Paper chromatography is an analytical method used to separate colored chemicals or substances. It is primarily used as a teaching tool, having been replaced by other chromatography methods, such as thin-layer chromatography.
This presentation contains all the topics related to column chromatography. That includes introduction, principle,apparatus, experimental aspects of column chromatography, application of column chromatography, advantage and disadvantage of column chromatography with reference.
This presentation contains all the topics related to column chromatography. That includes introduction, principle,apparatus, experimental aspects of column chromatography, application of column chromatography, advantage and disadvantage of column chromatography with reference.
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
What is Chromatography?
Applications of Chromatography
Types of Chromatography
1- Column Chromatography
2- Planar chromatography
Paper Chromatography
Gas Chromatography
Detectors
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)Abhishek Gupta
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way
It is also known as planar chromatography or Flat-bed chromatography.
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
What is Chromatography?
Applications of Chromatography
Types of Chromatography
1- Column Chromatography
2- Planar chromatography
Paper Chromatography
Gas Chromatography
Detectors
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)Abhishek Gupta
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way
It is also known as planar chromatography or Flat-bed chromatography.
INTRODUCTION TO PAPER CHROMATOGRAPHY
Cellulose filter paper is often used as the statioary phase in the paper chromatography. Since it is hydrophillic, it is usually covered with thin film of water. The procedure is often regarded as liquid-liquid cromatography
Other liquids can be encorporated in place of water, thus provides different type of stationary phase. Eg. Paper treated with silicone or paraffin oil permits reverse phase-paper chromatography, in which mobile phase is a polar solvent.
There are some commercially available papers that contain an adsorbent or an ion-exchange resin, thus permmits adsorption and ion-exhange paper chromatography.
PRINCIPLE OF CHROMATOGRAPHY
This is type of partition chromatography in which the substance are distributed between two liquids that is one is the stationary liquid (usually water) which is held in the fibres of paper and called the stationary phase; the other is the moving liquid or developing solvent and called the mobile phase. The components of mixture to be separated migrates at different rates as its solubility between two phases and appear as spot at different points on the paper.
In this technique, a drop of the test solution is applied as a small spot on a filter paper and the spot is dried. The paper is kept in close chamber and the edge of filter is dipped into a solvent called as developing solvent. As soon as filter paper gets liquids via capillary action and reaches to the spot of the test solution then various substances are moved by solvent with various speeds. When solvent move up to suitable height (15-18) the paper is dried and various spot are visualised by suitable reagent called visualising reagent.
MIGRATION PARAMETERS
1) RF VALUE(RETENSION FACTOR) :- It is ratio of the solute’s distance travelled to solvent’s distance travelled.
It is constant for a given substance, provided the conditions of chromatographic system are kept constant with respect to tempreture, type of paper, duration and direction of development, nature and the shape and the size of the wick used (i.e., radial chromatography), the amount of liquid in the reservoir, humidity etc.
The Rf of of a substance depends upon a number of factors which are:
The solvent employed
The medium used for separation i.e., the quality of paper chromatography
The nature of mixture
The tempreture
Size of vessel in which operation has been carried out
It is possible to compare the Rf Values of different substances keeping above factor constant
types of paper chromatogtaphy
ascending, descending, ascending-descending, radial , two diamentional chromatography
Chromatography : A seperation techniqueSHIVANEE VYAS
Chromatography is a method of seperating mixture of components into individual components through equlibrium distribution between two phases.
Each chromatographic method essentially consists of 2 phases a staionary phase and a mobile phase.
Stationary phase : solid or liquid
Mobile phase : liquid or gas
Paper chromatography is an analytical method used to separate coloured chemicals or substances.It is now primarily used as a teaching tool, having been replaced in the laboratory by other chromatography methods such as thin-layer chromatography (TLC).
CALIBRATION OF HPLC
Pressure Test.
Drift and Noise
Column oven and sample cooler
Pump by flow rate accuracy measurement.
Pump by gradient flow measurement.
UV-Vis / PDA detector by reference energy check.
Introduction
Measures of central tendency
Correlation:
Regression
Probability
Parametric test
Non Parametric test
Graphs:
Designing of Methodology
Regression Modeling
Introduction to Practical components of Industrial and Clinical Trials Problems:
Design and Analysis of experiments
COLUMN CHROMATOGRAPHY - SEPERATION OF THE MIXTURE OF COMPONENTS IJN TO INDIVIDUAL COMPONENTS BY USING STATIONARY PHASE AND MOBILE PHASE UPON THE USING OF COLUMN
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Historically, drugs were discovered by identifying the active ingredient from traditional remedies or by serendipitous discovery, as with penicillin. More recently, chemical libraries of synthetic small molecules, natural products or extracts were screened in intact cells or whole organisms to identify substances that had a desirable therapeutic effect in a process known as classical pharmacology. After sequencing of the human genome allowed rapid cloning and synthesis of large quantities of purified proteins, it has become common practice to use high throughput screening of large compounds libraries against isolated biological targets which are hypothesized to be disease-modifying in a process known as reverse pharmacology. Hits from these screens are then tested in cells and then in animals for efficacy
Historically, drugs were discovered by identifying the active ingredient from traditional remedies or by serendipitous discovery, as with penicillin. More recently, chemical libraries of synthetic small molecules, natural products or extracts were screened in intact cells or whole organisms to identify substances that had a desirable therapeutic effect in a process known as classical pharmacology. After sequencing of the human genome allowed rapid cloning and synthesis of large quantities of purified proteins, it has become common practice to use high throughput screening of large compounds libraries against isolated biological targets which are hypothesized to be disease-modifying in a process known as reverse pharmacology. Hits from these screens are then tested in cells and then in animals for efficacy
In microbiology, the term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment, for example in water or soil flora, or from living beings with skin flora, oral flora or gut flora, in order to identify the microbe(s) of interest. Historically, the laboratory techniques of isolation first developed in the field of bacteriology and parasitology (during the 19th century), before those in virology during the 20th century. Methods of microbial isolation have drastically changed over the past 50 years, from a labor perspective with increasing mechanization, and in regard to the technology involved, and hence speed and accuracy.
Nutrient media – A source of amino acids and nitrogen (e.g., beef, yeast extract). This is an undefined medium because the amino acid source contains a variety of compounds with the exact composition being unknown
The word MICROBIOLOGY describes exactly what the discipline is: the study of small living things. MICRO = small, BIO = living, and LOGY = to study. Microbiology (or specifically, bacteriology) is still a very young science and not yet completely understood.
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose.
Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds from a mixture
High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).
How to Split Bills in the Odoo 17 POS ModuleCeline George
Bills have a main role in point of sale procedure. It will help to track sales, handling payments and giving receipts to customers. Bill splitting also has an important role in POS. For example, If some friends come together for dinner and if they want to divide the bill then it is possible by POS bill splitting. This slide will show how to split bills in odoo 17 POS.
Model Attribute Check Company Auto PropertyCeline George
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The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
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Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
Students, digital devices and success - Andreas Schleicher - 27 May 2024..pptxEduSkills OECD
Andreas Schleicher presents at the OECD webinar ‘Digital devices in schools: detrimental distraction or secret to success?’ on 27 May 2024. The presentation was based on findings from PISA 2022 results and the webinar helped launch the PISA in Focus ‘Managing screen time: How to protect and equip students against distraction’ https://www.oecd-ilibrary.org/education/managing-screen-time_7c225af4-en and the OECD Education Policy Perspective ‘Students, digital devices and success’ can be found here - https://oe.cd/il/5yV
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Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
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http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
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2. PAPER CHROMATOGRAPHY
•
•
Paper Chromatography (PC) was first introduced by
German scientist Christian Friedrich Schonbein
(1865).
PC is considered to be the simplest and most widely
used of the chromatographic techniques because of
its applicability to isolation, identification and
quantitative determination of organic and inorganic
compounds.
2
3. PAPER CHROMATOGRAPHY
• ANALYSIS OF UNKNOWN SUSTANCES
It is carried out mainly by the flow of solvents
on specially designed filterpaper.
There are two types of paperchromatography,
theyare:
3
4. PAPER CHROMATOGRAPHYTYPES
1.PAPER ADSORPTION CHROMATOGRAPHY
Paper impregnated with silica or alumina acts as
adsorbent (stationary phase) and solvent as
mobile phase.
2.PAPER PARTITION CHROMATOGRAPHY
Moisture / Water present in the pores of
cellulose fibers present in filter paper acts as
stationary phase & another mobile phase is used
as solvent
In general P.C – Paper Partition
Chromatography
4
5. PRINCIPLE OF SEPERATION
The principle of separation is mainly partition
rather than adsorption.
Cellulose layers in filter paper contains moisture
which acts as stationary phase & organic
solvents/buffers are used as mobile phase
5
7. PAPER CHROMATOGRAPHY
• STATIONARY PHASE AND PAPERS USED
Whatman filter papers of different grades like
No.1, No.2, No.3, No.4, No.20, No.40, No.42 etc
are used. In general this paper contains 98-99%
of α-cellulose, 0.3 – 1% β -cellulose
Factors that governs the choice of paper:
» Nature of Sample and solvents used.
» Based on Quantitative or Qualitative analysis.
» Based on thickness of the paper.
7
8. PAPER CHROMATOGRAPHY
•
8
•
Modified Papers – acid or base washed filter
paper, glass fiber type paper.
Hydrophilic Papers – Papers modified with
methanol, formamide, glycol, glycerol etc.
•
•
Hydrophobic papers – acetylation of OH groups
leads to hydrophobic nature, hence can be used
for reverse phase chromatography.
Impregnation of silica, alumna, or ion exchange
resins can also be made.
9. PREPARATION OF PAPER
• Cut the paper into
desired shape and size
depending upon work
to be carried out.
•
•
The starting line is
marked on the paper
with an ordinary
pencil 5cm from the
bottom edge.
On the staring line
marks are made 2cm
apart from each other.
9
10. PAPER CHROMATOGRAPHY
• Preparation of the solution
• Choice of suitable solvent for making solution is very
important. Pure solutions can be applied direct on
the paper but solids are always dissolved in small
•
quantity of a suitable solvent.
Biological tissues are treated with suitable solvents
and their extracts obtained. Proteins can be
precipitated with alcohol and salts can be removed by
treatment with ion exchange resin.
10
11. PAPER CHROMATOGRAPHY
APPLICATION OF SAMPLE
The sample to be applied is dissolved in the mobile
phase and applied as a small spot on the origin line,
using capillary tube or micropipette.
very low concentration is used to avoid larger zone
• The spot is dried on the filter paper and is placed in
developing chamber.
11
12. Choice of the Solvent
12
• The commonly employed solvents are the polar
solvents, but the choice depends on the nature of the
substance to be separated.
• If pure solvents do not give satisfactory separation, a
mixture of solvents of suitable polarity may be
applied.
13. PAPER CHROMATOGRAPHY
•
•
•
•
MOBILE PHASE
Pure solvents, buffer solutions or mixture of solvents
Examples- Hydrophilic mobile phase
Isopropanol: ammonia:water 9:1:2
•
•
Methanol
N-butanol
: water
: glacial acetic acid : water
4:1
4:1:5
Hydrophobic mobile phases
dimethyl ether: cyclohexane
kerosene : 70% isopropanol
13
14. CHROMATOGRAPHIC CHAMBER
The chromatographic chamber are made up of many
materials like glass, plastic or stainless steel.
Glass tanks are preferred most. They are available in
various dimensional size depending upon paper
length and development type.
The chamber atmosphere should be saturated with
solvent vapor.
14
15. PAPER CHROMATOGRAPHY
DEVELOPMENT TECHNIQUE
• Paper is flexible when compared to glass plate
used in TLC, several types of development are
possible which increases the ease of operation.
15
•
• The paper is dipped in solvent in such a manner
that the spots will not dip completely into the
solvent.
The solvent will rise up and it is allowed to run
2/3rd of paper height for better and efficient result.
16. PAPER CHROMATOGRAPHY
• Different types of development tech. are
•
1) ASCENDING DEVELOPMENT (go up)
Like conventional type, the solvent flows against
gravity. The spots are kept at the bottom portion of
paper and kept in a chamber with mobile phase
solvent at the bottom.
16
17. PAPER CHROMATOGRAPHY
•
17
2) DESCENDING TYPE (a downward slope)
• This is carried out in a special chamber where the
solvent holder is at the top. The spot is kept at the
•
top and the solvent flows down the paper.
In this method solvent moves from top to bottom so
it is called descending chromatography.
• ADVANTAGE IS THAT, DEVELOPMENT IS FASTER
18. PAPER CHROMATOGRAPHY
3)ASCENDING – DESCENDING DEVELOPMENT
A hybrid of above two technique is called
ascending-descending chromatography.
Only length of separation increased, first ascending
takes place followed by descending
18
19. PAPER CHROMATOGRAPHY
4)CIRCULAR / RADIAL DEVELOPMENT
Spot is kept at the centre of a circular paper. The
solvent flows through a wick at the centre & spreads
in all directions uniformly.
19
20. PAPER CHROMATOGRAPHY
DRYING OF CHROMATOGRAM
• After the solvent has moved a certain distance for
certain time the chromatogram is taken out from the
tank & position of the solvent front is marked with a
pencil.
• They are dried by cold or hot air depending on
volatility of solvents. A simple hair dryer is a
convenient device to dry chromatograms.
20
21. DETECTING / VISUALISING AGENTS
If the substance are colored they are visually
detected easily.
But for colorless substance, Physical and chemical
methods are used to detect the spot.
(d) Non specific methods ( Physical methods)
E.g. iodine chamber method,
UV chamber for fluorescent compounds – at 254
or at 365nm.
PAPER CHROMATOGRAPHY
21
23. Following detecting tech. can also be
categorized as
•
•
•
1) Destructive techniques
Specific spray reagents, samples destroyed before
detection e.g. – ninhydrin reagent
2) Non-destructive techniques
•
•
For radio active materials - Geiger Muller counter
uv chamber, iodine chamber
QUANTITATIVE ESTIMATIONS
The method can be divided into two main groups
1. Direct techniques-
2. Indirect techniques-
23
24. PAPER CHROMATOGRAPHY
• Direct Measurement Method
• (i) Comparison of visible spots
• A rough quantitative measurements
• Component in a mixture can be carried out by comparing
the intensity and size of the spot with a standard
substance.
• (ii) Photo densitometry
• The method is used with the chromatograms of colored
compound, instrument which measures quantitatively
the density of the spots.
24
25. PAPER CHROMATOGRAPHY
•
•
25
(iii) Fluorimetry
The compound to be determined by fluorimetry must
be fluorescent or convertible into fluorescent
derivatives.
•
•
•
•
(iv) Radiotracer Method
The compound containing radioactive element is
labeled and treated with locating reagent. Using
Geiger Muller counter.
(v) Polarographic & Conductometric methods
Used to measure the amount of material in the spot
26. PAPER CHROMATOGRAPHY
• Indirect Measurement Method
26
• In this technique, the spots are cut into portions and
eluted with solvents. This solution can be analyzed
by any techniques of analysis like spectrophotometry,
electrochemical methods, etc.
27. Rf VALUE (Retardation Factor)
In paper
chromatography the
results are represented
by Rf value which
represent the movement
or migration of solute
relative to the solvent
front.
27
28. Factors affecting Rf VALUE
28
•
•
•
i. The temperature
ii. The purity of the solvents used
iii.The quality of the paper, adsorbents & impurities
present n the adsorbents
•
•
•
•
iv.Chamber saturation techniques, method of drying
& development
v. The distance travelled by the solute & solvent
vi.Chemical reaction between the substances being
partitioned.
vii. pH of the solution
29. Sources of Error
•
•
1. Error during application of the spots
Apply minimum volume of the concentrated solution
in order to avoid diffusion through the paper which
leads to poor separation
•
•
•
•
•
•
Spots should be approximately of the same diameter.
2. Development
Improper adjustment of the paper in the tank leads
to this error so the paper should be held vertically.
Do chamber saturation
3. Detection
The sprayi ng methods affect the finalresult
30. APPLICATIONS
• Separation of mixtures of drugs
•
•
Separation of carbohydrates, vitamins, antibiotics,
proteins, etc.
Identification of drugs
•
•
Identification of impurities
Analysis of metabolites of drugs in blood , urine ….
ADVANTAGES OF P.C
Simple ,rapid ,inexpensive ,excellent resolving
power
PRECAUTIONS IN P.C
Establishing the vapor solvent equilibrium
Stability of solvent mixture is first ensured