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HL Chemistry - Option A : Modern Analytical Chemistry. 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.

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HL Chemistry - Option A : Modern Analytical Chemistry

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Hl chemistry option a modern analytical chemistry l.jpg

HL Chemistry - Option A :Modern Analytical Chemistry

Chromatography


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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.


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Stationary Phase: Alumina

Acidic: -Al-OH

Neutral: -Al-OH + -Al-O-

Basic: -Al-O-


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Stationary Phase: Silica (SiO2)


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Distribution Coefficient (Equilibrium Distribution )

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


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Some Types of Chromatography

  • Liquid Column Chromatography (Reverse Phase too)

  • High Pressure (performance) Liquid Chromatograph (HPLC)

  • Paper Chromatography

  • Thin-layer Chromatography (TLC)

  • Gas Liquid Chromatography


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LIQUID COLUMN CHROMATOGRAPHY

A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.

With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.


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Diagram of Simple Liquid Column Chromatography


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BASIC LIQUID CHROMATOGRAPHY

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)


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LIQUID SOLID CHROMATOGRAPHY

The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel.


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LIQUID SOLID CHROMATOGRAPHY


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WATER-SOLUBLE VITAMINS


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WATER-SOLUBLE VITAMINS


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LIQUID-LIQUID CHROMATOGRAPHY

The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible in the solvent (Mobile) phase.

Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation.


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Chromatography Schematic


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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).


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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.


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MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS


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Chromatography of Amino Acids


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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.


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SOLVENTS

Polar Solvents

Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile

Non-polar Solvents

N-Decane > N-Hexane > N-Pentane > Cyclohexane


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SELECTING AN OPERATING MODE

Sample TypeLC Mode

Positional isomersLSC or LLC

Moderate Polarity Molecules LSC or LLC

Compounds with Similar FunctionalityLSC or LLC

Ionizable SpeciesIEC

Compounds with Differing SolubilityLLC

Mixture of Varying Sized Molecules GCC


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1.Ultraviolet Detector200-400nm 254 nm2.Reflective Index DetectorUniversal Detector

Detectors


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Liquid Chromatography Set Up


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HPLC Chromatography

  • Pump System. Mobil phase pressures up to 6000 psi are necessary to achieve reasonable column elution times (~ minutes). Typical flow rates are 0.1 to 10 mL/minute.

  • Injection System. Used to introduce small samples (0.1 to 500 µL) into the carrier stream under high pressure.

  • Reservoirs (Solvents). Multiple solvents are necessary for performing gradient elution's (i.e. changing the polarity of the mobil phase during a run).

  • Chromatographic Column. Typically 10-30 cm in length containing a packing of 5-10 µm diameter. Many types of columns are available, depending on the type of liquid chromatography desired.

  • Detector. Many types are available including UV, IR, refractive index, fluorescence, conductivity, mass spectrometry, and electrochemical. Diode array detectors are used when wavelength scans are desired.


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Schematic of an HPLC System


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HPLC System


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Pump System

Desirable Features:

  • Must generate pressures up to 6,000 psi

    • To allow for separation in reasonable time frames

  • Flow-rates range from 0.1 to 10 mL/minute

  • Limited pulsing in the system

    • Many HPLC systems have a dual pump system to minimize pulsing

  • Flow control and reproducibility < 0.5%

  • Corrosion resistance


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Sample Injection System

Used to introduce small samples (0.001 to 0.5 mL) into the carrier stream under high pressure


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HPLC Detectors

  • No universal or versatile detector

  • Types

    • General – respond to mobil phase bulk properties which vary in the presence of solutes (e.g. refractive index)

    • Specific – respond to some properties of the solute (not possessed by the mobil phase (e.g. UV adsorption)

    • “Hyphenated” detector – LC-MS


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Absorbance Detectors

  • The UV/Vis source usually comes from a monochromator so the wavelength can be selected, or scanned.

  • Absorbance increases as eluate passes through the cell.

  • If wavelength scanning is desired, the flow is stopped long enough for the scan to take place.

  • It’s possible to have the same setup using IR light, although not as common since many useful solvents are not IR transparent.


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Diode Array Detector


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HPLC Detectors


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HPLC Column

  • Must operate in high pressure

    • Usually constructed of metals

  • Typical dimensions

    • 10-30 cm long

    • 1-3 cm ID

  • Contains packing material which holds the stationary phase

    • Many types exist

    • Typical packing materials are 5-10 µm in diameter

  • Guard column used to extend life of main column


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Type of HPLC Depends on:

  • Molecular weight of solute

  • Water solubility of the solute

  • Polarity of the solute

  • Ionic/non-ionic character of the solute


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Separation Principles in HPLC

General Rule of Thumb:

Polarity of analytes ≈ polarity of stationary phase ≠ polarity of mobile phase

To achieve good separation, the analytes should interact with the stationary phase, but not too strongly or the retention time will become very long


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Reversed order

of elution

Increasing Mobil phase Polarity, Decreases Elution Time


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Typical Applications of HPLC Chromatography


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HPLC of Orange Juice Compounds


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How to Increase HPLC Resolution

1.Increase column length

2.Decrease column diameter

3.Decrease flow-rate

4.Pack column uniformly

5.Use uniform stationary phase (packing material)

6.Decrease sample size

7.Select proper stationary phase

8.Select proper mobile phase

9.Use proper pressure

10.Use gradient elution


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Separating Proteins from Mixtures

In order to understand and study proteins it is essential to separate them from the biological fluid.

Proteins can be separated from each other based on differences in physical properties

Due to different amino acid sequences proteins differ in solubility, size, charge, and binding affinity and can be separated on either of these properties.

The inside of a cell. White shapes are proteins (several 10s of thousands per cell).


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Water, Chemical bonds and groups

Amino acids, pH dependence

Protein primary sequence, peptide bonds, secondary structures


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Protein studies: Understanding protein structure and function relationships

All proteins have a distinctive 3D structural conformation

This unique structure enables its function

Amino acid sequence determines structure

A major goal of biochemistry is to determine how amino acid sequences specify the 3D conformations of proteins and to catalogue all proteins in cells.

Characterization

cell


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Protein purification: general experimental setup

Column chromatography

Characterization

Homogenize

Centrifugation


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UV

time

Gel permeation chromatography: separating on basis of size

Mixture of proteins

  • A mixture of proteins in a small volume is applied to a column filled with porous beads

  • Because large proteins cannot enter the beads, they emerge sooner than do small ones

  • A detector (e.g. UV) is used to detect protein fragments

  • Fragments are collected separately


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Affinity Chromatography: separating on the basis of affinity

X

X

X

X

X

X

X

X

X

X

X

X

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X

  • To separate proteins that recognize a chemical group X

  • X is covalently attached to beads that are packed in a column

  • Sample of proteins is added

  • Washed with buffer to remove non specifically bound protein

  • Eluted with high concentration of soluble X


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Separation on the basis of charge

All proteins are charged. Their charges depend on the relative number of acid andbasic amino acids in their primary structure.

All proteins have a pH value where they are uncharged: the isolelectic point (pI)

H2N- Met Ala Asn Cys His Glu Ser Thr Glu Arg-COOH


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Ionic amino acids


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Separation on the basis of charge (continued)

His: 6.0

Glu: 4.1

Arg: 12.5

N-terminal amine: 8.0

C-terminal acid: 3.1

For this peptide:

pI=pKa/N= 6.3

Positively charged at pH < 6.3

Negatively charged at pH > 6.3

H2N- Met Ala Asn Cys His Glu Ser Thr Glu Arg-COOH


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Ion Exchange Chromatography: separation on basis of net charge

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  • Positive or negatively charged resin can be used for separation of positive or negatively charged proteins

  • Sample of proteins is added

  • Washed with buffer to remove non specifically bound protein

  • Elute with increasing concentration of salt

  • Proteins with highest net charge come of last


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Why hydrogels are used for protein separations

1.2.

3.

  • Correct protein folding in aqueous environment

  • Proteins can denature on surfaces

  • Hydrogels are >90% water, good environment for proteins


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Compare Reverse Phase toNormal Phase Column Chromatography

In Normal Phase Liquid Chromatography:

The 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 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.


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Reverse Phase Column Chromatography

  • The stationary phase (column packing) is nowNON-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


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Reverse phase chromatography

Silica is alkylated with long chain hydrocarbon groups, using 18

carbons long. This is usually referred to as C-18 silica.


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Summary of Methodology

One of the main aims of biochemistry is to characterize and catalogue all proteins in the cell

We have discussed some important tools for separating proteins based on physical properties such as size, affinity, charge.

Chromatography methods: ion exchange, affinity, gel permeation chromatography

Electrophoresis: iso electric focusing, SDS PAGE, 2D gels (in the Biochemistry lecture series)


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Overview of Paper Chromatography

  • Works on principle of Partition.

  • Separates dried liquid samples.

  • Mobile phase is solvent used.

  • Stationary phase is water bound to surface of paper.

  • Advantage : its cheap!


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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.

Important Concepts in Paper Chromatography


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Paper/TLC

Chromatography

Animation


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Simple Example of Paper Chromatography using “Sharpie” Pens


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Dye Separation in a Black “Sharpie”

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


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Thin Layer Chromatography

  • Works mainly on principle of adsorption.

  • Mobile phase is the solvent

  • Stationary phase is the solid on the plate.


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TLC vs. Column Chromatography

  • Thin-layer chromatography and column chromatography and are different types of liquid chromatography.

  • The mobile (moving) phase is a liquid. The stationary phase is usually silica or alumina. This phase is very polar.

  • The principle of operation is the same!


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Thin Layer Chromatography

The surface of a plate consists of a very thin layer of silica on a plastic or

aluminum backing. The silica is very polar. This is the stationary phase. Material is spotted at the origin (bottom) of the TLC plate.

The plate is placed into a glass jar with a small amount of a solvent in a glass jar. This solvent acts as the moving phase.

The plate is removed from the bottle when the solvent is close to the top of the plate.

The spots are visualized (explanation to follow).

Non-polar compounds will be less strongly attracted to the plate and will spend

more time in the moving phase. This compound will move faster and will appear

closer to the top of the plate.

Polar compounds will be more strongly attracted to the plate and will spend less

time in the moving phase and appear lower on the plate.


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Thin-Layer Chromatography: A Two-Component Mixture

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Increasing Development Time


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Thin-Layer Chromatography: Determination of Rf Values

Rf of component A =

dA

dS

Rf of component B =

dB

dS

The Rf value is a decimal fraction, generally only reported to two decimal places


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Thin-Layer Chromatography: Qualitative Analysis


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Visualization Method

  • The previous slide shows colored spots. Most of the time, the spots won’t show unless they are visualized!

  • Vizualization is a method that is used to render the TLC spots visible.

  • A visualization method can be:

    • Ultraviolet light

    • Iodine vapors to stain spots

    • Colored reagents to stain spots

    • Reagents that selectively stain spots while leaving others unaffected.


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TLC Advantages

Advantages over paper:

  • Its faster

  • It gives a better separation.

  • It is more versatile as the solid on the plate can be varied.


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Uses of TLC

  • To determine how many components there are in a mixture (is it really pure?)

  • To determine the best solvent conditions for separation on a column

  • To identify the substances being studied

  • To monitor the composition of fractions collected from column chromatography

  • To monitor the progress of a reaction


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Gas-Liquid Chromatography

  • Works on principle of Partition.

  • Mobile phase is the carrier gas.

  • Stationary phase is oil bound to surface of beads within the column.


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Most Common Stationary Phases

  • 1.Separation of mixture of polar compounds

  • Carbowax 20M (polyethylene glycol)

  • 2.Separation of mixtures of non-polar compounds

  • OV101 or SE-30 (polymer of methylsilicone)

  • Methylester of fatty acids

  • DEGS (diethylene glycol succinate)


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Gas-Liquid Chromatography


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Gas-Liquid Chromatography

  • Retention time is used to identify a component of a mixture. It depends on:-

  • The temperature of the column.

  • The length of the column.

  • The material used to pack the column.

  • The gas pressure.


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Gas –Liquid Chromatography

  • The area under a peak is proportional to the amount of substance present.


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H

RESET

Air

Hydrogen

Gas Carrier

Gas Chromatography

Filters/Traps

Data system

  • gas system

  • inlet

  • column

  • detector

  • data system

Regulators

Syringe/Sampler

Inlets

Detectors

Column


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Schematic Diagram of Flame Ionization Detector

Collector

Detector electronics

 - 220 volts

Flame

Chassis ground

Jet

Signal output

Column


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Gas-Liquid Chromatography

  • Gas-Liquid Chromatography is often combined with mass spectroscopy. The GC separates the components then the MS analyses them.


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One possible Use of GC:

SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS


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Gas Chromatogram of Methyl Esters of Fatty Acids


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Another GC Use:

TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS


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GLC ADVANTAGES

1.Very good separation

2.Time (analysis is short)

3.Small sample is needed - ml

4.Good detection system

5.Quantitatively analyzed


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DISADVANTAGES OF GAS CHROMATOGRAPHY

Material has to be volatilized at 250º C without decomposition!


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Summary of Some Chromatographic Techniques


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