High Performance Adsorption Chromatography

High-Performance Adsorption Chromatography: Here the analyte species (components 

to be analysed) are adsorbed onto the surface of a polar packing. The stationary phase 

consists of finely divided solid particles packed inside a steel tube. If the component mixture 

is eluted through this tube with the mobile phase, different components present in the mixture 

adsorb to different degrees of strength and they become separated as the mobile phase moves 

steadily through the column. The nature of the  adsorption involves the interaction of polar 

molecules with a very polar solid stationary phase. The stationary phase could be silica gel or 

alumina. This method is extensively used for the separations of relatively non-polar, water-

insoluble organic compounds (since polar molecules will be adsorbed on to the column 

momentarily). 

One particular application is in resolving isomeric mixtures such as meta- and 

para-substituted benzene derivatives.  

liquid chromatography LC / HPLC

LIQUID CHROMATOGRAPHY (LC/HPLC)  

Principle:  Early liquid chromatography  was carried out in long glass columns with wide 

diameter. The diameters of the stacked particles inside the column were of the order of 150- 

200 microns range. Even then, the flow rates (eluent time) of the mobile phase with the 

analyte were very slow and separation times were long - often several hours!. With the 

advent of latest technology the  particle diameters were reduced as small as to 10 microns 

with replacement of glass columns with steel ones. The flow rate of the mobile phase was 

improved by applying high pressure to the column using pumps and hence the performance 

was improved. This development led the  instrument to be mostly called as  “High-

Performance Liquid  Chromatography” or  “High-Pressure Liquid Chromatography” 

(HPLC). Though HPLC retains major of the credits to the analytical side, the earlier one of 

simple Liquid Chromatography still finds applications in the preparative purposes.  

The HPLC technique can be divided into four main categories depending on the nature of the 

processes that occur at the columns as follow:

High-Performance Adsorption Chromatography: Here the analyte species (components

to be analysed) are adsorbed onto the surface of a polar packing. The stationary phase

consists of finely divided solid particles packed inside a steel tube. If the component mixture

is eluted through this tube with the mobile phase, different components present in the mixture

adsorb to different degrees of strength and they become separated as the mobile phase moves

steadily through the column. The nature of the  adsorption involves the interaction of polar

molecules with a very polar solid stationary phase. The stationary phase could be silica gel or

alumina. This method is extensively used for the separations of relatively non-polar, water-

insoluble organic compounds (since polar molecules will be adsorbed on to the column

momentarily). One particular application is in resolving isomeric mixtures such as meta- and

para-substituted benzene derivatives.

 High-Performance Partition Chromatography:  It is the most widely used liquid

chromatographic procedures to separate most kinds of organic molecules. Here the

components present in the analyte mixture distribute (or partition) themselves between the

mobile phase and stationary phase as the  mobile phase moves through the column. The

stationary phase actually consists of a thin liquid film either adsorbed or chemically bonded

to the surface of finely divided solid particles. Of these the latter is considered more

important and has a distinct stability advantage. It is not removed from the solid phase either

by reaction or by heat and hence it is more popular. It finds wide applications in various

fields, viz., pharmaceuticals, bio-chemicals, food products, industrial chemicals, pollutants,

forensic chemistry, clinical medicine, etc.

High-Performance Ion-Exchange Chromatography:  This method is used to separate

mixtures of ions (organic or inorganic),  and finds its application mostly in protein

separations. The stationary phase consists of very small polymer resin “beads” which have

many ionic bonding sites on their surface, termed as Ion Exchange Resins. This resin can be

either an anion exchange resin, which possesses positively charged sites to attract negative

ions, or a cation exchange resin, which possesses negatively charge sites to attract positive

ions. If the analyte mixture which contains mixture of ions is introduced into the column

packed with suitable ion-exchange resin, selected ions will be attached or bonded on to the

resin, thus being separated from other species that do not bond. Later, these attached ions can

be dislodged from the column by repeated elution with a solution that contains an ion that

competes for the charged groups on the resin surface, in other words, which has high affinity

for the charged sites on the resin than the analyte ions. Thus the analyte ions get exchanged

and separated from the column.

High Performance Size Exclusion Chromatography:  This technique is for separating

dissolved species on the basis of their size and particularly applicable to high-molecular-

weight species like oligomers and polymers to determine their  relative sizes and molecular

weight distributions. Here, the stationary phase is polymer resin, which contains small pores.

If the components to be separated are passed through the column the small sized particles can

easily enter into these pores and their mobility is retarded. Whereas the large sized particles,

which can’t enter into these pores can come out of the column fast and elude first. Thus the

separation of various sized particles is possible  through variations in the elution time. It is

classified into two categories based on the nature of the columns and their packing as:

Gel Filtration Chromatography - which uses hydrophilic packing  to separate polar species

and uses mostly aqueous mobile phases. This  technique is mostly used to identify the

molecular weights of large sized proteins & bio-molecules.

Gel Permeation Chromatography -  which uses hydrophobic packing to separate nonpolar

species and uses nonpolar organic solvents. This technique is used to identify the molecular

weights of polymers.

Instrumentation:  The basic HPLC system consists of  a solvent (mobile phase) reservoir,

pump, degasser, injection device, column and  detector. The pump draws the mobile phase

from the reservoir and pumps it to the column through the injector. At the end of the column

(effluent end), a detector is positioned. Mostly UV absorption detector is used. In the case of

analytical studies, after the detection the eluents are collected in waste bottles. In the case of

preparative studies the eluents are fractionally collected for further studies. Most of the

HPLC design will be the same as described for all the four main groups previously described.

However, there can be differences in selecting the specific detectors for particular type of

analysis, say for example, with ion-exchange chromatography, detectors commonly used are

conductivity detectors for obvious reasons. Other important detectors for HPLC separations

include refractive index detector, fluorescence  detector and mass selective detector. The

following is the most generalised outlay of the HPLC system:

Disadvantages:  Column performance is very sensitive, which depends on the method of

packing. Further, no universal and sensitive detection system is available.

ANALYSIS THROUGH CHROMATOGRAPHY

ANALYSIS THROUGH CHROMATOGRAPHY 

The technique through which the chemical components present in complex mixtures are 

separated, identified and determined is termed as chromatography. This technique is widely 

used like spectroscopy and is a very powerful tool not only for analytical methods but also 

for preparative methods. Compounds of high grade purity can be obtained by this method. 

Chromatography can be simply defined as follows: 

“It is the technique in which the components of a mixture are separated based upon the 

rates at which they are carried or moved through a stationary phase (column) by a gaseous 

or liquid mobile phase”.  

Based on the mobile phase this technique can be simply classified into two categories as: 

Liquid Chromatography and Gas Chromatography.  The column which holds the stationary 

phase (which in the form of small particles of the diameter of the order in microns), plays 

unique role in these processes. Usually silica is the base material for producing this phase.

Electron Spin Resonance Spectroscopy ESR

Electron Spin Resonance Spectroscopy (ESR):

The basic principle of electron spin resonance spectroscopy is  that, electrons always have a

spin and thus have a magnetic moment. Thus, the magnetic resonance theory applies to

electrons too like that of nuclei, as in NMR. Especially this technique is of high value when it

comes to the compounds which contain odd electrons, i.e. those substances which have

paramagnetic behaviour (if electrons are paired as in bonded  orbital then their mutual

spinning will cancel each other and there will be no response for the applied magnetic field,

whereas, if it is unpaired then it can align with the applied magnetic field and the feasibility 

of getting ESR spectra is higher). Thus, the  principle and the instrumentation are much 

similar to that of NMR technique. It is also referred as,  Electron Magnetic Resonance 

(EMR) or Electron Paramagnetic Resonance (EPR) spectroscopy. 

It is mostly used as a potential technique to study the formation and lifetime of free radicals, 

which are the major intermediates in most of the organic reactions. Another important 

application is in the estimation of trace amounts of paramagnetic ions, particularly in 

biological works like, Mn2+, Mg2+  etc. 

Nuclear Magnetic Resonance Spectroscopy NMR

Nuclear Magnetic Resonance Spectroscopy (NMR) : 

Principle :  In NMR substances absorb energy in the radio frequency region of the electro-

magnetic spectrum under influence of a strong magnetic field. It is a well known fact that the 

nuclei of the atoms bonded to each other in molecules spin on an axis like a top. Since nuclei 

are positively charged, this spin will create a  small magnetic field. If an external magnetic 

field is applied to these nuclei this magnetic  field will split into two energy levels. The 

energy difference is very small and corresponds to radiofrequency energy which is unique for 

every molecule and will give the information regarding the nature of the compounds and the 

presence of various functional groups and their environment.  

Since this technique is mostly measures the spinning of the hydrogen nuclei (almost all the 

organic compounds contain hydrogen atoms!), it is sometimes referred as Proton Magnetic 

Resonance (PMR) spectroscopy. 

Instrumentation :  The instrumentation for this technique includes powerful magnet, radio-

frequency signal generator, amplifier, detector, etc. The following is the outline of the 

instrument: 

Applications :  The application lies mostly in the identification and structural analysis of 

organic compounds and thus, it is mostly a tool for qualitative  analysis.  It gives valuable 

information regarding the position of the functional groups in a molecule and provides 

distinguished spectra for the isomer. Much  precise information on the structure of the 

compounds can be obtained using the same technique with other magnetic nuclei like C 13

, 

O17

, the instrumentation being the same except that the sweep of the magnetic field is varied. 

  

Disadvantages: Very expensive and the instrumentation is complex and needs exceptional 

skills to operate. Its sensitivity ranges from moderate to poor, however, can get clear 

information using C13

 or O17 

NMR. The usage of the solvents  is limited and in most of the 

situations deuterated solvents are required. 

Microwave Spectroscopy

Microwave Spectroscopy : 

This technique is actually an extension to IR spectroscopy. Microwave region lies at the far 

infra-red region of the electromagnetic spectrum and its absorption by molecules give rise to  

change in the  rotational energies  of the molecules. 

In IR spectroscopy, the molecules are subjected to changes in vibrational energies; the energy required for making changes at rotational levels is lesser than that for vibrational levels. Though the principles are same to 

that of IR, the instrumentation is slightly different and it requires samples in gaseous state for 

the analysis. 

Its applications are limited to smaller and simpler molecules since larger 

molecules will have interactions between the rotational energy levels within the molecule 

through various bonds they have. Besides qualitative analysis, this technique can be applied for conformational analysis  of simpler compounds (study of stereo chemistry of the compounds). 

Fourier Transform Raman Spectroscopy FT-Raman

Fourier Transform Raman Spectroscopy (FT-Raman): 

Principle : This technique is complementary to FT-IR and is a scattering technique, whereby a 

laser beam (near-IR region) is directed to the sample and the scattered radiation is collected. 

Most of the scattered radiation has the same wavenumber as that of the incident laser beam, 

however a fraction will be having a different wavenumber. This is the Raman signal and 

characteristic of particular functional group. Raman spectroscopy finds applications in 

identifying organic compounds containing non-polar bonds such as carbon - carbon double 

bonds or aromatic rings (weak dipoles).  

Instrumentation : The instrumentation comprises of exciting laser normally in near-IR region,  

Rayleigh filter, beam splitter, detector, etc. Data collection and processing are akin to IR  

including the Fourier transformations.  

Applications : The applications are similar to FT-IR and gives useful information on the non-

polar bonds, i.e. bonds with null or reduced dipole moment. Water is a good solvent for FT-

Raman. 

Disadvantages : Signal strength is normally weak, and liquid samples give poor signals. Heat 

sensitive samples can’t be analyzed, since local heating will damage the samples. Dark 

colored samples can’t be analyzed. 

Fourier Transform Infrared Spectroscopy FT-IR

Fourier Transform Infrared Spectroscopy (FT-IR): 

Principles :   It involves the absorption of electromagnetic radiation in the infrared region of 

the spectrum which results in changes in the vibrational energy of molecule. Since, usually 

all molecules will be having vibrations in the form of stretching, bending, etc., the absorbed 

energy will be utilised in changing the energy levels associated with them. It is a valuable 

and formidable tool in identifying organic compounds which have polar chemical bonds 

(such as OH, NH, CH, etc.) with good charge separation (strong dipoles).  

Instrumentation :  It was originally designed as a double beam spectrophotometer comprising 

IR source (red hot ceramic material), grating monochromator, thermocouple detector, cells 

made of either sodium chloride or potassium bromide materials, etc. In this process the light 

is dispersed by the monochromator. But, this type of basic design for IR measurements has 

been outdated. Instead a newer technique termed Fourier Transform-Infrared (FT-IR) has 

been in practice. This technique utilises a single beam of un-dispersed light and has the 

instrument components similar to the previous one. 

In FT-IR, the un-dispersed light beam is passed through the sample and the absorbances at all 

wavelengths are received at the detector simultaneously. A computerized mathematical 

manipulation (known as “Fourier Transform”) is performed on this data, to obtain absorption 

data for each and every wavelength. To perform this type of calculations interference of light 

pattern is required for which the FT-IR instrumentation contains two mirrors, one fixed and 

one moveable with a beam splitter in between them. Before scanning the sample a reference 

or a blank scanning is required. The following is the simplified design of the instrument: 

Applications :  It finds extensive use in the identification and structural analysis of organic 

compounds, natural products, polymers, etc. The presence of particular functional group in a 

given organic compound can be identified.  Since every functional group has unique 

vibrational energy, the IR spectra can be seen as their fingerprints. 

Disadvantages :  Samples containing mixture of substances can not be analysed. Since the 

sample holders and beam splitter, are made  of moisture sensitive materials like sodium 

chloride or potassium bromide (KBr), special cells are required for aqueous samples (e.g. 

KRS-5, ZnSe, etc.). Water is a bad solvent for IR spectral works.

Fluorometry Molecular Fluorescence

Fluorometry:  Molecular Fluorescence 

Principle :  This technique utilises the phenomenon  of molecular fluorescence, the theory 

behind this is exactly the same that has been discussed under atomic fluorescence but through 

the excitation of bonded electrons. Here, most often the irradiating light is in the range of 

ultraviolet and visible.  

Instrumentation :  The instrumental set-up comprises of a UV/Visible source, two 

monochromators, detector and recorder. The fluorescence exhibited by the sample is 

measured at right angles to the incident beam. The following is the basic set-up: 

Applications :  The applications of this technique are limited and it offers quantitative 

estimations of those compounds like benzene and fused benzene ring systems. Inorganic 

metals can also be analysed by the ability of  them to form complexes with the ligands. It 

finds uses in the analysis of foods for vitamin content, since vitamins like riboflavin, niacin, 

etc., exhibit fluorescence.

Only limited  compounds show the fluorescence hence this technique is relatively free of any interference and is very sensitive.

Disadvantages : The application is very limited as relatively a few substances exhibit
flourescence.

Ultraviolet Visible Spectroscopy UV/Vis

Ultraviolet - Visible Spectroscopy (UV/Vis): 

Principle :  It involves the absorption of electromagnetic radiation by the substances in the

ultraviolet and visible regions of the spectrum. This will result in changes in the electronic

structure of ions and molecules through the excitations of bonded and non-bonded electrons.

Instrumentation :  It consists of a dual light source viz., tungsten lamp for visible range and

deuterium lamp for ultraviolet region, grating monochromator, photo-detector, mirrors and

glass or quartz cells.

NOTE:  For measurements to be made under visible region both glass and quartz cells can be 

used. For the measurements under ultraviolet region, only quartz cell should be used, since, 

glass cells absorb ultraviolet rays.

There are two types of instrumental designs for this technique as single beam and double

beam spectrophotometers. However double beam spectrophotometers are widely used and

following is the outline of the instrument:

Applications :   It is the most widely used technique  for quantitative molecular analysis, for

this Beer-Lambert law is applied. Sometimes it is used in conjunction with other techniques

such as NMR, IR, etc., in the identification and structural analysis of organic compounds. For

qualitative analysis it provides valuable information through the absorption spectrum which

is unique for a given compound.

Disadvantages :  Samples should be in solution. Mixture of substances poses difficult to

analyse and requires prior separation. Interference from the sample’s matrix makes the

measurement difficult.

Atomic Fluorescence Fluorometry

Fluorometry : Atomic Fluorescence 

This technique is not widely used though its counterpart -  the molecular fluorescence is 

applied well to the analytical studies.

 The principle of atomic fluorescence is that when atoms are elevated to higher energy levels, they  sometimes return to the ground state through a 

pathway, which has several intermediate electronic states, before reaching to the actual 

ground state.

 Such series of fall through the electronic levels accompany by light emission - 

which is atomic fluorescence.

 The intensity of this emitted light is measured at right angles to 

the incident light and related to concentration.

Uses are similar to AAS and AES. 

Plasma Emission Spectroscopy

Plasma Emission Spectroscopy : 

Principle :  Mostly referred as Inductively Coupled Plasma (ICP) Emission Spectroscopy, is 

also an atomic emission technique, most closely related to the preceded flame photometry 

except that the atoms and ions present in the sample are excited in high temperature gas 

plasma.  Since the plasma provides very high  temperature and hence the energy, almost all 

the atoms present in the sample can be excited with this technique ending up with high 

efficiency (a hotter source increases both atomization  efficiency and excitation efficiency). 

Thus, the emissions from the atoms would  be more intense and even very small 

concentrations of metals/metal ions can be detected and accurately measured. 

Instrumentation:  This is basically an emission spectrometer comprising nebulizer, RF coil, 

ICP Source (Argon plasma), monochromator, detector and recorder. 

A plasma source or jet is a flame-like system of ionized, very hot flowing argon gas. At high 

temperatures (≈ 6000 K) a gas such as argon will contain a high proportion of ions and free 

electrons constituting plasma (This ionisation is initiated by “Tesla” coil). Additional energy 

may be supplied to the electrons in the plasma by the application of an external 

electromagnetic field through RF coil. By collisions between the electrons and other species 

in the plasma this additional energy is uniformly distributed. As the collisions increase, the 

energy transfer becomes more efficient, which leads to a substantial temperature 

enhancement to a range of 8000 - 10000 K. It is the temperature at which the samples are 

introduced and analysed. 

Applications : Similar to atomic emission spectroscopy but it covers very widespread for both 

qualitative and quantitative analysis of metals and some non-metals too, at trace levels. 

Because of the high temperature and homogeneity of the source, it offers better signal 

stability and hence the analytical precision. The technique when utilising an optical emission 

detector is termed as Inductively Coupled Plasma – Optical Emission Spectrometer  (ICP-

OES) and if it utilises a mass spectrometer (refer section 9.6) as detector then it is termed as 

Inductively Coupled Plasma – Mass Spectrometer (ICP-MS). 

Disadvantages:  Samples require dissolution before analysis. Instrumentation is complex and 

requires high operator’s skill and is very expensive. 

Atomic Emission Spectroscopy AES

Atomic Emission Spectroscopy (AES): 

Principle :  This is simply called as ‘Flame Photometry’, and measures the atoms excited by a

flame  (temperature range: 2000 – 31000

 K) and not by light source as in the atomic

absorption case. After excitation, atoms will readily lose the gained energy and revert back to

the ground state and the emission occurs. It is that emission that actually being measured.

The wavelengths of the emitted light will almost be similar as those that were absorbed in the

atomic absorption, since exactly the same energy transitions occur, except in the order of

reverse!    9

Instrumentation :  A simple flame photometer consists of burner, nebulizer, monochromator,

detector and recorder. The following is the simplified figure:

Applications :  It is used exclusively in  the quantitative determination of metals in solution,

especially alkali and alkaline earth in the given samples. The principle is like that described

for atomic absorption. Qualitative determination is also possible as  each element emits its

own characteristic line spectrum.

Disadvantages :  Intensity of emission is very sensitive to changes in flame temperature.

Usually, spectral interference and self-absorption are also  encountered which affects the

precision of the measurement. Further, a linear plot of absorbance against concentration is

not always obtained.

Atomic Absorption Spectroscopy AAS

Atomic Level 

Atomic Absorption Spectroscopy  AAS : 

Principle: The sample is vaporized by aspiration of solution into a flame or evaporation from 

electrically heated surface (temperature range: 1800 – 31000

 K). At this condition where the 

individual atoms co-exist, a beam of light is passed through them. The atoms will absorb in 

the visible and ultraviolet region resulting in changes in electronic structure (excited state). 

So, the resultant light beam coming out of the sample will be missing the light in the 

corresponding wave length, which is a measure of the characteristics of the sample. 

Instrumentation :  Sources emitting radiation characteristic of element of interest (hollow - 

cathode lamp), flame or electrically heated furnace, monochromator, detector 

(photomultiplier) and  recorder. The following is the simplified outline of the 

instrumentation: 

Applications : This is the most widely used technique for the quantitative determination of 

metals at trace levels (0.1 to 100ppm), which present in various materials. It utilizes Beer  - 

Lambert Law  for the analysis and a standard curve is obtained by plotting absorbance  vs 

concentration of the samples taken. The usual procedure is to prepare a series of standard 

solutions over a concentration  range suitable for the sample to be analysed. Then, the 

standards and the samples are separately aspirated into the flame, and the absorbances are 

read from the instrument. The plot will give the useful linear range and the concentrations of 

the samples can be found out from the plot. 

Disadvantages : Sample must be in solution or at least volatile. Individual source lamp and 

filters needed for each element, since, each metal has its own characteristic absorption.

Processes in Spectroscopy

ANALYSIS THROUGH SPECTROSCOPY 

 Processes in Spectroscopy 

The interaction of the light (electro-magnetic radiation) with a substance and the subsequent 

energy transfer ends with three main processes namely: 

Absorption:

The process by which the energy of the light (in the form of photons) is transferred to the 

atom or molecule raising them from the ground state to an excited state 

Fluorescence: 

The absorbed energy is rapidly lost to the surroundings by collisions within the system and 

relax back to the ground state. Sometimes the energy is not lost in this way but is re-emitted a 

few milli seconds later, which is referred as fluorescence 

Emission:          

If the substances (atoms or molecules) are heated to high temperatures (in a flame or in an 

electric discharge) the electrons are exited to higher energy levels. Later, they relax to the 

ground state with the emission of radiation, the magnitude of which is more or less equivalent 

to absorbed energy   

Most of the analytical techniques are based on the light interactions with the substances and 

utilise any of the above three associated processes. Substances interact with light differently 

at various wavelengths and hence different types of analysis & instruments. The entire 

spectrum of light can be represented as below. Since, light has both electrical and magnetic 

components, this representation is referred as an ‘Electro-Magnetic Spectrum’: 

The following is short comparison between Ultra Violet (UV), Visible (Vis) and Infra Red  

(IR) ranges for the energy, frequency and wavelength:

Energy:   UV > Vis > IR 

Frequency:   UV > Vis > IR 

Wavelength:   UV < Vis < IR         

The symbol for the wavelength is  “lambda” (λ) and the unit is either nanometer (nm) or 

micrometer (or micron, μm). The symbol for frequency is “nu” (μ) and the unit is either hertz 

or sec-1

A parameter closely related to frequency is the wave number, which has the symbol 

“nu bar” ( υ ) and the unit is cm-1

There are two levels by which the substances can interact with the light as, atomic level and 

molecular level and hence the corresponding techniques: 

CLASSIFICATION OF THE ANALYTICAL TECHNIQUES

CLASSIFICATION OF THE ANALYTICAL TECHNIQUES 

In a broad sense the techniques for the chemical analysis can be classified as follows. Though 

this classification doesn’t include few other techniques like radiochemical analyses, 

bioanalytical methods and some of the physical methods, it is more than sufficient to start 

with, since it covers almost all our Departmental analytical equipment under common pool:  

ANALYSIS THROUGH SPECTROSCOPY 

ANALYSIS THROUGH CHROMATOGRAPHY 

ANALYSIS THROUGH THERMAL ENERGY 

ANALYSIS THROUGH X-RAY TECHNIQUES 

ANALYSIS THROUGH MICROSCOPY  

ANALYSIS THROUGH ELECTROCHEMICAL TECHNIQUES 

ANALYSIS THROUGH MISCELLANEOUS TECHNIQUES 

This classification is based on the interactions of molecules with various forms of energy like 

electro-magnetic radiation, heat (thermal energy) and with  matters like electrons. Each 

technique has specific principle, mode of operation, advantages and disadvantages. 

Denigés' reagent for qualitative analysis

The Denigés' reagent is a reagent used for qualitative analysis.

Denigés' reagent is used to detect isolefin or tertiary alcohols which can be easily dehydrated to form isoolefin in the presence of acid. Treatment of solutions containing either isolefin or tertiary alcohols with this reagent will result in the formation of a solid yellow or red precipitate

How Synthesis Denigés' reagent

Despite the different stoichiometry in these mixtures which varies the concentration of the reagent, they all follow the same idea of adding HgO to distilled water and concentrated sulfuric acid. The Denigés' reagent is ultimately mercury(II) sulfate in an aqueous solution.

• 5 grams of mercury(II) oxide (HgO) is dissolved in 40 mL of distilled water. The mixture is slowly stirred, while 20 mL of concentrated sulfuric acid is added. After adding an additional 40 mL of distilled water, the solution is stirred until the HgO is completely dissolved.

• The Denigés' reagent can also be prepared by dissolving 5 grams of HgO in 20 mL of concentrated sulfuric acid and 100 mL of distilled water.

• The Denigés' reagent can be modified by using nitric acid in place of sulfuric acid

Benedict reagent Benedict's solution Benedict's test

Benedict reagent Benedict's solution Benedict's test

What is Benedict's Reagent?

Benedict's reagent also called Benedict's solution or Benedict's test) is a chemical reagent.

Benedict's reagent is used as a test for the presence of reducing sugars. This includes all monosaccharides and the disaccharides, lactose and maltose.

 Even more generally, Benedict's test will detect the presence of aldehydes (except aromatic ones), and alpha-hydroxy-ketones, including those that occur in certain ketoses. Thus, although the ketose fructose is not strictly a reducing sugar, it is an alpha-hydroxy-ketone, and gives a positive test because it is converted to the aldoses glucose and mannose by the base in the reagent.

The copper sulphate in Benedict's solution reacts with reducing sugars.

One litre of Benedict's reagent can be prepared from 100 g of anhydrous sodium carbonate, 173 g of sodium citrate and 17.3 g of copper(II) sulfate pentahydrate.

It is often used in place of Fehling's solution.

Benedict's reagent contains blue copper(II) ions (Cu²⁺) which are reduced to copper(I) ions (Cu⁺). These are precipitated as red copper(I) oxide which is insoluble in water.

This reagent is used to test for sugars. 
The disacarides (Sucrose, Lactose and Maltose) doesn't give a positive result with just the reagent, but there is a round about method I'm going to show in a future video

Model of a section of the molecules DNA

Model of a section of the molecules DNA In the form of a double helix in the three-dimensional space

DNA that contains genetic instructions that describe the biological evolution of living organisms and most viruses that also contains the genetic instructions needed to perform vital functions of all living organisms.

Baeyer's reagent

Baeyer's reagent : used in organic chemistry as a qualitative test for the presence of unsaturation, such as double bonds or triple bond .

The bromine test is also able to determine the presence of unsaturation.

Baeyer's reagent is an alkaline solution of potassium permanganate, which is a powerful oxidant Reaction with double or triple bonds (-C=C- or -C≡C-) in an organic material causes the color to fade from purplish-pink to brown.

 It is a syn addition reaction.

 Aldehydes and formic acid (and formic acid esters) also give a positive test

The brown solid manganese(IV) oxide precipitates out of solution when potassium permanganate reacts with a double or triple bond.

Notes : Baeyer's reagent, named after the German organic chemist Adolf von Baeyer

Instrumental methods of analytical chemistry

Instrumental analysis is a field of analytical chemistry that investigates analytes using scientific instruments

Block diagram of an analytical instrument showing the stimulus and measurement of response

Spectroscopy
Mass spectrometry
Crystallography
Electrochemical analysis
Thermal analysis
Separation
Hybrid techniques
Microscopy
Lab-on-a-chip

classical or wet chemistry methods of analytical chemistry

Classical or Wet Chemistry Methods of Analytical Chemistry

Qualitative analysis

A qualitative analysis determines the presence or absence of a particular compound, but not the mass or concentration. 

Chemical tests

There are numerous qualitative chemical tests, for example, the acid test for gold and the Kastle-Meyer test for the presence of blood.

Flame test

Inorganic qualitative analysis generally refers to a systematic scheme to confirm the presence of certain, usually aqueous, ions or elements by performing a series of reactions that eliminate ranges of possibilities and then confirms suspected ions with a confirming test. Sometimes small carbon containing ions are included in such schemes. With modern instrumentation these tests are rarely used but can be useful for educational purposes and in field work or other situations where access to state-of-the-art instruments are not available or expedient.

Gravimetric analysis

Gravimetric analysis involves determining the amount of material present by weighing the sample before and/or after some transformation. A common example used in undergraduate education is the determination of the amount of water in a hydrate by heating the sample to remove the water such that the difference in weight is due to the loss of water.

Volumetric analysis

Titration involves the addition of a reactant to a solution being analyzed until some equivalence point is reached. Often the amount of material in the solution being analyzed may be determined. Most familiar to those who have taken college chemistry is the acid-base titration involving a color changing indicator. There are many other types of titrations, for example potentiometric titrations. These titrations may use different types of indicators to reach some equivalence point.