Double beam filter photometer

Double beam filter photometer:


double beam filter photometer
double beam filter photometer


It consists of a source of light (tungsten filament lamp), lens to make the light beam parallel, filter of wavelength selection, cuvette with sample holder for keeping the solution to be analyzed, mirror for incident the part of light beam onto reference photocell, two photocells (one as reference and other as measuring), potentiometers for zero and span adjustments and a recording device (galvanometer).

In double beam photometer, the light rays from the source are made parallel and passed through a filter. It is divided into two parts; one part passes through the sample solution cuvette and falls on the measuring photocell and the other part passes directly onto the reference photocell. The galvanometer receives opposing currents from the two photocells.

Steps in experiment:

1)      With the lamp off, the galvanometer is adjusted to zero mechanically.

2)      The potentiometer R1 is adjusted for T=1 or A=0.

3)      Then with lamp on blank solution is placed in the light path of measuring photocell and potentiometer R2 is adjusted until the galvanometer reads zero.

4)      The solution to be analyzed is then placed in the light path of measuring photocell and R1 is adjusted until the galvanometer reads zero, keeping R2 unchanged. The absorbance or transmittance can be read directly on the scale of potentiometer R1. Since the potentiometer R1 is calibrated in terms of transmittance and absorbance.

In double beam filter photometer errors due to fluctuations of the lamp intensity are minimized also the scale of potentiometer R1 can be made much larger in size than the scale of meter in single beam filter photometer.

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Single beam filter photometer

Single beam filter photometer:


single beam filter photometer
single beam filter photometer


It consists of a source of light (tungsten filament lamp), lens to make the light beam parallel, filter of wavelength selection, cuvette with sample holder for keeping the solution to be analyzed , detector (photocell) and reading device (galvanometer or micro-ammeter).

The tungsten filament lamp gives the light radiation. This light is incident on the lens which makes it a parallel beam of light. This parallel beam of light is passed through the sample solution after passing through a filter. The sample absorbs some light energy, transmitting the other. This transmitted light falls on the photocell that generates the photocurrent. This photo-current is recorded by the galvanometer or micro ammeter which is having transmittance scale, since the photometer is directly proportional to the transmitted light, the transmittance scale is linear.

Steps in experiment:

1)      With photocell darkened, the meter is adjusted to zero by zero adjustment.

2)      The blank or reference solution is inserted in the path of light beam and light intensity is adjusted by means of rheostat in series with lamp. With this adjustment the meter reading is brought to 100 scale divisions.

3)      Solutions of both standard and unknown samples are inserted in place of blank and the reading of the specimen relative to the blank is recorded.

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High Pressure Liquid Chromatography (HPLC)

High Pressure Liquid Chromatography (HPLC):

In this post we are going to learn construction and working of high pressure liquid chromatography and some basic types of high pressure liquid chromatography (HPLC).

Basically a high pressure liquid chromatography (HPLC) is a method used to separate the components in a mixture to identify and quantify each component.

Construction and working of High Pressure Liquid Chromatography


high pressure liquid chromatography HPLC
high pressure liquid chromatography HPLC

A high pressure liquid chromatography consists of following major components:

1)      A high pressure pumps system to force the liquid mobile phase through the column.

2)      Gradient elution or solvent programmer.

3)      The sample injection system.

4)      Column in thermo stated oven.

5)      Detector and recorder.

In HPLC (High Pressure Liquid Chromatography) the sample to be analyzed is injected in the column as the mobile phase. This mobile phase flows over the stationary phase in the column. This causes separation of the sample components. These components leave the column at different time and reach at the detector. The detector detects the components and gives the signal to the recorder. The recorder shows the chromatograph as shown in the figure.

The peak position determines the component and the peak amplitude determines the concentration of the compound in the sample.

Sample injection system in High Pressure Liquid Chromatography (HPLC):

There are three methods of sample injection as follows:

1)      Syringe injection method.

2)      Sampling loops method.

3)      Automatic injection method.

  • Syringe injection method:

In this method the sample is introduced through a self sealing elastomeric septum, for this purpose micro syringe designed to withstand pressure up to 1500 psi are used. In stop flow injections, the flow of solvent is stopped momentarily and a fitting at the column head is removed. Then the sample is injected directly onto the head of column packing. After replacing the fitting, the system is again pressurized. This method is simple but the reproducibility of the result can’t be obtained.

  • Sampling loop method:

Sampling loop method
Sampling loop method

The diagram shows sampling loop configuration. These valve devices are the integral part of high pressure liquid chromatography (HPLC) equipment and have interchangeable loops providing a choice of sample sizes from 5 to 500ul. Sampling loops of this type permit the introduction of samples at pressure up to 7000psi. Micro sample injection valves with sampling loops having volumes of 0.5 to 5 ul are also available.

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Liquid Chromatography Principle & its Types

Hi friends, in this post we are going to discuss about Liquid Chromatography and its classification. Later on we will discuss each type of liquid chromatography in detail.

The Liquid Chromatography is classified as follows:

  1. Thin layer chromatography
  2. Paper chromatography
  3. Ion exchange
  4. Column chromatography : Column chromatography is again classified as :
  • liquid/ liquid (Partition)
  • Liquid/ solid (Adsorption)
  • Gel permeation

Introduction to various types of Liquid Chromatography:

Now we will see all above types of Liquid Chromatography in detail as follows:

1)      Thin layer chromatography:

Separation of black ink on a TLC plate (This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license.)

In Thin Layer Chromatography the stationary adsorbents are applied to a planar glass or plastic surface and the mobile phase (sample to be analyzed) is allowed to flow over the stationary phase. For separation of components in the sample anyone of the following techniques is used:

2)      Paper chromatography:

Paper chromatography
Paper chromatography (This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license.)

Paper partition chromatography makes use of strips or hollow cylinders of filter paper to hold the solid and liquid phases. In this system, the drops of the sample solutions are applied to number of parallel strips placed at few inches from the end of each test paper and allowed to dry. These strips of paper are then placed in a chromatographic chamber with a saturating and equilibrating vapor and hung from a solvent downward can be timed and the components can be measured.

3)      Column Chromatography:

a)      Liquid/ liquid chromatography: In this method, the liquid stationary phase is retained on the surface of packing by physical adsorption and the sample (mobile phase) is allowed to percolates through the column (stationary phase).

b)      Liquid/ solid (Adsorption) chromatography: In this method, the stationary phase is formed by a solid adsorbent, generally silica and alumina in powdered form. The sample solution is allowed to percolate through the stationary phase in the column.

c)       Gel permeation chromatography: In this method, the separation is based on molecular size and shape. The gel permeation column is packed with a stationary phase in the form of a gel contacting pores of a specific size. The sample solution is allowed to flow over the column bed then the sample penetrates the pores in the packing gel (depending upon the size and shape of the molecules). The large molecules don’t penetrate the gel and leave the column earlier. For more details on Gel permeation click here.


4)      Ion Exchange Chromatography:

In ion exchange chromatography, the exchange of ions between solution and solid insoluble in contact with solution takes place. The ion exchange process is reversible. In this process, when a sample is introduced at the top of the ion exchange column, the sample ions are displaced into solution again and then re-exchange on to the resin. This process continuous till the sample ions leave the column. Now the various sample ions corresponding to the different components in the sample are hold on to the resin to different extent. This cause the different time of passage through the column for different ions and separation of the sample components is achieved. For more details on Ion exchange chromatography click here.

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What is Gas Chromatography?

This post provides basic information about what is gas chromatography, construction and working of gas chromatography and finally some applications of gas chromatography.

Definition of Gas Chromatography:

Chromatography is a physical method of separation of the compounds of a mixture by distribution between two phases out of which one is a stationary bed of large surface area and the other is a fluid phase which percolates through or along the stationary phase.

Construction and working principle of gas chromatography:

The basic gas chromatograph consists of following parts

  1. Carrier gas supply with pressure regulator and flow monitor
  2. Sample injection system.
  3. Chromatographic column
  4. Thermal compartment
  5. Detection system
  6. Strip chart recorder

Diagram of Gas Chromatography:

Gas Chromatography
Gas Chromatography

In gas chromatography, the carrier gas is stored in a cylinder at a controlled pressure. The carrier is stored in a cylinder at controlled pressure. The pressure regulator is fitted on the cylinder. This gas is passed to sample injection port at a fixed, controlled flow rate through a floe regulator. The sample injection port is maintained at a certain temperature T1. The temperature T1 is selected such that the rapid vaporization of solute takes place but it doesn’t have a thermal degradation.

The sample injector injects the gas/ liquid sample through syringe. The solute vapor mixes rapidly with the carrier gas and flows into the column. In the column, the different solutes in the vaporized sample are separated from each other depending upon their interaction with the column packing. The column is operated at temperature T2. The resolution efficiency obtained with a particular column is dependent of this temperature T2. A detector is placed at the end of the column. The separated solutes reach the detector individually.

The detector then produces the electrical signals corresponding to each solute quantity in the sample. This detector output is connected to the strip chart recorder which plots the signals amplitude versus time. This plot is known as gas chromatograph. From this gas chromatograph the components of the mixture and their concentration is identified.

A typical gas chromatograph is as shown below:

strip chart gas chromatograph
strip chart gas chromatograph

Gas chromatography Applications:

  1. Using gas chromatography, substances that vaporize below ca. 300 °C (and therefore are stable up to that temperature) can be measured quantitatively.
  2. Gas chromatography can be used to analyze content of chemical product like measuring the quality of products in the chemical industries.
  3. It can also be used for measuring toxic substances in soil, water or air.
  4. Gas Chromatography is used extensively in forensic science.

Related topics:

Liquid chromatography
Gas chromatography
Liquid Chromatograph (HPLC)
Flame ionization detector

Watch following video to understand what is gas chromatography? and Working principle of Gas chromatography. what is Gas chromatography gcse?


Working of Argon Ionization Detector

Argon ionization detector:

In this article we are going to learn how argon ionization detectors works?



Working of argon ionization detector:

For the argon ionization detector, argon gas is used as the carrier gas. Argon ionization detector consists of two electrodes placed parallel to each other. A potential difference is applied across them. The effluent from the column is allowed to enter the ionization detector.Now as the carrier gas is non conductive there is no current under normal conditions.

A radioactive source is placed in the approach region of electrodes. The rays emitted by the radioactive source excite the argon atoms and electrons are produced. Further under the influence of applied potential these electrons accelerate and collide with the argon atoms and raise them to metastable state. Such metastable state argon atom collides with argon atom from the effluent. Then these argon atoms form the effluents. Then these argon atoms are ionized and became conducting. This results in a current proportional to the ions. Thus this current is proportional to the quantity of organic compound in a sample.

This argon ionization detector is sensitive to most of the organic and insensitive to H20, oxygen, CO2 and hydrocarbon like methane.

Advantages of the argon ionization detector:

1)      Argon ionization detector is sensitive to most of the organic and inorganic compounds.

2)      Argon ionization detector has high linear dynamic range (10^5).

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Working of Electron Capture Detector

Electron capture detector:

This post provides an information about electron capture detector. We will also see the construction and working of electron capture detector.



Construction and working of electron capture detector:

The diagram shows electron capture detector used in chromatographic analysis of environmental samples. This detector operates similar to proportional counter used for X-ray measurement. In this detector, the effluent from the column is passed over a β emitter (Ni-63). An electron from β emitter causes ionization of carrier gas and production of a burst of electrons. In the absence of organic compounds, a constant standing current between a pair of electrodes results from this ionization process. However in presence of organic compounds the current decreases significantly. The response of this detector is nonlinear unless the applied potential across the detector is pulsed.

The electron capture detector is highly sensitive molecules containing electro-magnetive functional groups like halogens, peroxides, nitro groups; it is insensitive to the functional groups like amines, alcohols, and hydrocarbons. Hence it is a powerful tool in the determination of chlorinated insecticides.

Advantages of electron capture detector:

  • 1)      Electron capture detector is highly sensitive.
  • 2)      Non destructive to the sample.

Disadvantages of the electron capture detector:

  • 1)      Highly non linear response.
Electron capture detector
Electron capture detector
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Thermal conductivity detectors

Thermal conductivity detectors (TCD):

This post provides an information about thermal conductivity detector which are widely used in chromatography.



The diagram shows the cross sectional view of one of the temperature sensitive elements in a thermal conductivity detector. This detector is based upon the changes in the thermal conductivity of the gas stream. It is also called as katharometer. The sensing element in this device is an electrically heated element whose temperature at constant electrical power depends upon the thermal conductivity of the surrounding gas. The heated element may be a fine platinum, gold, tungsten, wire or semiconducting thermistor. The resistance of the wire measure of thermal conductivity of the gas.

The next diagram shows the arrangement of detector elements in a typical detector unit.

thermal conductivity sensor

In the above system, two pairs of elements are employed. One pair is located in the flow of effluent from the column and the other pair is located in the gas stream ahead of the sample injection chamber (reference). The resistances of the pairs are compared by incorporating them into two arms of the Wheatstone bridge.

The thermal conductivities of He and H2 are roughly 6 times greater than those of most of the organic compounds. Thus in presence of even small amounts of organic compounds, a relatively high decrease in thermal conductivity of the column effluent takes place and thus the detector undergoes a rise in temperature.

Advantages of the thermal conductivity detector:

  1.  Simplicity
  2.  Large linear dynamic range
  3.  General response to organic and inorganic samples
  4.  Non destructive character

Limitations of the thermal conductivity detector:

  1. Low sensitivity, which requires large sample size.
  2. Thermal conductivity detector is costly.

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Flame ionization detector

Flame ionization detector (FID):

Hi friends, today we will learn flame ionization detector and its working.


The diagram shows the flame ionization detector used in gas chromatography. It consists of a burner in which the effluent in the burner is mixed with hydrogen and air. This mixture is then ignited electrically. Most of the organic compounds when paralyzed at the temperature of H2/air flame, they produce ions and electrons which can conduct electrically through flame. A potential of few hundreds volts is applied across a burner tip and collector electrode located above the flame. The resulting current is then directed into a high impedance operational amplifier circuit for the measurement. Now as the number of ions produced in the flame is near about proportional to the number of carbon atoms, the current produced is also proportional to the carbon atoms in the sample.

Flame Ionization Detector
Flame Ionization Detector

The flame ionization detector is powerful detector for the analysis of organic samples because of insensitivity of flame ionization detector towards the functional groups like carbonyl, alcohol, halogen and amine as well as the noncombustible gases like H2O, CO2, NOx. It is particularly used for the detection of pollutants in natural water sample.

Advantages of flame ionization detector:

  • 1)      High sensitivity (10^-13 gm/sec.)
  • 2)      Large linear response range (10^7)
  • 3)      Low noise
  • 4)      Rugged and easy to use

Limitations of flame ionization detector:

1)      Destructive to the sample.

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