher concentration of strong acid passed through the greater affinity molecule, such as sodium, will form the resin, reversing equilibrium and convert the resin back to an acidic form. Generally it is possible to return either ion exchange resin column to a desired starting form by passing a large excess of the desired ion at very high concentration through the resin

What makes it unique

Careful selection of the ionic composition of the eluent, and the gradual adjustment of its strength during elution using a controlled gradient The components of a mixture of ions can be induced to separate just as the components of a mixture separated by partitionion chromatography The parameters controlling the relative residence of the analyte or other eluent ions is the resin stationary phase or the ionic solution mobile phase 1) both the relative selectivity of the resin for the ions and their relative concentrations in each phase 2) In ion exchange, selectivity resides in relative ion-pairing interaction strengths only in the stationary phase

Relative Affinity of Ions

The higher the charge the higher the affinity
Na+ < Ca2+ < Al3+ and Cl- < SO42-

The Ion with the greatest size and charge has the highest affinity
Li+ < Na+ < K+ < Cs+ < Be2+ < Mg2+ < Cu2+ and F- < Cl- < Br- < I-

Ion Exchange Chromatography

The parameters controlling the residence times of analyte or other eluent ions in the resin stationary phase or ionic solution mobile phase are 1) both the relative selectivity of the resin for the ions 2) their relative concentrations in

Applications of IEC
In cell and molecular biology, ion exchange chromatography is used to separate different proteins out of an eluant. areas of research such as the environment, industry, commercial products of organic molecules without UV-vis absorption

Gel Filtration Chromatography

Original Article

Intensive versus Conventional Glucose Control in Critically Ill Patients

The NICE-SUGAR Study Investigators

N Engl J Med Volume 360(13):1283-1297 March 26, 2009

 Gel filtration (chromatography), is also known as molecular sieve chromatography. Gel filtration chromatography separates molecules according to their size and shape. The stationary phase consists of beads containing pores that span a relatively narrow size range.

 Smaller molecules spend more time inside the beads than larger molecules and therefore elute later (after a larger volume of mobile phase has passed through the column).

Size exclusion chromatography Gel filtration chromatography

y Contains porous beads y Separates according to size and shape y Larger proteins excluded from the small pores y Quaternary structure determination, & Mr estimation using a standard curve (log Mr vs elution volume) Fibrous proteins Spherical vs rod-shaped proteins

Types of gels used

The gels used as molecular sieves are cross linked polymers. They are uncharged and inert i.e. dont bind or react with the materials being analyzed. Three types of gels are used:

Types of gels cont

1. Dextran: is a homopolysaccharide of glucose residues. its prepared with various degrees of cross-linking to control pore size. Its bought as dry beads, the beads swell when water is added. The trade name is sephadex. Its mainly used for separation of small peptides and globular proteins with small to average molecular mass.

Types of gels cont

2. Polyacrylamide:these gels are prepared by cross linking acrylamide with N,Nmethylene bis acrylamide. The pore size is determined by the degree of cross-linking. The separation properties of polyacrylamide gels are mainly the same as those of dextrans. They are sold as bio-gel P. They are available in wide range of pore sizes.

Types of gels cont

3. Agarose: linear polymers of D-galactose and 3,6 anhydro-1-galactose. It forms a gel thats held together with H bonds. Its dissolved in boiling water and forms a gel when its cold. The concentration of the material in the gel determines the pore size. The pores of agarose gel are much larger than those of sephadex or bio-gel p. Its useful for analysis or separation of large globular proteins or long linear molecules such as DNA

 The gel filtration material that will be used in the experiment below is called Sephadex G-75 and it will separate molecules with molecular weights from 3,000 to 70,000. Molecules with molecular weights larger than 70,000 will be excluded from the beads.

AFFINITY CHROMATOGRAPHY

Affinity History
1930s, first developed by Arne Wilhelm Tiselius, won the Nobel Prize in 1948 Used to study enzymes and other proteins Relies on the affinity of various biochemical compounds with specific properties ex) enzymes for their substrates antibodies for their antigens

Affinity chromatography y Separates by specific interactions y Contains a ligand: enzyme substrate receptor hormone antigen antibody (His)6 protein Ni2+

So now what.
The Sample is injected into the equilibrated affinity chromatography column Only the substance with affinity for the ligand are retained on the column The substance with no affinity to the ligand will elute off The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent

Specificity of Affinity Chromatography

Specificity is based on three aspect of affinity
1) the matrix 2) the ligand 3) the attachment of the ligands to the matrix

Matrix
The matrix simply provides a posre structure to increase the surface area to which the molecule can bind This has been what kept the Affinity Chromatography from being developed earlier and useful to the scientific community The matrix must be activated for the ligand to bind to it but still able to retain its own activation towards the target molecule

Matrix
Amino, hydroxyl, carbonyl and thio groups located with the matrix serve as ligand binding sites Matrix are made up of agarose and other polysaccharides The matrix also must be able to withstand the decontamination process of rinsing with sodium hydroxide or urea

Ligand
The Ligand binds only to the desired molecule within the solution The ligand attaches to the matrix which is made up of an inert substance The ligand should only interact with the desired molecule and form a temporary bond The ligand/molecule complex will remain in the column, eluting everything else off The ligand/molecule complex dissociates by changing the pH

So many ligands so little time

The chosen ligand must bind strongly to the molecule of interest If the ligand can bind to more than onel molecule in the sample a technique, negative affinity is performed - this is the removal of all ligands, leaving the molecule of interest in the column - done by adding different ligands to bind to the ligands within the column

Applications
Used in Genetic Engineering Production of Vaccines And Basic Metabolic Research

Uses for Chromatography

Real-life examples of uses for chromatography:

Pharmaceutical Company determine amount of each chemical

found in new product

Hospital detect blood or alcohol levels in a patients blood

stream

Law Enforcement to compare a sample found at a crime scene

to samples from suspects

Environmental Agency determine the level of pollutants in the

water supply

Manufacturing Plant to purify a chemical needed to make a

product