Chapter 4 bio 300 obe

Chapter 4: Techniques in Biochemical Analysis1

Chapter 4: Techniques in Biochemical Analysis

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BIO 300BIO 300BIOLOGICAL TECHNIQUES AND SKILLSBIOLOGICAL TECHNIQUES AND SKILLS

SARINI BINTI AHMAD WAKIDSARINI BINTI AHMAD WAKIDFACULTY OF APPLIED SCIENCEFACULTY OF APPLIED SCIENCE

BIO 300BIO 300BIOLOGICAL TECHNIQUES AND SKILLSBIOLOGICAL TECHNIQUES AND SKILLS

SARINI BINTI AHMAD WAKIDSARINI BINTI AHMAD WAKIDFACULTY OF APPLIED SCIENCEFACULTY OF APPLIED SCIENCE


3

CHAPTER 4CHAPTER 4Techniques in Biochemical Techniques in Biochemical

AnalysisAnalysis

Chapter 4: Techniques in Biochemical Analysis 4

Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components.

Separate

• Analyze

• Identify

• Purify

• QuantifyComponentsMixture

What is Chromatography?


Chromatography

Chromatography is a method of separating a mixture of molecules depending on their distribution between a mobile phase and a stationary phase.

The mobile phase (also known as solvent) may be either liquid or gas.

The stationary phase (also known as sorbent) can be either a solid or liquid, a liquid stationary phase is held stationary by a solid.

The solid holding the liquid stationary phase is the support or matrix.

The molecules in the mixture to be separated are the solutes.


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Chromatography is used by scientists to:

• Analyze – examine a mixture, its components, and their relations to one another

• Identify – determine the identity of a mixture or components based on known components

• Purify – separate components in order to isolate one of interest for further study

• Quantify – determine the amount of the a mixture and/or the components present in the sample

Uses for Chromatography


Uses for ChromatographyReal-life examples of uses for

chromatography:

• Pharmaceutical Company – determine amount of each chemical found in new product

• Hospital – detect blood or alcohol levels in a patient’s blood stream

• Law Enforcement – to compare a sample found at a crime scene to samples from suspects

• Environmental Agency – determine the level of pollutants in the water supply

• Manufacturing Plant – to purify a chemical needed to make a product


Detailed Definition:Chromatography is a laboratory technique that

separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass.

Terminology:• Differential – showing a difference, distinctive

• Affinity – natural attraction or force between things

• Mobile Medium – gas or liquid that carries the components (mobile phase)

• Stationary Medium – the part of the apparatus that does not move with the sample (stationary phase)

Definition of Chromatography


Simplified Definition:Chromatography separates the

components of a mixture by their distinctive attraction to the mobile phase and the stationary phase.

Explanation:• Compound is placed on stationary phase• Mobile phase passes through the stationary

phase• Mobile phase solubilizes the components• Mobile phase carries the individual

components a certain distance through the stationary phase, depending on their attraction to both of the phases

Definition of Chromatography


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Illustration of Chromatography

Components

Affinity to Stationary Phase

Affinity to Mobile Phase

Blue ---------------- Insoluble in Mobile Phase

Black

Red

Yellow

Mixture Components

Separation

Stationary Phase

Mobile Phase


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• Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)

• Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a

liquid or of solid beads (stationary phase)

• Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)

• Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

Types of Chromatography


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Types of chromatography

• Partition chromatography• Adsorption chromatography• Gel filtration• Ion exchange chromatography


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(A) uses charge, (B) uses pores, and (C) uses covalent bonds to create the differential affinities among the mixture components for the stationary phase.


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Partition chromatography

• The distribution of solutes between two immiscible phases.

• The solute will distribute it self between the two phases according to its solubility in each phase, this is called partitioning.


Examples of partition chromatography

The two most common types of partition chromatography are thin layer chromatography and paper chromatography.

In both cases the stationary phase is a liquid bound to a matrix. In paper chromatography the stationary phase are water molecules

bound to a cellulose matrix. In TLC, the stationary phase is the solvent added to the support to

form the thin layer so the solvent gets bound to the matrix (support).

Partition chromatography is mainly used for separation of molecules of small molecular weight.


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Paper chromatography

• The cellulose support contains a large amount of bound water.

• Partitioning occurs between the bound water which is the stationary phase and the solvent which is the mobile phase.


Experimental procedure for paper chromatography A small volume of a solution of a mixture to be separated or identified is

placed at a marked spot (origin) on a sheet or strip of paper and allowed to dry.

The paper is then placed in a closed chamber and one end is immersed in a suitable solvent.

The solvent is drawn (moved) through the paper by capillary action. As the solvent passes the origin, it dissolves the sample and moves the

components in the direction of flow. After the solvent front has reached a point near the other end of the paper,

the sheet or strip is removed and dried. The spots are then detected and their positions marked. The ratio of the distance moved by a solute to the distance moved by the

solvent = Rf.

The Rf. is always less than one.


Chromatogram Once a sample is applied on TLC or paper, it’s called

chromatogram. Paper chromatogram can be developed either by

ascending or descending solvent flow. Descending chromatography is faster because gravity

helps the solvent flow. Disadvantages : it’s difficult to set the apparatus. Ascending is simple and inexpensive compared with

descending and usually gives more uniform migration with less diffusion of the sample "spots".


Detection of spots

Spots in paper chromatograms can be detected in 4 different ways:

1. By their natural color

2. By their fluorescence

3. By their chemical reactions that take place after the paper has been sprayed with various reagents for example: during paper chromatography of amino acids, the chromatograms are sprayed with ninhydrin.

4. By radioactivity


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Identification of spots

• The spots are usually identified by comparing of standards of known Rf values.


Thin layer chromatography Paper chromatography uses paper which can be

prepared from cellulose products only. In TLC, any substance that can be finely divided

and formed into a uniform layer can be used. Both organic and inorganic substances can be

used to form a uniform layer for TLC. Organic substances include: cellulose, polyamide,

polyethylene Inorganic: silica gel, aluminum oxide and

magnesium silicate


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TLC

• The stationary phase is the solvent used to form a layer of sorbent spread uniformly over the surface of a glass or plastic plate


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Advantages of TLC over paper chromatography• Greater resolving power because

there is less diffusion of spots.• Greater speed of separation• Wide choice of materials as

sorbents


The separation of compounds by chromatography depends on several factors:

Partition of a solute between a moving solvent phase and a stationary aqueous phase. The solute moves in the direction of a solvent flow at a rate determined by the solubility of the solute in the moving phase. Thus a compound with high mobility is more attracted to the moving organic phase than to the stationary phase.


Cont..

Ion exchange effect: any ionized impurities in the support medium will tend to bind or attract oppositely charged ions (solutes) and will therefore reduce the mobility of these solutes.

Temperature: Since temperature can effect the solubility of the solute in a given solvent temperature is also an important factor.


The molecular weight of a solute also affects the solubility and hence chromatographic performance.

Adsorption of compound (solute) onto support medium: Although the support medium (silica gel) is theoretically inert, this isn't always the case. If a solute tends to bind to the support medium this will slow down its mobility in the solvent system.

The composition of the solvent: since some compounds are more soluble in one solvent than in the other, the mixture of solvents used will affect the separation of compounds.


Expression of the results

The term "Rf" (relative flow) is used to express the performance of a solute in a given solvent system /support medium. The term Rf value may be defined as the ratio of the distance the compound migrates to the distance the solvent migrates. Rf value is constant for a particular compound, solvent system and insoluble matrix.

Rf= Distance of migration of solute

Distance moved by solvent


Rf values qualitative results of TLC

expressed as fractions of 1.0 can be expressed from Rf values (eg Rf x 100) no more than two decimal places

due to inaccuracy of physical measurement may not be reproducible

only give an indication of possible nature of unknown complete identification only obtained if spot is eluted and

micro-scale physical measurements done (MS, UV, IR) standard references should always be used on same

plate for comparison most sprays produce differential colours of fluorescence colour test provides extra evidence with distance

migration



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Principles of Paper Chromatography

• 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.


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Paper Chromatography Experiment

What Color is that Sharpie?


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Overview of the Experiment

Purpose: To introduce students to the principles and terminology of chromatography and demonstrate separation of the dyes in Sharpie Pens with paper chromatography.

Time Required: Prep. time: 10 minutesExperiment time: 45 minutes


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• 6 beakers or jars• 6 covers or lids • Distilled H2O• Isopropanol• Graduated cylinder• 6 strips of filter paper• Different colors of

Sharpie pens• Pencil• Ruler• Scissors• Tape

Materials List


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Preparing the Isopropanol Solutions

• Prepare 15 ml of the following isopropanol solutions in appropriately labeled beakers:

- 0%, 5%, 10%, 20%, 50%, and 100%


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Preparing the Chromatography Strips

• Cut 6 strips of filter paper

• Draw a line 1 cm above the bottom edge of the strip with the pencil

• Label each strip with its corresponding solution

• Place a spot from each pen on your starting line


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Developing the Chromatograms

• Place the strips in the beakers

• Make sure the solution does not come above your start line

• Keep the beakers covered• Let strips develop until

the ascending solution front is about 2 cm from the top of the strip

• Remove the strips and let them dry


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Developing the Chromatograms

Chapter 4: Techniques in Chapter 4: Techniques in Biochemical AnalysisBiochemical Analysis 3939

Developing the Developing the ChromatogramsChromatograms


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Observing the Observing the ChromatogramsChromatograms

Concentration of Isopropanol

0% 20% 50% 70% 100%


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Black Dye


0% 20% 50% 70% 100%

1. Dyes separated – purple and black

2. Not soluble in low concentrations of isopropanol

3. Partially soluble in concentrations of isopropanol >20%


Blue DyeBlue Dye


0% 20% 50% 70% 100%

1. Dye separated – blue2. Not very soluble in low

concentrations of isopropanol

3. Completely soluble in high concentrations of isopropanol


Green DyeGreen Dye


0% 20% 50% 70% 100%

1. Dye separated – blue and yellow

2. Blue – Soluble in concentrations of isopropanol >20%

3. Yellow – Soluble in concentrations of isopropanol >0%


Red DyeRed Dye1. Dyes separated – red and yellow1. Dyes separated – red and yellow

2. Yellow –soluble in low concentrations of isopropanol and 2. Yellow –soluble in low concentrations of isopropanol and

less soluble in high concentrations of isopropanolless soluble in high concentrations of isopropanol


0% 20% 50% 70% 100%

3. Red – slightly soluble in low concentrations of isopropanol, and more soluble in concentrations of isopropanol >20%


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Alternative Experiments

• Test different samples:– Other markers, pens, highlighters– Flower pigments– Food Colors

• Test different solvents:– Other alcohols: methanol, ethanol,

propanol, butanol

• Test different papers:– Coffee filters– Paper towels– Cardstock– Typing paper


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Chromatography Chromatography InstrumentsInstruments

Chromatography Chromatography InstrumentsInstruments


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• Chromatography techniques• Affinity Chromatography (AC)• Hydrophobic Interaction Chromatography (HIC)• Ion Exchange Chromatography (IEC)• Gel Filtration (GF)• Capillary electrochromatography (CEC)


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Affinity Chromatography

Surface bound with

Epoxy, aldehyde or aryl ester groups

Metal Interaction Chromatography

Surface bound with

Iminodiacetic acid + Ni2+/Zn2+/Co2+


(Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)


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Metal Interaction Chromatography (AC)

Points to Note:

1. Avoid chelating agents

2. Increasing incubation time

3. Slow gradient elution

(www.qiagen.com)



Binding Capacity (mg/ml) medium 12mg of histag proteins (MW= 27kDa)

Depends on Molecular weight

Degree of substitution /ml medium~15mol Ni2+

Backpressure ~43psiChange the guard column filter



Biopolymer (phenyl agarose - Binding Surface)

Driving force for hydrophobic adsorptionWater molecules surround the analyte and the binding surface.

When a hydrophobic region of a biopolymer binds to the surface of a mildly hydrophobic stationary phase, hydrophilic water molecules are effectively released from the surrounding hydrophobic areas causing a thermodynamically favorable change in entropy.

Temperature plays a strong role

Ammonium sulfate, by virtue of its good salting-out properties and high solubility in water is used as an eluting buffer

Hydrophobic Interaction Chromatography

Hydrophobic region



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Fractogel matrix is a methacrylate resin upon which polyelectrolyte Chains (or tentacles) have been grafted. (Novagen)

Ion Exchange Chromatography

Globular Protein

Deformation due to interaction with conventional ion exchanger

Maintenance of conformation while interacting with tentacle ion exchanger

(www.novagen.com)


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Gel Filtration

(http://lsvl.la.asu.edu/resources/mamajis/chromatography/chromatography.html)


Capillary Electrochromatography

• CEC is an electrokinetic separation technique

• Fused-silica capillaries packed with stationary phase

• Separation based on electroosmotically driven flow

• Higher selectivity due to the combination of chromatography and

electrophoresis

Fused silica tube filled with porous methacrylamide-stearyl methacrylate-

dimethyldiallyl ammonium chloride monolithic polymers, 80 x 0.5mm i.d.,

5.5kV. High Plate count ~ 400,000

Height Equivalent to a Theoretical Plate /Plate Count (HETP) H = L/Nnumber of plates N = 16(t/W)2 where L = column length, t = retention time, and W = peak width at baseline

(http://www.capital-hplc.co.uk)


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CEC columns AC, IEC columns

CEC column NP, RP columns


Schematic of a Multi-dimensional Separation System


Fast Protein Liquid Chromatograph (FPLC)

1

2

3

5

4

• No air bubbles (Priming)• Use degassed buffers

Injector Module

Column Inlet

DetectorFractionCollector

(www.pharmacia.com)


Chromatography systems

ÄKTAprime:

simple automated purification

ÄKTAFPLC: high performance

purification of proteins & other biomolecules

ÄKTApurifier: high performance

purification and characterizationÄKTAexplorer: for fast method

development and scale-upÄKTApilot: rapid process

development and pilot-scale

ÄKTAxpress: for high throughput tagged

protein purification


High Performance Liquid Chromatography (HPLC)

What is HPLC? Types of Separations Columns and Stationary Phases Mobile Phases and Their Role in Separations Injection in HPLC Detection in HPLC

Variations on Traditional HPLC Ion Chromatography Size Exclusion Chromatography


What is HPLC? High Performance Liquid Chromatography

High Pressure Liquid Chromatography (usually true]

Hewlett Packard Liquid Chromatography (a joke)

High Priced Liquid Chromatography (no joke)

HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time!

Probably the most widely practiced form of quantitative, analytical chromatography practiced today due to the wide range of molecule types and sizes which can be separated using HPLC or variants of HPLC!!




Types of HPLC Separations (partial list) Normal Phase: Separation of polar analytes by partitioning onto a polar,

bonded stationary phase.

Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase.

Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica)

Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support.

Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a “maze” of tunnels in the stationary phase.





What does the analyst do? Select the correct type of separation for the analyte(s) of interest, based on the sample type (among other factors).

Select an appropriate column (stationary phase) and mobile phase

Select an appropriate detector based on whether universal or compound-specific detection is required or available

Optimize the separation using standard mixtures

Analyze the standards and sample



Columns and Stationary Phases. HPLC is largely the domain of packed columns

some research into microbore/capillary columns is going on. Molecules move too slowly to be able to reach and therefore

“spend time in” the stationary phase of an open tubular column in HPLC. In solution, not the gas phase Larger molecules in HPLC vs. GC (generally)

Stationary phases are particles which are usually about 1 to 20 m in average diameter (often irregularly shaped) In Adsorption chromatography, there is no additional phase

on the stationary phase particles (silica, alumina, Fluorosil). In Partition chromatography, the stationary phase is coated

on to (often bonded) a solid support (silica, alumina, divinylbenzene resin)



Stationary Phases Polar (“Normal” Phase):

Silica, alumina Cyano, amino or diol terminations on the bonded phase

Non-Polar (“Reversed Phase”) C18 to about C8 terminations on the bonded phase Phenyl and cyano terminations on the bonded phase

Mixtures of functional groups can be used!!

Packed particles in a column require: Frits at the ends of the column to keep the particles in Filtering of samples to prevent clogging with debris High pressure pumps and check-valves Often a “Guard Column” to protect the analytical column


Optimization of Separations in HPLC Correct choice of column so the above equilibrium has some

meaningful (non-infinity, non-zero) equilibrium constants. Correct choice of mobile phase Decision on the type of mobile phase composition

constant composition = isocratic varying composition = gradient elution

Determination if flow rate should be constant usually it is

Decision on heating the column heating HPLC columns can influence the above equilibrium….



The Mobile Phase in HPLC... Must do the following:

solvate the analyte molecules and the solvent they are in be suitable for the analyte to transfer “back and forth”

between during the separation process

Must be: compatible with the instrument (pumps, seals, fittings,

detector, etc) compatible with the stationary phase readily available (often use liters/day) of adequate purity

spectroscopic and trace-composition usually! Not too compressible (causes pump/flow problems)

Free of gases (which cause compressability problems)


Typical HPLC Pump (runs to 4,000+ psi)



Polarity Index for Mobile Phases….. The polarity index is a measure of the relative polarity of a solvent.

It is used for identifying suitable mobile phase solvents. The more polar your solvent is, the higher the index. You want to try to choose a polarity index for your solvent (or

solvent mixture) that optimizes the separation of analytes usually the index is a starting point the polarity of any mixture of solvents to make a mobile phase can be

modeled to give a theoretical chromatogram Usually, optimization of solvent composition is experimental

A similar number is the Eluent Strength (Eo] Increasing eluent strength or polarity index values mean increasing

solvent polarity. Remember, the analyte(s) and samples must be mobile phase and

stationary phase compatible!




Optimization of Mobile Phase Polarity…

Changing the mobile phase composition alters the separation.


Isocratic versus Gradient Elution Isocratic elution has a constant mobile phase composition

Can often use one pump! Mix solvents together ahead of time! Simpler, no mixing chamber required Limited flexibility, not used much in research

mostly process chemistry or routine analysis.

Gradient elution has a varying mobile phase composition Uses multiple pumps whose output is mixed together

often 2-4 pumps (binary to quarternary systems) Changing mobile phase components changes the polarity index

can be used to subsequently elute compounds that were previously (intentionally) “stuck” on the column

Some additional wear on the stationary phase Column has to re-equiluibrate to original conditions after each run

(takes additional time).





Injection in HPLC Usually 5 to 1000 L volumes, all directly onto the column

not much worry about capacity since the columns have a large volume (packed).

Injector is the last component before the column(s) A source of poor precision in HPLC

errors of 2-3 %RSD are due just to injection other errors are added to this due to capillary action and the small dimensions/cavities inside

the injector 6-PORT Rotary Valve is the standard manual injector Automatic injectors are available Two positions, load and inject in the typical injector Injection loop internal volume determines injection volume.


LOAD (the sample loop)

Inject (move the sampleloop into the mobilephase flow)



Detection in HPLC Numerous Types (some obscure) Original HPLC Detectors were common laboratory

instruments such as spectrophotometers, etc. Must be solvent -compatible, stable, etc. Universal

respond to all analytes Analyte Specific

respond to specific properties of analytes Non-destructive

most Destructive

ELSD, MS and a few others.



Standard Absorbance Detector…. Single Beam UV-VIS instrument with a flow-through cell (cuvette) Can use any UV-VIS with a special flow cell

Extra connections lead to band-broadening if UV-VIS is far from HPLC column exit.

Usually utilize typical UV-VIS lamps and 254 nm default wavelenth Can be set to other wavelengths (most) Simple filter detectors no longer widely used

adjustable wavelength units are cost-effective Non-destructive, not-universal

not all compounds absorb light can pass sample through several cells at several different wavelenghts

Usually zeroed at the start of each run using an electronic software command. You can have real-time zeroing with a reference cell.



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SPECTROSCOPYSPECTROSCOPY SPECTROSCOPYSPECTROSCOPY


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Definition• Spectroscopy - The study of the

interaction of electromagnetic

radiation with matter


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Introduction• Spectroscopy is an analytical technique

which helps determine structure.• It destroys little or no sample.• The amount of radiation absorbed by

the sample is measured as wavelength is varied.


Major Types of Spectroscopy Infrared (IR) spectroscopy measures the bond vibration

frequencies in a molecule and is used to determine the functional group.

Mass spectrometry (MS) fragments the molecule and measures the masses.

Nuclear magnetic resonance (NMR) spectroscopy detects signals from hydrogen atoms and can be used to distinguish isomers.

Ultraviolet (UV) spectroscopy uses electron transitions to determine bonding patterns.


Introduction of Spectrometric Analyses

The study how the chemical compound interacts with different wavelenghts in a given region of electromagnetic radiation is called spectroscopy or spectrochemical analysis.

The collection of measurements signals (absorbance) of the compound as a function of electromagnetic radiation is called a spectrum.


101

Energy AbsorptionThe mechanism of absorption energy is different in the Ultraviolet, Infrared, and Nuclear magnetic resonance regions. However, the fundamental process is the absorption of certain amount of energy.

The energy required for the transition from a state of lower energy to a state of higher energy is directly related to the frequency of electromagnetic radiation that causes the transition.


Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

Wavelength (nm)

Spectral Distribution of Radiant Energy


Electromagnetic SpectrumElectromagnetic Spectrum


Electromagnetic SpectrumElectromagnetic Spectrum


V = Wave Number (cm-1)

Wave Length

C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.

= Frequency of Radiation (cycles/sec)

The energy of photon:

h (Planck's constant) = 6.62 x 10-27 (Ergsec)

V =C

E = h = hC

C

= C =

Electromagnetic Radiation


106

Equation Definitions• E = energy (Joules, ergs)• c = speed of light (constant) = wavelength• h = Planck’s constant = “nu” = frequency (Hz)• nm = 10-9 m• Å = angstrom = 10-10 m


Visible

Ultra violet

Radio

Gamma ray

Hz

cmcm-1Kcal/mol eV

Type

Quantum Transition

Type

spectroscopy

Type

Radiation

Frequency

υ

Wavelength

λ

Wave

Number VEnergy

9.4 x 107 4.9 x 106 3.3 x 1010 3 x 10-11 1021

9.4 x 103 4.9 x 102 3.3 x 106 3 x 10-7 1017

9.4 x 101 4.9 x 100 3.3 x 104 3 x 10-5 1015

9.4 x 10-1 4.9 x 10-2 3.3 x 102 3 x 10-3 1013

9.4 x 10-3 4.9 x 10-4 3.3 x 100 3 x 10-1 1011

9.4 x 10-7 4.9 x 10-8 3.3 x 10-4 3 x 103 107

X-ray

Infrared

Micro-wave

Gamma ray emission

X-ray absorption, emission

UV absorption

IR absorption

Microwave absorption

Nuclear magnetic resonance

Nuclear

Electronic (inner shell)

Molecular vibration

Electronic (outer shell)

Molecular rotation

Magnetically induced spin states

Spectral Properties, Application and Interactions of Electromagnetic Radiation



109

Spectrum of Radiation


110

Visible Light


111

Visible Light

Red

Orange

Yellow

Green

Blue

Indigo

Violet

R

O

Y

G

B

I

V

700 nm

650 nm

600 nm

550 nm

500 nm

450 nm

400 nm


Dispersion of Polymagnetic Light with a Prism

Polychromatic Ray

Infrared

RedOrange

Yellow

Green

Blue

Violet

Ultraviolet

monochromatic Ray

SLIT

PRISM

Polychromatic Ray Monochromatic Ray

Prism - Spray out the spectrum and choose the certain wavelength( that you want by slit.


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Ultra Violet Spectrometry

The absorption of ultraviolet radiation by molecules is dependent upon the electronic structure of the molecule.

So the ultraviolet spectrum is called electronic spectrum.


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INTRODUCTION TO INTRODUCTION TO SPECTROPHOTOMETRYSPECTROPHOTOMETRY


115

SpectrophotometrySpectrophotometry

• Spectrophotometry: An analytical Spectrophotometry: An analytical method using several spectra (lights). method using several spectra (lights). (State each spectrum used in (State each spectrum used in spectrophotometry.)spectrophotometry.)

• Spectrophotometer: An instrument for Spectrophotometer: An instrument for measuring absorbance that uses a measuring absorbance that uses a monochromatormonochromator to select the to select the wavelength.wavelength.


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SpectrophotometrySpectrophotometry

-Advantages of spectrophotometers-Advantages of spectrophotometers

i.i. relatively inexpensive relatively inexpensive

ii.ii. inexpensiveinexpensive

iii.iii. easy to maintaineasy to maintain

iv.iv. portableportable


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BACKGROUNDBACKGROUNDwhite light is observed, what is actually seen is a

mixture of all the colors of light

Why do some substances appear colored?

When this light passes through a substance, certain energies (or colors) of the light are absorbed while other color(s) are allowed to pass

through or are reflected by the substance.

If the substance does not absorb any light, it appears white (all light is reflected) or colorless (all light is transmitted). A solution appears a

certain color due to the absorbance and transmittance of visible light. For example, a blue solution appears blue because it is absorbing all of

the colors except blue.


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BACKGROUNDBACKGROUND


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BACKGROUNDBACKGROUND• The amount of light absorbed by a solution is The amount of light absorbed by a solution is

dependent on the ability of the compound to dependent on the ability of the compound to absorb light (absorb light (molar absorptivitymolar absorptivity), the distance ), the distance through which the light must pass through the through which the light must pass through the sample sample (path length(path length) and the ) and the molar molar concentrationconcentration of the compound in the solution. of the compound in the solution.

• If the same compound is being used and the If the same compound is being used and the path length is kept constant, then the path length is kept constant, then the absorbance is directly proportional to the absorbance is directly proportional to the concentration of the sample.concentration of the sample.


120

SpectrophotometerSpectrophotometer • A spectrophotometer is used to provide a A spectrophotometer is used to provide a

source of light of certain energy source of light of certain energy (wavelength) and to measure the quantity (wavelength) and to measure the quantity of the light that is absorbed by the sample. of the light that is absorbed by the sample.

Light Bulb Prism Filter Slit Sample Detector


121

Spectrophotometer Spectrophotometer • The basic operation of the spectrophotometer includes a white light The basic operation of the spectrophotometer includes a white light

radiation source that passes through a radiation source that passes through a monochromatormonochromator. The . The monochromator is either a prism or a monochromator is either a prism or a diffraction grating diffraction grating that separates that separates the white light into all colors of the visible spectrum. After the light is the white light into all colors of the visible spectrum. After the light is separated, it passes through a separated, it passes through a filter filter (to block out unwanted light, (to block out unwanted light, sometimes light of a different color) and sometimes light of a different color) and a a slitslit (to narrow the beam of (to narrow the beam of light--making it form a rectangle). Next the beam of light passes through light--making it form a rectangle). Next the beam of light passes through the the samplesample that is in the sample holder. The light passes through the that is in the sample holder. The light passes through the sample and the unabsorbed portion strikes a sample and the unabsorbed portion strikes a photodetectorphotodetector that that produces an electrical signal which is proportional to the intensity of the produces an electrical signal which is proportional to the intensity of the light. The signal is then converted to a light. The signal is then converted to a readable output readable output that is used in that is used in the analysis of the sample. the analysis of the sample.

Light Bulb Prism Filter Slit Sample Detector


Spectrophotometer

An instrument which can measure the absorbance of a sample at any wavelength.

Light Lens Slit Monochromator

Sample Detector Quantitative Analysis

Slits


123PGCC CHM 103 SinexPGCC CHM 103 Sinex

Io I

Cell withPathlength, b,

containing solution

lightsource detector

blank where Io = I

concentration 2concentration 1

b

with sample I < Io

The process of light being absorbed by a solution

As concentration increased, less light was transmitted (more light absorbed).


Beer – Lambert Law

Glass cell filled with concentration of solution (C)

IILight

0

As the cell thickness increases, the transmitted intensity of light of I decreases.


R- Transmittance

R = I0 - Original light intensity

I- Transmitted light intensity

% Transmittance = 100 x

Absorbance (A) = Log

= Log = 2 - Log%T

Log is proportional to C (concentration of solution) and is

also proportional to L (length of light path through the solution).

I

I0

I

I0

I0

I

1

T

I

I0


A CL = ECL by definition and it is called the Beer - Lambert Law.

A = ECL

A = ECL

E = Molar Extinction Coefficient ---- Extinction Coefficient of a solution containing 1g molecule of solute per 1 liter of solution


E =Absorbance x Liter

Moles x cm

UNITS

A = ECL

A = No unit (numerical number only)

E = Liter

Cm x Mole

L = Cm C = Moles/Liter

A = ECL = (Liter

Cm x Mole) x

Mole

Literx Cm


PGCC CHM 103 SinexPGCC CHM 103 Sinex

The BLANKThe BLANK

The blank contains The blank contains allall substances substances expect the analyte.expect the analyte.

Is used to set the absorbance to zero:Is used to set the absorbance to zero:

AAblankblank = 0 = 0 This removes any absorption of light This removes any absorption of light

due to these substances and the cell.due to these substances and the cell. All measured absorbance is due to All measured absorbance is due to

analyte.analyte.


129PGCC CHM 103 SinexPGCC CHM 103 Sinex

Beer’s LawBeer’s Law

A = abcA = abc

where a – molar absorptivity, b – where a – molar absorptivity, b – pathlength, and c – molar concentrationpathlength, and c – molar concentration

See the See the Beer’s Law Simulator


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SpectrophotometerSpectrophotometer

The spectrophotometer displays this quantity in one of two ways:

(1)Absorbance -- a number between 0 and 2

(2) Transmittance -- a number between 0 and 100%.

The sample for a spectral analysis is prepared by pouring it into a cuvette which looks similar to a small test tube. A cuvette is made using a special optical quality glass that will itself absorb a minimal amount of the light. It is also marked with an indexing line so that it can be positioned in the light beam the same way each time to avoid variation due to the differences in the composition of the glass


131

Fundamentals of Spectrophotometry Absorption of Light

Beer’s Law The relative amount of a certain wavelength of light absorbed (A) that passes

through a sample is dependent on:- distance the light must pass through the sample (cell path length - b)- amount of absorbing chemicals in the sample (analyte concentration – c)- ability of the sample to absorb light (molar absorptivity - )

Increasing [Fe2+]

Absorbance is directly proportional to concentration of Fe+2


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3.) Beer’s Law Absorbance is useful since it is directly related to the analyte concentration,

cell pathlength and molar absorptivity. This relationship is known as Beer’s Law

where: A = absorbance (no units)= molar absorptivity (L/mole-cm)b = cell pathlength (cm)c = concentration of analyte (mol/L)

Beer’s Law allows compounds to be quantified by their ability to absorb light, Relates directly to concentration (c)

A = abc


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4.) Absorption Spectrum By choosing different wavelengths of light (A vs. B) different compounds

can be measured

A B


134

Fundamentals of Spectrophotometry Spectrophotometer

1.) Basic Design An instrument used to make absorbance or transmittance measurements is

known as a spectrophotometer


135

Single Beam Spectrophotometer


136

Dual Beam Spectrophotometer


137


1.) Basic Design Light Source: provides the light to be passed through the sample

- Tungsten Lamp: visible light (320-2500 nm)

- Deuterium Lamp: ultraviolet Light (160-375 nm)

In presence of arc, some of the electrical energy is absorbed by D2 (or H2) which results in the disassociation of the gas and release of light

D2 + Eelect D*2 D’ + D’’ + h(light produced)

Excited state

Low pressure (vacuum)

Tungsten Filament

- based on black body radiation:heat solid filament to glowing, light emitted will be characteristic of temperature more than nature of solid filament


138


1.) Basic Design Wavelength Selector (monochromator): used to select a given wavelength

of light from the light source- Prism:

- Filter:


139


1.) Basic Design Wavelength Selector (monochromator): used to select a given wavelength

of light from the light source- Reflection or Diffraction Grating:


140


1.) Basic Design Sample Cell: sample container of fixed length (b).

- Usually round or square cuvet- Made of material that does not absorb light in the wavelength range of

interest

1. Glass – visible region

2. Quartz – ultraviolet

3. NaCl, KBr – Infrared region


141

Cuvettes (sample holder)

• Polystyrene– 340-800 nm

• Methacrylate– 280-800 nm

• Glass– 350-1000 nm

• Suprasil Quartz– 160-2500 nm


142

Spectrophotometer

1.) Basic Design Light Detector: measures the amount of light passing through the

sample.- Usually works by converting light signal into electrical signal

Fundamentals of Spectrophotometry

Photomultiplier tubeProcess: a) light hits photoemissive cathode and e- is emitted. b) an emitted e- is attracted to electrode #1 (dynode 1), which is 90V more positive. Causes several more e- to be emitted. c) these e- are attracted to dynode 2, which is 90V more positive then dynode 1, emitting more e-. d) process continues until e- are collected at anode after amplification at 9 dynodes. e) overall voltage between anode and cathode is 900V. f) one photon produces 106 – 107 electrons. g) current is amplified and measured


Applications of Spectrophotometry

Quantitative Applications

• Usually using UV-Vis• IR can be used

- Environmental applications; analysis waters & waste waters

- Clinical applications: analysis of glucose

- Industrial analysis; analysis of iron content in food

- Forensic applications: Determination of blood alcohol


Advantage of Advantage of spectrophotometer over spectrophotometer over colorimetercolorimeter

can be used to profile printers & scanners, measure colors "in can be used to profile printers & scanners, measure colors "in the wild", measure your illuminationthe wild", measure your illumination

colorimeter measures only 3 points on the specturm (RGB), colorimeter measures only 3 points on the specturm (RGB), while a spectrophotometer measures while a spectrophotometer measures manymany points across the points across the entire spectrumentire spectrum

colorimeters use a single type of light (such as incandescent or colorimeters use a single type of light (such as incandescent or pulsed xenon) Spectrophotometers can compensate for this shift, pulsed xenon) Spectrophotometers can compensate for this shift, making spectrophotometers a superior choice for accurate, making spectrophotometers a superior choice for accurate, repeatable color measurement.repeatable color measurement.


145

Sample Cells

UV Spectrophotometer

Quartz (crystalline silica)

Visible Spectrophotometer

Glass


146

Light Sources

UV Spectrophotometer

1. Hydrogen Gas Lamp

2. Mercury Lamp

Visible Spectrophotometer

1. Tungsten Lamp


147

Chemical Structure & UV Absorption

Chromophoric Group ---- The groupings of the molecules which contain the electronic system which is giving rise to absorption in the ultra-violet region.


148

UV Spectrometer Application

Protein

Amino Acids (aromatic)

Pantothenic Acid

Glucose Determination

Enzyme Activity (Hexokinase)


149

Flurometric Application

Thiamin (365 nm, 435 nm)

Riboflavin

Vitamin A

Vitamin C


150

Visible Spectrometer Application

Niacin

Pyridoxine

Vitamin B12

Metal Determination (Fe)

Fat-quality Determination (TBA)

Enzyme Activity (glucose oxidase)


Major Types of Light Spectroscopy

Absorption spectroscopy Measures amount of light absorbed Most common, non-destructive Concentration, pH measures, purity, ID

Atomic emission spectroscopy Measures light emitted from burned sample Elemental analysis

Fluorescence spectroscopy Samples fluoresce when they emit at higher than what they absorb Measures solvent interactions, distances, molecular shape, and motion

Circular Dichroism spectroscopy Absorption of circular polarized light Chiral compound identification

Transmission spect. (colorimetry)


Introduction

Atomic absorption is the absorption of light by free atoms. An atomic absorption spectrophotometer is an instrument that uses this principle to analyze the concentration of metals in solution. The substances in a solution are suctioned into an excited phase where they undergo vaporization, and are broken down into small fragmented atoms by discharge, flame or plasma.


Atomic Emission Spectroscopy

By exposing these atoms to such temperatures they are able to “jump” to high energy levels and in return, emit light. The versatility of atomic absorption an analytical technique (Instrumental technique) has led to the development of commercial instruments. In all, a total of 68 metals can be analyzed.


Advantages of AA

Determination of 68 metals Ability to make ppb determinations on major components of a

sample Precision of measurements by flame are better than 1% rsd.

There are few other instrumental methods that offer this precision so easily.

AA analysis is subject to little interference. Most interference that occurs have been well studied and

documented. Sample preparation is simple (often involving only dissolution in

an acid) Instrument easy to tune and operate


Atomic emission spectrometer


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