INSTRUMENTAL ANALYSIS

CLS 332

Course Contents:• Chapter 1 (3 Weeks):

• Spectrophotometry• Photometry• Basic Characteristics of Light• Beer’s Law• Design & Application of a UV-Visible Spectrophotometer

• Chapter 2 (2 Weeks):• Flame Photometry• Principles• Components of a Flame Photometer• Applications

• Chapter 3 (2 Weeks):• Fluorometry• Principles of Fluorescence• Factors Affecting Fluorescence• Components of a Fluorometer• Applications

• Chapter 4 (3 Weeks):• Chromatography• Principles• Thin-Layer Chromatography (TLC)• Gel Chromatography• Ion-Exchange Chromatography (IEC)• High-Performance Liquid Chromatography (HPLC)

• Chapter 5 (2 Weeks):• Electrophoresis• Principles and Applications to Biological Fluids

• Chapter 6 (2 Weeks):• Autoanalyzers in the Clinical Chemistry Lab• Principles, Advantages, and Applications

Introduction to Spectrophotometry

-The concept of photometry

-Basic characteristics of light

-Types of electromagnetic rays

-Beer’s Law

-Spectrophotometric techniques

-UV Visible Spectrophotometer

-Components of a Spectrophotometer

-Applications & Assays

Fluorometry

-The concept of Fluorescence

-Components of a Fluorometer

-Fluorometry v/s Spectrophotometry

-Fluorometric assays

-Applications

Flame Photometry

-Principles of Flame Photometry

-Components of a Flame Photometer

-Flame Photometric assays

-Applications

Light Scattering Phenomenon

-Turbidity and Nephelometry

-Nephelometer v/s Spectrophotometer

Electrophoretic Techniques

-Introduction & Principles

-Buffers, support materials

-Endosmosis

-Factors affecting the migration rate

-Clinical Applications of electrophoresis

Chromatographic Techniques

-General Principles

-Physical basis of separation

-Adsorption phenomenon

-Gel filtration

-Ion-Exchange chromatography

-Thin-Layer Chromatography

-High-Performance Liquid Chromatography (HPLC)

-Gas Chromatography (GS)

Automated Analytical Procedures

-Basic Concepts, operation & troubleshooting

-Continuous Flow Analysis

-Discrete Analysers

-Single & Multichannel instruments

-Centrifugal Analysers

Chapter 1: Spectrophotometry

•Photometry: Photometry means the measurement of light. In the lab, light intensity is usually measured after the passage of light of a specific wavelength through a colored solution. The light absorbed by the solution is limited to a narrow portion of the spectrum.

• Principles:

• When light of a specific wavelength strikes a solution, part of the light is absorbed by the solute particles of the solution. The rest of the light is transmitted through the solution and can be quantitated when it falls on a photodetector film.

• The amount of light absorbed by the particles in the solution is proportional to the concentration of the particles.

• Characteristics of Light:• Light is a form of electromagnetic radiation that travels in

waves. The wavelength is a function of the energy of the ray.

• High energy gamma rays associated with nuclear energy have many short wavelengths in order of 0.1nm. At the other end of the scale we have low energy radiowaves where λ = 25cm or more. Visible light seen by the human eye constitutes a small portion of electromagnetic radiation and its λ is limited in the range 380-750 nm. A good spectrophotometer measures light from 180 or 200 nm in the UV region to app. 1000 nm in the near infrared region. (see table).

• Wavelengths of various types of radiation:

Type of Radiation Approximate λ

ϒ rays < 0.1 nm

X rays 0.1-10 nm

UV rays < 380 nm

Visible rays 380-750 nm

Infrared rays > 750 nm

Radiowaves Over 25 cm

Visible Light:• Visible light that appears white is a mixture of different

colors. Each color is bond has a specific range of λ. The color components of visible light (known as the spectrum) can be illustrated by allowing visible light to go through a prism:

• Thus, the color of light is afunction of its wavelength. As the wavelength changes withinthe visible range, an alteration incolor is detected.

• Objects that appear to the eyecolored absorb light of a specificwavelength and reflect other parts of the visible spectrum thatappear to the eye as a certainshade of color. (see table)

Approximate λColor of Light Absorbed

Color of Light Detected by the Eye

400-435 nm Violet Green-Yellow

435-500 nm Blue Yellow

500-570 nm Green Red

570-600 nm Yellow Blue

600-630 nm Orange Green-Blue

630-725 nm Red Green

Principles of Spectrophotometry• Spectrophotometry takes advantage of the property of

colored solution to absorb light of a specific λ. Example: To measure the solute concentration of a blue solution, light of about 590 nm λ is passed through it. The amount of yellow light absorbed by the solute particles of the solution varies directly with the concentration of the blue particles in the solution.

• Beer’s Law: When light of an appropriate λ stikes a cuvette containing a colored sample, some of the light is absorbed by the particles in the solution. The rest of the light is transmitted through the sample to a photodetector film. The proportion of the light that reaches the detector is known as percent transmittance (%T) and is mathematically represented by:

• %T = It/I0 x 100 where

I0 = Intensity of light striking the sample

It = Intensity of light transmitted through the sample

• As the concentration of the solute particles in the colored solution is increased, It and consequently %T is decreased. This is because the intensity of light absorbed is increased.

• The relationship between concentration and %T however is not linear. A graph of %T against concentration of the solution appears as shown below:

• However, if log %T is plotted against concentration, a straight line is observed.

• Log %T is equivalent to absorbance and this explains the linear relationship between absorbance and concentration.

Application of Beer’s Law:• In spectrophotometric assays procedures, the absorbance

of an unknown concentration of a particular constituent is compared to that of a known concentration (standard). The standard is reacted in the same way as the unknown to produce a colored solution.

• We will give a specific example to illustrate the design of a spectrophotometric assay. This is the assay of amylase in serum.

Assay Requested: Spectrophotometric Assay of the Activity of the Enzyme Amylase in a Serum Sample.• To construct the assay we need to know the nature of the

chemistry of the enzymatically catalyzed reaction• Starch Maltose

• To quantitate amylase activity in serum we need to incubate an aliquot of serum (source of amylase) with the substrate starch and quantitate the amount of maltose formed/unit time.

• One unit of enzyme activity is defined as the amount of enzyme required to convert 1 mole of substrate into product per minute at optimal conditions of temperature and pH.

• Thus, one measures enzyme activity and not concentration. This is because the enzyme concentration is very low and there is no sensitive methodology that can measure such low concentrations.

• There are 2 phases of the amylase assay in serum:

1. Stage 1: Incubation of the Serum Sample with the Substrate of the Enzyme

• The incubation tube will contain the following ingredients:• The reaction is started by addition of the serum sample• The incubation: at 37oC for 15 minutes. This is sufficient time to allow all

of the starch to be hydrolyzed into maltose. At the end of the 5 minutes, 2 drops of TCA are added to stop the reaction.

2. Stage 2: Formation of the Color Complex• At the end of the 15 minute incubation period, the tube is taken out

of the water bath and a color reagent is (3 ml, dinitrosalicylate) is added. The tube is now incubated at 100oC for 5 minutes. This will allow a maltose-DNS complex deep orange in color to form.

• Maltose + DNS Maltose-DNS Complex

• The aim of the assay is to measure the amount of maltose that has been formed as a result of the catalytic activity of amylase on starch over the 15 minute incubation period.

Stage 1: Incubation of the Serum Sample with the Substrate of the Enzyme

• To do that, the maltose (colorless) is converted to a colored complex. The intensity of the color (absorbance) at a specific wavelength (540 nm) will be proportional to the concentration of maltose in the assay mixture.

• As the color develops, the tube is taken out of the water bath, and cooled, and the absorbance of an aliquot of it is measured at 540 nm against a DNS blank.

• This absorbance is recorded and needs to be translated into a maltose concentration.

• To do this, a standard calibration curve for maltose must be constructed.

Construction of a Standard Calibration Curve for Maltose• Increasing volumes of a stock maltose solution (1 mg/ml)

are aliquoted into a series of tubes as shown below:

Tube Volume of Starch Sol. (ml)

Maltose (mg) Water (ml) DNS

1 0.2 0.2 0.8 3 ml

2 0.4 0.4 0.6 3 ml

3 0.6 0.6 0.4 3 ml

4 0.8 0.8 0.2 3 ml

5 1 1 – 3 ml

Blank 0 0 1 3 ml

• The tubes are now placed in a water bath for 5 minutes at 100oC to allow color development.

• Aliquots of each tube are used to measure absorbance at 540 nm against the reagent blank.

• The data is then used to plot a standard calibration curve of absorbance against mg of maltose:

•

• Calculations: The standard calibration curve is used to translate the absorbance value obtained for the sample into mg of maltose. As shown on the curve and assuming that the test sample gave a 0.5 absorbance value then this will translate into 0.48 mg of maltose. Thus:

0.48 mg maltose are formed/min

= 0.48/15 mg/min

= 0.48/(15x342) mmoles/min

= (0.48x1000)/(15x342) umoles/min = units of enzyme

More specifically this is equal to:

= 0.093 umoles/min/20 ul sample

= 0.093 x 50 x 1000 umoles/min/l

= 4650 U/l

Spectrophotometric Determination of Glucose

• Laboratory measurement of glucose is based mainly on the fact that G is an excellent reducing agent and can easily be oxidized. Therefore, most of the available methods are based on the oxidation of G and subsequent quantitation of the products formed.

• Glucose in a serum sample can be oxidized in the presence of glucose oxidase:

• G + O2 + H2O Gluconic acid + H2O2

• The hydrogen peroxide formed as a result of the reaction is used in turn to oxidize o-Dianisidine in the presence of the enzyme peroxidase

• 2H2O2 + o-dianisidine oxidized dianisidine (Brown)

• The intensity of the brown color complex is now measured using a spectrophotometer at 450 nm. The intensity of the color (abs.) is directly proportional to the G concentration in the sample.

• Reagents: Can be prepared in the lab:1. Glucose oxidase & peroxidase solution, consisting of 500 U of

glucose oxidase and 100 U of peroxidase dissolved in 100 ml of distilled water

2. o-Dianisidine hydrochloride solution consisting of 50 mg o-dianisidine hydrochloride dissolved in 20 ml H2O

3. Glucose standard: 100 mg/dl

• We can prepare a working solution as follows:• 100 ml of glucose oxidase and peroxide• 1.6 ml of o-dianisidine hydrochloride in a light-protected bottle

This solution is prepared fresh and used immediately

• Procedure of assay: use 5 tubes labeled as blank, standard, and patient• To blank add 25 ul water• To std add 25 ul G std. sol.• To patient add 25 ul patient serum• Add 5 ml G working solution to each tube• Mix & incubate all tubes for 45 mins at RT or 30 mins at 37oC

• Avoid direct light by covering tubes• Read absorbance at 450nm against the blank within 30 mins

• Calculations:

Glucose mg/dl = [abs (pt)/abs (std)] x std value

Components of a Spectrophotometer• The spectrophotometer is regarded as the workhorse of

the clinical chemistry lab

• The components of many spectrophotometers are basically the same. They consist of a power supply that provides current at the proper voltage and the components are as follows:1. A lamp as a bright source of light

2. A monochromator (diffraction grating as a prism) that provides and isolates the desired wavelength

3. A sample holder or cuvette

4. A photodetector film which produces a current in response to light falling on it

5. A readout system (digital or meter)

Light Source: This is usually a tungsten lamp to provide light in the visible range (350-750nm) or a deuterium or hydrogen lamp to provide light in the UV range (below 350nm; 200-350nm)

Cuvettes: Capacity 3-4 ml. Smaller volume cuvettes are available. Glass cuvettes are used for reading absorbance in the visible range. Quartz or fused silica are used for reading absorbance in the UV range.

• Convenient Accessories of Spectrophotometers

a) Flush out cuvettes (each sample can be automatically introduced and flushed out of the cuvette after it has been read)

b) The cuvette holder is thermostated to provide a certain temperature. This is done either by water facketing or heating coils.

c) Monitoring of the progress of a spectrophotometric assay with time. This is known as continuous or kinetic monitoring. It is possible to show a linear increase in the absorbance of a colored solution with time by attaching a recorder to the spectrophotometer.

• NAD-Linked Enzyme Assays

The rate of formation of NAD(P)H is equivalent to the rate of formation of B (oxoglutarate)

The amount of NADH formed is monitored spectrophotometrically by measuring the increase in the absorbance of an aliquot of the reaction mixture at 280nm (UV range)

Flame Photometry (Flame Emission Spectrophotometry)

• The invention of the flame photometry technique revolutionized the determination of serum Na+ and K+ concentrations, which prior to that took 2-3 days to obtain results.

• Principle:• The aspiration into a flame of a sample (such as serum)

containing Na+ and K+ causes evaporation of the water and allows the salt molecules to be dissociated by heat to an atomic vapor. A small percentage of the Na+ and K+ atoms is transformed to an excited state by the absorption of discrete packets of energy.

• This energy causes displacement of orbital electrons to higher energy levels. The excitation of the atoms is however temporary. The atoms immediately return to the ground state which they were in.

• During this return process, there will be release of the absorbed energy in the form of light. The emitted light is of a wavelength specific for each element, and its intensity is can be quantitated under carefully controlled conditions.

• Components of a Flame Photometer:

The last three components of a flame photometer are similar to those of a spectrophotometer. The flame however takes place of the light source and sample compartment

Analysis of Samples

a) Dilute the sample (serum or urine) into a solution of a non-ionic detergent. The detergent reduces the viscosity of the sample solution and allows better aspiration.

b) The diluted sample is aspirated into a premix chamber by passing a stream of air at high velocity over the open end of a capillary tube that dips into the sample solution.

c) The heat of the flame evaporates the water and vaporises the atoms into an atomic vapor. This allows the electrons to jump to higher energy orbitals and thus the atoms become excited.

d) The light emitted by the excited atoms as they return to their original ground state is measured at a wavelength specific for that element.

-Glass cuvettes are used for excitation at wavelengths > 350nm. Otherwise, quartz or silica gel is required.

Factors Affecting Fluorescence:

1. pH: Changes in pH may induce changes in the ionic state of a molecule which result in changes in fluorescing properties.

2. Temperature: Increased temperature enhances molecular motion, increases molecular collisions, and thus decreases fluorescence.

3. Length of time of light exposure: Because molecules are excited more quickly than they fluoresce, there will be an increase in the number of excited molecules present with time and consequently an increase in fluorescence. Hence, short bursts of light exposure are used.

4. Concentration: Fluorescence increases with concentration only in dilute solutions. In concentrated solutions, the light emitted may be absorbed by other molecules of the same compound as it passes through the solution in the cuvette. Thus, a significant decrease in fluorescence results (This phenomenon is known as Quenching).

• Clinical Applications: Fluorometry is used when great sensitivity is required. This is for assay of:

1. Drugs

2. Hormones

3. Vitamins

4. Amino Acids

Fluorometry• Principles: Some molecules have the ability to fluoresce,

or emit light, after exposure to light of a specific wavelength. Light is emitted within a brief time (< 10-8 sec), and is of lower energy (longer wavelength) than the light absorbed.

• The intensity of fluorescence varies directly with the concentration of solute particles in the solution.

• The sensitivity of fluorometric assays is about a thousand times higher than that of spectrophotometric assays.

• Since light of specific wavelength is required for excitation of a given molecule, and the emitted (or fluorescent) light is also of a wavelength characteristic of that molecule, then fluorometric analysis is more specific than spectrophotometric analysis

• The process of fluorescence is illustrated in the figure (next slide).

• A molecule at the ground state energy level is excited by light absorption to a higher energy level (E2). Vibrational energy losses (such as collision & heat loss) drop the molecule to a lower energy level. However this energy level (E1), is still higher than the ground state, and the molecule is still in an excited form. No light emission accompanies this drop. But as the molecule quickly drops and returns to its more stable ground state energy level, light is emitted. This is known as fluorescence.

• The number of molecules that have the ability to fluoresce is limited. These are usually cyclic molecules with conjugated double bonds (-C=C-C=C-).

-Some molecules that cannot fluoresce by themselves may be chemically converted to fluorescent derivatives. This is usually accomplished by adding side chains. Eg: addition of an amino (NH2) group enhances fluorescence.

Components of a Fluorometer• This differs from a spectrophotometer by having two

monochromators. One regulates the wavelength striking the sample (excitation wavelength), and the other isolates the wavelength emitted from the sample (emission wavelength). Thus, EλEλ.

• The exciter lamp is usually either a mercury arc discharge lamp or a xenon arc tube for providing light in the UV range. If no excitation at wavelengths less than 350nm, a tungsten lamp is adequate.

• The monochromators are both diffraction gratings in a fluorescence spectrophotometer, or filters in a simple filter fluorometer.

• The primary filter provides a wavelength for excitation; the secondary filter isolates the emission wavelength.