VARIOUS LABORATORY INSTRUMENTS IN BIOCHEMISTRY

Presented by:Dr.Kusum Bala Jain2nd Yr ResidentDept. of BiochemistryGMC Kota.

CENTRIFUGE

CENTRIFUGECentrifugation is a separation technique by which

particles of different shape,size and density are separated based

upon their sedimentation rate by spinning under the influence of centrifugal force.(extra gravitational force)

The instrument used to hold the sample and generate the centrifugal force is called as centrifuge.centrifuge is made up of rotor.

.A rotor can hold the samle tube and spin along its own axis at different speeds to generate centrifugal force.Speed of rotor spin denoted as rpm.

Urinometer

•A Urinometer is a simple piece of equipment for determining urine specific gravity.

•A typical urinometer is composed of a float, a weight, and a stem. The float is an air-filled glass tube, ending in the weight and the stem.

•The weight is a bulb filled with ball bearings embedded in a red solid, probably a glue of some sort. The glass stem has calibrated graduations and numbers marked off to indicate specific gravity measurements.

•It is placed in a tube of urine, and where the meniscus of the urine reaches displays the specific gravity of the urine.

•Correction for temperature has to be done.

•A urinometer is typically used in medical diagnostic labs

•It is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. 

•The various constituents of the mixture travel at different speeds, causing them to separate. •The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation.

•Chromatography may be preparative or analytical.

•The purpose of preparative chromatography is to

separate the components of a mixture for more

advanced use (and is thus a form of purification).

•Analytical chromatography is done normally with

smaller amounts of material and is for measuring

the relative proportions of analytes in a mixture.

ELECTROPHORESIS

Factors affecting electrophoretic mobilityChargeSizeShape

COLORIMETER

COLORIMETRYCOLORIMETRY

How Do We Use This Principle?How Do We Use This Principle?

Perform a Chemical Reaction with the Element to Perform a Chemical Reaction with the Element to be Analyzed that Results in a colored Compound be Analyzed that Results in a colored Compound

of that Element after Absorbing Light.of that Element after Absorbing Light.

Measure the AmountMeasure the Amountof Light Absorbed.of Light Absorbed.

COLORIMETRYCOLORIMETRY

1.The Chemistry Involved.1.The Chemistry Involved.2. The Length of Light Travel.2. The Length of Light Travel.3. The Amount (Concentration) of 3. The Amount (Concentration) of

Absorbing MaterialAbsorbing Material..

The Amount of Light AbsorbedThe Amount of Light AbsorbedIs Related To:Is Related To:

CONCENTRATION CAN BE COLORIMETRICALLYCONCENTRATION CAN BE COLORIMETRICALLYDETERMINED IF:DETERMINED IF:

1. Able to chemically develop a color with that substance and only that substance

2. The developed color obeys (follows) Beer’s Law over a reasonable range of concentrations

3. The developed color must be stable for reasonable length of time, reproducible, and sensitive to small changes in concentration

4. All loss of transmitted light must be from absorbance by substance measured (developed color)

5. All of substance present in sample must be available for reaction with color developing agent

6. Able to measure amount of light absorbed

•A colorimeter is a device used in colorimetry. In scientific fields the word generally refers to the device that measures the absorbance of particular wavelengths of light by a specific solution.

•This device is most commonly used to determine the concentration of a known solute in a given solution by the application of the Beer-Lambert law, which states that the concentration of a solute is proportional to the absorbance.

1) Wavelength selection, (2) Printer button, (3) Concentration factor adjustment, (4) UV mode selector (Deuterium lamp), (5) Readout, (6) Sample compartment, (7) Zero control (100% T), (8) Sensitivity switch, (9)ON/OFF switch[2]

The essential parts of a colorimeter are:•a light source (often an ordinary low-voltage filament lamp)•an adjustable aperture•a set of colored filters•a cuvette to hold the working solution•a detector (usually a photocell) to measure the transmitted light•a meter to display the output from the detector

Filters•Changeable optics filters are used in the colorimeter to select the wavelength of light which the solute absorbs the most, in order to maximize accuracy. The usual wavelength range is from 400 to 700nanometers (nm).•If it is necessary to operate in the ultraviolet range (below 400 nm) then some modifications to the colorimeter are needed. In modern colorimeters the filament lamp and filters may be replaced by several light-emitting diodes of different colors.

Cuvettes•Main article: Cuvette•In a manual colorimeter the cuvettes are inserted and removed by hand. An automated colorimeter (as used in an AutoAnalyzer) is fitted with a flowcell through which solution flows continuously.

Output• The output from a colorimeter may be

displayed by an analogue or digital meter and may be shown as transmittance (a linear scale from 0-100%) or as absorbance (a logarithmic scale from zero to infinity).

• The useful range of the absorbance scale is from 0-2 but it is desirable to keep within the range 0-1 because, above 1, the results become unreliable due to scattering of light.

• In addition, the output may be sent to a chart recorder, data logger, or computer.

SPECTROPHOTOMETER

PRINCIPLE- Beer Lambert Law is concerned with light absorption in

relation to solution concentration and cell path length.

- It states that the intensity of a ray of monochromatic light decreases exponentially as the concentration of the absorbing medium increases.

- In other words, the more dissolved substance you have in a solution, the more light that will be absorbed, and the less light that will be transmitted through the solution.

Absorbance, Transmittance, and Reflection. A spectrophotometer measures how light interacts with atoms or molecules in a sample.

Parts of a Spectrophotometer

• Lamp• Prism• Sample holder• photomultiplier• Display

How a Spectrophotometer Works

•White light hits grating or prism

•Light is split into colors of the rainbow

•Wavelength knob directs different colors toward sample

How a UV Spectrophotometer Works. Similar to a VIS spectrophotometer, the UV spec shines ultraviolet light or visible light on a sample, and a detector measures the amount of light that passes through, or is absorbed by, the sample.

Colors of Light in the Visible Spectrum. Humans can see light with wavelengths of about 350 to 700 nm.

How Concentration Affects Absorbance. If a sample has twice as many molecules as another, it can absorb twice as much light. This is true at any wavelength. It is important to know a sample’s wavelength of maximum light absorbance, so that the difference in absorbance due to concentration is obvious.

FLUROMETER

•A fluorometer or fluorimeter is a device used to measure parameters of fluorescence, its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light.•These parameters are used to identify the presence and the amount of specific molecules in a medium. •Modern fluorometers are capable of detecting fluorescent molecule concentrations as low as 1 part per trillion.•Fluorescence analysis can be orders of magnitude more sensitive than other techniques. Application:•includechemistry/biochemistry, medicine, environmental monitoring.

Principle Fluorescence is the emission of visible light

by a substance that has absorbed light of a different wavelength. The emitted photon has a longer wavelength and lesser energy.This phenomenon is called as fluorescence.

Quantification and Characterization of fluorescent compounds by measuring intensity of fluorescence using fluorimeter called as fluorimetry.

Uses of flurometerTo quantify amino acids and peptides by

labelling with an extrinsic fluor,acridine orange.

For localization of enzymes in cells and metals in metalloproteins.

For conformation alanalysis of enzymes proteins and nucleic acid.

In cell sorting and cell counting.To detect malignant cells To quantify catecholamine.quinidine,and

porphyrin.

SEMI AUTOANALYZER

Semi AutoanalyserHere the samples and reagents are mixed

and read manually .Based on colorimetry principle.

Disadvantages are:More amount of sample is neededTime consumingNeed technical expertisation

Purpose of Autoanalyzers

An autoanalyzer sequentially measures blood chemistry through

series of steps of

• mixing, • reagent reaction and • colorimetric measurements.

consists of different module including:

•a sampler, pump, mixing coils, optional sample treatments dialysis, distillation, heating, etc,

•a detector, and data generator.

•Most continuous flow analyzers depend on color reactions using a flow through colorimeter

Principle of operation

In Segmented Flow Analyzers (SFA), the sample is mixed with small reproducible volumes of the required reagents air bubbles are introduced into the flow, creating about 20 - 100 segments of liquid for each sample

The sample / reagent mixture flows through mixing coils (heated coils) a color proportional to the amount of analyte in each sample is developed

The samples with developed color flow through a colorimeter to measure the color

It consists of

Sampler: Aspirates samples, standards, wash solutions

into the systemProportioning pump:

Mixes samples with the reagents so that proper chemical color reactions can take place, which are then read by the colorimeter

Dialyzer: The purpose of a dialyzer is to separate the

analyte from interfering substances such as protein, whose large molecules do not go through the dialysis membrane but go to a separate waste stream

The analyte infuses through the diaphragm into a separate flow path going on to further analysis

It consists of

Heating bath: Controls temperature (typically at 37 °C),

as temp is critical in color developmentColorimeter:

Monitors the changes in optical density of the fluid stream flowing through a tubular flow cell. Color intensities proportional to the substance concentrations are converted to equivalent electrical voltages .

Recorder: Displays the output information in a

graphical form.

Block diagram

ELECTROLYTE ANALYZER

•It is a very sensitive in vitro assay technique used to measure concentrations of antigens (for example, hormone levels in the blood) by use of antibodies. As such, it can be seen as the inverse of a radiobinding assay, which quantifies an antibody by use of corresponding antigens.

•Although the RIA technique is extremely sensitive and extremely specific, requiring specialized equipment, it remains among the least expensive methods to perform such measurements. It requires special precautions and licensing, since radioactive substances are used.

Radioimmunoassay (RIA)

Method:

To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine, such as I-125, attached to tyrosine.

This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, these two specifically bind to one another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added.

This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites.

As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen.

The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter

Radioimmunoassay Procedure

Applications of Radioimmunoassays

EndocrinologyInsulin, HCG, VasopressinDetects Endocrine DisordersPhysiology of Endocrine Function

PharmacologyMorphineDetect Drug Abuse or Drug PoisoningStudy Drug Kinetics

Applications of Radioimmunoassays

EpidemiologyHepatitis B

Clinical ImmunologyAntibodies for Inhalant AllergensAllergy Diagnosis

OncologyCarcinoembryonic AntigenEarly Cancer Detection and Diagnosis

CHEMILUMINESCENCEEmission of light with limited emission of heat (luminescence), as the result of a chemical reaction.

[A] + [B] → [◊] → [Products] + light

[A], [B]: reactants[◊]: excited intermediate

For example, if [A] is luminol and [B] is hydrogen peroxide in the presence of a suitable catalyst we have:

luminol + H2O2 →3-APA[◊] →3-APA + light

Where:3-APA is 3-aminophthalate3-APA[◊] is the excited state producing light as it decays to a lower energy level.

CHEMILUMINISCENCE

Luminol and peroxidase before adding H2O2

Chemiluminiscence after addition H2O2

68

What is AAS ?Atomic absorption spectroscopy is a quantitative method of analysis that is applicable to many metals and a few nonmetals.

The technique was introduced in 1955 by Walsh in Australia

IntroductionIntroduction

69

PRINCIPLEPRINCIPLE

A much larger number of the gaseous metal atoms will normally remain in the ground state.

These ground state atoms are capable of absorbing radiant energy of their own specific resonance wavelength.

If light of the resonance wavelength is passed through a flame containing the atoms , then part of the light will be absorbed.

The extent of absorption will be proportional to the number of ground state atoms present in the flame.

70

What is AAS ?

• An atomic absorption spectrophotometer consists of a light source, a sample compartment and a detector.

Light SourceLight Source DetectorDetector

SampleSampleCompartmentCompartment

72

Fuel and oxidant

flame

Air – acetylene

Air- propane

Air- hydrogen

Nitrous oxide – acetylene

Auxiliary oxidant

Fuel

77

InstrumentationInstrumentation

Line source

Monochromator Detector

Read-outNebulize

r

Schematic diagram of a flame spectrophotomer

Atomization

BackgroundThe impact of a stream of high energy

electrons causes the molecule to lose an electron forming a radical cation.A species with a positive charge and one

unpaired electron

+ e-C H

H

HH H

H

H

HC + 2 e-

Molecular ion (M+)

m/z = 16

BackgroundThe impact of the stream of high energy electrons can also break the molecule or the radical cation into fragments.

(not detected by MS)

m/ z = 29

molecular ion (M+) m/ z = 30

+ C

H

H

H

+ H

HH C

H

H

C

H

H

H C

H

H

C

H

H

H C

H

H

+ e-H C

H

H

C

H

H

H

m/z = 15

BackgroundOnly cations are detected.

Radicals are “invisible” in MS.

The amount of deflection observed depends on the mass to charge ratio (m/z).Most cations formed have a charge of +1 so

the amount of deflection observed is usually dependent on the mass of the ion.

Fragmentation PatternsThe impact of the stream of high energy

electrons often breaks the molecule into fragments, commonly a cation and a radical.

Bonds break to give the most stable cation.

Mass Spectrometer can be combined with gas chromatography to analyze mixtures of compounds. GC separates the components of the mixture.Each component is analyzed by the Mass

Spectrometer.

ARTERIAL BLOOD GAS ANALYZER

pH meter

pH meterIt is an electronic device used for measuring

the pH which is the concentration of Hydrogen ions in an aqueous solution.

The pH meters work in liquids though special probes are sometimes also used to measure the pH of semi-solid substances.

Typical pH meter consists of a special measuring probe (a glass electrode) connected to an electronic meter that measures and displays the pH reading.

pH MeterA sample is placed in a cup and the glass

probe at the end of the retractable arm is placed in it.

The probe is connected to the main box.There are two electrodes inside the probe

that measure voltage.One is contained in liquid with fixed pH.The other measures the acidity of the sample

through the amount of H+ ions.

pH MeterA voltmeter in the probe measures the

difference between the voltages of the two electrodes.

The meter then translates the voltage difference into pH and displays it on the screen.

Before taking a pH measurement the meter must be calibrated using a solution of known pH.

Effect of Temperature and BuffersTemperature compensation is contained

within the instrument because pH electrodes are temperature sensitive.

Temperature compensation only corrects for the change in the output of the electrode, not for the change in the actual solution.

Buffers are solutions that have constant pH values and the ability to resist changes in pH.

They are used to calibrate the pH meter.

THERMAL CYCLER

The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR).

 Thermal cyclers may also be used in laboratories to facilitate other temperature-sensitive reactions, including but not limited to restriction enzyme digestion or rapid diagnostics.

 The device has a thermal block with holes where tubes holding the reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps.

HbA1c Analyzer

HPLCChromatography is a physical process whereby

components ( solutes ) of a sample mixture are separated by their differential distribution between stationary & mobile phases .

Planar & column are two basic forms of chromatography .

High performance liquid chromatography is a form of column chromatography .

contd During column chromatography process

mobile phase carries the sample through the column containing stationary phase .

As the mobile phase flows through the stationary phase the solutes may

1) Reside only on stationary phase ( no migration ) ,

2) Reside only in the mobile phase ( migration with mobile phase ) ,

3) Distribute between 2 phases ( differential migration)

The basis of all forms of chromatography is

partition or distribution coefficient ( Kd )

Principle of HPLCThe limit to the length of the column is due the

problem of peak broadening .

The number of theoretical plates is related to the surface area of the stationary phase therefore smaller the particle size of the stationary phase , the better is the resolution.

The Smaller the paritcle size , the greater is the resistance to flow of the mobile phase .

contdThe resistance in flow causes back pressure in

the column that is sufficient to damage the matrix structure of the stationary phase .

The new smaller particle size stationary phases that can withstand high pressures causes dramatic development in the column chromatography .

Instrumentation The increased resolution achieved in

HPLC compared to classical chromatography is primarily the result of adsorbents of very small particle size ( less then 20µm )& large surface areas .

The smallest gel beads used in gel exclusion chromatography are superfine grade with diameters of 20-50µm .

A combination of high pressure & adsorbents of smaller size leads to high resolution power & short analysis time in HPLC .

(1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in "inject position", (6') Switching valve in "load position", (7) Sample injection loop, (8) Pre-column (guard column), (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector.

contdRepeated application of highly impure samples

such as sera , urine , plasma or whole blood are preferably deproteinated because they decrease the resolving power of the column .

To prevent the above problem a guard column is frequently installed between the injector & the analytical column .

HPLC with Mass spectrometerNarrow-bore columns (1-2 mm) are used for in

this application . Liquid chromatography-mass spectrometry

(LC-MS, or alternatively HPLC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry.

HPLC with Mass spectrometerNarrow-bore columns (1-2 mm) are used for in

this application . Liquid chromatography-mass spectrometry

(LC-MS, or alternatively HPLC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry.

Application of HPLCHPLC has had big impact on separation of

oligopeptides & proteins .FPLC a modified version useful in separation of

proteins .HPLC coupled with electrochemical detector is

useful in assay of catecholamines ,vitamins (AD&E ,niacin , thiamine) & antioxidants .

HPLC has role in quantification of various hemoglobins in hemoglobinopathies .

HPLC coupled with MS is useful in measuring cortisol in blood & saliva .

contdHPLC is useful in cytokine measurement .Useful in assay of HbA1c .Useful in assay of fructosamine .5 – hydroxy idole acetic acid & serotonin can be

assayed.The pharmaceutical industry regularly employs

Reverse Phase HPLC to qualify drugs before their release.

Assay of plasma & urinary catecholamines , plasma & urinary metanephrines

contdFor diagnosis of different porphyrias .

Thyroxine , uric acid .

Nucleic acid analysis, oliginucleotides , steroids , amino acids , serotonin , measurement of isoenzymes .