Factors Affecting Resolution in Chromatography

 

As we all know, Chromatography is a science of separation of mixtures into its components. There are several types of chromatography, each differing in the kind of stationary phase and mobile phase they use. There are many chromatographic techniques like HPLC, HPTLC and GC.

Let us understand what is the resolution in Thin Layer Chromatography?

Many times we use the term ‘Resolution’ but what exactly does it mean in TLC/HPTLC?      In Chromatography resolution is ability (of the method) to separate two peaks. In Thin Layer Chromatography or High-Performance Thin Layer Chromatography, as it is a visual technique, we can also say it is the ability of the method to separate two bands or two peaks. Resolution is termed good when there is a difference of Rf 0.05 between two bands. Ideal resolution is considered when there is base to base separation between adjacent bands.

What are the factors affecting resolution in high performance thin layer chromatography?

HPTLC is modern version of TLC wherein the principle remains the same but the main difference between TLC and HPTLC is, HPTLC practice is fully automated and GLP/USP/EP compliant.

Factors to consider while using HPTLC

CAMAG HPTLC system has diverse applications in many fields like herbal for drug analysis, pharmaceutical analysis, food analysis and cosmetic ingredients analysis.

While doing HPTLC, following factors should be considered that can affect resolution and play major role in developing suitable/good method for specific sample.

• Sample preparation/Extraction procedure optimization:

Sample preparation is one of the foremost important steps in chromatography. For analysis of botanicals, the procedure is standardized by USP (United States Pharmacopoeia). Sometimes modification is necessary depending on aim of analysis.

e.g., Alkaloids may require addition of few drops of conc. ammonia for higher extraction efficiency and improved detection limits. Amino acids and sugars in sample may require acid hydrolysis i.e., addition of conc. HCl to the sample. Oil samples should be dissolved in nonpolar solvent like hexane, toluene or dichloromethane etc.

• Application pattern:

Application pattern significantly affects resolution in HPTLC. USP chapter 1064 has defined parameter selection pattern like humidity control, band length, chamber configuration, solvent front etc. which when followed as per guidelines ensures reproducible results.

• Concentration and application volume:

While working on botanicals, USP set protocol should be followed for sample preparation as far as possible. If the same is not working, concentration and volume should be optimized or sample preparation methods can be modified in order to achieve good resolution.

Concentration and volume play a significant role in achieving resolution. If the plate is overloaded with sample volume/conc., it will show poor separation although the mobile/stationary phase or other chromatographic conditions are suitable.

• Choice of mobile phase:

Depending on the analyte chemistry mobile phase should be selected/optimized. Polarity of the sample/analyte of interest should be checked or if not known it should be run in different polarity mobile phases to check the polarity nature of the sample to be tested.

Polar, midpolar and nonpolar mobile phases can be selected first to check the polarity of the sample and accordingly it can be optimized depending on the response of the analyte in these respective mobile phases.

• Choice of Stationary phase:

Stationary phase should be selected depending on the nature of the mobile phase and aim of the analysis. For most of the samples, Silica gel 60F254 plates do work but as per need, stationary phase can be changed. E.g., For sugar analysis, cellulose plates can be used. Analysis of anthocyanins, oil samples can be done on RP (Reverse phase) plates.

• Saturation time: 

As per USP protocol saturation should be given for at least 20 minutes or while doing method development trials can be taken without saturation as well. Sometimes resolution achieved is better when there is no saturation at all. This is part of R & D during method development.

• Selection of Derivatizing reagent and process:

If the sample/analyte does not show any response in UV or fluorescence, a suitable derivatizing reagent should be selected in order to detect the analyte. After analysis, images can be recorded at 254 nm, 366 nm and in white light using a suitable HPTLC photo documentation system like CAMAG visualizer.

There are universal and specific derivatizing reagents available for different components/analytes. According to suitability /nature of sample derivatizing reagent should be selected. Derivatization can be done by dipping or spraying technique. Technique used also affects results of analysis.

Generally, in manual spraying, if not done properly, due to excess reagent dripping occurs and the analyte band is sometimes disturbed or washed away with excess reagent. Thus, the right choice of derivatizing reagent and derivatization technique affects the chromatographic results.

Anchrom Enterprises Pvt. Ltd is one of the leaders in HPTLC analysis. Please contact us at lab@anchrom.in for HPTLC analysis of plant extracts, drugs, ingredients in cosmetics, and forensic science.