Qualitative and Quantitative Analysis

qualitative and quantitative analysis

Russian scientist Tswett in 1906 used a glass columns packed with divided CaCO3(calcium carbonate) to separate plant pigments extracted by hexane. The pigments after separation appeared as colour bands that can come out of the column one by one.

Tswett was the first to use the term "chromatography" derived from two Greek words "Chroma" meaning color and "graphein" meaning to write. Invention of Chromatography by M. Tswett

Ether Chromatography

Colors Chlorophyll

CaCO3

5 *Definition of chromatography

*Tswett (1906) stated that „chromatography is a method in which the components of a mixture are separated on adsorbent column in a flowing system”.

*IUPAC definition (International Union of pure and applied Chemistry) (1993): Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction.

*Principles of Chromatography

* Any chromatography system is composed of three components :

* Stationary phase * Mobile phase * Mixture to be separated

The separation process occurs because the components of mixture have different affinities for the two phases and thus move through the system at different rates. A component with a high affinity for the mobile phase moves quickly through the chromatographic system, whereas one with high affinity for the solid phase moves more slowly. *Forces Responsible for Separation

* The affinity differences of the components for the stationary or the mobile phases can be due to several different chemical or physical properties including:

* Ionization state * Polarity and polarizability * Hydrogen bonding / van der Waals’ forces * Hydrophobicity * Hydrophilicity

* The rate at which a sample moves is determined by how much time it spends in the mobile phase. *Classification of chromatographic methods

Chromatographic methods are classified according to:

A – Mechanism of separation:

The mechanism of separation depends mainly on the nature of the stationary phase. Based on separation mechanisms chromatography can be classified into: * 1- Adsorption Chromatography:

It is the oldest technique. Separation is due to difference in the adsorption power of mixture components. The stationary phase is a solid with adsorption characters. Silica gel and alumina are the most common stationary phase in adsorption chromatography. * 2- Partition Chromatography:

Separation is due to difference in solubility of components in two immiscible liquids. The stationary phase is a liquid thin film on an inert solid support. The stationary liquid is usually more polar than the mobile phase. Cellulose powder and wetted silica gel are examples of supports in partition chromatography that carry film of water act as stationary phase. * 3- Ion Exchange Chromatography (IEC):

It is used for separation of charged molecules. The stationary phase is an ion exchange resin to which a cationic or anionic groups are covalently bonded. Ions of opposite charges (counter ions) in the mobile phase will be attracted to the resin and compete with the components of the mixture for the charged group on the resin. * 4- Molecular Exclusion ( Size Exclusion) Chromatography:

Separation is based on molecular size. Stationary phase is a material of controlled pore size.

Molecules that are very small in relation to the pore size all behave similarly and these small molecules are also not separated.

Medium sized molecules are separated based on how far they penetrate into the gel beads. * 5- Affinity Chromatography:

The separation is based on the affinity of proteins to specific ligands such as enzymes. The ligand is attached to suitable polysaccharide polymer such as cellulose - agarose – dextran.

B- According to the nature of the mobile and stationary phase:

In this regard chromatography is classified into:

1- Liquid Chromatography (LC): The mobile phase is liquid.

2- Gas Chromatography (GC) The mobile phase is an inert gas nitrogen or helium. C- According to the technique (methods of holding the Stationary Phase):

1- Planar or Plane Chromatography: *In this type the stationary phase is used in the form of layer. Plane chromatography is additionally classified into: a- Thin Layer Chromatography (TLC): The stationary phase is spread on glass or plastic or aluminum sheets. b- Paper Chromatography (PC): A specific type of papers is used as stationary phase.

2- Columnar or Column Chromatography (CC): The stationary phase is held in to a tube made of glass or metal (gel – ion exchange – adsorption).

D- ACCORDING TO PURPOSE OF USE:

QUALITITATIVE CHROMATOGRAPHY In this case Chromatography can be used to:

1- Confirm the absence or probable presence of certain constituent in the sample under investigation 2- Give an idea about the complexity of the mixture and the least number of compounds present. 3- Check purity and identity of any compound.

QUANTITATIVE CHROMATOGRAPHY The development of modern instruments enable the use of chromatography to determine the amount of any component in a mixture as absolute amount or relative to another component HPLC/ GC/ HPTLC can be used for there applications. quantitative and qualitative liqiud chromatography

*Thin layer chromatography

*qualitative analysis After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes moved the TLC plate at different rates, separation is achieved. 2.0 cm R (A) = = 0.40 f 5.0 cm

Solvent Front R (B) = 3.0 cm = 0.60 f 5.0 cm Distance solvent migrated = 5.0 cm 4.0 cm Distance A 0.8 cm R (C) = = 0.16 migrated = 3.0 cm f 5.0 cm

Distance B migrated = 2.0 cm 4.0 cm 3.0 cm R (D) = = 0.80 f 5.0 cm

Distance C migrated = 0.8 cm 0.8 cm R (U ) = 3.0 cm = 0.60 f 1 5.0 cm Origen x x x x x A B U C D 0.8 cm R (U ) = = 0.16 f 2 5.0 cm

The Rf retardation factor is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin)

The three substances have the same Rf

1 2 3 sample

standards The three substances have the same colour

1 2 3 sample The three substances are…invisible

1 2 3 sample *Visualization Method

*Most of the time, the spots won’t show unless they are visualized!

*Visualization is a method that is used to render the TLC spots visible.

*A visualization method can be: *Ultraviolet light *Colored reagents to stain spots *ULTRAVIOLET LAMP

After developing a TLC plate, the first analysis technique should always be UV light.

This technique is fast. When using "F254 silica gel" TLC plates (silica gel that fluoresces with a 254 nm absorption) these compounds will appear as dark spots (because they "block" the fluorescence by absorbing the UV light) on a green background.

Colored reagents to stain spots

 A TLC stain is used in TLC development to reveal compounds that are not visible by UV. Also, selective detection of compounds is possible by choosing the appropriate TLC stain.

*TLC SCANER

Classical densitometry uses monochromatic light and a slit of selectable length and width to scan the tracks of a chromatogram, measuring the diffusely reflected light. The CAMAG TLC Scanner uses the entire spectral range from 190 to 900 nm with high spectral selectivity for data acquisition. Absorption spectra for substance identification and for selection of the most suitable measurement wavelength can be recorded within this range.

Thin-Layer Chromatography: Qualitative Analysis

1 2 3 sample Densytometr can automatically record spectra as soon as all peak positions are known.

The difference beetwen spectra allows us to confirm the identity of analytes in a sample. However, some spectra have small differences and cannot be absolute confirmations by themselves. Analytical chemists use both retention time and spectra to determine a probability of identifying a chemical in a sample.

Caffeine spectrum Paracetamol spectrum

The Mass Spectrometer In order to measure the characteristics of individual molecules, a mass spectrometer converts them to ions so that they can be moved about and manipulated by external electric and magnetic fields. The three essential functions of a mass spectrometer, and the associated components, are: 1. A small sample is ionized, usually to cations by loss of an electron. The Ion Source 2. The ions are sorted and separated according to their mass and charge. The Mass Analyzer 3. The separated ions are then measured, and the results displayed on a chart. The Detector

Sample

+ _

Ionizer Mass Analyzer Detector Sample

+ _ *Mass Spec Principles Ionizer Mass Analyzer Detector

The versatile instrument to extract compounds from a TLC/HPTLC plate and feed them into a mass spectrometer for substance identification or structure elucidation. http://www.google.pl/url?sa=i&source=imag es&cd=&cad=rja&docid=wGxtCaD2i- aHdM&tbnid=lYU4DocqZYlOKM:&ved=0C AgQjRwwADhI&url=http%3A%2F%2Fwww .sciencedirect.com%2Fscience%2Farticle %2Fpii%2FS0021967312013398&ei=ws2b Ub3HBoTCtAb_nYDIBg&psig=AFQjCNFT YgLztE_wmKaWEkYYS5v6Aj3u_Q&ust=1 369251650161134 quantitative analysis

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HPLC *What does HPLC means?

High Performance Liquid Chromatography

High Pressure Liquid Chromatography

High Price Liquid Chromatography

High Patience Liquid Chromatography Smaller column particle size can improve chromatographic resolution, but increased solvent delivery pressure is needed.

Mobile phase is the most important parameter in HPLC. Type of mobile phase used may have a big effect on the retention. It can promote or suppress an ionization of the analyte molecules, and it also can shield an accessible residual silanol or any other active adsorption centers on the adsorbent surface. The most common solvent reservoirs are as simple as glass bottles with tubing connecting them to the pump inlet.

High-pressure pumps are needed to push the mobile phase through the packed stationary phase.

An injector for an HPLC system should provide injection of the liquid sample within the range of 0.1-100 mL of volume with high reproducibility and under high pressure (up to 4000 psi). For liquid chromatography, liquid samples can be directly injected and solid samples need only to be diluted in the appropriate solvent.

There are many different types of detectors that can be used for HPLC.

2.0 cm R (A) = = 0.40 f 5.0 cm

Solvent Front R (B) = 3.0 cm = 0.60 f 5.0 cm Distance solvent migrated = 5.0 cm 4.0 cm Distance A 0.8 cm R (C) = = 0.16 migrated = 3.0 cm f 5.0 cm

Distance B migrated = 2.0 cm 4.0 cm 3.0 cm R (D) = = 0.80 f 5.0 cm

Distance C migrated = 0.8 cm 0.8 cm R (U ) = 3.0 cm = 0.60 f 1 5.0 cm Origen x x x x x A B U C D 0.8 cm R (U ) = = 0.16 f 2 5.0 cm

The Rf retardation factor is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin)

Each peak is labeled with retention time. Retention time indicates how long it takes for a compound to come out of the HPLC column. CAPACITY FACTOR (k): is one way to measure sample retention; bands which come out in the chromatogram at the column dead time have a k-value of zero. Later bands have k-values that increase with band retention time. Values of k for each band or compound are constant if experimental conditions do not change. k does not change when flow rate or column dimensions are changed. k can change when mobile phase composition, stationary phase chemistry, or temperature change.

Thin layer chromatography HPLC chromatogram

The data-acquisition system of most HPLC systems is a computer. The computer integrates the response of the detector to each component and places it into a chromatograph that is easy to read and interpret. Other more advanced features can also be applied to a chromatographic system. These features include computer-controlled automatic injectors, multi- pump gradient controllers and sample fraction collectors.

The column or stationary phase is the core of any chromatographic system. Columns are commercially available in different lengths, bore sizes and packing materials. The use of the correct combination of length and packing material in correlation with the appropriate mobile phase can assist in the most effective separation of a sample compound *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

Separations Based on Polarity

*As shown in Figure, classes of molecules can be ordered by their relative retention into a range or spectrum of chromatographic polarity from highly polar to highly non-polar. *Molecules with similar chromatographic polarity tend to be attracted to each other; those with dissimilar polarity exhibit much weaker attraction, if any, and may even repel one another. This becomes the basis for chromatographic separation modes based on polarity. To design a chromatographic separation system we create competition for the various compounds contained in the sample by choosing a mobile phase and a stationary phase with different polarities. Then, compounds in the sample that are similar in polarity to the stationary phase [column packing material] will be delayed because they are more strongly attracted to the particles. Compounds whose polarity is similar to that of the mobile phase will be preferentially attracted to it and move faster. Normal-Phase HPLC In his separations of plant extracts, Tswett was successful using a polar stationary phase with a much less polar [non-polar] mobile phase. This classical mode of chromatography became known as normal phase.

The stationary phase is polar and retains the polar yellow dye most strongly. The relatively non-polar blue dye is won in the retention competition by the mobile phase, a non-polar solvent, and elutes quickly. Since the blue dye is most like the mobile phase [both are non-polar], it moves faster. It is typical for normal-phase chromatography on silica that the mobile phase is 100% organic; no water is used. Reversed-Phase HPLC The term reversed-phase describes the chromatography mode that is just the opposite of normal phase, namely the use of a polar mobile phase and a non-polar [hydrophobic] stationary phase.

Now the most strongly retained compound is the more non-polar blue dye, as its attraction to the non-polar stationary phase is greatest. The polar yellow dye, being weakly retained, is won in competition by the polar, aqueous mobile phase, moves the fastest through the bed, and elutes earliest like attracts like. Today, because it is more reproducible and has broad applicability, reversed-phase chromatography is used for approximately 75% of all HPLC methods. Most of these protocols use as the mobile phase an aqueous blend of water with a miscible, polar organic solvent, such as acetonitrile or methanol. This typically ensures the proper interaction of analytes with the non-polar, hydrophobic particle surface. A C18–bonded silica [sometimes called ODS] is the most popular type of reversed- phase HPLC packing.

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 -O-Si Si CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS) MOBILE PHASE

Figure. (A) 30% MeCN: 70% 20mM Phosphate, pH 7. (B) 50% MeCN: 50% 20mM Phosphate, pH 7. (C) 80% MeCN: 20% 20mM Phosphate, pH 7. STATIONARY PHASE HPLC used for Qualitative Analysis

HPLC used for Quantitative Analysis concentration ug/L 0,6 0,9 1,2 1,5

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*Electrophoresis

One of the key elements in a gel electrophoresis system is the gel itself. Polyacrylamide and agarose gels formed as Second key element is buffer. The layers are the principal mediums for electrical current in an electrophoresis. electrophoresis cell is carried The gel matrix acts as an medium reducing largely by the ions supplied by diffusion, so that separated sample buffer, which also maintain proper components remain positioned pH and provide a medium for heat in sharp zones. In addition, the gel acts as a dissipation. molecular sieve, separating molecules according to their size. Third key is voltage.

Cathode (-) Anode (+) + High H high OH- *Factors Affecting Electrophoresis

Charge of molecule depends on its dissociation at pH of the buffer solution used. As a charge of molecule increases, its migration rate also increases. Note that depending on the nature of the net charge, the charged particles will migrate to the cathode or to the anode.

Electrophoresis of positively charged particles (cations) is called cataphoresis, while electrophoresis of negatively charged particles (anions) is called anaphoresis.

start

anode catode

As a charge of molecule increases, its migration rate also increases. The size of molecules influence their migration rate: small molecules travel faster, large molecules travel slower.

start

anode catode The shape of molecules influence their migration rate. Globular molecule exhibit a different (higher) migration rate from that of fibrous one.

DNA        – +  Buffer Properties  Electrical Field Characteristics  Temperature Effects

* Qualitative analysis Can be used to separate the size of:

*DNA

*RNA

*Protein

A technique used by scientists to distinguish between individuals of the same species using

only samples of their DNA *

*The process of DNA fingerprinting was invented by Alec Jeffreys at the University of Leicester in 1985.

DNA molecules are very long

They may consist of millions of base pairs

In order to study the structure of DNA, the molecules are broken up into smaller fragments by enzymes called restriction enzymes

Restriction enzymes do not break up the DNA molecule randomly but ‘cut’ it at particular sites 3 For example, a restriction enzyme called EcoR1* ‘recognises’ the base sequence CAATTC and cuts it between the two As

recognised

--C-C-G-C-A-G-C-T-G-T-C-A-A-T-T-C-T-C-T-C-C-G-G-A-T-C-C-A

cut --C-C-G-C-A-G-C-T-G-T-C-A A-T-T-C- T-C-T-C-C-G-G-A-T-C-C-A-

Other restriction enzymes cut the DNA in different places and so produce fragments which are easier to analyse

--C-C-G-C-A-G-C-T-G-T-C-A A-T-T-C-T-C-T-C-C-G-G-A-T-C-C-C-A-

--C-C-G-C-A-G C-T-G-T-C-A A-T-T-C-T-C-C-G G-A-T-C-C-C-A- Genetic fingerprinting

90% or more of DNA does not carry nucleotide triplets that code for proteins

The non-coding DNA is often called ‘junk DNA’ but this only means that its functions have not yet been discovered

Some of the non-coding regions consist of repeated sequences of nucleotides

For example -C-A-T-G-C-A-T-G-C-A-T-G-C-A-T-G- *

The number of repeats in any one section of DNA varies from one individual to the next

Since these sections do not code for proteins (and, therefore are not genes) there is no observable difference in these individuals Particular repeat sequences can be ‘cut out’ by restriction enzymes

For example

restriction enzyme cuts

here……………and…..….…..here

-CATCCACGACATGCATGCATGCATGCCACATCCA-

here…….…..…..………and…...….…………..here

-CCACGACATGCATGCATGCATGCATGCATGCCACAT- The fragments can be separated using gel electrophoresis

The separation takes place in a sheet of a firm but jelly-like substance (a ‘gel’)

Samples of the DNA extracts are placed in shallow cavities (‘wells’) cut into one end of the gel

A voltage is applied to opposite ends of the gel

DNA has a negative charge and moves slowly towards the positive end

The shorter fragments travel through the gel faster than the longer fragments DNA extract added well

gelatinous sheet

solution DNA samples placed in wells cut in gel thin slab of Voltage supply gel negative electrode

positive DNA fragments electrode + Move from negative To positive A sample with the shorter DNA fragments travels through the gel faster than a sample with the larger fragments * Appearance of separated fragments on gel

These bands will contain the shorter DNA fragments

These bands will contain the longer DNA fragments

starting positions

*After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. DNA may be visualized using ethidium bromide which, when intercalated into DNA, fluoresce under ultraviolet light.

*Photographs can be taken of gels, often using a Gel Doc system. Genetic fingerprinting

DNA analysis can be used for catching criminals, establishing parentage, finding how closely organisms are related and many other applications.

The pattern of bands in a gel electrophoresis is known as a genetic fingerprint or a ‘genetic profile’

If a genetic fingerprint found in a sample of blood or other tissue at the scene of a crime matches the genetic fingerprint of a suspect, this can be used as evidence

A DNA sample can be obtained from the suspect using blood, cheek epithelial cells taken from the mouth lining or even the cells clinging to the root of a hair Suppose that…………

….there is a chance of 1 in 10 that this fragment occurs in many individuals…

…and.there is a chance of 1 in 20 that this fragment occurs in many individuals…

…and.there is a chance of 1 in 10 that this fragment occurs in many individuals…

…and.there is a chance of 1 in 30 that this fragment occurs in many individuals, but…

…the probability of all 4 bands matching in any person other than the suspect is

1 in 10 x 1 in 20 x 1 in 10 x 1 in 30

= 1 in 10 x 20 x 10 x 30 That is 1 in 60,000

When a larger number of bands is involved, the probability that the suspect is not guilty becomes one in many thousands* * *Uses: Criminology

*Comparing blood samples on defendant’s clothing to determine if it belongs to victim

*Uses: Paternity Mom F1 F2 child – Genetic fingerprint of … DNA  1 mom 4 child 2 possible father 1

3 possible father 2

There is a match between 3 of the child’s restriction fragments and one of the mother’s.

There is also a match between the child’s + fragments and one from possible father 2. Neither of the child’s restriction fragments match those of possible father 1

* *Uses: Medical diagnostic

*Comparing normal allele to disease allele

chromosome chromosome with with normal disease-causing allele 1 allele 2

Example: test for Huntington’s disease DNA analysis of Huntington disease. Each lane shows a different person's DNA: two bands in the normal (N) range show someone is unaffected. One band in the H range predicts the person will get Huntington disease. * Uses: Evolutionary relationships

*Comparing DNA samples from different organisms to measure evolutionary relationships

turtle snake rat squirrel fruitfly – 1 2 3 4 5 1 2 3 4 5 DNA 

+ *Capillary electrophoresis