Chromatographic techniques

Table of Contents

• Introduction and Background
• Chromatographic techniques and traditional techniques
• Why chromatographic techniques are required for analysis?
• Traditional techniques vs chromatographic techniques
• Advantages of chromatographic techniques
• Applications of chromatographic techniques
• Conclusion

Introduction and Background

Chromatographic techniques are the backbone of pharmaceuticals development. Pharmaceuticals development is impossible without the use of chromatographic techniques. However, most of the chemists don’t know why chromatographic techniques are widely used in pharma industries. That is why I decided to share my skill-based knowledge on ” Need for Chromatographic techniques”. Having read this article all your doubts will be cleared and you will be able to answer questions like:

• What is chromatographic techniques?
• What are the different types of chromatographic techniques?
• Why chromatographic techniques are widely used in the Pharmaceutical industry?
• Which chromatographic techniques are widely used in pharma industries?
• What are the differences between chromatographic techniques and traditional techniques?
• Why pharmaceuticals development is not possible using traditional techniques?
• What are the advantages of chromatographic techniques?

Chromatographic techniques and traditional techniques

Traditional techniques

The following techniques are used as traditional techniques

• Chemical method of analysis and
• Spectroscopic method of analysis

The above methods are not specific and selective; it means the result obtained by the above method may or may not be correct. That is why pharmaceutical development is impossible using these traditional techniques.

Chemical method

Generally following volumetric titrations are widely used for the pharmaceuticals analysis:

• Acid base titration
• Oxidation -reduction titration
• Iodometric titration
• Iodimetric titration
• Sodium nitrite titration
• Complexometric titration
• Non aqueous titration or perchloric titration

Note: Gravimetric methods are not correct that is why this technique is rarely used in the pharmaceutical analysis

Spectroscopic methods

Generally following spectroscopic methods are widely used for the pharmaceuticals analysis:

• Ultraviolet spectroscopy (UV) and
• Infrared spectroscopy (IR)

Chromatographic techniques

The term chromatography was given by Tswett in 1906, he also published some papers on liquid chromatography. In 1941, Martin and Synge tried to separate amino acids in wool samples using a column packed with silica gel and a mixture of Butanol and chloroform as a mobile phase. He also explained the theoretical aspect of procedure and got Nobel Prize in 1952. Since that time LC technique has become a versatile technique available for chemists because of its simplicity and capacity for high resolution.

Types of chromatographic techniques:

• Complete chromatography
• Continuous chromatography

Complete chromatography

Following techniques come under the complete chromatography:

• Thin layer chromatography or TLC and
• Paper chromatography or PC

Thin layer chromatography or TLC

It is widely used in industries for qualitative analysis during drug development e.g. identification tests, related substances tests, purity tests, monitoring the reaction (during drug development) etc. This technique is specific and selective and accepted by all regulatory agencies.

Acceptance criteria of TLC

The Rf value of the principal spot obtained from the test solution correspond to that obtained from the standard solution

Paper chromatography or PC

It is used in industries for qualitative analysis e.g. identification tests, related substances tests, purity tests etc. This technique is specific and selective and accepted by all regulatory agencies. This technique is specific and selective and accepted by all regulatory agencies.

Acceptance criteria of PC

The Rf value of the principal spot obtained from the test solution correspond to that obtained from the standard solution

Continuous chromatography

Following techniques are widely used in the continuous chromatography

• High – pressure liquid chromatography or HPLC
• Gas chromatography or GC

Both HPLC and GC are specific and selective methods and are widely used in the industries for both qualitative and quantitative analysis.

Why chromatographic techniques are required for analysis?

Case study-1

Let us consider a sample containing 4 – methyl benzoic acid and 2 – methyl benzoic acid in the ratio of 50:50. How content each will be calculated?

Using chemical method

Since both 4 – 4-methyl benzoic acid and 2 – methyl benzoic acid are acids hence can be estimated by using acid-base titration. Since both molecules 4 – methyl benzoic acid and 2 – methyl benzoic acid contain -COOH group and hence both molecules will be titrated simultaneously. Hence the correct result will not be obtained and consequently traditional /chemical titration method is not suitable for such molecule

Using chromatographic/HPLC method

Since the chromatographic method is selective and specific and hence one can get two peaks (about 50% each) in the chromatogram. That is why the chromatographic method is correct for such molecules

Case study-2

Let us consider a sample contains phenol and Benzoic acid in ration of 10 :90. How content each will be calculated using traditional method and chromatographic method?

Using chemical method

Phenol contains the -OH group and Benzoic acid contains -COOH group and hence can be estimated by acid-base titration. But acid base titration will not give the correct result because each -OH group and -COOH group will be titrated simultaneously. Hence it will be difficult to distinguish between each and consequently correct results will not be obtained. Therefore, the traditional /chemical titration method is not suitable for such molecules

Using chromatographic/HPLC method

Since chromatographic method is selective and specific in nature and hence one can get two peaks (about 10% Phenol and 90% for Benzoic acid) in the chromatogram. That is why chromatographic method is correct for such molecules

Traditional techniques vs chromatographic techniques

Advantages of chromatographic techniques

• Specific and selective. It means there is not any interference/merging of peaks
• Helpful in controlling the quality of drug at different stages
• Accepted by all regulatory agencies
• Method can be easily validated

Applications of chromatographic techniques

• Identification test
• Assay
• Related substances
• Chiral purity
• Content test

Conclusion

The chromatographic technique is the backbone of pharmaceutical analysis. That is why it is widely used in the industries. Now I hope all your doubts related to the use of chromatographic technique and traditional techniques have been cleared. For any doubts or questions related to this post write in the comment section and I will answer the same on a priority basis.

References:

• IP
• USP

Abbreviations’

• TLC: Thin layer chromatography
• PC: Paper chromatography
• GC: Gas chromatography
• HPLC: High pressure liquid chromatography