instrumental methods in spice industry

INSTRUMENTATION

1.SPECTROPHOTOMETER:

1. CURCUMIN (Colour value)

PROCEDURE:

Weigh 0.2 gram turmeric powder in a 250 ml. RB flask, Add 75 ml acetone. Cover the RBF with aluminium foil paper to prevent light absorption. Reflux for 1 hour in a mantle using water condenser at 10^oC. Then cotton filter to a 250 ml. dark standard flask and make up with acetone. Then bowl pipette 5 ml to 50 ml standard flask and make up using acetone.

The Curcumin content is detected by spectrophotometer it contains two quvettes, one for acetone and one for solution which is used to make up the solution. The wave length of Curcumin is 420 nm. Adjust to that wave length and make the reading to 0. Then find the value of solution.

CALCULATION:

Absorbance x dilution factor

Factor derived from Curcumin standard x wt. of sample taken

2. PIPERINE (Pepper)

PROCEDURE:

Take 0.5 gm. Sample in 250ml RBF. Add 70 ml SDA No.3A denatured alcohol. (i.e. 20: 1 - ethanol: methanol solution) the flask is covered with aluminium foil. Reflex for one hour at 20^oC allow cooling to room temperature and making upto 250 ml. Use extraordinary filter paper and 250 ml amber colour std. flask. From this solution pipette 2 ml and make upto 50 ml. It is filled in cuvette and kept in spectrophotometer and the reading is noted at the wavelength of 342 nm.

CACULATION:

Absorbance x dilution factor

Std. of Piperine x weight of sample

3. CAPSANTHIN (Colour value)

PROCEDURE:

Accurately weigh 1 gm of ground chilly sample into a 100 ml volumetric flask. Add 75 ml extra pure acetone into this flask. Keep this inside a dark cupboard for 16 hours or overnight to allow the particles to settle. Take 5 ml into 100 ml volumetric flask. Make up with acetone and transfer a portion of the extract using Millipore and pour into quvette and keep in spectrophotometer and measure the absorbance (A) at 460 nm with acetone.

CALCULATION:

Absorbance x 60.4 x 10

Weight of sample taken

2.CHROMATOGRAPHY

OPERATION:

The sample to be analyzed is introduced in small volume to the stream of mobile phase. The analyte's motion through the column is slowed by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time; the retention time under particular conditions is considered a reasonably unique identifying characteristic of a given analyte. The use of smaller particle size column packing (which creates higher backpressure) increases the linear velocity (speed) giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. 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 analyte components, or compounds such as trifluoroacetic acid which acts as an ion pairing agent.

1. AFLATOXIN (CHILLY)

INSTRUMENT CONFIGRATION:

· Shimadzu HPLC with following configuration

· Liquid chromatograph pump

· Fluorescent detector

· Rheodine injector

· c- 18 250 mm 170 Armstrong silica column

· CMB- 20 alite system controller

· Data processor- LC solution

INSTRUMENT CONDITION:

· Mobile phase flow rate: 1.2 ml/min

· Total run time : 20 min

· Temperature : 25⁰C

MOBILE PHASE:

· Ethanol: Water (45:55)

AIM:

To detect the presence of Aflatoxin in chilly

PROCEDURE:

Take 25 gm sample and 5 gm sample into the blending jar. Add 100 ml 80% methanol. This is blended for 80 sec, filtered through fluted filter paper. From this solution 100 ml passes through immuno affinity column. 20 ml water washing, elute with 1ml methanol. This is collected in cuvette (1 ml). This colour solution is injected in HPLC and a graph is obtained which shows the absence or presence of Aflatoxin.

2. CAPSAICIN (Pungency)

INSTRUMENT CONDITION:

· Mobile phase flow rate: 1 ml/min

· Total run time : 20 min

· Temperature : 25⁰C

MOBILE PHASE:

· Acetonitrile: Water: Acetic acid

AIM:

To find the percentage of capsaicin in chilly powders

PROCEDURE:

Take 12.5 gm of chilly powder into a RBF and add 100 ml alcohol. Reflux for 3 hours in multimantle. Cool at room temperature. Filter it using whattmann No.1 filter paper, and then using Millipore. It is a device which has two kinds of filters. Collect the sample and inject through the inject port system.

3. SUDAN DYE (Adulteration)

INSTRUMENT CONFIGRATION:

· Shimadzu HPLC with following configuration:

· Liquid chromatograph pump

· UV-Visible detector

· Rheodine injector

· SCL-10 A VP system controller

· C- 18 250 nm 100 Armstrong 5 micron silican column

· Data processor- LC solution

INSTRUMENT CONDITION:

· Mobile phase flow rate: 1 ml/min

· Total run time : 20 min

· Temperature : 25⁰C

MOBILE PHASE:

· Acetonitrile: Water (95:5)

· Water: Acetic acid (100:16.5)

AIM:

To detect the presence of Sudan dye on chilly

PROCEDURE:

Take 10 gm sample into 250 ml conical flask then add 100 ml CH₃CN. This solution is stirred for 1 hour using magnetic stirrer. Then filter through whattmann No.1 filter paper from this solution pipette 5 ml make up to 50 ml using acetonitrile. Then it Millipore filtered. Take this sample and inject into the HPLC. A graph is obtained which shows the absence or presence of Sudan dye.

GAS CHROMATOGRAPHY

Gas-liquid chromatography (GLC), or simply gas chromatography (GC), is a type of chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen, and the stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside glass or metal tubing, called a column. The instrument used to perform gas chromatographic separations is called a gas chromatograph (also: aerograph, gas separator).

Gas Chromatography is different from other forms of chromatography (HPLC, TLC, etc.) because the solutions travel through the column in a gas state. The interactions of these gaseous analyte with the walls of the column (coated by different stationary phases) causes different compounds to elute at different times called retention time. The comparison of these retention times is the analytical power of GC. This makes it very similar to high performance liquid chromatography.

GC analysis

A gas chromatograph is a chemical analysis instrument for separating chemicals in a complex sample. A gas chromatograph uses a flow-through narrow tube known as the column, through which different chemical constituents of a sample pass in a gas stream (carrier gas, mobile phase) at different rates depending on their various chemical and physical properties and their interaction with a specific column filling, called the stationary phase. As the chemicals exit the end of the column, they are detected and identified electronically. The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time (retention time). Other parameters that can be used to alter the order or time of retention are the carrier gas flow rate, and the temperature.

In a GC analysis, a known volume of gaseous or liquid analyte is injected into the "entrance" (head) of the column, usually using a micro syringe (or, solid phase micro extraction fibers, or a gas source switching system). As the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the adsorption of the analyte molecules either onto the column walls or onto packing materials in the column. The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the analyte mixture are separated as they progress along the column and reach the end of the column at different times (retention time). A detector is used to monitor the outlet stream from the column; thus, the time at which each component reaches the outlet and the amount of that component can be determined. Generally, substances are identified (qualitatively) by the order in which they emerge (elute) from the column and by the retention time of the analyte in the column.