INSTRUMENTAL TECHNIQUES IN FOOD ANALYASIS : High Performance Liquid Chromatography.

 High Performance Liquid Chromatography.

Objective: To study instrumentation, Working and applications of HPLC in Food Analysis.

Introduction:

High performance liquid chromatography or commonly known as HPLC is an analytical technique used to separate, identify or quantify each component in a mixture.

The mixture is separated using the basic principle of column chromatography and then identified and quantified by spectroscopy.

In the 1960s the column chromatography LC with its low-pressure suitable glass columns was further developed to the HPLC with its high-pressure adapted metal columns.

HPLC is thus basically a highly improved form of column liquid 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.

Principle:

The separation principle of HPLC is based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase (packing material of the column). Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary phase. The component which has more affinity towards the adsorbent, travels slower. The component which has less affinity towards the stationary phase travels faster. Since no 2 components have the same affinity towards the stationary phase, the components are separated.

Types of HPLC

A. Based on modes of chromatography

1.   Normal phase mode
2.   Reverse phase mode

B. Based on principle of separation

1.  Adsorption chromatography
2.  Ion exchange chromatography
3.  Ion pair chromatography
4.  Size exclusion(or)Gel permeation chromatography
5.  Affinity chromatography
6.  Chiral phase chromatography

 E. Based on the type of analysis

1.  Qualitative analysis
2.  Quantitative analysis

INSTRUMENTATION

Components of HPLC consists of:

1.  Solvent reservoir (Mobile phase)

2.  High pressure pump

3. Sample inlet device

4. Column

5.  Detector

6.  Recorder

1.  Solvent reservoir (Mobile phase): The solvent system or mobile phase used in HPLC is usually a mixture of an organic solvent (e.g. Methanol, IPA and water). The mobile phase in HPLC refers to the solvent being continuously applied to the column or stationary phase. The mobile phase acts as a carrier to the sample solution.

A sample solution is injected into the mobile phase of an assay through the injector port. As a sample solution flows through a column with the mobile phase, the components of that solution migrate according to the non-covalent interaction of the compound with the column. The mobile phase must be filtered (to prevent solods  from depositing at the column head) and degassed (to remove air bubbles).

Characteristics of mobile phase: it should possess following characteristics:

·       It should be pure

·       Having low viscosity

·       Should be chemically inert

·       Having low price

·       Compatible with detector

·       Having good solubility with the sample

 2 .  The Pump

The development of HPLC led to the development of the pump system. The role of the pump is to force a liquid (mobile phase) through the liquid chromatograph at a specific flow rate a pump can deliver a constant mobile phase. The pump is positioned in the most upper stream of the liquid chromatograph system and generates a flow of eluent from the solvent reservoir into the system. High- pressure generation is a “standard” requirement of pumps besides which, it should also to be able to provide a consistent pressure at any condition and a controllable and reproducible flow rate. Most pumps used in current LC systems generate the flow by back-and-forth motion of a motor-driven piston (reciprocating pumps). Because of this piston motion, it produces “pulses”.

The solvents or mobile phases used must be passed through the column at high pressure at about 1000 to 3000 psi this is because as the particle size of stationary phase is around 5-10µ, so the resistance to the flow of solvent is high.

3. Sample injection device: 

     An injector is placed next to the pump. The simplest method is to use a syringe, and the sample is introduced to the flow of eluent. The most widely used injection method is based on sampling loops.

The use of the autosampler (auto-injector) system is also widely used that allows repeated injections in a set scheduled-timing.

The injector serves to introduce the liquid sample into the flow stream of the mobile phase. May be by auto-sampler or manual.

4.   Degasser

•   The eluent used for HPLC analysis may contain gases such as oxygen that are non-visible to our eyes. When gas is present in the eluent, this is detected as noise and causes an unstable baseline.

• Degasser uses special polymer membrane tubing to remove gases. The numerous very small pores on the surface of the polymer tube allow the air to go through while preventing any liquid to go through the pore.

5.  Column

Column Of  HPLC

          The heart of a HPLC system is the column. The column contains the particles that              contains the stationary phase. The mobile phase is pumped through the column by a              pump. Solvents must be degassed to eliminate formation of bubbles.

The main separation is performed inside the column. The recent columns are often prepared in stainless steel housing, instead of glass columns.

The packing material generally used is silica or polymer gels compared to calcium carbonate. The eluent used for LC varies from acidic to basic solvents. Most column housing is made of stainless steel since stainless is tolerant towards a large variety of solvents.

6.  Column Heater

The LC separation is often largely influenced by the column temperature.  In order to obtain repeatable results, it is important to keep consistent temperature conditions.

Also for some analysis, such as sugar and organic acid, better resolutions can be obtained at elevated temperatures (50 to 80°C). Thus columns are generally kept inside the column oven (column heater).

7.   Detector:

Separation of analytes is performed inside the column, whereas a detector is used to observe the obtained separation.

The composition of the eluent is consistent when no analyze is present. While the presenceof analyte changes the composition of the eluent. What detector does is to measure these differences. This difference is monitored as a form of an electronic signal. There are different types of detectors available.

      Advantages of HPLC

• Speed, high resolution, sensitivity

•  Efficiency

• Automation and accuracy

• Versatile and extremely precise when it comes to identifying and               quantifying chemical components.

• Complete recovery of sample

            Limitations of HPLC

·   Cost: Despite its advantages, HPLC can be costly, requiring large quantity of expensive organics.

     ·  Complexity

· HPLC does have low sensitivity for certain compounds, and some cannot be      detected as they are irreversibly adsorbed.

·  Volatile substances are better separated by gas chromatography.

         Applications of HPLC

Very applicable to separating substances like–

·       Amino acids

·       Proteins

·       Nucleic acids

·       Carbohydrates

·       Terpenoids

·       Antibiotics

·       Steroids

      Inorganic salts