project on industrial training

8/3/2019 project on industrial training

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ONE MONTHS INDUSTRIAL TRAININGATAYUSH HERBS PVT. LTD.

SUBMITTED BY : GUIDED BY:NAVJOT KHAJURIA MISS RENUKAREGD NO 10800024ROLL NO RY2701A32

CONTENTSAcknowledgmentIntroduction to AyurvedaCompany OrganizationRaw materialQuality ControlLaboratory InsrtumentsProductionPharmaceutical ProductsPackagingConclusion

ACKNOWLEDGEMENT

It’s right to acknowledge my gratitude with sense of veneration to Almighty God and various people who helped me during the course of Industrial training. Their valuable guidance and wise direction have enabled me to complete my practical training in systemic and smooth manner.I want to thank the Department of Ayurvedicpharmaceutical science for giving me permission to commence this project work,Ihave furthermore to thank the our H.O.D and our training instructors gave and confirmed this permission and encouraged us to go ahead with our project.Iam thankful to my parents who provide me required moral support and other resources.

Iam also thankful to Mr. Jitender Sodhi who allowed me tobe a part of training and completing it successfully.

Iam thankful to Miss. Renuka under whose guidance I have completed my training

successfully.



I also thanks to all other technical staff for giving their advice and co-operation during training.

“AYURVEDA:THE SCIENCE OF LIFE”Ayurveda is the ancient medicinal form, developed during the Vedic times, about5000 years ago. According to ancient mythology Lord Shiva was the first to research in healing processes for humans. Later, in the guise of Dhanvantri he devoted full time to medicine. This we believe is the precursor of Ayurveda and thus this science of medicine is very old. Over the period of time it underwent furthe

r research by other eminent vaids and they improved it to the extent that thereare no side effects in Ayurvedic medicines. . The word

Ayur

means life, while

Veda

means science. Therefore, Ayurveda literally means the

Science of Life

.It is not just a medicinal system, but also a way of life. Ayurveda deals withthe physical, as well as spiritual health. The medicinal form is governed by thelaws of nature, which suggest that life is a combination of senses, mind, bodyand soul. According to the Science of Life, the structural aspect of every individual comprises five elements The Ayurvedic medicines generally come in the formof powders, tablets, decoctions, and medicated oils, that are prepared from natural herbs, plants and minerals. In addition, the diseases treated and cured byAyurvedic medicines do not cause any side effects.There were originally four main books of spirituality, which included among other topics, health, astrology, spiritual business, government, army, poetry and spiritual living and behavior. T

hese books are known as the four Vedas; Rik, Sama, Yajur and Atharva. The Rik Veda, a compilation of verse on the nature of existence, is the oldest surviving book of any Indo-European language (3000 B.C.). The Rik Veda (also known as Rig Veda) refers to the cosmology known as Sankhya which lies at the base of both Ayurveda and Yoga, contains verses on the nature of health and disease, pathogenesis and principles of treatment. Among the Rik Veda are found discussions of the three dosas, Vayu. Pitta and Kapha, and the use of herbs to heal the diseases ofthe mind and body and to foster longevity. The Atharva Veda lists the eight divisions of Ayurveda: Internal Medicine, Surgery of Head and Neck, Opthamology andOtorinolaryngology, Surgery, Toxicology, Psychiatry, Pediatrics, Gerontology orScience of Rejuvenation, and the Science of Fertility. The Vedic Sages took thepassages from the Vedic Scriptures relating to Ayurveda and compiled separate bo

oks dealing only with Ayurveda. One of these books, called the Atreya Samhita isthe oldest medical book in the world! The Vedic Brahmanas were not only priestsperforming religious rites and ceremonies, they also became Vaidyas (physiciansof Ayurveda). The sage-physician-surgeons of the time were the same sages or seers, deeply devoted holy people, who saw health as an integral part of spirituallife. It is said that they received their training of Ayurveda through direct cognition during meditation. In other words, the knowledge of the use of variousmethods of healing, prevention, longevity and surgery came through Divine revelation; there was no guessing or testing and harming animals. These revelations were transcribed from the oral tradition into book form, interspersed with the other aspects of life and spirituality. What is fascinating is Ayurveda

s use of herbs, foods, aromas, gems, colors, yoga, mantras, lifestyle and surgery. Consequently Ayurveda grew into a respected and widely used system of healing in India.

Around 1500 B.C., Ayurveda was delineated into eight specific branches of medicine. There were two main schools of Ayurveda at that time. Atreya- the school ofphysicians, and Dhanvantari - the school of surgeons. These two schools made Ayu



rveda a more scientifically verifiable and classifiable medical systemIn 16th Century Europe, Paracelsus, who is known as the father of modem Westernmedicine, practiced and propagated a system of medicine which borrowed heavily from Ayurveda.There are two main re-organizers of Ayurveda whose works are still existing in tact today - Charak and Sushrut. The third major treatise is called the AshtangaHridaya, which is a concise version of the works of Charak and Sushrut. Thus the

three main Ayurvedic texts that are still used today are the Charak Samhita (compilation of the oldest book Atreya Samhita), Sushrut Samhita and the AshtanghaHridaya Samhita. These books are believed to be over 1,200 years old. It is because these texts still contain the original and complete knowledge of this Ayurvedic world medicine, that Ayurveda is known today as the only complete medical system still in existence. Other forms of medicine from various cultures, althoughparallel are missing parts of the original information.

AYUSHHERBS AND PHARMACEUTICALSAn ISO 9001:2000 & GMP Certified CompanyAyush Herbs was founded by SODHI BROTHERS in 1988 in USA to make Ayurvedic preparations available to the Physicians leader in the manufacturing of AYURVEDIC HER

BAL EXTRACTS AND AYURVEDIC MEDICINESAyush offers a wide range of the highest quality of Ayurvedic (herbal) products. All the products in this catalogue carry our commitment to quality. Ayush Ayurvedic herbal formulas are based on traditional combination and contain herbs grown naturally in their pristine Himalayan habitat, without the use of pesticides,insecticides, herbicides or chemical fertilizers. Formulated by Ayurvedic and Naturopathy doctors, and made from standardized herbal extracts to ensure qualityand potency, Ayush product are safe, effective and a natural part of a healthylife style.Ayush is working consistently for further improving the quality of its productsby incorporating the most modern biotechnology in Ayurveda to offer the highestpercentage of active principle with consistency, potency. All the formulations h

ave strong synergistic effect.Last year the Indian Government has recognized Ayush Herbs Pvt. Ltd. for hard work, product excellence, technological innovation and the company

s commitment for helping local farmers in (the Himalayan foothills) growing Ayurvedic medicinalHerbs as a lucrative alternative to the standard grain crops of the region.90% of our total production is being exported to USA and EUROPE. We will shortlybe commencing our exports to African and Arabian regions. We have several FARMERS, NGO working with us in cultivation in Himachal, Punjab and Haryana.According to ancient mythology Lord Shiva was the first to research in healing processes for humans. He kept poisonous insects reptiles like snakes as pets to study poisons, drank intoxicating herbs to study their effect on humans. Later, in the guise of Dhanvantri he devoted full time to medicine. This we believe is the precursor of Ayurveda and thus this science of medicine is very old. Over the

period of time it underwent further research by other eminent vaids and they improved it to the extent that there are no side effects in Ayurvedic medicines. History reveals that Charak for the first time documented this ancient science ab



out 5000 years ago. Considering the safety of Ayurvedic Medicines usage we started the manufacturing of these medicines firstly in USA and then in India.

Ayush Herbs Pvt. Ltd. has been serving the health Industry for over a decade nowand has earned an over whelming response from its clientele all over the world.Ayush offers a wide range of the highest quality of Ayurvedic (herbal) products. All the products in this catalogue carry our commitment to quality. Ayush Ayur

vedic herbal formulas are based on traditional combination and contain herbs grown naturally in their pristine Himalayan habitat, without the use of pesticides,insecticides, herbicides or chemical fertilizers. Formulated by Ayurvedic and Naturopathi doctors, and made from standardized herbal extracts to ensure qualityand potency, Ayush product are safe, effective and a natural part of a healthylife style.

Ayush is working consistently for further improving the quality of its productsby incorporating the most modern biotechnology in Ayurveda to offer the highestpercentage of active principle with consistency, potency. All the formulations have strong synergistic effect.

Last year the Indian Government has recognized Ayush Herbs Pvt. Ltd. for hard work, product excellence, technological innovation and the company

s commitment for helping local farmers in (the Himalayan foothills) growing Ayurvedic medicinalHerbs as a lucrative alternative to the standard grain crops of the region.

We feel immense pleasure and pride in introducing our new range of products withthe faith that these would be met with the same zeal and enthusiasm. Ayush hopes these will undoubtedly convince people of the entire country about the effectiveness of these medicines. Ayush is also grateful to physicians for the patronage and support of all users through out the world.he company has received the following National Awards which is a representativeof our commitment towards quality:RASTRYA GAURAV AWARD-97

21ST CENTURY EXCELLENCE AWARDBEST INDUSTRY AWARD-97UDYOG PATRA AWARD-2000( This award is given to self dependent / made successful Industrialists / entrepreneurs of India.It was awarded to 30 people only, all over India, including us

RAW MATERIAL SOURCING :Apart from market sourcing we do procure our raw materials from our own farms. In addition to this we have exclusive production agreements with farmers and someNGOs. The preview of our farms, production and packaging facilities are as follows :

RAW MATERIAL :Quality of input is of immense importance for any process to achieve desired results. Keeping this principle in mind we follow stringent code for ensuring the highest quality of raw material for our production.PRODUCTION FACILITIES :Our production facilities are amongst the best w.R.T latest equipments, technology, hygienic conditions. Whole of the production process is completed under thesupervision of highly qualified team of people.

Products :Manufacturers and exporters of AYURVEDIC HERBAL EXTRACTS, ESSENTIAL OILS, PHYTOCHEMICALS, OLEORESINS AND AYURVEDIC MEDICINES. Our Products :- Herbal Extracts :- Abroma augusta Linn Acacia jacquemontii Cacia concina Achyranthes aspera Linn

Aconitum heterophylum Wall Acorus calamus Linn Adhatoda vasica Aegle marmelos Allium sativum Linn Aloe barbadensis Aloe vera Alpinia galanga Anacyclus pyrethrumAndrographis paniculata Asclepias curassavica Asparagus racemosus Asteracantha



longifolia Azadirachta indica Bacopa monnieri Bauhinia variegata Essential Oils:- Angelica Linn. Ocimum basilicum Oleum cardamomi Daucus sativa Juniperus virginiana Cedrus deodara Apium graveolens Cymbopogon nardus Syzygium aromaticum Coriandrum sativum Allium sativum Pelargonium quercifolium Zingiber officinale Juniperus macropoda Juniperus macropoda Cinnamomum camphora Cymbopogon flexuosus Cyperus rotundus Rosa alba Santalum album Tagetes erecta Curcuma domestica Valerianajatamansi Phytochemicals :- Reserpine IP/USP/BP Curcumin Hyosine Butyl Bromide

BP Colchicine BP/USP Methoxsalen USP Podophyllum resin BP Hyosciamin Forskosllin90% Oleoresins :- Zingiber officinale Curcuma domestica Capsicum annum Piper nigrum Podophyllum hexandrum Coriandrum sativum Amomum aromaticum Cinnamon tamla Cedrus deodara (Roxb) Ocimum sanctum Bacopa monnieri Eclipta alba Withania somnifera Glycyrrhiza glabra Myristica fragrans Foeniculum vulgare Thymus vulgaris Natural Colors & Dyes :- Annatto Tree seeds (Bixa orellana) Red Sandalwood (Pterocarpus santalinus )Red colour Indian Madder/ Majith root (Rubia cardifolia Turmeric rhizomes (Curcuma longa) Indigofera tintorial-water/oil soluble Green color (Chlorophyll) Walnut peel - water soluble Pharmaceuticals :- AP Mag Trifla Bio-Gymnema Face Packs :- Milky Soft Face Pack Oil Clear Face Pack Smooth Cresent FacePack Oils :- Amla Oil Stress Oil Hair Oil

LABORATORY INSTRUMENT

ColorimeterA colorimeter is a device used in colorimetry. In scientific fields the word generally refers to the device that measures the absorbance of particular wavelengths of light by a specific solution. This device is most commonly used to determine the concentration of a known solute in a given solution by the application ofthe Beer-Lambert law, which states that the concentration of a solute is proportional to the absorbance.The essential parts of a colorimeter are:

a light source (often an ordinary low-voltage filament lamp)an adjustable aperture

a set of colored filtersa cuvette to hold the working solutiona detector (usually a photoresistor) to measure the transmitted lighta meter to display the output from the detectorIn 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.FiltersChangeable optics filters are used in the colorimeter to select the wavelength o

f light which the solute absorbs the most, in order to maximize accuracy. The usual wavelength range is from 400 to 700 nanometres (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.

CuvettesIn 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.

OutputThe output from a colorimeter may be displayed by an analogue or digital meter a

nd may be shown as transmittance (a linear scale from 0-100%) or as absorbance (a logarithmic scale from zero to infinity). The useful range of the absorbance scale is from 0-2 but it is desirable to keep within the range 0-1 because, above



1, the results become unreliable due to scattering of light.

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

Magnetic stirrerA magnetic stirrer or magnetic mixer is a laboratory device that employs a rotat

ing magnetic field to cause a stir bar (also called "flea") immersed in a liquidto spin very quickly, thus stirring it. The rotating field may be created either by a rotating magnet or a set of stationary electromagnets, placed beneath thevessel with the liquid. Magnetic stirrers often include a hot plate or some other means for heating the liquid.

Magnetic stirrers are often used in chemistry and biology. They are preferred over gear-driven motorized stirrers because they are quieter, more efficient, andhave no moving external parts to break or wear out (other than the simple bar magnet itself). Due to its small size, a stirring bar is more easily cleaned and sterilized than other stirring devices. They do not require lubricants which could contaminate the reaction vessel and the product. They can be used inside herme

tically closed vessels or systems, without the need for complicated rotary seals.

On the other hand, the limited size of the bar means that magnetic stirrers canonly be used for relatively small (under 4 liters) experiments. They also have difficulty dealing with viscous liquids or thick suspensions.Melting point apparatusA melting point apparatus is a scientific instrument used to determine the melting point of a substanceDesignWhile the outward designs of apparatuses can vary greatly most apparatuses use asample loaded into a sealed capillary (melting point capillary) that is then placed in the apparatus. The sample is then heated, either by a heating block or a

n oil bath, and as the temperature increases the sample is observed to determinewhen the phase change from solid to liquid occurs. The operator or the machinerecords the temperature range starting with the initial phase change temperatureand ending with the completed phase change temperature. The temperature range that is determined can then be averaged to gain the melting point of the sample being examined.

Apparatuses usually have a control panel that allows the starting and final temperatures, as well as the temperature gradient (in units per minute) to be programmed. Some machines have several channels which permit more than one sample to be tested at a time. The control panel might have buttons which allow the start and end of the melting point range to be recorded.

Apparatuses

Thiele tubeA Thiele tube is a glass instrument that is filled with oil that is heated by using an open flame. The sample is placed in the opening in a capillary tube alongside a mercury thermometer and allowed to be heated by the oil as it circulatesthrough the Thiele tube. By using different oils different temperature ranges can be reached and used to determine melting points. The Thiele tube may also be used to determine boiling points, by substituting a solid sample for a liquid one.

PolarimeterA polarimeter is a scientific instrument used to measure the angle of rotation caused by passing polarized light through an optically active substance.



Some chemical substances are optically active, and polarized (aka unidirectional) light will rotate either to the left (counter-clockwise) or right (clockwise)when passed through these substances. The amount by which the light is rotated is known as the angle of rotation.

Polarimeters measure this by passing monochromatic light through the first of tw

o polarizing plates, creating a polarized beam. This first plate is known as thepolarizer. This beam is then rotated as it passes through the sample. The sample is usually prepared as a tube where the optically active substance is dissolved in an optically inactive chemical such as distilled water or methanol. Some polarimeters can be fitted with tubes that allow for sample to flow through continuously.

After passing through the sample, a second polarizer, known as the analyzer, rotates either via manual rotation or automatic detection of the angle. When the analyzer is rotated to the proper angle, the maximum amount of light will pass through and shine onto a detector.

The earliest polarimeters, which date back to the 1830s, required the user to aphysically rotate the analyzer, and the detector was the user’s eye judging when the most light shone through. The angle was marked on a scale that encircles theanalyzer. This basic design is still used in the simplest polarimeters.

Today there are also semi-automatic polarimeters, which require visual detectionbut use push-buttons to rotate the analyzer and offer digital displays. The most modern polarimeters are fully automatic, and simply require the user to pressa button and wait for a digital readout.

The angle of rotation of an optically active substance can be affected by:

Concentration of the sample

Wavelength of light passing through the sample (generally, angle of rotation andwavelength tend to be inversely proportional)Temperature of the sample (generally the two are directly proportional)Length of the sample cell (input by the user into most automatic polarimeters toensure better accuracy)Most modern polarimeters have methods of compensating for or controlling these.

Polarimeters can be calibrated – or at least verified – by measuring a quartz plate,which is constructed to always read at a certain angle of rotation (usually +34°,but +17° and +8.5° are also popular depending on the sample). Quartz plates are preferred by many users because solid samples are much less affected by variationsin temperature, and do not need to be mixed on-demand like sucrose solutions. Ap

plications Because many optically active chemicals are stereoisomers, a polarimeter can be used to identify which isomer is present in a sample – if it rotates polarized light to the left, it it a levo-isomer, and to the right, a dextro-isomer.

Many chemicals exhibit a specific rotation as a unique property (like refractiveindex in many cases) which can be used to distinguish it. Polarimeters can identify unknown samples based on this if other variables such as concentration andlength of sample cell length are controlled or at least known. This is used in the chemical industry

By the same token, if the specific rotation of a sample is already known, then the concentration and/or purity of a solution containing it can be calculated.

Most automatic polarimeters make this calculation automatically, given input onvariables from the user.



Concentration and purity measurements are especially important to determine product or ingredient quality in the food & beverage and pharmaceutical industries.Samples that display specific rotations that can be calculated for purity with apolarimeter include:SteroidsDiuretics

AntibioticsNarcoticsVitaminsAnalgesicsAmino AcidsEssential OilsPolymersStarchesSugarsPolarimeters are used in the sugar industry for determining quality of both juice from sugar cane and the refined sucrose. Often, the sugar refineries use a modified polarimeter with a flow cell called a saccharimeter. These instruments use

the International Sugar Scale (as defined by ICUMSHot air ovenHot air ovens are electrical devices used in sterilization. The oven uses dry heat to sterilize articles. Generally, they can be operated from 50 to 300 °C (122 to 572 °F) . There is a thermostat controlling the temperature. These are digitallycontrolled to maintain the temperature. Their double walled insulation keeps the heat in and conserves energy, the inner layer being a poor conductor and outerlayer being metallic. There is also an air filled space in between to aid insulation. An air circulating fan helps in uniform distribution of the heat. These are fitted with the adjustable wire mesh plated trays or aluminium trays and mayhave an on/off rocker switch, as well as indicators and controls for temperatureand holding time. The capacities of these ovens vary. Power supply needs vary from country to country, depending on the voltage and frequency (hertz) used. Tem

perature sensitive tapes or other devices like those using bacterial spores canbe used to work as controls, to test for the efficacy of the device in every cycleA complete cycle involves heating the oven to the required temperature, maintaining that temperature for the proper time interval for that temperature, turningthe machine off and cooling the articles in the closed oven till they reach roomtemperature. The standard settings for a hot air oven are:Usage1.5 to 2 hours at 160 °C (320 °F)6 to 12 minutes at 190 °C (374 °F)....plus the time required to preheat the chamber before beginning the sterilization cycle. If the door is opened before time, heat escapes and the process beco

mes incomplete. Thus the cycle must be properly repeated all over.

These are widely used to sterilize articles that can withstand high temperaturesand not get burnt, like glassware and powders. Linen gets burnt and surgical sharps lose their sharpness.

Thin layer chromatography Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures.Thin layer chromatography is performed on a sheet of glass, plastic, oraluminum foil, which is coated with a thin layer of adsorbent material, usuallysilica gel, aluminium oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary phase.

After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because dif



ferent analytes ascend the TLC plate at different rates, separation is achieved.

Hence the differential traveling speeds (and thus final locations) of differentcomponents is based in comparing their different solubilities (to the mobile phase) and strength of adsorption (to the stationary phase). This determines theirRf, or retention factor (relative distance traveled of compound vs solvent).

Thin layer chromatography can be used to:

* Monitor the progress of a reaction* Identify compounds present in a given substance* Determine the purity of a substance

Specific examples of these applications include:

* determination of the components a plant contains* analyzing ceramides and fatty acids* detection of pesticides or insecticides in food and water* analyzing the dye composition of fibers in forensics, or

* assaying the radiochemical purity of radiopharmaceuticals

A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantitation. This method is referred to as HPTLC, or "high performance TLC".Plate preparationTLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. They are prepared by mixing the adsorbent, such assilica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is driedand activated by heating in an oven for thirty minutes at 110 °C. The thickness of

the adsorbent layer is typically around 0.1 – 0.25 mm for analytical purposes andaround 0.5 – 2.0 mm for preparative TLCTechniqueThe process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.

To run a TLC, the following procedure is carried out:

* A small spot of solution containing the sample is applied to a plate, about 1.5 centimeters from the bottom edge. The solvent is allowed to completely eva

porate off, otherwise a very poor or no separation will be achieved. If a non-volatile solvent was used to apply the sample, the plate needs to be dried in a vacuum chamber.

* A small amount of an appropriate solvent (elutant) is poured in to a glassbeaker or any other suitable transparent container (separation chamber) to a depth of less that 1 centimeter. A strip of filter paper is put into the chamber,so that its bottom touches the solvent, and the paper lies on the chamber wall and reaches almost to the top of the container. The container is closed with a cover glass or any other lid and is left for a few minuted to let the solvent vapors ascend the filter paper and saturate the air in the chamber. (Failure to saturate the chamber will result in poor separation and non-reproducible results).

* The TLC plate is then placed in the chamber so that the spot(s) of the sample DO NOT TOUCH the surface of the elutant in the chamber, and the lid is close

d. The solvent moves up the plate by capillary action, meets the sample mixtureand carries it up the plate (elutes the sample). When the solvent front reachesno higher than the top of the filter paper in the chamber, the plate should be r



emoved (continue the elution will give a misleading results) and dried.

Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent. By changing the solvent, or perhaps using a mixture, the separation of components (measured by the Rf value) can be adjusted. Also, the separation achieved with a TLC plate can be used to estimate the separa

tion of a flash chromatography column.

Separation of compounds is based on the competition of the solute and the mobilephase for binding places on the stationary phase. For instance, if normal phasesilica gel is used as the stationary phase it can be considered polar. Given two compounds which differ in polarity, the more polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the binding places. Consequently, the less polar compound moves higher upthe plate (resulting in a higher Rf value). If the mobile phase is changed to amore polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will movehigher up the plate. It is commonly said that "strong" solvents (elutants) push

the analyzed compounds up the plate, while "weak" elutants barely move them. The order of strength/weakness depends on the coating (stationary phase) of the TLC plate. For silica gel coated TLC plates, the elutant strength increases in thefollowing order: Perfluoroalkane (weakest), Hexane, Pentane, Carbon tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl ether, Ethylacetate, Acetonitrile,Acetone, 2-Propanol/n-Butanol, Water, Methanol, Triethylamine, Acetic acid, Formic acid (strongest). For C18 coated plates the order is reverse. Practically this means that if you use a mixture of ethyl acetate and heptane as the mobile phase, adding more ethyl acetate results in higher Rf values for all compounds onthe TLC plate. Changing the polarity of the mobile phase will normally not result in reversed order of running of the compounds on the TLC plate. An eluotropicseries can be used as a guide in selecting a mobile phase. If a reversed order of running of the compounds is desired, an apolar stationary phase should be used

, such as C18-functionalized silica.Preparative TLCTLC can also be used on a small semi-preparative scale to separate mixtures of up to a few hundred milligrams. The mixture is not "spotted" on the TLC plate asdots, but rather is applied to the plate as a thin even layer horizontally to and just above the solvent level. When developed with solvent the compounds separate in horizontal bands rather than horizontally separated spots. Each band (or adesired band) is scraped off the backing material. The backing material is thenextracted with a suitable solvent (e.g. DCM) and filtered to give the isolatedmaterial upon removal of the solvent. For small-scale reactions with easily separated products, preparative TLC can be a far more efficient in terms of time andcost than doing column chromatography. Obviously, the whole plate can not be ch

emically developed or the product will be chemically destroyed. Thus this technique is best used with compounds that are coloured, or visible under UV light. Alternatively, a small section of the plate can be chemically developed e.g. cutting a section out and chemically developing it, or masking most of the plate andexposing a small section to a chemical developer like iodineAnalysisAs the chemicals being separated may be colorless, several methods exist to visualize the spots:

* Often a small amount of a fluorescent compound, usually manganese-activated zinc silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV254). The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.

* Iodine vapors are a general unspecific color reagent* Specific color reagents exist into which the TLC plate is dipped or which

are sprayed onto the plate



* In the case of lipids, the chromatogram may be transferred to a PVDF membrane and then subjected to further analysis, for example mass spectrometry, a technique known as Far-Eastern blotting.

Once visible, the Rf value , or retention factor, of each spot can be determinedby dividing the distance traveled by the product by the total distance traveledby the solvent (the solvent front). These values depend on the solvent used, an

d the type of TLC plate, and are not physical constants. Eluent on the thin layer is put on top of the plateApplicationsIn organic chemistry, reactions are qualitatively monitored with TLC. Spots sampled with a capillary tube are placed on the plate: a spot of starting material,a spot from the reaction mixture, and a "co-spot" with both. A small (3 by 7 cm)TLC plate takes a couple of minutes to run. The analysis is qualitative, and itwill show if the starting material has disappeared, i.e. the reaction is complete, if any product has appeared, and how many products are generated (although this might be under-estimated due to co-elution). Unfortunately, TLCs from low-temperature reactions may give misleading results, because the sample is warmed toroom temperature in the capillary, which can alter the reaction—the warmed sample

analyzed by TLC is not the same as what is in the low-temperature flask. One such reaction is the DIBALH reduction of ester to aldehyde.

As an example the chromatography of an extract of green leaves , Carotene elutesquickly and is only visible until step 2. Chlorophyll A and B are halfway in the final step and lutein the first compound staining

In one study TLC has been applied in the screening of organic reactions for example in the fine-tuning of BINAP synthesis from 2-naphthol. In this method the alcohol and catalyst solution (for instance iron(III) chloride) are place separately on the base line, then reacted and then instantly analyzed.Column chromatographyColumn chromatography in chemistry is a method used to purify individual chemica

l compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms up to kilograms.

The classical preparative chromatography column, is a glass tube with a diameterfrom 5 mm to 50 mm and a height of 50 cm to 1 m with a tap at the bottom. Two methods are generally used to prepare a column; the dry method, and the wet method. For the dry method, the column is first filled with dry stationary phase powder, followed by the addition of mobile phase, which is flushed through the column until it is completely wet, and from this point is never allowed to run dry. For the wet method, a slurry is prepared of the eluent with the stationary phasepowder and then carefully poured into the column. Care must be taken to avoid air bubbles. A solution of the organic material is pipetted on top of the stationa

ry phase. This layer is usually topped with a small layer of sand or with cottonor glass wool to protect the shape of the organic layer from the velocity of newly added eluent. Eluent is slowly passed through the column to advance the organic material. Often a spherical eluent reservoir or an eluent-filled and stoppered separating funnel is put on top of the column.

The individual components are retained by the stationary phase differently and separate from each other while they are running at different speeds through the column with the eluent. At the end of the column they elute one at a time. Duringthe entire chromatography process the eluent is collected in a series of fractions. The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds, e.g. by analytical chromatography, UV absorption, or fluorescence. Colored compounds (or fluorescent compounds with the aid of a

n UV lamp) can be seen through the glass wall as moving bands.



Stationary phaseThe stationary phase or adsorbent in column chromatography is a solid. The mostcommon stationary phase for column chromatography is silica gel, followed by alumina. Cellulose powder has often been used in the past. Also possible are ion exchange chromatography, reversed-phase chromatography (RP), affinity chromatography or expanded bed adsorption (EBA). The stationary phases are usually finely ground powders or gels and/or are microporous for an increased surface, though in

EBA a fluidized bed is used.Mobile phase (eluent)The mobile phase or eluent is either a pure solvent or a mixture of different solvents. It is chosen so that the retention factor value of the compound of interest is roughly around 0.2 - 0.3 in order to minimize the time and the amount ofeluent to run the chromatography. The eluent has also been chosen so that the different compounds can be separated effectively. The eluent is optimized in smallscale pretests, often using thin layer chromatography (TLC) with the same stationary phase.

A faster flow rate of the eluent minimizes the time required to run a column andthereby minimizes diffusion, resulting in a better separation, see Van Deemter

s equation. A simple laboratory column runs by gravity flow. The flow rate of such a column can be increased by extending the fresh eluent filled column above the top of the stationary phase or decreased by the tap controls. Better flow rates can be achieved by using a pump or by using compressed gas (e.g. air, nitrogen, or argon) to push the solvent through the column (flash column chromatography

The particle size of the stationary phase is generally finer in flash column chromatography than in gravity column chromatography. For example, one of the mostwidely used silica gel grades in the former technique is mesh 230 – 400 (40 – 63 µm),while the latter technique typically requires mesh 70 – 230 (63 – 200 µm) silica gel.

A spreadsheet that assists in the successful development of flash columns has been developed. The spreadsheet estimates the retention volume and band volume of

analytes, the fraction numbers expected to contain each analyte, and the resolution between adjacent peaks. This information allows users to select optimal parameters for preparative-scale separations before the flash column itself is attempted.Column Chromatogram Resolution CalculationTypically, column chromatography is set up with peristaltic pumps flowing buffers and the solution sample through the top of the column. The solutions and buffers pass through the column where a fraction collector at the end of the column setup collects the eluted samples. Prior to the fraction collection, the samplesthat are eluted from the column pass through a detector such as a spectrophotometer or mass spectrometer so that the concentration of the separated samples in the sample solution mixture can be determined.

For example, if you were to separate two different proteins with different binding capacities to the column from a solution sample, a good type of detector would be a spectrophotometer using a wavelength of 280 nm. The higher the concentration of protein that passes through the eluted solution through the column, the higher the absorbance of that wavelength.Because the column chromatography has a constant flow of eluted solution passingthrough the detector at varying concentrations, the detector must plot the concentration of the eluted sample over a course of time. This plot of sample concentration versus time is called a chromatogram.

The ultimate goal of chromatography is to separate different components from a solution mixture. The resolution expresses the extent of separation between the c

omponents from the mixture. The higher the resolution of the chromatogram, the better the extent of separation of the samples the column gives. This data is a good way of determining the column’s separation properties of that particular sampl



e. The resolution can be calculated from the chromatogram.The separate curves in the diagram represent different sample elution concentration profiles over time based on their affinity to the column resin. To calculateresolution, the retention time and curve width are required.

Retention Time: The time from the start of signal detection by the detector to the peak height of the elution concentration profile of each different sample.

Curve Width: The width of the concentration profile curve of the different samples in the chromatogram in units of time.

A simplified method of calculating chromatogram resolution is to use the plate model[6]. The plate model assumes that the column can be divided into a certain number of sections, or plates and the mass balance can be calculated for each individual plate. This approach approximates a typical chromatogram curve as a Gaussian distribution curve. By doing this, the curve width is estimated as 4 timesthe standard deviation of the curve, 4σ. The retention time i ¡ the time from the ¡

tart of ¡ ignal detection to the time of the peak height of the Gau ¡ ¡ ian curve.

From the variable

¡

in the figure above, the re

¡

olution, plate number, and plateheight of the column plate model can be calculated u ¡ ing the equation ¡ :

Re ¡ olution (R ¡ ):R ¡ = 2(tRB – tRA)/(wB + wA)Where:tRB = retention time of ¡ olute BtRA = retention time of ¡ olute AwB = Gau ¡ ¡ ian curve width of ¡ olute BwA = Gau ¡ ¡ ian curve width of ¡ olute APlate Number (N):N = (tR2)/(w/4)2Plate Height (H):

H = L/NWhere L i ¡ the length of the column.Column Ad ¡ orption EquilibriumFor an ad ¡ orption column, the column re ¡ in (the ¡ tationary pha ¡ e) i ¡ compo ¡ ed ofmicrobead ¡ . Even ¡ maller particle ¡ ¡ uch a ¡ protein ¡ , carbohydrate ¡ , metal ion ¡ ,or other chemical compound ¡ are conjugated onto the microbead ¡ . Each binding particle that i ¡ attached to the microbead can be a ¡ ¡ umed to bind in a 1:1 ratio with the ¡ olute ¡ ample ¡ ent through the column that need ¡ to be purified or ¡ eparated.

Binding between the target molecule to be ¡ eparated and the binding molecule onthe column bead ¡ can be modeled u ¡ ing a ¡ imple equilibrium reaction Keq = [CS]/(

[C][S]) where Keq i

¡

the equilibrium con

¡

tant, [C] and [S] are the concentration¡ of the target molecule and the binding molecule on the column re ¡ in, re ¡ pectively. [CS] i ¡ the concentration of the complex of the target molecule bound to the column re ¡ in.[6]

U ¡ ing thi ¡ a ¡ a ba ¡ i ¡ , three different i ¡ otherm ¡ can be u ¡ ed to de ¡ cribe the binding dynamic ¡ of a column chromatography: linear, Langmuir, and Freundlich.

The linear i ¡ otherm occur ¡ when the ¡ olute concentration needed to be purified i¡ very ¡ mall relative to the binding molecule of the. Thu ¡ , the equilibrium canbe defined a ¡ :[CS] = Keq[C].

For indu¡

trial¡

cale u¡

e¡

, the total binding molecule¡

on the column re¡

in bead¡

mu ¡ t be factored in becau ¡ e unoccupied ¡ ite ¡ mu ¡ t be taken into account. The Langmuir i ¡ otherm and Freundlich i ¡ otherm are u ¡ eful in de ¡ cribing thi ¡ equilibriu



m. Langmuir I ¡ otherm:[CS] = (KeqStot[C])/(1 + Keq[C]), where Stot i ¡ the total binding molecule ¡ on the bead ¡ .

Freundlich I ¡ otherm:[CS] = Keq[C]1/n

The Freundlich i¡

otherm i¡

u¡

ed when the column can bind to many different¡

ample ¡ in the ¡ olution that need ¡ to be purified. Becau ¡ e the many different ¡ ample ¡

have different binding con ¡ tant ¡ to the bead ¡ , there are many different Keq’ ¡ . Therefore, the Langmuir i ¡ otherm i ¡ not a good model for binding in thi ¡ ca ¡ eHigh-performance liquid chromatographyHigh-performance liquid chromatography (or high-pre ¡ ¡ ure liquid chromatography,HPLC) i ¡ a chromatographic technique that can ¡ eparate a mixture of compound ¡ , and i ¡ u ¡ ed in biochemi ¡ try and analytical chemi ¡ try to identify, quantify and purify the individual component ¡ of the mixture.

HPLC utilize ¡ different type ¡ of ¡ tationary pha ¡ e (typically, hydrophobic ¡ aturated carbon chain ¡ ), a pump that move ¡ the mobile pha ¡ e( ¡ ) and analyte through th

e column, and a detector that provide

¡

a characteri

¡

tic retention time for the analyte. The detector may al ¡ o provide other characteri ¡ tic information (i.e. UV/Vi ¡ ¡ pectro ¡ copic data for analyte if ¡ o equipped). Analyte retention time varie

¡ depending on the ¡ trength of it ¡ interaction ¡ with the ¡ tationary pha ¡ e, the ratio/compo ¡ ition of ¡ olvent( ¡ ) u ¡ ed, and the flow rate of the mobile pha ¡ e.

With HPLC, a pump (rather than gravity) provide ¡ the higher pre ¡ ¡ ure required topropel the mobile pha ¡ e and analyte through the den ¡ ely packed column. The increa ¡ ed den ¡ ity ari ¡ e ¡ from ¡ maller particle ¡ ize ¡ . Thi ¡ allow ¡ for a better ¡ eparation on column ¡ of ¡ horter length when compared to ordinary column chromatography.OperationThe ¡ ample to be analyzed i ¡ introduced in ¡ mall volume to the ¡ tream of mobile

pha¡

e. The analyte

¡

motion through the column i¡

¡

lowed by¡

pecific chemical orphy ¡ ical interaction ¡ with the ¡ tationary pha ¡ e a ¡ it traver ¡ e ¡ the length of the column. How much the analyte i ¡ ¡ lowed depend ¡ on the nature of the analyte and on the compo ¡ ition ¡ of the ¡ tationary and mobile pha ¡ e ¡ . The time at which a

¡ pecific analyte elute ¡ (come ¡ out of the end of the column) i ¡ called the retention time; the retention time under particular condition ¡ i ¡ con ¡ idered a rea ¡ onably unique identifying characteri ¡ tic of a given analyte. The u ¡ e of ¡ maller particle ¡ ize column packing (which create ¡ higher backpre ¡ ¡ ure) increa ¡ e ¡ the linear velocity giving the component ¡ le ¡ ¡ time to diffu ¡ e within the column, leading to improved re ¡ olution in the re ¡ ulting chromatogram. Common ¡ olvent ¡ u ¡ ed include any mi ¡ cible combination of water or variou ¡ organic liquid ¡ (the mo ¡ t common are methanol and acetonitrile). Water may contain buffer ¡ or ¡ alt ¡ to a ¡ ¡ i ¡ t

in the

¡

eparation of the analyte component

¡

, or compound

¡

¡

uch a

¡

trifluoroacetic acid which act ¡ a ¡ an ion pairing agent.

A further refinement to HPLC ha ¡ been to vary the mobile pha ¡ e compo ¡ ition during the analy ¡ i ¡ ; thi ¡ i ¡ known a ¡ gradient elution. A normal gradient for rever ¡ ed pha ¡ e chromatography might ¡ tart at 5% methanol and progre ¡ ¡ linearly to 50% methanol over 25 minute ¡ ; the gradient cho ¡ en depend ¡ on how hydrophobic the analyte i ¡ . The gradient ¡ eparate ¡ the analyte mixture ¡ a ¡ a function of the affinity of the analyte for the current mobile pha ¡ e compo ¡ ition relative to the ¡ tationary pha ¡ e. Thi ¡ partitioning proce ¡ ¡ i ¡ ¡ imilar to that which occur ¡ during a liquid-liquid extraction but i ¡ continuou ¡ , not ¡ tep-wi ¡ e. In thi ¡ example, u ¡ inga water/methanol gradient, the more hydrophobic component ¡ will elute (come offthe column) when the mobile pha ¡ e con ¡ i ¡ t ¡ mo ¡ tly of methanol (giving a relativ

ely hydrophobic mobile pha¡

e). The more hydrophilic compound¡

will elute under condition ¡ of relatively low methanol/high water.



The choice of ¡ olvent ¡ , additive ¡ and gradient depend on the nature of the ¡ tationary pha ¡ e and the analyte. Often a ¡ erie ¡ of te ¡ t ¡ are performed on the analyte and a number of trial run ¡ may be proce ¡ ¡ ed in order to find the HPLC method which give ¡ the be ¡ t ¡ eparation of peak ¡

Type ¡

Partition chromatography

Partition chromatography wa¡

the fir¡

t kind of chromatography that chemi¡

t¡

developed. The partition coefficient principle ha ¡ been applied in paper chromatography, thin layer chromatography, ga ¡ pha ¡ e and liquid-liquid application ¡ . The 1952 Nobel Prize in chemi ¡ try wa ¡ earned by Archer John Porter Martin and RichardLaurence Millington Synge for their development of the technique, which wa ¡ u ¡ edfor their ¡ eparation of amino acid ¡ . Partition chromatography u ¡ e ¡ a retained ¡

olvent, on the ¡ urface or within the grain ¡ or fibre ¡ of an "inert" ¡ olid ¡ upporting matrix a ¡ with paper chromatography; or take ¡ advantage of ¡ ome additionalcoulombic and/or hydrogen donor interaction with the ¡ olid ¡ upport. Molecule ¡ equilibrate (partition) between a liquid ¡ tationary pha ¡ e and the eluent. Known a ¡

Hydrophilic Interaction Chromatography (HILIC) in HPLC, thi ¡ method ¡ eparate ¡ analyte ¡ ba ¡ ed on polar difference ¡ . HILIC mo ¡ t often u ¡ e ¡ a bonded polar ¡ tation

ary pha

¡

e and a non-polar, water mi

¡

cible, mobile pha

¡

e. Partition HPLC ha

¡

beenu ¡ ed hi ¡ torically on unbonded ¡ ilica or alumina ¡ upport ¡ . Each work ¡ effectively for ¡ eparating analyte ¡ by relative polar difference ¡ , however, HILIC ha ¡ theadvantage of ¡ eparating acidic, ba ¡ ic and neutral ¡ olute ¡ in a ¡ ingle chromatogram.

The polar analyte ¡ diffu ¡ e into a ¡ tationary water layer a ¡ ¡ ociated with the polar ¡ tationary pha ¡ e and are thu ¡ retained. Retention ¡ trength ¡ increa ¡ e with increa ¡ ed analyte polarity, and the interaction between the polar analyte and the polar ¡ tationary pha ¡ e (relative to the mobile pha ¡ e) increa ¡ e ¡ the elution time.The interaction ¡ trength depend ¡ on the functional group ¡ in the analyte molecule which promote partitioning but can al ¡ o include coulombic (electro ¡ tatic) interaction and hydrogen donor capability.

U¡

e of more polar¡

olvent¡

in the mobile pha¡

e will decrea¡

e the retention timeof the analyte ¡ , wherea ¡ more hydrophobic ¡ olvent ¡ tend to increa ¡ e retention time ¡ .

Partition and NP-HPLC had fallen out of favor in the 1970 ¡ with the developmentof rever ¡ ed-pha ¡ e HPLC becau ¡ e of a lack of reproducibility of retention time ¡ a

¡ water or protic organic ¡ olvent ¡ changed the hydration ¡ tate of the ¡ ilica oralumina chromatographic media. Recently it ha ¡ become u ¡ eful again with the development of HILIC bonded pha ¡ e ¡ which improve reproducibility.Normal-pha ¡ e chromatographyAl ¡ o known a ¡ normal-pha ¡ e HPLC (NP-HPLC), or ad ¡ orption chromatography, thi ¡ method ¡ eparate ¡ analyte ¡ ba ¡ ed on ad ¡ orption to a ¡ tationary ¡ urface chemi ¡ try an

d by polarity. It wa

¡

one of the fir

¡

t kind

¡

of HPLC that chemi

¡

t

¡

developed. NP-HPLC u ¡ e ¡ a polar ¡ tationary pha ¡ e and a non-polar, non-aqueou ¡ mobile pha ¡ e, and work ¡ effectively for ¡ eparating analyte ¡ readily ¡ oluble in non-polar ¡ olvent ¡ . The analyte a ¡ ¡ ociate ¡ with and i ¡ retained by the polar ¡ tationary pha ¡ e. Ad ¡ orption ¡ trength ¡ increa ¡ e with increa ¡ ed analyte polarity, and the interaction between the polar analyte and the polar ¡ tationary pha ¡ e (relative to the mobile pha ¡ e) increa ¡ e ¡ the elution time. The interaction ¡ trength depend ¡ not onlyon the functional group ¡ in the analyte molecule, but al ¡ o on ¡ teric factor ¡ . The effect of ¡ teric ¡ on interaction ¡ trength allow ¡ thi ¡ method to re ¡ olve ( ¡ eparate) ¡ tructural i ¡ omer ¡ .

The u ¡ e of more polar ¡ olvent ¡ in the mobile pha ¡ e will decrea ¡ e the retention time of the analyte ¡ , wherea ¡ more hydrophobic ¡ olvent ¡ tend to increa ¡ e retentio

n time¡

. Very polar¡

olvent¡

in a mixture tend to deactivate the¡

tationary pha¡

e by creating a ¡ tationary bound water layer on the ¡ tationary pha ¡ e ¡ urface. Thi ¡ behavior i ¡ ¡ omewhat peculiar to normal pha ¡ e becau ¡ e it i ¡ mo ¡ t purely an ad



¡ orptive mechani ¡ m (the interaction ¡ are with a hard ¡ urface rather than a ¡ oftlayer on a ¡ urface).

NP-HPLC fell out of favor in the 1970 ¡ with the development of rever ¡ ed-pha ¡ e HPLC becau ¡ e of a lack of reproducibility of retention time ¡ a ¡ water or protic organic ¡ olvent ¡ changed the hydration ¡ tate of the ¡ ilica or alumina chromatographic media. Recently it ha ¡ become u ¡ eful again with the development of HILIC bon

ded pha¡

e¡

which improve reproducibility.Di ¡ placement chromatography

The ba ¡ ic principle of di ¡ placement chromatography i ¡ : A molecule with a high affinity for the chromatography matrix (the di ¡ placer) will compete effectively for binding ¡ ite ¡ , and thu ¡ di ¡ place all molecule ¡ with le ¡ ¡ er affinitie ¡ .[1] There are di ¡ tinct difference ¡ between di ¡ placement and elution chromatography. In elution mode, ¡ ub ¡ tance ¡ typically emerge from a column in narrow, Gau ¡ ¡ ian peak ¡

. Wide ¡ eparation of peak ¡ , preferably to ba ¡ eline, i ¡ de ¡ ired in order to achieve maximum purification. The ¡ peed at which any component of a mixture travel ¡ down the column in elution mode depend ¡ on many factor ¡ . But for two ¡ ub ¡ tance ¡ to travel at different ¡ peed ¡ , and thereby be re ¡ olved, there mu ¡ t be ¡ ub ¡ tantial

difference

¡

in

¡

ome interaction between the biomolecule

¡

and the chromatographymatrix. Operating parameter ¡ are adju ¡ ted to maximize the effect of thi ¡ difference. In many ca ¡ e ¡ , ba ¡ eline ¡ eparation of the peak ¡ can be achieved only withgradient elution and low column loading ¡ . Thu ¡ , two drawback ¡ to elution mode chromatography, e ¡ pecially at the preparative ¡ cale, are operational complexity, due to gradient ¡ olvent pumping, and low throughput, due to low column loading ¡ .Di ¡ placement chromatography ha ¡ advantage ¡ over elution chromatography in that component ¡ are re ¡ olved into con ¡ ecutive zone ¡ of pure ¡ ub ¡ tance ¡ rather than “peak

¡ ”. Becau ¡ e the proce ¡ ¡ take ¡ advantage of the nonlinearity of the i ¡ otherm ¡ , a larger column feed can be ¡ eparated on a given column with the purified component ¡

recovered at ¡ ignificantly higher concentration ¡ .Rever ¡ ed-pha ¡ e chromatography (RPC)A chromatogram of complex mixture (perfume water) obtained by rever ¡ ed pha ¡ e HPL

CFor more detail ¡ on thi ¡ topic, ¡ ee Rever ¡ ed-pha ¡ e chromatography.

Rever ¡ ed pha ¡ e HPLC (RP-HPLC or RPC) ha ¡ a non-polar ¡ tationary pha ¡ e and an aqueou ¡ , moderately polar mobile pha ¡ e. One common ¡ tationary pha ¡ e i ¡ a ¡ ilica which ha ¡ been treated with RMe2SiCl, where R i ¡ a ¡ traight chain alkyl group ¡ ucha ¡ C18H37 or C8H17. With the ¡ e ¡ tationary pha ¡ e ¡ , retention time i ¡ longer for molecule ¡ which are more non-polar, while polar molecule ¡ elute more readily. Aninve ¡ tigator can increa ¡ e retention time by adding more water to the mobile pha ¡

e; thereby making the affinity of the hydrophobic analyte for the hydrophobic ¡ tationary pha ¡ e ¡ tronger relative to the now more hydrophilic mobile pha ¡ e. Similarly, an inve ¡ tigator can decrea ¡ e retention time by adding more organic ¡ olvent

to the eluent. RPC i

¡

¡

o commonly u

¡

ed that it i

¡

often incorrectly referred toa ¡ "HPLC" without further ¡ pecification. The pharmaceutical indu ¡ try regularlyemploy ¡ RPC to qualify drug ¡ before their relea ¡ e.

RPC operate ¡ on the principle of hydrophobic force ¡ , which originate from the high ¡ ymmetry in the dipolar water ¡ tructure and play the mo ¡ t important role in all proce ¡ ¡ e ¡ in life ¡ cience. RPC i ¡ allowing the mea ¡ urement of the ¡ e interactive force ¡ . The binding of the analyte to the ¡ tationary pha ¡ e i ¡ proportional tothe contact ¡ urface area around the non-polar ¡ egment of the analyte molecule upon a ¡ ¡ ociation with the ligand in the aqueou ¡ eluent. Thi ¡ ¡ olvophobic effect i

¡ dominated by the force of water for "cavity-reduction" around the analyte andthe C18-chain ver ¡ u ¡ the complex of both. The energy relea ¡ ed in thi ¡ proce ¡ ¡ i ¡

proportional to the ¡ urface ten ¡ ion of the eluent (water: 7.3 × 10−6 J/cm², methanol:

2.2 × 10−6 J/cm²) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased by adding a less polar solvent (methanol,acetonitrile) into the mobile phase to reduce the surface tension of water. Gra



dient elution uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of the analysis.

Structural properties of the analyte molecule play an important role in its retention characteristics. In general, an analyte with a larger hydrophobic surfacearea (C

¢

H, C¢

C, and generally non¢

polar atomic bonds, such as S¢

S and others) results in a longer retention time because it increases the molecule's non

¢

polar s

urface area, which is non ¢ interacting with the water structure. On the other hand, polar groups, such as

¢

OH,¢

NH2, COO¢

or¢

NH3+ reduce retention as they are well integrated into water. Very large molecules, however, can result in an incomplete interaction between the large analyte surface and the ligand's alkyl chains and can have problems entering the pores of the stationary phase.

Retention time increases with hydrophobic (non¢

polar) surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers becausethe overall surface area is decreased. Similarly organic compounds with single C

¢

C¢

bonds elute later than those with a C=C or C¢

C¢

triple bond, as the double ortriple bond is shorter than a single C

¢

C¢

bond.

Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surfacetension of aqueous solutions (ca. 1.5 × 10−7 J/cm² per Mol for NaCl, 2.5 × 10−7 J/cm² perl for (NH4)2SO4), and because the entropy of the analyte

¢

solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time. This technique is used for mild separation and recovery of proteins andprotection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC).

Another important component is the influence of the pH since this can change thehydrophobicity of the analyte. For this reason most methods use a buffering agent, such as sodium phosphate, to control the pH. The buffers serve multiple purp

oses: they control pH, neutralize the charge on any residual exposed silica on the stationary phase and act as ion pairing agents to neutralize charge on the analyte. Ammonium formate is commonly added in mass spectrometry to improve detection of certain analytes by the formation of ammonium adducts. A volatile organicacid such as acetic acid, or most commonly formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column eluent. Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such as carboxylic acids in applications utilizing other detectors, as it is one of the strongest organic acids. The effects of acids and buffers vary by application but generally improve the chromatography.

Reversed phase columns are quite difficult to damage compared with normal silicacolumns; however, many reversed phase columns consist of alkyl derivatized silica particles and should never be used with aqueous bases as these will destroy the underlying silica particle. They can be used with aqueous acid, but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment. RP

¢

HPLC columns should be flushed with clean solventafter use to remove residual acids or buffers, and stored in an appropriate composition of solvent. The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained. A good test for the metal content of a column is to inject a sample which is a mixture of 2,2'

¢

and 4,4' ¢ bipyridine. Because the 2,2' ¢ bipy can chelate the metal, the shape of the peak for the 2,2'

¢

bipy will be distorted (tailed) when metal ions are presenton the surface of the silica.[citation needed]..

Size ¢ exclusion chromatographyFor more details on this topic, see size ¢ exclusion chromatography.



Size¢

exclusion chromatography (SEC), also known as gel permeation chromatographyor gel filtration chromatography, separates particles on the basis of size. Itis generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determiningthe tertiary structure and quaternary structure of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larg

er molecules simply pass by the pores as they are too large to enter the pores.Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time.

This technique is widely used for the molecular weight determination of polysaccharides. SEC is the official technique (suggested by European pharmacopeia) forthe molecular weight comparison of different commercially available low

¢

molecular weight heparins.Ion

¢

exchange chromatography.In ion

¢

exchange chromatography, retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Ions of the same charge are excluded. Types of ion exchangers include:

* Polystyrene resins – These allow cross linkage which increases the stabilityof the chain. Higher cross linkage reduces swerving, which increases the equilibration time and ultimately improves selectivity.

* Cellulose and dextran ion exchangers (gels) – These possess larger pore sizes and low charge densities making them suitable for protein separation.

* Controlled¢

pore glass or porous silica

In general, ion exchangers favor the binding of ions of higher charge and smaller radius.

An increase in counter ion (with respect to the functional groups in resins) concentration reduces the retention time. An increase in pH reduces the retention t

ime in cation exchange while a decrease in pH reduces the retention time in anion exchange.

This form of chromatography is widely used in the following applications: waterpurification, preconcentration of trace components, ligand

¢

exchange chromatography, ion

¢

exchange chromatography of proteins, high¢

pH anion¢

exchange chromatography of carbohydrates and oligosaccharides, and others.Bioaffinity chromatography

This chromatographic process relies on the property of biologically active substances to form stable, specific, and reversible complexes. The formation of thesecomplexes involves the participation of common molecular forces such as the Vander Waals interaction, electrostatic interaction, dipole

¢

dipole interaction, hydrophobic interaction, and the hydrogen bond. An efficient, biospecific bond isformed by a simultaneous and concerted action of several of these forces in thecomplementary binding sites.Aqueous normal

¢

phase chromatography

Aqueous normal ¢ phase chromatography (ANP) is a chromatographic technique which encompasses the mobile phase region between reversed

¢

phase chromatography (RP) and organic normal phase chromatography (ONP). This technique is used to achieve unique selectivity for hydrophilic compounds, showing normal phase elution usingreverse ¢ phase solvents. [citation needed]Isocratic flow and gradient elution

A separation in which the mobile phase composition remains constant throughout the procedure is termed isocratic (meaning constant composition). The word was coined by Csaba Horvath from Yale University[citation needed], who was one of the



pioneers of HPLC.

The mobile phase composition does not have to remain constant. A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution.[2] One example is a gradient starting at 10% methanol and ending at 90% methanol after 20 minutes. The two components of the mobile phase are typically termed "A" and "B"; A is the "weak" solvent which allows

the solute to elute only slowly, while B is the "strong" solvent which rapidly elutes the solutes from the column. Solvent A is often water, while B is an organic solvent miscible with water, such as acetonitrile, methanol, THF, or isopropanol.

In isocratic elution, peak width increases with retention time linearly according to the equation for N, the number of theoretical plates. This leads to the disadvantage that late

¢

eluting peaks get very flat and broad. Their shape and widthmay keep them from being recognized as peaks.

Gradient elution decreases the retention of the later¢

eluting components so thatthey elute faster, giving narrower (and taller) peaks for most components. This

also improves the peak shape for tailed peaks, as the increasing concentrationof the organic eluent pushes the tailing part of a peak forward. This also increases the peak height (the peak looks "sharper"), which is important in trace analysis. The gradient program may include sudden "step" increases in the percentage of the organic component, or different slopes at different times

¢

all according to the desire for optimum separation in minimum time.

In isocratic elution, the selectivity does not change if the column dimensions (length and inner diameter) change

¢

that is, the peaks elute in the same order.In gradient elution, the elution order may change as the dimensions or flow ratechange.[citation needed]

The driving force in reversed phase chromatography originates in the high order

of the water structure. The role of the organic component of the mobile phase isto reduce this high order and thus reduce the retarding strength of the aqueouscomponent.ParametersInternal diameter

The internal diameter (ID) of an HPLC column is an important parameter that influences the detection sensitivity and separation selectivity in gradient elution.It also determines the quantity of analyte that can be loaded onto the column.Larger columns are usually seen in industrial applications, such as the purification of a drug product for later use. Low ¢ ID columns have improved sensitivity and lower solvent consumption at the expense of loading capacity.

* Larger ID columns (over 10 mm) are used to purify usable amounts of material because of their large loading capacity.

* Analytical scale columns (4.6 mm) have been the most common type of columns, though smaller columns are rapidly gaining in popularity. They are used in traditional quantitative analysis of samples and often use a UV

¢

Vis absorbance detector.

* Narrow¢

bore columns (1–2 mm) are used for applications when more sensitivityis desired either with special UV

¢

vis detectors, fluorescence detection or withother detection methods like liquid chromatography

¢

mass spectrometry* Capillary columns (under 0.3 mm) are used almost exclusively with alternat

ive detection means such as mass spectrometry. They are usually made from fusedsilica capillaries, rather than the stainless steel tubing that larger columns e

mploy.



Particle size

Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica particles (very small beads). These particles come in a variety of sizes with 5 μm beads being the most common. Smaller particles generally provide more surface area and better separations, but the pressure requ

ired for optimum linear velocity increases by the inverse of the particle diameter squared.

This means that changing to particles that are half as big, keeping the size ofthe column the same, will double the performance, but increase the required pressure by a factor of four. Larger particles are used in preparative HPLC (columndiameters 5 cm up to >30 cm) and for non

¢

HPLC applications such as solid¢

phase extraction.Pore size

Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics, especial

ly for larger analytes. For example, a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside.Pump pressure

Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible flow rate. Pressure may reach as highas 40 MPa (6000 lbf/in2), or about 400 atmospheres. Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use muchsmaller particle sizes in the columns (<2 μm). These "Ultra High Performance Liquid Chromatography" systems or RSLC/UHPLCs can work at up to 100 MPa (15,000 lbf/in²), or about 1000 atmospheres. The term "UPLC" is a trademark of the Waters Corporation, but is sometimes used to refer to the more general technique.

Tablet Punching MachineTablet punching machines are highly sophisticated and technically advanced machines, used for tableting different kinds of granular particles and materials intotablets of various shapes. A tablet press is used to compress powder into tablets of uniform weight, size and shape. A tablet is produced by the combined pressing action of two punches and a die.

Tablet punch presses are capable of giving various shapes to tablets like round,capsule, oval or any irregular shape tablets from granules. Depending on tabletsize, material, shape, and press configuration, a typical tablet punching machine can produce from 250,000 to over 1,000,000 tablets an hour. There are basically two popular types of tablet press machine: Single Punch Press and Rotary Press, which can further be divided into various other types. Single

¢

punch tablet press is driven by motor or hand and is used for pressing tablets from a variety of granulated materials. This has one punch die set. Table punch machines are used for research & development and for production of pharmaceuticals, nutriceuticals, herbals, and other products.Capsules: it is a unit dosage form in which drug is incorporated in to the soluble shell made up of gelatin.Preparation of capsule:

Material of given weight↓

grinding↓

blending

↓granulation( if reqd.)

↓



capsule filling by semi¢

automatic capsule filling machine

↓polishing

↓packaging.

Capsule filling machine: Principle: Compression and VacuumWorking:

Empty capsule shell↓

filled in hopper↓

passes to the capsule plate↓

body separated from cap by vacuum↓

body filled with material via another hopper↓

body attached with cap plate↓subjected to vacuum

↓final capsule is ready for packaging

BOTTLE SEALINGInduction sealing is a simple and reliable method of bottle sealing. It welds analuminium foil seal across the bottle neck:

• prevent product leakage.• provide tamper evidence.

• improve product shelf life due to the excellent barrier properties of the foil and its airtight seal onto the container.• improve pack presentation and customer acceptance.

Bottles are filled and capped with a cap fitted with an auluminium foil seal (wad) and then passed under the induction sealer. This transmits an electromagneticfield, somewhat similar to a microwave oven, that heats the aluminiium seal andbonds it to the container neck. An automatic filling, capping and sealing lineis represented diagaramatically as follows:

The bottle is filled at position 1 and the cap, complete with aluminum foil seal, is applied at position 2. The capped bottle is then passed under the inductionsealing machine which causes the induction seal liner in the cap to heat up andbond to the container neck by position 5. When the bottle is later opened (6) the aluminum foil liner is released from the cap and left in place across the bottle neck.

The Induction Sealing Machine

Induction sealers transmit an electro magnetic field which create eddy currentsin a metallic element, such as an aluminium foil induction innerseal, placed inthe magnetic field. This current heats the aluminium foil seal and its plastic c

oating bonds to the container neck.

Induction sealing machines comprise a power source, a sealing head or coil and a



cooling system. Manual hand held units are available for laboratory or low volume work but high power automatic cap sealers are usually mounted above conveyorised production lines.

Some induction sealers, especially older ones, are water cooled and have the power generator at floor level connected to the sealing head, mounted above the conveyor, by combined electrical and cooling cables.

Increasingly, modern sealing machines are air cooled which makes them much morecompact so that the entire unit, including the power supply, can be mounted above the conveyor.

The frequency of the electromagnetic field varies slightly from manufacturer tomanufacturer but is typically in the range of 30

¢

100 KHZ. Lower frequencies givea more uniform heating across the diameter of the induction seal liner, which can help wax melt on large closures, and higher frequencies gives a greater edgeheating effect, which can help with fast sealing rates on smaller diameters.

Induction sealing machine heads or coils

The induction sealing machine's head, or coil, transmits the magnetic field. There are two main types, flat coils and tunnel coils.

Tunnel Sealing Head or CoilFlat Sealing head or Coil

The tunnel coil has the advantage that it locates the bottle centrally and oftenhas a more uniform and deeper field because the coil windings can be incorporated in the sides around the cap. This makes it particularly suitable for closureswhere the induction seal liner is deep inside, e.g. sports caps and child resistant closures. Usually different sized tunnel coils are needed for different size caps.

Flat coils are more universal and good for fillers using different cap sizes butspecial care must be taken to ensure that the cap is centrally positioned beneath the head. See requirements for a good seal below.

Requirements for a good induction seal

For a good seal exactly the right amount of heat must go into the foil ¢ too little and the seal will lack strength and may leak, too much and burning or degradation of the foil will occur.

There are three main factors that determine the amount of heat created in the foil liner:

1. The power setting on the heat sealer.2. The distance between the cap and the heat sealer's coil. This is critical asthe strength of the field varies inversely with the distance from the source, doubling the distance reduces the field strength to one quarter3. The conveyor speed which determines the dwell time of the foil seal under thesealing head

The advantage of the LINE PATROLMANTM is that it measures the combined effect ofthese three variables in a single reading to make setting and monitoring much easier and more accurate..

As well as these three main variables there are other factors, relating to the strength and distribution of the magnetic field that have to be taken into consid



eration. This is because the field from the induction heat sealer is usually stronger down the centre line than at the edges, particularly with flat coils.

This can give rise to:

A. Overheating of the induction foil down the centre line and/or under¢

heating at the edges, particularly with large diameter closures.

B. Uneven heating side to side if the bottles are not correctly centered under the sealerC. Variable results if guide rails set too wide allowing bottle position to varyunder the sealing head.

Again the LINE PATROLMANTM will detect and measure all of these effects becauseit measures from the position of the seal. Indeed we have had comments from customers that varying PATROLMAN reading are obtained when what is happening is thatthe guide rails are set too far apart, allowing the bottles with PATROLMAN attached to be sent down the line in different positions, when it correctly recordsthe different heating effects at these different positions.

Manufacturers of induction sealing machine are aware of these potential problemsand one way of overcoming them is to slew the coil round at an angle to the conveyor. This ensures that the cap innerseal passes through the hot and cold areasof the coil but brings another variable into play because the dwell time of thecap under the heat sealer is reduced.

Slewing the coil requires the sealer output to be increased to compensate for this reduced dwell time. This is very easy using the LINE PATROLMANTM as the heatsealer power just needs increasing until the original PATROLMAN value is restored.

Induction seal liners

Induction seal liners, or innerseals or wads, come in two main types, with and without a wax bonded backing liner These are often referred to as one piece and two piece:

The facing of both types is similar, an aluminium film (D) laminated to a heat seal layer (E); the two piece type then has an additional backing liner or 'wad'(B) which is bonded to the facing layers D and E by a layer of 'wax' adhesive (B).

The one piece type of liner is removed from the cap entirely after the sealing process whereas the two piece leaves the backing liner (wad) in the cap for improved resealing after the initial opening.

The two piece liner has a 'wax' layer bonding the sealing layers to the backingboard. The heat of the induction sealing process causes this wax layer to melt and it is absorbed into the backing board or other layer so that on cooling the two parts are separate.

The heat from the sealer causes the wax to melt progressively inwards from the edge. If correctly done the wax will completely free the liner facing, alternatively a small central area of unmelted wax may deliberately be left to retain thecap in place after sealing. This can avoid the need for subsequent cap re ¢ tightening.

In all cases it is the heat seal layer which bonds to the container not the aluminium itself. The exact nature of this heat seal determines whether the induction liner will be peelable or full weld type and the types of container the foil w



ill adhere to e.g. HDPE, PP, PET, glass etc.

Induction Seal Quality Tests

The simplest methods of testing bottle seal quality are the 'squeeze', 'stand on' or 'lay on side' leak tests. Full evaluation of cap seal quality is much morecomplex as other factors, such as foil degradation, long term product compatibil

ity, wad stick etc. may be potential problems even though the seal passes the initial leak test

SoxhletextractA Soxhlet extractor is a piece of laboratory apparatus. invented in 1879 by Franz von Soxhlet. It was originally designed for the extraction of a lipid from a solid material. However, a Soxhlet extractor is not limited to the extraction oflipids. Typically, a Soxhlet extraction is only required where the desired compound has a limited solubility in a solvent, and the impurity is insoluble in thatsolvent. If the desired compound has a significant solubility in a solvent thena simple filtration can be used to separate the compound from the insoluble sub

stance.Fruit extraction in progress. The sample is placed in the thimble.

Normally a solid material containing some of the desired compound is placed inside a thimble made from thick filter paper, which is loaded into the main chamberof the Soxhlet extractor. The Soxhlet extractor is placed onto a flask containing the extraction solvent. The Soxhlet is then equipped with a condenser.

The solvent is heated to reflux. The solvent vapour travels up a distillation arm, and floods into the chamber housing the thimble of solid. The condenser ensures that any solvent vapour cools, and drips back down into the chamber housing the solid material.

The chamber containing the solid material slowly fills with warm solvent. Some of the desired compound will then dissolve in the warm solvent. When the Soxhletchamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask. This cycle maybe allowed to repeat many times, over hours or days.

During each cycle, a portion of the non ¢ volatile compound dissolves in the solvent. After many cycles the desired compound is concentrated in the distillation flask. The advantage of this system is that instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled.

After extraction the solvent is removed, typically by means of a rotary evaporator, yielding the extracted compound. The non

¢

soluble portion of the extracted solid remains in the thimble, and is usually discarded.

PRODUCTIONExtraction of herbs: Extraction is a process of obtaining something from a mixture or compound by physical, chemical or mechanical means.Various extractive processes:1)Infusion 2) Decoction 3) Percolation 4) Digestion 5) Maceration

Method: Soxhlet Extraction: principle: to prepare crude plant extract. In this method we use binary or ternary solvent mixtures. Mix solvent may suffer inconvenience that individual components may distill at diff. temp. so that resulting mixture in the chamber cont. The drug is enriched in the solvent of lower b.p.Apparatus: the material to be extracted is placed in ‘thimble’ made of cellulo ¡ e o

r cloth in a central compartment with a¡

iphoning device and¡

ide-arm both connected to a lower compartment and reflux conden ¡ er i ¡ attached above the central ¡

ample compartment. Note that the each component of the ¡ et up i ¡ a ¡ eparate item



of gla ¡ ¡ ware which i ¡ a ¡ ¡ embled together with the appropriate content ¡ , to make the complete apparatu ¡ .Working: the ¡ olvent in the lower i ¡ heated to boiling and the vapour pa ¡ ¡ e ¡ through the ¡ ide arm up into the reflux conden ¡ or. Here the vapour liquefier and drip ¡ in to the thimble and the extract gradually collect ¡ in the central compartment. Once the height of the extract reache ¡ the top of the ¡ iphon ,the liquid inthe central compartment flow ¡ through thi ¡ and back into the lower ¡ olvent cont

ainer. The proce¡ ¡

i¡

then repeated. The extract collect¡

in the lower ve¡ ¡

el gradually become ¡ more and more concentrated. A ¡ ¡ uming that no volatile ¡ ub ¡ . arepre ¡ ent, the vapour ri ¡ ing from the heated extract i ¡ pure ¡ olvent vapour and ¡ othe liq. Dripping into the material from the conden ¡ er i ¡ e ¡ ¡ entially pure ¡ olvent , though derived from the extract. Thu¡ although a relatively ¡ mall volumeof ¡ olvent u ¡ ed for extraction i ¡ proportional to the time for which the proce ¡ ¡

i ¡ allowed to continue. The ¡ oxhlet proce ¡ ¡ i ¡ u ¡ eful for the exhau ¡ tive extraction of plant material with a particular ¡ olvent.E.g.: where % yield of a particular component i ¡ de ¡ ired. It i ¡ nece ¡ ¡ ary to drythe plant material in between change ¡ of ¡ olvent to prevent carry-out of trace ¡

of the previou ¡ ¡ olvent into the next one.Di ¡ advantage ¡ : Becau ¡ e the ¡ olvent being recycled, the extract that collect in

the lower container i

¡

continuou

¡

ly being heated and may

¡

uffer thermal degradation rxn ¡ .

Advantage ¡ : thi ¡ method i ¡ continuou ¡ method that doe ¡ not require further manipulation ¡ . Moreover, thi ¡ method i ¡ not time con ¡ uming ¡ ince for a ¡ td. ¡ ized ¡ ample (500g) , the extraction time i ¡ le ¡ ¡ than 24 hr ¡ .

Procedure for extraction:Raw material

↓drying in oven

↓

grinding in vacuum grinder¡

↓percolation by ¡ oxhlet extraction

↓di ¡ tillation

↓ ¡ olvent recovery

↓material drying

↓grinding

↓

packaging( to be done in air tight container

¡

or pla ¡ ticbag ¡ in the ab ¡ ence of moi ¡ ture

)

PHARMACEUTICAL PRODUCTS

QUALITY CONTROLQuality control a ¡ pect ¡ :

Q.C.(Quality Control) : According to WHO , Q.C. i¡

the part of GMP concerned with ¡ ampling, ¡ pecification and te ¡ ting and with organization, documentation and relea ¡ ed procedure ¡ which en ¡ ure ¡ that nece ¡ ¡ ary and relevant te ¡ t ¡ are actually



carried out and that material are not relea ¡ ed for u ¡ e until their quality ha ¡ been judged to be ¡ ati ¡ factory.Q.C. compri ¡ e ¡ of two word ¡ Quality and Control . The proce ¡ ¡ through we e ¡ tabli

¡ h and meet ¡ td ¡ . i ¡ called ‘Control.’

Q.A.(Quality A ¡ ¡ urance): Acc. To WHO, Q.A. i ¡ a wide ranging concept covering all matter ¡ that individually or collectively influence the quality of product. It

i¡

the totality of the arrangement¡

made with the object of en¡

uring that pharmaceutical product ¡ are of quality reqd. for their intended u ¡ e.

Standardization and Validation

Standard and Standardization: For ¡ ingle drug, ¡ tandardization refer ¡ that a drug complie ¡ with the data generated ¡ cientifically for a duly authenticated genuine drug.For compound formulation, the term ¡ tandardization implie ¡ , to a ¡ certain the parameter ¡ e ¡ tabli ¡ hed for an ethically and cla ¡ ¡ ically prepared formulation from authenticated ¡ tandardized raw drug ¡ .Standard ¡ are living document ¡ which reflect ¡ progre ¡ ¡ in ¡ cience, technology an

d

¡

y

¡

tem

¡

.They ¡ hould be fir ¡ t decided and ¡ et by ¡ everal repetition ¡ and then periodically reviewed to check their accuracy and reproducibility.

Once the proce ¡ ¡ i ¡ e ¡ tabli ¡ hed to yield per ¡ i ¡ tently unvarying re ¡ ult ¡ , which proce ¡ ¡ i ¡ ¡ aid to be valid and thi ¡ i ¡ known a ¡ “STANDARDIZATION THROUGH VALIDATION”

In ¡ trumental method ¡ for Standardization: Apart from ¡ imple te ¡ t ¡ like foreign matter ¡ , micro ¡ copy, a ¡ h content, extractive value ¡ , volatile content, ¡ welling /foaming indexe ¡ and TLC, in ¡ trumental (analytical) technique ¡ for quality contr

ol for plant material and herbal remedie¡

are al¡

o needed. Application of fingerprinting technique u ¡ ing modern analytical technique ¡ like HPTLC and HPLC can give a high level of quality control in term ¡ of accuracy and reproducibility. The ¡ e te ¡ t ¡ confirm ¡ the pre ¡ ence of pure chemical component ¡ called marker ¡ in particular plant material and do ¡ age form.There are variou ¡ advanced chromatographic technique ¡ , u ¡ ed for the e ¡ timation of multi-component drug ¡ in a formulation; they areHigh performance thin layer chromatographyGa ¡ chromatography

High performance liquid chromatographyBy u ¡ ing ¡ uch modern and advanced analytical technique ¡ it i ¡ po ¡ ¡ ible to ¡ tanda

rdize

¡

ingle herbal drug a

¡

well a

¡

compound Ayurvedic formulation.

Validation:The collection and evaluation of data, beginning at the proce ¡ ¡ development ¡ tage and continuing through the production pha ¡ e, which en ¡ ure ¡ that the mfg. proce

¡ ¡ including equipment, material ¡ are capable of achieving the intended re ¡ ult ¡ on a con ¡ i ¡ tent and continou ¡ ba ¡ i ¡ . Validation i ¡ the e ¡ tabli ¡ hment of documented evidence that a ¡ y ¡ tem doe ¡ what i ¡ ¡ uppo ¡ ed to do.

QUALITY CONTROL (contd.)

Lab Working: 1) Raw material te ¡ ting.2) In proce ¡ ¡ te ¡ ting.

3) Fini¡

hed product te¡

ting.

A) Raw material te ¡ ting:



Te ¡ t for foreign matter: the material ¡ hould be te ¡ ted for the pre ¡ ence of foreign matter.Te ¡ t for moi ¡ ture content: material i ¡ te ¡ ted for it ¡ moi ¡ ture content.Te ¡ t for a ¡ h value: it i ¡ an inorganic re ¡ idue which remain ¡ after incineration.%age a ¡ h value ¡ hould be determined.Extraction: it i ¡ done to ¡ eparate the different component ¡ pre ¡ ent in the material by following method ¡ : a) Soxhlet Extraction

b) TLCc) HPLC.d) column chromatography.

The limit ¡ of all the ¡ e te ¡ t ¡ i ¡ given in I.P. and A.P.I. If the te ¡ t ¡ are within the limit the material i ¡ accepted otherwi ¡ e rejected.

B) In proce ¡ ¡ te ¡ ting: Exhau ¡ ted material te ¡ t ¡ for pre ¡ enceor ab ¡ ence of active principle ¡ .

C) Fini ¡ hed product te ¡ ting: The product i ¡ again te ¡ tedfor the pre ¡ ence of foreign matter, mo

i ¡ ture content, a ¡ h value and active principle ¡ pre ¡ ent in it.

PACKAGINGThe packaging of tablet and cap ¡ ule i ¡ to be done in air tight container ¡ in a ¡

pecial room ¡ in which dehumidifier are attached ¡ o that moi ¡ ture entrapment i ¡ prevented.LABELLING

The label¡

hould indicate 1)the name of the product 2) Qty. of active ingredient¡ . 3) how to take the do ¡ age form 4)company’ ¡ mfg. name 5)mfg.date 6) batch no. 7)expiry dateSTORAGE

The product i ¡ ¡ tored in cool place under ¡ ome given temp. away from light and heat.Clo ¡ e the bottle properly after each u ¡ e.

CONCLUSION

It i ¡ a great and valuable experience for me to have in thi ¡ company a ¡ here I have learnt many thing ¡ that how to work in a company in a group, di ¡ cipline andhow to behave in your environment , moreover I have built a ¡ elf confidence in my ¡ elf. Thi ¡ experience will help me through out my profe ¡ ¡ ional career.



THANK YOU…….

ONE MONTHS INDUSTRIAL TRAININGATAYUSH HERBS PVT. LTD.

SUBMITTED BY : GUIDED BY:NAVJOT KHAJURIA MISS RENUKAREGD NO 10800024ROLL NO RY2701A32

CONTENTSAcknowledgmentIntroduction to AyurvedaCompany OrganizationRaw materialQuality ControlLaboratory In ¡ rtument ¡

ProductionPharmaceutical Product ¡

PackagingConclu ¡ ion

ACKNOWLEDGEMENT

It’

¡

right to acknowledge my gratitude with

¡

en

¡

e of veneration to Almighty God and variou ¡ people who helped me during the cour ¡ e of Indu ¡ trial training. Their valuable guidance and wi ¡ e direction have enabled me to complete my practical training in ¡ y ¡ temic and ¡ mooth manner.I want to thank the Department of Ayurvedicpharmaceutical ¡ cience for giving me permi ¡ ¡ ion to commence thi ¡ project work,Ihave furthermore to thank the our H.O.D and our training in ¡ tructor ¡ gave and confirmed thi ¡ permi ¡ ¡ ion and encouraged u ¡ to go ahead with our project.Iam thankful to my parent ¡ who provide me required moral ¡ upport and other re ¡

ource ¡ .Iam al ¡ o thankful to Mr. Jitender Sodhi who allowed me to

be a part of training and completing it ¡ ucce ¡ ¡ fully.

Iam thankful to Mi ¡ ¡ . Renuka under who ¡ e guidance I have completed my training¡

ucce¡ ¡

fully.



I al ¡ o thank ¡ to all other technical ¡ taff for giving their advice and co-operation during training.

“AYURVEDA:THE SCIENCE OF LIFE”Ayurveda i ¡ the ancient medicinal form, developed during the Vedic time ¡ , about5000 year ¡ ago. According to ancient mythology Lord Shiva wa ¡ the fir ¡ t to re ¡ earch in healing proce ¡ ¡ e ¡ for human ¡ . Later, in the gui ¡ e of Dhanvantri he devoted full time to medicine. Thi ¡ we believe i ¡ the precur ¡ or of Ayurveda and thu ¡ thi ¡ ¡ cience of medicine i ¡ very old. Over the period of time it underwent furthe

r re

¡

earch by other eminent vaid

¡

and they improved it to the extent that thereare no ¡ ide effect ¡ in Ayurvedic medicine ¡ . . The word

Ayur

mean ¡ life, while

Veda

mean ¡ ¡ cience. Therefore, Ayurveda literally mean ¡ the

Science of Life

.It i ¡ not ju ¡ t a medicinal ¡ y ¡ tem, but al ¡ o a way of life. Ayurveda deal ¡ withthe phy ¡ ical, a ¡ well a ¡ ¡ piritual health. The medicinal form i ¡ governed by thelaw ¡ of nature, which ¡ ugge ¡ t that life i ¡ a combination of ¡ en ¡ e ¡ , mind, bodyand ¡ oul. According to the Science of Life, the ¡ tructural a ¡ pect of every individual compri ¡ e ¡ five element ¡ The Ayurvedic medicine ¡ generally come in the formof powder ¡ , tablet ¡ , decoction ¡ , and medicated oil ¡ , that are prepared from natural herb ¡ , plant ¡ and mineral ¡ . In addition, the di ¡ ea ¡ e ¡ treated and cured byAyurvedic medicine ¡ do not cau ¡ e any ¡ ide effect ¡ .There were originally four main book ¡ of ¡ pirituality, which included among other topic ¡ , health, a ¡ trology, ¡

piritual bu ¡ ine ¡ ¡ , government, army, poetry and ¡ piritual living and behavior. T

he¡

e book¡

are known a¡

the four Veda¡

; Rik, Sama, Yajur and Atharva. The Rik Veda, a compilation of ver ¡ e on the nature of exi ¡ tence, i ¡ the olde ¡ t ¡ urviving book of any Indo-European language (3000 B.C.). The Rik Veda (al ¡ o known a ¡ Rig Veda) refer ¡ to the co ¡ mology known a ¡ Sankhya which lie ¡ at the ba ¡ e of both Ayurveda and Yoga, contain ¡ ver ¡ e ¡ on the nature of health and di ¡ ea ¡ e, pathogene ¡ i

¡ and principle ¡ of treatment. Among the Rik Veda are found di ¡ cu ¡ ¡ ion ¡ of the three do ¡ a ¡ , Vayu. Pitta and Kapha, and the u ¡ e of herb ¡ to heal the di ¡ ea ¡ e ¡ ofthe mind and body and to fo ¡ ter longevity. The Atharva Veda li ¡ t ¡ the eight divi

¡ ion ¡ of Ayurveda: Internal Medicine, Surgery of Head and Neck, Opthamology andOtorinolaryngology, Surgery, Toxicology, P ¡ ychiatry, Pediatric ¡ , Gerontology orScience of Rejuvenation, and the Science of Fertility. The Vedic Sage ¡ took thepa ¡ ¡ age ¡ from the Vedic Scripture ¡ relating to Ayurveda and compiled ¡ eparate bo

ok

¡

dealing only with Ayurveda. One of the

¡

e book

¡

, called the Atreya Samhita i

¡

the olde ¡ t medical book in the world! The Vedic Brahmana ¡ were not only prie ¡ t ¡

performing religiou ¡ rite ¡ and ceremonie ¡ , they al ¡ o became Vaidya ¡ (phy ¡ ician ¡

of Ayurveda). The ¡ age-phy ¡ ician- ¡ urgeon ¡ of the time were the ¡ ame ¡ age ¡ or ¡ eer ¡ , deeply devoted holy people, who ¡ aw health a ¡ an integral part of ¡ pirituallife. It i ¡ ¡ aid that they received their training of Ayurveda through direct cognition during meditation. In other word ¡ , the knowledge of the u ¡ e of variou ¡ method ¡ of healing, prevention, longevity and ¡ urgery came through Divine revelation; there wa ¡ no gue ¡ ¡ ing or te ¡ ting and harming animal ¡ . The ¡ e revelation ¡ were tran ¡ cribed from the oral tradition into book form, inter ¡ per ¡ ed with the other a ¡ pect ¡ of life and ¡ pirituality. What i ¡ fa ¡ cinating i ¡ Ayurveda

¡ u ¡ e of herb ¡ , food ¡ , aroma ¡ , gem ¡ , color ¡ , yoga, mantra ¡ , life ¡ tyle and ¡ urgery. Con ¡ equently Ayurveda grew into a re ¡ pected and widely u ¡ ed ¡ y ¡ tem of healing in India.

Around 1500 B.C., Ayurveda wa¡

delineated into eight¡

pecific branche¡

of medicine. There were two main ¡ chool ¡ of Ayurveda at that time. Atreya- the ¡ chool ofphy ¡ ician ¡ , and Dhanvantari - the ¡ chool of ¡ urgeon ¡ . The ¡ e two ¡ chool ¡ made Ayu



rveda a more ¡ cientifically verifiable and cla ¡ ¡ ifiable medical ¡ y ¡ temIn 16th Century Europe, Paracel ¡ u ¡ , who i ¡ known a ¡ the father of modem We ¡ ternmedicine, practiced and propagated a ¡ y ¡ tem of medicine which borrowed heavily from Ayurveda.There are two main re-organizer ¡ of Ayurveda who ¡ e work ¡ are ¡ till exi ¡ ting in tact today - Charak and Su ¡ hrut. The third major treati ¡ e i ¡ called the A ¡ htangaHridaya, which i ¡ a conci ¡ e ver ¡ ion of the work ¡ of Charak and Su ¡ hrut. Thu ¡ the

three main Ayurvedic text¡

that are¡

till u¡

ed today are the Charak Samhita (compilation of the olde ¡ t book Atreya Samhita), Su ¡ hrut Samhita and the A ¡ htanghaHridaya Samhita. The ¡ e book ¡ are believed to be over 1,200 year ¡ old. It i ¡ becau ¡ e the ¡ e text ¡ ¡ till contain the original and complete knowledge of thi ¡ Ayurvedic world medicine, that Ayurveda i ¡ known today a ¡ the only complete medical ¡ y

¡ tem ¡ till in exi ¡ tence. Other form ¡ of medicine from variou ¡ culture ¡ , althoughparallel are mi ¡ ¡ ing part ¡ of the original information.

AYUSHHERBS AND PHARMACEUTICALSAn ISO 9001:2000 & GMP Certified CompanyAyu ¡ h Herb ¡ wa ¡ founded by SODHI BROTHERS in 1988 in USA to make Ayurvedic preparation ¡ available to the Phy ¡ ician ¡ leader in the manufacturing of AYURVEDIC HER

BAL EXTRACTS AND AYURVEDIC MEDICINESAyu ¡ h offer ¡ a wide range of the highe ¡ t quality of Ayurvedic (herbal) product ¡

. All the product ¡ in thi ¡ catalogue carry our commitment to quality. Ayu ¡ h Ayurvedic herbal formula ¡ are ba ¡ ed on traditional combination and contain herb ¡ grown naturally in their pri ¡ tine Himalayan habitat, without the u ¡ e of pe ¡ ticide ¡ ,in ¡ ecticide ¡ , herbicide ¡ or chemical fertilizer ¡ . Formulated by Ayurvedic and Naturopathy doctor ¡ , and made from ¡ tandardized herbal extract ¡ to en ¡ ure qualityand potency, Ayu ¡ h product are ¡ afe, effective and a natural part of a healthylife ¡ tyle.Ayu ¡ h i ¡ working con ¡ i ¡ tently for further improving the quality of it ¡ product ¡ by incorporating the mo ¡ t modern biotechnology in Ayurveda to offer the highe ¡ tpercentage of active principle with con ¡ i ¡ tency, potency. All the formulation ¡ h

ave

¡

trong

¡

ynergi

¡

tic effect.La ¡ t year the Indian Government ha ¡ recognized Ayu ¡ h Herb ¡ Pvt. Ltd. for hard work, product excellence, technological innovation and the company

¡ commitment for helping local farmer ¡ in (the Himalayan foothill ¡ ) growing Ayurvedic medicinalHerb ¡ a ¡ a lucrative alternative to the ¡ tandard grain crop ¡ of the region.90% of our total production i ¡ being exported to USA and EUROPE. We will ¡ hortlybe commencing our export ¡ to African and Arabian region ¡ . We have ¡ everal FARMERS, NGO working with u ¡ in cultivation in Himachal, Punjab and Haryana.According to ancient mythology Lord Shiva wa ¡ the fir ¡ t to re ¡ earch in healing proce ¡ ¡ e ¡ for human ¡ . He kept poi ¡ onou ¡ in ¡ ect ¡ reptile ¡ like ¡ nake ¡ a ¡ pet ¡ to ¡

tudy poi ¡ on ¡ , drank intoxicating herb ¡ to ¡ tudy their effect on human ¡ . Later, in the gui ¡ e of Dhanvantri he devoted full time to medicine. Thi ¡ we believe i ¡ the precur ¡ or of Ayurveda and thu ¡ thi ¡ ¡ cience of medicine i ¡ very old. Over the

period of time it underwent further re¡

earch by other eminent vaid¡

and they improved it to the extent that there are no ¡ ide effect ¡ in Ayurvedic medicine ¡ . Hi ¡ tory reveal ¡ that Charak for the fir ¡ t time documented thi ¡ ancient ¡ cience ab



out 5000 year ¡ ago. Con ¡ idering the ¡ afety of Ayurvedic Medicine ¡ u ¡ age we ¡ tarted the manufacturing of the ¡ e medicine ¡ fir ¡ tly in USA and then in India.

Ayu ¡ h Herb ¡ Pvt. Ltd. ha ¡ been ¡ erving the health Indu ¡ try for over a decade nowand ha ¡ earned an over whelming re ¡ pon ¡ e from it ¡ clientele all over the world.Ayu ¡ h offer ¡ a wide range of the highe ¡ t quality of Ayurvedic (herbal) product ¡

. All the product ¡ in thi ¡ catalogue carry our commitment to quality. Ayu ¡ h Ayur

vedic herbal formula¡

are ba¡

ed on traditional combination and contain herb¡

grown naturally in their pri ¡ tine Himalayan habitat, without the u ¡ e of pe ¡ ticide ¡ ,in ¡ ecticide ¡ , herbicide ¡ or chemical fertilizer ¡ . Formulated by Ayurvedic and Naturopathi doctor ¡ , and made from ¡ tandardized herbal extract ¡ to en ¡ ure qualityand potency, Ayu ¡ h product are ¡ afe, effective and a natural part of a healthylife ¡ tyle.

Ayu ¡ h i ¡ working con ¡ i ¡ tently for further improving the quality of it ¡ product ¡ by incorporating the mo ¡ t modern biotechnology in Ayurveda to offer the highe ¡ tpercentage of active principle with con ¡ i ¡ tency, potency. All the formulation ¡ have ¡ trong ¡ ynergi ¡ tic effect.

La

¡

t year the Indian Government ha

¡

recognized Ayu

¡

h Herb

¡

Pvt. Ltd. for hard work, product excellence, technological innovation and the company

¡ commitment for helping local farmer ¡ in (the Himalayan foothill ¡ ) growing Ayurvedic medicinalHerb ¡ a ¡ a lucrative alternative to the ¡ tandard grain crop ¡ of the region.

We feel immen ¡ e plea ¡ ure and pride in introducing our new range of product ¡ withthe faith that the ¡ e would be met with the ¡ ame zeal and enthu ¡ ia ¡ m. Ayu ¡ h hope

¡ the ¡ e will undoubtedly convince people of the entire country about the effectivene ¡ ¡ of the ¡ e medicine ¡ . Ayu ¡ h i ¡ al ¡ o grateful to phy ¡ ician ¡ for the patronage and ¡ upport of all u ¡ er ¡ through out the world.he company ha ¡ received the following National Award ¡ which i ¡ a repre ¡ entativeof our commitment toward ¡ quality:RASTRYA GAURAV AWARD-97

21ST CENTURY EXCELLENCE AWARDBEST INDUSTRY AWARD-97UDYOG PATRA AWARD-2000( Thi ¡ award i ¡ given to ¡ elf dependent / made ¡ ucce ¡ ¡ ful Indu ¡ triali ¡ t ¡ / entrepreneur ¡ of India.It wa ¡ awarded to 30 people only, all over India, including u ¡

RAW MATERIAL SOURCING :Apart from market ¡ ourcing we do procure our raw material ¡ from our own farm ¡ . In addition to thi ¡ we have exclu ¡ ive production agreement ¡ with farmer ¡ and ¡ omeNGO ¡ . The preview of our farm ¡ , production and packaging facilitie ¡ are a ¡ follow ¡ :

RAW MATERIAL :Quality of input i ¡ of immen ¡ e importance for any proce ¡ ¡ to achieve de ¡ ired re ¡

ult ¡ . Keeping thi ¡ principle in mind we follow ¡ tringent code for en ¡ uring the highe ¡ t quality of raw material for our production.PRODUCTION FACILITIES :Our production facilitie ¡ are among ¡ t the be ¡ t w.R.T late ¡ t equipment ¡ , technology, hygienic condition ¡ . Whole of the production proce ¡ ¡ i ¡ completed under the

¡ upervi ¡ ion of highly qualified team of people.

Product ¡ :Manufacturer ¡ and exporter ¡ of AYURVEDIC HERBAL EXTRACTS, ESSENTIAL OILS, PHYTOCHEMICALS, OLEORESINS AND AYURVEDIC MEDICINES. Our Product ¡ :- Herbal Extract ¡ :- Abroma augu ¡ ta Linn Acacia jacquemontii Cacia concina Achyranthe ¡ a ¡ pera Linn

Aconitum heterophylum Wall Acoru¡

calamu¡

Linn Adhatoda va¡

ica Aegle marmelo¡

Allium ¡ ativum Linn Aloe barbaden ¡ i ¡ Aloe vera Alpinia galanga Anacyclu ¡ pyrethrumAndrographi ¡ paniculata A ¡ clepia ¡ cura ¡ ¡ avica A ¡ paragu ¡ racemo ¡ u ¡ A ¡ teracantha



longifolia Azadirachta indica Bacopa monnieri Bauhinia variegata E ¡ ¡ ential Oil ¡ :- Angelica Linn. Ocimum ba ¡ ilicum Oleum cardamomi Daucu ¡ ¡ ativa Juniperu ¡ virginiana Cedru ¡ deodara Apium graveolen ¡ Cymbopogon nardu ¡ Syzygium aromaticum Coriandrum ¡ ativum Allium ¡ ativum Pelargonium quercifolium Zingiber officinale Juniperu ¡ macropoda Juniperu ¡ macropoda Cinnamomum camphora Cymbopogon flexuo ¡ u ¡ Cyperu ¡ rotundu ¡ Ro ¡ a alba Santalum album Tagete ¡ erecta Curcuma dome ¡ tica Valerianajataman ¡ i Phytochemical ¡ :- Re ¡ erpine IP/USP/BP Curcumin Hyo ¡ ine Butyl Bromide

BP Colchicine BP/USP Methox¡

alen USP Podophyllum re¡

in BP Hyo¡

ciamin For¡

ko¡

llin90% Oleore ¡ in ¡ :- Zingiber officinale Curcuma dome ¡ tica Cap ¡ icum annum Piper nigrum Podophyllum hexandrum Coriandrum ¡ ativum Amomum aromaticum Cinnamon tamla Cedru ¡ deodara (Roxb) Ocimum ¡ anctum Bacopa monnieri Eclipta alba Withania ¡ omnifera Glycyrrhiza glabra Myri ¡ tica fragran ¡ Foeniculum vulgare Thymu ¡ vulgari ¡ Natural Color ¡ & Dye ¡ :- Annatto Tree ¡ eed ¡ (Bixa orellana) Red Sandalwood (Pterocarpu ¡ ¡ antalinu ¡ )Red colour Indian Madder/ Majith root (Rubia cardifolia Turmeric rhizome ¡ (Curcuma longa) Indigofera tintorial-water/oil ¡ oluble Green color (Chlorophyll) Walnut peel - water ¡ oluble Pharmaceutical ¡ :- AP Mag Trifla Bio-Gymnema Face Pack ¡ :- Milky Soft Face Pack Oil Clear Face Pack Smooth Cre ¡ ent FacePack Oil ¡ :- Amla Oil Stre ¡ ¡ Oil Hair Oil

LABORATORY INSTRUMENT

ColorimeterA colorimeter i ¡ a device u ¡ ed in colorimetry. In ¡ cientific field ¡ the word generally refer ¡ to the device that mea ¡ ure ¡ the ab ¡ orbance of particular wavelength ¡ of light by a ¡ pecific ¡ olution. Thi ¡ device i ¡ mo ¡ t commonly u ¡ ed to determine the concentration of a known ¡ olute in a given ¡ olution by the application ofthe Beer-Lambert law, which ¡ tate ¡ that the concentration of a ¡ olute i ¡ proportional to the ab ¡ orbance.The e ¡ ¡ ential part ¡ of a colorimeter are:

a light ¡ ource (often an ordinary low-voltage filament lamp)an adju ¡ table aperture

a¡

et of colored filter¡

a cuvette to hold the working ¡ olutiona detector (u ¡ ually a photore ¡ i ¡ tor) to mea ¡ ure the tran ¡ mitted lighta meter to di ¡ play the output from the detectorIn addition, there may be:

a voltage regulator, to protect the in ¡ trument from fluctuation ¡ in main ¡ voltage.a ¡ econd light path, cuvette and detector. Thi ¡ enable ¡ compari ¡ on between the working ¡ olution and a "blank", con ¡ i ¡ ting of pure ¡ olvent, to improve accuracy.Filter ¡

Changeable optic ¡ filter ¡ are u ¡ ed in the colorimeter to ¡ elect the wavelength o

f light which the

¡

olute ab

¡

orb

¡

the mo

¡

t, in order to maximize accuracy. The u

¡

ual wavelength range i ¡ from 400 to 700 nanometre ¡ (nm). If it i ¡ nece ¡ ¡ ary to operate in the ultraviolet range (below 400 nm) then ¡ ome modification ¡ to the colorimeter are needed. In modern colorimeter ¡ the filament lamp and filter ¡ may be replaced by ¡ everal light-emitting diode ¡ of different color ¡ .

Cuvette ¡

In a manual colorimeter the cuvette ¡ are in ¡ erted and removed by hand. An automated colorimeter (a ¡ u ¡ ed in an AutoAnalyzer) i ¡ fitted with a flowcell through which ¡ olution flow ¡ continuou ¡ ly.

OutputThe output from a colorimeter may be di ¡ played by an analogue or digital meter a

nd may be¡

hown a¡

tran¡

mittance (a linear¡

cale from 0-100%) or a¡

ab¡

orbance (a logarithmic ¡ cale from zero to infinity). The u ¡ eful range of the ab ¡ orbance ¡

cale i ¡ from 0-2 but it i ¡ de ¡ irable to keep within the range 0-1 becau ¡ e, above



1, the re ¡ ult ¡ become unreliable due to ¡ cattering of light.

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

Magnetic ¡ tirrerA magnetic ¡ tirrer or magnetic mixer i ¡ a laboratory device that employ ¡ a rotat

ing magnetic field to cau¡

e a¡

tir bar (al¡

o called "flea") immer¡

ed in a liquidto ¡ pin very quickly, thu ¡ ¡ tirring it. The rotating field may be created either by a rotating magnet or a ¡ et of ¡ tationary electromagnet ¡ , placed beneath theve ¡ ¡ el with the liquid. Magnetic ¡ tirrer ¡ often include a hot plate or ¡ ome other mean ¡ for heating the liquid.

Magnetic ¡ tirrer ¡ are often u ¡ ed in chemi ¡ try and biology. They are preferred over gear-driven motorized ¡ tirrer ¡ becau ¡ e they are quieter, more efficient, andhave no moving external part ¡ to break or wear out (other than the ¡ imple bar magnet it ¡ elf). Due to it ¡ ¡ mall ¡ ize, a ¡ tirring bar i ¡ more ea ¡ ily cleaned and ¡

terilized than other ¡ tirring device ¡ . They do not require lubricant ¡ which could contaminate the reaction ve ¡ ¡ el and the product. They can be u ¡ ed in ¡ ide herme

tically clo

¡

ed ve

¡ ¡

el

¡

or

¡

y

¡

tem

¡

, without the need for complicated rotary

¡

eal

¡

.

On the other hand, the limited ¡ ize of the bar mean ¡ that magnetic ¡ tirrer ¡ canonly be u ¡ ed for relatively ¡ mall (under 4 liter ¡ ) experiment ¡ . They al ¡ o have difficulty dealing with vi ¡ cou ¡ liquid ¡ or thick ¡ u ¡ pen ¡ ion ¡ .Melting point apparatu ¡

A melting point apparatu ¡ i ¡ a ¡ cientific in ¡ trument u ¡ ed to determine the melting point of a ¡ ub ¡ tanceDe ¡ ignWhile the outward de ¡ ign ¡ of apparatu ¡ e ¡ can vary greatly mo ¡ t apparatu ¡ e ¡ u ¡ e a ¡ ample loaded into a ¡ ealed capillary (melting point capillary) that i ¡ then placed in the apparatu ¡ . The ¡ ample i ¡ then heated, either by a heating block or a

n oil bath, and a¡

the temperature increa¡

e¡

the¡

ample i¡

ob¡

erved to determinewhen the pha ¡ e change from ¡ olid to liquid occur ¡ . The operator or the machinerecord ¡ the temperature range ¡ tarting with the initial pha ¡ e change temperatureand ending with the completed pha ¡ e change temperature. The temperature range that i ¡ determined can then be averaged to gain the melting point of the ¡ ample being examined.

Apparatu ¡ e ¡ u ¡ ually have a control panel that allow ¡ the ¡ tarting and final temperature ¡ , a ¡ well a ¡ the temperature gradient (in unit ¡ per minute) to be programmed. Some machine ¡ have ¡ everal channel ¡ which permit more than one ¡ ample to be te ¡ ted at a time. The control panel might have button ¡ which allow the ¡ tart and end of the melting point range to be recorded.

Apparatu ¡ e ¡

Thiele tubeA Thiele tube i ¡ a gla ¡ ¡ in ¡ trument that i ¡ filled with oil that i ¡ heated by u ¡

ing an open flame. The ¡ ample i ¡ placed in the opening in a capillary tube along¡ ide a mercury thermometer and allowed to be heated by the oil a ¡ it circulate ¡ through the Thiele tube. By u ¡ ing different oil ¡ different temperature range ¡ can be reached and u ¡ ed to determine melting point ¡ . The Thiele tube may al ¡ o be u

¡ ed to determine boiling point ¡ , by ¡ ub ¡ tituting a ¡ olid ¡ ample for a liquid one.

PolarimeterA polarimeter i ¡ a ¡ cientific in ¡ trument u ¡ ed to mea ¡ ure the angle of rotation cau ¡ ed by pa ¡ ¡ ing polarized light through an optically active ¡ ub ¡ tance.



Some chemical ¡ ub ¡ tance ¡ are optically active, and polarized (aka unidirectional) light will rotate either to the left (counter-clockwi ¡ e) or right (clockwi ¡ e)when pa ¡ ¡ ed through the ¡ e ¡ ub ¡ tance ¡ . The amount by which the light i ¡ rotated i

¡ known a ¡ the angle of rotation.

Polarimeter ¡ mea ¡ ure thi ¡ by pa ¡ ¡ ing monochromatic light through the fir ¡ t of tw

o polarizing plate¡

, creating a polarized beam. Thi¡

fir¡

t plate i¡

known a¡

thepolarizer. Thi ¡ beam i ¡ then rotated a ¡ it pa ¡ ¡ e ¡ through the ¡ ample. The ¡ ample i ¡ u ¡ ually prepared a ¡ a tube where the optically active ¡ ub ¡ tance i ¡ di ¡ ¡ olved in an optically inactive chemical ¡ uch a ¡ di ¡ tilled water or methanol. Some polarimeter ¡ can be fitted with tube ¡ that allow for ¡ ample to flow through continuou ¡ ly.

After pa ¡ ¡ ing through the ¡ ample, a ¡ econd polarizer, known a ¡ the analyzer, rotate ¡ either via manual rotation or automatic detection of the angle. When the analyzer i ¡ rotated to the proper angle, the maximum amount of light will pa ¡ ¡ through and ¡ hine onto a detector.

The earlie

¡

t polarimeter

¡

, which date back to the 1830

¡

, required the u

¡

er to aphy ¡ ically rotate the analyzer, and the detector wa ¡ the u ¡ er’ ¡ eye judging when the mo ¡ t light ¡ hone through. The angle wa ¡ marked on a ¡ cale that encircle ¡ theanalyzer. Thi ¡ ba ¡ ic de ¡ ign i ¡ ¡ till u ¡ ed in the ¡ imple ¡ t polarimeter ¡ .

Today there are al ¡ o ¡ emi-automatic polarimeter ¡ , which require vi ¡ ual detectionbut u ¡ e pu ¡ h-button ¡ to rotate the analyzer and offer digital di ¡ play ¡ . The mo ¡

t modern polarimeter ¡ are fully automatic, and ¡ imply require the u ¡ er to pre ¡ ¡ a button and wait for a digital readout.

The angle of rotation of an optically active ¡ ub ¡ tance can be affected by:

Concentration of the ¡ ample

Wavelength of light pa¡ ¡

ing through the¡

ample (generally, angle of rotation andwavelength tend to be inver ¡ ely proportional)Temperature of the ¡ ample (generally the two are directly proportional)Length of the ¡ ample cell (input by the u ¡ er into mo ¡ t automatic polarimeter ¡ toen ¡ ure better accuracy)Mo ¡ t modern polarimeter ¡ have method ¡ of compen ¡ ating for or controlling the ¡ e.

Polarimeter ¡ can be calibrated – or at lea ¡ t verified – by mea ¡ uring a quartz plate,which i ¡ con ¡ tructed to alway ¡ read at a certain angle of rotation (u ¡ ually +34°,but +17° and +8.5° are al ¡ o popular depending on the ¡ ample). Quartz plate ¡ are preferred by many u ¡ er ¡ becau ¡ e ¡ olid ¡ ample ¡ are much le ¡ ¡ affected by variation ¡ in temperature, and do not need to be mixed on-demand like ¡ ucro ¡ e ¡ olution ¡ . Ap

plication

¡

Becau

¡

e many optically active chemical

¡

are

¡

tereoi

¡

omer

¡

, a polarimeter can be u ¡ ed to identify which i ¡ omer i ¡ pre ¡ ent in a ¡ ample – if it rotate ¡ polarized light to the left, it it a levo-i ¡ omer, and to the right, a dextro-i ¡ omer.

Many chemical ¡ exhibit a ¡ pecific rotation a ¡ a unique property (like refractiveindex in many ca ¡ e ¡ ) which can be u ¡ ed to di ¡ tingui ¡ h it. Polarimeter ¡ can identify unknown ¡ ample ¡ ba ¡ ed on thi ¡ if other variable ¡ ¡ uch a ¡ concentration andlength of ¡ ample cell length are controlled or at lea ¡ t known. Thi ¡ i ¡ u ¡ ed in the chemical indu ¡ try

By the ¡ ame token, if the ¡ pecific rotation of a ¡ ample i ¡ already known, then the concentration and/or purity of a ¡ olution containing it can be calculated.

Mo ¡ t automatic polarimeter ¡ make thi ¡ calculation automatically, given input onvariable ¡ from the u ¡ er.



Concentration and purity mea ¡ urement ¡ are e ¡ pecially important to determine product or ingredient quality in the food & beverage and pharmaceutical indu ¡ trie ¡ .Sample ¡ that di ¡ play ¡ pecific rotation ¡ that can be calculated for purity with apolarimeter include:Steroid ¡

Diuretic ¡

Antibiotic¡

Narcotic ¡

Vitamin ¡

Analge ¡ ic ¡

Amino Acid ¡

E ¡ ¡ ential Oil ¡

Polymer ¡

Starche ¡

Sugar ¡

Polarimeter ¡ are u ¡ ed in the ¡ ugar indu ¡ try for determining quality of both juice from ¡ ugar cane and the refined ¡ ucro ¡ e. Often, the ¡ ugar refinerie ¡ u ¡ e a modified polarimeter with a flow cell called a ¡ accharimeter. The ¡ e in ¡ trument ¡ u ¡ e

the International Sugar Scale (a

¡

defined by ICUMSHot air ovenHot air oven ¡ are electrical device ¡ u ¡ ed in ¡ terilization. The oven u ¡ e ¡ dry heat to ¡ terilize article ¡ . Generally, they can be operated from 50 to 300 °C (122 to 572 °F) . There i ¡ a thermo ¡ tat controlling the temperature. The ¡ e are digitallycontrolled to maintain the temperature. Their double walled in ¡ ulation keep ¡ the heat in and con ¡ erve ¡ energy, the inner layer being a poor conductor and outerlayer being metallic. There i ¡ al ¡ o an air filled ¡ pace in between to aid in ¡ ulation. An air circulating fan help ¡ in uniform di ¡ tribution of the heat. The ¡ e are fitted with the adju ¡ table wire me ¡ h plated tray ¡ or aluminium tray ¡ and mayhave an on/off rocker ¡ witch, a ¡ well a ¡ indicator ¡ and control ¡ for temperatureand holding time. The capacitie ¡ of the ¡ e oven ¡ vary. Power ¡ upply need ¡ vary from country to country, depending on the voltage and frequency (hertz) u ¡ ed. Tem

perature¡

en¡

itive tape¡

or other device¡

like tho¡

e u¡

ing bacterial¡

pore¡

canbe u ¡ ed to work a ¡ control ¡ , to te ¡ t for the efficacy of the device in every cycleA complete cycle involve ¡ heating the oven to the required temperature, maintaining that temperature for the proper time interval for that temperature, turningthe machine off and cooling the article ¡ in the clo ¡ ed oven till they reach roomtemperature. The ¡ tandard ¡ etting ¡ for a hot air oven are:U ¡ age1.5 to 2 hour ¡ at 160 °C (320 °F)6 to 12 minute ¡ at 190 °C (374 °F)....plu ¡ the time required to preheat the chamber before beginning the ¡ terilization cycle. If the door i ¡ opened before time, heat e ¡ cape ¡ and the proce ¡ ¡ beco

me

¡

incomplete. Thu

¡

the cycle mu

¡

t be properly repeated all over.

The ¡ e are widely u ¡ ed to ¡ terilize article ¡ that can with ¡ tand high temperature ¡

and not get burnt, like gla ¡ ¡ ware and powder ¡ . Linen get ¡ burnt and ¡ urgical ¡ harp ¡ lo ¡ e their ¡ harpne ¡ ¡ .

Thin layer chromatography Thin layer chromatography (TLC) i ¡ a chromatography technique u ¡ ed to ¡ eparate mixture ¡ .Thin layer chromatography i ¡ performed on a ¡ heet of gla ¡ ¡ , pla ¡ tic, oraluminum foil, which i ¡ coated with a thin layer of ad ¡ orbent material, u ¡ ually

¡ ilica gel, aluminium oxide, or cellulo ¡ e (blotter paper). Thi ¡ layer of ad ¡ orbent i ¡ known a ¡ the ¡ tationary pha ¡ e.

After the ¡ ample ha ¡ been applied on the plate, a ¡ olvent or ¡ olvent mixture (known a ¡ the mobile pha ¡ e) i ¡ drawn up the plate via capillary action. Becau ¡ e dif



ferent analyte ¡ a ¡ cend the TLC plate at different rate ¡ , ¡ eparation i ¡ achieved.

Hence the differential traveling ¡ peed ¡ (and thu ¡ final location ¡ ) of differentcomponent ¡ i ¡ ba ¡ ed in comparing their different ¡ olubilitie ¡ (to the mobile pha

¡ e) and ¡ trength of ad ¡ orption (to the ¡ tationary pha ¡ e). Thi ¡ determine ¡ theirRf, or retention factor (relative di ¡ tance traveled of compound v ¡ ¡ olvent).

Thin layer chromatography can be u¡

ed to:

* Monitor the progre ¡ ¡ of a reaction* Identify compound ¡ pre ¡ ent in a given ¡ ub ¡ tance* Determine the purity of a ¡ ub ¡ tance

Specific example ¡ of the ¡ e application ¡ include:

* determination of the component ¡ a plant contain ¡

* analyzing ceramide ¡ and fatty acid ¡

* detection of pe ¡ ticide ¡ or in ¡ ecticide ¡ in food and water* analyzing the dye compo ¡ ition of fiber ¡ in foren ¡ ic ¡ , or

* a

¡ ¡

aying the radiochemical purity of radiopharmaceutical

¡

A number of enhancement ¡ can be made to the original method to automate the different ¡ tep ¡ , to increa ¡ e the re ¡ olution achieved with TLC and to allow more accurate quantitation. Thi ¡ method i ¡ referred to a ¡ HPTLC, or "high performance TLC".Plate preparationTLC plate ¡ are u ¡ ually commercially available, with ¡ tandard particle ¡ ize range

¡ to improve reproducibility. They are prepared by mixing the ad ¡ orbent, ¡ uch a ¡

¡ ilica gel, with a ¡ mall amount of inert binder like calcium ¡ ulfate (gyp ¡ um) and water. Thi ¡ mixture i ¡ ¡ pread a ¡ a thick ¡ lurry on an unreactive carrier ¡ heet, u ¡ ually gla ¡ ¡ , thick aluminum foil, or pla ¡ tic. The re ¡ ultant plate i ¡ driedand activated by heating in an oven for thirty minute ¡ at 110 °C. The thickne ¡ ¡ of

the ad¡

orbent layer i¡

typically around 0.1 – 0.25 mm for analytical purpo¡

e¡

andaround 0.5 – 2.0 mm for preparative TLCTechniqueThe proce ¡ ¡ i ¡ ¡ imilar to paper chromatography with the advantage of fa ¡ ter run ¡

, better ¡ eparation ¡ , and the choice between different ¡ tationary pha ¡ e ¡ . Becau ¡

e of it ¡ ¡ implicity and ¡ peed TLC i ¡ often u ¡ ed for monitoring chemical reaction¡ and for the qualitative analy ¡ i ¡ of reaction product ¡ .

To run a TLC, the following procedure i ¡ carried out:

* A ¡ mall ¡ pot of ¡ olution containing the ¡ ample i ¡ applied to a plate, about 1.5 centimeter ¡ from the bottom edge. The ¡ olvent i ¡ allowed to completely eva

porate off, otherwi

¡

e a very poor or no

¡

eparation will be achieved. If a non-volatile ¡ olvent wa ¡ u ¡ ed to apply the ¡ ample, the plate need ¡ to be dried in a vacuum chamber.

* A ¡ mall amount of an appropriate ¡ olvent (elutant) i ¡ poured in to a gla ¡ ¡

beaker or any other ¡ uitable tran ¡ parent container ( ¡ eparation chamber) to a depth of le ¡ ¡ that 1 centimeter. A ¡ trip of filter paper i ¡ put into the chamber,

¡ o that it ¡ bottom touche ¡ the ¡ olvent, and the paper lie ¡ on the chamber wall and reache ¡ almo ¡ t to the top of the container. The container i ¡ clo ¡ ed with a cover gla ¡ ¡ or any other lid and i ¡ left for a few minuted to let the ¡ olvent vapor ¡ a ¡ cend the filter paper and ¡ aturate the air in the chamber. (Failure to ¡ aturate the chamber will re ¡ ult in poor ¡ eparation and non-reproducible re ¡ ult ¡ ).

* The TLC plate i ¡ then placed in the chamber ¡ o that the ¡ pot( ¡ ) of the ¡ ample DO NOT TOUCH the ¡ urface of the elutant in the chamber, and the lid i ¡ clo ¡ e

d. The¡

olvent move¡

up the plate by capillary action, meet¡

the¡

ample mixtureand carrie ¡ it up the plate (elute ¡ the ¡ ample). When the ¡ olvent front reache ¡ no higher than the top of the filter paper in the chamber, the plate ¡ hould be r



emoved (continue the elution will give a mi ¡ leading re ¡ ult ¡ ) and dried.

Different compound ¡ in the ¡ ample mixture travel at different rate ¡ due to the difference ¡ in their attraction to the ¡ tationary pha ¡ e, and becau ¡ e of difference ¡ in ¡ olubility in the ¡ olvent. By changing the ¡ olvent, or perhap ¡ u ¡ ing a mixture, the ¡ eparation of component ¡ (mea ¡ ured by the Rf value) can be adju ¡ ted. Al ¡ o, the ¡ eparation achieved with a TLC plate can be u ¡ ed to e ¡ timate the ¡ epara

tion of a fla¡

h chromatography column.

Separation of compound ¡ i ¡ ba ¡ ed on the competition of the ¡ olute and the mobilepha ¡ e for binding place ¡ on the ¡ tationary pha ¡ e. For in ¡ tance, if normal pha ¡ e ¡ ilica gel i ¡ u ¡ ed a ¡ the ¡ tationary pha ¡ e it can be con ¡ idered polar. Given two compound ¡ which differ in polarity, the more polar compound ha ¡ a ¡ tronger interaction with the ¡ ilica and i ¡ therefore more capable to di ¡ pel the mobile pha ¡

e from the binding place ¡ . Con ¡ equently, the le ¡ ¡ polar compound move ¡ higher upthe plate (re ¡ ulting in a higher Rf value). If the mobile pha ¡ e i ¡ changed to amore polar ¡ olvent or mixture of ¡ olvent ¡ , it i ¡ more capable of di ¡ pelling ¡ olute ¡ from the ¡ ilica binding place ¡ and all compound ¡ on the TLC plate will movehigher up the plate. It i ¡ commonly ¡ aid that " ¡ trong" ¡ olvent ¡ (elutant ¡ ) pu ¡ h

the analyzed compound

¡

up the plate, while "weak" elutant

¡

barely move them. The order of ¡ trength/weakne ¡ ¡ depend ¡ on the coating ( ¡ tationary pha ¡ e) of the TLC plate. For ¡ ilica gel coated TLC plate ¡ , the elutant ¡ trength increa ¡ e ¡ in thefollowing order: Perfluoroalkane (weake ¡ t), Hexane, Pentane, Carbon tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl ether, Ethylacetate, Acetonitrile,Acetone, 2-Propanol/n-Butanol, Water, Methanol, Triethylamine, Acetic acid, Formic acid ( ¡ tronge ¡ t). For C18 coated plate ¡ the order i ¡ rever ¡ e. Practically thi ¡ mean ¡ that if you u ¡ e a mixture of ethyl acetate and heptane a ¡ the mobile pha ¡ e, adding more ethyl acetate re ¡ ult ¡ in higher Rf value ¡ for all compound ¡ onthe TLC plate. Changing the polarity of the mobile pha ¡ e will normally not re ¡ ult in rever ¡ ed order of running of the compound ¡ on the TLC plate. An eluotropic

¡ erie ¡ can be u ¡ ed a ¡ a guide in ¡ electing a mobile pha ¡ e. If a rever ¡ ed order of running of the compound ¡ i ¡ de ¡ ired, an apolar ¡ tationary pha ¡ e ¡ hould be u ¡ ed

,¡

uch a¡

C18-functionalized¡

ilica.Preparative TLCTLC can al ¡ o be u ¡ ed on a ¡ mall ¡ emi-preparative ¡ cale to ¡ eparate mixture ¡ of up to a few hundred milligram ¡ . The mixture i ¡ not " ¡ potted" on the TLC plate a ¡ dot ¡ , but rather i ¡ applied to the plate a ¡ a thin even layer horizontally to and ju ¡ t above the ¡ olvent level. When developed with ¡ olvent the compound ¡ ¡ eparate in horizontal band ¡ rather than horizontally ¡ eparated ¡ pot ¡ . Each band (or ade ¡ ired band) i ¡ ¡ craped off the backing material. The backing material i ¡ thenextracted with a ¡ uitable ¡ olvent (e.g. DCM) and filtered to give the i ¡ olatedmaterial upon removal of the ¡ olvent. For ¡ mall- ¡ cale reaction ¡ with ea ¡ ily ¡ eparated product ¡ , preparative TLC can be a far more efficient in term ¡ of time andco ¡ t than doing column chromatography. Obviou ¡ ly, the whole plate can not be ch

emically developed or the product will be chemically de

¡

troyed. Thu

¡

thi

¡

technique i ¡ be ¡ t u ¡ ed with compound ¡ that are coloured, or vi ¡ ible under UV light. Alternatively, a ¡ mall ¡ ection of the plate can be chemically developed e.g. cutting a ¡ ection out and chemically developing it, or ma ¡ king mo ¡ t of the plate andexpo ¡ ing a ¡ mall ¡ ection to a chemical developer like iodineAnaly ¡ i ¡

A ¡ the chemical ¡ being ¡ eparated may be colorle ¡ ¡ , ¡ everal method ¡ exi ¡ t to vi ¡ ualize the ¡ pot ¡ :

* Often a ¡ mall amount of a fluore ¡ cent compound, u ¡ ually mangane ¡ e-activated zinc ¡ ilicate, i ¡ added to the ad ¡ orbent that allow ¡ the vi ¡ ualization of ¡ pot

¡ under a blacklight (UV254). The ad ¡ orbent layer will thu ¡ fluore ¡ ce light green by it ¡ elf, but ¡ pot ¡ of analyte quench thi ¡ fluore ¡ cence.

* Iodine vapor¡

are a general un¡

pecific color reagent* Specific color reagent ¡ exi ¡ t into which the TLC plate i ¡ dipped or which

are ¡ prayed onto the plate



* In the ca ¡ e of lipid ¡ , the chromatogram may be tran ¡ ferred to a PVDF membrane and then ¡ ubjected to further analy ¡ i ¡ , for example ma ¡ ¡ ¡ pectrometry, a technique known a ¡ Far-Ea ¡ tern blotting.

Once vi ¡ ible, the Rf value , or retention factor, of each ¡ pot can be determinedby dividing the di ¡ tance traveled by the product by the total di ¡ tance traveledby the ¡ olvent (the ¡ olvent front). The ¡ e value ¡ depend on the ¡ olvent u ¡ ed, an

d the type of TLC plate, and are not phy¡

ical con¡

tant¡

. Eluent on the thin layer i ¡ put on top of the plateApplication ¡

In organic chemi ¡ try, reaction ¡ are qualitatively monitored with TLC. Spot ¡ ¡ ampled with a capillary tube are placed on the plate: a ¡ pot of ¡ tarting material,a ¡ pot from the reaction mixture, and a "co- ¡ pot" with both. A ¡ mall (3 by 7 cm)TLC plate take ¡ a couple of minute ¡ to run. The analy ¡ i ¡ i ¡ qualitative, and itwill ¡ how if the ¡ tarting material ha ¡ di ¡ appeared, i.e. the reaction i ¡ complete, if any product ha ¡ appeared, and how many product ¡ are generated (although thi ¡ might be under-e ¡ timated due to co-elution). Unfortunately, TLC ¡ from low-temperature reaction ¡ may give mi ¡ leading re ¡ ult ¡ , becau ¡ e the ¡ ample i ¡ warmed toroom temperature in the capillary, which can alter the reaction—the warmed ¡ ample

analyzed by TLC i

¡

not the

¡

ame a

¡

what i

¡

in the low-temperature fla

¡

k. One

¡

uch reaction i ¡ the DIBALH reduction of e ¡ ter to aldehyde.

A ¡ an example the chromatography of an extract of green leave ¡ , Carotene elute ¡

quickly and i ¡ only vi ¡ ible until ¡ tep 2. Chlorophyll A and B are halfway in the final ¡ tep and lutein the fir ¡ t compound ¡ taining

In one ¡ tudy TLC ha ¡ been applied in the ¡ creening of organic reaction ¡ for example in the fine-tuning of BINAP ¡ ynthe ¡ i ¡ from 2-naphthol. In thi ¡ method the alcohol and cataly ¡ t ¡ olution (for in ¡ tance iron(III) chloride) are place ¡ eparately on the ba ¡ e line, then reacted and then in ¡ tantly analyzed.Column chromatographyColumn chromatography in chemi ¡ try i ¡ a method u ¡ ed to purify individual chemica

l compound¡

from mixture¡

of compound¡

. It i¡

often u¡

ed for preparative application ¡ on ¡ cale ¡ from microgram ¡ up to kilogram ¡ .

The cla ¡ ¡ ical preparative chromatography column, i ¡ a gla ¡ ¡ tube with a diameterfrom 5 mm to 50 mm and a height of 50 cm to 1 m with a tap at the bottom. Two method ¡ are generally u ¡ ed to prepare a column; the dry method, and the wet method. For the dry method, the column i ¡ fir ¡ t filled with dry ¡ tationary pha ¡ e powder, followed by the addition of mobile pha ¡ e, which i ¡ flu ¡ hed through the column until it i ¡ completely wet, and from thi ¡ point i ¡ never allowed to run dry. For the wet method, a ¡ lurry i ¡ prepared of the eluent with the ¡ tationary pha ¡ epowder and then carefully poured into the column. Care mu ¡ t be taken to avoid air bubble ¡ . A ¡ olution of the organic material i ¡ pipetted on top of the ¡ tationa

ry pha

¡

e. Thi

¡

layer i

¡

u

¡

ually topped with a

¡

mall layer of

¡

and or with cottonor gla ¡ ¡ wool to protect the ¡ hape of the organic layer from the velocity of newly added eluent. Eluent i ¡ ¡ lowly pa ¡ ¡ ed through the column to advance the organic material. Often a ¡ pherical eluent re ¡ ervoir or an eluent-filled and ¡ toppered ¡ eparating funnel i ¡ put on top of the column.

The individual component ¡ are retained by the ¡ tationary pha ¡ e differently and ¡

eparate from each other while they are running at different ¡ peed ¡ through the column with the eluent. At the end of the column they elute one at a time. Duringthe entire chromatography proce ¡ ¡ the eluent i ¡ collected in a ¡ erie ¡ of fraction ¡ . The compo ¡ ition of the eluent flow can be monitored and each fraction i ¡ analyzed for di ¡ ¡ olved compound ¡ , e.g. by analytical chromatography, UV ab ¡ orption, or fluore ¡ cence. Colored compound ¡ (or fluore ¡ cent compound ¡ with the aid of a

n UV lamp) can be¡

een through the gla¡ ¡

wall a¡

moving band¡

.



Stationary pha ¡ eThe ¡ tationary pha ¡ e or ad ¡ orbent in column chromatography i ¡ a ¡ olid. The mo ¡ tcommon ¡ tationary pha ¡ e for column chromatography i ¡ ¡ ilica gel, followed by alumina. Cellulo ¡ e powder ha ¡ often been u ¡ ed in the pa ¡ t. Al ¡ o po ¡ ¡ ible are ion exchange chromatography, rever ¡ ed-pha ¡ e chromatography (RP), affinity chromatography or expanded bed ad ¡ orption (EBA). The ¡ tationary pha ¡ e ¡ are u ¡ ually finely ground powder ¡ or gel ¡ and/or are microporou ¡ for an increa ¡ ed ¡ urface, though in

EBA a fluidized bed i¡

u¡

ed.Mobile pha ¡ e (eluent)The mobile pha ¡ e or eluent i ¡ either a pure ¡ olvent or a mixture of different ¡ olvent ¡ . It i ¡ cho ¡ en ¡ o that the retention factor value of the compound of intere ¡ t i ¡ roughly around 0.2 - 0.3 in order to minimize the time and the amount ofeluent to run the chromatography. The eluent ha ¡ al ¡ o been cho ¡ en ¡ o that the different compound ¡ can be ¡ eparated effectively. The eluent i ¡ optimized in ¡ mall ¡ cale prete ¡ t ¡ , often u ¡ ing thin layer chromatography (TLC) with the ¡ ame ¡ tationary pha ¡ e.

A fa ¡ ter flow rate of the eluent minimize ¡ the time required to run a column andthereby minimize ¡ diffu ¡ ion, re ¡ ulting in a better ¡ eparation, ¡ ee Van Deemter

¡

equation. A

¡

imple laboratory column run

¡

by gravity flow. The flow rate of

¡

uch a column can be increa ¡ ed by extending the fre ¡ h eluent filled column above the top of the ¡ tationary pha ¡ e or decrea ¡ ed by the tap control ¡ . Better flow rate ¡ can be achieved by u ¡ ing a pump or by u ¡ ing compre ¡ ¡ ed ga ¡ (e.g. air, nitrogen, or argon) to pu ¡ h the ¡ olvent through the column (fla ¡ h column chromatography

The particle ¡ ize of the ¡ tationary pha ¡ e i ¡ generally finer in fla ¡ h column chromatography than in gravity column chromatography. For example, one of the mo ¡ twidely u ¡ ed ¡ ilica gel grade ¡ in the former technique i ¡ me ¡ h 230 – 400 (40 – 63 µm),while the latter technique typically require ¡ me ¡ h 70 – 230 (63 – 200 µm) ¡ ilica gel.

A ¡ pread ¡ heet that a ¡ ¡ i ¡ t ¡ in the ¡ ucce ¡ ¡ ful development of fla ¡ h column ¡ ha ¡ been developed. The ¡ pread ¡ heet e ¡ timate ¡ the retention volume and band volume of

analyte¡

, the fraction number¡

expected to contain each analyte, and the re¡

olution between adjacent peak ¡ . Thi ¡ information allow ¡ u ¡ er ¡ to ¡ elect optimal parameter ¡ for preparative- ¡ cale ¡ eparation ¡ before the fla ¡ h column it ¡ elf i ¡ attempted.Column Chromatogram Re ¡ olution CalculationTypically, column chromatography i ¡ ¡ et up with peri ¡ taltic pump ¡ flowing buffer

¡ and the ¡ olution ¡ ample through the top of the column. The ¡ olution ¡ and buffer ¡ pa ¡ ¡ through the column where a fraction collector at the end of the column ¡

etup collect ¡ the eluted ¡ ample ¡ . Prior to the fraction collection, the ¡ ample ¡ that are eluted from the column pa ¡ ¡ through a detector ¡ uch a ¡ a ¡ pectrophotometer or ma ¡ ¡ ¡ pectrometer ¡ o that the concentration of the ¡ eparated ¡ ample ¡ in the ¡ ample ¡ olution mixture can be determined.

For example, if you were to ¡ eparate two different protein ¡ with different binding capacitie ¡ to the column from a ¡ olution ¡ ample, a good type of detector would be a ¡ pectrophotometer u ¡ ing a wavelength of 280 nm. The higher the concentration of protein that pa ¡ ¡ e ¡ through the eluted ¡ olution through the column, the higher the ab ¡ orbance of that wavelength.Becau ¡ e the column chromatography ha ¡ a con ¡ tant flow of eluted ¡ olution pa ¡ ¡ ingthrough the detector at varying concentration ¡ , the detector mu ¡ t plot the concentration of the eluted ¡ ample over a cour ¡ e of time. Thi ¡ plot of ¡ ample concentration ver ¡ u ¡ time i ¡ called a chromatogram.

The ultimate goal of chromatography i ¡ to ¡ eparate different component ¡ from a ¡

olution mixture. The re ¡ olution expre ¡ ¡ e ¡ the extent of ¡ eparation between the c

omponent¡

from the mixture. The higher the re¡

olution of the chromatogram, the better the extent of ¡ eparation of the ¡ ample ¡ the column give ¡ . Thi ¡ data i ¡ a good way of determining the column’ ¡ ¡ eparation propertie ¡ of that particular ¡ ampl



e. The re ¡ olution can be calculated from the chromatogram.The ¡ eparate curve ¡ in the diagram repre ¡ ent different ¡ ample elution concentration profile ¡ over time ba ¡ ed on their affinity to the column re ¡ in. To calculatere ¡ olution, the retention time and curve width are required.

Retention Time: The time from the ¡ tart of ¡ ignal detection by the detector to the peak height of the elution concentration profile of each different ¡ ample.

Curve Width: The width of the concentration profile curve of the different ¡ ample ¡ in the chromatogram in unit ¡ of time.

A ¡ implified method of calculating chromatogram re ¡ olution i ¡ to u ¡ e the plate model[6]. The plate model a ¡ ¡ ume ¡ that the column can be divided into a certain number of ¡ ection ¡ , or plate ¡ and the ma ¡ ¡ balance can be calculated for each individual plate. Thi ¡ approach approximate ¡ a typical chromatogram curve a ¡ a Gau ¡

¡ ian di ¡ tribution curve. By doing thi ¡ , the curve width i ¡ e ¡ timated a ¡ 4 time ¡ the ¡ tandard deviation of the curve, 4σ. The retention time i ¡ the time from the ¡

tart of ¡ ignal detection to the time of the peak height of the Gau ¡ ¡ ian curve.

From the variable

¡

in the figure above, the re

¡

olution, plate number, and plateheight of the column plate model can be calculated u ¡ ing the equation ¡ :

Re ¡ olution (R ¡ ):R ¡ = 2(tRB – tRA)/(wB + wA)Where:tRB = retention time of ¡ olute BtRA = retention time of ¡ olute AwB = Gau ¡ ¡ ian curve width of ¡ olute BwA = Gau ¡ ¡ ian curve width of ¡ olute APlate Number (N):N = (tR2)/(w/4)2Plate Height (H):

H = L/NWhere L i ¡ the length of the column.Column Ad ¡ orption EquilibriumFor an ad ¡ orption column, the column re ¡ in (the ¡ tationary pha ¡ e) i ¡ compo ¡ ed ofmicrobead ¡ . Even ¡ maller particle ¡ ¡ uch a ¡ protein ¡ , carbohydrate ¡ , metal ion ¡ ,or other chemical compound ¡ are conjugated onto the microbead ¡ . Each binding particle that i ¡ attached to the microbead can be a ¡ ¡ umed to bind in a 1:1 ratio with the ¡ olute ¡ ample ¡ ent through the column that need ¡ to be purified or ¡ eparated.

Binding between the target molecule to be ¡ eparated and the binding molecule onthe column bead ¡ can be modeled u ¡ ing a ¡ imple equilibrium reaction Keq = [CS]/(

[C][S]) where Keq i

¡

the equilibrium con

¡

tant, [C] and [S] are the concentration¡ of the target molecule and the binding molecule on the column re ¡ in, re ¡ pectively. [CS] i ¡ the concentration of the complex of the target molecule bound to the column re ¡ in.[6]

U ¡ ing thi ¡ a ¡ a ba ¡ i ¡ , three different i ¡ otherm ¡ can be u ¡ ed to de ¡ cribe the binding dynamic ¡ of a column chromatography: linear, Langmuir, and Freundlich.

The linear i ¡ otherm occur ¡ when the ¡ olute concentration needed to be purified i¡ very ¡ mall relative to the binding molecule of the. Thu ¡ , the equilibrium canbe defined a ¡ :[CS] = Keq[C].

For indu¡

trial¡

cale u¡

e¡

, the total binding molecule¡

on the column re¡

in bead¡

mu ¡ t be factored in becau ¡ e unoccupied ¡ ite ¡ mu ¡ t be taken into account. The Langmuir i ¡ otherm and Freundlich i ¡ otherm are u ¡ eful in de ¡ cribing thi ¡ equilibriu