1

Chromatography Definition:

Chromatography is a technique for separating a sample into various fractions. The heart of any chromatography is the stationary phase

(liquid or solid). The stationary phase is attached to a support, a solid inert material. The sample (gas, liquid or solid which may or may

not dissolved in solvents) is carried across the stationary phase by a mobile phase (gas or liquid). The sample components undergo a

series of exchanges (partitions) between the two phases due to the differences in their chemical and physical properties. These

differences govern the rate of movement (called migration) of the individual components.

Purpose of Chromatography:

Analytical - determine chemical composition of a sample

Preparative - purify and collect one or more components of a sample

Classification of Chromatographic Methods

Chromatography is classified according to three ways:

1. According to the physical state of the mobile phase:

Liquid chromatography: This subdivided according to the stationary phase into liquid-liquid or liquid-solid chromatography.

Gas chromatography: This subdivided according to the stationary phase into Gas-liquid or Gas-solid chromatography.

2. According to the method of contact between the mobile phase and stationary phase:

Column chromatography: the stationary phase is placed in a column through which the mobile phase moves under the influence of

gravity or pressure. The stationary phase is either a solid or a thin, liquid film coating on a solid particulate packing material or

the column’s walls.

Planar chromatography: the stationary phase coats a flat glass, metal, or plastic plate and is placed in a reservoir containing the

mobile phase which moves by capillary action carrying with it the sample components

3. According to the chemical or physical mechanism responsible for separating the sample’s constituents.( attractive forces)

Adsorption chromatography: for polar non-ionic compounds

Ion Exchange chromatography: for ionic compounds

- Anion: analyte is anion; bonded phase has positive charge

- Cation: analyte is cation; bonded phase has negative charge

Partition chromatography: based on the relative solubility of analyte in mobile and stationary phases

- Normal: analyte is non-polar organic; stationary phase MORE polar than the mobile phase

- Reverse: analyte is polar organic; stationary phase LESS polar than the mobile phase

Size Exclusion chromatography: stationary phase is a porous matrix.

2

Planner chromatography

1- Paper chromatography (PC)

Principle

In paper chromatography, the mobile phase (solvent) carries the components of the sample on the stationary phase (filter paper)

separating them according to the differences in the migration rate (depends on the molecular weight , polarity and adsorption ability)

Components

For one-dimensional paper chromatography, either ascending or descending development can be carried out in simple units. Descending

development is more often used because it is faster and more suitable for long paper sheets.

- The stationary phase (filter paper) - The mobile phase (solvent may be in a reservoir)

Procedures

1. Make the initial line on the paper.

2. Apply the solvent alone on the initial line.

3. Wait till the solvent migration is stopped, then make the final line.

4. Spot the sample, and then apply the solvent either in ascending or descending or concentric manner.

5. In case of colored sample: Calculate the rate of flow (Rf) directly then compare it with stander in order to know the unknown

sample (qualitatively).

6. In case of the colorless sample: use UV-lamb to detect the spot position then determine the (Rf).

Rf depends on the temp., solvent, type of paper

Rf = distance of sample migration / distance of solvent migration

Applications

1. Separation of amino acids

2. Separation of the plant pigments

Advantages

1. Simple

2. Cheap

Disadvantages

1. Time consuming.

2. Need high quantity of sample.

3. With weak solvent power.

4. limited use

5. Difficulty detection of spots

6. Difficulty isolation of separated substances.

3

2- Thin layer chromatography (TLC)

Principle (As in paper chromatography)

Components

1- glass or plastic plate: as a support to the stationary phase

2- stationary phase (silica gel, alumina or agar)

3- mobile phase solvent system

Procedures

(a) Preparing the plate

1- Prepare a glass plate. for example (20X20)Cm

2- Dissolve suitable amount of the silica gel in water path.

3- Spread it on glass plate homogeneously. Then wait till solidification.

(b) Running the sample 1- Make the initial and final line as in PC

2- Spot the sample on the initial line and then apply the solvent either in ascending or descending or concentric manner

3- For example: add the plate on a tank containing the solvent (ascending) where the solvent move by the capillary action carrying

with it the components of the sample.

4- The plate is removed from the tank and dried. (Additional separation can be achieved in two dimensional TLC)

5- According to the sample type ( if colored, if colorless, if florescent …) , identification (qualitative) occurs either by Rf , UV,

spraying colored reagent or autoradiography.

6- Compare it with stander in order to know the unknown sample components.

7- We can separate the sample by cutting the silica layer by spatula, then dissolve it with the same solvent then filter for further

purification.

Applications

1- Environmental application from water analysis (especially pesticides) to plant residues

2- Pharmaceutical applications from stability and impurity studies to drug monitoring in biological fluid

3- Biomedical compounds (organic acids, lipids, carbohydrates and steroids)

4- Food analysis from carcinogens, drug residues , preservatives and flavors

Disadvantages

1- Time consuming

2- Limited to non-volatile compounds

3- Less accurate and less sensitive

Advantages

1- Need small quantity of sample.

2- With greater solvent power.

3- easy detection of spots

4- easy isolation of separated substances

4

Column chromatography

1- Gel filtration chromatography (Size-exclusion chromatography)

Principle

This technique separate proteins according to their size and shape, as they pass through a stationary phase (cross-linked polymer

=sephadex) by the help of mobile phase (without binding). Larger proteins or molecules, which can not penetrate the sephadex pores,

move around the sephadex in space between them faster than the smaller molecules which may penetrate the sephadex pores taking long

time to elute from the column.

Components

1. column: as a support to the stationary phase

2. stationary phase (pours matrix in the form of spherical particles, stable, inert e.g. sephadex or agarose)

3. mobile phase (buffer system)

Procedures

1. (loading step): spherical particles of the sephadex are packed into the column

2. (Sampling step): sample is applied to the column

3. Buffer (mobile phase) and sample move through the column. The sample components diffuse in and out

of the pores of the matrix (sephadex) according to their size.

4. Larger proteins or molecules move faster than the smaller molecules and leave the column first

5. Separation completed as the entire buffer volume is passed.

Applications

1. Separation of neutral proteins and larger molecules including polymers and biomolecules according to size.

2. The determination of formula weights.

Disadvantages

1. limited applications

2. low purification

Advantages

1. provides a rapid means for separating larger molecules

2. Use only one buffer (coast effective)

3. Do not need elution step because there are no bonds formed.

Lecture (2)

5

Relation between pH, Pi & ion exchange

pH: is aquantitative description of the acidity of

an aqueous solution.

pH= -Log[H]+ =Log 1/[H]

+

Isoelectric point (Pi): is the point at which the

protein charge reach zero.

If pH > Pi use anion exchange

If pH < Pi use cation exchange

pH = pK + Log conjugated base / conjugated acid

2- Ion-exchange chromatography

Principle

Ion exchange chromatography separates molecules (proteins) according to their differences between the overall charges. The proteins to

be separated must have a charge opposite to that of stationary phase in order to bind.

Ion exchange has two types according to the stationary phase charge:

1. Cation-exchanger: in which the stationary phase is charged negatively in order to binds with positive molecules (cations)

2. Anion-exchanger: in which the stationary phase is charged positively in order to binds with negative molecules (anions)

Cation-exchange chromatography Cation-exchange chromatography can be classified as: either strong or weak. A strong cation exchanger contains strong acid which

stable along pH1-14. Whereas, weak cation exchanger contains weak acid which loss its charge as the pH decrease below 4-5 The sample must be charged positive in order to bind with the negative matrix (strong or weak acid). H

+

Anion-exchange chromatography Anion-exchange chromatography can be classified as: either strong or weak. A strong anion exchanger contains strong base which

stable along pH1-14. Whereas, weak anion exchanger contains weak base which loss its charge as the pH increase over 9 The sample must be charged negative in order to bind with the positive matrix (strong or weak base).OH

-

Components

1. The column containing the stationary phase (anion or cation exchanger) on suitable matrix

2. washing and eluting buffer

3. pump to withdrew the buffer

4. Detector

Procedures

Before carry out the process, you must answer two important questions:

a) What is the sample charge? If +Ve, use cation exchanger. if –Ve, use anion exchanger

b) What is the suspected strength of the charge? If weak +Ve, use weak cation exchanger, if strong +Ve, use strong cation

exchanger, if weak –Ve, use weak anion exchanger, if strong –Ve use strong anion exchanger.

basicفإذاا كاتإم مع إح اضماضإه ايمينيإة , ؟ طبعا من خالل تركيبه(قوية او ضعيفة)او موجبة( قوية اوضعيفة)كيف تستطيع معرفة ان هذا البروتين يحمل شحنة سالبة : ملحوظة

الشحنةومن خالل عدد ايضماض تستطيع توقع قوة او ضعف . سيكون موجب acidicسيكون سالب وااا كاتم

e.g. the sample is weak negative proteins. So we will use anion exchanger contain weak base.

1. (loading step): the column is packed with the matrix that charged with weak positive charge by adding weak base e.gDEAE-

cellulose (stationary phase)

2. (Sampling step): apply the sample in the column: the negatively charged proteins bind to positively charged matrix whereas;

the positively charged proteins flow down to the exterior. Some negative charged contaminants can bind to matrix.

3. (Washing step): apply washing buffer (Tris-HCL) to remove the contaminants remaining the target proteins.

4. (Elution step): now, we need to separate the target proteins from the matrix, so we apply an eluting buffer that has the same

charge of protein in order to substitutes it (ion exchange). Separation can be done also by ion exclusion and ion pairing.

5. (Gradient step): make gradient elution with different buffer till you obtain 100% correct proteins. i.e. repeat washing and

eluting steps with different buffer

6. (detection step): after separation carry out detection by electrophoresis

Applications

(Separation of Ions and ionized sp) القاعدة هنا

1. Separation and detection of ions and ionized species.

2. separation and purification of components from mixture

3. identification of ionic impurities

Disadvantages

1. Analytes can be misidentified

2. Analytes are performed sequentially

3. Analysis consume eluent

Advantages

1. Selective to charge

2. Separation and detection of ions and ionized species

Note

Lecture (5)

6

3- Affinity chromatography

Principle

Affinity chromatography depends on separation of proteins or molecules by their binding specificities. So the proteins of interest bind

specifically to a ligand (which attached to the matrix) such as antigen-antibody interaction.

Components

1. stationary phase: affinity matrix (ligand attached to matrix)

2. suitable column

3. mobile phase: buffer

Procedures

1. (Loading step): the column is packed with suitable matrix to which ligand (or antigen) has been coupled.

2. (Sampling step): sample (may be serum) is applied to the column. The protein or antibody of interest will bind tightly to ligand

(because they are specific to each others) whereas; other contaminants similar to target proteins bind loosely.

3. (Washing step): the unwanted proteins or antibodies do not bind to ligands so; it is easy to be washed off. Also the

contaminants that bind loosely washed off by another buffer.

4. (Elutiion step): proteins or antibodies that bind tightly to ligands are eluted by eluting buffer.

5. (Detection step): after separation carry out detection to ensure the correct proteins.

Applications

(Separation of samples that can carry out KEY & LOCK mechanism with stationary phase) القاعدة هنا

1. Separation of biomacromolecules from other biological compounds.

o Separation of proteins

o Separation of mRNA (poly A) from RNAs using oligo dT cellulose (poly T)

2. it can be used to determine dissociation constants of ligands and molecules

Advantages

1. it is more specific method for the target analyte

2. simple and practical

3. fast

Lecture (4)

7

4- Gas chromatography (GC)

Principle

In gas chromatography (GC) the sample, which may be a gas or liquid, is injected into a stream of an inert gaseous mobile phase (often

called the carrier gas). The sample is carried by mobile phase through a packed or capillary column where the sample’s components

separate based on their ability to distribute themselves between the mobile and stationary phases.

Components Gas chromatography has two types according to the stationary phase:

a) Gas-liquid chromatography b) gas-solid chromatography

The main components are:

1. Gas supply: it is usually helium, hydrogen or nitrogen. This serves as mobile phase that moves the sample towards column.

2. Injector: it is a hollow heated glass cylinder where the sample is introduced and vaporized into the mobile phase stream.

3. The column: it is the heart of GC. It is coated with either solid or liquid stationary phase.

4. Oven: it contain the column where the temp. can be controlled giving constant heat along the run.

5. Detector: it detects the separating components passing from the column. Detectors have many types e.g. flame ionization

detector, photo-ionization detector, helium ionization detector …ect

6. Data recorder: it plots the signal from the detector over time. The time and height of the signal can identify the compound

Procedures

1. The sample is introduced and vaporized to GC through the injector port.

2. The gas supply is opened to provide carrier gas (mobile phase) that carries the vaporized components of sample to column.

3. The motion of carrier gas and sample in column is inhibited by adsorption of the sample components on stationary phase.

4. The rate at which the molecules pass along the column depends on the strength of the adsorption, which depends on the type

of molecules. So separation occurs according to this differences of the retention time (RT)

5. The detector monitors the outlet stream from the column in form of signals. The time of signal appearance (RT) (qualitative

detection) and its height (quantitative detection) can identify the compound and its concentration.

6. Spectroscopy is required for confirmation

(RT: is the time required for a compound to travel from the injector to the detector)

Applications

(The sample must has sufficient volatility and thermal stability to be estimated in GC) القاعدة هنا

1. Separation and identification of compound in mixture qualitatively and quantitatively

2. It is used in industry for analysis of row material, intermediate and final products.

3. It is used in environmental, pharmaceutical, forensic and clinical applications.

Disadvantages

1. limited to volatile samples

2. not suitable for thermally labile samples

3. some sample may require intensive preparation

4. requires spectroscopy

Advantages

1. Fast analysis. 2. High resolution. 3. Sensitive detector. 4. Require small sample.

5. Non-destructive 6. Highly accurate quantification. 7. Automated system.

Factors affecting GC separation

1. Volatility of compounds: low volatile move faster

2. Polarity of compound: polar move slow

3. Column temp.: high temp. increase movement

4. Column length: long column slow movement

5. flow rate of gas: speeding up a carrier gas will speed all components

Lecture (5)

8

5- High performance liquid chromatography (HPLC)

Principle

In liquid chromatography, the sample is separated into its components according to the differences in their partitioning or adsorption

behavior between a liquid mobile phase and stationary phase. When an effective column is used in LC, the technique called high

performance LC (HPLC).

Components

1. The pump: to pressurize the liquid mobile phase and force the mobile phase to the column.

2. Solvent reservoir: to hold a liquid mobile phase.

3. Injector: to introduce small volume of sample mixture under high pressure to mobile phase stream.

4. High efficiency column: it is the heart of the system because separation occurs there. It consists of stainless steel tube that

contains packing (stationary phase). According to the type of packing, HPLC may be:

a) Normal phase HPLC: with polar stationary phase and non-polar mobile phase. Separation based on polarity where the

polar sample adsorbed on the polar stationary phase. (separate polar samples)

b) Reversed phase HPLC: with a non-polar stationary phase and polar mobile phase. Separation is based on the non-

polarity where the non-polar sample adsorbed on the non-polar stationary phase. (separate non-polar samples)

c) Size exclusion HPLC: with porous st. phase and polar to non polar mobile phase.(separate non-polar to ionic sample)

d) Ion-exchange HPLC: with ion exchange resins st. phase and buffer mobile phase. (separate highly polar sample)

5. Detector: an optical sensor that detects the separating components passing from the column

6. Data recorder: it plots the signal from the detector over time. The time and height of the signal can identify the compound

Procedures

1. The sample is introduced through injector (that applies high pressure) to mobile phase stream.

2. The pump is opened to aspirate the mobile phase from the solvent reservoir and force it towards the column.

3. The mobile phase carries with it the sample components to the column.

4. In the column: separation occurs due to different adsorption abilities between sample components.

5. Elution occurs by continuous flowing of mobile phase through stationary phase.

6. The detector detect compound as they pass from the column.

7. The computer system (Data recorder) plots the signal from the detector over time. The time (RT) and height (conc.) of the

signal can identify the compound

Applications

(For polar and non-polar samples) القاعدة هنا

1. Identification and purification of environmental samples.

2. Identification and purification of pharmaceutical samples.

3. Identification and purification of forensic samples

4. Identification and purification of clinical samples

5. Identification and purification of food and flavor samples.

Disadvantages

1. Involve combination of mass spectrometry for accurate identification.

2. Only one sample can be analyzed at a time.

3. Require training in order to optimize conditions of separation

4. Sample preparation is often required.

Advantages

1. Separate wide variety of samples.

2. Can be used in many fields (clinical, environmental…)

3. It is widely accepted separation technique for both sample analysis and purification.

Factors affecting HPLC separation

1. Sample polarity. NP-HPLC & RP-HPLC اشرح ضسب النوع 2. Sample size. RT increase with decrease size

3. added salts: RT increase with salt addition in RP-HPLC 4. Alkyl chain: RT increase with long chain in RP-HPLC

Lecture (6)

9

Electrophoresis Principle

Electrophoresis means the migration of charged particles in a liquid medium under the influence of an electric field.

When an electric field is applied to a medium containing charged particles or molecules (e.g. DNA or protein), the negatively charged

molecules migrate towards the positive electrode (anode) and vice versa. After separation (according to difference in charge and mass)

permanent fixation of the fractions at the position to which they migrate is done. Bands are then stained in order to visualize them.

Components

1. Power supply: provide stable direct current, and has controls for both voltage and current output. (cathode & anode)

2. Support medium: It is the heart of the system where separation occurs there. Its function is to provide an inert porous medium

for the electrolytes solution. Zone Electrophoresis is classified according to the support medium type. Support media may be

Thin sheet (of paper, cellulose acetate or silica) or Gel (of starch, agarose or polyacrylamide that separate samples according

to the charge and size) e.g. cellulose acetate electrophoresis, polyacrylamide gel electrophoresis.

3. Buffer: It serves as a multifunctional component in the electrophoretic process as it: a) carries the applied current

(electrophoresis buffer) b) establish the pH at which electrophoresis is performed (gel buffer). c) Determine the electric charge

of the sample (sample buffer). There are several considerations must be done to select buffer: A) the buffer must be not interact

with sample. B) The ionic strength and concentration of buffer must be suitable for sample. C) It must allow the sample to be

charged not denaturated.

4. Stains: It is used to visualize and locate the separated protein and nucleic acid fractions e.g. Coomassie Brilliant Blue (CBB).

(Tracking dye such as Bromophenol Blue (BB) is often used to see the sample movement on gel (do not stain sample bands) that

enables us to terminate the process when the bands reach lower buffer reservoir. It moves faster than any macromolecules).

Procedures (e.g. Poly-Acrylamide Gel Electrophoresis) SDS-PAGE Laemmle (1970)

A) Gel preparation:

Fist: prepare the following solutions as follow:

1- Acrylamide solution. 2- Separating gel buffer. 3- Stacking gel buffer. 4- SDS solution. 5- Initiator 6-Catalyst

Acrylamide 30g Tris 18.16g Tris 6g SDS 10g APS 0.5g TEMED

Bis-acrylamide 2g in 100ml Bidist. H2O in 100ml Bidist. H2O in 100ml bidist.H2O in 5ml bidist.H2O

In 100ml Bidist. H2O pH adjusted to 8.8 pH adjusted to 6.8

Second: prepare the separating and stacking gel as follow:

Separating gel: (3.5ml from 1) + (2.5ml from 2) + (0.1ml from 4) + (0.05ml from 5) + (10μl from 6)

Stacking gel: (0.6ml from 1) + (1.25ml from3) + (0.05ml from 4) + (0.05ml from 5) + (5μl from 6)

Note: These gels are polymer. We can control their pores though the concentration of their constituents .high concentration decrease

pores size, and become suitable for passing low molecular weight proteins and vice versa.

B) Pouring the gel in electrophoresis unit:

1. The separating gel is transferred to the gel glass sandwich. Wait till polymerization (25min) (take care with air bubbles)

2. The stacking gel is then transferred over separating gel. The comb is inserted into the top then removed after polymerization

C) Sample application:

1. The sample is homogenized by sample buffer (Tris + SDS+ 2-mercapto ethanol + BB + sucrose) then centrifuged.

2. 25μl of supernatant is applied side by side in the wells inside the gel.

3. 15μl of protein marker is applied in the last well for comparisons with unknown bands.

4. The upper and lower buffer reservoir is filled with electrophoresis buffer (Tris + glycine + SDS)

D) Running of samples: The power is switched on. Wait till the bands reach at lower end of gel (stopping gel) then switch off.

E) Detection and quantification: 1. The plate is removed, dried then stained to see the bands.

2. Compare the separating unknown bands with the known marker bands.

(The process can carry out without SDS in certain samples: Native-PAGE)

Applications

1. Gel electrophoresis is used in quantitative analysis in molecular biology and genetics.

2. Nucleic acids carry negative charge on their suger-phosphte backbone so they migrate into gel with similar rates and separation

is done due to different molecular size. Agarose gel electrophoresis is suitable for DNA and RNA analysis.

3. Proteins have different charges, so they charged negatively (denaturated) by SDS in order to migrate into the gel with similar

rates and separation is done due to different molecular size. Polyacrylamide gel electrophoresis is suitable for protein analysis

4. Other different types of electrophoresis have many applications in many fields.

Factors affecting electrophoresis

1. The sample: a) Charge: migration increase with charge increase b) Size: migration decrease with size increase

2. The support media: a) if adsorption, migration decrease b) if molecular sieving, migration increase

3. The buffer: a)composition, bad buffer decrease migration b) concentration increase, migration decrease c) pH affect ionization

4. The electric field: a) Voltage: migration increase with its increase b) Current: migration increase with its increase c) Resistance:

migration decrease with its increase

5. The heat: migration increase with its increase due to fall in resistance.

Lecture (7)

10

Types of electrophoresis

Electrophoresis has two mains types according to the support media:

(I) Thin sheet electrophoresis: it separates samples according to the charge. Support medium is thin sheet.

1. Paper electrophoresis: used in past to separate charged samples (support medium is thin sheet of paper)

2. Cellulose acetate electrophoresis: it suitable for separation of radio-labeled substances especially for clinical investigations

(support medium is cellulose acetate that is prepared by treating cellulose with acetic anhydride)

3. Thin layer electrophoresis (TLE): as in TLC but the plate is placed in electrophoresis unit (support medium is silica)

(II) Gel electrophoresis: it separates samples according to the charge and molecular size. Support medium is gel.

1. Continuous gel electrophoresis:

a) Starch gel electrophoresis: it is prepared heating and cooling starch in suitable buffer.

b) Agar/Agarose gel electrophoresis: Agar composed of agaropectin and agarose. Separation based on charge only

c) Polyacrylamide gel electrophoresis (PAGE): it is made from acrylamide monomers copolymerized with the cross linker

N,N`methylenebisacrylamide in presence of ammonium persulphate and TEMED as catalyst. Separation based on

molecular size (molecular sieving). It has two types:

Native-PAGE: under non-denaturating conditions.

SDS-PAGE: under denaturating conditions.

2. Discontinuous gel electrophoresis:

3. Two dimensional gel electrophoresis

Gradient gel electrophoresis:

a) Isoelectric focusing

b) Pulse-field gel electrophoresis

c) Capillary electrophoresis

1D 2D

1. separation according to molecular weight

2. depends on gradient Acrylamide

1. separation according to isoelectric point of protein

2. depends on gradient pH

Native PAGE SDS- PAGE

1. used to determine total proteins

2. detergent not used to avoid deformation of proteins

1. used to determine fragments of protein

2. detergents are used to do fragmentation of proteins

Material Function

1. Acrylamide and Bisacrylamide

2. separating gel

3. stacking gel

4. sodium dodecyl sulfate (SDS)

5. Amm. Per sulfate (APS)

6. TEMED

7. Ampholyne

To form a net structure through which, proteins will sieve according to their size

Provide an inert porous medium for separation

To press all sample bands in one line in order to run with each other

To provide a negative charge to proteins

Initiate the reaction between Acrylamide and Bisacrylamide

Increase or catalyst the reaction of between Acrylamide and Bisacrylamide

To establish a pH gradient on electrophoresis unit before running proteins. When running

proteins, they move on till reaching a pH corresponding to its isoelectric point.

11

Mass Spectrometry (MS)

Principle

Mass Spectrometry can identify molecules according to measurements of their mass/charge (m/e) ratio where each

molecule has specific m/e ratio. It carries out three main functions: 1- Ionization: to convert the sample in to ions. 2- Analysis: to resolve

these ions on the basis of their m/e ratio. 3- Detection: to detect the m/e ration of sample.

Components The main components of any types of MS are: 1- Ionization source. 2- The Analyzer. 3- The detector. According to

the ionization mechanism, MS has several types. The usual methods of ionization (bombardment of gaseous sample by a beam of very

high energetic electrons) do not used to detect proteins because they caused their rapid decomposition. So we need to use a high

resolution MS that has safe ionization methods (MALDI or ESI) of proteins without decomposing or affecting them e.g. MALDI-TOF

MS & ESI MS & MS/MS.

A) MALDI-TOF MS

(Matrix-Assisted Laser Desorption-Ionization / Time Of Flight Mass Spectrometry)

Principle The principle is as the normal MS but the ionization here is by MALDI, in which proteins are placed in a light-

absorption matrix, and then with a short pulse of laser light, the proteins are ionized and then desorbed from the matrix into the analyzer

(TOF) without decomposition.

Components

1- Ionization source (MALDI): it is a combination of a) laser wavelength and b) matrix

A. Laser: it provides energy that incident on the matrix containing sample. It ionize the matrix first then the matrix transfer part of its

energy to the sample (protein) thus ionizing them without any destruction.

B. Matrix: it serves: a) as source of H+ ions that is transferred to protein thus ionizing them b) to isolate the analyte molecules from

one another to avoid direct ionization by laser c) as an absorbing medium for the UV light, thus converting laser energy to

electronic energy which may be used for desorption and ionization .

2- The Analyzer (TOF): the Time Of Flight mass analyzer measures the time that required by ions of different masses to move from the

ion source (MALDI) to the detector (higher the mass of ion, lower its velocity and takes long time to leave TOF tube). This needs that

the starting time (time at which the ions leave the ion source) is well defined. TOF may be: A) Linear TOF. B) Reflectron TOF. The

Reflectron is used to compensate for the difference in flight times of the same m/e ions.

3- The Detector: All types of detectors for MS require a surface on which the ions impinge and the charge is neutralized. As a result of

this, electric current is developed and then amplified then converted into a signal recorded on the computer system. The detector may

be: a) Faraday Cup, b) Electron multiplier, c) Array detectors.

Procedures

1. A micromole of sample is introduced (either directly or via HPLC, GC or capillary electrophoresis) into the sample chamber.

2. The sample components are ionized by the MALDI

3. The ions are resolved according to their m/e ratio in the TOF analyzer

4. from the analyzer, ions fall on the detector that give electric current that analyzed in computer to give a signal.

Applications

The main application of the MALADI-TOF MS is to study the proteome. Proteome is protein complement

expressed by the genome of an organism under certain conditions. The study of all proteins called Proteomics. Proteomics is very

important to know which proteins are made of?, what is the 3D structure?, where are they? What do they do so it have many

applications in many fields such as: 1- in biotechnology (e.g. analysis of proteins &peptides) 2- in pharmaceutical (e.g. drug discovery)

3- in Clinical (e.g. hemoglobin analysis) 4- environmental (e.g. water quality) 5-in geological (e.g. oil composition) 6- in biochemistry

(e.g. reaction monitoring, amino acid sequences, protein structure, molecular weight measurement….)

Advantages

1. it is very soft ionization method with short measuring time and now sample consumption

2. it does not produce high fragmentations due to presence of matrix, so it is safe for protein analysis

3. it give accurate mass measurement of molecules

4. it used for oligonucleotide sequencing and protein/peptide identification

Disadvantages

1. Very expensive, where it cost higher than 500000 $.

2. It needs a trained person.

3. The sample must be purified and needs high care in its preparation.

Lecture (8)

12

B) ESI-MS/ MS

Electrospray Ionization/ Tandem Mass Spectrometry

Principle

The principle is as the normal MS but the ionization here is by ESI, in which the proteins in solution (protein ions +

solvent) are forced directly from the liquid to gas phase giving ions that introduced without being decomposed to the analyzer. The

analyzer here is tandem mass spectrometry in which the first MS sort and weight the ionized sample that undergoes further

fragmentation by collision cell then the m/e ratios of all charged fragments are determined by the second MS. Finally the detector carries

out its usual function.

Components

1. Ionization source (ESI): in which the proteins that exist as ions

(either by adding H+ to protein or removing H

+ from protein) in

large amount of volatile solvent are pushed through charged

glass capillary that leads to formation of small droplets (protein

ions surrounded by solvent). Then the solvent is evaporated (by

the action of high pressure, Temp and coulomic fission)

leaving the protein ions alone that are introduced

nondestructively into analyzer. Here, electrons have neither

been added or removed.

2. The analyzer: Tandem mass (MS/MS) it is a combination of

two MS with a collision cell between them:

A. First MS: the ionized sample is sorted according to their molecular weights. ( الترتيب هنا قبل تكسير كل الروابط )

B. Collision cell: the sample is further fragmented by high-energy with a small amount of noble gas e.g. He in order to break all

peptide bonds between all amino acids. So the amino acids become free

C. Second MS: it sorts all the charged fragments and measures their m/e ratios. ( تكسير كل الروابط دالترتيب هنا بع)

3. The detector: As in MALDI-TOF MS

Procedures e.g. protein analysis

1. A solution containing the protein of interest is treated with

protease or chemical reagent to hydrolyze it to shorter peptides

2. the mixture is injected into the device

3. it ionized by ESI and then introduced to first MS

4. The combination of first MS, Collision cell & second MS

contribute to measure the target m/e ratios as explained previously.

5. from the Tandem MS, ions fall on the detector that give electric current that analyzed in computer to give a signal

It is very useful for sequencing the amino acids where:

The typical fragmentation occurs either from C-terminal (the charge is retained on -COOH terminal) that called y-type or from N-

terminal (the charge is retained on –NH2 terminal) that called B-type.

Example:

H2N-Arg-Lys-cyst-Val-Isoleu-Ala-COOH

Break at N-terminal Break at C-terminal +H2N-Arg-Lys-cyst-Val-Isoleu Ala-COOH

+

+H2N-Arg-Lys-cyst-Val Isoleu-Ala-COOH

+

+H2N-Arg-Lys Val-Isoleu-Ala-COOH

+

+H2N-Arg cyst-Val-Isoleu-Ala-COOH

+

Comparison

ESI MALDI

1. generate multiply charged ions

2. readily coupled to LC

3. used with variety of mass analyzers

4. combined with low energy collision induced dissociation CID

1. generate singly charged ions

2. off-line coupling to LC

3. used mainly with TOF

4. combined with high energy CID

Lecture (9)

ESI

Tandem MS

with ESI

ابدأ Cاو N ال ي يتح الكسر من جاتب

13

Flame

Flame

A-1) Flame Atomic Absorption spectroscopy

The atomic absorption spectroscopy has two types: (1) Flame atomic absorption spectroscopy. (Single & double beam)

(2) Flameless atomic absorption spectroscopy

Principle

In Flame atomic absorption spectroscopy, there are three main steps:

1. An energy source as flame converts the sample to an atomic vapor (not excited but merely dissociated from its bonds and

placed in ground state) so, neutral atom of low energy ready to absorb radiation (step 3)

2. The wavelengths produced by this atomic vapor are characteristic of the element. (qualitative)

3. The amount of light absorbed by the vaporized sample is proportional to the concentration of the element. (quantitative)

Components

1. Power supply: is the source of radiation energy

2. Hollow Cathode Lamb (HCL): it is a good source of atomic line emission.

It consists of a) two electrodes (cathode that made of metal of analyte

& anode), b) Vacuum glass filled with inert gas. c) Quartz window.

When HCL is connected to the power supply, the Ne is excited to Ne+ that attracted to cathode (while electrons collected by

anode). At cathode, Ne+ impact on its surface giving excited metal ions that emit photon radiation when they return to ground

state. These radiation photons pass to the sample (that present in ground stat i.e. ready to absorb energy)

3. Modulator: serves as a) split the light from HCL onto two beams b) distinguish between resonance line and absorbed line.

C) Converts current from HCL into alternating current

4. Flame: serves as a) atomization of the sample into free atoms which depends on the temp where at low temp: the sample reach

the preheated region forming large droplets that decrease absorption, but at high temp reach the heated region forming small

droplets that increase the absorption b) nebulization of the sample into small droplets.

We can avoid its problems by: a) controlling the gas flow b) maintaining steady flame c) Cleaning its head

5. Monochromator: isolate specific emission line of the element from other lines. i.e. allow passing specific λ

6. Detector: detect the energy absorbed by the sample. It is photomultipliers tubes that convert the energy absorbed into current.

It has two types: single and double beam system. The two systems have the same components.

Procedures

e.g. Determination of the Zinc

1. Light the hollow cathode lamp for zinc and D2-lamb if such background correction is used (Pressure reducing regulator for

acetylene and air.), (adjust for λ for Zn)

2. Position the Monochromator at λ 241 (for Zn), and choose slit height "HIGH"

3. Adjust the burner head to assure that the centre of the light beam passes over the burner slot.

4. Light the flame and regulate the flow of fuel and oxidant to produce an oxidizing flame. (lean blue)

5. Aspirate the calibration blank and establish a zero point. Then Aspirate stander solution and make a calibration curve.

6. Aspirate sample and obtain its concentration from the calibration curve. تأتي بتركيزها من المنحنى القياسي, وبمعرفة ايمتصاص للعينة

Applications: for metals analysis (e.g. Ca,Cu, Fe. Pb, Cd, Mn, Co...), in any field (clinical, environmental…)

Factors affecting F-AAS: 1) Rate of aspiration. 2) Degree of sample decomposition. 3) Temperature.

4) Position of light in flame. 5) Solvent use & inter chemical effect

Interference There is four main interferences occur with F-AAS:

1. Chemical interference: occurs when a chemical substance react with sample giving more stable complexes that can not be

dissociate by flame e.g. presence of PO4-3

during Ca+2

analysis. To avoid this, use La to displace Ca+2

from the complex.

2. Ionization interference: occurs when atoms in the flame become ionized instead of remaining in GS due to high energy of heat.

This can be eliminated by: a) addition of substance that provide excess free electrons and easily ionized b) lowering flame temp

3. Matrix interference: occurs when the composition of standers varies from sample due to: a) sample contains protein but stander

does not b) difference in viscosity c) solvent composition d) and salt conc. between them. This can be eliminated by: a) using

protein containing calibrator to match viscosity & sample composition b) dilution to minimize the difference.

4. Spectral interference: occurs when there is non specific absorption by materials other than the sample due to undissociated

molecular complexes and solid formed in the flame. It is eliminated by a) using clean burner head b) using background

correction.

Lecture (10)

Power

supply

Sample

Prism

Detector

Bakr

Modulator

Slit

Slit

14

A-2) Flameless Atomic Absorption spectroscopy

Principle

The same principle as F-AAS, but here the burner flame is displaced with graphite furnace that insures uniform heating for the sample,

increase sensitivity and lower detection limit where the graphite furnace is heated electronically to temp up to 2700 at which the trace

and heavy metals are detected.

Components

The components are the same as in F-AAS with exception the presence of graphite furnace instead of flame burner.

1) Power supply. 2) HCL. 3) Modulator. 4) Graphite furance. 5) Monochromator. 6) Detector.

Advantages and disadvantages للمعرفة

1. flame atomic absorption spectroscopy:

2. furance atomic absorption spectroscopy:

3. general comparison : (مهح)

flame atomic absorption spectroscopy Furnace (flameless) atomic absorption spectroscopy

5. flame for atomization

6. Hcl for excitation

7. less sensitivity

8. higher detection limit

9. unlimited temperature

5. graphite furance for atomization

6. Hcl for excitation

7. high sensitivity

8. lower detection limit

9. limited temperature

The sensitivity of flameless (furance) atomic absorption is higher than that of flame atomic absorption. This is because the use of

furance instead of flame results in:

1. Insure uniform heating of sample.

2. The graphite furance is heated electronically up to 2700 C ْ at which trace elements and heavy metals can be detected.

3. Lower detection limit where we can determine the very low concentration of the element (0,001ppm).

Note:

15

Radiation

Excitation

Atomization

Evaporation

B) Atomic Emission spectroscopy (AES)

(Flame photometry) Principle

Flame photometry is based on the measurement of intensity of radiation emitted from excited alkali atoms. At high temp of the flame,

the atoms are excited (A*) and then after few seconds, they returned back to the ground state (A°) resulting in emitted radiation that can

be selected at specific wavelength and detected by the detector.

Components

1. Flame: its design facilitates the nebulization of the sample into small droplets that can be easily evaporated into fuel oxidant

mixture. At high temp the flame functions are 1- evaporation of solvent, 2- dissociation of sample into free atoms (atomization)

3- excitation of free atoms into excited state (not ionized). The flame process takes place as follow:

The atomization and excitation of the sample depends on the flame temp where:

Temp of flame increases, atomization increases, excitation increases & intensity of emitted light increases.

If the temp is very higher than the required temp, ionization (removal of electrons) will be occurred not excitation

2. Monochromator: it used to select the emitted light at specific wavelength for the test element (i.e. λ-filter). It may be prism

3. Detector: it is a photocell which measures the intensity of emitted light from the sample by the conversion of the photo energy

to electrical energy. If the intensity is weak, photomultiplier detector is used to amplify intensity.

Lens Monochromator

Factors affecting the intensity of emitted light

1. Concentration of the sample: it increases, the intensity increases

2. Rate of spraying sample

3. Temperature of the flame. Temp increases, excitation increases & intensity increase

4. position of sprayed sample in the flame

Application

1. In general, it is very useful for detection of alkali metals such as Na, K, Li, Cs…so it is important in clinical field.

Comparison

flame atomic absorption spectroscopy atomic emission spectroscopy

1. flame for atomization

2. Hcl for excitation

3. major ions in ground state

4. the detector measures the light absorbed

5. more suitable for heavy metals

1. flame for atomization and excitation

2. no Hcl

3. major ions in excited state

4. the detector measures the light emitted

5. more suitable for alkali metals

Lecture (11)

Sample - fuel &

oxidant

Nebulization

Evaporation

Atomization

Excitation

By the flame large droplets are converted into small that

can be atomized by flame temp into free atoms that excited

then return back to ground state resulting in emission of

radiation energy that can be measured by detector.

Large droplets may not remain in the hottest part of flame

long time enough to be excited. This can be avoided by

passing the droplets through very narrow chamber.

Flame

Detector

16

Enzyme linked immunosorbent assay

(ELISA) Principle

It is antigen-antibody interaction, one of them is synthetic (kit) while another is natural (sample), the reaction medium is an inert

surface, usually a 96-well polystyrene plate. This reaction does not be seen, so it is important to use enzyme linked-antibody and the

substrate of this enzyme in order to change its color if the reaction occurs. The arrangement of Antigen, Antibody, Enzyme-linked

antibody and substrate on well give three main mechanisms: 1-Indirect, 2-Sandwitch & 3-competitive mechanisms

Components

1. An inert surface, usually a 96-well polystyrene plate

2. Antigen & antibody (one in kit, another in sample according to the mechanism used)

3. Enzyme-linked antibody 4. Substrate specific to the enzyme. 5.washing buffer

Procedures

A- Indirect method 1. The synthetic antigens (kit) are attached to the well plate though the active sites on the well

2. The remained active sites on the well are blocked by adding BSA for 0.5-1h. Then washing occurs to remove excess BSA.

3. The serum (with primary Ab) is added. The target antibodies react with specific antigens forming Ag/Ab complex.

4. The excess unbounded antibodies are washed. (second wash)

5. The enzyme-linked antibodies (secondary Ab) are added to bind with Ag/Ab complex forming Ag /Ab/ Ab-E complex.

6. Washing to remove the excess Ab-E in case of +Ve result or to remove the all Ab-E in case of –Ve result. اهح خطوة

7. The substrate specific to E is added. After short incubation, the color is developed in case of +Ve results.

(1) + (2) + (3) + Color

Ag Ab Ab-E

B- Sandwich method 1. The plate well contains primary antibodies ready for use.

2. The serum (with Ag) is added. The target antigens react with specific antibodies forming Ab/Ag complex

3. The excess unbounded antigens are washed. (first wash)

4. The enzyme-linked antibodies (secondary Ab) are added to bind with Ab/Ag complex forming Ab/Ag/Ab-E complex which

called Sandwich complex.

5. Washing to remove the excess Ab-E in case of +Ve result or to remove the all Ab-E in case of –Ve result. اهح خطوة

6. The substrate specific to E is added. After short incubation, the color is developed in case of +Ve results

(1) + (2) + (3) + Color

Ab Ag Ab-E

C- Competitive method 1. The plate well contains primary antibodies ready for use

2. A known concentration of enzyme-linked antigen. Ag-E and serum (with Ag) are added

3. The Ag-E and Ag compete for binding with Ab forming either Ab/Ag-E complex or Ab/Ag complex

4. The excess unbounded antigens are washed.

5. The substrate specific to E is added. After short incubation, measure the total intensity of color and determine the target Ag by

subtract the resulted intensity from known total intensity

Example:

You added known amount of AG-E (that give intensity = 100nm when reacted with known volume of substrate).

After reaction, the intensity became 40nm. So this indicate that there were similar Ag (from serum) competed with Ag-E on the binding

with Ab, So target Ag = Total intensity – resulted intensity. In this example, the target Ag conc. = 60nm

Washing

+ Color

Failed to bind

Lecture (12)