Etiket Arşivleri: PRECIPITATION

Gravimetric Analysis and Precipitation Equilibria

Gravimetric Analysis and Precipitation Equilibria

Dr. A.K.M. Shafiqul Islam & Dr. Zarina Zakaria


The term gravimetric pertains to a Weight Measurement.

Gravimetric method is one in which the analysis is completed by a weighing operation.

Gravimetric Analysis is a group of analytical methods in which the amount of analyte is determined by the measurement of the mass of a pure substance containing the analyte.

Gravimetric Methods can also be defined as quantitative methods based on the determining the mass of a pure compound to which the analyte is chemically related.

Example for Precipitation:-

Calcium can be determined gravimetrically by precipitation of calcium oxalate and ignition of the oxalate ion to calcium oxide.

  Ca2+  +  C2O42- →CaC2O4

  CaC2O4 → CaO  + CO2  + CO

The precipitate thus obtained are weighed and the mass of calcium oxide is determined.

Example for Volatilisation:-

The analyte or its decomposition products are volatilised at a suitable temperature. The volatile product is then collected and weighed, i.e. the mass of the product is indirectly determined from the loss in mass of the sample.


Water can be separated from most inorganic compounds by ignition, the evolved water can then be absorbed on any one of several solid desiccants. The weight of water evolved may be calculated from the gain in weight of the absorbent.

Gravimetric Analysis

Gravimetric analysis is potentially more accurate and more precise than volumetric analysis.

Gravimetric analysis avoids problems with temperature fluctuations, calibration errors, and other problems associated with volumetric analysis.

But there are potential problems with gravimetric analysis that must be avoided to get good results.

Proper lab technique is critical

Steps in a Gravimetric Analysis

1.Preparation of the solution





6.Drying or ignition



Downstream Processing


Bioseparation and Downstream processing cost:

(1) 50%  (drug, protein, biological product)

(2) 80% (recombinant protein)

Ratio of recovery to fermentation cost

(1)  2.0 (enzyme)

(2)  1.0 penicillin

(3)  0.16 (ethanol)


Parameters to consider in selecting downstream processes


Plate and filter press

most suitable for fermentation broths with a low solids content and low resistance to filtration.

used as a ‘polishing‘ device in breweries to filter out residual yeast cells following initial clarification by centrifugation or rotary vacuum filtration.

It may also be used for collecting high value solids.

Because of high labour costs and the time involved in dismantling, cleaning and reassembly, not be used when removing large quantities of worthless solids from a broth.

increased temperature would lower media density but is of little practical use with fermentation broths), the diameter of the cells (could be increased by coagulation/flocculation) and the viscosity of the liquid.

In practice, the cells are usually very small, of low density and are often suspended in viscous media.

Liquid shear

Liquid shear is widely used in large scale enzyme purification

Dures. High-pressure homogenizers used in the processing of milk and other products the food industry have proved to be very effective microbial cell disruption

Solid shear

Pressure extrusion of frozen micro-organisms at around – 25 C through a small orifice.

Disruption is due to a combination of liquid shear through a narrow orifice and the presence of ice crystals.

It was possible to obtain 90% disruption (single passage of S. Cerevisiae, 10 kg yeast cell paste h -1.

This technique might be ideal for microbial products which are very temperature labile.

Agitation with abrasives

Mechanical cell disruption can also be achieved in a disintegrator containing a series of rotating discs and a charge of small beads.

The beads are made of glass, alumina ceramics and some titanium compounds

Dissipation of heat generated in the mill is one of the major problems in scale up ( use a cooling jacket.)

Freezing and thawing

Freezing and thawing of a microbial cell paste will inevitably cause ice crystals to form and their expansion followed by thawing will lead to some subsequent disruption of cells.

It is slow and has not often been used as a technique on its own( used in combination with other techniques. ) ( ie α-Glucosidase from S. Cerevisiae)


High frequency vibration (- 20 kHz) at the tip of an ultrasonication probe leads to cavitation, and shock waves thus produced cause cell disruption. very effective on a small scale, unsuitable for large-scale operations.

Power requirements are high, there is a large heating effect so cooling is needed, the probes have a short working life and are only effective over a short range.

Osmotic shock

Osmotic shock caused by a sudden change in salt concentration will cause disruption of a number of cell types.

the effect on microbial cells is normally minimal.

a successful technique for the extraction of luciferase from Photobacterium fischeri

(provided that the desired enzyme will tolerate a pH of 11.5 to 12.5 for 20 to 30 minutes.)

Used to extract L-asparaginase

Enzyme treatment

There are a number of enzymes which hydrolyse specific bonds in cell walls of a limited number of micro-organisms.

Enzymes shown to have this activity include lysozyme (from  hen egg white, commercially available)and enzyme extracts from leucocytes,

one of the most gentle methods available,

unfortunately it is relatively expensive and the presence of the enzyme(s) may complicate futher downstream purification processes.

Enzymes may also be used as a pretreatment to partially hydrolyse cell walls prior to cell disruption by mechanical methods.


chromatographic techniques are used to isolate and purify relatively low concentrations of metabolic products.

Depending on the mechanism by which the solutes may be differentially held in a column, the techniques can be grouped as follows:

(a) Adsorption chromatography.

(b) Ion-exchange chromatography.

(c) Gel permeation chromatography.

(d) Affinity chromatography.

(e) Reverse phase chromatography.

(f) High performance liquid chromatography.

Ultrafiltration, solutes of high molecular weight are retained when the solvent and low molecular weight solutes are forced under hydraulic pressure (around 7 atmospheres)through a membrane of a very fine pore size.

examples: for the recovery of bio-molecules: viruses, enzymes,Antibiotics.

Reverse osmosis, the solvent molecules are forced by an applied pressure to flow through a semi-permeable membrane in the opposite direction to that dictated by osmotic forces, and hence is termed reverse osmosis.

It is used for the concentration of smaller  molecules than is possible by ultrafiltration.

Concentration polarization is again a problem and must be controlled by increased turbulence at the membrane surface.

Liquid membranes

Liquid membranes are insoluble liquids (e.g. an organic solvent) which are selective for a given solute and separate two other liquid phases.

Extraction takes place by the transport of solute from one liquid to the other.


(a) Large area for extraction.

(b) Separation and concentration are achieved in one step.

(c) Scale-up is relatively easy.

Used in lactic and citric acid extractions


The drying of any product (including  biological products) is often the last stage of a manufacturing process

-spray dryer( commonly used , economic)

-drum dryer-

-fluidized bed dryer ( common in pharmaceutical ind)

-freeze dryer( suitable for heat sensitive materials)



Gravimetric Analysis and Precipitation Equilibria


1. Precipitation gravimetry (oldest)

2. Electrogravimetry

3. Volatilization gravimetry and Thermogravimetry

4. Gravimetric titrimetry

5. Particulate gravimetry

When signal is mass of a precipitate, the method is called precipitation gravimetry. For example, determination of Cl– by precipitating it as AgCl.

Electrogravimetry: the analyte is deposited on one electrode in an electrochemical cell. For example

• oxidation of Pb2+, and its deposition as PbO2 on a Pt anode or

• reduction of Cu2+ to Cu and its electrodeposition on a Pt cathode, for direct analysis for Cu2+.

Volatilization gravimetry: when thermal or chemical energy is used to remove a volatile species. For example, determining moisture content using thermal energy to vaporize H2O. Also carbon and hydrogen in an organic compound may be determined by combustion with O2 to CO2 and H2O.

Gravimetric titrimetry, mass of titrant intead of its volume is measured. (Mass measurements are much more accurate and precise)

Finally, in particulate gravimetry the analyte is determined following its re-moval from the sample matrix by filtration or extraction. The determination of sus-pended solids is oneexample of particulate gravimetry.

Precipitation Gravimetric Analysis

• Gravimetric Analysis – one of the most accurate and precise methods of macro-quantitative analysis.

• Analyte is selectively converted to an insoluble form and precipitated quantitatively from its solution.

• Precipitate is treated to make it easily filterable and then filtered, dried and finally its mass is measured.

• Analyte mass is then calculated on the basis of the chemical composition of ppt and its mass.

Why use gravimetric analysis?

– Conducted with simple apparatus.

– Interpretation of results is easy – readings are directly related to analyte amount.

– Provides very accurate and precise results – in fact gravimetric results are used to check the accuracy of other methods.