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

• 1915 R.M Willstatter, German Chemist win Nobel Prize for similar experiement

• 1922 L.S Palmer, American scientist used Tswett’s techniques on various natural products

• 1931 Richard Kuhn used chromatography to separate isomers of polyene pigments; this is the first known acceptance of chromatographic methods

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, co-recipient, with Richard L. M. Synge, of the 1952 Nobel Prize in chemistry, "for their invention of partition chromatography."

• Liquid Chromatography was first discovered in 1903 by M.S.Tswett.

• In the mid-1950's, the growth of today’s technique of HPLC was much slower than the GLC.

• In the late 1970's, researchers began to use relatively short columns with small particles to achieve faster separations with better efficiencies and lower back pressures. This method became known as high performance liquid chromatography and is extensively used today.

What on Earth did scientist do before Chromatography?

-Extraction is based on the difference in solubility material is grounded, placed with a solvent which dissolves soluble compounds. A second extract solvent . The mixture is placed in a separatory funnel-Crystallization also based on the difference of solubility. The solubility is solved in a fixed volume of solvent. The purified compound crystallizes as solution cools, evaporates or diffuses

-Distillilation separates components based on their volatility typically via vaporization-condensation method

Filtration separate components of a mixture based on their particle size. Used most often to separate a liquid from a solid

اجزاء کروماتوگرافی

فازی است که جداسازی بر روی آن :فاز ساکنانجام می شود.

فازی است که جداسازی توسط آن :فاز متحرکانجام می شود.

شناسایی یا تعیین مقدار :نمونه

کروماتوگرافی اصول

کروماتوگرافی انواع

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

Kinds of Chromatography

1. Liquid Column Chromatography

 

2. Gas Liquid Chromatography

 

3. Thin-layer Chromatography

13

Column Chromatography

Gas Liquid Chromatography (GLC)Gas Liquid Chromatography (GLC)

High Performance Liquid Chromatography (HPLC)High Performance Liquid Chromatography (HPLC)

Paper Chromatography and Thin Layer Chromatography (TLC)

The 4 basic liquid chromatography modes are named according to the mechanism involved:

 1. Liquid/Solid Chromatography (adsorption chromatography)

A. Normal Phase LSC

B. Reverse Phase LSC

 2. Liquid/Liquid Chromatography (partition chromatography)

A. Normal Phase LLC

B. Reverse Phase LLC

 3. Ion Exchange Chromatography

 4. Gel Permeation Chromatography (exclusion chromatography)

FOUR BASIC LIQUID CHROMATOGRAPHY

CHROMATOGRAPHY

Chromatography basically involves the separation of mixtures due to differences in

the distribution coefficient (equilibrium distribution) of sample components between

2 different phases.

One of these phases is a mobile phase and the other is a stationary phase.

Definition:

 

Different affinity of these 2 components to stationary phase causes the separation.

Concentration of component A in stationary phase

Concentration of component A in mobile phase

Distribution Coefficient (Equilibrium Distribution )

Principles of Chromatography

• Chromatography is used when there is a difference in the retention times of different components

• Two types of phases

1) Stationary phase

2) mobile phases

• Properties of Chromatographic Properties

1) immiscible stationary and mobile phases

2) an arrangement where a mixture is depositied at one end of the

stationary phase

3) flow of the mobile phase towards the other end of the stationary

phase

4) different rates of partitioning for each component

5) means for visualizing the separation of each component

Paper Chromatography

Paper chromatography. Molecules separate as they move up the paper. The distance that the molecules travel depends on their size and solubility in the solvent.

19

Thin Layer and Paper Chromatography

A solution of a mixture is applied as a spot/band at the bottom of the plate and allowed to travel with the solvent up the

plate.

A B C A+B+C

standards Mixedstandard

s

Unknown +

standards

•• •• •• •• •• ••

A+B+C ??

كروماتوگراصفحه فی

ای

(TLC) غشاء روی بر کروماتوگرافی نازک

A B

Rf= A/B

• 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.

Principles of Paper Chromatography

• TLC involves spotting a dilute solution (1%) of sample TLC involves spotting a dilute solution (1%) of sample on one end of a small sheet that has been coated with on one end of a small sheet that has been coated with silica gel (SiOsilica gel (SiO22) or alumina (Al) or alumina (Al22OO33), known as the ), known as the

stationary adsorbent phase.stationary adsorbent phase.• The sheet is placed upright inside a jar in a small pool The sheet is placed upright inside a jar in a small pool

of solvent. As the solvent rises up the sheet by of solvent. As the solvent rises up the sheet by capillary action, the components travel at different capillary action, the components travel at different rates based on competing interactions with the mobile rates based on competing interactions with the mobile (solvent) and adsorbent phases.(solvent) and adsorbent phases.

• SiOSiO22 is used for separation of more polar compounds is used for separation of more polar compounds

while while AlAl22OO33 is used in the separation of non is used in the separation of non-polar -polar compounds.compounds.

Thin-Layer Chromatography Thin-Layer Chromatography (TLC)(TLC)

Thin-Layer Chromatography

Thin-layer chromatography. Molecules separate as they move through the silica gel. Thin-layer chromatography is used to separate small molecules, such as amino acids.

Thin-Layer Chromatography (TLC)Thin-Layer Chromatography (TLC)

• A polar solvent will carry a polar compound farther while a non-A polar solvent will carry a polar compound farther while a non-polar solvent will carry a non-polar compound farther.polar solvent will carry a non-polar compound farther.

• RRf f value is the ratio of the distance the spot travels from the value is the ratio of the distance the spot travels from the

origin to the distance the solvent travels.origin to the distance the solvent travels.

Detectors آشکارسازها

• 1) UV lamp

• 2) Iodine

• 3) Specific reagents

TLC

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

Affinity Chromatography. Separating molecules based on shape is often done using antibody resin. Antibodies recognize only certain antigens and will bind those and pull them out of solution (fraction #3).

Affinity Chromatography

Surface bound with

Epoxy, aldehyde or aryl ester groups

Metal Interaction Chromatography

Surface bound with

Iminodiacetic acid + Ni2+/Zn2+/Co2+

Affinity Chromatography

How do they get those iddy bitty molecules in there?

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

Applications

• Used in Genetic Engineering

• Production of Vaccines

• And Basic Metabolic Research

Ion-Exchange Chromatography

Ion Exchange Resin. Resins are manufactured with ions attached. The ions present a certain degree of positive or negative charge, depending on the buffer pH.

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+OH-

NH(-R)2+OH- + B- Res-NH(-R)2+B- +OH-

MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS

SO3-

SO3-

Na+

COO-

H3N+

Na+

COOHH3N

+

pH2

pH4.5

Ion-exchange Resin

ION-EXCHANGE CHROMATOGRAPHY

Separation in Ion-exchange Chromatography is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing).

SO3

-Na

+

GEL-PERMEATION CHROMATOGRAPHY

Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution. Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules.

Hemoglobin A1c

The HgA1c is the gold standard for diabetes care. To help prevent diabetes complications, the HgA1c should be maintained at 7% or below according to the Diabetes Control and Complications Trial (DCCT) and United Kingdom Prospective Diabetes Study (UKPDS) findings.

According to the United Kingdom Prospective Diabetes Study for every percentage point decrease in HbA1c (e.g. 9 to 8 percent), there was a 35 percent reduction in the risk of micro vascular complications. (ADA, Clinical Practice Recommendations, 2002).

HbA1c: the blood test with a memory

What is HbA1c?

Hemoglobin is a protein that makes your red blood cells red-colored.

When hemoglobin picks up glucose from your bloodstream, the hemoglobin becomes glycosylated.

Glycosylated hemoglobin is HbA1c. The HbA1c test measures the percentage of HbA1c in your blood—a number that corresponds to your average blood glucose for the previous 3 months.

HbA1c in your bloodstream.

• The hemoglobin A1C percentage is a way of looking at average blood sugar control over a period of 3 months.

• When plasma glucose is episodically elevated over time, small amounts of hemoglobin A are nonenzymatically glycosylated to form HbA1C.

• Red blood cells live 90 to 120 days. This means that

once sugar has combined with the hemoglobin in red blood cells, the hemoglobin A1C stays in the blood for 90 to 120 days. This means the amount of hemoglobin A1C in blood reflects how often and how high the blood sugar has been over the past 3 months.

• The hemoglobin A1C percentage rises as the average plasma glucose level rises.

Depending on the assay method, HbA1 represent approximately 3-6 % of total hemoglobin in nodiabetics, and as much as 20% or more in poorly controlled people with diabetes. Hemoglobin may become glycated at sites other than the ends of the beta chains, e.g., at lysine residues (HbA0). These modifications, which represent almost 50% of the total GHb, do not alter the charge of the hemoglobin molecule,

thus are not detected by methods based on charge differencesbetween glycated and non-glycated hemoglobin.

The minor hemoglobin components first recognised because some of them showed differences in

electrical charge; they were called "fast haemoglobin"

because they showed less positive charge at neutral pH, and migrated more rapidlythen hemoglobin A when placed in an electrical field

Hemoglobin NomenclatureHbA The major form of hemoglobin, a native, unmodified tetramer consisting of two α and two β chains.HbA0 The major component of HbA, identified by its chromatographic and electrophoretic properties. Post translational modifications, including glycosylation, do exist in this fraction but do not significantly affect the charge properties of the protein.

HbA1 Post translationally modified more negatively charged forms of HbA, as detected by chromatographic and electrophoretic methods.

HbA1a1, HbA1a2 Chromatographically distinct components of HbA1.HbA1b, HbA1c

HbA1c Adduct of glucose attached to the β-chain terminal valine residue by a ketoamine linkage.

Pre-HbA1c A labile form of glycosylated hemoglobin containing glucose attached by aldimine linkage at the β-chain terminal valine residue.

"Fast" The total HbA1 fraction, which, because of a more negative hemoglobin(s) charge, migrates more rapidly toward the anode in electrophoresis and elutes earlier in cation-exchange chromatography than does HbA0.

Glycosylated Haemoglobin modified by glucose at α-and β-chain terminal valine residues and ε-amino groups of lysine residues.

Glycated (glycosylated) haemoglobin(s) A generic term of hemoglobin containing glucose and(or) other carbohydrate

Terminology

• Elution - washing of the mixture

• Eluent - additional solvents used for

elution

• Effluent - exiting fluid stream

• Residency - time spent on column

• Stationary Phase - • Mobile Phase - fluid carrying the mixture of

analytes

• Paper chromatography – a form of chromatography that uses filter paper as the solid phase, and allows molecules to separate based on size or solubility in a solvent

• Thin-layer chromatography – a separation technique that involves the separation of small molecules as they move through a silica gel

• Chromatograph – the medium used in chromatography (ie, paper, resin, etc.) through which the molecules of interest move and separate

• Gel-filtration chromatography – a type of column chromatography that separates proteins based on their size using size-exclusion beads; also called size-exclusion chromatography

• Ion-exchange chromatography – a separation technique that separates molecules based on their overall charge at a given pH

• Affinity chromatography – a type of column chromatography that separates proteins based on their shape or attraction to certain types of chromatography resin

• Hydrophobic-interaction chromatography – column chromatography that separates molecules based on their hydrophobicity (aversion to water)

• Elution – when a protein or nucleic acid is released from column chromatography resin

• Cation exchange – a form of ion-exchange chromatography in which positively charged ions (cations) are removed by a positively charged resin

Vocabulary