MEVE 018: Unit 02 - Basic Chromatography

-

UNIT 2: BASIC CHROMATOGRAPHY

2.0 Introduction

Chromatography is an essential analytical tool used extensively in chemical, biological, and environmental sciences. It allows the separation, identification, and quantification of components in a mixture based on their different interactions with a stationary and a mobile phase. This unit introduces the basic principles of chromatography, its classification, and the common chromatographic techniques used in laboratories.

2.1 Objectives

After studying this unit, learners will be able to:

  • Understand the fundamental principle of chromatography.
  • Classify various chromatographic techniques.
  • Describe the working of different types of chromatography.
  • Compare their applications in analytical chemistry.
  • Appreciate the importance of chromatography in environmental analysis and biochemical research.

2.2 Classification of Chromatographic Techniques

Chromatographic techniques can be classified based on several factors:

A. Based on Physical State of Mobile Phase:

  • Gas Chromatography (GC) – Uses gas as the mobile phase.
  • Liquid Chromatography (LC) – Uses liquid as the mobile phase.

B. Based on Mode of Separation:

  • Adsorption Chromatography – Separation based on differences in adsorption to the stationary phase.
  • Partition Chromatography – Based on solubility differences.
  • Ion Exchange Chromatography – Based on charge interactions.
  • Size Exclusion Chromatography – Based on molecular size.
  • Affinity Chromatography – Based on specific binding interactions.

C. Based on Technique Type:

  • Planar Chromatography – Stationary phase is on a flat surface (e.g., TLC, Paper Chromatography).
  • Column Chromatography – Stationary phase is packed in a column.

2.3 Thin Layer Chromatography (TLC)

Principle:

TLC separates compounds based on their differential movement on a stationary phase (usually silica gel) under the influence of a solvent (mobile phase).

Procedure:

  1. A small spot of the mixture is placed near the bottom of a TLC plate.
  2. The plate is placed in a solvent chamber.
  3. The solvent moves up the plate by capillary action, carrying the components with it.
  4. Different compounds move at different rates and appear as separate spots.

Applications:

  • Rapid analysis of organic compounds.
  • Monitoring chemical reactions.
  • Detecting pesticides or contaminants in food.

2.4 Paper Chromatography

Principle:

Relies on partitioning between water held in the cellulose fibers of the paper (stationary phase) and a mobile phase solvent.

Types:

  • Ascending Chromatography – Solvent rises up the paper.
  • Descending Chromatography – Solvent flows down.

Steps:

  1. Apply sample on filter paper.
  2. Dip the end in the solvent.
  3. Allow separation and visualize the spots using UV light or chemical reagents.

Applications:

  • Separation of amino acids, sugars.
  • Plant pigment analysis.

2.5 Gas Chromatography (GC)

Principle:

Volatile compounds are separated based on their partitioning between a gas mobile phase and a stationary phase inside a column.

Components:

  • Carrier Gas (e.g., Helium, Nitrogen).
  • Injector.
  • Column (packed or capillary).
  • Detector (e.g., Flame Ionization Detector – FID).
  • Data processor.

Applications:

  • Environmental monitoring (e.g., air pollutants).
  • Analysis of essential oils and hydrocarbons.
  • Drug testing and forensic science.

2.6 Ion Exchange Chromatography

Principle:

Separation is based on reversible exchange of ions between charged resin beads and ions in solution.

Types:

  • Cation Exchange – Binds positive ions.
  • Anion Exchange – Binds negative ions.

Procedure:

  1. Load sample onto column packed with ion exchange resin.
  2. Elute ions using a gradient of increasing ionic strength or pH.

Applications:

  • Purification of proteins and nucleotides.
  • Water softening and desalination.
  • Analysis of soil and water minerals.

2.7 Size Exclusion Chromatography (SEC)

Principle:

Molecules are separated based on their size. Larger molecules elute first as they are excluded from the pores of the stationary phase.

Stationary Phase:

Porous beads (e.g., dextran, agarose).

Applications:

  • Separation of proteins, polysaccharides.
  • Determination of molecular weight.
  • Polymer analysis.

2.8 Affinity Chromatography

Principle:

Based on specific interactions between a ligand and a target molecule (e.g., antibody-antigen, enzyme-substrate).

Procedure:

  1. Ligand is immobilized on a solid support.
  2. Target binds specifically to ligand while other substances are washed away.
  3. Elution with competitive molecule or change in pH.

Applications:

  • Purification of enzymes, antibodies, receptors.
  • Studying molecular interactions.
  • Diagnostic and therapeutic product preparation.

2.9 Let Us Sum Up

Chromatography is a powerful technique used for separating and analyzing components in complex mixtures. Various chromatographic methods like TLC, paper chromatography, GC, ion exchange, SEC, and affinity chromatography offer diverse options based on physical and chemical principles. These techniques are vital in environmental analysis, biochemical research, pharmaceuticals, and industrial processes. Understanding their principles and applications enables precise and efficient analysis of organic and inorganic compounds.

Keywords

  • Chromatography-Analytical technique for separating components of a mixture.
  • Stationary Phase-The fixed phase that interacts with components in a sample.
  • Mobile Phase-The fluid that carries the sample through the stationary phase.
  • TLC (Thin Layer Chromatography)-Separation on a thin layer of adsorbent like silica gel.
  • Paper Chromatography-Separation using a paper as the stationary phase.
  • Gas Chromatography (GC)-Technique using gas as a mobile phase to separate volatile substances.
  • Ion Exchange Chromatography-Separation based on ionic charges using resins.
  • Size Exclusion Chromatography-Separation based on molecular size using porous beads.
  • Affinity Chromatography-Highly specific separation using biological interactions (e.g., ligand binding).

Comments

Post a Comment

Popular Posts

Jcert Class 8 Daffodil Chapter 1a: The Naive Friends Solutions

-
Chapter 1a : The Naive Friends Answer the following questions: 1. Which questions made Keshav and Shyama curious about the egg? Ans.         How did the chick come out? How big would the eggs? How many? What colours would they? What did they eat? Such types of questions made Keshav and Shyama curious about the egg. 2. What three things did the children do to protect the eggs? Ans.         The children done three things are: (i)          Adjustment of food. (ii)        Adjustment of water. (iii)      Adjustment of roof. 3. Why did Keshav think of making a cushion for the eggs? Ans.         When Keshav saw the egg on direct sun light, he thaught making a cushion for the eggs. 4. What excuse did Keshav give for not showing the eggs to Shyama? Ans.     ...
Read more

Jcert Class 8 भाषा मंजरी Chapter 3 मित्रता Solutions

-
अध्याय - 3 : मित्रता 1. आप किस प्रकार कह सकते हैं कि मित्रों के चुनाव की उपयुक्तता पर हमारे जीवन की सफलता निर्भर करती है? उ. मित्रों के चुनाव की उपयुक्तता पर हमारे जीवन की सफलता निर्भर करती है क्योंकि संगति का गुप्त प्रभाव हमारे आचरण पर पड़ता है और यदि हमें अच्छी संगति, अच्छे मित्र मिलते हैं तो वे हमें सफलता की ओर ले जाते हैं जबकि बुरी संगति हमारे जीवन को खाक में मिला देती है।  2. 'विश्वासपात्र मित्र जीवन की एक औषध है।' आशय स्पष्ट कीजिए। उ. 'विश्वासपात्र मित्र जीवन की एक औषध है'- ऐसा इसलिए कहा गया है कि यदि हमें विश्वासी मित्र मिल गया तो हम अपने जीवन में चाहे जैसा भी कदम उठाएँ, उस कदम की आलोचना करके वह हमें सही दिशा की ओर उन्मुख करेगा। हम अपनी गलती रूपी बीमारी को उसकी आलोचना रूपी औषधि से दूर कर सकेंगे।  3. मित्र का चुनाव करते समय हमें किन बातों का ध्यान रखना चाहिए? उ. मित्र का चुनाव करते समय हमें निम्नलिखित बातों का ध्यान रखना चाहिए हमारे अच्छे विचारों (संकल्पों) को दृढ़ करने वाला हो। हमें दोषों और त्रुटियों से बचाए। हमारे सत्य, पवित्रता ...
Read more

Jcert Class 8 भाषा मंजरी Chapter 8 अमरूद का पेड Solutions

-
अध्याय - 8 : अमरूद का पेड 1. लेखक को क्यों लगता है कि इन सब पिछड़े खयालातों का हमारे घर में गुजर नहीं हो सकेगा। उ. अमरूद के पेड़ को देखकर सबसे पहले बाबू कन्हैयालाल की बूढ़ी पत्नी ने एक दिन टोका कि पश्चिम की तरफ अगर मकान का मुखड़ा हो और सामने ही अमरूद का पेड़ तो 'राम-राम बड़ा अशुभ होता है और माँ के चेहरे पर थोड़ा-सा भय दिखा पर लेखक को विश्वास था कि इन सब पिछड़े खयालातों का हमारे घर में गुजर नहीं हो सकेगा क्योंकि उसके घर के लोग आधुनिक विचारधारा के थे उसकी माँ सत्याग्रह के दिनों में अजमेर में लम्बा जेल कर चुकी है। पिताजी खुद राजनैतिक सामाजिक उदारता वाले आदमी थे। बुआ ने विवाह नहीं किया था, पढ़ने-लिखने में ही अपनी सम्पूर्ण जिन्दगी लगा दी और समाज कोई चुनौती देने का साहस नहीं कर सका। साथ ही परिवार के सभी लोगों में पिछड़ेपन के प्रति भर्त्सना का भाव था। 2. अमरूद के पेड़ की छाया किस प्रकार लेखक को उनके भाई-बहनों के साथ जोड़ती है ? उ. अमरूद के पेड़ की छाया लेखक को उसके भाई-बहनों के साथ जोड़ती है। वे सभी अमरूद की छाया में बेंत की कुर्सियों पर बैठ चर्चाएँ करते थे जिससे उनके बीच...
Read more