BIOLOGICAL TECHNIQUES (BIO 204)

BIOLOGICAL TECHNIQUES (BIO 204)
COLLEGE OF BASIC AND APPLIED SCIENCES DEPARTMENT OF BIOLOGICAL SCIENCES BIOLOGICAL TECHNIQUES (BIO 204) Ibadin F. H. (Ph.D) Adebami, G.E Rabiu, O. R Ayodele O. O. BIOLOGICAL TECHNIQUES by Ibadin F. H. (Ph.D), Adebami, G.E, Rabiu, O. R, Ayodele O. O. is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

PREPARATION OF MICROSCOPE SLIDES

Introduction The main methods of placing samples onto microscope slides are wet mount, dry mount, smear, squash and staining.

Dry Mount: The dry mount is the most basic technique: simply position a thinly sliced section on the center of the slide and place a cover slip over the sample. Dry mounts are ideal for observing hair, feathers, airborne particles such as pollens and dust as well as dead matter such as insect and aphid legs or antennae. Opaque specimens require very fine slices for adequate illumination. Since they are used for primarily inorganic and dead matter, dry mounts can theoretically last indefinitely.

Wet Mount: Used for aquatic samples, living organisms and natural observations, wet mounts suspend specimens in fluids such as water, brine, glycerin and immersion oil. A wet mount requires a liquid, tweezers, pipette and paper towels.

To prepare the wet mount:
Place a drop of fluid in the centre of the slide Position sample on liquid, using tweezers At an angle, place one side of the cover slip against the slide making contact with outer edge of the liquid drop Lower the cover slowly, avoiding air bubbles Remove excess water with the paper towel

Smear Slides: Smear slides require two or more flat, plain slides, cover slips, pipette and tissue paper: Pipe a liquid sample such as blood or slime onto a slide Using the edge of the second slide, slowly smear the sample creating a thin, even coating Put a cover slip over the sample, careful not to trap air bubbles Remove excess liquid Ideally, smears should dry naturally in an environment of moderate, steady temperature. The angle of the smearing slide determines the length of the smear; a steeper angle creates a shorter smear. For samples such as blood, begin by backing the smearing slide into the sample and then push across the slide, pulling the blood in the opposite direction to create a smooth layer. A thicker slide can be created with two drops, but only with the blood of mammals as the erythrocytes lack a nucleus allowing cells to be amassed in multiple layers.

Squash Slides: Designed for soft samples, squash slides begin by preparing a wet mount; place lens tissue over the cover glass; gently press down, careful not to destroy the sample or break the cover glass, and squash the sample; remove excess water.

Staining: A variety of methods exist for staining microscope slides, including non-vital or in vitro stains of non-living cells and vital or in vivo stains of living tissue. Staining provides contrast through color that reveals structural details undetected in other slide preparations. Staining solutions such as iodine, methylene blue and crystal violet can be added to wet or dry mounts. A simple staining method: Add a drop of staining solution on the edge of one side of the cover slip Position the edge of a paper towel on the opposite end Allow dye to be pulled across the specimen Stains are especially useful in the fields of histology, virology and pathology, allowing researchers to study and diagnose diseases, identify gram positive and negative bacteria as well as examine detailed attributes of a variety of cells.

BIOLOGICAL DRAWINGS

BIOLOGICAL DRAWINGS A good biological drawings are important in that they help you become more familiar with the subject through careful attention to the smallest detail. Drawings allow you to improve your observational skills which is the essence of good science. Good drawings are those that simplify, emphasize, summarize, and clarify all at once. For these reasons, attention to the smallest detail is very important. Biological drawings are not meant to be artistic masterpieces, but are more like graphic notes that help record a set of observations. As such, these observations must be completed in class with the specimen directly observable.

Format For A Good Biological drawings
1. Drawings must always be done on blank paper using a sharp pencil and a good eraser where necessary. 2. Drawings must have an underlined title, centred at the top of the page. The title must indicate the cell/tissue/organ type, type of the section being viewed, and the stain used. 3. The date of the assignment and your name must appear in the upper right corner of the page.

4. The drawing itself must be:
Large; Drawn just left of centre on the page; Proportional such that all components of the drawing are proportional to the specimen itself; Stippled to show contrast and detail. Shading must never be used; Drawn with a sharp pencil. Sketch lines are not permitted. There should not be any open circles. All lines have a distinct beginning and end; and Very detailed and should not contain any artefacts.

5. The labels must: Be lined up and found on the right side of the drawing only; Be printed with the first letter capitalized. Labels are pluralized, where appropriate; and Have lines that point precisely to the structure being labelled. Label lines are drawn with a ruler, do not cross, and do not end in an arrow.

6. At the bottom right corner of the drawing is where you will indicate the appropriate dimensions and relative sizes. For drawings of observations made through a microscope, you must indicate: i. Total Magnification ii. Diameter of Field iii. Estimated Size of Specimen (length and width) For all other drawings, you must indicate the size of your drawing relative to the size of the actual specimen. Example: i. Standard x 2 (drawing is twice as big as the specimen) ii. Standard x 1 (drawing is the same size as the specimen) iii. Standard x 0.5 (drawing is half as big as the specimen)

7. At the bottom of your drawing, you should indicate additional observations about things like:
Behaviours Colours Questionable features (i.e. artefacts), etc.

MICROTOMY

WHAT IS MICROTOME? The term Microtome was derived from the Greek mikros, meaning “small”, and temnein, meaning “to cut”) Microtome: Basic instrument used in microtomy. Mechanical device for cutting thin uniform slices of tissue – sections. Microtomy : is the means by which tissue can be sectioned and attached to a surface for further microscopic examination.

History The earliest form of microtomy was the freehand sectioning of fresh or fixed material using a sharp razor. The section produced, could, with practice, be quite thin and translucent.

Santiago Ramón y Cajal managing the microtome, 1884-1887

Minot’s rotating microtome
Charles Sedgwick Minot (1852–1914)

Types of microtomes Based on the mechanism: Rocking Rotary Rocking
Base-sledge Sliding Freezing Vibrating Saw Cryostat Ultra Laser

Rocking microtome:

Name derived from the rocking action of the cross arm.
Oldest in design, cheap , simple to use. Extremely reliable. Very minimum maintenance.

Mechanism of action: Knife is fixed, the block of the tissue moves through an arc to strike the knife. Between strokes the block is moved towards the knife for the required thickness of sections by means of a ratchet operated micrometer thread. Steady backward and forward movement of the handle gives ribbons of good sections.

Disadvantage: Size of the block that can be cut is limited.
Sections are cut in a curved plane: Microtomes designed to cut perfectly flat sections; the block moving through an arc at right angles to the knife edge are available. Light instrument : advisable to fit it into a tray which is screwed to the bench, or to place it on a damp cloth to avoid movement during cutting.

Rotary microtome First machine designed by Professor Minot, hence often referred to as the “Minot Rotary”.

Mechanism: The hand wheel rotates through 360 degree moving the specimen vertically past the cutting surface and returning it to the starting position. Block holder is mounted on a steel carriage which moves up and down in grooves and is advanced by a micrometer screw- cutting perfectly flat sections.

Manual (completely manipulated by the operator).
Semi-automated (one motor to advance either the fine or coarse hand – wheel) Fully automated ( two motors that drive both the fine and the coarse advance hand-wheel) Mechanism of block advancement: retracting or non retracting. Retracting action moves the tissue block away from the knife on upstroke, producing a flat face to the tissue block.

Advantages: Ability to cut thin 2-3 mm sections. Easy adaptation to all types of tissues ( hard, fragile, or fatty) sectioning. Ideal for cutting serial sections: large number of sections from each block. Cutting large blocks Cutting angle of knife is adjustable. Large and heavier knife used-less vibration when cutting hard tissue. Heavier and more stable .

Sledge microtome

Originally designed for cutting sections of very large blocks of tissue (eg. whole brains)
Used primarily for: Large blocks,hard tissues,whole mounts. Especially useful in neuropathology and ophthalmic pathology.

Mechanism of action: The block holder is mounted on a steel carriage which slides backwards and forwards on guides against a fixed horizontal knife.

Advantages: Heavy , very stable, not subject to vibration. Knife large(24 cm in length) and usually wedge shaped –less vibration . Adjustable knife holding clamps allow tilt and angle of the knife to the block to be easily set – used for cutting celloidin sections by setting the knife obliquely paraffin wax embedded sections are more easily cut .

Disadvantages Slower in use than rocker or rotary microtome- true only when change from one instrument to another is made . With practice, sections from routine paraffin blocks can be cut as quickly as on any other type of microtome.

Sliding microtome Designed for cutting celloidin-embedded tissue blocks. The knife or blade is stationary, specimen slides under it during sectioning. Also used for paraffin –wax embedded sections.

Freezing microtome Gives best results for cutting frozen sections.
Machine is clamped to the edge of a bench and connected to a cylinder of CO2 by means of a specially strengthened flexible metal tube.

Freezing microtome Knife freezing attachment is supplied with most machines. Separately controlled flow of CO2 on the edge of the knife - to delay the thawing of sections on the knife and make it possible to transfer them directly from knife to slides. Sections thickness gauge is graduated in units of 5 micrometer instead of 1micrometer.

Vibrating microtome Designed to cut tissue which has not been fixed, processed or frozen. Used in enzyme histochemistry, ultrastructural, histochemistry. During sectioning, the tissue is immersed in either water ,saline or fixative. It is cut by a vibrating razor blade , at thickness generally greater than used for paraffin wax. Tissues are cut at a very slow speed to avoid disintegration.

Microtome knives Developed to fit specific types of microtomes and cope with different degrees of hardness of tissues and embedding media. Paraffin-wax embedded tissues knives are made of steel. Resin-embedded tissue is normally cut using glass knives.

Knives are classified according to their shape when viewed in profile as:
Wedge. Planoconcave. Biconcave. Tool edge or D profile.

Wedge : Originally designed for cutting frozen sections
Gives great rigidity to the knife Used for cutting all types of section on any microtome. Plano-concave: Used primarily for cutting nitrocellulose –embedded tissues. Available with varying degrees of concavity.

Biconcave : Classical knife shape introduced by Heiffor. Used with the rocking microtome. Relatively easy to sharpen. Less rigid , prone to more vibrations. With gradual adoption of more substantial microtomes , this knife design has lost popularity. Tool edge(D-profile): Called ‘chisel edge’, similar to a woodworker’s chisel. Used primarily to section exceptionally hard tissue. Decalcified dense cortical bone. Undecalcified bone. Stouter than conventional knives to give added rigidity. Edge may be coated with tungsten-carbide for increased life.

Disposable blades Used for routine microtomy and cryotomy.
Provide a sharp cutting edge, produce flawless 2-4 mm sections. Disposable blade holders incorporated into the microtome or an adapter. Blade is coated with PTFE (polytetrafluoroethylene) allowing ribbons to be sectioned with ease. Over-tightening the disposable blade in the clamping device may cause cutting artifact such as thick and thin sections.

Glass and diamond knives
Used in electron microscopy and with plastic resin- embedded blocks.

Knife angles Clearance angle: angle formed by a line drawn along the block surface and the lower bevel of the knife. Rake angle: angle between the upper bevel of the knife and a line at 90 degrees to the block surface. Angles associated with the knife edge. A:rake angle; b:bevel ; c:clearance angle.

Sharpness or acuity:reflection of light by the knife edge when viewed under the microscope.
Figures of 0.3and 0.1 micrometer – necessary for maximal acuity or sharpness. Bevel angle: angle of facets which meet to form the edge. Vary between 15 and 35 degrees. Longer facets and smaller bevel would give rise to a keener edge.

Microtome knife sharpening
Manual procedure or automatic procedure. 1) Abrasive grinding of the facets [HONING] 2)Polishing [STROPPING] Abrasive grinding of the facets [HONING] Naturally occuring slabs of stone with varying abrasive properties: Stones : belgian black vein and arkansas, Aloxite and carborundum-composites. Lubricated with soapy water or light oil during use.

Abrasive grinding of the facets [HONING]
Glass plates: hand sharpening Readily available ,cheap Surface roughened to enable particles of abrasive to adhere to the glass . Easily cleaned after use. Copper and bronze plates: automatic knife sharpening machines. Expensive , superior properties

Abrasives Aluminium oxide(alumina) Iron oxide(Jeweller’s rouge)
Silicon carbide Diamond

Manual method Knife with a ‘back’ effectively raises the non cutting edge up off the hone. Back of the knife is ground simultaneously with the edge , hence reserved for use only with that particular knife.

Manual method Hone is placed on the bench on a non-skid surface (damp cloth) to prevent moving during honing. Small quantity of light oil or soapy water applied to the hone and smeared over the surface. Abrasive is applied to the glass or metal plate. Knife with handle and backing sheath is laid on the hone with cutting edge facing away from the operator , heel roughly in the centre of the nearest end of the hone.

Manual method Handle of the knife is held between the thumb and the forefinger . Thumb and forefinger of other hand rest on the other end of the knife to ensure even pressure along the whole edge of the knife. Knife is pushed forward diagonally from heel to toe ,turned over on its back and moved across the hone until the heel is in the centre with the cutting edge leading and then brought back diagonally. It is then turned over on its back and moved across the hone to its original position completing figure of eight movement.

Stropping Process of polishing an already fairly sharp edge.
Types of strop: best strops made from hide from the rump of the horse marked ‘shell horse’. 2 types: flexible(hanging) and rigid. Flexible type: Back of the strop is made of canvas and is intended to support the leather during stropping. Strops should be kept soft by applying a small quantity of vegetable oil into the back of the leather

Stropping Strops should be kept free from grit and dust. Rigid type:
Single leather strop stretched over a wooden frame to give a standard tension or a block of wood about 12x2x2 inches in size having a handle at one end with four grades of leather or even a soft stone cemented on each side. The sides of these strops are numbered and the knife is stropped on No1, then No2 and so on finishing on the finest leather.

Automatic knife sharpners

Automatic knife sharpeners
Two basic designs available. 1) knife is held vertically with revolving sharpening wheels grinding the cutting edge. 2) knife is held horizontally against the surface of a slowly rotating flat plate. Plates – glass , copper or bronze charged with an abrasive. Glass plates need to be roughened before use to allow the abrasive particles to be held more easily in place. Copper and bronze plates used in conjunction with diamond paste, 6micrometer particle size being most appropriate for rough sharpening, and 1 micrometer for fine polishing.

Stropping Technique: Knife is laid on the near end of the strop with the cutting edge towards the operator (opposite direction to that used in honing.) Knife held with forefinger and thumb to facilitate easy rotation at end of each stroke. Action is exact opposite to that used in honing,using full length of the strop and stropping evenly the whole of the blade.

Microtomy- paraffin wax
Factors involved in producing good paraffin-wax sections : Temperature: Tissues are more easily sectioned at a lower temperature than that of the atmosphere. Lowering temperature brings tissues of differing composition to a more uniform consistency,degree of hardness-ensures a uniform cutting process. Blocks are cooled by keeping , face down on ice-tray (2-3min).

Knife angle Greater the rake angle(flatter the knife)more likely is a smooth plastic flow type cutting action. Higher rake angles are more suitable to softer tissues Lower rake angles for harder tissues.

Speed of cutting Slant Soft tissues are cut more easily at a slow speed. Hard tissues are cut easily at a little fast rate. If sections are cut at too fast speed, compression will become more marked. If cut too slowly, difficult to maintain the rhythmic action required. Commonly used to refer to the relationship of the knife edge to the block when cutting nitrocellulose- embedded tissue on a sliding microtome. Advantages: larger area of the edge is employed. Resistance to cutting force is applied more gently.

Paraffin section cutting
Equipment required: Microtome. Flotation(water bath) Slide drying oven or hot plate Fine pointed or curved forceps. Sable or camel haired brush. Scalpel. Slide rack. Clean slides. Teasing needle. Ice tray. Chemical resistant pencil or pen.

Cutting technique Insert apprropriate knife in the knife-holder of the microtome and screw it tightly in position. Correctly set the adjustable knife angles. Fix the block in the block holder of the microtome Move the block holder forward or upward until the paraffin wax is almost touching the knife edge. Ensure that the whole surface of the block will move parallel to the edge of the knife, Trim the excess wax from the block surface and expose the tissue,advance the block by setting the thickness to about 15 micrometer. Care should be taken not to trim too coarsely as A)Small biopsies may be lost. B) tissue in the block may be torn giving rise to considerable artefact.

C) unsuspected small foci of calcification may cause tears in the tissue and nicks in the knife.
Once the surface of tissue has been revealed proceed to trim the next block. Replace the trimming edge by a sharp one and check it is tightly secured. Reset the thickness gauge to 4-5 micrometer. Insert the block to be cut and tighten securely. Bring the block face up until it nearly touches the knife edge.

Cutting difficult tissue
Alternate sections being thick and thin. Only part of the tissue being cut. Sections extremely compressed. Divided into two groups: 1) tissue exceptionally hard and tough. 2) fragmentation of tissue occurs as it is cut.

Cutting dificult tissue
Hard tissues Decreasing the rake angle. Resharpening the knife. Softening agents: solution of 4 % phenol,Mollifex(British drug houses Ltd) soak the block for minutes Fragmentation of tissue: Blood clots and lymphoid tissues : increasing the rake angle. coating the block with celloidin by a camel hair brush in between the sections.

Serial sections Necessary to cut and preserve every section from a piece of tissue or from a specific area . Required: To identify small ulcer Presence of malignant cells tracking along a lymphatic or neural sheath. Scarce organisms such as acid fast bacilli. In embryology.

Problems and solutions for paraffin section.
Trim block until parallel. Replace balde. Trim away excess paraffin. Re-orient block Problem: Ribbon/consecutive sections curved. Block edges not parallel Dull blade edge. Excessive paraffin. Tissue varying in consistency

Solutions Problem: Thick and thin sections
Remove excess paraffin Increase clearance angle. Check for faults in microtome. Tighten block and blade Problem: Thick and thin sections Paraffin too soft for tissue Insufficient clearance angle Faulty microtome mechanisms Blade or block loose in holder.

Solutions Re-embed in lower melting point paraffin. Warm surface of block Clean blade and back of blade holder Adjust to optimal angle. Problem: Sections will not form ribbons Paraffin too hard for sectioning. Debris on knife edge. Clearance angle incorrect.

Problem: sections attach to block on return stroke
Insufficient clearance angle. Debris on blade edge. Debris on block edge. Static electricity on ribbon. Solutions Increase clearance angle. Clean blade edge. Trim edges of block Humidify the air around the microtome. Place static guard or dryer sheets near microtome.

Problem: incomplete section
Incomplete impregnation of tissue with paraffin. Tissue incorrectly embedded. Sections superficially cut. Solutions Re-process tissue block. Re-embed tissue;make sure orientation is correct and tissue is flat in mould. Re-face block,cut deeper into the tissue.

Problem: chatter-thick and thin zones parallel to blade edge
Knife or block loose in holder Excessive knife tilt Paraffin too hard for sectioning. Calcified areas in tissue. Overhydration of tissue. Dull blade. Solutions Clean blade edge to remove excess paraffin. Replace or use new area of blade. Tighten the blade levers. Reduce angle. Rehydrate . Re-embed in fresh paraffin.

Problem: Solutions Splitting of sections at right angles to knife edge
Nicks in blade. Hard particles in tissue. Hard particles in paraffin. Solutions Use different part of blade or replace. Calcium deposit-surface decal. Mineral or other particle- remove with fine sharp pointed forceps

References Cellular pathology technique – C.F.A.Culling.
Histological techniques- J.D.Bancroft.

CYTOLOGICAL TECHNIQUES

Cytology Cytology is the study of cells, their origin, structure, function, and pathology. Microscopic examination of cells Very valuable diagnostic tool, inexpensive, quick and easy and involves little or no risk to the patient.

Cell – Prokaryote and Eukaryote

Cell – Animal and Plant Cells

Specimen collection methods - Imprints

Specimen collection methods - Scrapings

Specimen collection methods - Swabs

Specimen collection methods – Fine Needle Biopsy

Cytology specimen preparation
Squash Preparation Needle / Starfish Preparation Blood Smear Technique

Stains and staining in cytology
Romanowsky type stains (Wright’s, Giemsa, Leishman, Diff- Quik®) - They provide good nuclear detail, excellent cytoplasmic detail and infectious organisms are readily visualised. It may also be used for wet fixed slides, but are primarily applied to air- dried smears. Supravital stains (toluidine blue, Methylene blue) - provide excellent nuclear detail but poor cytoplasmic detail and are typically reserved for evaluation of reticulocyte identification Papanicolaou stains - provide excellent nuclear detail and adequate cytoplasmic detail. It is recommended for the staining of alcohol fixed cytology slides.

Cytology techniques Microscopy Fluorescence microscopy
Phase-contrast microscopy Dark field microscopy Confocal microscopy Transmission electron microscopy Cytometry

Cytology Fluorescent Microscope Phase contrast microscope
Dark field Microscope

Cytology Confocal microscope Transmission Electron Microscope

Cytometry The counting of cells, especially blood cells using a cytometer or haemocytometer. The counting and measuring of cells, especially the counting and analysis of cell size, morphology, and other characteristics, traditionally performed using a standardized glass slide or small glass chamber of known volume.

Haemocytometer

Coulter counter

Flow cytometer

PHOTOMETRY AND COLORIMETRY

OUTLINE Photometry Colorimetry Electromagnetic radiation
Photometric Instruments - Colorimeter - Spectrophotometer

PHOTOMETRY Photometry is the measurement of electromagnetic radiation weighted by the human eye's response. This response changes with wavelength, and to an extent, from person to person. When light is passed through a coloured solution, certain wavelengths are selectively absorbed giving a plot of the absorption spectrum of the compound in solution. The wavelength at which maximum absorption is called the absorption maximum (λmax) of that compound. The light that is not absorbed is transmitted through the solution and gives the solution its colour.

Photometric instruments measure transmittance, which is defined as follows:
Intensity of the emergent (or transmitted) light Ie Transmittance (T)= = Intensity of the incident light Io Transmittance is usually expressed on a range of 0 to 100%. If the concentration of the substance in solution is increased linearly, or if the path length that the light beam has to traverse is increased, transmittance falls exponentially. Thus, absorbance is defined so that it is directly proportional to the concentration of the substance. Absorbance, A =log 1/T=logIo/Ie

Absorbance has no units
Absorbance has no units. Photometric instruments electronically convert the measured transmittance to absorbance values. Photometry is therefore based on two laws: 1. Beer's Law: This states that when a parallel beam of monochromatic light passes through a solution, the absorbance (A) of the solution is directly proportional to concentration(c) of the compound in the solution. 2. Lambert's Law: This law states that the amount of light transmitted decreases with increase in thickness of the layer of colored solution i.e. each successive layer of the solution absorbs a constant proportion of the light entering the solution, although the absolute amount entering each layer diminishes progressively.

Therefore, absorbance is directly proportional to the thickness or length of the light path (l) through the solution. Thus, A α c (Beer's law) ) A α l (Lambert's law) ) By combining 1) and 2) , we get A = εcl ε, the proportionality constant is termed the molar absorption coefficient It is specific for a given substance at a given wavelength. It is the absorbance of a one molar solution of a substance with a light path of one centimetre (if c is expressed in mol/L).

Beer's law applies to only dilute solutions and in practice the concentrations of the solutions that are used in photometry are usually in the mmol/L range. In colorimetry, the absorption coefficient is not usually used. Concentration of an unknown solution can be determined by using equation 1, which is derived as follows: Absorbance of test sample (At) = ε x concentration of test (Ct) x l Absorbance of standard sample (As) = ε x concentration of standard (Cs) x l Hence, Absorbance of Test sample (At) Conc. of Test (Ct) = X Conc. of standard (Cs) Absorbance of Standard sample (As)

The light path, l, is usually kept constant in photometric measurements at 1 cm. This is the diameter of the tube (called the cuvette) containing the solution. The concentration of the compound in the test sample is obtained by comparing its absorbance with that of a known concentration of a standard solution. Ideally, a series of standards of known concentration are prepared to obtain a standard (calibration) curve. This helps to determine the range of concentrations over which Beer's law is obeyed. Appropriate blanks exclude the absorbance contributed by the solvents and reagents used- i.e. by anything other than the compound of interest.

COLORIMETRY Colorimetry is the determination of the concentration of a substance by measurement of relative absorption of light or transmitters with respect to a known concentration of the substance. When a beam of radiant energy falls upon a substance, the energy of the beam is partially altered by reflection, refraction, diffraction or absorption and the remaining energy may be transmitted through the substance. The term colorimetry originates from the times when measurements were done by comparing the color of a component under investigation with a standard color by the eye.

Although this principle is still the basis of modern techniques, the measurement with the eye has been replaced by the measurement of photons with a photon detector. From this the term photometry came about. This technique is covered by a more generic term called spectrometry, which refers to all techniques that are based upon the production or interaction of electromagnetic radiation with matter. The concerned matter may produce or absorb electromagnetic radiation, from which the terms emission and absorption spectrometry originate. Further spectroscopic techniques are characterized according to the type of electromagnetic radiation (spectrum) involved e.g. X-ray, UV, visible.

Nowadays spectrometric methods are the most applied analytical techniques in the world.
Note: Colorimetry uses the basic principles of photometry but the solutions have to be coloured, i.e. they must absorb light in the visible range. Colourless compounds are converted into coloured compounds using chemical reactions. Under defined reaction conditions, the quantity of colour formed is proportional to the quantity of the original colourless compound.

ELECTROMAGNETIC RADIATION
Electromagnetic radiation is a form of energy that is produced by oscillating electric and magnetic disturbance, or by the movement of electrically charged particles traveling through a vacuum or matter. The electric and magnetic fields come at right angles to each other and combined wave moves perpendicular to both magnetic and electric oscillating fields. Electron radiation is released as photons, which are bundles of light energy that travel at the speed of light as quantized harmonic waves. This energy is then grouped into categories based on its wavelength into the electromagnetic spectrum.

These electric and magnetic waves travel perpendicular to each other and have certain characteristics, including amplitude, wavelength, and frequency. General Properties of all electromagnetic radiation: Electromagnetic radiation can travel through empty space. Most other types of waves must travel through some sort of substance. For example, sound waves need either a gas, solid, or liquid to pass through in order to be heard. The speed of light is always a constant. (Speed of light : x 108 m s-1) Wavelengths are measured between the distances of either crests or troughs. It is usually characterized by the Greek symbol λ.

Light as a Wave form The light that we see is just a small region of electromagnetic spectrum, which human eyes are capable of sense. This region is called “visible light”. Due to fusion reaction in the sun; when two hydrogen atoms fused with each other, they form a helium molecule. In this process some mass is removed in the form of energy. This energy is partly electric and partly magnetic, so it is called an electromagnetic radiation. When the visible light is reflected from any object and touches the sensors of our eyes, we can see the objects. In the process of reflection- out of multiple colors of the radiation, some colors are absorbed and some are reflected.

The color which is reflected is the color what we can see.
For example, all the frequencies of white light (all colors) are absorbed by the black colored car, while all the frequencies of white light (all colors) are reflected by white colored car. So, black colored car is hotter than white colored car during summer. Characteristics of Waves An Electromagnetic wave

Amplitude: This is the distance from the maximum vertical displacement of the wave to the middle of the wave. This measures the magnitude of oscillation of a particular wave. Thus, the amplitude is basically the height of the wave. Larger amplitude means higher energy and lower amplitude means lower energy. Amplitude is important because it tells the intensity or brightness of a wave in comparison with other waves. Wavelength: Wavelength (λ) is the distance of one full cycle of the oscillation.

Longer wavelength waves such as radio waves carry low energy; this is why one can listen to the radio without any harmful consequences. Shorter wavelength waves such as x-rays carry higher energy that can be hazardous to the health. Consequently lead aprons are worn to protect the body from harmful radiation when undergoing X-rays. The wavelength frequently relationship is characterized by: c = λν where: c is the speed of light, λ, is wavelength, and ν is frequency.

Shorter wavelength means greater frequency, and greater frequency means higher energy.
Wavelengths are important because they show the type of wave involved. Different wavelengths and frequencies

Frequency: Frequency is defined as the number of cycles per second, and is expressed as sec-1 or Hertz (Hz). Frequency is directly proportional to energy and can be express as: E = hν where • E is energy, • h is Planck's constant, (h= x J), and • ν is frequency. Period: Period (T) is the amount of time a wave takes to travel one wavelength; it is measured in seconds (s).

Velocity: The velocity of wave in general is expressed as: Velocity = λν For Electromagnetic wave, the velocity in vacuum is 2.99×108m/s or 186,282186,282 miles/second. Electromagnetic spectrum

Spectrum of visible light
Visible Light is the only part of the electromagnetic spectrum that humans can see with an unaided eye. This part of the spectrum includes a range of different colors that all represent a particular wavelength. Rainbows are formed in this way; light passes through matter in which it is absorbed or reflected based on its wavelength. Thus, some colors are reflected more than other, leading to the creation of a rainbow.

The color regions of the Visible Spectrum

Fig.: EM spectrum with Radiation types

Exercises: What is the wavelength of a wave with a frequency of 4.28 Hz? What is the frequency of a wave with a wavelength of 200 cm? What is the frequency of a wave with a wavelength of 500 pm? What is the wavelength of a wave with a frequency of × 105 Hz? 5. A radio transmits a frequency of 100 Hz. What is the wavelength of this wave?

PHOTOMETRIC INSTRUMENTS
A. COLORIMETER 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 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. A colorimeter measures the intensity of light transmitted through a coloured solution. It uses light only in the visible range.

Ordinary light from a tungsten lamp is passed through a suitable filter to obtain light of a desired wavelength, which is then passed through the solution. Transmitted light falls on the sensitive surface of selenium photocell which generates a current proportional to the light intensity. The cell is connected to a galvanometer, which is used to read out percentage transmission or absorbance. There are two types of colorimeters: color densitometers: measure the density of primary colors, and color photometer: measure the color reflection and transmission.

Essential parts of a colorimeter:
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 photoresistor) to measure the transmitted light; a meter (analog or digital) to display the output from the detector in terms of transmittance or absorbance.

In addition, there may be:
a voltage regulator, to protect the instrument from fluctuations in mains voltage; a second light path, cuvette and detector. This enables comparison between the working solution and a "blank", consisting of pure solvent, to improve accuracy. Some colorimeters are portable and useful for on-site tests, while others are larger, bench-top instruments useful for laboratory testing. Filters: changeable optics filters are used in the colorimeter to select the wavelength which the solute absorbs the most, in order to maximize accuracy.

The usual wavelength range is from 400 to 700 nanometers (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: 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.

Working Principle: The colorimeter is based on Beer-Lambert's law, according to which the absorption of light transmitted through the medium is directly proportional to the medium concentration. In a colorimeter, a beam of light with a specific wavelength is passed through a solution via a series of lenses, which navigate the colored light to the measuring device. This analyzes the color compared to an existing standard. A microprocessor then calculates the absorbance or percent transmittance. To determine the concentration of an unknown sample, several sample solutions of a known concentration are first prepared and tested.

The concentrations are then plotted on a graph against absorbance, thereby generating a calibration curve. The results of the unknown sample are compared to that of the known sample on the curve to measure the concentration. Applications: Colorimeters are widely used to monitor the growth of a bacterial or yeast culture. They are used to measure and monitor the color in various foods and beverages, including vegetable products and sugar. Certain colorimeters can measure the colors that are used in copy machines, fax machines and printers.

They are used in testing water quality by screening chemicals such as chlorine, fluoride, cyanide, dissolved oxygen, iron, molybdenum, zinc and hydrazine. They are also used to determine the concentrations of plant nutrients such as ammonia, nitrate and phosphorus in soil. They are used in drug analysis to identify substandard and counterfeit drugs. They are also used by the food industry, and by manufacturers of paints and textiles.

Criteria of Colorimetric Analysis:
1. Specificity of the color reaction: Very few reactions are specific for a particular substance, but many give colors for a small group of related substances only, i.e. they are selective. By introducing other complex-forming compounds, by alternating the oxidation states, and by controlling the pH, close approximation to specificity may often be obtained. 2. Proportionality between color and concentration: For visual colorimeters it is important that the color intensity should increase linearly with the concentration of the substance to be determined. That is, it is desirable that the system should follow the Beer's law even when the photoelectric colorimeters are used.

3. Stability of the color: The color produced should be sufficiently stable to permit an accurate readings to be taken. This also applies to those reactions in which colors tend to reach maximum after a time; the period of maximum color must be long enough for precise measurements to be made. The influence of other substances must be known, and the influence of experimental conditions (temperature, pH, stability in air, etc.) 4. Reproducibility: the colorimetric procedure must give reproducible results under specific experimental conditions. 5. Clarity of the solution: The solution must be free from precipitate if comparison is to be made, with a clear standard.

Turbidity scatters light as well as absorb it.
6. High Sensitivity: The color reaction should be highly sensitive, particularly when minute amounts of substances are to be determined. It is also desirable that the reaction product absorbs strongly in the visible rather than the ultraviolet; the interfering effect of other substances in the ultraviolet is usually more pronounced. Deviation in Beer - Lambert’s Law: If a substance is following the beer’s law, a plot of absorbance against concentration gives a straight line which is passing from the origin, and the slope is “ab”. But some time, there is a deviation from the straight line.

If the absorbance value is greater than desired, the deviation is said to be positive (+ve) deviation. But if the absorbance value is less than desired, the deviation is termed negative (-ve) deviation.

There are two major errors for these deviations:
Instrumental Errors: The possible cause of instrumental errors are as follows: Fluctuation in electricity. Weak or malfunctioning source of light. Faulty arrangement of filter/monochromator. scattering of light inside the instrument. Faulty placement (fixing) of slit. Control knobs not working according to inner instrumentation Sensitivity of detector is low or malfunctioning. 2. Chemical Errors: The cause of chemical errors include the following: Presence of bacteria

Cloudy solution Pigmentation of the solution Acid-Base reaction taking place in the solution Association-Dissociation reaction of the solution Polarization reaction taking place in the solution Unstable colour of the solution

SPECTROPHOTOMETER A spectrophotometer works on the same principle as a colorimeter but it is more sensitive and sophisticated. There are light sources that emit light in the ultraviolet, visible and infrared regions of the spectrum. The wavelength is selected using a prism or diffraction grating and narrower bandwidths can be selected. Since light in the ultraviolet and infrared ranges is also emitted, the compound to be estimated does not necessarily has to be coloured and can be measured directly if they significantly absorb even at these wavelengths. This offers a significant advantage over the colorimeter, which is restricted only in the visible range.

Fig.: Components of spectrophotometer

Instrumentation of spectrophotometer:
Source of light: The light source must be fulfilling the following requisitions: The light coming from the source must be having proper intensity. The light source must be having all the frequencies of light, so that the required frequency can be acquired. The light source must be stable. It must not change with time.

For different region of light, following lamps (light sources) can be used:
EM Region Light source Wavelength 1 U.V. / near IR region Hydrogen or Deuterium discharge Lamp nm 2 Visible region Tungsten Lamp nm 3 IR region Nernst glover ,00,000 nm

2. Filters and Monochromators:
Filters and monochromators allow the radiation with only specific wavelength or specific wavelength to be transmitted, while other radiations are absorbed. a. Filters: Filters allow only a small section of frequency to pass through and all others are absorbed. Filters are used mostly in colorimeter. They are made up of glass or gelatin. b. Monochromators: these are optical devices used for selecting a specific wavelength from a range of frequency. They are of two types: i) Slit, and ii) Dispersive element. For dispersion of light, prism or grating or both can be used.

3. Cuvette: In all the spectrophotometric analysis
3. Cuvette: In all the spectrophotometric analysis. the absorption of light is measured. The type of the cell used in the analysis must not absorb light or the absorption must be minimum. Thus, for different region of spectrum – different material could be used as follows: For UV range: Quartz cuvette For visible range: glass cuvette For IR range: NaCl, KBr, nujol cuvette.

4. Detector: The light which is initiated from the source and pass through cuvette is measured by the detector. Absorption or transmission of the light can be measured by detector. Mainly three type of detectors can be used: 1. Photovoltaic Cell (Barrier layer Cell) 2. Photo tubes (Photo emissive tube) 3. Photo multiplier tubes The measurement of absorption or transmission is recorded in digital form in recorder.

Types of spectrophotometers:
Based on the type of monochromator, spectrophotometers can be classified as: Grating spectrophotometers: In these spectrophotometers, the monochromator is a diffraction grating, which disperses the white light into a continuous spectrum. By turning the wavelength adjustment knob, the grating is rotated and different parts of the spectrum are allowed to fall on the photocell. In this manner, the desired wavelength can be selected. Fig: Grating monochromator

b. Prism spectrophotometers: The prisms may be made of glass or quartz.
Light from a tungsten filament is focused on the entrance slit and it passes through a glass prism that forms an extended spectrum. Only the light that falls on the exit slit can pass through the cuvette and illuminate the photocell. The monochromatic light obtained here is much more pure than that produced by light filters, i.e. a much narrower band of wavelength is present in the incident beam of light. Different photocells are used: For short wavelength range of the instrument ( nm) For the longer wavelengths (600 to 1000nm).

The total wavelength range at which measurements can be made includes:
the visible spectrum (400 to 750nm), and extends on each side into the near ultraviolet (360 to 450 nm), and the near infrared (750 to 1000nm). It is therefore possible to estimate substances that are more or less colourless in the visible region, but which absorb light in the ultraviolet or infrared regions. Fig.: Prism monochromator

Two types of spectrophotometer are commercially available:
1. Single beam spectrophotometer 2. Double beam spectrophotometer Single beam Spectrophotometer Double beam Spectrophotometer

Uses of Spec. A spectrophotometer can be used for routine analysis in clinical chemistry. By using a narrower bandwidth than is available with ordinary filters, the absorbance is often higher and the relation between absorbance and concentration remains linear over a wide range. If very dilute colour is to be measured (e.g. in serum iron determinations), then a spectrophotometer is usually required. It can also be used for analytical methods depending on ultraviolet absorption e.g. serum enzyme assays, assays of glucose, urea, uric acid, e.tc.

CHROMATOGRAPHY

OUTLINE Introduction Key terms in Chromatography
Classification of Chromatographic Methods -Planar Chromatography -Column Chromatography High Performance Liquid Chromatography (HPLC)

INTRODUCTION Chromatography is a technique in which the components of a mixture are separated based on differences in the rates at which they are carried through a fixed or stationary phase by a gaseous or liquid mobile phase. It is a widely used method that allows the separation, identification and determination of the chemical components in complex mixtures. No other separation method is as powerful and generally applicable as chromatography. In chromatography, components of a mixture are carried through a stationary phase by the flow of a mobile phase, and separation are based on differences in migration rates among the mobile-phase components.

Key Terms in Chromatography:
Stationary phase: This is fixed in place either in a column or in a planar surface . Mobile phase: This moves over or through the stationary phase, carrying with it the analyte mixture. The mobile phase may be a gas, a liquid, or a supercritical fluid. Elution: This is the process in which solutes are washed through a stationary phase by the movement of a mobile phase. The mobile phase that exits the column is termed the ‘eluate’. Eluent: This is a solvent used to carry the components of a mixture through the stationary phase.

Adsorption: Interaction of solute molecules (or atoms or ions) with the surface of the stationary phase (note that it is different from absorption where the molecules fill the pores of a solid). Elution time: The time taken for a solute to pass through the system. A solute with a short elution time travels through the stationary phase rapidly, i.e. it elutes fast. Normal phase: “Unmodified” stationary phase where POLAR solutes interact strongly and run slowly. Reverse phase: “Modified” stationary phase where POLAR solutes run fast i.e. reverse order. 9. Resolution: Degree of separation of different solutes.

In principle, resolution can be improved by using a longer stationary phase, finer stationary phase (e.g. column packing or TLC plate coating) or slower elution. 10. Stationary phase: The part of the chromatography system that is fixed in place. Most commonly a solid e.g. the packing in column chromatography or gas chromatography or the coating on a chromatographic plate. 11. Rf value: distance travelled by solute distance travelled by solvent. Rf = retardation factor. The Rf value has to be between 0 and 1 (by definition), and it is typically reported to two decimal places. 12. Chromatogram: This is a plot of some function of solute concentration versus the elution time or elution volume. It is useful for both qualitative and quantitative analysis.

The position of the peaks on the time axis can be used to identify the components of the sample, while the area under the peaks provide a quantitative measure of the amount of each species. Fig.: Chromatogram

CLASSIFICATION OF CHROMATOGRAPHIC METHODS
Chromatography can be classified by various ways: Based on interaction of solute with the stationary phase: Adsorption chromatography: This is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes.

Partition chromatography:
This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid.

2. Based on chromatographic bed shape:
This is the major criteria for classification of chromatographic methods. There are two basic types: i. Column chromatography Planar chromatography In column chromatography, the stationary phase is held in a narrow tube, and the mobile phase is forced through the tube under pressure or by gravity. In planar chromatography, the stationary phase is supported on a flat plate or in the pores of a paper, while the moves through the stationary phase by capillary action or under the influence of gravity.

Planar Chromatography
Planar chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography). Different compounds in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. The specific Retention factor (Rf) of each chemical can be used to aid in the identification of an unknown substance.

Paper chromatography:
This technique involves placing a small dot or line of sample solution onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and sealed. As the solvent rises through the paper, it meets the sample mixture which starts to travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and the compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far.

Thin layer chromatography (TLC):
This is a widely employed laboratory technique and is similar to paper chromatography. Instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. For better resolution and to allow for quantification, high-performance TLC can be used.

Column Chromatography
This is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample. Classification: column chromatography fall into three categories based on the nature of mobile phase: liquid, gas, and supercritical fluid.

Classification of Column Chromatographic methods
General Classification Specific Method Stationary Phase Type of Equilibrium Gas Chromatography (GC) Gas-liquid (GLC) Liquid adsorbed or bonded on a solid surface. Partition between gas and liquid. Gas-solid Solid Adsorption Liquid Chromatography (LC) Liquid-liquid or partition. Liquid adsorbed or bonded to a solid surface. Partition between immiscible liquids. Liquid-solid or Adsorption. Solid. Ion exchange. Ion-exchange resin. Ion exchange Size exclusion. Liquid in interstices of a polymeric solid. Partition/sieving. Affinity. Group-specific liquid bonded to a solid surface. Partition between surface liquid and mobile liquid. Supercritical fluid chromatography (SFC) Mobile phase: supercritical fluid. Organic species bonded to a solid surface. Partition between supercritical fluid and bonded surface.

QUIZ List the components of a colorimeter with their functions.
Define the following: i. Eluent ii. Chromatogram iii. Stationary phase iv. Mobile phase v. Retention factor (Rf)

Ion exchange chromatography:
In this type of chromatography, anions or cations are covalently attached to a resin (the stationary solid phase). Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.

Molecular exclusion chromatography
This is also known as gel permeation or gel filtration. This type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to their sizes. The pores are normally small and exclude the larger solute molecules, but allow smaller molecules to enter the gel, causing them to flow through a larger volume. Thus, the larger molecules to pass through the column at a faster rate than the smaller ones.

Fig. Gel filtration chromatography

Affinity chromatography
Affinity chromatography is based on selective non-covalent interaction between an analyte and specific molecules. It is often used in biochemistry in the purification of proteins bound to tags. These fusion proteins are labeled with compounds such as His-tags, biotin or antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained. Affinity chromatography often utilizes a biomolecule's affinity for a metal (Zn, Cu, Fe, etc.). Columns are often manually prepared.

Immobilized Metal Affinity Chromatography (IMAC) is useful to separate biomolecules based on the relative affinity for the metal. Often these columns can be loaded with different metals to create a column with a targeted affinity. Fig. Affinity chromatography

Supercritical fluid chromatography
In this type of chromatography, the mobile phase is a fluid above and relatively close to its critical temperature and pressure. A supercritical fluid is any substance at a temperature above its critical point, where distinct liquid and gas phases do not exist. It can effuse through solids like a gas, and dissolve materials like a liquid. In addition, close to the critical point, small changes in pressure or temperature result in large changes in density.

Experimental apparatus of supercritical fluid chromatography

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
The acronym HPLC, coined by the late Prof. Csaba Horvath for his 1970 Pittcon paper, originally indicated the fact that high pressure was used to generate the flow required for liquid chromatography in packed columns. Thus, High pressure Liquid Chromatography. Advance in technology brought about change in name to more sophisticated High performance liquid Chromatography which is now one of the most powerful tools in analytical chemistry. It has the ability to separate, identify, and quantitate the compounds that are present in any sample that can be dissolved in a liquid.

This separation is then analyzed by a detector to yield results.
HPLC is an efficient type of chromatography that uses a high pressure gradient, rather than simply gravity, to propel a sample through a column. A sample is injected, and then a pump containing high amounts of pressure helps to move the sample along a packed column, where it is separated and quantitated as individual components. This separation is then analyzed by a detector to yield results. Fig: HPLC Setup

HPLC COMPONENTS AND THEIR FUNCTIONS
There are 5 major components of HPLC: 1. Pump: The role of the pump is to force the mobile phase through the liquid chromatograph at a specific flow rate, expressed in milliliters per min (mL/min). Normal flow rates in HPLC are in the 1-to 2-mL/min range. Typical pumps can reach pressures in the range of psi (400-to 600-bar). During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).

2. Injector: The injector serves to introduce the liquid sample into the flow stream of the mobile phase. Typical sample volumes are 5-to 20-microliters (μL). The injector must also be able to withstand the high pressures of the liquid system. An auto sampler (the automatic version) is used when there are many samples to analyze or when manual injection is not practical 3. Column: Considered the “heart of the chromatograph” the column’s stationary phase separates the sample components of interest using various physical and chemical parameters. The small particles inside the column are what cause the high backpressure at normal flow rates.

The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatograph. 4. Detector: The detector can see (detect) the individual molecules that come out (elute) from the column. A detector serves to measure the amount of those molecules so that the operator can quantitatively analyze the sample components. The detector provides an output to a recorder or computer that result in the liquid chromatogram (i.e., the graph of the detector response). Since sample compound characteristics can be very different, several types of detectors have been developed.

Examples include: UV-absorbance detector, fluorescence detector, and evaporative-light-scattering detector (ELSD). The most powerful approach is the use multiple detectors in series. For example, a UV and/or ELSD detector may be used in combination with a mass spectrometer (MS) to analyze the results of the chromatographic separation. This provides, from a single injection, more comprehensive information about an analyte. The practice of coupling a mass spectrometer to an HPLC system is called LC/MS. For sugar separation, the column is connected to a differential Refractometer which serves as the detector.

5. Computer: Frequently called the data system, the computer not only controls all the modules of the HPLC instrument, but it takes the signal from the detector and uses it to determine the time of elution (retention time) of the sample components (qualitative analysis) and the amount of sample (quantitative analysis). ISOCRATIC AND GRADIENT ELUTION Two basic elution modes are used in HPLC: The first is called isocratic elution; In this mode, the mobile phase, either a pure solvent or a mixture, remains the same throughout the run. The second type is called gradient elution, where the mobile phase composition changes during the separation.

Fig. Isocratic LC System

Fig. High Pressure Gradient System

USES OF CHROMATOGRAPHY
Chromatography has gained immense popularity in the past few years in almost every field. It is widely used in the field of chemistry, industry, medicines. It also has applications in the field of biological research. Chromatography is used to figure out the relation of different mixtures with one another. It is very effective technique to test the purity of the sample. For quantification of mixture present in a sample. In pharmaceutical companies large amount of pure chemicals for making further medicines is prepared by using chromatography. It is also very important in determination of authenticity of medicine.

In forensic science it helps in solving many cases by detecting residual burnt particles and flammable chemicals present in the body parts in case of fire or explosions. Paper chromatography and Gas chromatography are employed in finger print, DNA RNA analysis. It is used in the food industries for analysis of different additives in the food. For example milk is consumed all over the world. The common adulterant that can be added in milk is pyruvic acid. Pyruvic acid is derived from lactic acid bacteria. Chromatography is employed to check the quality of milk. Chromatography is also used to separate the contaminants, traces of harmful chemicals and other micro-organisms in food.

Gas chromatography is used in the manufacture and separation of essential oils.
It is used in industries for separating different components whose amounts can range from milligrams to tons. Thin layer chromatography is used to check and remove Polychlorinated biphenyls, pesticides and insecticides in ground water and fish contaminated by these. Environmental and governmental agencies also use chromatography to test drinking water.

COLLECTION AND PRESERVATION OF BIOLOGICAL SPECIMEN AND HERBARIUM TECHNIQUES

INTRODUCTION THE PROCESS OF ACQUIRING ANIMALS FOR BIOLOGICAL SPECIMENS
Field Study It is always better when biologists choose to learn about organisms in their natural habitats. You can see how they live and relate to others organisms and their impact on their environment. For this reason it is always advisable to do a field trip to partly see where such organisms inhabit, and then collect the organisms.

Collection of Specimens
Specimens that can be found readily in our immediate environment include rats, leaves, seeds, fruits, flowers, flies, fish, birds, lizards, snakes, toads, frogs, butterflies, mosquitoes, grasshoppers, ants, snails, scorpion, ants, worms, etc. Collection of these different specimens naturally vary with the type of organism and in which state you want to have it and the kind of study you intend to do.

Collection of live animals
Live animals are generally collected from aquatic and land areas. For collection visit a nearby aquatic body (e.g. pond) and land area (e.g. Park/Field). Collection of Aquatic Invertebrates Sources The sources can be ponds, lakes, rivers for fresh water animals and ocean/sea coasts for marine animals. However, in this section, as an example we will study the collection of animals from ponds.

Materials Required Nylon nets (Fine weave for small animals and coarse weave for large animals), large clean jars or buckets, shallow white pans or papers. Method 1. Take a clean bucket or a jar and fill it up to about half with the pond water from which you are going to collect the samples. 2. With a trowel, scoop a little amount of mud from the wet edge of the pond and put it in the bucket or jar having pond water. 3. Also put one or two small submerged branches of aquatic plants in the bucket or jar. 4. Take the suitable net and sweep through the water in the pond. You have to sweep more than once.

5. Take out the net. You will see the specimens trapped in the net
5. Take out the net. You will see the specimens trapped in the net. Transfer the specimen into the bucket or the jar. 6. Take some extra pond mud, submerged branches or aquatic plants along with some pond water and carry to the laboratory for subsequent use, if needed. 7. In the lab, transfer the live specimens into shallow white pans or place them on a large sheet of paper and spread them out for study.

Insect Nets (Sweep nets)

Collection of Earthworms
These annelids can easily be collected from the soil having organic matter especially at night and after a rain, when they come out at the surface of the soil. In dry season earthworms are not easily available. So they are preserved whenever available for use in dry season Sources: Rich garden soils, lawns, agricultural fields especially after a rainy day or night. Material Required: A bucket, flashlight/torch light (for night collection), blunt-end forceps Blunt end forceps

Method: Visit the collection site. Put some moist soil in the bucket. Pick up the worms with blunt-end forceps and put them in the bucket. Use flashlight/torch light if collection is to be done in the night. Take the worms to the laboratory. Collection of Insects Sources: Terrestrial insects are found in gardens especially during flowering seasons, in the fields and indoors. Aquatic insects can be collected from water bodies like ponds, lakes etc. There are several methods of collecting insects but in this section you will collect terrestrial insects by three methods using: a net, light trap and aspiration.

An aspiration for capturing insects

Methods (a) Sweep Net Method: This method is suitable for collecting many insects. Materials Required: Insect-collecting net and killing jar Steps: 1. Go to the garden/field and identify the insects to be collected. 2. Approach the specimen(s) very quietly. You should try to avoid chasing the insects overtly as it would alert the insects and make them fly/run away. 3. Sweep the net through the herbage over the specimen(s). You might have to sweep more than once. 4. When the insect(s) is trapped in the net, twist the net or your wrist so that net is closed and the specimen is not able to escape. 5. Transfer the collected insects into the killing jar.

(b) Light Trap Method In this method the collector is not required to be present. It is mainly used for nocturnal insects like moths, midges, some beetles and winged termites. Materials Required: Light sources such as an electric bulb (200 W) or a lantern lamp, a large shallow container such as a basin sauce pan, white paper sheet and Killing jar. Steps 1. Select an area where insects are abundance 2. Hang the light source with the help of a hook. 3. Put the white paper as lining in the shallow container and set the container below the light sources so that electric lamp is shining in the middle of the container. (In the absence of an electric light keep a lantern lamp in the middle of the container)

5. Transfer the collected insects into the killing jar.
4. Soon the insects will be attracted by the light and fall into the container. (In case the shallow basin saucepan is not available you can keep a collecting jar fitted with a cone made of white sheet under the light source. The most efficient light source for insect-trapping is a mercury vapour lamp) 5. Transfer the collected insects into the killing jar. Insect Collecting Jar

(c) Aspirator An aspirator is a simple suction device used for collection of small insects such as mosquitoes, thrips, sandflies etc. It is made up of the following: 1. A transparent vial made of glass or plastic (transparent plastic is preferably used). Rubber stopper with two holes 2. Two glass tubes each with a bend Rubber tube 3. Small piece of muslin cloth Steps: 1. Insert the two glass tubes (intake and suction tube) through the two holes in the stopper. 2. At one end of one glass tube attach a rubber tube. Cover the other end of this tube by tying a piece of muslin cloth. This tube acts as a suction tube. The other tube is the intake tube. 3. To the open end of the vial fix the rubber stopper (with inserted tubes). The stopper should be tightly fixed in the vial. The end of the suction tube that is covered by muslin cloth should be inside the vial. The aspirator is now ready for use.

4. Place the aspirator with the outer end of its intake tube facing the insect(s) and suck through the rubber tube. The suction creates a partial vacuum in the vial there by drawing the insect through the intake tube. The muslin cloth tied on the inner end of suction tube will prevent the entry of insects into this tube. 5. Plug the outer end of the intake tube to prevent the escaping of the insects caught in the vial and then transfer the collected insects into the killing jar.

THE PROCESS OF ACQUIRING PLANTS FOR BIOLOGICAL SPECIMENS
Collection of Lower Plants Care is to be taken when collecting plant samples, and this is particularly done with the use of a vasculum, polythene bags or in bottles. You will need a pair of secateurs for cutting hard material, a sharp knife for cutting soft parts, pick for digging out underground parts like roots and rhizomes, scalpel and forceps for separating those plants which grow attached to the barks of trees and rocks. The stems and roots are cut into pieces of size about 3 cm long with the aid of sharp razor or knife. Bryophytes are made free from soil particles and debris before storing in some preservative. The smaller leaves can be preserved as such and larger ones can be cut in pieces, and then preserved.

A Vasculum A Secateurs A Pick

ii) Sterilize spatula by swirling it in spirit and then flaming it.
A Scalpel A Spatula Collection of Algae Sources: Algae occur widely on the soil surface and below it, on the bark of trees, in fresh water, sea water, and a variety of other habitats. Collection from Bark: i) In case of bark algae pick up the algae patches from the tree trunk with the help of iron spatula. ii) Sterilize spatula by swirling it in spirit and then flaming it. iii) Store various samples collected in separate sterilized bottles after fixing and labeling in their respective shelves.

Collection from Fresh Water:
i) Collect the fresh water algae at the spot in sterilized specimen tubes containing some habitat water. ii) Never fill the container more than a quarter so that the quantity of oxygen present in the water may be sufficient for respiration. iii) Fix the material, label them and keep in respective shelves. Collection from Sea Water: i) Collection of marine algae is best done during low tides as during this period these are mostly in their reproduction stages. ii) Collect marine algae in large bottles. iii) Fix them and label them and keep them in their respective shelves

Collection of Bryophytes
Bryophytes occur in nature attached to wet soil, rocks and bark of living and dead trees, wood and humus rich in organic substances. I) Scrape the bryophytes from the place of occurrence with the help of sharp scalpel or knife and keep them in polythene bags within which they remain alive to a number of days. ii) Keep these bags loosely tied and in damp condition in laboratory. iii) Wash the soil growing species with ordinary water to remove soil particles and dirt attached to plant. iv) Keep the bags under illumination at 0oC – 5oC to keep the plant alive for a longer duration. v) Fix the material, label and keep it in the cupboard. Algae Specimen in tubes

Collection of Pteridophytes
Pteridophytes are spore-producing vascular plants. They possess the vascular tissues xylem and phloem. They grow in variable habitats. Most of the pteridophytes are terrestrial while a few are epiphytes and some of them are found in aquatic habitats. i) Collect the pteridophytes from natural habitats in mature spore producing stage. ii) Collect the plants with or without strobili or mature sporophyll in polythene bags loosely tied at mouth. iii) If the material is large cut them into pieces, fix label and keep them in a cupboard. Collection of Gymnosperms Gymnosperm belong to seed plants but the seeds are naked with a very conspicuous and independent sporophyte which is the main plant and have very reduced gametophyte dependent on the sporophyte. They have xeric characteristics also. i) Collect the root, stem, and leaves of male and female gametophytes of the plant. ii) Cut the material into small pieces of 3 cm, fix them, label them and keep them in a cupboard. iii) You can collect the dry fruits and cones of gymnosperms and preserve them as such.

Preparing Specimens for Laboratory Studies
Upon collection or procurement of your specimens, it is necessary to bring them into the laboratory so that they could be well studied. It is often important for the specimens to be kept for a considerable length of time for sufficient studies to be made. For this reason, it is necessary to know how best to keep them preserved. The same methods may not apply to all types of specimens. For example, bones will not be stored the same way as worms. It is however important for you to know that some storage and preservation may be necessary. Killing, mounting and display of insect specimens The insect to be killed is transferred to a bottle contain the killing agent, such as ethyl acetate, chloroform, ether, tetrachloroethane etc. However, the safe and most efficient agent is ethyl acetate.

Materials required include an empty glass bottle with an air-tight lid (you can take a jam or Horlicks bottle), ethyl acetate, cotton, blotting paper and forceps. Steps: i) Soak a wad of cotton in ethyl acetate. You must hold this cotton wad with forceps and not with hands. ii) Place the soaked cotton at the bottom of the bottle and cover it with a piece of blotting paper. Blotting paper is used to avoid the direct contact of the specimens with the chemical because it will wet the specimens and spoil them.

iii) Transfer the insects into the bottle and close tightly
iii) Transfer the insects into the bottle and close tightly. The insects should be taken out within 20 mins. to prevent them from being decolourised and get unduly hardened. The bottle should not be over crowded, and different bottles should be used for different types of insects. iv) The bottles should be labelled ‘poison’ and kept out of reach. Bottles that are no longer in use should be buried. Mounting of the insects After being killed the insects are pinned with the help of entomological pins on the pinning board. You can also prepare entomological pins with sewing needles and coloured beads. Take thin sewing needles, heat the eye of needle on a spirit lamp flame and insert the heated end into a coloured bead, which forms the needle head. Mounting Direct mounting: Mounting should be done immediately the insect is dead, using the following steps: i) The entomological pin is pushed through the thorax region of the insect. However, the exact point in the body of the insect through which the pin should pass differs in the different groups of insects.

ii) Insert the pin vertically through the body or sloping in such a way that the front part of the body is raised very slightly. iii) Push the specimen up in the pin until it’s back slightly away from the top so that it does not have any contact with the back of the insect body. iv) Mount the pinned insects on the board or on a pinning block. Take care to mount the insects uniformly so that specimens can be examined and compared easily.

This method is especially used for mounting small and dried insects.
Point Mounting: This method is especially used for mounting small and dried insects. Take the following steps i) Take a stiff card paper and cut triangles from it. For a smaller insect the size of the triangle can be 6 mm long, 2 mm wide at base and 0.5 mm wide at the apex (tip). However, the size of the triangle varies depending upon the size of the insect. ii) Attach the dried specimens to the apical tip of the triangle with the help of a quick drying adhesive like quick-fix. The best places on the insect body for adhesion can be at the sides of thorax below the wings, margin of the tergum and above or between the bases of the legs. iii) Insert the entomological pin in the broader end of the triangle and pin this triangle with mounted insect on the display board. Point mounting of insect

Spreading To display the head, abdomen, wings and legs you have to spread the freshly killed insects on the spreading board. In the freshly killed insects the internal parts are soft to allow the pin in and appendages are pliable. The pin is pushed through the thorax region. A spreading board is available in the market but can also be made locally. A simple method is to take a thick sheet of cork or thermocol and cut a groove in it for the body of the insect. Steps: i) The insect is placed in such a way that the body and thorax of insect rest in the groove of the board. ii) One end of a narrow strip of setting paper is pinned at the front end on each side of the insect body. iii) The fore wings on the back are drawn forward and each pinned on either side with a fine pin inserted behind one of the strong veins in the wings. iv) The hind wings are also spread like this and pinned.

v) When the wings are correctly placed the paper strips can be taken over the wings and their other end is pinned on the back of the insect body so that both the wings are held by paper strips and setting pins. vi) The antennae are also spread symmetrically and pinned under the narrow strip. vii) Legs (appendages) are also spread and pinned on both sides under the strips. Care should be taken that while spreading, the joints and the shape of the appendages remain intact. viii) If the abdomen is inclined to fall into the groove it can be supported by crossed pins placed beneath it. After the pinning and spreading the specimens are dried for few weeks in the open or in drying chambers and stored.

2. Host plant, crop or the area from where it is found.
Displaying Once the specimens are collected and spread, they should be given permanent labels. These labels should be small and made of white card. The following information should be there on the label of each specimen: 1. Name of the insect. 2. Host plant, crop or the area from where it is found. 3. Locality from where it is found. 4. Date. 5. Collector's name The ink used for writing should be permanent and not spoiled when in contact with any type of liquid. Butterfly on a Spreading Board Mounted insects

The spread board along with spread insects with labels should be displayed in wooden boxes with glass tops. The mounted insects should also be stored in closed boxes. You must keep naphthalene balls in the storing or displaying boxes used for insect specimens. In case these precautions are not taken the specimen insects can get spoiled or eaten by other insects/small animals.

PRESERVATION OF ANIMAL MATERIAL
For preserving taxonomic material such as laboratory and museum study specimens, different preservation methods can be considered. In the field, there may be limited access to materials and equipment necessary, so preliminary preservation with more simple methods may be necessary before final preparation as a permanent collection specimen.

Examples: procedures for preliminary preservation of a whole animal
Short-term storage without preservation (of freshly dead animals needed for mounting or skin preparation) Small animals in a cold to moderate climate may be stored without refrigeration in the shade for 4 -5 hrs. After this period, in warmer climate sooner, the viscera will begin to decompose. Preparing Specimens for Laboratory Studies Upon collection or procurement of your specimens, it is necessary to bring them into the laboratory so that they could be well studied. It is often important for the specimens to be kept for a considerable length of time for sufficient studies to be made. For this reason, it is necessary to know how best to preserve them. The same methods may not apply to all types of specimens. For example, bones will not be stored the same way as worms. It is however important for you to know that some storage and preservation may be necessary.

Preservation and Storage
Apart from preservation and storage, you might also need to prepare the specimen for the kind of study that is desired. For example, if you want to study the structure of a section through the root of a plant, you need a microscope to view the details of the sections. Besides, you need to make a slide. Specimen preservation means a long term preservation of organisms either plant or animal in the best possible condition, so that it can be accessed in future as reference collection for scientific purposes. For reference collections, mammals can be prepared as a variety of specimens. The condition of the specimen may determine possible ways to preserve it; if for instance decomposition of the skin has loosened the hair of a carcass so much that it can easily be pulled out or removed by rubbing, it will be very difficult or impossible to produce a study skin or mounted specimen.

The some of the most usual types of specimens are:
Entire fluid-preserved animals (for studying anatomy and histology; fluid preservation may change the fur colour) 2) Study of skins with accompanying skulls / partial skeletons (some bones remain in the skin), for studying pelage colour, hair quality and moulting patterns, 3) Mounted skins with accompanying partial or entire skeleton (some bones may remain in the skin, dependent on the method of preservation) or freeze-dried specimens, 4) Entire skeletons, for instance for studying anatomy, geographic variation or for age determination (entire skeletons are poorly represented in collections, so it is recommended that preparation of at least one male and one female skeleton per species.

Many chemical methods are used to preserve both vertebrate and invertebrate specimens.
Why specimens are preserved? a) Taxonomic reasons b) For detailed examination. c) For morphological study of particular animals as each and every animal can't be in researcher’s vicinity. d) For zoological museum collection Steps for Specimen Preservation 1. Killing and relaxing of alcohol, 2. Fixation (stops cellular respiration, kills bacteria within the organism, a good penetrating ability) 3. Storage in bottles, jar vials, trays etc.

Types of Specimen 1) Entire fluid-preserved animals Purpose: (for studying anatomy and histology; fluid preservation may change the fur colour) 2) Study skins with skulls / partial skeletons (some bones in skin) Purpose: for studying colour, hair quality and moulting patterns. 3) Mounted skins with partial or entire skeleton (some bones may remain in the skin, dependent on the method of preservation) or freeze-dried specimens. 4) Entire skeletons, for instance for studying anatomy, geographic variation or for age determination.

Preservatives and Their Usage
1) Formalin (Fixative mostly) Formalin is the commercial name of a solution of formaldehyde gas (CH2O) in water. Formalin must be diluted with water before it is used as a preservative. A strength of 10% formalin is best for most purposes. If the original strength is 40%, it should be mixed at a ratio of nine parts water to one part formalin Usage: It is used for vertebrates only. It is avoided for long-term storage since it is acidic and difficult to handle. Mostly formalin is used where colour is important since alcohol dissolves most colours almost immediately. It penetrates more rapidly, and internal organs remain in better condition.

Procedure: Dilution Conc. formalin (100%) = Water saturated with 40% formaldehyde 10% formalin = 4% formaldehyde (Used for preservation) 2% formalin with seawater for small specimen ii. Mix one part concentrated formalin to nine parts water. Fill about two-thirds the bottle's volume with 10% formalin. As formalin is acidic, it should be buffered by adding a pinch or two of sodium bicarbonate.

Precaution: Inhalation of formalin fumes is harmful and causes extreme discomfort to nose and eyes. Contact with fluid causes severe irritation to the skin Contact with sore or raw spots results in extreme pain. It is carcinogen. Hand should be rinsed after usage. Storage: It should be kept in safe, water-tight, spill-proof bottles It should always be clearly labelled.

2. Industrial Alcohol (for both fixing and storage)
Usage: Alcohol is usually not used for killing and fixing vertebrates. But of course used for long-term storage. Colour of specimen is lost immediately. A teaspoonful of glycerin in a quart of alcohol helps to preserve natural colours and to keep integuments flexible. Alcohol usually comes in the 95% concentrated form. For long-term preservation, 70-75% strength is used.

Warning: Alcohol is usually safe to handle, it can however cause irritation to the skin in cases of prolonged contact. Always rinse hands with water after working with alcohol. Industrial alcohol is toxic and should never be drunk. Alcohol is highly flammable. Never work with this fluid in the vicinity of open flames. It is rapidly evaporated, and receptacles holding it should be securely covered at all times, and not be opened unnecessarily.

3. Isopropyl Alcohol It is cheap and easy to obtain. There are different strengths available (70% and 90%), so if you use isopropyl you will have to dilute it to a 40% alcohol solution. Isopropyl alcohol can be hard on the specimens and tends to make them brittle over time. Buffering: It can be buffered with a few drops of glycerin or a pinch of calcium carbonate tablets (crush the tablets to a powder and add).

4. Ethyl Alcohol (for invertebrates)
A better solution for long term storage of invertebrate specimens is in an 80% solution of ethyl alcohol. Ethyl alcohol can be found in painting supplies. It is labelled as denatured alcohol. It should also be buffered with glycerin/antacid tablets.

VERTEBRATES SPECIMEN PRESERVATION
1. Fishes After capture Fishes are placed in 10% formalin for quick killing (painful). It is not needed to relax fish. Fishes dies with its fin well spread-out, and the body straight and well-stretched. Examination and counting of fin rays and scales is quite easy on such well-preserved material. For 30cm fish, the following is used i. Formalin - 1 week (fix soft tissue) ii. Water - 1 day (leach out the formalin) iii. Alcohol - long term storage The length of time for each step may have to be increased with increasing size of specimens.

2. Herptiles (Reptiles and Amphibians)
Herptiles are individually kept in plastic & then killed by freezing or chloroform. Shallow trays are used for herptile specimens. Snakes should be coiled up, and frogs are to be placed on their bellies with their limbs set at right angles to their body. Depending on the size of the specimen, the time necessary for complete fixation can be different, from 2 days for small salamanders to a month for something like alligators, salamanders and turtle. Then added to water for a day or two and then in 75% alcohol for long-term storage.

3. Birds and Mammals: Usually birds and mammals are skinned. Skull or Skeletal mounts and flesh can be removed by several means such as boiling or using dermested beetles. Once bones are defleshed they can be placed in a bleach or hydrogen peroxide solution to whiten. Then the bones are allowed to dry and placed in a bag or box with complete label tied to skull if possible.

INVERTEBRATE SPECIMEN PRESERVATION
The easiest way to preserve these animals is to use alcohol. One should be aware of which kind of alcohol they are using as each animal requires a different concentration for preservation. Most invertebrates, however, will be kept in bottles, and sets of tubes or jars for preservation 1. Coelenterates Coelenterates are difficult to preserve. They should be preserved in 70% alcohol or 5% formalin. Hydra can be quickly fixed in Bouin's fluid (warm, not hot)

2. Platyhelminthes (Flatworms)
Bouin fluid is used for fixation. Paraffin is the best long-term preserving and storage medium of all flatworms. 3. Echinoderms Echinoderms are narcotised by the addition of magnesium sulphate or menthol to the sea water in which they live. When completely insensitive to stimuli such as pricking they should be transferred to 70% alcohol for preservation and storage.

4. Arthropods They are easy to process, as they are killed immediately and stored in alcohol. Crabs and prawns may also be killed in formalin, but this renders their joints hard and brittle The larger arthropods (especially those with hard exoskeletons) sometimes need to be injected with 10% formalin to prevent them from rotting. Industrial alcohol is used for most arthropods. Insects, crustaceans and arachnids can be simply dropped into alcohol for immediate preservation.

Liquid hand sanitizer can be used for insects
Liquid hand sanitizer can be used for insects. Hand sanitizer is gelled alcohol, hence the usage. Specimen will float inside the vials and do not sink or move despite any amount of handling. It is best to kill the insects in an alcohol solution then transfer them to the hand sanitizer for preservation. The gel will break down over time and become liquid, so it should be replaced occasionally.

5. Molluscs Relaxed mollusk is used for good preservation. Newly killed molluscs could first be fixed in buffered 10% formalin for two or three days, and then transferred to 75% alcohol after soaking for a few hours in water. Molluscs can also be relaxed in a solution of magnesium chloride, but this does not work very well with land molluscs.

Precautionary Measures
a.) Do not crowd living animals in small containers - this will result in damage to their appendages. b.) Features important in the taxonomic study of fish, for example, are easily damaged with contact even after preservation. c.). Live crabs before preservation should be kept individually as some species will damage each other and other animals which will distort their morphological features. A reasonable number (about 95%) of the museums of the world use ethanol (drinking or grain alcohol) for long term preservation. Over 4.9% use isopropyl (rubbing alcohol), while percent use methanol, or wood alcohol.

HERBARIUM TECHNIQUES A herbarium is a collection of preserved plants stored, catalogued, and arranged systematically for scientific study by professionals and amateurs. Herbaria are a vital reference library to aid in current plant identification and future taxonomy. The written data accompanying the specimen is as important as the specimen itself. A specimen with no data has no scientific value. The data provides evidence of where the specimen was found, who found it and when it was collected. Herbarium specimens are datasets, providing information relating to taxonomy, classification, chemistry, curatorial practice, flowering/fruiting times, morphology and physiology.

Well preserved plant specimens can also be used to provide samples of DNA, other biological compounds and to validate scientific observations. Herbaria are used to aid plant identification, to help understand biodiversity and used in support of conservation, ecology and sustainable development. The long-term preservation of this material is essential for future generations. Collections can date back hundreds of years and so conserving this material raises challenges that must be met as the collections are an essential resource for ecologists, scientists, geographers and historians. A Herbarium may include some or all of the collections.

Methods of preparation of herbarium specimens
The preparation of a herbarium involves (i) Field visit (ii) Collection of specimens (iii) Drying (iv) Mounting on a herbarium sheet, (v) Preservation (vi) Labelling and (vii) Proper storage

(a) Field visits and specimen collection
A complete specimen possesses all parts including root system, flowers and fruits. Therefore, regular field visits are necessary to obtain information at every stage of growth and reproduction of a plant species. In the fields, the tools required are mainly trowel (digger) for digging roots, scissors and knife for cutting twigs, a stick with a hook for collection of parts of tall trees, a field note book, polythene bag, old newspaper and magazines A trowel

The specimens selected should be vigorous, typical specimens
The specimens selected should be vigorous, typical specimens. Insect-damaged plants should be avoided. Specimens should be representative of the population, but should include the range of variation of the plants. Roots, bulbs, and other underground parts should be carefully dug up, and the soil removed with care. Make sure the specimen includes flowers and/or fruits. It may be a good idea to collect extra flowers and fruit for identification purposes.

In collecting large herbs, shrubs and trees, different types of foliage, flowers and fruits should be collected from the same plant. Collect sufficient material to fill an herbarium sheet (450 x 300 mm) and still leave enough room for the label. Plants too large for a single sheet may be divided and pressed as a series of sheets. Herbarium sheet

Bark and wood samples are often desirable additions when collecting woody plants. There are special requirements for the identification of some plants. A Eucalyptus specimen, where possible, should include mature leaves, juvenile leaves, buds, fruits, and bark. Other general hints for collecting are: 1) Bulky plants or parts can often be halved or sliced before pressing. Odd fragments - bark, fruits or seeds - should be kept in numbered or labelled envelopes or packets with the main specimen.

2) Very bushy twigs should be pruned to make a flatter specimen, in such a way that it is obvious where pieces have been broken off. 3) Spiny plants may first be placed under a board and stood on before pressing to prevent tearing the paper. 4) Succulent plants need to be killed first by soaking in methylated spirits for minutes. Bulbs should also be killed, or may sprout on herbarium sheet.

5) Water plants must be floated out in a dish of water and lifted out on a sheet of stiff white paper slipped under them in the water; dry excess water, then press the plant in the usual way leaving it on the white paper on which it can remain permanently stuck. A piece of waxed paper over the top of the plant will prevent it adhering to the drying paper. 6) Tall rosette plants and grasses may be pressed complete by bending them once or more into the shape of a "V", "N" or "M".

7) Dioecious plants should be represented by both sexes.
8) Palms - several herbarium sheets are necessary to show the various portions of the leaf, inflorescence and fruit of these species. Photographs of the tree and of each part are essential. 9) Cones of some gymnosperms may need to be enclosed in a wire mesh to prevent them falling apart.

b) Pressing and Care of Specimens ( drying)
Specimens should be pressed as quickly as possible after collection. If this is not possible, specimens may be stored in plastic bags preferably wrapped in damp (but not wet) papers. Bags should not be packed tightly, and should be kept cool and moist. Make sure that each bag is correctly labelled for locality. Place each specimen, with numbered tie-on tag attached, in a fold of several sheets of newspaper, and place in the press. If necessary, occasionally add a sheet of corrugated cardboard to act as a ventilator. As you fill the press, try to keep it level to allow even distribution of pressure.

This may mean the use of alternate corners of the fold for bulky roots and other parts, or packing around a bulky specimen with foam. Close the press and exert pressure with the straps. The plants in the press should be dried fairly quickly, in a warm place if possible. The specimens must not be left in damp papers or they will go moldy. It is therefore necessary to go through the press daily during the first few days and change the plants into dry newspapers. Then continue to inspect press daily and change newspapers as necessary until the plants are dry. Delicate plants and petals may be lost in changing and should be kept in tissue-paper (or toilet-paper) folders throughout changes. A properly dried plant specimen is brittle.

(c) Mounting:

The dried specimens are mounted on herbarium sheets of standard size (41 x 29 cm). Mounting is done with the help of glue, adhesive or cello-tape. The bulky plant parts like dry fruits seeds, cones etc. are dried without pressing and are put in small envelops called fragment packets. Succulent plants are not mounted on herbarium sheets but are collected in 4% formalin or FAA (Formalin Acetic Alcohol).

Preservation The mounted specimens are sprayed with fungicides like 2% solution of mercuric chloride.

(e) Labelling A label is pasted or printed on the lower right hand corner. The label should indicate the information about the locality, altitude, habit, date and time of collection, name of collector, common name, complete scientific name etc. The collection is also recorded in the field notebook together with information about that collection. As much as possible of the following data should be included: Exact locality - a good plain language description, and latitude and longitude. Altitude.

Nature of the habitat - type of soil, topography, slope, aspect.
Associated species, vegetation type. The plant proper - record features which will not be evident from the pressed specimen e.g. whether it is a tree or shrub, height, branching, notes on root system, odour, etc., as well as those features which may be lost on drying e.g. flower colour and odour. BIOLOGICAL TECHNIQUES by Ibadin F. H. (Ph.D), Adebami, G.E, Rabiu, O. R, Ayodele O. O. is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

BIOLOGICAL TECHNIQUES (BIO 204)

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BIOLOGICAL TECHNIQUES (BIO 204)

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