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No. Title Writer Date
2 PGS-504 Amitava Rakshit 2012-10-17
1 PGS-506 Amitava Rakshit 2012-10-17

PGS-504   2012-10-17 16:05:44
Amitava Rakshit Basics of Laboratory Safety
Common laboratory rules and regulations

Safety glasses, goggles, face shield, and shoes (no sandals) must be worn at all times. Normal prescription lenses are insufficient! .Keep your work space clean and tidy. The working space, desk drawers, cabinets, and instruments must be kept neat and clean at all times. Use common sense and do not rush in the laboratory. Never be complacent about chemicals or chemical reactions. Common sense and consideration for fellow workers must be exercised rigorously and constantly.

In accord with the rules with regard to waste disposal, there are several protocols which one should observe. Clean solvent waste should be placed in the RED solvent waste can in the back of the laboratory. Other liquid waste will the sorted as organic or aqueous and put into appropriate waste containers in the waste hood in the back of the laboratory. Other solid chemical waste (including heavily contaminated towels) will be disposed of in canisters in the waste hood.
Normal trash can be thrown into the waste bins.


Work in the chemistry laboratory involves the use of inflammable solvents, some corrosive and toxic chemicals, and apparatus which, if used improperly, can cause minor to severe injury. All work with solvents and chemicals must be performed in the fume hoods NOT on the benchtop. Safety glasses and shoes must be worn at all times while in the laboratory.
A. Solvents
1. Never heat inflammable solvents, even small amounts, with or near a flame. As for refluxing or distillation, never place solvents in an open beaker. Pouring solvents in the vicinity of a flame is extremely hazardous. Use an oil bath, steam bath, water bath, heating mantle, or hot plate as a heat source whenever possible.
2. Ethyl ether and petroleum ether (bp 30-60°) are especially dangerous. Never heat them on a hot plate; always use a water or steam bath, and collect the distillate in an ice-cooled flask. In the case of ethyl ether, the receiver should be a filtering or distilling flask connected to the condenser with a cork and with a piece of rubber tubing leading from the side tube on the flask to the floor. This allows the heavy ether vapors to spread along the floor instead of the desktop where they may be ignited by burners.
3. If an inflammable solvent is spilled, have all workers at the desk turn off their burners and clean it up immediately using a cloth. Wring the solvent from the cloth into the solvent waste can and then rinse the cloth in the sink with much water. Use gloves.
4. If acetone is used to aid in drying glassware, use it sparingly and not near a flame.
5. Inflammable solvents which you may have contact with are: ether, ligroin (petroleum ether), cyclohexane, toluene, xylene, alcohols, ethyl acetate, carbon disulfide, acetone, dioxane, etc. If in doubt about the inflammability of a solvent, assume that it is hazardous.
6. Benzene and chlorinated solvents are toxic. In some cases, the toxic effect is cumulative. Avoid contact with the skin and inhalation of solvent vapors.
7. Many organic solvents freely permeate latex gloves commonly used in laboratories, and are therefore inadequate protection of the skin from solvent vapors. Thicker neoprene or butyl rubber gloves are recommended.
B. Chemicals
1. Especially corrosive substances which give off noxious fumes (e.g., bromine, acetyl chloride, benzyl chloride, phosphorus trichloride, acetic anhydride, fuming nitric and sulfuric acids, etc.) should be handled in the hoods. Use proper gloves. Do not spill these chemicals on yourself or on the desktops. They will cause very painful burns. Bromine is especially bad. Do not put any of these in organic waste cans.
2. Over the last several years a number of organic compounds have been confirmed as carcinogens and the list is steadily growing. It is best to assume that all chemicals are toxic, and possibly carcinogenic.
3. Sodium and potassium metals react explosively with water. They are rapidly corroded by the atmosphere and should be stored in kerosene or oil. These metals should not be allowed to come into contact with the skin. They may be handled with dry filter paper or tweezers. Unused pieces of metal may be destroyed by dropping into 95% ethyl alcohol, or they may be returned to the bottle. Avoid all contact between chlorinated solvents and sodium or potassium.

C. Apparatus
1. Approved safety glasses, goggles, or a face shield must be worn at all times when in the lab. Normal prescription lenses are insufficient due to the possibility of explosion.
2. When inserting tubing or thermometers into bored stoppers, it is wise to take some simple precautions. The tubing and stopper should be held by a towel, so that if the tubing breaks the towel will reduce the impact of the jagged edge. Very serious cuts have resulted from carelessness in inserting tubes in stoppers.
3. Closed systems are liable to explode if heated. Never carry out an atmospheric pressure distillation in a closed system.
4. Do not support apparatus on books, boxes, pencils, etc. Use large, strong wooden blocks, rings, or lab jacks. Assemblies with a high center of gravity (as when a reagent is added through the top of a condenser) should be assembled and operated with much care.
5. Use glass-stirring rods with care for breaking up solids. They are liable to break.
6. Do not evacuate Erlenmeyer flasks larger than 50 mL . They may collapse.
7. Oil baths and melting point baths can cause severe burns if spilled. Make sure they are well supported. Be especially careful not to get water into oil baths. We will use electric heating mantles in preference to oil baths when possible.
8. Dewar flasks and vacuum desiccators, because they are evacuated, implode easily when tipped over or dropped. Make sure the ones you use are wound on the outside with friction tape or are contained in protective shields, so they will not shower glass around the laboratory if broken.
D. Accidents
1. Fire. Personal safety is most important. If a person’s clothing catches on fire, he/she needs help. Prevent him/her from running. If he/she is close enough, put him/her under the safety shower because it is more effective than a blanket. If not, make him/her lie down and smother the flames by rolling, wrapping with lab coats, blankets, towels, etc. Never turn a carbon dioxide extinguisher on a person.
If a fire breaks out, turn off all burners and remove solvents if time allows. There are carbon dioxide extinguishers in the laboratory and the positions and operation of these should be known. Point the extinguisher at the base of the flames. Very small fires can be put out with a damp towel by smothering. Only after the safety of all is assured should the matter of extinguishing the fire be considered.
Because a few seconds delay can result in very serious injury, every person in the laboratory should plan in advance what he/she will do in case of such an emergency.
2. Chemicals. If corrosive chemicals are spilled on the clothing, immediate showering (with clothing on) is the best remedy. Safety showers are located by each door. If chemicals are spilled on the skin, wash them off with large volumes of water. Bromine should be washed off with water and the skin then massaged with ethanol or glycerine. Do no apply a burn ointment. If the chemical is spilled in the eye, it should immediately be washed out thoroughly with water using the eyewash sprayer in the sinks. If acid was involved, a weak solution of sodium bicarbonate in an eyecup should then be used. If a base, boric acid is effective.
If corrosive chemicals are spilled on the desk, dilute them with a large volume of water and then neutralize with sodium bicarbonate if an acid, or dilute acetic acid if a base.
Flame photometer
Principles of operation
Flame photometry relies upon the fact that the compounds of the alkali and alkaline earth metals can be thermally dissociated in a flame and that some of the atoms produced will be further excited to a higher energy level. When these atoms return to the ground state they emit radiation which lies mainly in the visible region of the spectrum. Each element will emit radiation at a wavelength specific for that element. The table below gives details of the measurable atomic flame emissions of the alkali and alkaline earth metals in terms of the emission wavelength and the colour produced.
Element Emission Wavelength (nm) Flame Colour
Sodium (Na) 589 Yellow
Potassium (K) 766 Violet
Barium (Ba) 554 Lime Green
Calcium (Ca) 622 Orange
Lithium (Li) 670 Red

Over certain ranges of concentration the intensity of the emission is directly proportional to the number of atoms returning to the ground state. This is in turn proportional to the absolute quantity of the species volatized in the flame, i.e. light emitted is proportional to sample concentration.It can be seen that if the light emitted by the element at the characteristic wavelength is isolated by an optical filter and the intensity of that light measured by a photo-detector, then an electrical signal can be obtained proportional to sample concentration. Such an electrical signal can be processed and the readout obtained in an analogue or digital form.
A simple flame photometer consists of the following basic components:
a) The burner: a flame that can be maintained in a constant form and at a constant temperature.
b) Nebuliser and mixing chamber: a means of transporting a homogeneous solution into the flame at a steady rate.
c) Simple colour filters (interference type): a means of isolating light of the wavelength to be measured from that of extraneous emissions.
d) Photo-detector: a means of measuring the intensity of radiation emitted by the flame.
The analysis of alkali and alkaline earth metals by flame photometry has two major advantages:
i. Their atoms reach the excited state at a temperature lower than that at which most
other elements are excited.
ii. Their characteristic wavelengths are easily isolated from those of most other elements
due to wide spectral separation.
The analysis of Na, K, Li, Ba and Ca are typically determined at low temperatures, i.e. 1500- 2000°C, therefore suitable fuel mixtures are propane/air, butane/air and natural gas/air.
Atomic absorption spectroscopy (AAS)
Atomic absorption spectroscopy (AAS) is a spectroanalytical procedure for the qualitative and quantitative determination of chemical elements employing the absorption of optical radiation (light) by free atoms in the gaseous state. In analytical chemistry the technique is used for determining the concentration of a particular element (the analyte) in a sample to be analyzed. AAS can be used to determine over 70 different elements in solution or directly in solid samples. Atomic absorption spectrometry was first used as an analytical technique, and the underlying principles were established in the second half of the 19th century by Robert Wilhelm Bunsen and Gustav Robert Kirchhoff.
The technique makes use of absorption spectrometry to assess the concentration of an analyte in a sample. It requires standards with known analyte content to establish the relation between the measured absorbance and the analyte concentration and relies therefore on Beer-Lambert Law. In short, the electrons of the atoms in the atomizer can be promoted to higher excited state for a short period of time (nanoseconds) by absorbing a defined quantity of energy (radiation of a given wavelength). This amount of energy, i.e., wavelength, is specific to a particular electron transition in a particular element. In general, each wavelength corresponds to only one element, and the width of an absorption line is only of the order of a few picometers (pm), which gives the technique its elemental selectivity. The radiation flux without a sample and with a sample in the atomizer is measured using a detector, and the ratio between the two values (the absorbance) is converted to analyte concentration or mass using Beer-Lambert Law.
In order to analyze a sample for its atomic constituents, it has to be atomized. The atomizers most commonly used nowadays are flames and electrothermal (graphite tube) atomizers. The atoms should then be irradiated by optical radiation, and the radiation source could be an element-specific line radiation source or a continuum radiation source. The radiation then passes through a monochromatic in order to separate the element-specific radiation from any other radiation emitted by the radiation source, which is finally measured by a detector.

Fig Schematic of an atomic-absorption spectroscopy
Although other atomizers, such as heated quartz tubes, might be used for special purposes, the atomizers most commonly used nowadays are (spectroscopic) flames and electrothermal graphite tube) atomizers.
Flame atomizers
The oldest and most commonly used atomizers in AAS are flames, principally the air-acetylene flame with a temperature of about 2300 °C and the nitrous oxide (N2O)-acetylene flame with a temperature of about 2700 °C. The latter flame, in addition, offers a more reducing environment, being ideally suited for analytes with high affinity to oxygen.
Liquid or dissolved samples are typically used with flame atomizers. The sample solution is aspirated by a pneumatic nebulizer, transformed into an aerosol, which is introduced into a spray chamber, where it is mixed with the flame gases and conditioned in a way that only the finest aerosol droplets (< 10 μm) enter the flame. This conditioning process is responsible that only about 5% of the aspirated sample solution reaches the flame, but it also guarantees a relatively high freedom from interference.
On top of the spray chamber is a burner head that produces a flame that is laterally long (usually 5-10 cm) and only a few mm deep. The radiation beam passes through this flame at its longest axis, and the flame gas flow-rates may be adjusted to produce the highest concentration of free atoms. The burner height may also be adjusted, so that the radiation beam passes through the zone of highest atom cloud density in the flame, resulting in the highest sensitivity. The processes in a flame include desolvation, vaporization, atomization and ionization. Each of these stages includes the risk of interference in case the degree of phase transfer is different for the analyte in the calibration standard and in the sample.
Electrothermal atomizers
Electrothermal using graphite tube atomizers uses a wide variety of graphite tube designs have been used over the years, the dimensions nowadays are typically 20-25 mm in length and 5-6 mm inner diameter. With this technique liquid/dissolved, solid and gaseous samples may be analyzed directly. A measured volume (typically 10-50 μL) or a weighed mass (typically around 1 mg) of a solid sample are introduced into the graphite tube and subject to a temperature program. This typically consists of drying, pyrolysis, atomization and cleaning. This technique has the advantage that any kind of sample, solid, liquid or gaseous, can be analyzed directly. Its sensitivity is 2-3 orders of magnitude higher than that of flame AAS, so that determinations in the low μg L-1 range (for a typical sample volume of 20µL) and ng g-1 range (for a typical sample mass of 1 mg) can be carried out.
Radiation sources

Radiation sources are defined as those devices and their associated apparatus components which
produce electromagnetic radiation.
Hollow cathode lamps
Hollow cathode lamps (HCL) are the most common radiation source in LS AAS. Inside the sealed lamp, filled with argon or neon gas at low pressure, is a cylindrical metal cathode containing the element of interest and an anode. A high voltage is applied across the anode and cathode, resulting in an ionization of the fill gas. The gas ions are accelerated towards the cathode and, upon impact on the cathode, sputter cathode material that is excited in the glow discharge to emit the radiation of the sputtered material, i.e., the element of interest. Most lamps will handle a handful of elements, i.e. 5-8. A typical machine will have two lamps, one will take care of five elements and the other will handle four elements for a total of nine elements analyzed.
Electrodeless discharge lamps
Electrodeless discharge lamps (EDL) contain a small quantity of the analyte as a metal or a salt in a quartz bulb together with an inert gas, typically argon, at low pressure. The bulb is inserted into a coil that is generating an electromagnetic radio frequency field, resulting in a low-pressure inductively coupled discharge in the lamp. The emission from an EDL is higher than that from an HCL, and the line width is generally narrower, but EDLs need a separate power supply and might need a longer time to stabilize.
Deuterium lamps
Deuterium HCL or even hydrogen HCL and deuterium discharge lamps are used in LS AAS for background correction purposes. The radiation intensity emitted by these lamps is decreasing significantly with increasing wavelength, so that they can be only used in the wavelength range between 190 and about 320 nm.
Continuum sources
When a continuum radiation source is used for AAS, it is necessary to use a high-resolution monochromator, as will be discussed later. In addition it is necessary that the lamp emits radiation of intensity at least an order of magnitude above that of a typical HCL over the entire wavelength range from 190 nm to 900 nm. A special high-pressure xenon short arc lamp, operating in a hot-spot mode has been developed to fulfill these requirements.
Theoretical Concepts and Definitions

Several related terms are used to define the amount of light absorption which has taken place. The ‘‘transmittance’’ is defined as the ratio of the final intensity to the initial intensity.
T = I/Io
Transmittance is an indication of the fraction of initial light which passes through the flame cell to fall on the detector. The ‘‘percent transmission’’ is simply the transmittance expressed in percentage terms.
%T = 100 x I/Io
The ‘‘percent absorption’’ is the complement of percent transmission defining the percentage of the initial light intensity which is absorbed in the flame.
%A = 100 - %T
These terms are easy to visualize on a physical basis. The fourth term, ‘‘absorbance’’, is purely a mathematical quantity.
A = log (Io/I)
Absorbance is the most convenient term for characterizing light absorption in absorption spectrophotometry, as this quantity follows a linear relationship with concentration.
Beer’s Law defines this relationship:
A = abc
where ‘‘A’’ is the absorbance; ‘‘a’’ is the absorption coefficient, a constant which is characteristic of the absorbing species at a specific wavelength; ‘‘b’’ is the length of the light path intercepted by the absorption species in the absorption cell; and ‘‘c’’ is the concentration of the absorbing species.

Figure Concentration versus absorbance

This equation simply states that the absorbance is directly proportional to the concentration of the absorbing species for a given set of instrumental conditions. This directly proportional behavior between absorbance and concentration is observed in atomic absorption. When the absorbances of standard solutions containing known concentrations of analyte are measured and the absorbance data are plotted against concentration, a calibration relationship is established. Over the region where the Beer’s Law relationship is observed, the calibration yields a straight line. As the concentration and absorbance increase, nonideal behavior in the absorption process can cause a deviation from linearity, as shown. After such a calibration is established, the absorbance of solutions of unknown concentrations may be measured and the concentration determined from the calibration curve. In modern instrumentation, the calibration can be made within the instrument to provide a direct readout of unknown concentrations.
18.3 Inductively coupled plasma spectroscopy
Inductively coupled plasma spectroscopy (ICP), also referred to as inductively coupled plasma optical emission spectrometry, is an analytical technique used for the detection of trace metals. It is a type of emission spectroscopy that uses the inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. The intensity of this emission is indicative of the concentration of the element within the sample.
The ICP is composed of two parts: the ICP and the optical spectrometer. The ICP torch consists of 3 concentric quartz glass tubes. The output or "work" coil of the radio frequency (RF) generator surrounds part of this quartz torch. Argon gas is typically used to create the plasma.
When the torch is turned on, an intense electromagnetic field is created within the coil by the high power radio frequency signal flowing in the coil. This RF signal is created by the RF generator which is, effectively, a high power radio transmitter driving the "work coil" the same way a typical radio transmitter drives a transmitting antenna. The argon gas flowing through the torch is ignited with a Tesla unit that creates a brief discharge arc through the argon flow to initiate the ionization process. Once the plasma is "ignited", the Tesla unit is turned off.
The argon gas is ionized in the intense electromagnetic field and flows in a particular rotationally symmetrical pattern towards the magnetic field of the RF coil. A stable, high temperature plasma of about 7000 K is then generated as the result of the inelastic collisions created between the neutral argon atoms and the charged particles.

Figure ICP torch
A peristaltic pump delivers an aqueous or organic sample into a nebulizer where it is changed into mist and introduced directly inside the plasma flame. The sample immediately collides with the electrons and charged ions in the plasma and is itself broken down into charged ions. The various molecules break up into their respective atoms which then lose electrons and recombine repeatedly in the plasma, giving off radiation at the characteristic wavelengths of the elements involved.
In some designs, a shear gas, typically nitrogen or dry compressed air is used to 'cut' the plasma flame at a specific spot. One or two transfer lenses are then used to focus the emitted light on a diffraction grating where it is separated into its component wavelengths in the optical spectrometer. In other designs, the plasma impinges directly upon an optical interface which consists of an orifice from which a constant flow of argon emerges, deflecting the plasma and providing cooling while allowing the emitted light from the plasma to enter the optical chamber. Still other designs use optical fibers to convey some of the light to separate optical chambers.

Figure Schematic of an ICP system
Within the optical chamber(s), after the light is separated into its different wavelengths (colors), the light intensity is measured with a photomultiplier tube or tubes physically positioned to "view" the specific wavelength(s) for each element line involved, or, in more modern units, the separated colors fall upon an array of semiconductor photodetectors such as charge coupled devices (CCDs). In units using these detector arrays, the intensities of all wavelengths (within the system's range) can be measured simultaneously, allowing the instrument to analyze for every element to which the unit is sensitive all at once. Thus, samples can be analyzed very quickly.
The intensity of each line is then compared to previously measured intensities of known concentrations of the elements, and their concentrations are then computed by interpolation along the calibration lines.
Advantages and Disadvantages
Advantages of using an ICP include its ability to identify and quantify all elements with the exception of Argon; since many wavelengths of varied sensitivity are available for determination of any one element, the ICP is suitable for all concentrations from ultratrace levels to major components; detection limits are generally low for most elements with a typical range of 1 - 100 g / L. Probably the largest advantage of employing an ICP when performing quantitative analysis is the fact that multielemental analysis can be accomplished, and quite rapidly. A complete multielement analysis can be undertaken in a period as short as 30 seconds, consuming only 0.5 ml of sample solution. Although in theory, all elements except Argon can be determined using and ICP, certain unstable elements require special facilities for handling the radioactive fume of the plasma. Also, an ICP has difficulty handling halogens--special optics for the transmission of the very short wavelengths become necessary.
ICP is often used for analysis of trace elements in soil, and it is for that reason it is often used in forensics to ascertain the origin of soil samples found at crime scenes or on victims etc. Taking one sample from a control and determining the metal composition and taking the sample obtained from evidence and determine that metal composition allows a comparison to be made. ICP is also used for motor oil analysis. Analyzing used motor oil reveals a great deal about how the engine is operating.
Gas Liquid Chromatography (GLC)
GLC Instrumentation
• Gas cylinder and regulator
o Nitrogen
o Helium
o Argon
o Hydrogen
• Injection port
• Column
o Gas-solid - adsorption
o Gas-liquid - High boiling point stationary phase
o Glass, steel, capillary glass
• Column oven
• Detector
o Flame ionization (FID) general purpose - modest sensitivity
o Nitrogen phosphorus FID specific for N and P
o Electron capture (EC)
o Mass spectrometry (MS)
• Recorder

Fig Diagram illustrating a Typical GLC System
Gas chromatography - specifically gas-liquid chromatography - involves a sample being vapourised and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.
Instrumental components
Carrier gas
The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependant upon the type of detector which is used. The carrier gas system also contains a molecular sieve to remove water and other impurities.
Sample injection port
For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a "plug" of vapour - slow injection of large samples causes band broadening and loss of resolution. The most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column. The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample. For packed columns, sample size ranges from tenths of a microliter up to 20 microliters. Capillary columns, on the other hand, need much less sample, typically around 10-3 mL. For capillary GC, split/splitless injection is used. Have a look at this diagram of a split/splitless injector;
The injector can be used in one of two modes; split or splitless. The injector contains a heated chamber containing a glass liner into which the sample is injected through the septum. The carrier gas enters the chamber and can leave by three routes (when the injector is in split mode). The sample vapourises to form a mixture of carrier gas, vapourised solvent and vapourised solutes. A proportion of this mixture passes onto the column, but most exits through the split outlet. The septum purge outlet prevents septum bleed components from entering the column.
There are two general types of column, packed and capillary (also known as open tubular). Packed columns contain a finely divided, inert, solid support material (commonly based on diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.
Column temperature
For precise work, column temperature must be controlled to within tenths of a degree. The optimum column temperature is dependant upon the boiling point of the sample. As a rule of thumb, a temperature slightly above the average boiling point of the sample results in an elution time of 2 - 30 minutes. Minimal temperatures give good resolution, but increase elution times. If a sample has a wide boiling range, then temperature programming can be useful. The column temperature is increased (either continuously or in steps) as separation proceeds.
There are many detectors which can be used in gas chromatography. Different detectors will give different types of selectivity. A non-selective detector responds to all compounds except the carrier gas, a selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound. Detectors can also be grouped into concentration dependant detectors and mass flow dependant detectors. The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample Dilution of with make-up gas will lower the detectors response. Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gas. Have a look at this tabular summary of common GC detectors:
Detector Type Support gases Selectivity Detectability Dynamic range
Flame ionization (FID) Mass flow Hydrogen and air Most organic cpds. 100 pg 107
Thermal conductivity (TCD) Concentration Reference Universal 1 ng 107
Electron capture (ECD) Concentration Make-up Halides, nitrates, nitriles, peroxides, anhydrides, organometallics 50 fg 105
Nitrogen-phosphorus Mass flow Hydrogen and air Nitrogen, phosphorus 10 pg 106
Flame photometric (FPD) Mass flow Hydrogen and air possibly oxygen Sulphur, phosphorus, tin, boron, arsenic, germanium, selenium, chromium 100 pg 103
Photo-ionization (PID) Concentration Make-up Aliphatics, aromatics, ketones, esters, aldehydes, amines, heterocyclics, organosulphurs, some organometallics 2 pg 107
Hall electrolytic conductivity Mass flow Hydrogen, oxygen Halide, nitrogen, nitrosamine, sulphur
High-performance liquid chromatography
High-performance liquid chromatography, HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.
High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster.It also allows to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture.The other major improvement over column chromatography concerns the detection methods which can be used. These methods are highly automated and extremely sensitive.
HPLC typically utilizes different types of stationary phases, a pump that moves the mobile phase(s) and analyte through the column, and a detector to provide a characteristic retention time for the analyte. The detector may also provide additional information related to the analyte. Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase. It is a form of liquid chromatography that utilizes smaller column size, smaller media inside the column, and higher mobile phase pressures.

Figure Schematic of HPLC
With HPLC, a pump provides the higher pressure required to move the mobile phase and analyte through the densely packed column. The increased density arises from smaller particle sizes. This allows for a better separation on columns of shorter length when compared to ordinary column chromatography.
The column and the solvent
Confusingly, there are two variants in use in HPLC depending on the relative polarity of the solvent and the stationary phase.
Normal phase HPLC
This is essentially just the same as in thin layer chromatography or column chromatography. Although it is described as "normal", it isn't the most commonly used form of HPLC.
The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for example. A typical column has an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to 250 mm.Polar compounds in the mixture being passed through the column will stick longer to the polar silica than non-polar compounds will. The non-polar ones will therefore pass more quickly through the column.
Reversed phase HPLC
In this case, the column size is the same, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in them. A polar solvent is used - for example, a mixture of water and an alcohol such as methanol.In this case, there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. There won't be as much attraction between the hydrocarbon chains attached to the silica and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent.
Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules, for example. They therefore spend less time in solution in the solvent and this will slow them down on their way through the column.That means that now it is the polar molecules that will travel through the column more quickly.Reversed phase HPLC is the most commonly used form of HPLC.
The sample to be analyzed is introduced, in small volumes, into the stream of mobile phase. The solution moved through the column is slowed by specific chemical or physical interactions with the stationary phase present within the column. The velocity of the solution depends on the nature of the sample and on the compositions of the stationary (column) phase. The time at which a specific sample elutes (comes out of the end of the column) is called the retention time; the retention time under particular conditions is considered an identifying characteristic of a given sample. The use of smaller particle size column packing (which creates higher backpressure) increases the linear velocity giving the components less time to diffuse within the column, improving the chromatogram resolution. Common solvents used include any miscible combination of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the sample components, or compounds such as trifluoroacetic acid which acts as an ion pairing agent.
Injection of the sample
Injection of the sample is entirely automated.
Retention time
The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound.
Different compounds have different retention times. For a particular compound, the retention time will vary depending on:
• the pressure used (because that affects the flow rate of the solvent)
• the nature of the stationary phase (not only what material it is made of, but also particle size)
• the exact composition of the solvent
• the temperature of the column
The detector
There are several ways of detecting when a substance has passed through the column. A common method which is easy to explain uses ultra-violet absorption.Many organic compounds absorb UV light of various wavelengths.
Interpreting the output from the detector
The output will be recorded as a series of peaks - each one representing a compound in the mixture passing through the detector and absorbing UV light. Peaks can also be used as a way of measuring the quantities of the compounds present. The area under the peak is proportional to the amount of compound which has passed the detector, and this area can be calculated automatically by the computer linked to the display.

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