Overview
The biochemistry text that every medical student must own--now in full color!
Comprehensive, concise, and up-to-date, Harper's is unrivaled in its ability to clarify the link between biochemistry and the molecular basis of health and disease.
The Twenty-Eighth Edition has undergone sweeping changes -- including a conversion to full-color artwork and the substantial revision and updating of every chapter -- all to reflect the latest advances in knowledge and technology and to make the text as up-to-date and clinically relevant as possible. Combining outstanding full-color illustrations with integrated coverage of biochemical diseases and clinical information, Harper's Illustrated Biochemistry offers an organization and clarity not found in any other text on the subject.
Striking just the right balance between detail and brevity, Harpers Illustrated Biochemistry is essential for USMLE review and is the single best reference for learning the clinical relevance of a biochemistry topic.
NEW to this edition:
* Full-color presentation, including 600+ illustrations
* Every chapter opens with a Summary of the Biomedical Importance and concludes with a Summary reviewing the topics covered
* Two all-new chapters: "Free Radicals and Antioxidant Nutrients" and "Biochemical Case Histories" which offers an extensive presentation of 16 clinical conditions
* A new appendix containing basic clinical laboratory results and an updated one with a list of important websites and online journals
* NEW or updated coverage of important topics including the Human Genome Project and computer-aided drug delivery
Table of contents
1. Biochemistry and Medicine
2. Water & PH
3. Amino Acids and Peptides
4. Proteins: Determination of Primary Structure
5. Proteins: Higher Orders of Structure
6. Proteins: Myoglobin and Hemoglobin
7. Enzymes: Mechanisms of Action
8. Enzymes: Kinetics
9. Enzymes: Regulation of Activities
10. Bioenergetics: The Role of ATP
11. Biologic Oxidation
12. The Respiratory Chain & Oxidative Phosphorylation
13. Carbohydrates of Physiologic Significance
14. lipids of Physiologic Significance
15. Overview of Metabolism
16. The Citric acid Cycle: The Catabolism of Acetyl-CoA
17. Glycolysis and Oxidation of Pyruvate
18. Metabolism of Glycogen
19. Gluconeogenesis and Control of the Blood Glucose
20. The Pentose Phosphate Pathway & other Pathways of Hexose Metabolism
21. Biosynthesis of Fatty Acids
22. Oxidation of Fatty Acids: Ketogenesis
23. Metabolism of Unsaturated Fatty Acids & Eicosanoids
24. Metabolism of Acyglycerols and Sphingolipids
25. Lipid Transport And Storage
26. Cholesterol Synthesis, Transport and Excretion
27. Integration of Metabolism- the Provision of Metabolic Fuels
28. Biosynthesis of Nutritionally Nonessential Amino Acids
29. Catabolism of Proteins & of Amino Acid Nitrogen
30. Catabolism of the Carbon Skeletons of Amino Acids
31. Conversion of Amino Acids
32. Porphyrins & Bile Pigments
33. Nucleotides
34. Metabolism of Purine & Pyrimidine Nucleotides
35. Nucleic Acid Structure and Function
36. DNA Organization, Replication and Repair
37. RNA Synthesis, Processing and Modifications
38. Protein Synthesis and Genetic Code
39. Regulation of Gene Expression
40. Molecular Genetics, Recombinant DNE and Genomic Technology
41. Membranes: Structure and Function
42. The Diversity of Endocrine System
43. Hormone Action and Signal Transduction
44. Nutrition, Digestion and Absorption
45. Vitamins and Minerals
46. Intracellular Traffic and Sorting of Proteins
47. Glycoprotein
48. The Extracellular Matrix
49. Muscle and the Cytoskeleton
50. Plasma Proteins and Immunoglobulins
51. Homeostasis and Thrombosis
52. The Red and White Blood Cells
53. Metabolism of Xenobiotics
54. The Human Genome Project
Biographical note
Robert K. Murray, MD, PhD,
Professor (Emeritus) of Biochemistry,
University of Toronto
David A. Bender, PhD,
Sub-Dean, University College Medical School,
Senior Lecturer in Biochemistry,
Department of Structural and Molecular Biology and
Division of Medical Education,University College London
Kathleen M. Botham, PhD, DSc,
Professor of Biochemistry, Royal Veterinary
College, University of London
Peter J. Kennelly, PhD,
Professor and Head, Department of Biochemistry,
Virginia Polytechnic Institute and State University,
Blacksburg, Virginia
Victor W. Rodwell, PhD,
Emeritus Professor of Biochemistry, Purdue University, West Lafayette,
Indiana
P. Anthony Weil, PhD,
Professor of Molecular Physiology and Biophysics,
Vanderbilt University School of Medicine,
Nashville, Tennessee
Co-Authors:
Daryl K. Granner, MD,
Emeritus Professor of Molecular Physiology and Biophysics
and Medicine,
Vanderbilt University, Nashville, Tennessee
Peter L. Gross MD, MSc, FRCP(C),
Associate Professor, Department of Medicine,
McMaster University
Frederick W. Keeley, PhD,
Associate Director and Senior Scientist, Research Institute,
Hospital for Sick Children, Toronto, and Professor,
Department of Biochemistry, University of Toronto
Peter A. Mayes, PhD, DSc,
Emeritus Professor of Veterinary Biochemistry, Royal Veterinary College, University of London
Margaret L. Rand, PhD,
Associate Senior Scientist, Hospital for Sick Children, Toronto,
and Professor, Departments of Laboratory Medicine and Pathobiology
and Department of Biochemistry, University of Toronto
download link
http://rapidshare.com/files/282803148/H_BC_28ed_lycan.rar
torrent link
http://freshwap.net/torrents/dl/2991880
Monday, February 22, 2010
Harper's Biochemistry 28th edition
Monday, February 15, 2010
Carbohydrate Chemistry
Introduction
Carbohydrates have the general molecular formula (CH2O)n, and thus were once thought to represent "hydrated carbon".
All sugars are very soluble in water because of their many hydroxyl groups.
They are called sugars because they are sweet in taste & crystalline in nature.
Sugars are the most important source of energy for many cells.
Definition:
Hydrates of carbon Cn(H2O)n
o {exceptions:
Acetic acid, C2H4O2 & lactic Acid, C3H6O3 Formaldehyde, CH2Oare not carbohydrates but
Deoxy Ribose C6H12O5 is a carbohydrate
carbohydrates are polyhydroxy aldehydes or ketones or compounds, which yields these on hydrolysis.
Classification:
- Classified as
- Monosaccharides
- Disaccharides
- Oligosaccharides
- Polysaccharides
- Monosaccharides
Monosaccharides
- Simple sugars that cannot be hydrolyzed to yield another Carbohydrate.
- Classification
- Based on the functional group
- They are classified as ALDOSES (-CHO) and KETOSES (=C=O)
- They are classified as ALDOSES (-CHO) and KETOSES (=C=O)
- Based on the number of Carbon atoms as
- Triose (C3)
- Tetrose(C4)
- Pentose(C5)
- Hexose(C6)
- Heptose(C7)
- Nonose (C9)
- Triose (C3)
- Examples
- Trioses - C3H6O3
- Aldotriose
- Glyceraldehyde
- Glyceraldehyde
- Ketotriose
- Dihydroxy acetone
- Dihydroxy acetone
- Tetroses - C4H8O4
Aldotetrose
Erythrose
Ketotetrose
Erythrulose
- Pentoses - C5H10O5
Aldopentose
Ribose
Ketopentose
Ribulose
- Hexoses -C6H12O6
Aldohexose
Glucose
Mannose
Galactose
Ketohexose
Fructose
- Heptoses - C7H14O7
Sedoheptulose
- Nonoses - C9H18O9
Sialic acid or Neuramic acid
- Simple sugars that cannot be hydrolyzed to yield another Carbohydrate.
Disaccharides
- Disaccharides are made up of two monosaccharides joined by Glycosidic bond.
- The disaccharides on hydrolysis gives two monosaccharides
- Examples
- Biologically important Disaccharides are
- Maltose
- Sucrose
- Lactose
- Maltose
- Others include
- Cellobiose
- Trehalose
- Cellobiose
- Disaccharides are made up of two monosaccharides joined by Glycosidic bond.
- Maltose C3H6O3
- Reducing in nature
- the major degradation product of starch
- composed of 2 glucose monomers in an alpha-(1,4) glycosidic bond.
- Reducing in nature
- Lactose C3H6O3
- Milk Sugar
- Reducing in nature
- consists of galactose and glucose in a beta-(1,4) glycosidic bond.
- Milk Sugar
- Sucrose C3H6O3
- Cane sugar and sugar beets
- Non-Reducing in nature
- composed of glucose and fructose through an alpha-(1,2)beta-glycosidic bond.
- Cane sugar and sugar beets
Oligosaccharides
- They are made up of 3 to 10 Monosaccharides joined by glycosidic bonds.
- They on hydrolysis yields 3-10 monosaccharides
- Examples
- Maltotriose
Glucose + Glucose + Glucose
Degradation product of starch hydrolysis
- Raffinose
A trisaccharide that occurs in sugar beets and cotton seeds and cereals
- Stachyose
A tetrasaccharide found in the tubers of the Chinese artichoke
- They are made up of 3 to 10 Monosaccharides joined by glycosidic bonds.
Polisaccharides
- Are madeup of more than 10 monosaccharides
- They yields more than 10 Monosaccharides on hydrolysis.
- Classified in to TWO types
- Homopolysaccharides
- Heteropolysaccharides
- Homopolysaccharides
- Are madeup of more than 10 monosaccharides
Homopolysaccharides (Homoglycans)
- Homopolysaccharides are polymer of a single type of Monosaccharide.
- Classified in to
- Storage Homopolysaccharides
- Structural Homopolysaccharides
- Storage Homopolysaccharides
- Homopolysaccharides are polymer of a single type of Monosaccharide.
Storage Homopolysaccharides
- Examples
- Starch
- Polymer of Glucose
- Source : Potato,tapioca,rice,wheat,&grains
- Importance :Plant storage form of Energy
- Gives blue colour with iodine
- Polymer of Glucose
- Amylose
- Unbranched starch
- Contains alpha 1-4 linkages
- Unbranched starch
- Amylopectin
- Branched starch (at every 18-22 Glucose)
- Contains both alpha 1-4 and alpha 1-6 linkages
- Branched starch (at every 18-22 Glucose)
- Glycogen
- Polymer of Glucose
- Source : Liver (5%) and muscle
- Branched (at every 8-10 Glucose)
- Importance :Animal Storage form of Energy
- Polymer of Glucose
Structural Homopolysaccharides
- Examples
- Cellulose
- Polymer of glucose
- Contains beta 1-4 linkages
- Considered as dietary fiber undigested by man
- Polymer of glucose
- Chitin
- Polymer of N-Ac-Glucosamine
- Present in the Exoskeleton of Insects
- Polymer of N-Ac-Glucosamine
Other homopolysaccharides
- Inulin
- Fructose
- Source : various bulbs&tubers(onion,garlic)
- Used for :Renal clearance studies.
- Source : various bulbs&tubers(onion,garlic)
Hetroplysaccharides (Hetroglycans)
- polymer of two or more types of sugar residues
- Examples
- polymer of two or more types of sugar residues
- Agar
- Polymer of : Glu +Gal + other
- Obtained from Sea weeds
- A colloidal extract of algae; used especially in culture media and as a gelling agent in foods (undigested by bacteria, dissolves at 100 oC and upon cooling sets into a Gel)
- A colloidal extract of algae; used especially in culture media and as a gelling agent in foods (undigested by bacteria, dissolves at 100 oC and upon cooling sets into a Gel)
MUCOPOLYSACCHARIDES (MPS) or GLYCOSAMINOGLYCANS (GAG)
- Are Carbohydrates containing Uronic Acid & Amino Sugars.
- Examples
- Are Carbohydrates containing Uronic Acid & Amino Sugars.
Hyaluronic acid
- A viscous mucopolysaccharide found in the connective tissue space and the synovial fluid of movable joints and the humours of the eye; a cementing and protective substance
Heparin
- A polysaccharide produced in basophils (especially in the lung and liver) and that inhibit the activity of thrombin in coagulation of the blood.
Chondroitin SO4
- Inground substance of connective tissues (cartilage, tendon, bone)
Keratan SO4
- In cornea & tendons.
Carbohydrates
CARBOHYDRATES
Carbohydrates have the general molecular formula (CH2O)n, and thus were once thought to represent "hydrated carbon". All sugars are very soluble in water because of their many hydroxyl groups. They are called sugars because they are sweet in taste & crystalline in nature. Although not as concentrated a fuel as fats, sugars are the most important source of energy for many cells. Carbohydrates provide the bulk of the calories (4 kcal/gram)
1)Definition:
a)Hydrates of carbon Cn(H2O)n{exceptions: Acetic acid, C2H4O2 & lactic Acid, C3H6O3 Formaldehyde, CH2Oare not carbohydrates but Rahnose,C6H12O5 is.}
b)carbohydrates are polyhydroxy aldehydes or ketones or compounds, which yields these on hydrolysis.
a)Monosaccharides:simple sugars that cannot be hydrolyzed to yield Carbohydrates
Name | Aldose | Ketose | Formula |
Trioses | Glyceraldehyde | Dihydroxy acetone | C3H6O3 |
Tetroses | Erythrose | Erytrulose | C4H8O4 |
Pentoses | Ribose | Ribulose | C5H10O5 |
Hexoses | Glucose,Mannose, Galactose | Fructose | C6H12O6 |
Heptoses | Sedoheptulose | C7H14O7 | |
Nonoses | Sialic acid or Neuramic acid | C9H18O9 |
b)Disaccharides: yields two molecules of Monosaccharides on hydrolysis
Name | Monosaccharides | Formula | Sources | Properties |
Maltose | Glucose + Glucose | C3H6O3 | Malt sugar, starch | Reducing |
Lactose | Glucose + Galactose | C3H6O3 | Milk Sugar | Reducing |
Sucrose | Glucose + Fructose | C3H6O3 | Cane sugar | Non-Reducing |
Sucrose: prevalent in sugar cane and sugar beets, is composed of glucose and fructose through an a-(1,2)b-glycosidic bond.
Lactose: is found exclusively in the milk of mammals and consists of galactose and glucose in a b-(1,4) glycosidic bond.
Maltose: the major degradation product of starch, is composed of 2 glucose monomers in an a-(1,4) glycosidic bond.
c)Oligosaccharides: yields 3 to 10 Monosaccharides on Hydrolysis.
Maltotriose | Glucose + Glucose + Glucose |
Raffinose | A trisaccharide that occurs in sugar beets and cotton seeds and cereals |
Stachyose | A tetrasaccharide found in the tubers of the Chinese artichoke |
d)Polisaccharides: yields more than 10 Monosaccharides on hydrolysis. They are of two types.
i)Homopolysaccharides (Homoglycans) Polymer of a single type of Monosaccharide.
Name | Monosacc. | Sources | Importance |
Starch | Glucose | Potato,tapioca,rice,wheat,&grains | Plant source of Energy |
Amylose | Glucose | Un branched starch similar to cellulose, containing alpha(1->4) linkages | |
Amylopectin | Glucose | Branched starch containing both 1,4 and 1,6 linkages. | |
Glycogen | Glucose | 5% of liver mass> branched & compact | Animal storage of Glucose |
Inulin | Fructose | various bulbs&tubers(onion,garlic) | Renal clearance studies. |
Dextrins | Glucose | Product. ofstarch digestion | White dextrin is used in pharmaceutical preparations |
Chitin | N-Ac-Glucosamine | Exoskeleton of Insects |
ii)Hetroplysaccharides (Hetroglycans): polymer of two or more types of sugar residues
Agar | Glu +Gal + other | Obtained from Sea weeds |
A colloidal extract of algae; used especially in culture media and as a gelling agent in foods (undigested by bacteria, dissolves at 100 oC and upon cooling sets into a Gel) |
iii)MUCOPOLYSACCHARIDES (MPS) or GLYCOSAMINOGLYCANS (GAG)
Are Carbohydrates containing Uronic Acid & Amino Sugars.
Name | Functions |
Hyaluronic acid | A viscous mucopolysaccharide found in the connective tissue space and the synovial fluid of movable joints and the humours of the eye; a cementing and protective substance |
Heparin | A polysaccharide produced in basophils (especially in the lung and liver) and that inhibit the activity of thrombin in coagulation of the blood. |
Chondroitin SO4 | Inground substance of connective tissues (cartilage, tendon, bone) |
Keratan SO4 | In cornea & tendons. |
e)Isomerism
i)D & L Isomerism
oThe configuration of the asymmetric carbon atom ( carbon atom whose 4 valencies are filled up by 4 different groups)farthest from the aldehydic or ketonic group
oIf –OH group is attached to the right then 'D' series & left then 'L' series.
ii)Pyranose - Furanose ring Structure
oWhen ring structure were suggested by Haworth (projections)
oFive member ring structure is calledFuranose and Six number as Pyranose
iii)Alpha & Beta Anomerism
oThe anomeric carbon (which originally was a part of the aldo or keto group) forms a hemiacetal or hemiketal in the ring structure generating a new asymmetric centre.
oIf hydroxyl gp (-OH) of the anomeric carbon is to the right then it is -form and if to the left it -form.
iv)Epimerism
oEpimers differ in the position of the Hydroxyl gp at only one of their assymetric C-atom.
oD-Glucose & D-Galactsoe are C-4 Epimers
oD-Glucose & D-Mannose are C-2 epimers
v)Enantiomers
oStereo isomers that are mirror images of each other
oEg: D-Glyceraldehyde & L-Glyceraldehyde.
vi)Mutarotation
oWhen D-Glucose is crystallized at RT and fresh solution is prepared, its specific rotation of polarized light is +112 o; but after few hrs it changes to +52.5 o. This change in rotation is called Mutarotation. This b'se D-Glucose has two anomers & forms. (-D-Glucose has specific rotation of +112 o and -D-Glucose has +19 o at equilibrium 1/3 is form and 2/3 is form to get a specific rotation of +52.5 o
3)Other Physiologically important carbohydrates.
Sugar | Present where | Importance&clinical Significance |
D-Ribose | Nucleic acids | Nucleic acid co-enzymes. |
D-Ribulose | Metabolic Product | Pentose-PO4 pathway intermediate. |
D-Arabinose | Gum arabic, Plum & Cherry gum | Constituent of Glycoproteins |
D-Xylose | Wood gums, GAG's | Constituent of Glycoproteins |
D-Xylulose | Intermediate in Uronic acid pathway | Found in the Urine of patients with Essential pentosuria |
4)CHEMISTRY
a)Reactions
i)Benedict's Reaction
Contains Na2CO3, Na-Citrte,CuSO4
In alkaline medium (Na2CO3), the copper remains as cupric hydroxide in presence of sodium citrate
5 ml Benedict's reagent+ 0.5 ml Urine boil for 2 minutes. If sugar present copper is reduced to produce green, yellow, orange, red precipitate depending on the concentration of the sugar.
SugarEnediolCu++(cupric) CuSO 4(Reagent)
Sugar AcidCu+(cuprous) 2Cu(OH)[Yellow]Cu 2O [Red]
Chemical propertie | ||||||
1 | Reactions of aldehyde / keto group | |||||
Reactions | Reagents | Result | ||||
a. | Osazone formation | Sugar+Phenyl Hydrazine HCl+ Sodium Acetate (boil+cool) | Glucosazone-needle shapedLactosazone - Powder puff Maltosazone - Sunflower | |||
b. | Reduction → Alcohol | Sodium amalgum / Hydrogen | Glucose - sorbitolFructose - Manitol | |||
c. | Oxidation | |||||
I. | Mild (CHO → COOH) | Br2 H2O | Glucose - gluconic acid | |||
ii. | Srong (CHO & CH2OH to COOH | HNO3 | glucose - Glucosaccharic acid | |||
iii. | Oxdn of CH2OH alone | Enzymatic (living cells only) | Glucose to glucuronic acid | |||
d. | Tautomerisation( Inter conversions) | Weak alkaliBa (OH)2, Ca(OH)2 | Glucose ↔ fructose ↔ Mannose | |||
e. | Reduction Reactions | CuSO4, alkaline medium( cupric -> cuprous) | Benedicts, Fehlings & Barfoeds Test( Cuprous oxide- Red ppt) | |||
2 | Reactions of Alcoholic group | |||||
a. | Glycosides | 1→4 &1→6 bond in glycosidic bonds in glycogen | ||||
I. | Cardiac | Derivatives of DIGITALIS, | Drugs in cardiac insufficiency | |||
ii. | Oubin | strophanthus sp. | Sodium pump inhibitor (cardiac) | |||
iii. | Phlorbizin | Root/bark of apple tree | block transport of sugar | |||
iv. | Streptomycine | produced by the actinomycete Streptomyces griseus | Antibioticused to treat tuberculosis | |||
b. | Acetilation/Ester formation | Alcohol + acids | phosphates ( glucose -1-phosphate) | |||
c. | Dehydratin( Molisch, Selivanoffs, Bials tests) | Strong acids (Con.H2SO4) | Pentose→ Furfurals,Hexose→ Hydroxy methyl Furfural. | |||
TESTS | ||||||
Name of test | Substrate | Reagents | Result | |||
1 | Molisch's Test | Sugar | 3d Alfa-Naphthol in Methanol +H2SO4 (side) | Violet color in the Junction. | ||
2 | Iodine test | Starch | Dil. Iodine | Deep Blue Colour | ||
3 | Benedict's | Reducing Sugar(0.5 ml/ 8 dps) | CuSO4+Na2CO3+NaCitrate(5 ml) | Cuprous Oxide (Red Precipitate) | ||
4 | Fehling's Test | Reducing Sugar | CuSO4+KOH+NaK Tartrate | Cuprous Oxide (Red Precipitate) | ||
5 | Barfoed's test | Monosaccharide. | CuAcetate+Dil.AceticAcid | (Red Precipitate) in < 5' | ||
6 | Bial's Test | Pentose | Orcinol Hydrochloride (Boil) | Green | ||
7 | SelivanoffsTest | Ketoses | Dil. Resorcinol in Dil.HCl (heat) | Cherry red | ||
8 | Tollent's Test | Reducing Sugar | Ammoniacal Silver nitrate | Metalic Siver Mirror |
METABOLISM OF BILIRUBIN
METABOLISM OF BILIRUBIN
After 120 days of lifespan RBC's are broken down to release Hemoglobin
The hemoglobin then broken down to form Haem and Globin
Globin being a protein is used as such or converted to amino acids and taken to amino acid pool
The Haem is then broken down to release a porphyrin compound and Iron
The iron is taken to Iron pool and re utilized
The porphyrin ring opens and is converted to Biliveridin a green pigment
The biliveridin is immediately reduced to Bilirubin an yellow pigment
The formation of Bilirubin is taking place in the reticulo endothelial cells of Spleen, Bone marrow and
The Bilirubin thus formed being water insoluble is then attached with the transport protein albumin and is then transported to liver through the blood. This albumin Bilirubin complex is known as the Unconjugated Bilirubin or Indirect Bilirubin or Free Bilirubin
Liver.
In liver there are three processes taking place
- Uptake of Bilirubin in to liver cells (hepatocytes)
- Conjugation of Bilirubin with Glucuronic acid
- Excretion of Conjugated Bilirubin in to Bile
The albumin is removed form the Bilirubin and is taken in to the hepatocytes where they are conjugated.
The Bilirubin in the hepatocyte is conjugated with two molecules of the UDP Glucuronic acid to form the Bilirubin Di Glucuronide and is known as the Conjugated Bilirubin or Direct Bilirubin.
The conjugated Bilirubin being water soluble is excreted in to the bile and it reaches the small intestine
Small Intestine:
In small intestine the glucuronides are removed and the Bilirubin is converted to urobilinogen by the intestinal bacteria.
A part of the urobilinogen is reabsorbed back in to the liver through the portal circulation and reaches the liver and thus in to the general circulation. Then it is excreted in to the urine via kidneys. The urobilinogen in urine is the cause of yellow coloration to urine and when it is exposed to oxygen is converted to dark Urobilin.
The remaining urobilinogen in the small intestine is excreted in to the feces as Stercobilinogen. This Stercobilinogen in the stool is the cause of yellow coloration to the stool and when it is exposed to the oxygen it is converted to dark colored stercobilin.
Adsorption Chromatography
INTRODUCTION
- Adsorption chromatography is probably one of the oldest types of chromatography around.
- It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase.
- The equilibriation between the mobile and stationary phase accounts for the separation of different solutes.
PRINCIPLE
- Classic form of Chromatography by Tswett
Principle
- Based on the principle that certain solid materials, collectively known as adsorbents have the ability to hold molecules at their surface.
- This adsorbing process which involves weak non-ionic attractive forces of van der Waal's and hydrogen bond occurs at specific adsorption site.
- These sites have the ability to discriminate between the molecules and the adsorption of the molecule depends on the strength of the interaction.
- As elutes continuously pass down the column, difference in their binding eventually lead to the separation for the analytes.
- Binding depends on the functional groups present in the molecules
- Hydroxyl, aromatic groups - increase interaction with the binding surface
- Aliphatic groups - decrease interaction with the binding surface
- Hydroxyl, aromatic groups - increase interaction with the binding surface
STATIONARY PHSAE / ADSORBENT
Silica
- with Silanol groups (Si-OH) groups on its surface
- Are slightly acidic in nature
- Can interact with polar functional group of the analyte or eluent.
- The topology (arrangement of Si-OH explains their differential separation
- Silica is acidic - good for the separation of basic substances
- Available for both LPLC & HPLC
Alumina
- Available for both LPLC & HPLC
- Alumina is basic - good for the separation of acidic substances
Carbon
- Available for both LPLC & HPLC
MOBILE PHASE ELUENT
- Depends on k' (partition ratio / capacity ratio) of analyte. (which is the time spent by analyte in the stationary phase relative to the time spend in the mobile phase)
- Eluent with polarity comparable to the most polar compound in the analyte mixture is selected as mobile phase.
- Alcohol is selected for analytes with hydroxyl (-OH) groups
- Acetone/esters is selected for analytes with carbonyl groups (=C=O) groups
- Hexane / heptane /toluene (hydrocarbons) is selected for non polar analytes
- Mixture of solvents also can be used in gradient elution.
- Alcohol is selected for analytes with hydroxyl (-OH) groups
- Presence of small amounts of water in the MP is beneficial when silica is the stationary phase – which selectively block more active Silanol groups leaving a more selective population of weaker binding sites.
MODES/ APPLICATION -USES
Modes
- Column
- Thin layer
Application/USES
- Most commonly used to separate non ionic water insoluble substances
- Triglycerides
- PTH
- Aminoacids
- Vitamins
- Many drugs
- Triglycerides
TWO MAJOR TYPES
- Hydroxylapatite Chromatography
- Hydrophobic interaction Chromatography
HYDROXY(L)APATITE CHROMATOGRAPHY
- Also known as hydroxyapatite chromatography
- Adsorbent used
- Crystalline hydroxyapatite (Ca10(PO4)6(OH)2)
- Crystalline hydroxyapatite (Ca10(PO4)6(OH)2)
- Mechanism
- Involves both Ca & PO4 ions on the surface
- Involve dipole-dipole interaction
- Involve electrostatic interaction
- Involves both Ca & PO4 ions on the surface
APPLICATION/ USES OF HC
- Used to separate single stranded DNA from double stranded DNA
- Both single stranded and double stranded binds at low phosphate buffer concentration
- At increased buffer concentration single stranded DNA is selectively desorbed
- Further increase in buffer concentration desorbs double stranded DNA also
- Used of Cot Analysis
- So DNA can be separated from mixture of RNA and protein of cell Extracts.
COMMERCIAL
- Suitable for both LPLC & HPLC
- Crystalline or spheroidal hydroxy apatite is bonded to the agarose matrix
- Adsorption capacity is maximum around the neutral pH using 20 mM phosphate buffer
- Elution is done at 500 nM
HYDROPHOBIC INTERACTION CHROMATOGRAPHY
- Used to purify protein exploiting their surface hydrophobicity (non polar amino acids)
- In aqous solution these hydrophobic regions of proteins are covered with an ordered film of water molecules that effectivel mast these hydrophobic groups
- Exposed by the addition of salt ions
- Then these hydrophobic regions interact together and is the basis of salting out by the addition of ammonium sulphate
- If hydrophobic groups are attached to a suitable matrix the hydrophobic groups on the proteins will interact with them on the matrix to facilitate a protein-matrix attraction
- Most commonly used stationary phases are
- Alkyl (hexyl , Octyl)
- Phenyl groups
- Attached to Agarose matrix
- Alkyl (hexyl , Octyl)
Commercial
LPLC(HIC)
- Phenyl Sepharose , Phenyl SPW
HPLC (HIC)
- Biogel TSK phenyl, Spherogel TSK Phenyl
- Ammonium sulphate is present in the sample since it fractionated before analysis with it which help to expose the hydrophobic centers.
Elution process.
- Gradually decrease the ionic strength
- Increase the pH
- Selective displacement by displaces that has s stronger affinity for the stationary phase than has the protein
- Non ionic detergents like
- Tween 20
- Trinton –X100
- Tween 20
- Aliphatic Alcohols like
- Butanol
- Ethylene Glycol
- Butanol
- Aliphatic Amines
- Butylamine
- Butylamine
- Some of these elution condition may cause protein denaturation
- Protein purified -->Aldolase, Transferrin, Cytochrom c, thyroglobulin etc.